Details of a Researcher - SUZUKI Hiromu

Papers - SUZUKI Hiromu

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LINC02154 promotes cell cycle and mitochondrial function in oral squamous cell carcinoma.

Takeshi Niinuma, Hiroshi Kitajima, Tatsuya Sato, Toshifumi Ogawa, Kazuya Ishiguro, Masahiro Kai, Eiichiro Yamamoto, Yui Hatanaka, Iyori Nojima, Mutsumi Toyota, Akira Yorozu, Shohei Sekiguchi, Noritsugu Tohse, Masato Furuhashi, Hiroshi Ohguro, Akihiro Miyazaki, Hiromu Suzuki

Cancer science 2024.11 [International journal]

Long noncoding RNAs (lncRNAs) play pivotal roles in the development of human malignancies, though their involvement in oral squamous cell carcinoma (OSCC) remains incompletely understood. Using The Cancer Genome Atlas (TCGA) dataset, we analyzed expression of 7840 lncRNAs in primary head and neck squamous cell carcinoma (HNSCC) and found that upregulation of LINC02154 is associated with a poorer prognosis. LINC02154 knockdown in OSCC cell lines induced cell cycle arrest and apoptosis, and significantly attenuated tumor growth in vitro and in vivo. Notably, depletion of LINC02154 downregulated FOXM1, a master regulator of cell cycle-related genes. RNA pulldown and mass spectrometry analyses identified a series of proteins that could potentially interact with LINC02154, including HNRNPK and LRPPRC. HNRNPK stabilizes FOXM1 expression by interacting with the 3'-UTR of FOXM1 mRNA, which suggests LINC02154 and HNRNPK promote cell cycling by regulating FOXM1 expression. Additionally, LINC02154 positively regulates HNRNPK expression by inhibiting microRNAs targeting HNRPNK. Moreover, LINC02154 affects mitochondrial function by interacting with LRPPRC. Depletion of LINC02154 suppressed expression of mitochondrial genes, including MTCO1 and MTCO2, and inhibited mitochondrial respiratory function in OSCC cells. These results suggest that LINC02154 exerts its oncogenic effects by modulating the cell cycle and oxidative phosphorylation in OSCC, highlighting LINC02154 as a potential therapeutic target.

DOI PubMed

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma.

Fumika Okazaki, Akira Yorozu, Shohei Sekiguchi, Takeshi Niinuma, Reo Maruyama, Hiroshi Kitajima, Eiichiro Yamamoto, Kazuya Ishiguro, Mutsumi Toyota, Yui Hatanaka, Koyo Nishiyama, Kazuhiro Ogi, Masahiro Kai, Kenichi Takano, Shingo Ichimiya, Akihiro Miyazaki, Hiromu Suzuki

Scientific reports 14 ( 1 ) 27425 - 27425 2024.11 [International journal]

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

DOI PubMed

The molecular profile of gastric intraepithelial foveolar type neoplasia based on somatic copy number alterations and multiple mutation analysis.

Tamotsu Sugai, Noriyuki Uesugi, Mitsumasa Osakabe, Ryuya Yamamoto, Koichi Hamada, Michitaka Honda, Naoki Yanagawa, Hiromu Suzuki

Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association 27 ( 6 ) 1220 - 1228 2024.11 [Domestic journal]

BACKGROUND: Gastric foveolar type neoplasia is a rare histological variant of gastric tumors. It is very difficult to differentiate between benign and malignant intraepithelial foveolar neoplasia (IFN). Although limited molecular alterations have been identified in IFNs, somatic copy number alterations (SCNAs), which are linked to tumor progression, have not been systematically evaluated in IFN. METHODS: The aim of the present study was to comprehensively examine SCNAs using a SNP array in 37 cases of IFN, compared with intestinal type dysplasia, including 39 low grade (LGD) and 32 high grade dysplasia (HGD) cases. In addition, gene mutations were evaluated using a gene panel. Finally, we attempted to determine molecular profiles using a hierarchical clustering analysis. RESULTS: Two patterns could be categorized according to the SCNAs in 108 tumors examined: high (subgroup 1) and low (subgroup 2) frequencies of SCNAs. Although IFN and LGD were associated with subgroup 2, HGD was found in both subgroups. The median numbers of total SCNAs and copy number gains were higher in IFN or HGD than in LGD. In addition, the IFN genotype was characterized by altered genes located at 4p13-4q35.2, including RAP1GDS1 and LEF1, which may be associated with IFN development. Finally, no significant mutations were found in IFNs using a gene panel. CONCLUSIONS: The current molecular profiles of IFN may help elucidate the mechanisms of IFN development.

DOI PubMed

Serial single-cell RNA sequencing unveils drug resistance and metastatic traits in stage IV breast cancer.

Kazutaka Otsuji, Yoko Takahashi, Tomo Osako, Takayuki Kobayashi, Toshimi Takano, Sumito Saeki, Liying Yang, Satoko Baba, Kohei Kumegawa, Hiromu Suzuki, Tetsuo Noda, Kengo Takeuchi, Shinji Ohno, Takayuki Ueno, Reo Maruyama

NPJ precision oncology 8 ( 1 ) 222 - 222 2024.10 [International journal]

Metastasis is a complex process that remains poorly understood at the molecular levels. We profiled single-cell transcriptomic, genomic, and epigenomic changes associated with cancer cell progression, chemotherapy resistance, and metastasis from a Stage IV breast cancer patient. Pretreatment- and posttreatment-specimens from the primary tumor and distant metastases were collected for single-cell RNA sequencing and subsequent cell clustering, copy number variation (CNV) estimation, transcriptomic factor estimation, and pseudotime analyses. CNV analysis revealed that a small population of pretreatment cancer cells resisted chemotherapy and expanded. New clones including Metastatic Precursor Cells (MPCs), emerged in the posttreatment primary tumors in CNV similar to metastatic cells. MPCs exhibited expression profiles indicative of epithelial-mesenchymal transition. Comparison of MPCs with metastatic cancer cells also revealed dynamic changes in transcription factors and calcitonin pathway gene expression. These findings demonstrate the utility of single-patient clinical sample analysis for understanding tumor drug resistance, regrowth, and metastasis.

DOI PubMed

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.

Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshiyuki Yano, Hiromu Suzuki, Akihiro Miyazaki, Kohichi Takada, Hisashi Uhara, Masato Furuhashi, Fumihito Hikage

Cells 13 ( 18 ) 2024.09 [International journal]

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

DOI PubMed

Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas.

Hironori Aoki, Akira Takasawa, Eiichiro Yamamoto, Takeshi Niinuma, Hiro-O Yamano, Taku Harada, Toshiyuki Kubo, Akira Yorozu, Hiroshi Kitajima, Kazuya Ishiguro, Masahiro Kai, Akio Katanuma, Toshiya Shinohara, Hiroshi Nakase, Tamotsu Sugai, Makoto Osanai, Hiromu Suzuki

BMC gastroenterology 24 ( 1 ) 91 - 91 2024.03 [International journal]

BACKGROUND: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions. METHODS: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112). RESULTS: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components. CONCLUSION: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

DOI PubMed

A genome-wide study of gastric intramucosal neoplasia based on somatic copy number alterations, gene mutations, and mRNA expression patterns.

Yoshihiko Koike, Mitsumasa Osakabe, Ryo Sugimoto, Noriyuku Uesugi, Takayuki Matsumoto, Hiromu Suzuki, Naoki Yanagawa, Tamotsu Sugai

The journal of pathology. Clinical research 10 ( 2 ) e12368 2024.03 [International journal]

We performed comprehensive analyses of somatic copy number alterations (SCNAs) and gene expression profiles of gastric intramucosal neoplasia (IMN) using array-based methods in 97 intestinal-type IMNs, including 39 low-grade dysplasias (LGDs), 37 high-grade dysplasias (HGDs), and 26 intramucosal carcinomas (IMCs) with stromal invasion of the lamina propria to identify the molecular mechanism of IMN. In addition, we examined gene mutations using gene panel analyses. We used cluster analyses for exclusion of arbitrariness to identify SCNA patterns and expression profiles. IMNs were classified into two distinct subgroups (subgroups 1 and 2) based on SCNA patterns. Subgroup 1 showed a genomic stable pattern due to the low frequency of SCNAs, whereas subgroup 2 exhibited a chromosomal instability pattern due to the high frequencies of SCNAs and TP53 mutations. Interestingly, although the frequencies of LGD and HGD were significantly higher in subgroup 1 than in subgroup 2, IMC was commonly found in both types. Although the expression profiles of specific mRNAs could be used to categorise subgroups 1 and 2, no clinicopathological findings correlated with either subgroup. We examined signalling pathways specific to subgroups 1 and 2 to identify the association of each subgroup with signalling pathways based on gene ontology tree visualisation: subgroups 1 and 2 were associated with haem metabolism and chromosomal instability, respectively. These findings reveal a comprehensive genomic landscape that highlights the molecular complexity of IMNs and provide a road map to facilitate our understanding of gastric IMNs.

DOI PubMed

ACLP Activates Cancer-Associated Fibroblasts and Inhibits CD8+ T-Cell Infiltration in Oral Squamous Cell Carcinoma.

Shohei Sekiguchi, Akira Yorozu, Fumika Okazaki, Takeshi Niinuma, Akira Takasawa, Eiichiro Yamamoto, Hiroshi Kitajima, Toshiyuki Kubo, Yui Hatanaka, Koyo Nishiyama, Kazuhiro Ogi, Hironari Dehari, Atsushi Kondo, Makoto Kurose, Kazufumi Obata, Akito Kakiuchi, Masahiro Kai, Yoshihiko Hirohashi, Toshihiko Torigoe, Takashi Kojima, Makoto Osanai, Kenichi Takano, Akihiro Miyazaki, Hiromu Suzuki

Cancers 15 ( 17 ) 2023.08 [International journal]

We previously showed that upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in vascular endothelial cells promotes tumor angiogenesis. In the present study, we aimed to clarify the role of stromal AEBP1/ACLP expression in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis showed that ACLP is abundantly expressed in cancer-associated fibroblasts (CAFs) in primary OSCC tissues and that upregulated expression of ACLP is associated with disease progression. Analysis using CAFs obtained from surgically resected OSCCs showed that the expression of AEBP1/ACLP in CAFs is upregulated by co-culture with OSCC cells or treatment with TGF-β1, suggesting cancer-cell-derived TGF-β1 induces AEBP1/ACLP in CAFs. Collagen gel contraction assays showed that ACLP contributes to the activation of CAFs. In addition, CAF-derived ACLP promotes migration, invasion, and in vivo tumor formation by OSCC cells. Notably, tumor stromal ACLP expression correlated positively with collagen expression and correlated inversely with CD8+ T cell infiltration into primary OSCC tumors. Boyden chamber assays suggested that ACLP in CAFs may attenuate CD8+ T cell migration. Our results suggest that stromal ACLP contributes to the development of OSCCs, and that ACLP is a potential therapeutic target.

DOI PubMed

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

Nami Nishikiori, Kohichi Takada, Tatsuya Sato, Sho Miyamoto, Megumi Watanabe, Yui Hirakawa, Shohei Sekiguchi, Masato Furuhashi, Akira Yorozu, Kenichi Takano, Akihiro Miyazaki, Hiromu Suzuki, Hiroshi Ohguro

Cells 12 ( 17 ) 2023.08 [International journal]

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

DOI PubMed

TM4SF1-AS1 inhibits apoptosis by promoting stress granule formation in cancer cells

Hiroshi Kitajima, Reo Maruyama, Takeshi Niinuma, Eiichiro Yamamoto, Akira Takasawa, Kumi Takasawa, Kazuya Ishiguro, Akihiro Tsuyada, Ryo Suzuki, Gota Sudo, Toshiyuki Kubo, Kei Mitsuhashi, Masashi Idogawa, Shoichiro Tange, Mutsumi Toyota, Ayano Yoshido, Kohei Kumegawa, Masahiro Kai, Kazuyoshi Yanagihara, Takashi Tokino, Makoto Osanai, Hiroshi Nakase, Hiromu Suzuki

Cell Death & Disease ( Springer Science and Business Media LLC ) 14 ( 7 ) 2023.07

Abstract Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation.

DOI

Human Neuralized is a novel tumour suppressor targeting Wnt/β-catenin signalling in colon cancer.

Joo Mi Yi, Tae-Hong Kang, Yu Kyeong Han, Ha Young Park, Ju Hwan Yang, Jin-Han Bae, Jung-Soo Suh, Tae-Jin Kim, Joong-Gook Kim, Yan-Hong Cui, Hiromu Suzuki, Kohei Kumegawa, Sung Joo Kim, Yi Zhao, In Ja Park, Seung-Mo Hong, Joon-Yong Chung, Su-Jae Lee

EMBO reports e56335 2023.06 [International journal]

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/β-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic β-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic β-catenin, and reducing its cytoplasmic levels in a GSK3β- and β-TrCP-independent manner, indicating that NEURL-β-catenin interactions can lead to a disruption of the canonical Wnt/β-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/β-catenin signalling.

DOI PubMed

大腸癌転移に影響する腫瘍微小環境におけるmessenger RNA(mRNA)の発現

伊藤一洋, 刑部光正, 杉本亮, 山田峻, 佐藤綾香, 上杉憲幸, 柳川直樹, 佐藤孝, 鈴木拓, 菅井有

日本病理学会会誌 ( (一社)日本病理学会 ) 112 ( 1 ) 258 - 258 2023.03

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

Ayano Yoshido, Gota Sudo, Akira Takasawa, Hironori Aoki, Hiroshi Kitajima, Eiichiro Yamamoto, Takeshi Niinuma, Taku Harada, Toshiyuki Kubo, Hajime Sasaki, Kazuya Ishiguro, Akira Yorozu, Masahiro Kai, Akio Katanuma, Hiro-O Yamano, Makoto Osanai, Hiroshi Nakase, Hiromu Suzuki

Journal of gastroenterology and hepatology 38 ( 2 ) 301 - 310 2022.11 [International journal]

BACKGROUND AND AIM: The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. METHODS: Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. RESULTS: Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. CONCLUSIONS: Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

DOI PubMed

CXCL12 is expressed by skeletal muscle cells in tongue oral squamous cell carcinoma.

Akira Yorozu, Shohei Sekiguchi, Akira Takasawa, Fumika Okazaki, Takeshi Niinuma, Hiroshi Kitajima, Eiichiro Yamamoto, Masahiro Kai, Mutsumi Toyota, Yui Hatanaka, Koyo Nishiyama, Kazuhiro Ogi, Hironari Dehari, Kazufumi Obata, Makoto Kurose, Atsushi Kondo, Makoto Osanai, Akihiro Miyazaki, Kenichi Takano, Hiromu Suzuki

Cancer medicine 12 ( 5 ) 5953 - 5963 2022.10 [International journal]

BACKGROUND: The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs. METHODS: Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR. RESULTS: Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells. CONCLUSION: Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

DOI PubMed

Differential Expression in the Tumor Microenvironment of mRNAs Closely Associated with Colorectal Cancer Metastasis.

Kazuhiro Ito, Mitsumasa Osakabe, Ryo Sugimoto, Shun Yamada, Ayaka Sato, Noriyuki Uesugi, Naoki Yanagawa, Hiromu Suzuki, Tamotsu Sugai

Annals of surgical oncology 30 ( 2 ) 1255 - 1266 2022.10 [International journal]

BACKGROUND: Metastasis of colorectal cancer (CRC) is a major cause of CRC-related mortality. However, the detailed molecular mechanism of CRC metastasis remains unknown. A recent study showed that the tumor microenvironment, which includes cancer cells and the surrounding stromal cells, plays a major role in tumor invasion and metastasis. Identification of altered messenger RNA (mRNA) expression in the tumor microenvironment is essential to elucidation of the mechanisms responsible for tumor progression. This study investigated the mRNA expression of genes closely associated with metastatic CRC compared with non-metastatic CRC. METHODS: The samples examined were divided into cancer tissue and isolated cancer stromal tissue. The study examined altered mRNA expression in the cancer tissues using The Cancer Genome Atlas (TCGA) (377cases) and in 17 stromal tissues obtained from our laboratory via stromal isolation using an array-based analysis. In addition, 259 patients with CRC were enrolled to identify the association of the candidate markers identified with the prognosis of patients with stage 2 or 3 CRC. The study examined the enriched pathways identified by gene set enrichment analysis (GSEA) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) module in both the TCGA dataset and isolated stromal tissue. RESULTS: As a result, whereas tenascin-C, secreted phosphoprotein 1 and laminin were expressed in metastatic CRC cells, olfactory receptors (ORs) 11H1 and OR11H4 were expressed in stromal tissue cells isolated from metastatic CRC cases. Finally, upregulated expression of tenascin-C and OR11H4 was correlated with the outcome for CRC patients. CONCLUSION: The authors suggest that upregulated expression levels of tenascin-C and OR11H1 play an important role in CRC progression.

DOI PubMed

ASO Visual Abstract: Differential Expression in the Tumor Microenvironment of mRNAs Closely Associated with Colorectal Cancer Metastasis.

Kazuhiro Ito, Mitsumasa Osakabe, Ryo Sugimoto, Shun Yamada, Ayaka Sato, Noriyuki Uesugi, Naoki Yanagawa, Hiromu Suzuki, Tamotsu Sugai

Annals of surgical oncology 30 ( 2 ) 1267 - 1268 2022.10 [International journal]

DOI PubMed

Genome-wide analysis of colorectal cancer based on gene-based somatic copy number alterations during neoplastic progression within the same tumor.

Shun Yamada, Mitsumasa Osakabe, Noriyuki Uesugi, Naoki Yanagawa, Takayuki Matsumoto, Hiromu Suzuki, Tamotsu Sugai

Cancer medicine 2022.08 [International journal]

BACKGROUND: The objective of this study was to elucidate the association between neoplastic progression and somatic copy number alterations (SCNAs) occurring within the same colorectal cancer (CRC) tumor. METHODS: We investigated SCNAs to identify the progression from a high-grade intramucosal lesion (HGIL) to an invasive front lesion (IFL), via an invasive submucosal lesion (ISL), within the same tumor using a crypt isolation method combined with a SNP array. Immunohistochemistry was also performed. RESULTS: We identified 51 amplified genes that potentially promote progression from HGIL to ISL and 6 amplified genes involved in the progression from ISL to IFL. Of the 51 genes involved in HGIL to ISL progression, TORC1, MSLN, and STUB1, which are closely associated with CRC, were identified as candidate markers of submucosal invasion. However, no candidate genes were identified among the six genes associated with ISL to IFL progression. In addition, the number of total SCNAs and the number of gains were correlated with cancer progression (from HGIL to IFL). Finally, immunohistochemistry revealed higher expression of TORC1, MSLN, and STUB1 in ISL than in HGIL. CONCLUSIONS: These results suggest that specific SCNAs are required for acquisition of invasive ability in CRC, and the affected genes are potential markers of invasion.

DOI PubMed

Genome-wide analysis of mRNA expression identified the involvement of trefoil factor 1 in the development of sessile serrated lesions.

Tamotsu Sugai, Mitsumasa Osakabe, Makoto Eizuka, Yoshihito Tanaka, Shun Yamada, Naoki Yanagawa, Takayuki Matsumoto, Hiromu Suzuki

Pathology, research and practice 236 153987 - 153987 2022.08 [International journal]

Precursor lesions that progress into colorectal cancer (CRC) could be largely classified into sessile serrated lesions (SSLs), traditional serrated adenoma (TSA), and tubular adenoma (TA). We aimed to determine whether high expression of trefoil factor 1 (TFF1) is closely associated with serrated lesions, particularly SSLs. The samples were divided into the first (12 SSLs, 5 TSAs, and 15 TAs) and second cohorts (15 SSLs, 9 TSAs, and 15 TAs). First, we investigated TFF1 expression in isolated gland samples using array-based and reverse-transcription PCR. Second, we performed immunohistochemical analysis of TFF1 expression in paraffin-embedded tissues obtained from SSL, TSA, TA, and hyperplastic polyp (HP) samples. In addition, we compared TFF1 mRNA levels between SSLs and HPs. TFF1 expression was significantly higher in SSLs than in TSA and TA in both cohorts. Additionally, immunohistochemical staining of TFF1 in the HP, SSL, TSA, and TA samples revealed significant differences in the immunohistochemical scores of TFF1 among the four types of lesions (higher expression in SSLs than in the other three lesions). Finally, there were significant differences in TFF1 mRNA expression levels between SSLs and HPs in paraffin-embedded tissues. However, there was considerable overlap in the immunohistochemical scores and expression levels of TFF1 transcripts between SSLs and HPs. The current findings may help elucidate the molecular mechanisms involved in serrated lesion development. In addition, we suggest that despite the limited practical application, upregulation of TFF1 transcripts may help differentiate SSLs from other lesions.

DOI PubMed

A novel risk stratification model based on the Children's Hepatic Tumours International Collaboration-Hepatoblastoma Stratification and deoxyribonucleic acid methylation analysis for hepatoblastoma.

Takafumi Kondo, Shohei Honda, Hiromu Suzuki, Yoichi M Ito, Issei Kawakita, Kazuyoshi Okumura, Momoko Ara, Masashi Minato, Norihiko Kitagawa, Yukichi Tanaka, Mio Tanaka, Masato Shinkai, Tomoro Hishiki, Kenichiro Watanabe, Kohmei Ida, Atsushi Takatori, Eiso Hiyama, Akinobu Taketomi

European journal of cancer (Oxford, England : 1990) 172 311 - 322 2022.07 [International journal]

INTRODUCTION: Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) developed risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation. METHODS: HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisulfite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We investigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model. RESULTS: Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in ≥2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that ≥2 methylated genes was an independent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS. CONCLUSIONS: Our proposed stratification system considers individual risk in HB and may improve patient clinical management.

DOI PubMed

Genome-wide analysis of mRNA and microRNA expression in colorectal cancer and adjacent normal mucosa.

Yuma Ito, Mitsumasa Osakabe, Takeshi Niinuma, Noriyuki Uesugi, Ryo Sugimoto, Naoki Yanagawa, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki, Tamotsu Sugai

The journal of pathology. Clinical research 8 ( 4 ) 313 - 326 2022.07 [International journal]

mRNA expression varies in human cancers. Such altered mRNA expression is negatively regulated by the expression of microRNAs (miRNAs), which play an important role in human tumorigenesis. According to this theory, inverse mRNA/miRNA expression may be a direct driver of cancer development, and certain genetic events may occur prior to the development of any discernible histological abnormalities. We examined the inverse expression between mRNAs and their corresponding miRNAs in colorectal cancer (CRC) and adjacent normal mucosa and performed pathway analysis to identify mRNA/miRNA networks. The cancer samples were divided into first (20 cases) and second (24 cases) cohorts, and 48 samples were obtained from two sections of the normal mucosa adjacent to the tumors from the second cohort. We investigated mRNAs with commonly altered expression in CRC and adjacent normal mucosa using isolated cancer glands and normal crypts from the first cohort, compared with that of distal normal crypts, using an array-based method. As a result, significant inverse correlations between CEACAM1 and miRNA-7114-5p and between AK1 and miRNA-6780-5p were found in CRC and adjacent normal mucosa. We validated these correlations in the second cohort using RT-PCR. To confirm these findings, transfection and immunohistochemical assays were also performed, which verified the inverse correlation between CEACAM1 and miRNA-7114-5p. Our findings suggest that the inverse correlations between the CEACAM1/miRNA-7114-5p and possibly AK1/miRNA-6780-5p pairs play an important role in early CRC development, and may help identify potential molecular targets for early detection of CRC.

DOI PubMed

Cribriform-type adenocarcinoma of the colorectum: comprehensive molecular analyses of a distinctive histologic subtype of colorectal cancer.

Shun Yamada, Mitsumasa Osakabe, Makoto Eizuka, Mai Hashimoto, Noriyuki Uesugi, Naoki Yanagawa, Koki Otsuka, Hiromu Suzuki, Takayuki Matsumoto, Tamotsu Sugai

Carcinogenesis 43 ( 6 ) 601 - 610 2022.06 [International journal]

Colorectal adenocarcinoma (CRA) is characterized by marked heterogeneity and may be composed of an admixture of various histologic patterns, including well-formed gland and cribriform types. Although tumors displaying a prominent or predominant cribriform feature are frequently found in CRA, this type may contain specific histologic variants with a characteristic molecular alteration. We investigated the molecular features of 51 primary CRAs with a predominant cribriform histology using array-based analyses [somatic copy number alterations (SCNAs); mRNA expression]. Mutations (TP53, KRAS, PIK3CA and BRAF) and DNA methylation status were also analyzed. The crypt isolation method was used to obtain isolated tumor glands of each type separately. All patients were classified by their CRA histologic subtype into two groups: well-formed gland and cribriform. Next, we performed cluster analysis to stratify SCNA and mRNA expression patterns between the two subtypes. Two distinctive subgroups were stratified based on patterns of SCNA and mRNA expression and were correlated with each histologic subtype. The cribriform type was characterized by a high frequency of SCNA compared with that of the well-formed gland type and was closely associated with the expression of specific mRNAs. In addition, the frequency of KRAS mutation was significantly higher in the cribriform type than in the well-formed gland type. Finally, there was no difference in DNA methylation status between the two subtypes. Overall, these data suggest that the cribriform type provides important insights into colorectal carcinogenesis, suggesting specific potential histologic implications based on the molecular profile.

DOI PubMed

Comprehensive Analysis of microRNA Expression During the Progression of Colorectal Tumors.

Tamotsu Sugai, Ryo Sugimoto, Makoto Eizuka, Mitsumasa Osakabe, Shun Yamada, Naoki Yanagawa, Takayuki Matsumoto, Hiromu Suzuki

Digestive diseases and sciences 68 ( 3 ) 813 - 823 2022.06 [International journal]

BACKGROUND: No effective early diagnostic biomarkers are available for colorectal cancer (CRC). Therefore, we sought to identify new biomarkers that could identify CRC from progression as a pre-cancerous lesion to its invasive form. Recent studies have shown that microRNAs (miRs) are associated with the onset of cancer invasion and progression. AIMS: We hypothesized that the identification of miRs associated with CRC might be useful to detect this disease at early stages. METHODS: We conducted an integrated analysis of 79 isolated colorectal tumor glands, including adenomas, intramucosal cancers, and invasive CRCs that showed a microsatellite stable phenotype using GeneChip miRNA 4.0 microarray assays. The colorectal tumors we examined were divided into 2 cohorts (42 in the first cohort and 37 in the second cohort). RESULTS: First, cluster analysis was performed to stratify expression patterns of multiple miRs that were pooled according to the following criteria: fold change in expression (< -2.0 or > 2.0), p < 0.05, and mature miRs. As a result, the expression patterns of pooled miRs were subdivided into 3 subgroups that were correlated with tumor grade. Each subgroup was characterized by specific miRs. In addition, we found that specific miRs, including miR-140-3p and miR-378i, were closely associated with cancer invasion. Finally, we analyzed paired dysregulated miRs between adenomatous and cancerous components present within the same tumor. DISCUSSION: We showed that several miRs were dysregulated during progression from adenoma to intramucosal cancer. Specific miRs may have key roles in progression from intramucosal tumor to invasive CRC.

DOI PubMed

大腸癌および癌近傍正常粘膜のmessenger RNAとmicro RNAの網羅的解析

伊藤勇馬, 刑部光正, 杉本亮, 柳川直樹, 大塚幸喜, 佐々木章, 松本主之, 鈴木拓, 菅井有

日本病理学会会誌 ( (一社)日本病理学会 ) 111 ( 1 ) 312 - 312 2022.03

病態解明による大腸腫瘍診療の新展開早期大腸がん浸潤先進部の分子解析による新規バイオマーカー・治療標的の探索

須藤豪太, 鈴木拓, 仲瀬裕志

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 119 ( 臨増総会 ) A104 - A104 2022.03

Relevance of gene mutations and methylation to the growth of pancreatic intraductal papillary mucinous neoplasms based on pyrosequencing.

Go Asano, Katsuyuki Miyabe, Hiroyuki Kato, Michihiro Yoshida, Takeshi Sawada, Yasuyuki Okamoto, Hidenori Sahashi, Naoki Atsuta, Kenta Kachi, Akihisa Kato, Naruomi Jinno, Makoto Natsume, Yasuki Hori, Itaru Naitoh, Kazuki Hayashi, Yoichi Matsuo, Satoru Takahashi, Hiromu Suzuki, Hiromi Kataoka

Scientific reports 12 ( 1 ) 419 - 419 2022.01 [International journal]

We aimed to assess some of the potential genetic pathways for cancer development from non-malignant intraductal papillary mucinous neoplasm (IPMN) by evaluating genetic mutations and methylation. In total, 46 dissected regions in 33 IPMN cases were analyzed and compared between malignant-potential and benign cases, or between malignant-potential and benign tissue dissected regions including low-grade IPMN dissected regions accompanied by malignant-potential regions. Several gene mutations, gene methylations, and proteins were assessed by pyrosequencing and immunohistochemical analysis. RASSF1A methylation was more frequent in malignant-potential dissected regions (p = 0.0329). LINE-1 methylation was inversely correlated with GNAS mutation (r = - 0.3739, p = 0.0105). In cases with malignant-potential dissected regions, GNAS mutation was associated with less frequent perivascular invasion (p = 0.0128), perineural invasion (p = 0.0377), and lymph node metastasis (p = 0.0377) but significantly longer overall survival, compared to malignant-potential cases without GNAS mutation (p = 0.0419). The presence of concordant KRAS and GNAS mutations in the malignant-potential and benign dissected regions were more frequent among branch-duct IPMN cases than among the other types (p = 0.0319). Methylation of RASSF1A, CDKN2A, and LINE-1 and GNAS mutation may be relevant to cancer development, IPMN subtypes, and cancer prognosis.

DOI PubMed

Genome-Wide Analysis of microRNA and mRNA Expression in Colorectal Intramucosal Neoplasia and Colorectal Cancer With a Microsatellite-Stable Phenotype Based on Adenoma-Carcinoma Sequences.

Tamotsu Sugai, Mitsumasa Osakabe, Takeshi Niinuma, Ryo Sugimoto, Makoto Eizuka, Yoshihito Tanaka, Naoki Yanagawa, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki

Frontiers in oncology 12 831100 - 831100 2022 [International journal]

Background: Although MicroRNAs (miRNAs) play important roles in various biological processes, the biological functions of miRNAs are achieved through mRNAs. The aim of this study is to identify dysregulated miRNA/mRNA expression patterns in colorectal tumors. Methods: We examined 42 colorectal tumors [15 adenomas, 8 intramucosal cancers (IMCs), and 19 invasive colorectal cancers (CRCs)] with the microsatellite stable (MSS) phenotype (first cohort). The first cohort was used for genome-wide miRNA and mRNA expression arrays, whereas the second cohort (37 colorectal neoplasias) was used for validation analyses. Finally, we used 15 cases of "adenoma in/with carcinoma" to identify network patterns of miRNAs/mRNAs that were directly associated with neoplastic progression. In addition, simple regression analysis for array-based and RT-PCR analyses was performed to select candidate miRNA-mRNA pairs. Transfection of miRNA mimics was also performed to confirm whether target mRNA expression is affected by specific miRNAs. Results: Specific paired miRNA/mRNA networks, including hsa-miR-34a-5p/SLC12A2, hsa-miR-15b-5p/SLC12A2, hsa-miR-195-5p/SLC12A2, hsa-miRNA-502-3p/OLFM4, hsa-miRNA-6807-5p/ZG16, and hsa-miRNA 3064-5p/SH3BGRL3, were identified in samples of adenoma, IMC, and CRC with the MSS phenotype. In adenomatous lesions obtained from the same tumor with a carcinomatous lesion, we identified pairs of miRNA-130a-3p/HSPA8 and miRNA-22-3p/RP53 that were linked to multiple pathways. On the other hand, 2 pairs of miRNA/mRNA (miRNA-660-5p and miRNA-664a-5p/APP) were found in isolated carcinomatous glands. Ectopic expression of miRNA 3064-5p suppressed SH3BGRL3 expression. Conclusions: We found that networks based on specific pairs of miRNAs/mRNAs contribute to progression from adenomatous and carcinomatous lesions. Our results provide insights into the molecular tumorigenesis of colorectal tumors.

DOI PubMed

Comprehensive analyses of microRNA and mRNA expression in colorectal serrated lesions and colorectal cancer with a MSI phenotype.

Tamotsu Sugai, Mitsumasa Osakabe, Takeshi Niinuma, Makoto Eizuka, Yoshihito Tanaka, Shun Yamada, Naoki Yanagawa, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki

Genes, chromosomes & cancer 61 ( 3 ) 161 - 171 2021.11 [International journal]

MicroRNA (miRNA) expression is dysregulated in human tumors, thereby contributing to tumorigenesis through altered expression of mRNA. Thus, identification of the relationships between miRNAs and mRNAs is important for evaluating the molecular mechanisms of tumors. Additionally, elucidation of the molecular features of serrated lesions is essential in colorectal tumorigenesis. Here, we examined the relationships of miRNA and mRNA expressed in serrated lesions, including 26 sessile serrated lesions (SSLs), 12 traditional serrated adenomas (TSAs), and 11 colorectal cancers (CRCs) with a microsatellite instability (MSI) phenotype using crypt isolation. We divided the samples into the first and second cohorts for validation. Array-based expression analyses were used to evaluate miRNAs and mRNAs with opposite expression patterns in isolated tumor glands. In addition, we validated the relationships of miRNA/mRNA pairs in the second cohort using real-time polymerase chain reaction. We found that the expression of miRNA-5787 was correlated with reciprocal expression of 2 mRNAs, i.e., SRRM2 and POLR2J3, in SSL samples. In TSA samples, 2 pairs of miRNAs/mRNAs showing opposite expression patterns, i.e., miRNA-182-5p/ETF1 and miRNA-200b-3p/MYB, were identified. Ultimately, three pairs of miRNAs/mRNAs with opposite expression patterns, including miRNA-222-3p/SLC26A3, miRNA-6753-3p/FABP1, and miRNA-222-3p/OLFM4, were retained in CRC with an MSI phenotype. Finally, we performed transfection with an miR-222-3p mimic to confirm the expression of SLC26A3 and OLFM4; the results showed that ectopic expression of miR-222-3p moderately suppressed OLFM4 and downregulated SLC26A3 to some extent. Overall, our results provided basic insights into the evaluation of colorectal tumorigenesis of serrated lesions and CRC with an MSI phenotype. This article is protected by copyright. All rights reserved.

DOI PubMed

DLEU1 promotes oral squamous cell carcinoma progression by activating interferon-stimulated genes.

Yui Hatanaka, Takeshi Niinuma, Hiroshi Kitajima, Koyo Nishiyama, Reo Maruyama, Kazuya Ishiguro, Mutsumi Toyota, Eiichiro Yamamoto, Masahiro Kai, Akira Yorozu, Shohei Sekiguchi, Kazuhiro Ogi, Hironari Dehari, Masashi Idogawa, Yasushi Sasaki, Takashi Tokino, Akihiro Miyazaki, Hiromu Suzuki

Scientific reports 11 ( 1 ) 20438 - 20438 2021.10 [International journal]

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

DOI PubMed

大腸癌の腫瘍近傍分離正常腺管と分離癌腺管におけるmiRNA/mRNA発現状態の網羅的解析

刑部光正, 伊藤勇馬, 杉本亮, 上杉憲幸, 柳川直樹, 鈴木拓, 菅井有

日本癌学会総会記事 ( (一社)日本癌学会 ) 80回 [P14 - 3] 2021.09

エピジェネティクス阻害剤のGIST細胞におけるエピゲノム修飾、遺伝子発現への影響の統合解析

吉戸文乃, 須藤豪太, 北嶋洋志, 新沼猛, 甲斐正広, 原田拓, 佐々木基, 久保俊之, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 80回 [P14 - 7] 2021.09

Helicobacter pylori除菌後胃がんと背景胃粘膜におけるDNAメチローム解析

山本英一郎, 吉戸文乃, 須藤豪太, 三橋慧, 北嶋洋志, 新沼猛, 甲斐正広, 原田拓, 佐々木基, 久保俊之, 山野泰穂, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 80回 [P14 - 5] 2021.09

活性化マクロファージはIL-1β-SAA1 axisを介して早期大腸がんの浸潤を促進する

須藤豪太, 山本英一郎, 青木敬則, 高澤啓, 新沼猛, 久保俊之, 萬顕, 吉戸文乃, 北嶋洋志, 甲斐正広, 小山内誠, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 80回 [P14 - 3] 2021.09

頭頸部扁平上皮がんの腫瘍微小環境におけるAEBP1の解析

関口翔平, 萬顕, 山本英一郎, 新沼猛, 高澤啓, 須藤豪太, 小池和茂, 畠中柚衣, 吉戸文乃, 北嶋洋志, 甲斐正広, 小山内誠, 高野賢一, 宮崎晃亘, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 80回 [J14 - 5] 2021.09

内視鏡診療で目指す大腸腫瘍性病変に対するTranslational researchの確立

三宅高和, 吉井新二, 鈴木拓, 山野泰穂, 仲瀬裕志

日本消化器病学会北海道支部例会・日本消化器内視鏡学会北海道支部例会プログラム・抄録集 ( 日本消化器病学会-北海道支部 ) 129回・123回 26 - 26 2021.09

Regulatory roles of claudin-1 in cell adhesion and microvilli formation.

Kumi Takasawa, Akira Takasawa, Taishi Akimoto, Kazufumi Magara, Tomoyuki Aoyama, Hiroshi Kitajima, Taro Murakami, Yusuke Ono, Daisuke Kyuno, Hiromu Suzuki, Makoto Osanai

Biochemical and biophysical research communications 565 36 - 42 2021.08 [International journal]

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

DOI PubMed

Activated macrophages promote invasion by early colorectal cancer via an IL-1β-SAA1 axis.

Gota Sudo, Hironori Aoki, Eiichiro Yamamoto, Akira Takasawa, Takeshi Niinuma, Ayano Yoshido, Hiroshi Kitajima, Akira Yorozu, Toshiyuki Kubo, Taku Harada, Kazuya Ishiguro, Masahiro Kai, Akio Katanuma, Hiro-O Yamano, Makoto Osanai, Hiroshi Nakase, Hiromu Suzuki

Cancer science 112 ( 10 ) 4151 - 4165 2021.07 [International journal]

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA-sequencing (RNA-seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues, and detected significant upregulation of SAA1 in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Co-culture experiments using CRC cell lines and THP-1 cells suggested that interleukin 1β (IL-1β) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL-1β. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1-like/M2-like macrophages at SAA1-positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of MMP-9 in macrophages. Our data suggest that tumor-associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1, and that SAA1 may be a predictive biomarker and a useful therapeutic target.

DOI PubMed

A genome-wide analysis of the molecular alterations occurring in the adenomatous and carcinomatous components of the same tumor based on the adenoma-carcinoma sequence.

Tamotsu Sugai, Mitsumasa Osakabe, Wataru Habano, Yoshihito Tanaka, Makoto Eizuka, Ryo Sugimoto, Naoki Yanagawa, Takayuki Matsumoto, Hiromu Suzuki

Pathology international 71 ( 9 ) 582 - 593 2021.07 [International journal]

Identification of molecular alterations occurring in the adenomatous and carcinomatous components within the same tumor would greatly enhance understanding of the neoplastic progression of colorectal cancer. We examined somatic copy number alterations (SCNAs) and mRNA expression at the corresponding loci involved in the adenoma-carcinoma sequence in the isolated adenomatous and cancer glands of the same tumor in 15 cases of microsatellite-stable "carcinoma in adenoma," using genome-wide SNP and global gene expression arrays. Multiple copy-neutral loss of heterozygosity events were detected at 4q13.2, 15q15.1, and 14q24.3 in the adenomatous component and at 4q13.2, 15q15.1, and 14q24.3 in the carcinomatous component. There were significant differences in the copy number (CN) gain frequencies at 20q11.21-q13.33, 8q13.3, 8p23.1, and 8q21.2-q22.2 between the adenomatous and carcinomatous components. Finally, we found a high frequency of five genotypes involving CN gain with upregulated expression of the corresponding gene (RPS21, MIR3654, RSP20, SNORD54, or ASPH) in the carcinomatous component, whereas none of these genotypes were detected in the adenomatous component. This finding is interesting in that CN gain with upregulated gene expression may enhance gene function and play a crucial role in the progression of an adenoma into a carcinomatous lesion.

DOI PubMed

Immunohistochemical Examination is Highly Sensitive and Specific for Detection of the V600E BRAF Mutation in Colorectal Serrated Lesions.

Noriyuki Yamada, Makoto Eizuka, Ryo Sugimoto, Yoshihito Tanaka, Naoki Yanagawa, Hiroo Yamano, Hiromu Suzuki, Takayuki Matsumoto, Tamotsu Sugai

Applied immunohistochemistry & molecular morphology : AIMM 29 ( 6 ) 446 - 453 2021.07 [International journal]

Mutations in BRAF are important events in colorectal serrated lesions and specific genetic markers for the serrated pathway. However, examination of BRAF mutations is not easy in routine histopathologic analyses. Here, the authors examined 73 colorectal serrated lesions, including 21 hyperplastic polyps, 32 traditional serrated adenomas, and 30 sessile serrated lesions, for comparison of BRAF mutation status with immunopositive expression of the anti-BRAF V600E mutation-specific antibody VE1. Thirty-two tubular adenomas (TAs) were examined as controls. In addition, 5 examples of sessile serrated lesion with dysplasia were included. Mutations in BRAF (exon 15; V600E) and KRAS (exon 2) were analyzed in serrated lesions and TAs using pyrosequencing. Finally, the authors compared BRAF mutations with immunohistochemical expression of VE1 antibodies against the BRAF V600E mutation, which was examined based on quantitative analyses and correlations between semiquantitative (0, 1+, or 2+) and quantitative results in colorectal serrated lesions. The cut-off value of VE1 expression (32%) was set based on receiver operating characteristic curve analysis. In the current study, mutations in BRAF were well correlated with VE1 expression in serrated lesions, although no TAs without BRAF mutations were immunopositive. In contrast, serrated lesions and TAs with mutations in KRAS were not stained for VE1 expression. In serrated lesions, although the sensitivity was 96.2% to 100%, the specificity was 90.0% to 100%. In addition, there was also good correlation between semiquantitative and quantitative results. Analysis of BRAF V600E expression may be pathologically useful, particularly in routine histopathologic diagnosis.

DOI PubMed

An Integrated Epigenome and Transcriptome Analysis to Clarify the Effect of Epigenetic Inhibitors on GIST.

Takeshi Niinuma, Hiroshi Kitajima, Eiichiro Yamamoto, Reo Maruyama, Hironori Aoki, Taku Harada, Kazuya Ishiguro, Gota Sudo, Mutsumi Toyota, Ayano Yoshido, Masahiro Kai, Hiroshi Nakase, Tamotsu Sugai, Hiromu Suzuki

Anticancer research 41 ( 6 ) 2817 - 2828 2021.06 [International journal]

BACKGROUND/AIM: Epigenetic alterations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). To obtain further insight into the GIST epigenome, we analyzed genome-wide histone modification and DNA methylation in GIST cells. MATERIALS AND METHODS: To reverse epigenetic silencing, GIST-T1 cells were treated with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, and subsequently H3K4me3 levels, the DNA methylome, and the transcriptome were analyzed. RESULTS: Treatment with epigenetic inhibitors not only up-regulated genes with DNA methylation, but also genes related to interferon signaling. ChIP-seq analysis revealed that drug treatment up-regulated H3K4me3 levels in retrotransposons, including endogenous retroviruses (ERV). Finally, utilizing the omics data, we found that hypermethylation of MEG3 is a frequent event and an indicator of poorer prognosis in GIST patients. CONCLUSION: Epigenetic inhibitors may activate interferon signaling via viral mimicry in GIST cells. Moreover, epigenome data could be a useful resource to identify novel GIST-related genes.

DOI PubMed

Extracellular vesicles containing miR-146a-5p secreted by bone marrow mesenchymal cells activate hepatocytic progenitors in regenerating rat livers.

Norihisa Ichinohe, Masayuki Ishii, Naoki Tanimizu, Toru Mizuguchi, Yusuke Yoshioka, Takahiro Ochiya, Hiromu Suzuki, Toshihiro Mitaka

Stem cell research & therapy 12 ( 1 ) 312 - 312 2021.05 [International journal]

BACKGROUND: Small hepatocyte-like progenitor cells (SHPCs) appear to form transient clusters in rat livers treated with retrorsine (Ret) and 70% partial hepatectomy (PH). We previously reported that the expansion of SHPCs was amplified in Ret/PH-treated rat livers transplanted with Thy1+ cells derived from D-galactosamine-treated injured livers. Extracellular vesicles (EVs) produced by hepatic Thy1+ donor cells activated SHPCs via interleukin (IL)-17 receptor B signaling. As bone marrow-derived mesenchymal cells (BM-MCs) also express Thy1, we aimed to determine whether BM-MCs could also promote the growth of SHPCs. METHODS: BM-MCs were isolated from dipeptidyl-peptidase IV (DPPIV)-positive rats. BM-MCs or BM-MC-derived EVs were administered to DPPIV-negative Ret/PH rat livers, and the growth and the characteristics of SHPC clusters were evaluated 14 days post-treatment. miRNA microarrays and cytokine arrays examined soluble factors within EVs. Small hepatocytes (SHs) isolated from an adult rat liver were used to identify factors enhancing hepatocytic progenitor cells growth. RESULTS: The recipient's livers were enlarged at 2 weeks post-BM-MC transplantation. The number and the size of SHPCs increased remarkably in livers transplanted with BM-MCs. BM-MC-derived EVs also stimulated SHPC growth. Comprehensive analyses revealed that BM-MC-derived EVs contained miR-146a-5p, interleukin-6, and stem cell factor, which could enhance SHs' proliferation. Administration of EVs derived from the miR-146a-5p-transfected BM-MCs to Ret/PH rat livers remarkably enhanced the expansion of SHPCs. CONCLUSIONS: miR-146a-5p involved in EVs produced by BM-MCs may play a major role in accelerating liver regeneration by activating the intrinsic hepatocytic progenitor cells.

DOI PubMed

The continuum of transformation of TSA to low-grade neoplasia and cancer with BRAF mutation - A case with molecular analysis.

Hironori Aoki, Yasushi Adachi, Eiichiro Yamamoto, Yukinari Yoshida, Yoshifumi Ishii, Hiro-O Yamano, Hiromu Suzuki, Takao Endo

Pathology international 71 ( 5 ) 368 - 370 2021.05 [International journal]

DOI PubMed

Lynch症候群を背景とした多発微小大腸癌症例

柴田泰洋, 山野泰穂, 久保俊之, 吉井新二, 赤保内正和, 横山佳浩, 山川司, 風間友江, 山下健太郎, 能正勝彦, 須藤豪太, 山本英一郎, 鈴木拓, 杉田真太郎, 長谷川匡, 永塚真, 菅井有, 仲瀬裕志

日本大腸肛門病学会雑誌 ( (一社)日本大腸肛門病学会 ) 74 ( 5 ) 332 - 332 2021.05

Lynch症候群を背景とした多発微小大腸癌症例

日本大腸肛門病学会雑誌 ( (一社)日本大腸肛門病学会 ) 74 ( 5 ) 332 - 332 2021.05

A genome-wide study of the relationship between chromosomal abnormalities and gene expression in colorectal tumors.

Tamotsu Sugai, Mitsumasa Osakabe, Ryo Sugimoto, Makoto Eizuka, Yoshihito Tanaka, Naoki Yanagawa, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki

Genes, chromosomes & cancer 60 ( 4 ) 250 - 262 2021.04 [International journal]

The role of somatic copy number alterations (SCNAs) that occur in colorectal tumors is poorly understood. SCNAs are correlated with corresponding gene expression changes that may contribute to neoplastic progression. Thus, we examined SCNAs and the expression of messenger RNAs (mRNAs) located at corresponding loci in colorectal neoplasia, a progression model of human neoplasm. We used 42 colorectal neoplastic samples, including adenomas, intramucosal cancers (IMC) and invasive colorectal cancers (CRC) that were microsatellite stable (MSS) using a genome-wide SNP array and gene expression array (first cohort). In addition, validation analyses were examined (37 colorectal neoplasias). None of the mRNAs with a corresponding SCNA was found in the adenomas. However, three mRNAs, including ARFGEF2 at 20q13.13, N4BP2L2 at 13q13.1 and OLFM4 at 13q14.3 with a copy number (CN) gain at the corresponding locus were upregulated in IMCs of the first cohort. Moreover, upregulated expression of ARFGEF2 and OLFM4 was upregulated in the validation analysis. Finally, 28 mRNAs with gains of corresponding loci were pooled in invasive CRC of the first cohort. The mRNAs, including ACSS2 (20q11.22), DDX27 (20q13.13), MAPRE1 (20q11.21), OSBPL2 (20q11.22) and PHF20 (20q11.22-q11.23) with CN gains of the corresponding loci were identified in 28 mRNAs. Four of these mRNAs (DDX27, MAPRE1, OSBPL2 and PHF20) were upregulated in the invasive CRC in the validation analysis. We conclude that specific 13q and 22q CN gains with gene expression changes in the corresponding loci may play an important role in IMC cells' progression into invasive CRC.

DOI PubMed

Dual EZH2 and G9a inhibition suppresses multiple myeloma cell proliferation by regulating the interferon signal and IRF4-MYC axis.

Kazuya Ishiguro, Hiroshi Kitajima, Takeshi Niinuma, Reo Maruyama, Naotaka Nishiyama, Hitoshi Ohtani, Gota Sudo, Mutsumi Toyota, Hajime Sasaki, Eiichiro Yamamoto, Masahiro Kai, Hiroshi Nakase, Hiromu Suzuki

Cell death discovery 7 ( 1 ) 7 - 7 2021.01 [International journal]

Epigenetic mechanisms such as histone modification play key roles in the pathogenesis of multiple myeloma (MM). We previously showed that EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, and G9, a H3K9 methyltransferase, are potential therapeutic targets in MM. Moreover, recent studies suggest EZH2 and G9a cooperate to regulate gene expression. We therefore evaluated the antitumor effect of dual EZH2 and G9a inhibition in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed MM cell proliferation in vitro by inducing cell cycle arrest and apoptosis. Dual EZH2/G9a inhibition also suppressed xenograft formation by MM cells in vivo. In datasets from the Gene Expression Omnibus, higher EZH2 and EHMT2 (encoding G9a) expression was significantly associated with poorer prognoses in MM patients. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in MM cells. Notably, dual EZH2/G9a inhibition reduced H3K27/H3K9 methylation levels in MM cells and increased expression of endogenous retrovirus (ERV) genes, which suggests that activation of ERV genes may induce the IFN response. These results suggest that dual targeting of EZH2 and G9a may be an effective therapeutic strategy for MM.

DOI PubMed

Genome-wide analysis of microRNA to evaluate prognostic markers in isolated cancer glands and surrounding stroma in high-grade serous ovarian carcinoma.

Chie Sato, Mitsumasa Osakabe, Takayuki Nagasawa, Hiromu Suzuki, Hiroaki Itamochi, Tsukasa Baba, Tamotsu Sugai

Oncology letters 20 ( 6 ) 338 - 338 2020.12 [International journal]

The molecular mechanisms responsible for the progression of ovarian cancer remain incompletely understood. By targeting multiple cancer-related genes, microRNAs (miRNAs) have been identified as key regulators of cancer development and progression. In addition, the microenvironment, which constitutes cancer glands and the surrounding stromal tissue at the invasive front, has an important role in cancer progression. Using array-based analysis of 14 cases (cohort 1), the aim of the present study was to evaluate global miRNA expression in cancerous glands and surrounding stromal tissues (isolated using a crypt isolation method), in order to identify potential prognostic markers of high-grade serous carcinoma (HGSC). Reverse transcription-quantitative PCR was also used to verify the results in cohort 1 (14 cases) and in 16 additional HGSC cases (cohort 2; verification cohort). Firstly, miRNA expression levels were compared between HGSC and normal samples among both the isolated cancer gland and stromal tissue samples. Secondly, miRNA expression was compared between HGSC cases with recurrence and those without recurrence among the isolated cancer gland and stromal tissue samples. The results revealed six and seven miRNAs identified in both of the aforementioned comparisons in isolated cancer glands and surrounding stromal tissue, respectively. Furthermore, downregulation of miRNA-214-3p in isolated cancer glands and downregulation of miRNA-320c in the corresponding stromal tissue were associated with a decrease in disease-free survival (without recurrence) in cohort 2. These findings indicated that specific miRNAs expressed in cancer cells and surrounding stromal cells of HGSC may be potential biomarkers predicting patient prognosis.

DOI PubMed

The clinicopathological and molecular features of sporadic gastric foveolar type neoplasia.

Tamotsu Sugai, Noriyuki Uesugi, Wataru Habano, Ryo Sugimoto, Makoto Eizuka, Yasuko Fujita, Mitsumasa Osakabe, Yosuke Toya, Hiromu Suzuki, Takayuki Matsumoto

Virchows Archiv : an international journal of pathology 477 ( 6 ) 835 - 844 2020.12 [International journal]

Gastric intraepithelial foveolar type neoplasia (IEFN) is not well defined. In addition, atrophic mucosa (AM) is an important issue to consider when evaluating gastric tumorigenesis. Here, we assessed the clinicopathological characteristics and molecular alterations contributing to the development of IEFN compared with intestinal type neoplasia. We examined the clinicopathological and molecular features of 42 cases of IEFN with low-grade dysplasia (LGD) and those of 77 cases of intraepithelial intestinal type neoplasia (IEIN) with LGD. The clinicopathological and molecular features examined included the AM status, mucin phenotype expression, CDX2 expression, p53 overexpression, β-catenin intranuclear accumulation, microsatellite instability (MSI), DNA methylation status (low methylation epigenotype [LME], intermediate ME, or high ME), allelic imbalances (AIs), and APC promoter 1B mutations. There were no differences in the frequencies of AM and rates of CDX2 expression between IEFN and IEIN cases. Although no differences in the frequencies of p53 overexpression and MSI were observed between the two histological types, intranuclear expression of β-catenin was significantly higher in IEIN than in IEFN. In addition, although the rate of LME was significantly higher in IEFN cases than in IEIN cases, IEFN was characterized by AIs at multiple foci. Finally, mutation of the APC promoter 1B, which is a characteristic of gastric adenocarcinoma and proximal polyposis of the stomach (potentially resembling IEFN), was detected in only one IEFN case. These findings suggested that IEFN may be an independent entity in terms of molecular alterations including the presence of multiple AIs and LME.

DOI PubMed

【大腸鋸歯状病変の新展開】分類不能鋸歯状病変を由来としたTraditional serrated adenomaの癌化症例

山川司, 吉井新二, 市原真, 大和田紗恵, 柴田泰洋, 風間友江, 平山大輔, 久保俊之, 能正勝彦, 須藤豪太, 山本英一郎, 鈴木拓, 山野泰穂, 仲瀬裕志

胃と腸 ( (株)医学書院 ) 55 ( 13 ) 1631 - 1638 2020.12

＜文献概要＞患者は40歳代,女性.血便を主訴に近医を受診し,下部消化管内視鏡検査にて上部直腸(Ra)に径約35mmのLST-GMを認めた.本病変は辺縁に平坦部分を伴い複数の分葉構造を呈し,拡大所見では平坦部分は鋸歯状構造を伴ったII型様pitで構成されていたが,既存の分類には合致しない鋸歯状病変と考えられた.病変の大部分を占める中心の隆起部ではIV型pitを主体としcytological dysplasiaを疑った.病理組織像では平坦部の表層部分にわずかに鋸歯状構造は認めるが,深部はうねった管状腺管で腺腫様であり,免疫染色Ki-67では陽性細胞が散在し腺腫や既存の鋸歯状病変にも当てはまらない組織像であった.一方,隆起部では鋸歯状変化を呈するTSA様の領域と,絨毛状の形態をとりtubulovillous adenomaと考えられる領域が混在していた.また,隆起部の粘膜深部に細胞異型,構造異型が強く,腺管が密に増殖した領域があり,同部位でp53,Ki-67がびまん性の陽性を示しtub1相当の腺癌(Tis)と診断した.遺伝子解析では平坦部,隆起部いずれもKRAS変異陽性(G13D)で一連の病変と考えられた.本症例は内視鏡所見や免疫染色,遺伝子解析結果から既存の分類に当てはまらない鋸歯状病変を基盤として発育進展した病変と考え,ここに報告する.

Sigmoid colon tumor with G-nas mutation presenting unique morphology.

Gota Sudo, Hironori Aoki, Yuko Omori, Hiromu Suzuki, Hiroshi Nakase

Gastrointestinal endoscopy 92 ( 5 ) 1133 - 1135 2020.11 [Refereed] [International journal]

DOI PubMed

AEBP1の発現上昇は大腸がんの腫瘍血管新生を促進する

山本英一郎, 萬顕, 新沼猛, 北嶋洋志, 甲斐正広, 鈴木拓

電気泳動 ( 日本電気泳動学会 ) 64 ( Suppl. ) s38 - s38 2020.11

Prolonged oxidative stress and delayed tissue repair exacerbate acetaminophen-induced liver injury in aged mice.

Naoki Tanimizu, Norihisa Ichinohe, Hiromu Suzuki, Toshihiro Mitaka

Aging 12 ( 19 ) 18907 - 18927 2020.10 [International journal]

The liver gradually loses its regenerative capabilities with aging. However, it remains unknown whether aging affects drug-induced liver injury. Here, we used acetaminophen induced acute liver injury model to compare tissue injury and regeneration of aged mice (>80 weeks old) with young ones (8-10 weeks old). The mortality of aged mice after acetaminophen injury was higher than that of young mice. Transient increase of serum GOT and decrease of reduced glutathione (GSH) were not returned to original levels in aged mice even at 48 hours. In addition, Foxm1b and its targets Ccnd1 and Cdk1 were upregulated in young but not in aged mice after 48 hours. Moreover, an apoptosis-related gene, Cidea, was upregulated specifically in aged livers, which was consistent with increased number of TUNEL+ hepatocytes. Unexpectedly, damaged hepatocytes were retained in aged liver tissue, which may be caused by impaired recruitment of macrophages to the damaged area, without increases in Ccl2 after acetaminophen injury. Collectively, prolonged oxidative stress due to delayed recovery of GSH and the retention of damaged hepatocytes may suppress tissue repair and hepatocyte proliferation, resulting in exacerbation of acetaminophen injury in aged mice. Thus, aging is a risk factor conferring susceptibility against drug-induced liver injury.

DOI PubMed

分類不能鋸歯状病変を伴ったtraditional serrated adenoma癌化症例

山川司, 吉井新二, 市原真, 秋田浩太朗, 大和田紗恵, 横山佳浩, 柴田泰洋, 風間友江, 赤保内正和, 久保俊之, 仲地耕平, 能正勝彦, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 山野泰穂, 仲瀬裕志

Gastroenterological Endoscopy ( (一社)日本消化器内視鏡学会 ) 62 ( Suppl.2 ) 2150 - 2150 2020.10

分類不能鋸歯状病変を伴ったtraditional serrated adenoma癌化症例

Gastroenterological Endoscopy ( (一社)日本消化器内視鏡学会 ) 62 ( Suppl.2 ) 2150 - 2150 2020.10

頭頸部扁平上皮がんの微小環境におけるAEBP1の解析

萬顕, 関口翔平, 山本英一郎, 新沼猛, 高澤啓, 須藤豪太, 畠中柚衣, 北嶋洋志, 甲斐正広, 小山内誠, 宮崎晃亘, 高野賢一, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 PJ14 - 6 2020.10

AEBP1の発現上昇は大腸がんの腫瘍血管新生を促進する

山本英一郎, 萬顕, 新沼猛, 北嶋洋志, 須藤豪太, 甲斐正広, 高野賢一, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 OJ14 - 5 2020.10

早期大腸がん浸潤先進部の分子解析

須藤豪太, 山本英一郎, 青木敬則, 高澤啓, 新沼猛, 久保俊之, 萬顕, 北嶋洋志, 甲斐正広, 小山内誠, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 PJ14 - 8 2020.10

肝芽腫におけるDNAメチル化解析に基づく予後予測モデルの確立

近藤享史, 本多昌平, 鈴木拓, 荒桃子, 北河徳彦, 田中祐吉, 田中水緒, 新開真人, 檜山英三, 武冨紹信

日本小児血液・がん学会雑誌 ( (一社)日本小児血液・がん学会 ) 57 ( 4 ) 255 - 255 2020.10

非乳頭十二指腸腺腫、粘膜内癌におけるゲノム・エピゲノムの統合解析

澤田武, 佐々木泰史, 太田亮介, 津山翔, 八尾隆史, 中西宏佳, 山本英一郎, 久保田英嗣, 片岡洋望, 鈴木拓, 時野隆至, 源利成

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 OJ14 - 5 2020.10

新規癌抑制遺伝子SALL3はトリプルネガティブ乳癌の薬剤抵抗性に関与する

松下洋輔, 小松正人, 清谷一馬, 吉丸哲郎, 新沼猛, 鈴木拓, 本田純子, 井本逸勢, 丹黒章, 三好康雄, 笹三徳, 片桐豊雅

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 OJ14 - 7 2020.10

Dysregulation of microRNA expression during the progression of colorectal tumors.

Makoto Eizuka, Mitsumasa Osakabe, Ayaka Sato, Yasuko Fujita, Yoshihito Tanaka, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki, Tamotsu Sugai

Pathology international 70 ( 9 ) 633 - 643 2020.09 [International journal]

MicroRNAs (miRNAs) are potential biomarkers of neoplastic lesions, but additional information on dysregulated miRNA expression during progression of the adenoma-adenocarcinoma sequence may be helpful to identify the role of miRNAs in this sequence. We examined the expression levels of 13 miRNAs (hsa-miRNA-19a-3p, hsa-miRNA-21-5p, hsa-miRNA-27a-3p, hsa-miRNA-27b-3p, hsa-miRNA-31-5p, hsa-miRNA-34b-3p, hsa-miRNA-125b-5p, hsa-miRNA-143-3p, miRNA-191-5p, hsa-miRNA-193b-3p, hsa-miRNA-195-5p, hsa-miRNA-206 and hsa-let-7a-5p) that are closely associated with colorectal carcinogenesis in 40 conventional adenomas (tubular and tubulovillous adenomas), 20 intramucosal carcinomas (IMCs) and 60 invasive colorectal cancers (iCRCs) using reverse-transcription polymerase chain reaction. These 120 tumors were divided into two cohorts, that is, cohort 1 (60 cases) and cohort 2 (for validation; 60 cases). We analyzed the expression levels of these miRNAs in the first step (adenoma→IMC) and second step IMC→iCRC) of the adenoma-carcinoma sequence in both cohorts. Although no significant differences in the expression of any of the 13 miRNAs were found between adenomas and IMCs consistently in both cohorts, the expression levels of hsa-miRNA-125b-5p, hsa-miRNA-143-3p, and hsa-miRNA-206 were significantly upregulated in iCRC in both cohorts compared with those in IMC. The current results suggest that certain miRNAs, including hsa-miRNA-125b-5p, hsa-miRNA-143-3p and hsa-miRNA-206, are candidate markers that play critical roles in the progression of IMC to iCRC.

DOI PubMed

Traditional serrated adenoma has two distinct genetic pathways for molecular tumorigenesis with potential neoplastic progression.

Yoshihito Tanaka, Makoto Eizuka, Noriyuki Uesugi, Keisuke Kawasaki, Hiroo Yamano, Hiromu Suzuki, Takayuki Matsumoto, Tamotsu Sugai

Journal of gastroenterology 55 ( 9 ) 846 - 857 2020.09 [Domestic journal]

BACKGROUND: Recent studies have shown that traditional serrated adenoma (TSA) can be classified into BRAF and KRAS subtypes. Here, we examined the clinicopathological and molecular findings of 73 TSAs. MATERIALS AND METHODS: TSAs were subclassified into BRAF type (46 cases, type A) and KRAS type (27 cases, type B) and divided into polyp head (TSA component) and base (precursor component [PC]) to identify pathological and molecular differences between the two components. BRAF and KRAS mutations, microsatellite instability (MSI), and DNA methylation status of the TSA component and PC were analyzed. In addition, immunohistochemical expressions of annexin A10, MUC2, MUC5AC, MUC6, and CD10 were also examined. Finally, we compared endoscopic findings with histological features. RESULTS: We classified type As into 31 type A1s with mutation of the corresponding PC (42.5%) and 15 type A2s without mutation of the PC (20.5%). None of the corresponding PCs without KRAS mutation were observed in type Bs. MSI was not detected in the TSAs examined. There were significant differences in the frequency of annexin A10 and MUC5AC expression between the three subtypes. Furthermore, we compared the TSA component with the corresponding PC to identify the progression mechanism between the two components. Methylation status played an important role in the progression of type A1 from the corresponding PC, unlike type A2 and type B. Finally, specific endoscopic findings were well correlated with distinct histological findings. CONCLUSION: TSAs were heterogeneous tumors with two or three pathways to neoplastic progression.

DOI PubMed

Aggressive variant of splenic marginal zone lymphoma characterized using a cancer panel test and treated with rituximab-containing chemotherapy: A case report.

Kazuya Ishiguro, Yasushi Sasaki, Yoshitake Takagi, Takeshi Niinuma, Hiromu Suzuki, Takashi Tokino, Toshiaki Hayashi, Tohru Takahashi, Tetsuyuki Igarashi, Yoshihiro Matsuno

Medicine 99 ( 35 ) e21938 2020.08 [International journal]

RATIONALE: Aggressive variant of splenic marginal zone lymphoma (AV-SMZL) is a very rare disease that is often associated with TP53 mutations and has a poor prognosis. On the other hand, recent advances in genome sequencing techniques enable us to understand the molecular characteristics of rare cancers such as AV-SMZL. Here we present a case of AV-SMZL analyzed using a genetic test. PATIENT CONCERNS: A 66-year-old woman was admitted with splenomegaly and lymphocytosis. Computed tomography revealed marked splenomegaly without lymphadenopathy in any other areas. The serum soluble interleukin-2 receptor (sIL-2R) level was significantly elevated. Peripheral and bone marrow blood tests showed an increase in abnormal lymphocytes. DIAGNOSIS: A splenectomy revealed an SMZL pattern with increased numbers of large cells and mitotic cells and a high Ki-67 positivity rate, which led to a diagnosis of AV-SMZL. Although TP53 mutation was not detected, mutations in NOTCH2, NCOA4, PTEN, EPHA3, and KMT2D were identified. Among these, the mutations in NCOA4, PTEN, and EPHA3 were novel pathogenic mutations in SMZL, which suggests they may be related to the aggressiveness and persistence of the disease. INTERVENTIONS: The patient was administered a rituximab-containing regimen and rituximab-maintenance therapy. OUTCOMES: The patient continues to exhibit a complete response. LESSONS: This is a case of AV-SMZL in which a cancer panel test successfully detected genetic alterations that are potentially associated with its pathogenesis. These findings suggest that genetic analysis is useful for making diagnoses as well as for determining treatment strategies in AV-SMZL.

DOI PubMed

SSA/P由来が示唆された横行結腸SM癌の一例

久保俊之, 山野泰穂, 中村友哉, 柴田泰洋, 山川司, 風間友江, 赤保内正和, 吉井新二, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 永塚真, 菅井有, 杉田真太朗, 長谷川匡, 遠藤高夫, 仲瀬裕志

Gastroenterological Endoscopy ( (一社)日本消化器内視鏡学会 ) 62 ( Suppl.1 ) 1327 - 1327 2020.08

横行結腸に発生したcytological dysplasiaを伴う分類困難な鋸歯状病変の1例

秋田浩太朗, 山野泰穂, 柴田泰洋, 久保俊之, 吉井新二, 横山佳浩, 風間友江, 三橋慧, 能正勝彦, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 寺井琴美, 辻脇光洋, 長谷川匡, 永塚真, 菅井有, 仲瀬裕志

Gastroenterological Endoscopy ( (一社)日本消化器内視鏡学会 ) 62 ( Suppl.1 ) 1265 - 1265 2020.08

Superficially serrated adenoma(SuSA)の発がんが観察された微小大腸癌の1例

柴田泰洋, 山野泰穂, 久保俊之, 吉井新二, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 寺井琴美, 長谷川匡, 永塚真, 菅井有, 仲瀬裕志

Gastroenterological Endoscopy ( (一社)日本消化器内視鏡学会 ) 62 ( Suppl.1 ) 1265 - 1265 2020.08

Lynch症候群に合併した多発微小大腸癌の1例

柴田泰洋, 山野泰穂, 久保俊之, 吉井新二, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 寺井琴美, 長谷川匡, 永塚真, 菅井有, 仲瀬裕志

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 117 ( 臨増総会 ) A319 - A319 2020.07

新規大腸がん間質関連遺伝子の同定とがん微小環境における機能の解明

須藤豪太, 山本英一郎, 三橋慧, 久保俊之, 山野泰穂, 鈴木拓, 仲瀬裕志

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 117 ( 臨増総会 ) A318 - A318 2020.07

Lynch症候群に合併した多発微小大腸癌の1例

柴田泰洋, 山野泰穂, 久保俊之, 吉井新二, 山下健太郎, 須藤豪太, 山本英一郎, 鈴木拓, 寺井琴美, 長谷川匡, 永塚真, 菅井有, 仲瀬裕志

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 117 ( 臨増総会 ) A319 - A319 2020.07

Upregulation of adipocyte enhancer-binding protein 1 in endothelial cells promotes tumor angiogenesis in colorectal cancer.

Akira Yorozu, Eiichiro Yamamoto, Takeshi Niinuma, Akihiro Tsuyada, Reo Maruyama, Hiroshi Kitajima, Yuto Numata, Masahiro Kai, Gota Sudo, Toshiyuki Kubo, Toshihiko Nishidate, Kenji Okita, Ichiro Takemasa, Hiroshi Nakase, Tamotsu Sugai, Kenichi Takano, Hiromu Suzuki

Cancer science 111 ( 5 ) 1631 - 1644 2020.05 [Refereed] [International journal]

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT-PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.

DOI PubMed

大腸がんにおけるAEBP1の発現上昇は腫瘍血管新生を促進する

鈴木拓, 萬顕, 山本英一郎, 新沼猛, 北嶋洋志, 甲斐正広, 仲瀬裕志

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 57回 63 - 63 2020.04

消化器がん内視鏡診断・治療の最前線大腸腫瘍性病変に対するTranslational researchの確立を目指した内視鏡診療

赤保内正和, 吉井新二, 鈴木拓, 山野泰穂, 仲瀬裕志

日本消化器病学会北海道支部例会・日本消化器内視鏡学会北海道支部例会プログラム・抄録集 ( 日本消化器病学会-北海道支部 ) 126回・120回 34 - 34 2020.03

Hypermethylation of CSF3R is a novel cisplatin resistance marker and predictor of response to postoperative chemotherapy in hepatoblastoma.

Sunao Fujiyoshi, Shohei Honda, Masashi Minato, Momoko Ara, Hiromu Suzuki, Eiso Hiyama, Akinobu Taketomi

Hepatology research : the official journal of the Japan Society of Hepatology 2020.01 [Refereed] [International journal]

AIM: Most hepatoblastoma patients undergo pre/postoperative cisplatin treatment. Approximately 20% patients are cisplatin resistant, and show poor prognosis and high recurrence rates. However, some cisplatin-sensitive patients show early recurrence. We consider that a small population of cisplatin-resistant cells may remain after preoperative chemotherapy. Previous studies showed a correlation between DNA hypermethylation and hepatoblastoma progression. Here, we examined whether DNA hypermethylation was related to cisplatin resistance and could be a potential indicator for cisplatin as postoperative chemotherapy. METHODS: We extracted DNA from 43 resected hepatoblastoma tumors. Methylation array analyses were performed in 11 samples, including six cisplatin-sensitive and five cisplatin-resistant samples. We also performed cDNA microarray analysis in parental and cisplatin-resistant HuH6 cells. Through comparison of the datasets, we selected the strongest correlated cisplatin-resistant candidate gene. Using bisulfite pyrosequencing, the candidate gene methylation level was assessed in 38 cisplatin-sensitive patients after checking its usefulness as a substitute modality of methylation array. Correlations between the methylation status and clinical data were analyzed. RESULTS: CSF3R was the strongest correlated variable. Bisulfite pyrosequencing analysis also confirmed CSF3R was significantly hypermethylated in cisplatin-resistant patients. Among the 38 cisplatin-sensitive patients, recurrence curves showed that the CSF3R high methylation patients had significantly higher recurrence than CSF3R low methylation patients. The recurrence curve of methylation high patients was similar to that of cisplatin-resistant patients. CONCLUSIONS: Our findings suggested that CSF3R hypermethylation was related to cisplatin resistance in HB patients and could be a predictor of postoperative chemotherapy, and indicate that CSF3R high methylation patients should be treated with non-CDDP regimens.

DOI PubMed

Differential expression of microRNAs in colorectal cancer: Different patterns between isolated cancer gland and stromal cells

Ayaka Sato, Yasuko Fujita, Koki Otsuka, Akira Sasaki, Hiromu Suzuki, Takayuki Matsumoto, Tamotsu Sugai

Pathology International ( Blackwell Publishing ) 70 ( 1 ) 21 - 30 2020.01

Although microRNAs (miRNAs) play an important role in invasive tumor lesions, which involve cancer tissues mixed with stromal tissues, the differences in miRNA expression between cancer and stromal cells remain unclear. We selected 13 miRNAs and examined their differential expression patterns in cancer gland cells and surrounding stromal cells isolated from 24 colorectal cancer (CRC) specimens using a crypt isolation method. Although six miRNAs were upregulated in gland cells, only three were upregulated in the corresponding stromal cells, in the cancer compared with non-cancer specimens. Next, we examined the differences in miRNA expression between isolated cancer gland and stromal cells. Five miRNAs showed statistical differences in their cancer-related differential expression patterns between isolated cancer gland and stromal cells. We then compared these miRNA expression patterns in isolated cancer gland and stromal cells with those in fresh intact tumor tissues, consisting of cancer nests and stromal tissue, obtained from the 24 CRCs. The expression patterns of three miRNAs in the intact cancer tissue samples did not correspond with those in the isolated components. Identification of the expression patterns of miRNAs in both the cancer gland and stromal cell components of the tumor microenvironment greatly contributes to evaluating epigenetic regulation in CRC.

DOI PubMed

Self-Renewal Capability of Hepatocytic Parental Progenitor Cells Derived From Adult Rat Liver Is Maintained Long Term When Cultured on Laminin 111 in Serum-Free Medium.

Junichi Kino, Norihisa Ichinohe, Masayuki Ishii, Hiromu Suzuki, Toru Mizuguchi, Naoki Tanimizu, Toshihiro Mitaka

Hepatology communications 4 ( 1 ) 21 - 37 2020.01 [International journal]

In this study, we investigated how the ability of hepatocytic parental progenitor cells (HPPCs) to self-renew can be maintained and how laminin (LN) isoforms play an important role in their self-renewal and maturation. Hepatocytes isolated from adult rat livers were cultured on hyaluronic acid to form colonies consisting of CD44+ small hepatocytes, which could be passaged on dishes coated with Matrigel. When second-passage cells were plated on Matrigel, LN111, or LN511, HPPCs appeared on Matrigel and LN111 but not on LN511. We identified two types of cells among the second-passage cells: Small, round cells and large, flat ones were observed on Matrigel, whereas the former and latter ones were specifically attached on LN111 and LN511, respectively. We hypothesized that small and round cells are the origin of HPPC colonies, and the binding to LN111 could be key to maintaining their self-renewal capability. Among the integrins involved in LN binding, integrins α3 and β1 were expressed in colonies on LN111 more than in those on LN511, whereas β4 was more strongly expressed in colonies on LN511. Integrin α3highα6β1high cells could form HPPC colonies on LN111 but not on LN511, whereas integrin α6β1low cells could not on either LN111 or LN511. In addition, neutralizing anti-integrin β1 and anti-LN111 antibodies inhibited the passaged cells' ability to attach and form colonies on LN111 by HPPCs. Matrigel overlay induced second-passage cells growing on LN111 to increase their expression of hepatic functional genes and to form 3-dimensional colonies with bile canalicular networks, whereas such a shift was poorly induced when they were grown onLN511. Conclusion: These results suggest that the self-renewal capability of HPPCs depends on LN111 through integrin β1 signaling.

DOI PubMed

「消化管腫瘍学の最前線-臨床と基礎のブリッジング」-オミックス解析に基づいた消化管腫瘍分子腫瘍発生理論の新展開- 非乳頭十二指腸腫瘍におけるDNAメチル化と遺伝子変異の統合解析

澤田武, 太田亮介, 津山翔, 八尾隆史, 波佐谷兼慶, 海崎泰治, 中西宏佳, 吉田尚弘, 辻重継, 土山寿志, 湊宏, 山本英一郎, 久保田英嗣, 片岡洋望, 佐々木泰史, 源利成, 鈴木拓

日本消化管学会雑誌 ( (一社)日本消化管学会 ) 4 ( Suppl. ) 136 - 136 2020.01

【エピジェネティクスと疾患】DNAメチル化

新沼猛, 鈴木拓

医学のあゆみ ( 医歯薬出版(株) ) 272 ( 1 ) 4 - 9 2020.01

DNAのメチル化は最もよく知られたエピジェネティック修飾のひとつであり、発生、分化から加齢、がんを含む多くの疾患まで広範な生命現象に関連している。DNAメチル化にはメチル基転移酵素であるDNAメチルトランスフェラーゼ(DNMT)ファミリーが関与しており、DNA複製の際の維持メチル化やde novoメチル化を制御している。また、TETファミリー蛋白による能動的なDNA脱メチル化も、DNAメチル化を制御する重要な因子であることが知られている。また、DNAメチル化を介した遺伝子発現制御には、メチル化DNA結合蛋白など種々の分子群が関連している。これらの複数の機構が細胞特異的なDNAメチル化や遺伝子発現を規定している。本稿では、DNAメチル化制御に関連する分子の構築、調節機構について紹介し、さらに発生時におけるDNAメチル化レベルのダイナミックな変化について述べる。(著者抄録)

Significance of gene mutations in the Wnt signaling pathway in traditional serrated adenomas of the colon and rectum.

Hiroyoshi Nakanishi, Takeshi Sawada, Yasuharu Kaizaki, Ryosuke Ota, Hiromu Suzuki, Eiichiro Yamamoto, Hironori Aoki, Makoto Eizuka, Kenkei Hasatani, Naoki Takahashi, Satoko Inagaki, Masahide Ebi, Hiroyuki Kato, Eiji Kubota, Hiromi Kataoka, Satoru Takahashi, Takashi Tokino, Toshinari Minamoto, Tamotsu Sugai, Yasushi Sasaki

PloS one 15 ( 2 ) e0229262 2020 [Refereed] [International journal]

Recent studies have shown that colorectal serrated lesions, which include sessile serrated adenomas (SSAs) and traditional serrated adenomas (TSAs), are precursors of colorectal cancer. However, the molecular mechanisms underlying the carcinogenesis, particularly in TSAs, remain largely uncharacterized. To clarify their molecular and clinicopathological characteristics, we performed mutation and methylation analyses of cancer-associated genes in 78 serrated lesions, including TSAs, SSAs and microvesicular hyperplastic polyps. Target exon sequence analysis was performed with 39 genes, including genes known to be frequently mutated in colorectal cancers and/or serrated lesions. We also used bisulfite pyrosequencing to assess the methylation status of various cancer-associated genes and marker genes of the CpG island methylator phenotype (CIMP). The prevalence of mutations in genes associated with Wnt signaling was significantly higher in TSAs than SSAs (65% vs. 28%, p < 0.01). Among those, RNF43 mutations were observed in 38% of TSAs and 17% of SSAs. In immunohistochemical studies of 39 serrated lesions, the prevalence of abnormal nuclear β-catenin accumulation was significantly higher in TSAs (57%) than SSAs (8%) (P = 0.01). SMOC1 methylation was detected in 54% of TSAs but in no SSAs (p < 0.01). Additionally, SMOC1 methylation was more prevalent among TSAs with KRAS mutation (82%) than with BRAF mutation (38%, p = 0.03). Lesions with CIMP-high or RNF43 mutations were detected only in TSAs with BRAF mutation, suggesting two distinct carcinogenic pathways in TSAs. Mutations in genes associated with Wnt signaling play a greater role in the carcinogenesis of TSAs than SSAs.

DOI PubMed

肝細胞癌におけるBRD4阻害の抗腫瘍効果メカニズムの解析(Analysis of the anti-tumor mechanism of BRD4 inhibition in hepatocellular carcinoma)

佐々木基, 北嶋洋志, 新沼猛, 若杉英樹, 石黒一也, 萬顕, 須藤豪太, 畠平知, 阿久津典之, 山本英一郎, 甲斐正広, 佐々木茂, 仲瀬裕志, 鈴木拓

札幌医学雑誌 ( 札幌医科大学 ) 88 ( 1-6 ) 21 - 35 2019.12

BRD4を含むBETファミリー蛋白質はアセチル化ヒストンのリーダー蛋白質であり、転写調節において重要な役割を担っている。BRD4阻害剤は様々な悪性腫瘍において抗腫瘍効果を示す事が報告されている。今回我々は、肝細胞癌におけるBRD4阻害の抗腫瘍効果メカニズムの解明を目的とした。まずBRD4阻害剤JQ1の抗腫瘍効果を9種類の肝癌細胞株を用いて検証した結果、増殖抑制、細胞周期停止、アポトーシスの誘導が認められた。次に我々は、JQ1が遺伝子発現に与える影響をマイクロアレイによって解析した。その結果、JQ1が細胞周期やアポトーシス関連遺伝子に強い影響を与えること、そしてBCAT1、DDR1、GDF15、FANCD2、SENP1、TYRO3など複数の肝細胞癌関連遺伝子の発現を抑制することを見いだした。これらの遺伝子のプロモーター領域へのBRD4結合が確認されたことから、これらは肝細胞癌におけるBRD4の標的遺伝子と考えられた。今回の結果から、BRD4阻害剤は複数の癌関連遺伝子を抑制することで、肝細胞癌に対する抗腫瘍効果を示すと考えられた。(著者抄録)

Sessile serrated adenoma/polyp showed rapid malignant transformation in the final 13 months.

Sadahiro Amemori, Hiro-O Yamano, Yoshihito Tanaka, Kenjiro Yoshikawa, Hiro-O Matsushita, Ryo Takagi, Eiji Harada, Yuko Yoshida, Kazunori Tsuda, Bunichiro Kato, Eri Tamura, Makoto Eizuka, Tamotsu Sugai, Yasushi Adachi, Eiichiro Yamamoto, Hiromu Suzuki, Hiroshi Nakase

Digestive endoscopy : official journal of the Japan Gastroenterological Endoscopy Society 32 ( 6 ) 979 - 983 2019.11 [Refereed] [International journal]

Based on the concept of the adenoma-carcinoma sequence, most colorectal cancers are considered to arise from conventional adenomas. However, recent studies suggested that a subset of colorectal cancers develop through the serrated neoplastic pathway. It has also been documented that serrated polyps can rapidly transform into invasive cancers even when they are small in size. We now describe a case of a sessile serrated adenoma/polyp which had been followed up for 4 years but eventually showed rapid transformation into an advanced cancer accompanied by a remarkable morphological change within only 13 months. Retrospective genetic and epigenetic analyses showed microsatellite instability, CpG island methylator phenotype-positive, and BRAF mutation in the lesion, suggesting the tumor had developed through the serrated neoplastic pathway. This case may provide valuable information about the natural history of sessile serrated adenoma/polyps which eventually progress to advanced cancers.

DOI PubMed

非乳頭十二指腸腫瘍におけるDNAメチル化解析(Methylation analysis of non-ampullary duodenal precancerous and cancerous lesions)

澤田武, 太田亮介, 鈴木拓, 津山翔, 八尾隆史, 中西宏佳, 山本英一郎, 久保田英嗣, 片岡洋望, 佐々木泰史, 源利成

日本癌学会総会記事 ( 日本癌学会 ) 78回 P - 2036 2019.09

頭頸部扁平上皮がんの微小環境におけるAEBP1の解析(Analysis of AEBP1 in the microenvironment of head and neck squamous cell carcinoma)

萬顕, 山本英一郎, 須藤豪太, 沼田有斗, 新沼猛, 北嶋洋志, 甲斐正広, 小島隆, 高野賢一, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 78回 P - 1171 2019.09

SMOC1の大腸腫瘍診断マーカーとしての臨床的有用性の検討(Clinical usefulness of SMOC1 as a diagnostic marker of colorectal precancerous lesions)

青木敬則, 山本英一郎, 高澤啓, 新沼猛, 山野泰穂, 萬顕, 北嶋洋志, 甲斐正広, 小山内誠, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 78回 P - 1323 2019.09

新規大腸がん線維芽細胞関連遺伝子の同定(Identification of a novel cancer associated fibroblast-related gene in colorectal cancer)

須藤豪太, 山本英一郎, 萬顕, 新沼猛, 杉本亮, 北嶋洋志, 久保俊之, 畠中柚衣, 甲斐正広, 時野隆至, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 78回 P - 3102 2019.09

正常大腸粘膜のヒストン修飾異常とCIMP大腸腫瘍の発がんリスクの関連(Repressive histone mark in normal colon is associated with the risk of CRC with CpG island methylator phenotype)

山本英一郎, 須藤豪太, 久保俊之, 萬顕, 原田拓, 青木敬則, 北嶋洋志, 新沼猛, 甲斐正広, 菅井有, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 78回 J - 2035 2019.09

多発性骨髄腫における新規エピジェネティック併用療法の開発(Development of a new combinational epigenetic therapy of multiple myeloma)

石黒一也, 北嶋洋志, 新沼猛, 石田禎夫, 丸山玲緒, 池田博, 山本英一郎, 甲斐正広, 佐々木泰史, 時野隆至, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 78回 P - 2241 2019.09

UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells.

Takeshi Niinuma, Hiroshi Kitajima, Masahiro Kai, Eiichiro Yamamoto, Akira Yorozu, Kazuya Ishiguro, Hajime Sasaki, Gota Sudo, Mutsumi Toyota, Tomo Hatahira, Reo Maruyama, Takashi Tokino, Hiroshi Nakase, Tamotsu Sugai, Hiromu Suzuki

Clinical epigenetics 11 ( 1 ) 70 - 70 2019.05 [Refereed] [International journal]

BACKGROUND: Ubiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) is a major regulator of epigenetic mechanisms and is overexpressed in various human malignancies. In this study, we examined the involvement of UHRF1 in aberrant DNA methylation and gene silencing in colorectal cancer (CRC). RESULTS: CRC cell lines were transiently transfected with siRNAs targeting UHRF1, after which DNA methylation was analyzed using dot blots, bisulfite pyrosequencing, and Infinium HumanMethylation450 BeadChip assays. Gene expression was analyzed using RT-PCR and gene expression microarrays. Depletion of UHRF1 rapidly induced genome-wide DNA demethylation in CRC cells. Infinium BeadChip assays and bisulfite pyrosequencing revealed significant demethylation across entire genomic regions, including CpG islands, gene bodies, intergenic regions, and repetitive elements. Despite the substantial demethylation, however, UHRF1 depletion only minimally reversed CpG island hypermethylation-associated gene silencing. By contrast, the combination of UHRF1 depletion and histone deacetylase (HDAC) inhibition reactivated the silenced genes and strongly suppressed CRC cell proliferation. The combination of UHRF1 depletion and HDAC inhibition also induced marked changes in the gene expression profiles such that cell cycle-related genes were strikingly downregulated. CONCLUSIONS: Our results suggest that (i) maintenance of DNA methylation in CRC cells is highly dependent on UHRF1; (ii) UHRF1 depletion rapidly induces DNA demethylation, though it is insufficient to fully reactivate the silenced genes; and (iii) dual targeting of UHRF1 and HDAC may be an effective new therapeutic strategy.

DOI PubMed

DOT1L inhibition blocks multiple myeloma cell proliferation by suppressing IRF4-MYC signaling.

Kazuya Ishiguro, Hiroshi Kitajima, Takeshi Niinuma, Tadao Ishida, Reo Maruyama, Hiroshi Ikeda, Toshiaki Hayashi, Hajime Sasaki, Hideki Wakasugi, Koyo Nishiyama, Tetsuya Shindo, Eiichiro Yamamoto, Masahiro Kai, Yasushi Sasaki, Takashi Tokino, Hiroshi Nakase, Hiromu Suzuki

Haematologica 104 ( 1 ) 155 - 165 2019.01 [Refereed] [International journal]

Epigenetic alterations play an important role in the pathogenesis in multiple myeloma, but their biological and clinical relevance is not fully understood. Here, we show that DOT1L, which catalyzes methylation of histone H3 lysine 79, is required for myeloma cell survival. DOT1L expression levels were higher in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in normal plasma cells. Treatment with a DOT1L inhibitor induced cell cycle arrest and apoptosis in myeloma cells, and strongly suppressed cell proliferation in vitro The anti-myeloma effect of DOT1L inhibition was confirmed in a mouse xenograft model. Chromatin immunoprecipitation-sequencing and microarray analysis revealed that DOT1L inhibition downregulated histone H3 lysine 79 dimethylation and expression of IRF4-MYC signaling genes in myeloma cells. In addition, DOT1L inhibition upregulated genes associated with immune responses and interferon signaling. Myeloma cells with histone modifier mutations or lower IRF4/MYC expression were less sensitive to DOT1L inhibition, but with prolonged treatment, anti-proliferative effects were achieved in these cells. Our data suggest that DOT1L plays an essential role in the development of multiple myeloma and that DOT1L inhibition may provide new therapies for myeloma treatment.

DOI PubMed

Expression of Epithelial-Mesenchymal Transition Proteins in Pancreatic Anaplastic (Undifferentiated) Carcinoma.

Ishida K, Yamashita R, Osakabe M, Uesugi N, Yamada N, Nitta H, Fujishima F, Motoi F, Suzuki H, Shimamura H, Noda Y, Sawai T, Unno M, Sasano H, Sasaki A, Sugai T

Pancreas 48 ( 1 ) 36 - 42 2019.01 [Refereed] [International journal]

OBJECTIVES: The aim of this study was to identify an association of pancreatic anaplastic carcinoma (APC) with the epithelial-mesenchymal transition (EMT). METHODS: Resected APCs (n = 24) were examined to assess components of APCs, including carcinomatous, transitional, and sarcomatous regions. Analysis was performed based on the immunoreactivity of E-cadherin and 3 EMT-related proteins: Slug (zinc finger protein SNAI2), Twist (Twist-related protein 1), and Zeb1 (zinc finger E-box-binding homeobox 1). Expression score was determined based on staining intensity and stained area of the target cells. Finally, we performed a hierarchical clustering based on the expression pattern of E-cadherin and EMT-related proteins of the sarcomatous component. RESULTS: The expression score of E-cadherin decreased in the order of sarcomatous > transitional > carcinomatous components (P < 0.01). Although there were significant differences in the immunohistochemical scores of Slug, Twist, and Zeb1 between carcinomatous and transitional components (P < 0.01), the significant difference in immunohistochemical score of Zeb1 between transitional and sarcomatous components was found (P < 0.05). Furthermore, APCs were divided into 2 subgroups based on the expression patterns of E-cadherin and EMT-related proteins (hierarchical clustering analysis). Consequently, these subgroups were distinguished by Twist expression. CONCLUSIONS: Epithelial-mesenchymal transition plays an essential role in the pathogenesis of APC.

DOI PubMed

Integrated Analysis of the Endoscopic, Pathological and Molecular Characteristics of Colorectal Tumorigenesis.

Suzuki H, Yamamoto E, Yamano HO, Nakase H, Sugai T

Digestion 99 ( 1 ) 33 - 38 2019 [Refereed]

DOI PubMed

Molecular biology of colorectal cancer -mechanism of tumorigenesis and molecular subtypes-

Hiromu Suzuki, Eiichiro Yamamoto, Hiroshi Nakase

Journal of Japanese Society of Gastroenterology ( Japanese Society of Gastroenterology ) 116 ( 11 ) 859 - 866 2019

DOI PubMed

Surface microstructures are associated with mutational intratumoral heterogeneity in colorectal tumors.

Taku Harada, Eiichiro Yamamoto, Hiro-O Yamano, Hironori Aoki, Hiro-O Matsushita, Kenjiro Yoshikawa, Ryo Takagi, Eiji Harada, Yoshihito Tanaka, Yuko Yoshida, Makoto Eizuka, Akira Yorozu, Gota Sudo, Hiroshi Kitajima, Takeshi Niinuma, Masahiro Kai, Yasushi Sasaki, Takashi Tokino, Tamotsu Sugai, Hiroshi Nakase, Hiromu Suzuki

Journal of gastroenterology 53 ( 12 ) 1241 - 1252 2018.12 [Refereed] [Domestic journal]

BACKGROUND: Recent studies revealed that colorectal tumors are composed of genetically diverse subclones. We aimed to clarify whether the surface microstructures of colorectal tumors are associated with genetic intratumoral heterogeneity (ITH). METHODS: The surface microstructures (pit patterns) of colorectal tumors were observed using magnifying endoscopy, and biopsy specimens were obtained from respective areas when tumors exhibited multiple pit patterns. A total of 711 specimens from 477 colorectal tumors were analyzed for BRAF, KRAS and TP53 mutations using pyrosequencing and direct sequencing. A panel of cancer-related genes was analyzed through targeted sequencing in 7 tumors. RESULTS: Colorectal tumors with multiple pit patterns exhibited more advanced pit patterns and higher frequencies of KRAS and/or TP53 mutations than tumors with a single pit pattern. In tumors with multiple pit patterns, mutations were observed as public (common to all areas) or private (specific to certain areas), and private KRAS and/or TP53 mutations were often variable and unrelated to the pit pattern grade. Notably, invasive CRCs frequently exhibited public TP53 mutations, even in adenomatous areas, which is indicative of their early malignant potential. Targeted sequencing revealed additional public and private mutations in tumors with multiple pit patterns, indicating their single clonal origin. CONCLUSIONS: Our results suggest intratumoral pit pattern variation does not simply reflect the process of colorectal tumor evolution, but instead represents genetically diverse subclones, and this diversity may be associated with malignant potential.

DOI PubMed

Dysregulation of miRNA in chronic hepatitis B is associated with hepatocellular carcinoma risk after nucleos(t)ide analogue treatment.

Hideki Wakasugi, Hideaki Takahashi, Takeshi Niinuma, Hiroshi Kitajima, Ritsuko Oikawa, Naoki Matsumoto, Yuko Takeba, Takehito Otsubo, Masayuki Takagi, Yasushi Ariizumi, Michihiro Suzuki, Chiaki Okuse, Shogo Iwabuchi, Masayuki Nakano, Noriyuki Akutsu, Jong-Hon Kang, Takeshi Matsui, Norie Yamada, Hajime Sasaki, Eiichiro Yamamoto, Masahiro Kai, Yasushi Sasaki, Shigeru Sasaki, Yasuhito Tanaka, Hiroshi Yotsuyanagi, Takeya Tsutsumi, Hiroyuki Yamamoto, Takashi Tokino, Hiroshi Nakase, Hiromu Suzuki, Fumio Itoh

Cancer letters 434 91 - 100 2018.10 [Refereed] [International journal]

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk.

DOI PubMed

Molecular Profiling Based on KRAS/BRAF Mutation, Methylation, and Microsatellite Statuses in Serrated Lesions.

Sugai T, Eizuka M, Fujita Y, Kawasaki K, Yamamoto E, Ishida K, Yamano H, Suzuki H, Matsumoto T

Digestive diseases and sciences 63 ( 10 ) 2626 - 2638 2018.10 [Refereed] [International journal]

AIM: The aim of your study is to characterize serrated lesions according to their molecular patterns, specifically BRAF/KRAS mutation, methylation, and microsatellite statuses. We evaluated the molecular patterns of 163 serrated lesions, including 37 microvesicular hyperplastic polyps, 73 sessile serrated adenomas/polyps (SSA/Ps), 31 traditional serrated adenomas, and 22 SSA/Ps with cytological dysplasia/adenocarcinoma. METHODS: Mutations in BRAF (V600E)/KRAS (exon 2) and microsatellite status [microsatellite stability (MSS) vs. MSI] were examined using a pyrosequencer and the PCR-based microsatellite method, respectively. DNA methylation status was classified as low (LME), intermediate (IME), or high methylation epigenotype (HME) according to a PCR-based two-step method. In addition, mucin and annexin A10 expression was examined. Finally, we performed a hierarchical clustering analysis of the BRAF/KRAS mutation, DNA methylation, and microsatellite statuses. RESULTS: The molecular patterns observed in the serrated lesions could be divided into five subgroups: lesions characterized by (1) BRAF mutation, HME, and MSI; (2) BRAF mutation, HME, and MSS; (3) BRAF mutation, LME/IME, and MSS; (4) no BRAF/KRAS mutations, LME/IME, and MSS; and (5) KRAS mutation, LME/IME, and MSS. In addition, we demonstrated that these observed molecular patterns help identify the associations of the molecular patterns and markers (i.e., mucin and annexin A10) with the clinicopathological findings, including histological features and histological diagnosis. CONCLUSIONS: We suggest that the identified molecular patterns play an important role in the pathway of serrated lesion development.

DOI PubMed

MicroRNA-31 reflects IL-6 expression in cancer tissue and is related with poor prognosis in bile duct cancer.

Keisuke Ishigami, Katsuhiko Nosho, Hideyuki Koide, Shinichi Kanno, Kei Mitsuhashi, Hisayoshi Igarashi, Masahiro Shitani, Masayo Motoya, Yasutoshi Kimura, Tadashi Hasegawa, Hiroyuki Kaneto, Ichiro Takemasa, Hiromu Suzuki, Hiroshi Nakase

Carcinogenesis 39 ( 9 ) 1127 - 1134 2018.09 [Refereed] [International journal]

Bile duct cancer is a highly aggressive malignancy wherein early diagnosis is difficult and few treatment options are available. MicroRNA-31 (miR-31) is reported to be related with survival in patients with gastrointestinal cancers; however, the regulatory mechanism of miR-31 and association between miR-31 expression and survival in patients with bile duct cancer cases have not been established. Thus, we evaluated miR-31 expression in bile duct cancer tissues and assessed its relationship with prognosis. Additionally, we examined the effects of several cytokines on miR-31 expression. The study included 81 samples of bile duct cancer tissues. MiR-31 expression in bile duct cancer cells was significantly higher than that in normal bile duct epithelial cells (P = 0.038). There were no significant associations between miR-31 expression and clinical or pathological characteristics, except for tumour size (P = 0.012). In Kaplan-Meier analysis, high miR-31 expression was significantly associated with shorter survival (log-rank test, P = 0.0082). In multivariate Cox regression analysis, high miR-31 expression was significantly associated with prognosis (P = 0.043), independent of clinical or pathological features. Interleukin-6 (IL-6) significantly promoted miR-31 expression and cell proliferation in a dose-dependent manner, and the inhibition of STAT-3 signalling significantly suppressed miR-31 expression and cell proliferation. In conclusion, high expression was significantly associated with poor prognosis in bile duct cancer patients. The IL-6-STAT-3 signalling regulated bile duct cancer cell proliferation and miR-31 expression. Our findings suggest that miR-31 may be a promising biomarker that reflects IL-6 expression in bile duct cancer tissues and predicts poor prognosis.

DOI PubMed

新規大腸がん線維芽細胞関連遺伝子の同定(Identification of cancer-associated fibroblast-related genes in colorectal cancer)

沼田有斗, 山本英一郎, 萬顕, 新沼猛, 杉本亮, 北嶋洋志, 甲斐正広, 青木敬則, 須藤豪太, 時野隆至, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 77回 2189 - 2189 2018.09

大腸がんにおける腫瘍血管内皮関連遺伝子の同定(Identification and characterization of a tumor endothelium-related gene in colorectal cancer)

萬顕, 山本英一郎, 沼田有斗, 新沼猛, 北嶋洋志, 甲斐正広, 須藤豪太, 黒瀬誠, 時野隆至, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 77回 1574 - 1574 2018.09

肝細胞癌におけるBET阻害剤の抗腫瘍メカニズムの解析(Analysis of antitumor mechanism of BET inhibition in hepatocellular carcinoma)

佐々木基, 新沼猛, 北嶋洋志, 山本英一郎, 石黒一也, 若杉英樹, 萬顕, 須藤豪太, 甲斐正広, 鈴木拓, 仲瀬裕志

日本癌学会総会記事 ( 日本癌学会 ) 77回 1387 - 1387 2018.09

SMOC1のエピジェネティックなサイレンシングは大腸鋸歯状腺腫の発育進展に関与する(Epigenetic silencing of SMOC1 is associated with development of colorectal traditional serrated adenomas)

青木敬則, 山本英一郎, 高澤啓, 新沼猛, 山野泰穂, 萬顕, 北嶋洋志, 甲斐正広, 澤田典均, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 77回 1871 - 1871 2018.09

大腸鋸歯状病変における、遺伝子変異、コピー数変化、DNAメチル化の統合解析(Integrative analysis of gene mutations, copy number alterations and DNA methylation in colorectal serrated lesions)

澤田武, 中西宏佳, 佐々木泰史, 山本英一郎, 青木敬則, 永塚真, 高橋直樹, 太田亮介, 久保田英嗣, 片岡洋望, 源利成, 菅井有, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 1872 - 1872 2018.09

DOT1L阻害はIRF4-MYCシグナルの抑制を介して多発性骨髄腫細胞の増殖を抑制する(DOT1L inhibition blocks multiple myeloma cell proliferation by suppressing IRF4-MYC signaling)

石黒一也, 北嶋洋志, 新沼猛, 石田禎夫, 丸山玲緒, 池田博, 山本英一郎, 甲斐正広, 佐々木泰史, 時野隆至, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 988 - 988 2018.09

大腸腫瘍の表面構造が反映する腫瘍内不均一性(Microsurface structures are associated with mutational intratumoral heterogeneity in colorectal tumors)

山本英一郎, 山野泰穂, 青木敬則, 須藤豪太, 新沼猛, 甲斐正広, 佐々木泰史, 時野隆至, 菅井有, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 2405 - 2405 2018.09

慢性胃炎および胃がんに関連する長鎖non-coding RNAの同定と機能解析(Identification and characterization of a long non-coding RNA associated with chronic gastritis and gastric caner)

北嶋洋志, 丸山玲緒, 山本英一郎, 新沼猛, 甲斐正広, 佐々木泰史, 時野隆至, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 512 - 512 2018.09

トリプルネガティブ乳癌におけるSALL3の高頻度なエピジェネティックな不活化機構(Downregulation of SALL3 by recurrent genetic and epigenetic alterations is involved in triple negative breast cancers)

松下洋輔, 小松正人, 清谷一馬, 吉丸哲郎, 新沼猛, 鈴木拓, 本田純子, 井本逸勢, 丹黒章, 三好康雄, 笹三徳, 片桐豊雅

日本癌学会総会記事 ( 日本癌学会 ) 77回 1410 - 1410 2018.09 [Refereed]

Screening for long noncoding RNAs associated with oral squamous cell carcinoma reveals the potentially oncogenic actions of DLEU1.

Koyo Nishiyama, Reo Maruyama, Takeshi Niinuma, Masahiro Kai, Hiroshi Kitajima, Mutsumi Toyota, Yui Hatanaka, Tomohiro Igarashi, Jun-Ichi Kobayashi, Kazuhiro Ogi, Hironari Dehari, Akihiro Miyazaki, Akira Yorozu, Eiichiro Yamamoto, Masashi Idogawa, Yasushi Sasaki, Tamotsu Sugai, Takashi Tokino, Hiroyoshi Hiratsuka, Hiromu Suzuki

Cell death & disease 9 ( 8 ) 826 - 826 2018.08 [Refereed] [International journal]

Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

DOI PubMed

Subtypes of the Type II Pit Pattern Reflect Distinct Molecular Subclasses in the Serrated Neoplastic Pathway.

Hironori Aoki, Eiichiro Yamamoto, Hiro-O Yamano, Tamotsu Sugai, Tomoaki Kimura, Yoshihito Tanaka, Hiro-O Matsushita, Kenjiro Yoshikawa, Ryo Takagi, Eiji Harada, Michiko Nakaoka, Yuko Yoshida, Taku Harada, Gota Sudo, Makoto Eizuka, Akira Yorozu, Hiroshi Kitajima, Takeshi Niinuma, Masahiro Kai, Masanori Nojima, Hiromu Suzuki, Hiroshi Nakase

Digestive diseases and sciences 63 ( 7 ) 1920 - 1928 2018.07 [Refereed] [International journal]

BACKGROUND: Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood. AIMS: We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs. METHODS: A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing. RESULTS: Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers. CONCLUSIONS: These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

DOI PubMed

Translational regulation by miR-301b upregulates AMP deaminase in diabetic hearts

Yuki Tatekoshi, Masaya Tanno, Hidemichi Kouzu, Koki Abe, Takayuki Miki, Atsushi Kuno, Toshiyuki Yano, Satoko Ishikawa, Wataru Ohwada, Tatsuya Sato, Takeshi Niinuma, Hiromu Suzuki, Tetsuji Miura

Journal of Molecular and Cellular Cardiology ( Academic Press ) 119 138 - 146 2018.06 [Refereed]

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (> 50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3′UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.

DOI PubMed

Epigenetic silencing of miR-200b is associated with cisplatin resistance in bladder cancer.

Tetsuya Shindo, Takeshi Niinuma, Naotaka Nishiyama, Nobuo Shinkai, Hiroshi Kitajima, Masahiro Kai, Reo Maruyama, Takashi Tokino, Naoya Masumori, Hiromu Suzuki

Oncotarget 9 ( 36 ) 24457 - 24469 2018.05 [Refereed] [International journal]

In this study, we identified microRNAs (miRNAs) involved in cisplatin (CDDP) resistance in bladder cancer (BCa). After establishing CDDP-resistant BCa cell lines (T24RC and EJ138RC), TaqMan arrays revealed that members of the miR-200 family (miR-200b, miR-200a and miR-429) were downregulated in T24RC as compared to parental T24 cells. miR-200b was associated with CDDP sensitivity in BCa cells, and its downregulation was associated with CpG island hypermethylation. Pharmacological demethylation using 5-aza-2'-deoxycytidine restored miR-200b expression, and the combination of 5-aza-2'-deoxycytidine + CDDP strongly inhibited T24RC cell proliferation. Microarray analysis revealed that miR-200b + CDDP induced genes involved in CDDP sensitivity or cytotoxicity, including IGFBP3, ICAM1 and TNFSF10, in the resistant cells. Expression and DNA methylation of miR-200b were inversely associated in primary BCa, and low expression/high methylation was associated with poor overall survival. These results suggest downregulation of miR-200b is associated with CDDP resistance in BCa. Epigenetic silencing of miR-200b may be a marker of CDDP resistance and a useful therapeutic target for overcoming CDDP resistance in BCa.

DOI PubMed

Comprehensive molecular analysis based on somatic copy number alterations in intramucosal colorectal neoplasias and early invasive colorectal cancers

Tamotsu Sugai, Makoto Eizuka, Wataru Habano, Yasuko Fujita, Ayaka Sato, Ryo Sugimoto, Kouki Otsuka, Eiichiro Yamamoto, Takayuki Matsumoto, Hiromu Suzuki

Oncotarget ( Impact Journals LLC ) 9 ( 33 ) 22895 - 22906 2018.05 [Refereed]

It is unclear whether somatic copy number alterations (SCNAs) contribute to the development of colorectal cancer (CRC). Here, we aimed to identify the molecular profiles of early colorectal carcinogenesis based on SCNAs and determine the associations of other molecular abnormalities for the detection of neoplasia in both intramucosal neoplasia (IMN) and invasive CRC with invasion into the muscular layer without metastasis (early invasive CRC). A single nucleotide polymorphism array was used to examine 100 colorectal IMNs (low-grade adenoma [LGA], 40 high-grade adenoma [HGA], 25 intramucosal adenocarcinoma [IMA], 35) and early invasive CRC (20 tumors). In addition, genetic mutations (KRAS, BRAF), TP53 overexpression, microsatellite instability (MSI), and DNA methylation (low, intermediate, high) were examined. Hierarchical clustering analysis based on the SCNA pattern was carried out to identify molecular profiles in IMNs and early invasive CRC. Colorectal tumors were classified into three subgroups based on SCNA patterns. Subgroup 1 was characterized by multiple SCNAs, subgroup 3 was closely associated with infrequent SCNAs, and subgroup 2 was an intermediate subgroup in SCNA pattern between subgroups 1 and 3. Although mutations in KRAS were commonly found in all three subgroups, overexpression of TP53 was observed primarily in subgroup 1 and 2. DNA methylation showed a low/intermediate type. Finally, no MSI was detected. Each subgroup was correlated with histology (subgroup 1, early invasive CRC subgroup 2, LGA subgroups 2 and 3, HGA and IMA). Considerable SCNAs may be required for acquisition of invasive ability in CRC. Our results provide novel insights into early CRC.

DOI PubMed

Frequent downregulation of LRRC26 by epigenetic alterations is involved in the malignant progression of triple-negative breast cancer.

Yoshimasa Miyagawa, Yosuke Matsushita, Hiromu Suzuki, Masato Komatsu, Tetsuro Yoshimaru, Ryuichiro Kimura, Ayako Yanai, Junko Honda, Akira Tangoku, Mitsunori Sasa, Yasuo Miyoshi, Toyomasa Katagiri

International journal of oncology 52 ( 5 ) 1539 - 1558 2018.05 [Refereed] [International journal]

Triple-negative breast cancer (TNBC), defined as breast cancer lacking estrogen- and progesterone‑receptor expression and human epidermal growth factor receptor 2 (HER2) amplification, is a heterogeneous disease. RNA-sequencing analysis of 15 TNBC specimens and The Cancer Genome Atlas-TNBC dataset analysis identified the frequent downregulation of leucine-rich repeat-containing 26 (LRRC26), which negatively regulates nuclear factor-κB (NF-κB) signaling, in TNBC tissues. Quantitative polymerase chain reaction and bisulfite pyrosequencing analyses revealed that LRRC26 was frequently silenced in TNBC tissues and cell lines as a result of promoter methylation. LRRC26 expression was restored by 5-aza-2'-deoxycytidine (5'-aza-dC) treatment in HCC1937 TNBC cells, which lack LRRC26 expression. Notably, small interfering RNA-mediated knockdown of LRRC26 expression significantly enhanced the anchorage-independent growth, invasion and migration of HCC70 cells, whereas ectopic overexpression of LRRC26 in BT20 cells suppressed their invasion and migration. Conversely, neither knockdown nor overexpression of LRRC26 had an effect on cell viability in the absence of tumor necrosis factor-α (TNF-α) stimulation. Meanwhile, overexpression of LRRC26 caused the reduction of TNF-α-mediated NF-κB luciferase reporter activity, whereas depleting LRRC26 expression resulted in the upregulation of TNF-α-mediated NF-κB downstream genes [interleukin-6 (IL-6), IL-8 and C-X-C motif chemokine ligand-1]. Taken together, these findings demonstrate that LRRC26 is frequently downregulated in TNBC due to DNA methylation and that it suppresses the TNF-α-independent anchorage-independent growth, invasion and migration of TNBC cells. Loss of LRRC26 function may be a critical event in the aggressiveness of TNBC cells through a TNF-α/NF-κB-independent mechanism.

DOI PubMed

Frequent downregulation of LRRC26 by epigenetic alterations is involved in the malignant progression of triple-negative breast cancer.

Yoshimasa Miyagawa, Yosuke Matsushita, Hiromu Suzuki, Masato Komatsu, Tetsuro Yoshimaru, Ryuichiro Kimura, Ayako Yanai, Junko Honda, Akira Tangoku, Mitsunori Sasa, Yasuo Miyoshi, Toyomasa Katagiri

International Journal of Oncology 2018.03 [Refereed]

Triple-negative breast cancer (TNBC), defined as breast cancer lacking estrogen- and progesterone?receptor expression and human epidermal growth factor receptor 2 (HER2) amplification, is a heterogeneous disease. RNA-sequencing analysis of 15 TNBC specimens and The Cancer Genome Atlas-TNBC dataset analysis identified the frequent downregulation of leucine-rich repeat-containing 26 (LRRC26), which negatively regulates nuclear factor-κB (NF-κB) signaling, in TNBC tissues. Quantitative polymerase chain reaction and bisulfite pyrosequencing analyses revealed that LRRC26 was frequently silenced in TNBC tissues and cell lines as a result of promoter methylation. LRRC26 expression was restored by 5-aza-2'-deoxycytidine (5'-aza-dC) treatment in HCC1937 TNBC cells, which lack LRRC26 expression. Notably, small interfering RNA-mediated knockdown of LRRC26 expression significantly enhanced the anchorage-independent growth, invasion and migration of HCC70 cells, whereas ectopic overexpression of LRRC26 in BT20 cells suppressed their invasion and migration. Conversely, neither knockdown nor overexpression of LRRC26 had an effect on cell viability in the absence of tumor necrosis factor-α (TNF-α) stimulation. Meanwhile, overexpression of LRRC26 caused the reduction of TNF-α-mediated NF-κB luciferase reporter activity, whereas depleting LRRC26 expression resulted in the upregulation of TNF-α-mediated NF-κB downstream genes [interleukin-6 (IL-6), IL-8 and C-X-C motif chemokine ligand-1]. Taken together, these findings demonstrate that LRRC26 is frequently downregulated in TNBC due to DNA methylation and that it suppresses the TNF-α-independent anchorage-independent growth, invasion and migration of TNBC cells. Loss of LRRC26 function may be a critical event in the aggressiveness of TNBC cells through a TNF-α/NF-κB-independent mechanism.

DOI PubMed

Molecular profiling and genome-wide analysis based on somatic copy number alterations in advanced colorectal cancers

Tamotsu Sugai, Yayoi Takahashi, Makoto Eizuka, Ryo Sugimoto, Yasuko Fujita, Wataru Habano, Kouki Otsuka, Akira Sasaki, Eiichiro Yamamoto, Takayuki Matsumoto, Hiromu Suzuki

Molecular Carcinogenesis ( John Wiley and Sons Inc. ) 57 ( 3 ) 451 - 461 2018.03 [Refereed]

To characterize somatic alterations in colorectal cancer (CRC), we conducted a genome-scale analysis of 106 CRC specimens. We assessed comprehensive somatic copy number alterations (SCNAs) in these CRC specimens. In addition, we examined microsatellite instability (MSI low and high), genetic mutations (KRAS, BRAF, TP53, and PIK3CA), and DNA methylation status (classified into low, intermediate, and high type). We stratified molecular alterations in the CRCs using a hierarchical cluster analysis. The examined CRCs could be categorized into three subgroups using hierarchical cluster analysis. Tumors in subgroup 1 were characterized by a low frequency of SCNAs and a high frequency of MSI-high status, whereas tumors in subgroups 2 and 3 were closely associated with a high frequency of SCNAs. Tumors in subgroup 1 were preferentially present in the right-sided colon and showed frequent MSI-high status. Subgroup 3 was distinguished by specific alterations, including gains at 1q23-44, 1p11-36, 10q11-26, 10p11-13, 12q24-24, and 13q33-33. In contrast, tumors in subgroup 2 were characterized by copy-neutral LOH at 12p12-13, 1q24-25, and 10q22. In addition, KRAS mutations were more frequently found in subgroup 3 than in subgroup 1. TP53 mutations and intermediate levels of DNA methylation were common alterations in the three subgroups. SCNAs contributed to sporadic CRC, and there were three subgroups based on SCNAs that played a different role in driving the development of this disease.

DOI PubMed

Evaluation of Urinary DNA Methylation as a Marker for Recurrent Bladder Cancer: A 2-Center Prospective Study

Tetsuya Shindo, Takashi Shimizu, Masanori Nojima, Takeshi Niinuma, Reo Maruyama, Hiroshi Kitajima, Masahiro Kai, Naoki Itoh, Hiromu Suzuki, Naoya Masumori

Urology ( Elsevier Inc. ) 113 71 - 78 2018.03 [Refereed]

Objective: To clarify the clinical utility of urinary DNA methylation for detection of intravesical recurrence of non–muscle invasive BCa (NMIBC), we performed a 2-center prospective study. Patients and Methods: A series of 207 self-voided urine samples were prospectively collected from 132 patients with NMIBC who had undergone transurethral resection of BCa. Methylation of miRNA genes (miR-9-3, miR-124-2, miR-124-3, and miR-137) was analyzed using bisulfite pyrosequencing. The primary end point was detection of intravesical recurrence the secondary end point was prediction of late recurrence. The number of methylated genes (M-score) or quantitative level of methylation were compared with outcomes. Results: Twenty-six urine specimens were collected on the same day intravesical recurrence was detected, and 14 were collected from patients whose recurrences were found during the subsequent follow-up period (0-632 days, mean, 342.2 days). For detection of current recurrence, M-scores achieved 61.5% sensitivity and 74.0% specificity, and the area under the ROC curve was 0.71. Regarding prediction of late recurrence, patients with a high M-score (≥3) showed worse recurrence-free survival (P < .01). Multivariate analysis revealed that high M-scores were independently associated with current (P =.028) and late recurrence (P =.026). Elevated levels of urinary DNA methylation were also strongly associated with recurrence and radical cystectomy. Conclusion: Our data suggest that urinary methylation of miRNA genes may be a useful marker for detecting and predicting BCa recurrence.

DOI PubMed

Molecular profiling and comprehensive genome-wide analysis of somatic copy number alterations in gastric intramucosal neoplasias based on microsatellite status

Tamotsu Sugai, Makoto Eizuka, Noriyuki Arakawa, Mitsumasa Osakabe, Wataru Habano, Yasuko Fujita, Eiichiro Yamamoto, Hiroo Yamano, Masaki Endoh, Takayuki Matsumoto, Hiromu Suzuki

Gastric Cancer ( Springer Tokyo ) 21 ( 5 ) 1 - 11 2018.02 [Refereed]

Background: We attempted to identify the molecular profiles of gastric intramucosal neoplasia (IMN low-grade dysplasia, LGD high-grade dysplasia, HGD intramucosal cancer, IMC) by assessing somatic copy number alterations (SCNAs) stratified by microsatellite status (microsatellite stable, MSS microsatellite instable, MSI). Thus, microsatellite status was determined in 84 tumors with MSS status and 16 tumors with MSI status. Methods: One hundred differentiated type IMNs were examined using SCNAs. In addition, genetic mutations (KRAS, BRAF, PIK3CA, and TP53) and DNA methylation status (low, intermediate and high) were also analyzed. Finally, we attempted to identify molecular profiles using a hierarchical clustering analysis. Results: Three patterns could be categorized according to SCNAs in IMNs with the MSS phenotype: subgroups 1 and 2 showing a high frequency of SCNAs, and subgroup 3 displaying a low frequency of SCNAs (subgroup 1 > 2 > 3 for SCNA). Subgroup 1 could be distinguished from subgroup 2 by the numbers of total SCNAs (gains and losses) and SCN gains (subgroup 1 > 2). The SCNA pattern of LGD was different from that of HGD and IMC. Moreover, IMNs with the MSI phenotype could be categorized into two subtypes: high frequency of SCNAs and low frequency of SCNAs. Genetic mutations and DNA methylation status did not differ among subgroups in IMNs. Conclusion: Molecular profiles stratified by SCNAs based on microsatellite status may be useful for elucidation of the mechanisms of early gastric carcinogenesis.

DOI PubMed

Epigenetic silencing of SMOC1 in traditional serrated adenoma and colorectal cancer.

Hironori Aoki, Eiichiro Yamamoto, Akira Takasawa, Takeshi Niinuma, Hiro-O Yamano, Taku Harada, Hiro-O Matsushita, Kenjiro Yoshikawa, Ryo Takagi, Eiji Harada, Yoshihito Tanaka, Yuko Yoshida, Tomoyuki Aoyama, Makoto Eizuka, Akira Yorozu, Hiroshi Kitajima, Masahiro Kai, Norimasa Sawada, Tamotsu Sugai, Hiroshi Nakase, Hiromu Suzuki

Oncotarget 9 ( 4 ) 4707 - 4721 2018.01 [Refereed] [International journal]

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cancer (CRC) characterized by BRAF mutation and microsatellite instability. By contrast, the molecular characteristics of traditional serrated adenoma (TSAs) are not fully understood. We analyzed genome-wide DNA methylation in TSAs having both protruding and flat components. We identified 11 genes, including SMOC1, methylation of which progressively increased during the development of TSAs. SMOC1 was prevalently methylated in TSAs, but was rarely methylated in SSA/Ps (p < 0.001). RT-PCR and immunohistochemistry revealed that SMOC1 was expressed in normal colon and SSA/Ps, but its expression was decreased in TSAs. Ectopic expression of SMOC1 suppressed proliferation, colony formation and in vivo tumor formation by CRC cells. Analysis of colorectal lesions (n = 847) revealed that SMOC1 is frequently methylated in TSAs, high-grade adenomas and CRCs. Among these, SMOC1 methylation was strongly associated with KRAS mutation and CpG island methylator phenotype (CIMP)-low. These results demonstrate that epigenetic silencing of SMOC1 is associated with TSA development but is rarely observed in SSA/Ps. SMOC1 expression could thus be a diagnostic marker of serrated lesions, and SMOC1 methylation could play a role in neoplastic pathways in TSAs and conventional adenomas.

DOI PubMed

Molecular characterization and pathogenesis of gastrointestinal stromal tumor

Takeshi Niinuma, Hiromu Suzuki, Tamotsu Sugai

Translational Gastroenterology and Hepatology ( AME Publishing Company ) 2018 2 2018.01 [Refereed]

Most gastrointestinal stromal tumors (GISTs) harbor activating mutations in the receptor tyrosine kinase gene KIT or platelet-derived growth factor receptor alpha (PDGFRA), and the resultant activation of downstream signals plays a pivotal role in the development of GISTs. The sites of the tyrosine kinase gene mutations are associated with the biological behavior of GISTs, including risk category, clinical outcome and drug response. Mutations in RAS signaling pathway genes, including KRAS and BRAF, have also been reported in KIT/PDGFRA wild-type GISTs, though they are rare. Neurofibromin 1 (NF1) is a tumor suppressor gene mutated in neurofibromatosis type 1. Patients with NF1 mutations are at high risk of developing GISTs. Recent findings suggest that altered expression or mutation of members of succinate dehydrogenase (SDH) heterotetramer are causally associated with GIST development through induction of aberrant DNA methylation. At present, GISTs with no alterations in KIT, PDGFRA, RAS signaling genes or SDH family genes are referred to as true wild-type GISTs. KIT and PDGFRA mutations are thought as the earliest events in GIST development, and subsequent accumulation of chromosomal aberrations and other molecular alterations are required for malignant progression. In addition, recent studies have shown that epigenetic alterations and noncoding RNAs also play key roles in the pathogenesis of GISTs.

DOI PubMed

Analysis of the expression of cancer-associated fibroblast- and EMT-related proteins in submucosal invasive colorectal cancer.

Tamotsu Sugai, Noriyuki Uesugi, Yuriko Kitada, Noriyuki Yamada, Mitsumasa Osakabe, Makoto Eizuka, Ryo Sugimoto, Yasuko Fujita, Keisuke Kawasaki, Eiichiro Yamamoto, Hiroo Yamano, Hiromu Suzuki, Takayuki Matsumoto

Journal of Cancer 9 ( 15 ) 2702 - 2712 2018 [Refereed] [International journal]

Objective: Recent studies have shown that cancer-associated fibroblasts (CAFs) and the epithelial-mesenchymal transition (EMT) play important roles in the progression and metastasis of CRC. Although prediction of lymph node metastasis in submucosal invasive colorectal cancer (SiCRC) is important, the relationships of CAF and EMT with lymph node metastasis of SiCRC have not yet been examined. Here, we aimed to analyze the expression patterns of CAF- and EMT-related proteins in SiCRC. Materials and Methods: The expression of CAF-related markers, including α-smooth muscle actin, CD10, podoplanin, fibroblast specific protein 1, and adipocyte enhancer-binding protein 1, and EMT-related proteins [zinc finger protein SNAI2 (ZEB1) and twist-related protein 1 (TWIST1) in SiCRC with (n = 29) or without (n = 80) lymph node metastasis was examined by immunohistochemistry. We examined the expression patterns of biomarkers using hierarchical cluster analysis. Consequently, four subgroups were established based on the expression patterns of CAF- and EMT-related markers, and the associations of these subgroups with clinicopathological variables. Results: In multivariate analysis, subgroup 2, which was characterized by high expression of all markers, was correlated with lymph node metastasis (p < 0.01). Next, we examined the associations of individual biomarkers with lymph node metastasis. Multivariate analysis showed that moderately differentiated adenocarcinoma was significantly associated with lymph node metastasis (p < 0.05). Conclusions: Our findings showed that expression patterns of CAF markers and EMT-related proteins may allow for stratification of patients into risk categories for lymph node metastasis in SiCRC.

DOI PubMed

Endoscopic and molecular characterization of colorectal sessile serrated adenoma/polyps with cytologic dysplasia

Yoshihito Tanaka, Hiro-o Yamano, Eiichiro Yamamoto, Hiro-o Matushita, Hironori Aoki, Kenjiro Yoshikawa, Ryo Takagi, Eiji Harada, Michiko Nakaoka, Yuko Yoshida, Makoto Eizuka, Tamotsu Sugai, Hiromu Suzuki, Hiroshi Nakase

GASTROINTESTINAL ENDOSCOPY ( MOSBY-ELSEVIER ) 86 ( 6 ) 1131 - + 2017.12 [Refereed]

Background and Aims: Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. Methods: A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. Results: Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. Conclusions: Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs.

DOI PubMed

Identification and characterization of a metastatic suppressor BRMS1L as a target gene of p53.

Ryota Koyama, Miyuki Tamura, Takafumi Nakagaki, Tomoko Ohashi, Masashi Idogawa, Hiromu Suzuki, Takashi Tokino, Yasushi Sasaki

Cancer science 108 ( 12 ) 2413 - 2421 2017.12 [Refereed] [International journal]

The tumor suppressor p53 and its family members, p63 and p73, play a pivotal role in the cell fate determination in response to diverse upstream signals. As transcription factors, p53 family proteins regulate a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 family proteins are important for the regulation of cell invasion and migration. Microarray analysis showed that breast cancer metastasis suppressor 1-like (BRMS1L) is upregulated by p53 family proteins, specifically p53, TAp63γ, and TAp73β. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly indicate that BRMS1L is one of the mediators downstream of the p53 pathway, and that it inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results indicate that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer.

DOI PubMed

Identification and characterization of a metastatic suppressor BRMS1L as a target gene of p53

Ryota Koyama, Miyuki Tamura, Takafumi Nakagaki, Tomoko Ohashi, Masashi Idogawa, Hiromu Suzuki, Takashi Tokino, Yasushi Sasaki

CANCER SCIENCE ( WILEY ) 108 ( 12 ) 2413 - 2421 2017.12 [Refereed]

The tumor suppressor p53 and its family members, p63 and p73, play a pivotal role in the cell fate determination in response to diverse upstream signals. As transcription factors, p53 family proteins regulate a number of genes that are involved in cell cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 family proteins are important for the regulation of cell invasion and migration. Microarray analysis showed that breast cancer metastasis suppressor 1-like (BRMS1L) is upregulated by p53 family proteins, specifically p53, TAp63, and TAp73. We identified two responsive elements of p53 family proteins in the first intron and upstream of BRMS1L. These response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration; knockdown of BRMS1L by siRNA induced the opposite effect. Importantly, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Together, these results strongly indicate that BRMS1L is one of the mediators downstream of the p53 pathway, and that it inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results indicate that BRMS1L is involved in cancer cell invasion and migration, and could be a therapeutic target for cancer.

DOI PubMed

p53標的遺伝子BRMS1Lの同定と機能解析

佐々木泰史, 小山良太, 田村みゆき, 中垣貴文, 大箸智子, 井戸川雅史, 鈴木拓, 時野隆至

生命科学系学会合同年次大会 ( 生命科学系学会合同年次大会運営事務局 ) 2017年度 [1LBA - 057] 2017.12

Molecular alterations in colorectal adenomas and intramucosal adenocarcinomas defined by high-density single-nucleotide polymorphism arrays

Makoto Eizuka, Tamotsu Sugai, Wataru Habano, Noriyuki Uesugi, Yayoi Takahashi, Keisuke Kawasaki, Eiichiro Yamamoto, Hiromu Suzuki, Takayuki Matsumoto

JOURNAL OF GASTROENTEROLOGY ( SPRINGER JAPAN KK ) 52 ( 11 ) 1158 - 1168 2017.11 [Refereed]

We examined colorectal adenomas and intramucosal adenocarcinomas (IMAs) to develop a genome-wide overview of copy number alterations (CNAs) during colorectal tumorigenesis. We analysed CNAs using a high-resolution SNP array of isolated tumour glands obtained from 55 colorectal adenomas (35 low-grade adenomas and 20 high-grade adenomas) and 30 IMAs. Next, we examined whether frequent CNAs differed between low-grade and high-grade adenomas or high-grade adenomas and IMAs. Finally, we investigated the total lengths of the CNAs in low-grade adenomas, high-grade adenomas, and IMAs. Although no frequent CNAs were found in low-grade adenomas, the most frequent alterations of high-grade adenomas were gains of 7q11, 7q21 and 9p13 and loss of 5q14.3-35. High levels of gains were detected at 13q, 7q, 8p, 20q, 7p, 18p and 17p in IMAs. Although no frequent alteration differed between low-grade and high-grade adenomas, significant differences of gains at 13q, 17p and 18p were found between high-grade adenoma and IMAs. Although the total lengths of all CNAs (gains and losses), copy number gains, and losses of heterozygosity were significantly greater in high-grade adenomas than in low-grade adenomas, no significant differences in the lengths of CNAs were found between high-grade adenomas and IMAs. Genomic alterations play an essential role in early colorectal carcinogenesis. CNAs in colorectal tumours provide new insights for evaluation of colorectal tumorigenesis.

DOI PubMed

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model

Guang Hua Li, Shinya Akatsuka, Shan Hwu Chew, Li Jiang, Takahiro Nishiyama, Akihiko Sakamoto, Takashi Takahashi, Mitsuru Futakuchi, Hiromu Suzuki, Kunihiko Sakumi, Yusaku Nakabeppu, Shinya Toyokuni

PATHOLOGY INTERNATIONAL ( WILEY ) 67 ( 11 ) 564 - 574 2017.11 [Refereed]

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P=0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16(INKA) inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

DOI PubMed

Downregulation of miR-186 is associated with metastatic recurrence of gastrointestinal stromal tumors

Takeshi Niinuma, Masahiro Kai, Hiroshi Kitajima, Eiichiro Yamamoto, Taku Harada, Reo Maruyama, Takayuki Nobuoka, Toshirou Nishida, Tatsuo Kanda, Tadashi Hasegawa, Takashi Tokino, Tamotsu Sugai, Yasuhisa Shinomura, Hiroshi Nakase, Hiromu Suzuki

ONCOLOGY LETTERS ( SPANDIDOS PUBL LTD ) 14 ( 5 ) 5703 - 5710 2017.11 [Refereed]

Although dysregulation of microRNAs (miRNAs/miRs) is a common feature of human malignancies, its involvement in gastrointestinal stromal tumors (GISTs) is not fully understood. The present study aimed to identify the miRNAs that perform a role in GIST metastasis. miRNA expression profiles from a series of 32 primary GISTs were analyzed using microarrays, and miR-186 was observed to be downregulated in tumors exhibiting metastatic recurrence. Reverse transcription-quantitative polymerase chain reaction analysis of an independent cohort of 100 primary GISTs revealed that low miR-186 expression is associated with metastatic recurrence and a poor prognosis. Inhibition of miR-186 in GIST-T1 cells promoted cell migration. Gene expression microarray analysis demonstrated that miR-186 inhibition upregulated a set of genes implicated in cancer metastasis, including insulin-like growth factor-binding protein 3, AKT serine/threonine kinase 2, hepatocyte growth factor receptor, CXC chemokine receptor 4 and epidermal growth factor-containing fibulin-like extracellular matrix protein 1. These results suggest that the downregulation of miR-186 is involved in the metastatic recurrence of GISTs, and that miR-186 levels could potentially be a predictive biomarker for clinical outcome.

DOI PubMed

SMOC1のエピジェネティックなサイレンシングは大腸鋸歯状腺腫の発育進展に関連する

青木敬則, 山本英一郎, 高澤啓, 新沼猛, 山野泰穂, 原田拓, 萬顕, 北嶋洋志, 甲斐正広, 澤田典均, 仲瀬裕志, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 76回 P - 2227 2017.09

ヒストンメチル化酵素DOT1Lは多発性骨髄腫の治療標的となりうる

石黒一也, 佐々木基, 若杉英樹, 北嶋洋志, 新沼猛, 丸山玲緒, 甲斐正広, 池田博, 石田禎夫, 佐々木泰史, 時野隆至, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 76回 P - 2244 2017.09

口腔扁平上皮癌に関与する長鎖非コードRNAの同定

西山廣陽, 丸山玲緒, 新沼猛, 北嶋洋志, 荻和弘, 出張裕也, 宮崎晃亘, 甲斐正広, 佐々木泰史, 時野隆至, 平塚博義, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 76回 P - 1292 2017.09

Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer

Masahiro Kai, Eiichiro Yamamoto, Akiko Sato, Hiro-o Yamano, Takeshi Niinuma, Hiroshi Kitajima, Taku Harada, Hironori Aoki, Reo Maruyama, Mutsumi Toyota, Tomo Hatahira, Hiroshi Nakase, Tamotsu Sugai, Toshiharu Yamashita, Minoru Toyota, Hiromu Suzuki

MOLECULAR CARCINOGENESIS ( WILEY ) 56 ( 7 ) 1743 - 1752 2017.07 [Refereed]

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGK., was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGK. did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGK. mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGK. all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.

DOI PubMed

Analysis of molecular alterations in laterally spreading tumors of the colorectum

Tamotsu Sugai, Wataru Habano, Ryo Takagi, Hiroo Yamano, Makoto Eizuka, Noriyuki Arakawa, Yayoi Takahashi, Eiichiro Yamamoto, Keisuke Kawasaki, Syunichi Yanai, Kazuyuki Ishida, Hiromu Suzuki, Takayuki Matsumoto

JOURNAL OF GASTROENTEROLOGY ( SPRINGER JAPAN KK ) 52 ( 6 ) 715 - 723 2017.06 [Refereed]

Colorectal laterally spreading tumors (LSTs) are classified into LST-Gs and LST-NGs, according to macroscopic findings. In the present study, we determined the genetic and epigenetic alterations within colorectal LSTs and protruding adenomas. A crypt isolation method was used to isolate DNA from tumors and normal glands of 73 macroscopically verified colorectal LSTs (histologically defined adenomas; 38 LST-Gs and 35 LST-NGs) and 36 protruding adenomas. The DNA was processed using polymerase chain reaction (PCR) microsatellite assays, single-strand conformation polymorphism (SSCP) assays, and pyrosequencing to detect chromosomal allelic imbalance (AI), mutations in APC, KRAS, and TP53, and the methylation of MLH1, MGMT, CDKN2A, HPP1, RASSF2A, SFRP1, DKK1, ZFP64, and SALL4 genes. In addition, methylation status was examined using the following set of markers: MIN1, MINT2, MINT31, MLH1, and CDKN2A (with classification of negative/low and high). Microsatellite instability (MSI) was also examined. 5q AI and methylation of the SFRP1 and SALL4 genes were common molecular events in both LST-Gs and LST-NGs. Neither MSI nor mutations in BRAF ware observed in the LSTs. TP53 mutations were rarely found in LSTs. The frequencies of KRAS and APC mutations and the methylation levels of ZFP64, RASSF2A, and HPP1 genes were significantly higher in LST-Gs than in LST-NGs. Protruding adenomas showed alterations common to LST-Gs. Negative/low methylation status was common among the three types of tumors. Combined genetic and epigenetic data suggested that the molecular mechanisms of tumorigenesis were different between LST-Gs and LST-NGs.

DOI PubMed

Analysis of the DNA methylation level of cancer-related genes in colorectal cancer and the surrounding normal mucosa

Tamotsu Sugai, Masakazu Yoshida, Makoto Eizuka, Noriyuki Uesugii, Wataru Habano, Kouki Otsuka, Akira Sasaki, Eiichiro Yamamoto, Takayuki Matsumoto, Hiromu Suzuki

Clinical Epigenetics ( Springer Verlag ) 9 ( 1 ) 55 2017.05 [Refereed]

Background: Two molecular pathways promote the development of colorectal cancer (CRC). One is termed “microsatellite stable” (MSS) whereas the other is characterized by “microsatellite instability” (MSI or MIN). In addition, the CpG island methylation phenotype is known to be an important alteration as a third molecular type. Thus, DNA methylation is thought to provide potential biomarkers for assessment of cancer risk in normal mucosa. In addition, it is also known that colonic location is an important parameter in the development of CRC. Methods: We examined the surrounding normal mucosa in three parts of the colon. Next, we quantified DNA methylation levels of SFRP1, SFRP2, SFRP5, DKK2, DKK3, mir34b/c, RASSF1A, IGFBP7, CDKN2A, and MLH1 in isolated cancerous glands and crypts of normal colorectal mucosa adjacent to CRCs using a pyrosequencer. Results: DNA methylation levels of SFRP1, SFRP2, DKK2, and mir34b/c were significantly higher in CRCs with an MSS phenotype than in those with an MSI phenotype. The average level of methylation in normal crypts did not decrease with the distance from the tumor, irrespective of microsatellite status or the tumor location. DNA methylation levels in SFRP1 and SFRP2 genes in normal crypts were significantly higher in left-side than right-side CRC with an MSS phenotype. Finally, the genes were classified into three types based on the methylation frequencies in normal crypts, including type I (SFRP1 and SFRP2I), type II (DKK2 and mir34b/c), and type III (others). Conclusions: Our results showed that DNA methylation of SFRP1 and SFRP2 might be useful to predict cancer risk of surrounding normal mucosa. In addition, a field effect may be present in CRC, affecting both adjacent and non-adjacent normal mucosa.

DOI PubMed

Analysis of the DNA methylation level of cancer-related genes in colorectal cancer and the surrounding normal mucosa

Tamotsu Sugai, Masakazu Yoshida, Makoto Eizuka, Noriyuki Uesugii, Wataru Habano, Kouki Otsuka, Akira Sasaki, Eiichiro Yamamoto, Takayuki Matsumoto, Hiromu Suzuki

CLINICAL EPIGENETICS ( BIOMED CENTRAL LTD ) 9 2017.05 [Refereed]

Background: Two molecular pathways promote the development of colorectal cancer (CRC). One is termed "microsatellite stable" (MSS) whereas the other is characterized by "microsatellite instability" (MSI or MIN). In addition, the CpG island methylation phenotype is known to be an important alteration as a third molecular type. Thus, DNA methylation is thought to provide potential biomarkers for assessment of cancer risk in normal mucosa. In addition, it is also known that colonic location is an important parameter in the development of CRC. Methods: We examined the surrounding normal mucosa in three parts of the colon. Next, we quantified DNA methylation levels of SFRP1, SFRP2, SFRP5, DKK2, DKK3, mir34b/c, RASSF1A, IGFBP7, CDKN2A, and MLH1 in isolated cancerous glands and crypts of normal colorectal mucosa adjacent to CRCs using a pyrosequencer. Results: DNA methylation levels of SFRP1, SFRP2, DKK2, and mir34b/c were significantly higher in CRCs with an MSS phenotype than in those with an MSI phenotype. The average level of methylation in normal crypts did not decrease with the distance from the tumor, irrespective of microsatellite status or the tumor location. DNA methylation levels in SFRP1 and SFRP2 genes in normal crypts were significantly higher in left-side than right-side CRC with an MSS phenotype. Finally, the genes were classified into three types based on the methylation frequencies in normal crypts, including type I (SFRP1 and SFRP2I), type II (DKK2 and mir34b/c), and type III (others). Conclusions: Our results showed that DNA methylation of SFRP1 and SFRP2 might be useful to predict cancer risk of surrounding normal mucosa. In addition, a field effect may be present in CRC, affecting both adjacent and non-adjacent normal mucosa.

DOI

Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

Masayuki Ishii, Junichi Kino, Norihisa Ichinohe, Naoki Tanimizu, Takafumi Ninomiya, Hiromu Suzuki, Toru Mizuguchi, Koichi Hirata, Toshihiro Mitaka

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP ) 7 46177 2017.04 [Refereed]

The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomytreated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44(+) SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

DOI PubMed

Molecular subtypes of colorectal cancers determined by PCR-based analysis

Tamotsu Sugai, Makoto Eizuka, Yayoi Takahashi, Tomoyuki Fukagawa, Wataru Habano, Eiichiro Yamamoto, Risaburo Akasaka, Kouki Otuska, Takayuki Matsumoto, Hiromu Suzuki

Cancer Science ( Blackwell Publishing Ltd ) 108 ( 3 ) 427 - 434 2017.03 [Refereed]

Tumor tissue consists of a heterogeneous cell population. The allelic imbalance (AI) ratio, determined in isolated tumor glands, is a good index of tumor heterogeneity. However, associations of the patterns of AI and microsatellite instability (MSI) development, observed in most cases of colorectal cancer (CRC), with tumor progression have not been reported previously. In this study, we examined whether CRC genetic profiles stratified by a combination of the AI ratio and MSI facilitate categorization of CRC, and whether these genetic profiles are associated with specific molecular alterations in CRC. A crypt isolation method was used to isolate DNA from tumors and normal glands obtained from 147 sporadic CRCs. AI and MSI statuses were determined using PCR-based microsatellite analysis and stratified based on AI ratio and MSI status. DNA methylation status (high methylation, intermediate methylation and low methylation status and mutations in KRAS, BRAF, and TP53 were examined. In addition, mucin markers were immunostained. Based on this analysis, four subgroups were categorized. Subgroup 1 was characterized by a high MSI status and BRAF mutation subgroup 2 was closely associated with a high AI ratio, which accumulated during the early phases of colorectal carcinogenesis, and TP53 mutation subgroup 3 was associated with a low AI ratio, seen during the later phases of colorectal carcinogenesis, and KRAS mutation and subgroup 4 was defined as a minor subgroup. These results confirmed that classification of distinct molecular profiles provides important insights into colorectal carcinogenesis.

DOI PubMed

Genetic differences stratified by PCR-based microsatellite analysis in gastric intramucosal neoplasia

Tamotsu Sugai, Ryo Sugimoto, Wataru Habano, Masaki Endoh, Makoto Eizuka, Koudai Tsuchida, Eiichiro Yamamoto, Keisuke Kawasaki, Syunichi Yanai, Takayuki Matsumoto, Hiromu Suzuki

GASTRIC CANCER ( SPRINGER ) 20 ( 2 ) 286 - 296 2017.03 [Refereed]

Although genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood. We evaluated genetic differences in 158 IMNs, including 51 low-grade dysplasias, 58 high-grade dysplasias (HGDs), 30 intramucosal cancers (IMCs), and 19 mixed tumors (composed of IMC and HGD within the same tumor), using PCR-based microsatellite analysis [allelic imbalance (AI) and microsatellite instability (MSI)]. We classified the DNA methylation status as a hypermethylated epigenome, a moderately methylated epigenome, or a hypomethylated epigenome. In addition, p53 overexpression, beta-catenin nuclear localization, and mucin expression were also examined. From cluster analysis, the IMNs examined were categorized into four subgroups as follows. Tumors in subgroup 1 were characterized by MSI-high status, a hypermethylated epigenome, and loss or reduction of expression of MLH-1. Tumors in subgroup 2 showed a mixed pattern consisting of AI and MSI. In contrast, tumors in subgroup 3, which showed accumulation of multiple AIs, were closely associated with HGD, IMC, or mixed tumor and exhibited nuclear expression of beta-catenin. Tumors in subgroup 4, which were generally low-grade dysplasias, exhibited a low frequency of AIs and no MSI. Although the mucin phenotype was not correlated with any subgroup, expression of mucin was associated with some subgroups. Overexpression of p53 was common in all subgroups. The approach described herein was useful for studying genetic differences in IMNs. In addition, we suggest that stratification of genetic differences may help to identify genetic molecular profiles in IMNs.

DOI PubMed

Molecular subtypes of colorectal cancers determined by PCR-based analysis

Tamotsu Sugai, Makoto Eizuka, Yayoi Takahashi, Tomoyuki Fukagawa, Wataru Habano, Eiichiro Yamamoto, Risaburo Akasaka, Kouki Otuska, Takayuki Matsumoto, Hiromu Suzuki

CANCER SCIENCE ( WILEY ) 108 ( 3 ) 427 - 434 2017.03 [Refereed]

Tumor tissue consists of a heterogeneous cell population. The allelic imbalance (AI) ratio, determined in isolated tumor glands, is a good index of tumor heterogeneity. However, associations of the patterns of AI and microsatellite instability (MSI) development, observed in most cases of colorectal cancer (CRC), with tumor progression have not been reported previously. In this study, we examined whether CRC genetic profiles stratified by a combination of the AI ratio and MSI facilitate categorization of CRC, and whether these genetic profiles are associated with specific molecular alterations in CRC. A crypt isolation method was used to isolate DNA from tumors and normal glands obtained from 147 sporadic CRCs. AI and MSI statuses were determined using PCR-based microsatellite analysis and stratified based on AI ratio and MSI status. DNA methylation status (high methylation, intermediate methylation and low methylation status and mutations in KRAS, BRAF, and TP53 were examined. In addition, mucin markers were immunostained. Based on this analysis, four subgroups were categorized. Subgroup 1 was characterized by a high MSI status and BRAF mutation; subgroup 2 was closely associated with a high AI ratio, which accumulated during the early phases of colorectal carcinogenesis, and TP53 mutation; subgroup 3 was associated with a low AI ratio, seen during the later phases of colorectal carcinogenesis, and KRAS mutation; and subgroup 4 was defined as a minor subgroup. These results confirmed that classification of distinct molecular profiles provides important insights into colorectal carcinogenesis.

DOI

Genome-wide analysis of DNA copy number alterations in early and advanced gastric cancers

Noriyuki Arakawa, Tamotsu Sugai, Wataru Habano, Makoto Eizuka, Ryo Sugimoto, Risaburo Akasaka, Yosuke Toya, Eiichiro Yamamoto, Keisuke Koeda, Akira Sasaki, Takayuki Matsumoto, Hiromu Suzuki

MOLECULAR CARCINOGENESIS ( WILEY-BLACKWELL ) 56 ( 2 ) 527 - 537 2017.02 [Refereed]

To better understand progressive changes in gastric cancer (GC), early and advanced GCs (EGC and AGC, respectively) were examined for copy number alterations (CNAs). A crypt isolation method was used to isolate DNA from tumors and normal glands in 20 AGCs, and fresh tumor samples were obtained from 45 EGCs. We assessed CNAs for differentiated-type GCs using an Infinium HumanCytoSNP-12v2.1 BeadChip in EGCs and AGCs. The most frequent aberrations in EGC were gains at 8q23.3 (42.2%) and 8q23.2 (40%), and loss of heterozygosity (LOH) at 3p14.2 (24.2%), suggesting that these CNAs were involved in the development of EGC. On the other hand, the highest frequencies of gains in AGC were found at 8q24.21 (65%) and 8q24.3 (60%). The most frequent LOHs in AGC were at 11q24.3-25, 11q23.2-24.1, 11q14.1, and 12p11.21-13.33, whereas that in EGC was at 3p14.2. In addition, regions of copy-neutral LOHs in AGC were detected at 11q21, 11q13.3-14.3, 11q11, 11p13-15.3, 12q21.1, 12q12-13.3 and 5q33.3-35.1. Comparisons of gains in EGC and AGC showed significant differences at 12q22-q23.2, 12q21.33, 11p12, 11p14.1, 12q21.31-32.32, 3p12.3, 3p14.1, 10p15.1, 1q24.2 and 2q12.1. Copy neutral LOHs were significantly higher in AGC than in EGC at 14q32.11-32.33, 14q21.3, 14q11.2, 5q11.2, 5q 13.3, 14q21.1-23.2, 14q13.2-13.3, 5q12.1-12.3, 5q11.1, and 17p13.3. The total lengths of the CNAs were significantly greater in AGC than in EGC. We found that the pattern of CNAs in AGC was quite different from that in EGC. We suggest that increasing numbers of CNAs are associated with disease progression from EGC to AGC. (c) 2016 Wiley Periodicals, Inc.

DOI PubMed

DNA and Histone Methylation in Colon Cancer: Its Biological Impact and Clinical Implications

Hiromu Suzuki, Eiichiro Yamamoto, Hiroshi Nakase, Tamotsu Sugai

Cancer Drug Discovery and Development ( Humana Press Inc. ) ( 9783319597843 ) 461 - 487 2017

Colorectal cancers (CRCs) are thought to arise through accumulation of genetic and epigenetic alterations. CRC genomes exhibit dual-faceted DNA methylation abnormality, global hypomethylation with CpG island hypermethylation, and CRCs are classified into two groups based on whether their genomes exhibit microsatellite instability (MSI) or chromosomal instability (CIN). In addition, a subset of CRCs is characterized by concurrent hypermethylation of multiple CpG islands, known as the CpG island methylator phenotype (CIMP). Genomic instability and epigenetic alterations are tightly linked, and CRCs with MSI largely overlap CIMP-positive tumors, while CIN is associated with global DNA hypomethylation. Dysregulation of histone methylation and altered expression of histone modifying enzymes are also commonly observed in CRC, indicating their critical roles in CRC development. Evidence now suggests that DNA and histone methylation could potentially serve as biomarkers useful for CRC diagnosis, risk assessment and prediction of therapeutic effects and prognosis. Although many studies examining clinical applications are still at an early phase, it is anticipated that further investigation will lead to improved prevention and management of CRC.

DOI

TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells

Masahiro Kai, Takeshi Niinuma, Hiroshi Kitajima, Eiichiro Yamamoto, Taku Harada, Hironori Aoki, Reo Maruyama, Mutsumi Toyota, Yasushi Sasaki, Tamotsu Sugai, Takashi Tokino, Hiroshi Nakase, Hiromu Suzuki

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 11 ( 12 ) e0168281 2016.12 [Refereed]

Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2'-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2'-deoxycytidine in CRC cells.

DOI PubMed

Molecular differences in the microsatellite stable phenotype between left-sided and right-sided colorectal cancer

Yayoi Takahashi, Tamotsu Sugai, Wataru Habano, Kazuyuki Ishida, Makoto Eizuka, Koki Otsuka, Akira Sasaki, Takayuki Matsumoto, Takanori Morikawa, Michiaki Unno, Hiromu Suzuki

INTERNATIONAL JOURNAL OF CANCER ( WILEY-BLACKWELL ) 139 ( 11 ) 2493 - 2501 2016.12 [Refereed]

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left-sided CRC (LC) and right-sided CRC (RC) have not been clarified. To identify pathogenesis-related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR-bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR-bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR-single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome-wide study using a copy number alteration (CNA)-targeted single nucleotide polymorphism array was performed. Ninety-two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC. What's new? The classification of colorectal cancer (CRC) based on tumor location is simple, comprehensive, and consistent with recent attempts to characterize tumors by pathological and molecular features. Differences in the pathogenesis of microsatellite stable (MSS) sporadic CRCs between left-sided CRC (LC) and right-sided CRC (RC) have however not been clarified. Here, the authors found that TP53 mutations are closely associated with the development of LC whereas RC is characterized by KRAS mutations. Using an integrated genome-wide analysis, they also show significant differences in copy number alterations. The findings suggest a different molecular pathogenesis of the MSS phenotype between LC and RC.

DOI PubMed

口腔扁平上皮癌に関与する長鎖非コードRNAの同定

西山廣陽, 粂川昴平, 丸山玲緒, 新沼猛, 北嶋洋志, 荻和弘, 出張裕也, 甲斐正広, 宮崎晃亘, 佐々木泰史, 時野隆至, 平塚博義, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 J - 2018 2016.10

多発性骨髄腫に有効なヒストンメチル化阻害薬の探索

石黒一也, 北嶋洋志, 新沼猛, 丸山玲緒, 甲斐正広, 西山廣陽, 進藤哲哉, 池田博, 石田禎夫, 佐々木泰史, 時野隆至, 仲瀬裕志, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 P - 2096 2016.10

Epigenetic activation of LY6K predicts the presence of metastasis and poor prognosis in breast carcinoma

Hyun Kyung Kong, Sae Jeong Park, Ye Sol Kim, Kyoung Min Kim, Hyun-Woo Lee, Hyeok-Gu Kang, Yu Mi Woo, Eun Young Park, Je Yeong Ko, Hiromu Suzuki, Kyung-Hee Chun, Erwei Song, Kyu Yun Jang, Jong Hoon Park

ONCOTARGET ( IMPACT JOURNALS LLC ) 7 ( 34 ) 55677 - 55689 2016.08 [Refereed]

The role of lymphocyte antigen 6 complex, locus K (LY6K) in breast cancer has been studied, whereas the epigenetic control of LY6K transcription is not fully understood. Here, we report that breast cancer patients with increased LY6K expression had shorter disease-free and overall survival than the patients with low levels of LY6K by multivariate analysis. LY6K also was upregulated in breast cancer patients with distant metastases than those without distant metastases, downregulating E-cadherin expression. Furthermore, xenograft tumor volumes from LY6K knockdown nude mice were reduced than those of mice treated with control lentivirus. Interestingly, LY6K has a CpG island (CGI) around the transcription start site and non-CGI in its promoter, called a CGI shore. LY6K expression was inversely correlated with methylation in not only CGI but CGI shore, which are associated with histone modifications. Additionally, LY6K methylation was increased by the PAX3 transcription factor due to the SNP242 mutation in LY6K CGI shore. Taken together, breast cancer risk and metastasis were significantly associated with not only LY6K expression, but also methylation of CGI shore which induced by SNP242 mutation. Our results suggest that an understanding epigenetic mechanism of the LY6K gene may be useful to diagnose carcinogenic risk and predict outcomes of patients with metastatic breast cancer.

DOI PubMed

Plasticity of lung cancer stem-like cells is regulated by the transcription factor HOXA5 that is induced by oxidative stress

Hiroshi Saijo, Yoshihiko Hirohashi, Toshihiko Torigoe, Ryota Horibe, Akari Takaya, Aiko Murai, Terufumi Kubo, Toshimitsu Kajiwara, Tsutomu Tanaka, Yosuke Shionoya, Eri Yamamoto, Reo Maruyama, Munehide Nakatsugawa, Takayuki Kanaseki, Tomohide Tsukahara, Yasuaki Tamura, Yasushi Sasaki, Takashi Tokino, Hiromu Suzuki, Toru Kondo, Hiroki Takahashi, Noriyuki Sato

ONCOTARGET ( IMPACT JOURNALS LLC ) 7 ( 31 ) 50043 - 50056 2016.08 [Refereed]

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.

DOI PubMed

Clinical prognostic value of DNA methylation in hepatoblastoma: Four novel tumor suppressor candidates

Shohei Honda, Masashi Minato, Hiromu Suzuki, Masato Fujiyoshi, Hisayuki Miyagi, Masayuki Haruta, Yasuhiko Kaneko, Kanako C. Hatanaka, Eiso Hiyama, Takehiko Kamijo, Tadao Okada, Akinobu Taketomi

CANCER SCIENCE ( WILEY-BLACKWELL ) 107 ( 6 ) 812 - 819 2016.06 [Refereed]

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

DOI PubMed

Assessment of epigenetic alterations in early colorectal lesions containing BRAF mutations

Takeshi Sawada, Eiichiro Yamamoto, Hiro-o Yamano, Masanori Nojima, Taku Harada, Reo Maruyama, Masami Ashida, Hironori Aoki, Hiro-o Matsushita, Kenjiro Yoshikawa, Eiji Harada, Yoshihito Tanaka, Shigenori Wakita, Takeshi Niinuma, Masahiro Kai, Makoto Eizuka, Tamotsu Sugai, Hiromu Suzuki

ONCOTARGET ( IMPACT JOURNALS LLC ) 7 ( 23 ) 35106 - 35118 2016.06 [Refereed]

To clarify the molecular and clinicopathological characteristics of colorectal serrated lesions, we assessed the DNA methylation of cancer-associated genes in a cohort of BRAF-mutant precancerous lesions from 94 individuals. We then compared those results with the lesions' clinicopathological features, especially colorectal subsites. The lesions included hyperplastic polyps (n = 16), traditional serrated adenomas (TSAs) (n = 15), TSAs with sessile serrated adenomas (SSAs) (n = 6), SSAs (n = 49) and SSAs with dysplasia (n = 16). The prevalence of lesions exhibiting the CpG island methylator phenotype (CIMP) was lower in the sigmoid colon and rectum than in other bowel subsites, including the cecum, ascending, transverse and descending colon. In addition, several cancer-associated genes showed higher methylation levels within lesions in the proximal to sigmoid colon than in the sigmoid colon and rectum. These results indicate that the methylation status of lesions with BRAF mutation is strongly associated with their location, histological findings and neoplastic pathways. By contrast, no difference in aberrant DNA methylation was observed in normal-appearing background colonic mucosa along the bowel subsites, which may indicate the absence of an epigenetic field defect.

DOI PubMed

A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer

Kohei Kumegawa, Reo Maruyama, Eiichiro Yamamoto, Masami Ashida, Hiroshi Kitajima, Akihiro Tsuyada, Takeshi Niinuma, Masahiro Kai, Hiro-o Yamano, Tamotsu Sugai, Takashi Tokino, Yasuhisa Shinomura, Kohzoh Imai, Hiromu Suzuki

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP ) 6 26699 2016.05 [Refereed]

Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA.

DOI PubMed

Clinicopathological and molecular alterations in early gastric cancers with the microsatellite instability-high phenotype

Ryo Sugimoto, Tamotsu Sugai, Wataru Habano, Masaki Endoh, Makoto Eizuka, Eiichiro Yamamoto, Noriyuki Uesugi, Kazuyuki Ishida, Tomonori Kawasaki, Takayuki Matsumoto, Hiromu Suzuki

INTERNATIONAL JOURNAL OF CANCER ( WILEY-BLACKWELL ) 138 ( 7 ) 1689 - 1697 2016.04 [Refereed]

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)-high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal-type adenocarcinoma). Tumors were classified as MSI-high (45 cases), MSI-low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)-microsatellite assays and PCR-pyrosequencing to detect chromosomal allelic imbalances in multiple cancer-related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI-high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features.

DOI PubMed

Relationship Between Noncoding RNA Dysregulation and Epigenetic Mechanisms in Cancer

Hiromu Suzuki, Reo Maruyama, Eiichiro Yamamoto, Takeshi Niinuma, Masahiro Kai

LONG AND SHORT NONCODING RNAS IN CANCER BIOLOGY ( SPRINGER-VERLAG SINGAPORE PTE LTD ) 927 109 - 135 2016 [Refereed]

Epigenetic alterations, including aberrant DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, studies have demonstrated that short and long noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer, and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs act as tumor suppressors or oncogenes. In addition, emerging evidence suggests that dysregulation of long ncRNAs (lncRNAs) plays critical roles in tumorigenesis. And because ncRNAs are involved in regulating gene expression through interaction with epigenetic modifiers, their dysregulation appears causally related to epigenetic alterations in cancer. Dissection of the interrelationships between ncRNAs and epigenetic alterations has the potential to reveal novel approaches to the diagnosis and treatment of cancer.

DOI PubMed

【大腸鋸歯状病変の取り扱い】発生部位に基づいた大腸鋸歯状病変の臨床病理学的および分子病理学的検討

永塚真, 菅井有, 荒川典之, 高橋弥生, 杉本亮, 川崎啓祐, 梁井俊一, 中村昌太郎, 松下弘雄, 山野泰穂, 山本英一郎, 鈴木拓, 松本主之

胃と腸 ( (株)医学書院 ) 50 ( 13 ) 1709 - 1722 2015.12

左側と右側大腸間における大腸鋸歯状病変の臨床病理学的,分子遺伝学的検討を行った.対象として左側過形成性ポリープ(HP)27例,右側HP 18例,左側SSA/P 29例,右側SSA/P 32例を用いた.病理組織学的所見については陰窩の拡張,陰窩の分岐異常,陰窩腺底部の拡張・走行異常(腺底部の所見)の有無について検討した.MUC2,MUC5AC,MUC6,CD10,CDX-2,p53,MLH-1,MSH-2,MSH-6,PMS-2,ANNEXIN A10の発現を免疫組織化学的に検索した.分子解析はKRAS,BRAF変異解析とDNAメチル化解析を行った(高,中等度,低).病理組織学的所見ではSSA/Pで腺底部の所見が右側で有意に多く,各因子の組み合わせパターンでは左側で陰窩の拡張と分岐異常のパターンが多く,右側では3因子すべてのパターンが多かった.MUC6およびANNEXIN A10の発現は右側のSSA/Pで有意に多くみられた.BRAFはSSA/Pで発生部位による違いはなかったが,メチル化では高メチル化状態が右側SSA/Pで高く,中等度メチル化と低メチル化状態が左側のSSA/Pで多かった.HPは左側でKRAS変異の頻度が高かった以外は左右の差異はなかった.SSA/Pでは発生部位によって組織像,分子異常が異なっている可能性が示唆された.(著者抄録)

Epigenetic silencing of NTSR1 is associated with lateral and noninvasive growth of colorectal tumors

Seiko Kamimae, Eiichiro Yamamoto, Masahiro Kai, Takeshi Niinuma, Hiro-o Yamano, Masanori Nojima, Kennjiro Yoshikawa, Tomoaki Kimura, Ryo Takagi, Eiji Harada, Taku Harada, Reo Maruyama, Yasushi Sasaki, Takashi Tokino, Yasuhisa Shinomura, Tamotsu Sugai, Kohzoh Imai, Hiromu Suzuki

ONCOTARGET ( IMPACT JOURNALS LLC ) 6 ( 30 ) 29975 - 29990 2015.10 [Refereed]

Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (>= 20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portions revealed that NTSR1 is frequently methylated in the adenomatous portion, while methylation levels are generally lower in the cancerous portions. These results suggest that NTSR1 methylation is associated with lateral and noninvasive growth of colorectal tumors, while low levels of methylation may contribute to the malignant potential through activation of NTSR1. Our data also indicate that NTSR1 methylation may be a prognostic biomarker in CRC.

DOI PubMed

大腸癌における腫瘍血管内皮特異的遺伝子の新規同定

萬顕, 山本英一郎, 丸山玲緒, 津矢田明泰, 甲斐正広, 新沼猛, 檜森亮吾, 廣橋良彦, 西舘敏彦, 古畑智久, 平田公一, 菅井有, 鈴木拓

日本癌学会総会記事 ( 日本癌学会 ) 74回 E - 1169 2015.10

口腔扁平上皮癌の発生や進展において重要な役割を果たす長鎖非コードRNAの同定

西山廣陽, 粂川昂平, 丸山玲緒, 新沼猛, 竹田康佑, 荻和弘, 出張裕也, 宮崎晃亘, 甲斐正広, 佐々木泰史, 時野隆史, 平塚博義, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 74回 P - 3300 2015.10

Analysis of the molecular features of rectal carcinoid tumors to identify new biomarkers that predict biological malignancy

Kei Mitsuhashi, Itaru Yamamoto, Hiroyoshi Kurihara, Shinichi Kanno, Miki Ito, Hisayoshi Igarashi, Keisuke Ishigami, Yasutaka Sukawa, Mami Tachibana, Hiroaki Takahashi, Takashi Tokino, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Yasuhisa Shinomura, Hiroyuki Yamamoto, Katsuhiko Nosho

ONCOTARGET ( IMPACT JOURNALS LLC ) 6 ( 26 ) 22114 - 22125 2015.09 [Refereed]

Although gastrointestinal carcinoid tumors are relatively rare in the digestive tract, a quarter of them are present in the rectum. In the absence of specific tumor biomarkers, lymphatic or vascular invasion is generally used to predict the risk of lymph node metastasis. We, therefore, examined the genetic and epigenetic alterations potentially associated with lymphovascular invasion among 56 patients with rectal carcinoid tumors. We also conducted a microRNA (miRNA) array analysis. Our analysis failed to detect mutations in BRAF, KRAS, NRAS, or PIK3CA or any microsatellite instability (MSI); however, we did observe CpG island methylator phenotype (CIMP) positivity in 13% (7/56) of the carcinoid tumors. The CIMP-positive status was significantly correlated with lymphovascular invasion (P = 0.036). The array analysis revealed that microRNA-885 (miR-885)-5p was the most up-regulated miRNA in the carcinoid tumors with lymphovascular invasion compared with that in those without invasion. In addition, high miR-885-5p expression was independently associated with lymphovascular invasion (P = 0.0002). In conclusion, our findings suggest that miR-885-5p and CIMP status may be useful biomarkers for predicting biological malignancy in patients with rectal carcinoid tumors.

DOI PubMed

Low-Frequency IL23R Coding Variant Associated with Crohn's Disease Susceptibility in Japanese Subjects Identified by Personal Genomics Analysis

Kei Onodera, Yoshiaki Arimura, Hiroyuki Isshiki, Kentaro Kawakami, Kanna Nagaishi, Kentaro Yamashita, Eiichiro Yamamoto, Takeshi Niinuma, Yasuyoshi Naishiro, Hiromu Suzuki, Kohzoh Imai, Yasuhisa Shinomura

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 10 ( 9 ) e0137801 2015.09 [Refereed]

Background The common disease-common variant hypothesis is insufficient to explain the complexities of Crohn's disease (CD) genetics; therefore, rare variants are expected to be important in the disease. We explored rare variants associated with susceptibility to CD in Japanese individuals by personal genomic analysis. Methods Two-step analyses were performed. The first step was a trio analysis with whole-exome sequence (WES) analysis and the second was a follow-up case-control association study. The WES analysis pipeline comprised Burrows-Wheeler Aligner, Picard, Genome Analysis Toolkit, and SAMTOOLS. Single nucleotide variants (SNVs)/indels were annotated and filtered by using programs implemented in ANNOVAR in combination with identity-by-descent (IBD), subsequently were subjected to the linkage based, and de novo based strategies. Finally, we conducted an association study that included 176 unrelated subjects with CD and 358 healthy control subjects. Results In family members, 234,067-297,523 SNVs/indels were detected and they were educed to 106-146 by annotation based filtering. Fifty-four CD variants common to both individuals of the affected sib pair were identified. The linkage based strategy detected five candidate variants whereas the de novo based strategy identified no variants. Consequently, five candidates were analyzed in the case-control association study. CD showed a significant association with one variant in exon 4 of IL23R, G149R [rs76418789, P = 3.9E-5, odds ratio (OR) 0.21, 95% confidence interval (CI) 0.09-0.47 for the dominant model (AA + AG versus GG), and P = 7.3E-5, OR 0.21, 95% CI 0.10-0.48 for AG versus GG, and P = 7.2E-5, OR 0.23, 95% CI 0.10-0.50 for the allele model]. Conclusions The present study, using personal genomics analysis of a small CD pedigree, is the first to show that the low-frequency non-synonymous variant of IL23R, rs76418789, protects against CD development in Japanese subjects.

DOI PubMed

Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway

Miki Ito, Shinichi Kanno, Katsuhiko Nosho, Yasutaka Sukawa, Kei Mitsuhashi, Hiroyoshi Kurihara, Hisayoshi Igarashi, Taiga Takahashi, Mami Tachibana, Hiroaki Takahashi, Shinji Yoshii, Toshinao Takenouchi, Tadashi Hasegawa, Kenji Okita, Koichi Hirata, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura

INTERNATIONAL JOURNAL OF CANCER ( WILEY-BLACKWELL ) 137 ( 6 ) 1258 - 1268 2015.09 [Refereed]

Human gut microbiota is being increasingly recognized as a player in colorectal cancers (CRCs). Evidence suggests that Fusobacterium nucleatum (F. nucleatum) may contribute to disease progression and is associated with CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in CRCs; however, to date, there are no reports about the relationship between F. nucleatum and molecular features in the early stage of colorectal tumorigenesis. Therefore, we investigated the presence of F. nucleatum in premalignant colorectal lesions. In total, 465 premalignant lesions (343 serrated lesions and 122 non-serrated adenomas) and 511 CRCs were studied. We determined the presence of F. nucleatum and analyzed its association with molecular features including CIMP, MSI and microRNA-31 status. F. nucleatum was detected in 24% of hyperplastic polyps, 35% of sessile serrated adenomas (SSAs), 30% of traditional serrated adenomas (TSAs) and 33% of non-serrated adenomas. F. nucleatum was more frequently detected in CIMP-high premalignant lesions than in CIMP-low/zero lesions (p=0.0023). In SSAs, F. nucleatum positivity increased gradually from sigmoid colon to cecum (p=0.042). F. nucleatum positivity was significantly higher in CRCs (56%) than in premalignant lesions of any histological type (p<0.0001). In conclusion, F. nucleatum was identified in premalignant colorectal lesions regardless of histopathology but was more frequently associated with CIMP-high lesions. Moreover, F. nucleatum positivity increased according to histological grade, suggesting that it may contribute to the progression of colorectal neoplasia. Our data also indicate that F. nucleatum positivity in SSAs may support the colorectal continuum concept. What's new? Changes in the microbial community in the human gut may contribute to colorectal cancer (CRC). Of particular interest are changes in populations of Fusobacterium nucleatum, which previous work has shown to be abundant in CRC tissues. In this study, F. nucleatum was detected in CRCs, as well as in premalignant lesions with elevated CpG island methylator phenotype status. In sessile serrated adenomas, F. nucleatum increased steadily from the sigmoid colon to the cecum, supporting the existence of a colorectal continuum. F. nucleatum may become increasingly abundant with histological grade and may influence CRC progression.

DOI PubMed

Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene

Yuki Konishi, Hiroshi Hayashi, Hiromu Suzuki, Eiichiro Yamamoto, Hajime Sugisaki, Hiroko Higashimoto

ANALYTICAL BIOCHEMISTRY ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 484 66 - 71 2015.09 [Refereed]

Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight-by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R-2 = 0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R-2 = 0.864 and R-2 = 0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison. (C) 2015 Elsevier Inc. All rights reserved.

DOI PubMed

Contextual niche signals towards colorectal tumor progression by mesenchymal stem cell in the mouse xenograft model

Suguru Nakagaki, Yoshiaki Arimura, Kanna Nagaishi, Hiroyuki Isshiki, Masanao Nasuno, Shuhei Watanabe, Masashi Idogawa, Kentaro Yamashita, Yasuyoshi Naishiro, Yasushi Adachi, Hiromu Suzuki, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

JOURNAL OF GASTROENTEROLOGY ( SPRINGER JAPAN KK ) 50 ( 9 ) 962 - 974 2015.09 [Refereed]

The role of mesenchymal stem/stromal cells (MSCs) in tumorigenesis remains controversial. This study aimed to determine whether heterotypic interactions between MSCs and colon cancer cells can supply contextual signals towards tumor progression. Xenografts consisting of co-implanted human colorectal cancer cells with rat MSCs in immunodeficient mice were evaluated by tumor progression, angiogenic profiles, and MSC fate. Furthermore, we investigated how MSCs function as a cancer cell niche by co-culture experiments in vitro. Tumor growth progressed in two ways, either independent of or dependent on MSCs. Such cell line-specific dependency could not be explained by host immune competency. COLO 320 xenograft angiogenesis was MSC-dependent, but less dependent on vascular endothelial growth factor (VEGF), whereas HT-29 angiogenesis was not MSC-dependent, but was VEGF-dependent. MSCs and COLO 320 cells established a functional positive feedback loop that triggered formation of a cancer cell niche, leading to AKT activation. Subsequently, MSCs differentiated into pericytes that enhanced angiogenesis as a perivascular niche. In contrast, the MSC niche conferred an anti-proliferative property to HT-29 cells, through mesenchymal-epithelial transition resulting in p38 activation. In conclusion, MSCs demonstrate pleiotropic capabilities as a cancer cell or perivascular niche to modulate colorectal cancer cell fate in a cell line-dependent manner in a xenogeneic context.

DOI PubMed

Aberrant TET1 methylation closely associated with CpG island methylator phenotype in colorectal cancer

Ichimura Norihisa, Shinjo Keiko, Ohka Fumiharu, Katsushima Keisuke, Hatanaka Akira, Tojo Masayuki, Shimizu Yasuhiro, Yamamoto Eiichiro, Suzuki Hiromu, Kondo Yutaka

CANCER RESEARCH 75 2015.08 [Refereed]

DOI

Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer

Norihisa Ichimura, Keiko Shinjo, Byonggu An, Yasuhiro Shimizu, Kenji Yamao, Fumiharu Ohka, Keisuke Katsushima, Akira Hatanaka, Masayuki Tojo, Eiichiro Yamamoto, Hiromu Suzuki, Minoru Ueda, Yutaka Kondo

CANCER PREVENTION RESEARCH ( AMER ASSOC CANCER RESEARCH ) 8 ( 8 ) 702 - 711 2015.08 [Refereed]

Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMPP) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P < 0.0001) colorectal cancers. This trend was also observed in colon polyps (CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. (C) 2015 AACR.

DOI PubMed

Association of MicroRNA-31-5p with Clinical Efficacy of Anti-EGFR Therapy in Patients with Metastatic Colorectal Cancer

Hisayoshi Igarashi, Hiroyoshi Kurihara, Kei Mitsuhashi, Miki Ito, Hiroyuki Okuda, Shinichi Kanno, Takafumi Naito, Shinji Yoshii, Hiroaki Takahashi, Takaya Kusumi, Tadashi Hasegawa, Yasutaka Sukawa, Yasushi Adachi, Kenji Okita, Koichi Hirata, Yu Imamura, Yoshifumi Baba, Kohzoh Imai, Hiromu Suzuki, Hiroyuki Yamamoto, Katsuhiko Nosho, Yasuhisa Shinomura

ANNALS OF SURGICAL ONCOLOGY ( SPRINGER ) 22 ( 8 ) 2640 - 2648 2015.08 [Refereed]

Gene mutations in the pathway downstream of epidermal growth factor receptor (EGFR) are considered to induce resistance to anti-EGFR therapy in colorectal cancer (CRC). We recently reported that microRNA-31 (miR-31)-5p may regulate BRAF activation and play a role in the signaling pathway downstream of EGFR in CRC. Therefore, we hypothesized that miR-31-5p can be a useful biomarker for anti-EGFR therapy in CRC. We evaluated miR-31-5p expression and gene mutations [KRAS (codon 61 or 146), NRAS (codon 12, 13, or 61), and BRAF (V600E)] in the EGFR downstream pathway in 102 CRC patients harboring KRAS (codon 12 or 13) wild-type who were treated with anti-EGFR therapeutics. Progression-free survival (PFS) and overall survival (OS) were evaluated. KRAS (codon 61 or 146), NRAS, and BRAF mutations were detected in 6.9, 6.9, and 5.9 % patients, respectively. Compared with CRCs with at least one mutation (n = 20), significantly better PFS (P = 0.0003) but insignificantly better OS were observed in CRCs harboring all wild-type genes (KRAS, NRAS, and BRAF). High miR-31-5p expression was identified in 11 % (n = 11) patients and was significantly associated with shorter PFS (P = 0.003). In CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS (P = 0.027). High miR-31-5p expression was associated with shorter PFS in patients with CRC treated with anti-EGFR therapeutics. Moreover, in CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS, suggesting that it may be a useful and additional prognostic biomarker for anti-EGFR therapy.

DOI PubMed

A Screen for Epigenetically Silenced microRNA Genes in Gastrointestinal Stromal Tumors

Mai Isosaka, Takeshi Niinuma, Masanori Nojima, Masahiro Kai, Eiichiro Yamamoto, Reo Maruyama, Takayuki Nobuoka, Toshirou Nishida, Tatsuo Kanda, Takahiro Taguchi, Tadashi Hasegawa, Takashi Tokino, Koichi Hirata, Hiromu Suzuki, Yasuhisa Shinomura

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 10 ( 7 ) e0133754 2015.07 [Refereed]

Background Dysregulation of microRNA (miRNA) has been implicated in gastrointestinal stromal tumors (GISTs) but the mechanism is not fully understood. In this study, we aimed to explore the involvement of epigenetic alteration of miRNA genes in GISTs. Methods GIST-T1 cells were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which miRNA expression profiles were analyzed using TaqMan miRNA arrays. DNA methylation was then analyzed using bisulfite pyrosequencing. The functions of miRNAs were examined using MTT assays, wound-healing assays, Boyden chamber assays and Matrigel invasion assays. Gene expression microarrays were analyzed to assess effect of ectopic miRNA expression in GIST-T1 cells. Results Of the 754 miRNAs analyzed, 61 were significantly upregulated in GIST-T1 cells treated with 5-aza-dC plus PBA. Among those, 21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens. Transfection of miR-34a or miR-335 mimic molecules into GIST-T1 cells suppressed cell proliferation, and miR-34a also inhibited migration and invasion by GIST-T1 cells. Moreover, miR-34a downregulated a number of predicted target genes, including PDGFRA. RNA interference-mediated knockdown of PDGFRA in GIST-T1 cells suppressed cell proliferation, suggesting the tumor suppressive effect of miR-34a is mediated, at least in part, through targeting PDGFRA. Conclusions Our results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs.

DOI PubMed

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

Ying Su, Ashim Subedee, Noga Bloushtain-Qimron, Virginia Savova, Marcin Krzystanek, Lewyn Li, Andriy Marusyk, Doris P. Tabassum, Alexander Zak, Mary Jo Flacker, Mei Li, Jessica J. Lin, Saraswati Sukumar, Hiromu Suzuki, Henry Long, Zoltan Szallasi, Alexander Gimelbrant, Reo Maruyama, Kornelia Polyak

CELL REPORTS ( CELL PRESS ) 11 ( 10 ) 1549 - 1563 2015.06 [Refereed]

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

DOI PubMed

Molecular analysis of isolated tumor glands from endometrial endometrioid adenocarcinomas

Yasuko Suga, Tamotsu Sugai, Noriyuki Uesugi, Tomonori Kawasaki, Tomoyuki Fukagawa, Eiichiro Yamamoto, Kazuyuki Ishida, Hiromu Suzuki, Toru Sugiyama

PATHOLOGY INTERNATIONAL ( WILEY-BLACKWELL ) 65 ( 5 ) 240 - 249 2015.05 [Refereed]

We studied the extensive molecular alterations of endometrial endometrioid adenocarcinoma (EEA) using a crypt isolation method. We analyzed copy number variation (CNV) using a single nucleotide polymorphism (SNP) array, genetic mutations (KRAS, BRAF, p53, PIK3CA), DNA methylation and microsatellite instability (MSI) status. In addition, loss of PTEN protein expression was examined. Increased chromosome copy numbers of 1q21.2-44 (22%) and 10q11.21-23.31 (28%) were seen relatively frequently in EEA, and copy-neutral loss of heterozygosity (LOH) was also observed in 10q22.1-26.3 (22%). The CNV patterns of EEA were classified into four groups through hierarchical cluster analysis. Cluster 1 had many CNVs of 10q, and cluster 2 was characterized by MSI status. In cluster 3, increased CNVs of 1q were often seen. In cluster 4, p53 mutations were detected. KRAS and PIK3CA mutations and reduced PTEN protein expression were common to all groups. On the other hand, CpG island methylator phenotype (CIMP) was rare in all groups. The data indicated an association with chromosomal gain of 1q and 10q or 10q copy-neutral LOH in some cases. We suggest that EEA consists of four groups that are characterized with molecular alterations.

DOI PubMed

[Endoscopic diagnosis and clinical management for serrated lesions of the large intestine].

Yamano HO, Tanaka Y, Matsushita HO, Yoshikawa K, Takagi R, Harada E, Nakaoka M, Himori R, Yoshida Y, Tanaka D, Sato K, Imai Y, Sugai T, Eizuka M, Yamamoto E, Aoki H, Suzuki H

Nihon Shokakibyo Gakkai zasshi = The Japanese journal of gastro-enterology 112 ( 4 ) 676 - 682 2015.04 [Refereed] [Domestic journal]

DOI PubMed

Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis

Kei Mitsuhashi, Katsuhiko Nosho, Yasutaka Sukawa, Yasutaka Matsunaga, Miki Ito, Hiroyoshi Kurihara, Shinichi Kanno, Hisayoshi Igarashi, Takafumi Naito, Yasushi Adachi, Mami Tachibana, Tokuma Tanuma, Hiroyuki Maguchi, Toshiya Shinohara, Tadashi Hasegawa, Masafumi Imamura, Yasutoshi Kimura, Koichi Hirata, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura

ONCOTARGET ( IMPACT JOURNALS LLC ) 6 ( 9 ) 7209 - 7220 2015.03 [Refereed]

Recently, bacterial infection causing periodontal disease has attracted considerable attention as a risk factor for pancreatic cancer. Fusobacterium species is an oral bacterial group of the human microbiome. Some evidence suggests that Fusobacterium species promote colorectal cancer development; however, no previous studies have reported the association between Fusobacterium species and pancreatic cancer. Therefore, we examined whether Fusobacterium species exist in pancreatic cancer tissue. Using a database of 283 patients with pancreatic ductal adenocarcinoma (PDAC), we tested cancer tissue specimens for Fusobacterium species. We also tested the specimens for KRAS, NRAS, BRAF and PIK3CA mutations and measured microRNA-21 and microRNA-31. In addition, we assessed epigenetic alterations, including CpG island methylator phenotype (CIMP). Our data showed an 8.8% detection rate of Fusobacterium species in pancreatic cancers; however, tumor Fusobacterium status was not associated with any clinical and molecular features. In contrast, in multivariate Cox regression analysis, compared with the Fusobacterium species- negative group, we observed significantly higher cancer-specific mortality rates in the positive group (p = 0.023). In conclusion, Fusobacterium species were detected in pancreatic cancer tissue. Tumor Fusobacterium species status is independently associated with a worse prognosis of pancreatic cancer, suggesting that Fusobacterium species may be a prognostic biomarker of pancreatic cancer.

DOI PubMed

Clinicopathological and Molecular Characteristics of Serrated Lesions in Japanese Elderly Patients

Katsuhiko Nosho, Hisayoshi Igarashi, Miki Ito, Kei Mitsuhashi, Hiroyoshi Kurihara, Shinichi Kanno, Shinji Yoshii, Masashi Mikami, Hiroaki Takahashi, Takaya Kusumi, Masao Hosokawa, Yasutaka Sukawa, Yasushi Adachi, Tadashi Hasegawa, Kenji Okita, Koichi Hirata, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura

DIGESTION ( KARGER ) 91 ( 1 ) 57 - 63 2015 [Refereed]

Background: The population in Japan is aging more rapidly than in any other country. However, no studies have determined the characteristics of the large population of elderly patients with colorectal tumors. Therefore, we examined the clinicopathological and molecular features of these tumors in elderly patients. Methods: In total, 1,627 colorectal tumors (393 serrated lesions, 277 non-serrated adenomas and 957 colorectal cancers) were acquired from patients. Tumor specimens were analyzed for BRAF and KRAS mutations, CpG island methylator phenotype-specific promoters (CACNA1G, CDKN2A, IGF2 and RUNX3), IGFBP7, MGMT, MLH1 and RASSF2 methylation, microsatellite instability (MSI) and microRNA-31 (miR-31). Results: The frequency of elderly patients (aged >= 75 years) with sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that of those with other serrated lesions and non-serrated adenomas (p < 0.0001). In elderly patients, all SSAs were located in the proximal colon (particularly the cecum to ascending colon). High miR-31 expression, MLH1 methylation and MSI-high status were more frequently detected in SSAs from elderly patients than in those from non-elderly patients. In contrast, no significant differences were found between older age of onset and high-grade dysplasia for traditional serrated adenomas or non-serrated adenomas in any of these molecular alterations. Conclusion: In elderly patients, all SSAs were located in the proximal colon. Furthermore, cytological dysplasia and molecular alterations were more frequently detected in elderly patients with SSAs than in non-elderly patients. Thus, careful colonoscopic examinations of the proximal colon are necessary for elderly patients because SSAs in those patients may exhibit malignant potential. (C) 2015 S. Karger AG, Basel

DOI PubMed

Dietary patterns and the risk of systemic lupus erythematosus in a Japanese population: The Kyushu Sapporo SLE (KYSS) Study

Kiyohara C, Washio M, Horiuchi T, Takahashi H, Tada Y, Kobashi G, Asami T, Ide S, Atsumi T, Kodama H, Akashi K, Harada M, Tsukamoto H, Hotokebuchi T, Nagasawa K, Ushiyama O, Mori M, Oura A, Sinomura Y, Suzuki H, Yamamoto M, Abe T, Tanaka H, Yasuda S, Nogami N, Okamoto K, Sakamoto N, Sasaki S, Miyake Y, Yokoyama T, Inaba Y, Nagai M

International Medical Journal 22 ( 3 ) 110 - 115 2015 [Refereed]

MicroRNA-31 expression in relation to BRAF mutation, CpG island methylation and colorectal continuum in serrated lesions

Miki Ito, Kei Mitsuhashi, Hisayoshi Igarashi, Katsuhiko Nosho, Takafumi Naito, Shinji Yoshii, Hiroaki Takahashi, Masahiro Fujita, Yasutaka Sukawa, Eiichiro Yamamoto, Taiga Takahashi, Yasushi Adachi, Masanori Nojima, Yasushi Sasaki, Takashi Tokino, Yoshifumi Baba, Reo Maruyama, Hiromu Suzuki, Kohzoh Imai, Hiroyuki Yamamoto, Yasuhisa Shinomura

INTERNATIONAL JOURNAL OF CANCER ( WILEY-BLACKWELL ) 135 ( 11 ) 2507 - 2515 2014.12 [Refereed]

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p=0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.

DOI PubMed

Biological significance of the CpG island methylator phenotype

Hiromu Suzuki, Eiichiro Yamamoto, Reo Maruyama, Takeshi Niinuma, Masahiro Kai

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 455 ( 1-2 ) 35 - 42 2014.12 [Refereed]

Cancers exhibiting the CpG island methylator phenotype (CIMP) are found among a wide variety of human malignancies and represent a subclass of tumors showing concurrent hypermethylation of multiple CpG islands. These CIMP-positive tumors often exhibit characteristic molecular and clinicopathological features, suggesting CIMP represents a distinct carcinogenic pathway. However, marker genes to define CIMP have been largely inconsistent among studies, which has caused results to vary. Nonetheless, recent advances in genome-wide methylation analysis have enabled the existence of CIMP to be confirmed, and large-scale cancer genome analyses have begun to unravel the previously unknown molecular basis of CIMP tumors. CIMP is strongly associated with clinical outcome, suggesting it may be a predictive biomarker. (C) 2014 Elsevier Inc. All rights reserved.

DOI PubMed

Targeting of miR34a-NOTCH1 Axis Reduced Breast Cancer Stemness and Chemoresistance

Eun Young Park, EunSun Chang, Eun Ji Lee, Hyun-Woo Lee, Hyeok-Gu Kang, Kyung-Hee Chun, Yu Mi Woo, Hyun Kyung Kong, Je Yeong Ko, Hiromu Suzuki, Erwei Song, Jong Hoon Park

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 74 ( 24 ) 7573 - 7582 2014.12 [Refereed]

Human breast cancers include cancer stem cell populations as well as nontumorigenic cancer cells. Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. miRNAs regulate the expression of many target genes; therefore, dysregulation of miRNAs has been associated with the pathogenesis of human diseases, including cancer. However, a role for miRNA dysregulation in stemness and drug resistance has yet to be identified. Members of the miR34 family are reportedly tumor-suppressor miRNAs and are associated with various human cancers. Our results confirm that miR34a expression was downregulated in MCF7/ADR cells compared with MCF7 cells. We hypothesized that this reduction was due to the p53 (TP53) mutation in MCF7/ADR cells. In this study, we found that primary and mature miR34a were suppressed by treatment with p53 RNAi or the dominant-negative p53 mutant in MCF7 cells. Ectopic miR34a expression reduced cancer stemcell properties and increased sensitivity to doxorubicin treatment by directly targeting NOTCH1. Furthermore, tumors from nude mice treated with miR34a were significantly smaller compared with those of mice treated with control lentivirus. Our research suggests that the ectopic expression of miR34a represents a novel therapeutic approach in chemoresistant breast cancer treatment. (C)2014 AACR.

DOI PubMed

Analysis of DNA Methylation in Bowel Lavage Fluid for Detection of Colorectal Cancer

Taku Harada, Eiichiro Yamamoto, Hiro-o Yamano, Masanori Nojima, Reo Maruyama, Kohei Kumegawa, Masami Ashida, Kenjiro Yoshikawa, Tomoaki Kimura, Eiji Harada, Ryo Takagi, Yoshihito Tanaka, Hironori Aoki, Masayo Nishizono, Michiko Nakaoka, Akihiro Tsuyada, Takeshi Niinuma, Masahiro Kai, Kazuya Shimoda, Yasuhisa Shinomura, Tamotsu Sugai, Kohzoh Imai, Hiromu Suzuki

CANCER PREVENTION RESEARCH ( AMER ASSOC CANCER RESEARCH ) 7 ( 10 ) 1002 - 1010 2014.10 [Refereed]

Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the methylation of those three genes (M-score) achieved 82% sensitivity and 79% specificity, and the area under the ROC curve (AUC) was 0.834. The strong performance of this system was then validated in an independent test set (n = 153; AUC = 0.808). No significant correlation was found between M-score and the clinicopathologic features of the colorectal cancers. Our results demonstrate that DNA methylation in BLF specimens may be a useful biomarker for the detection of colorectal cancer. (C) 2014 AACR.

DOI PubMed

Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis

Yoko Takaki, Yoshimasa Saito, Azusa Takasugi, Kohta Toshimitsu, Shoji Yamada, Toshihide Muramatsu, Masaki Kimura, Kazuo Sugiyama, Hiromu Suzuki, Eri Arai, Hidenori Ojima, Yae Kanai, Hidetsugu Saito

CANCER SCIENCE ( WILEY-BLACKWELL ) 105 ( 10 ) 1254 - 1260 2014.10 [Refereed]

Non-alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus-independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR-122 in non-tumor LC at the age of 18weeks was significantly lower than that in LC at the age of 12weeks. Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR-122 is an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.

DOI PubMed

MicroRNA-31 expression in colorectal serrated pathway progression

Hironori Aoki, Katsuhiko Nosho, Hisayoshi Igarashi, Miki Ito, Kei Mitsuhashi, Takafumi Naito, Eiichiro Yamamoto, Tokuma Tanuma, Masafumi Nomura, Hiroyuki Maguchi, Toshiya Shinohara, Hiromu Suzuki, Hiroyuki Yamamoto, Yasuhisa Shinomura

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBLISHING GROUP INC ) 20 ( 34 ) 12346 - 12349 2014.09 [Refereed]

MicroRNAs have been increasingly recognized as useful biomarkers for colorectal cancers (CRC). We have recently observed that microRNA-31 (miR-31) expression is associated with BRAF mutation and prognosis in CRC. Moreover, high miR-31 expression is frequently detected in sessile serrated adenomas compared with hyperplastic polyps (HPs). These results suggest that miR-31 may contribute to the progression of serrated lesions. At a follow-up colonoscopy, we observed the case of a 75-year-old man with a 7-mm flat-elevated lesion in the cecum and diagnosed the lesion as an early invasive carcinoma with serrated features. Tissue specimens were obtained from the representative areas to compare the molecular alterations in the carcinoma component with those in the HP component. Higher miR-31 expression was observed in the carcinoma component (57-fold increase) and the HP component (8-fold increase) compared with the paired normal mucosa, suggesting that miR-31 may be one of the key molecules in serrated pathway progression. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

DOI PubMed

Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells

Masashi Idogawa, Tomoko Ohashi, Jun Sugisaka, Yasushi Sasaki, Hiromu Suzuki, Takashi Tokino

ONCOTARGET ( IMPACT JOURNALS LLC ) 5 ( 17 ) 7540 - 7548 2014.09 [Refereed]

p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

DOI PubMed

半導体シーケンサーを用いたがん関連遺伝子の網羅的解析(Comprehensive genomic analyses of cancer tissues by semiconductor-based next-generation sequencing)

佐々木泰史, 中垣貴文, 田村みゆき, 竹田康佑, 井戸川雅史, 大箸智子, 鈴木拓, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 73回 P - 2146 2014.09

口腔扁平上皮癌の発生や進展において重要な役割を果たす長鎖非コードRNAの同定(Identification of long non-coding RNAs potentially involved in oral squamous cell carcinoma)

西山廣陽, 丸山玲緒, 竹田康佑, 中垣貴文, 新沼猛, 荻和弘, 出張裕也, 甲斐正広, 宮崎晃亘, 佐々木泰史, 時野隆至, 平塚博義, 鈴木拓

日本癌学会総会記事 ( (一社)日本癌学会 ) 73回 P - 3277 2014.09

IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions

Takafumi Naito, Katsuhiko Nosho, Miki Ito, Hisayoshi Igarashi, Kei Mitsuhashi, Shinji Yoshii, Hironori Aoki, Masafumi Nomura, Yasutaka Sukawa, Eiichiro Yamamoto, Yasushi Adachi, Hiroaki Takahashi, Masao Hosokawa, Masahiro Fujita, Toshinao Takenouchi, Reo Maruyama, Hiromu Suzuki, Yoshifumi Baba, Kohzoh Imai, Hiroyuki Yamamoto, Shuji Ogino, Yasuhisa Shinomura

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBLISHING GROUP INC ) 20 ( 29 ) 10050 - 10061 2014.08 [Refereed]

AIM: To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions. METHODS: To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quantification of the relative methylation levels (scale 0%-100%) of IGF2 DMR0 and LINE-1, we used bisulfite pyrosequencing method. Tumor specimens were analyzed for microsatellite instability, KRAS (codons 12 and 13), BRAF (V600E), and PIK3CA (exons 9 and 20) mutations; MLH1 and MGMT methylation; and IGF2 expression by immunohistochemistry. RESULTS: The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas (with or without HGD) was as follows: mean 61.7, median 62.5, SD 18.0, range 5.0-99.0, interquartile range 49.5-74.4. The IGF2 DMR0 methylation level was divided into quartiles (Q1 >= 74.5, Q2 62.6-74.4, Q3 49.6-62.5, Q4 <= 49.5) for further analysis. With regard to the histological type, the IGF2 DMR0 methylation levels of SSAs (mean +/- SD, 73.1 +/- 12.3) were significantly higher than those of HPs (61.9 +/- 20.5), TSAs (61.6 +/- 19.6), and non-serrated adenomas (59.0 +/- 15.8) (P < 0.0001). The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level (r = -0.21, P = 0.0051). IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas (P < 0.0001). Multivariate logistic regression analysis also showed that IGF2 DMR0 hypomethylation was inversely associated with SSAs (P < 0.0001). The methylation levels of IGF2 DMR0 and LINE-1 in TSAs with HGD (50.2 +/- 18.7 and 55.7 +/- 5.4, respectively) were significantly lower than those in TSAs (61.6 +/- 19.6 and 58.8 +/- 4.7, respectively) (IGF2 DMR0, P = 0.038; LINE-1, P = 0.024). CONCLUSION: IGF2 DMR0 hypomethylation may be an infrequent epigenetic alteration in the SSA pathway. Hypomethylation of IGF2 DMR0 and LINE-1 may play a role in TSA pathway progression. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

DOI PubMed

Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid.

Suzuki H, Takashima Y, Ishiguri F, Yoshizawa N, Yokota S

Proteomes 2 ( 3 ) 323 - 340 2014.07 [Refereed]

DOI PubMed

Aberrant methylation of microRNA-34b/c is a predictive marker of metachronous gastric cancer risk

Ryo Suzuki, Eiichiro Yamamoto, Masanori Nojima, Reo Maruyama, Hiro-o Yamano, Kenjiro Yoshikawa, Tomoaki Kimura, Taku Harada, Masami Ashida, Takeshi Niinuma, Akiko Sato, Katsuhiko Nosho, Hiroyuki Yamamoto, Masahiro Kai, Tamotsu Sugai, Kohzoh Imai, Hiromu Suzuki, Yasuhisa Shinomura

JOURNAL OF GASTROENTEROLOGY ( SPRINGER JAPAN KK ) 49 ( 7 ) 1135 - 1144 2014.07 [Refereed]

Metachronous gastric cancer (GC) can develop after endoscopic resection of GC and cannot be predicted based on clinical signature. Aberrant DNA methylation in noncancerous gastric mucosa is strongly implicated in gastric carcinogenesis and could be a useful biomarker of GC risk. We evaluated the clinical utility of DNA methylation as a biomarker of metachronous GC risk. We carried out scheduled follow-up endoscopy in 129 patients after curative endoscopic resection of GC. Biopsy specimens were collected from noncancerous mucosa in the gastric antrum and body, after which quantitative methylation analysis of miR-34b/c, SFRP1, SFRP2, SFRP5, DKK2 and DKK3 was carried out using bisulfite pyrosequencing. The utility of the methylation for predicting the risk of metachronous GC development was assessed using Kaplan-Meier and Cox proportional hazards model analyses. During the follow-up period, 17 patients (13 %) developed metachronous GCs. The cumulative incidence of metachronous GC was significantly higher among patients with elevated miR-34b/c, SFRP2 and DKK2 methylation in their gastric body. MiR-34b/c showed the strongest association with the risk of metachronous GC, and the cumulative incidence of metachronous GC was much higher in the high-miR-34b/c-methylation group than the low-methylation group. Multivariate analysis adjusted for age, sex, H. pylori status and pathological findings showed miR-34b/c methylation in gastric body to be an independent predictor of metachronous GC risk. Our results suggest that methylation of miR-34b/c in the mucosa of the noncancerous gastric body may be a useful biomarker for predicting the risk of metachronous GC.

DOI PubMed

Identification and analysis of large intergenic non-coding RNAs regulated by p53 family members through a genome-wide analysis of p53-binding sites

Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Reo Maruyama, Lisa Kashima, Hiromu Suzuki, Takashi Tokino

HUMAN MOLECULAR GENETICS ( OXFORD UNIV PRESS ) 23 ( 11 ) 2847 - 2857 2014.06 [Refereed]

p53 is one of the most important known tumor suppressor genes, and it is inactivated in approximately half of human cancers. p53 family members execute various functions, such as apoptosis induction and cell cycle arrest, by modulating transcriptional regulation. Therefore, the direct transcriptional targets of the p53 family must be explored to elucidate the functional mechanisms of family members. To identify the direct transcriptional targets of p53 family members, we performed chromatin immunoprecipitation together with next-generation sequencing (ChIP-seq) and searched for p53-binding motifs across the entire human genome. Among the identified ChIP-seq peaks, approximately half were located in an intergenic region. Therefore, we assumed large intergenic non-coding RNAs (lincRNAs) to be major targets of the p53 family. Recent reports have revealed that lincRNAs play an important role in various biological and pathological processes, such as development, differentiation, stemness and carcinogenesis. Through a combination of ChIP-seq and in silico analyses, we found 23 lincRNAs that are upregulated by the p53 family. Additionally, knockdown of specific lincRNAs modulated p53-induced apoptosis and promoted the transcription of a gene cluster. Our results suggest that p53 family members, and lincRNAs constitute a complex transcriptional network involved in various biological functions and tumor suppression.

DOI PubMed

Mesenchymal Stem Cells Cancel Azoxymethane-Induced Tumor Initiation

Masanao Nasuno, Yoshiaki Arimura, Kanna Nagaishi, Hiroyuki Isshiki, Kei Onodera, Suguru Nakagaki, Shuhei Watanabe, Masashi Idogawa, Kentaro Yamashita, Yasuyoshi Naishiro, Yasushi Adachi, Hiromu Suzuki, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

STEM CELLS ( WILEY-BLACKWELL ) 32 ( 4 ) 913 - 925 2014.04 [Refereed]

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O-6-methylguanine (O(6)MeG) adducts through O(6)MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-beta signaling because such properties were completely abrogated by absorption of TGF-beta under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-beta-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. Stem Cells 2014;32:913-925

DOI PubMed

Association of microRNA-31 with BRAF mutation, colorectal cancer survival and serrated pathway

Katsuhiko Nosho, Hisayoshi Igarashi, Masanori Nojima, Miki Ito, Reo Maruyama, Shinji Yoshii, Takafumi Naito, Yasutaka Sukawa, Masashi Mikami, Wakana Sumioka, Eiichiro Yamamoto, Sei Kurokawa, Yasushi Adachi, Hiroaki Takahashi, Hiroyuki Okuda, Takaya Kusumi, Masao Hosokawa, Masahiro Fujita, Tadashi Hasegawa, Kenji Okita, Koichi Hirata, Hiromu Suzuki, Hiroyuki Yamamoto, Yasuhisa Shinomura

CARCINOGENESIS ( OXFORD UNIV PRESS ) 35 ( 4 ) 776 - 783 2014.04 [Refereed]

High miR-31 expression was associated with BRAF mutation and unfavourable survival in CRCs. Moreover, miR-31 inhibitor exhibited antitumour effect in colon cancer cell line, suggesting that miR-31 may be a useful biomarker and therapeutic target in CRC.BRAF is an important gene in colorectal cancers (CRCs) that is associated with molecular characterization and resistance to targeted therapy. Although microRNAs (miRNAs) are useful biomarkers of various cancers, the association between miRNA and BRAF in CRCs is undefined. Therefore, this study was conducted to identify a relationship between specific miRNA molecules and BRAF mutation in CRCs and serrated lesions. miRNA array was used for the measurement of 760 miRNAs in 29 CRCs. To assess the identified miRNAs, quantitative reverse transcriptionPCR was performed on 721 CRCs, 381 serrated lesions and 251 non-serrated adenomas. Moreover, proliferation and invasion assays were conducted using cell lines. miRNA array analysis revealed that microRNA-31 (miR-31)-5p was the most up-regulated miRNA in CRCs with mutated BRAF (V600E) compared with CRCs possessing wild-type BRAF (including cases with KRAS mutation). High miR-31 expression was associated with BRAF and KRAS mutations and proximal location (P < 0.0001). High miR-31 expression was related to cancer-specific mortality [multivariate hazard ratio 2.06, 95% confidence interval: 1.363.09, P 0.0008]. Functional analysis demonstrated that miR-31 inhibitor decreased cell invasion and proliferation. With regard to serrated lesions, high miR-31 expression was less frequently detected in hyperplastic polyps compared with other serrated lesions. In conclusion, associations were identified between miR-31, BRAF and prognosis in CRC. Transfection of miR-31 inhibitor had an antitumour effect. Thus, miR-31 may be a promising diagnostic biomarker and therapeutic target in colon cancers. Moreover, high miR-31 expression in serrated lesions suggested that miR-31 may be a key molecule in serrated pathway.

DOI PubMed

An updated review of gastric cancer in the next-generation sequencing era: Insights from bench to bedside and vice versa

Hiroyuki Yamamoto, Yoshiyuki Watanabe, Tadateru Maehata, Ryo Morita, Yoshihito Yoshida, Ritsuko Oikawa, Shinya Ishigooka, Shun-ichiro Ozawa, Yasumasa Matsuo, Kosuke Hosoya, Masaki Yamashita, Hiroaki Taniguchi, Katsuhiko Nosho, Hiromu Suzuki, Hiroshi Yasuda, Yasuhisa Shinomura, Fumio Itoh

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBLISHING GROUP INC ) 20 ( 14 ) 3927 - 3937 2014.04 [Refereed]

Gastric cancer (GC) is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. There is an increasing understanding of the roles that genetic and epigenetic alterations play in GCs. Recent studies using next-generation sequencing (NGS) have revealed a number of potential cancer-driving genes in GC. Whole-exome sequencing of GC has identified recurrent somatic mutations in the chromatin remodeling gene ARID1A and alterations in the cell adhesion gene FAT4, a member of the cadherin gene family. Mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) have been found in 47% of GCs. Whole-genome sequencing and whole-transcriptome sequencing analyses have also discovered novel alterations in GC. Recent studies of cancer epigenetics have revealed widespread alterations in genes involved in the epigenetic machinery, such as DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs and microRNAs. Recent advances in molecular research on GC have resulted in the introduction of new diagnostic and therapeutic strategies into clinical settings. The antihuman epidermal growth receptor 2 (HER2) antibody trastuzumab has led to an era of personalized therapy in GC. In addition, ramucirumab, a monoclonal antibody targeting vascular endothelial growth factor receptor (VEGFR)-2, is the first biological treatment that showed survival benefits as a single-agent therapy in patients with advanced GC who progressed after first-line chemotherapy. Using NGS to systematically identify gene alterations in GC is a promising approach with remarkable potential for investigating the pathogenesis of GC and identifying novel therapeutic targets, as well as useful biomarkers. In this review, we will summarize the recent advances in the understanding of the molecular pathogenesis of GC, focusing on the potential use of these genetic and epigenetic alterations as diagnostic biomarkers and novel therapeutic targets. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.

DOI PubMed

Fusobacterium in Colonic Flora and Molecular Features of Colorectal Carcinoma

Tomomitsu Tahara, Eiichiro Yamamoto, Hiromu Suzuki, Reo Maruyama, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Hiro-o Yamano, Tamotsu Sugai, Byonggu An, Imad Shureiqi, Minoru Toyota, Yutaka Kondo, Marcos R. H. Estecio, Jean-Pierre J. Issa

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 74 ( 5 ) 1311 - 1318 2014.03 [Refereed]

Fusobacterium species are part of the gut microbiome in humans. Recent studies have identified overrepresentation of Fusobacterium in colorectal cancer tissues, but it is not yet clear whether this is pathogenic or simply an epiphenomenon. In this study, we evaluated the relationship between Fusobacterium status and molecular features in colorectal cancers through quantitative real-time PCR in 149 colorectal cancer tissues, 89 adjacent normal appearing mucosae and 72 colonic mucosae from cancer-free individuals. Results were correlated with CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI), and mutations in BRAF, KRAS, TP53, CHD7, and CHD8. Whole-exome capture sequencing data were also available in 11 cases. Fusobacterium was detectable in 111 of 149 (74%) colorectal cancer tissues and heavily enriched in 9% (14/149) of the cases. As expected, Fusobacterium was also detected in normal appearing mucosae from both cancer and cancer-free individuals, but the amount of bacteria was much lower compared with colorectal cancer tissues (a mean of 250fold lower for Pan-fusobacterium). We found the Fusobacterium-high colorectal cancer group (FB-high) to be associated with CIMP positivity (P = 0.001), TP53 wild-type (P = 0.015), hMLH1 methylation positivity (P = 0.0028), MSI (P = 0.018), and CHD7/8 mutation positivity (P = 0.002). Among the 11 cases where whole-exome sequencing data were available, two that were FB-high cases also had the highest number of somatic mutations (a mean of 736 per case in FB-high vs. 225 per case in all others). Taken together, our findings show that Fusobacterium enrichment is associated with specific molecular subsets of colorectal cancers, offering support for a pathogenic role in colorectal cancer for this gut microbiome component. (C) 2014 AACR.

DOI PubMed

Colorectal Carcinomas With CpG Island Methylator Phenotype 1 Frequently Contain Mutations in Chromatin Regulators

Tomomitsu Tahara, Eiichiro Yamamoto, Priyanka Madireddi, Hiromu Suzuki, Reo Maruyama, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Hiro-o Yamano, Tamotsu Sugai, Yutaka Kondo, Minoru Toyota, Jean-Pierre J. Issa, Marcos R. H. Estecio

GASTROENTEROLOGY ( W B SAUNDERS CO-ELSEVIER INC ) 146 ( 2 ) 530 - + 2014.02 [Refereed]

BACKGROUND & AIMS: Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients' prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs. METHODS: We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort. RESULTS: We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate-dependent chromatin remodeling family. Somatic mutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P<.01) or CIMP-negative tumors (2 of 34 [6%]; P<.001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7. CONCLUSIONS: Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status.

DOI PubMed

The effect of IGF-I receptor blockade for human esophageal squamous cell carcinoma and adenocarcinoma

Yasushi Adachi, Hirokazu Ohashi, Arisa Imsumran, Hiroyuki Yamamoto, Yasutaka Matsunaga, Hiroaki Taniguchi, Katsuhiko Nosho, Hiromu Suzuki, Yasushi Sasaki, Yoshiaki Arimura, David P. Carbone, Kohzoh Imai, Yasuhisa Shinomura

TUMOR BIOLOGY ( SPRINGER ) 35 ( 2 ) 973 - 985 2014.02 [Refereed]

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and tumor development, and this pathway has not been well studied in human esophageal carcinomas. Esophageal cancer is one of the human cancers with the worst prognosis and has two main histologies: squamous cell carcinomas (ESCC) and adenocarcinoma (EAC). Previously, we have reported that detection of the IGF axis may be useful for the prediction of recurrence and poor prognosis of ESCC. We have also shown the successful therapy for several gastrointestinal cancers using recombinant adenoviruses expressing dominant negative IGF-IR (ad-IGF-IR/dn). The aim of this study is to develop potential targeted therapeutics to IGF-IR and to assess the effect of IGF-IR blockade in both of these types of esophageal cancer. We determined immunohistochemical expression of IGF-IR in a tissue microarray. We then assessed the effect of IGF-IR blockade on signal transduction, proliferation, apoptosis, and motility. Ad-IGF-IR/dn, a tyrosine kinase inhibitor, BMS-536924, and adenovirus expressing shRNA for IGF-IR were used. IGF-IR expression was common in both tumor types but not in normal tissues. IGF-IR was detected in metastatic sites at similar levels compared to the primary site. IGF-IR inhibition suppressed proliferation and colony formation in both cancers. IGF-IR blockades up-regulated both stress- and chemotherapy-induced apoptosis and reduced migration. Although IGF-IR/dn blocked ligand-induced activation of Akt-1 mainly, BMS-536924 effectively blocked both activation of Akt and MAPK. The IGF axis might play a key role in tumor progression of esophageal carcinomas. The IGF-IR targeting strategies might thus be useful anticancer therapeutics for human esophageal malignancies.

DOI PubMed

Epigenetic regulation of microRNA genes in colorectal cancer

Hiromu Suzuki, Eiichiro Yamamoto, Reo Maruyama

MicroRNA in Development and in the Progression of Cancer ( Springer New York ) 199 - 211 2014.01 [Refereed]

DNA methylation and histone modification are epigenetic changes that play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, studies have shown that microRNAs (miRNAs) play important roles in the development of colorectal cancer (CRC), and that epigenetic mechanisms are deeply involved in their dysregulation. Specifically, technological advances that enable comprehensive analysis of miRNA expression profiles and the epigenome in CRC cells have led to the identification of a large number of epigenetically regulated miRNAs. As with protein-coding genes, it appears that miRNA genes involved in regulating cancer-related pathways are silenced in association with CpG island hypermethylation and altered histone modification. Aberrant DNA methylation of miRNA genes is a potentially useful biomarker for detecting CRC or predicting its outcome. Moreover, re-expression of the miRNAs could be an effective approach to cancer therapy, and unraveling the relationship between epigenetic alteration and miRNA dysregulation may lead to the discovery of new therapeutic targets.

DOI

HER2 Expression and PI3K-Akt Pathway Alterations in Gastric Cancer

Yasutaka Sukawa, Hiroyuki Yamamoto, Katsuhiko Nosho, Miki Ito, Hisayoshi Igarashi, Takafumi Naito, Kei Mitsuhashi, Yasutaka Matsunaga, Taiga Takahashi, Masashi Mikami, Yasushi Adachi, Hiromu Suzuki, Yasuhisa Shinomura

DIGESTION ( KARGER ) 89 ( 1 ) 12 - 17 2014 [Refereed]

The anti-HER2 antibody trastuzumab has led to an era of personalized therapy in gastric cancer (GC). As a result, HER2 expression has become a major concern in GC. HER2 overexpression is seen in 7-34% of GC cases. Trastuzumab is an antibody that targets the HER2 extracellular domain and induces antibody-dependent cellular cytotoxicity and inhibition of the HER2 downstream signals. Mechanisms of resistance to trastuzumab have been reported in breast cancer. There are various mechanisms underlying trastuzumab resistance, such as alterations of HER2 structure or surroundings, dysregulation of HER2 downstream signal effectors and interaction of HER2 with other membrane receptors. The PI3K-Akt pathway is one of the main downstream signaling pathways of HER2. It is well known that PIK3CA mutations and phosphate and tensin homolog (PTEN) inactivation cause over-activation of the downstream signal without an upstream signal activation. Frequencies of PIK3CA mutations and PTEN inactivation have been reported to be 4-25 and 16-77%, respectively. However, little is known about the association between HER2 expression and PI3K-Akt pathway alterations in GC. We have found that HER2 overexpression was significantly correlated with pAkt expression in GC tissues. Furthermore, pAkt expression was correlated with poor prognosis. These results suggest that the PI3K-Akt pathway plays an important role in HER2-positive GC. Moreover, PIK3CA mutations and/or PTEN inactivation might affect the effectiveness of HER2-targeting therapy. Hence, it is necessary to clarify not only HER2 alterations but also PI3K-Akt pathway alterations for HER2-targeting therapy in GC. This review will introduce recent investigations and consider the current status of HER2-targeted therapy for treatment of GC. (C) 2014 S. Karger AG, Basel

DOI PubMed

AKR1B10, a Transcriptional Target of p53, Is Downregulated in Colorectal Cancers Associated with Poor Prognosis

Tomoko Ohashi, Masashi Idogawa, Yasushi Sasaki, Hiromu Suzuki, Takashi Tokino

MOLECULAR CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 11 ( 12 ) 1554 - 1563 2013.12 [Refereed]

p53 is one of the most important tumor suppressor genes, and it is frequently inactivated in various cancers. p53 modulates various cellular functions, such as apoptosis and cell-cycle arrest via transcriptional regulation. Recently, p53 has been reported to be involved in a wide range of cellular metabolic pathways, including glycolysis, oxidative phosphorylation, glutaminolysis, and the antioxidant response. To understand the functional mechanism of p53, it is important to find out the direct transcriptional targets of p53. In this study, aldo-keto reductase family 1, member B10 (AKR1B10) was identified as a direct target of the p53 family by cDNA microarray analysis after comparing the mRNA expression of control and H1299 cells that overexpressed with p53 family members. In addition, we found that the expression of AKR1B10 was significantly decreased in colorectal cancers and adenomas as compared with normal colon tissues. Knockdown of AKR1B10 significantly inhibited p53-induced apoptosis in colorectal cancer cells, whereas the overexpression of AKR1B10 enhanced p53-induced apoptosis and inhibited tumor proliferation in vivo. Furthermore, low expression of AKR1B10 in colon cancer patients was correlated with decreased survival and poor prognosis. These results suggest that decreased expression of AKR1B10 could disrupt the tumor suppressive function of p53, which result in decreased survival in colorectal cancer patients. In summary, AKR1B10 may be a novel prognostic predictor and a novel therapeutic target for colorectal cancer. Implications: AKR1B10, a transcriptional target of p53, is also a novel prognostic and therapeutic molecule in colorectal cancer. (C) 2013 AACR.

DOI PubMed

RASSF1A methylation indicates a poor prognosis in hepatoblastoma patients

Shohei Honda, Hisayuki Miyagi, Hiromu Suzuki, Masashi Minato, Masayuki Haruta, Yasuhiko Kaneko, Kanako C. Hatanaka, Eiso Hiyama, Takehiko Kamijo, Tadao Okada, Akinobu Taketomi

PEDIATRIC SURGERY INTERNATIONAL ( SPRINGER ) 29 ( 11 ) 1147 - 1152 2013.11 [Refereed]

The RAS association domain family protein 1 (RASSF1A) is known to be frequently inactivated by promoter hypermethylation in cancers. This study investigated the association of RASSF1A methylation with clinical outcomes in hepatoblastoma patients and whether it is correlated with the histological phenotype of hepatoblastoma tumors. Seventy-four hepatoblastoma tumors were obtained from patients enrolled in the Japanese study group for pediatric liver tumor protocol-2. From nine formalin-fixed, paraffin-embedded specimens, we extracted DNA by dissection under a light microscope. We examined the methylation status of the RASSF1A promoter region by bisulfite pyrosequencing. Twenty-five (33.8 %) hepatoblastoma tumors were classified as having methylated RASSF1A. The RASSF1A methylation was significantly associated with metastatic tumors and a poor prognosis. Despite the complete resection, five pretreatment extent of disease II tumors showed recurrence or distant metastasis postoperatively. Among these cases, four tumors were found to show RASSF1A methylation. When compared to histologically different types of cell, RASSF1A methylation values in samples of the normal liver, fetal type, and embryonal type, were significantly elevated in ascending order. We confirmed that RASSF1A methylation is a significant prognostic indicator in hepatoblastomas, and it may become a promising molecular marker to stratify patients into appropriate risk groups.

DOI PubMed

ゲノム網羅的p53結合領域解析によるp53ファミリーの転写標的となる大型介在性非コードRNA(LincRNA)の同定と機能解析(Identification of large intergenic non-coding RNAs regulated by p53 through a genome-wide analysis of p53 binding sites)

井戸川雅史, 大箸智子, 佐々木泰史, 丸山玲緒, 鈴木拓, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 72回 104 - 104 2013.10

p53によるXPO1の抑制機構とその意義(p53 negatively regulates the expression of the nuclear export protein XPO1, establishing a positive feedback loop)

佐々木泰史, 田村みゆき, 竹田康佑, 井戸川雅史, 丸山玲緒, 鈴木拓, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 72回 105 - 105 2013.10

食道癌におけるIGF-I受容体の発現と分子標的としての可能性(Insulin-like growth factor-I receptor(IGF-IR) in human esophageal carcinoma: the possibility as its molecular targeting)

松永康孝, 足立靖, 山本博幸, 大橋広和, 能正勝彦, 谷口博昭, 鈴木拓, 佐々木泰史, 有村佳昭, 遠藤高夫, 今井浩三, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 72回 156 - 156 2013.10

Methylation of a Panel of MicroRNA Genes Is a Novel Biomarker for Detection of Bladder Cancer

Takashi Shimizu, Hiromu Suzuki, Masanori Nojima, Hiroshi Kitamura, Eiichiro Yamamoto, Reo Maruyama, Masami Ashida, Tomo Hatahira, Masahiro Kai, Naoya Masumori, Takashi Tokino, Kohzoh Imai, Taiji Tsukamoto, Minoru Toyota

EUROPEAN UROLOGY ( ELSEVIER SCIENCE BV ) 63 ( 6 ) 1091 - 1100 2013.06 [Refereed]

Background: Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), although the mechanism is not fully understood. Objective: We aimed to explore the involvement of epigenetic alteration of miRNA expression in BCa. Design, setting, and participants: Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan array (Life Technologies, Carlsbad, CA, USA). Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples. Outcome measurements and statistical analysis: Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic performance of the miRNA gene panel. Results and limitations: Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, miR-124-2, miR-124-3, and miR-9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137: 68.7%; miR-124-2: 50.6%; miR-124-3: 65.1%; miR-9-3: 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion. Conclusions: Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection. (C) 2012 European Association of Urology. Published by Elsevier B. V. All rights reserved.

DOI PubMed

Brush border myosin Ia inactivation in gastric but not endometrial tumors

Rocco Mazzolini, Paulo Rodrigues, Sarah Bazzocco, Higinio Dopeso, Ana M. Ferreira, Silvia Mateo-Lozano, Elena Andretta, Stefan M. Woerner, Hafid Alazzouzi, Stefania Landolfi, Javier Hernandez-Losa, Irati MacAya, Hiromu Suzuki, Santiago Ramõn Y Cajal, Mark S. Mooseker, John M. Mariadason, Johannes Gebert, Robert M.W. Hofstra, Jaume Reventõs, Hiroyuki Yamamoto, Simo Schwartz Jr., Diego Arango

International Journal of Cancer 132 ( 8 ) 1790 - 1799 2013.04 [Refereed]

Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47 46.8%) but not endometrial (3/48 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A7A protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide-chase assay demonstrated that the mutant MYO1A7A protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non-MSI gastric primary tumors (Pearson's r = -0.46 p = 0.0003) but not in a cohort of 54 non-MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5-aza-2′- deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage. What's new? Brushborder myosin 1A (MYO1A), a monomeric ATP-dependent motor protein, was recently associated with tumor suppressor activity in the colon. Here, the authors demonstrate that MYO1A is one of the most frequently mutated genes (47%) in gastric tumors with microsatellite instability (MSI). Moreover, the promoter of MYO1A was found frequently methylated. This was unique to gastric tumors, as only background mutation rates in MYO1A (6%) were observed in endometrial tumors with MSI, highlighting profound differences in the tumorigenic processes of these organs. Copyright © 2012 UICC.

DOI PubMed

DNA methyltransferase 1 is essential for initiation of the colon cancers

Rena Morita, Yoshihiko Hirohashi, Hiromu Suzuki, Akari Takahashi, Yasuaki Tamura, Takayuki Kanaseki, Hiroko Asanuma, Satoko Inoda, Toru Kondo, Satoshi Hashino, Tadashi Hasegawa, Takashi Tokino, Minoru Toyota, Masahiro Asaka, Toshihiko Torigoe, Noriyuki Sato

EXPERIMENTAL AND MOLECULAR PATHOLOGY ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 94 ( 2 ) 322 - 329 2013.04 [Refereed]

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs. (C) 2012 Elsevier Inc. All rights reserved.

DOI PubMed

Spontaneous rupture of an advanced pancreatoblastoma: aberrant RASSF1A methylation and CTNNB1 mutation as molecular genetic markers.

Shohei Honda, Tadao Okada, Hisayuki Miyagi, Masatsugu Minato, Hiromu Suzuki, Akinobu Taketomi

Journal of pediatric surgery 48 ( 4 ) e29-32 - 32 2013.04 [Refereed] [International journal]

DOI PubMed

Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids

Norihiro Kato, Hiroyuki Yamamoto, Yasushi Adachi, Hirokazu Ohashi, Hiroaki Taniguchi, Hiromu Suzuki, Mayumi Nakazawa, Hiroyuki Kaneto, Shigeru Sasaki, Kohzoh Imai, Yasuhisa Shinomura

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBLISHING GROUP INC ) 19 ( 11 ) 1718 - 1727 2013.03 [Refereed]

AIM: To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers. METHODS: DNA was extracted from undiluted pancreatic and biliary fluids. As a surrogate for a genome-wide hypomethylation assay, levels of long interspersed nuclear element-1 (LINE-1) methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of 10 tumor-associated genes, aryl-hydrocarbon receptor repressor, adenomatous polyposis coli, calcium channel, voltage dependent, T type alpha 1G subunit, insulin-like growth factor 2, O-6-methyl-guanine-DNA methyltransferase, neurogenin 1, CDKN2A, runt-related transcription factor 3 (RUNX3), secreted frizzled-related protein 1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), was analyzed using MethyLight. To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1, human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 mu mol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h. After the treatment, UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction. RESULTS: Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease (58.7% +/- 4.3% vs 61.7% +/- 2.2%, P = 0.027; 53.8% +/- 6.6% vs 57.5% +/- 1.7%, P = 0.007); however, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids (45.4% +/- 5.5% vs 58.7% +/- 4.3%, P < 0.001). CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancers. Treatment with a demethylating agent, 5-AZA-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines. CONCLUSION: Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers. (C) 2013 Baishideng. All rights reserved.

DOI PubMed

Association between genomic alterations and metastatic behavior of colorectal cancer identified by array-based comparative genomic hybridization

Takeshi Sawada, Eiichiro Yamamoto, Hiromu Suzuki, Masanori Nojima, Reo Maruyama, Yoshihiro Shioi, Risaburo Akasaka, Seiko Kamimae, Taku Harada, Masami Ashida, Masahiro Kai, Yasushi Adachi, Hiroyuki Yamamoto, Kohzoh Imai, Minoru Toyota, Fumio Itoh, Tamotsu Sugai

GENES CHROMOSOMES & CANCER ( WILEY ) 52 ( 2 ) 140 - 149 2013.02 [Refereed]

Colorectal cancers (CRCs) exhibit multiple genetic alterations, including allelic imbalances (copy number alterations, CNAs) at various chromosomal loci. In addition to genetic aberrations, DNA methylation also plays important roles in the development of CRC. To better understand the clinical relevance of these genetic and epigenetic abnormalities in CRC, we performed an integrative analysis of copy number changes on a genome-wide scale and assessed mutations of TP53, KRAS, BRAF, and PIK3CA and DNA methylation of six marker genes in single glands isolated from 39 primary tumors. Array-based comparative genomic hybridization (array-CGH) analysis revealed that genomic losses commonly occurred at 3q26.1, 4q13.2, 6q21.32, 7q34, 8p12-23.3, 15qcen and 18, while gains were commonly found at 1q21.3-23.1, 7p22.3-q34, 13q12.11-14.11, and 20. The total numbers and lengths of the CNAs were significantly associated with the aberrant DNA methylation and Dukes' stages. Moreover, hierarchical clustering analysis of the array-CGH data suggested that tumors could be categorized into four subgroups. Tumors with frequent DNA methylation were most strongly enriched in subgroups with infrequent CNAs. Importantly, Dukes' D tumors were enriched in the subgroup showing the greatest genomic losses, whereas Dukes' C tumors were enriched in the subgroup with the greatest genomic gains. Our data suggest an inverse relationship between chromosomal instability and aberrant methylation and a positive association between genomic losses and distant metastasis and between genomic gains and lymph node metastasis in CRC. Therefore, DNA copy number profiles may be predictive of the metastatic behavior of CRCs. (c) 2012 Wiley Periodicals, Inc.

DOI PubMed

Epigenetic alteration and microRNA dysregulation in cancer

Hiromu Suzuki, Reo Maruyama, Eiichiro Yamamoto, Masahiro Kai

Frontiers in Genetics 4 258 2013 [Refereed]

MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation is a common feature of human cancer. Thanks to recent advances in the analysis of the cancer epigenome, we now know that epigenetic alterations, including aberrant DNA methylation and histone modifications, are major causes of miRNA dysregulation in cancer. Moreover, the list of miRNA genes silenced in association with CpG island hypermethylation is rapidly growing, and various oncogenic miRNAs are now known to be upregulated via DNA hypomethylation. Histone modifications also play important roles in the dysregulation of miRNAs, and histone deacetylation and gain of repressive histone marks are strongly associated with miRNA gene silencing. Conversely, miRNA dysregulation is causally related to epigenetic alterations in cancer. Thus aberrant methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Given that many of the silenced miRNAs appear to act as tumor suppressors through the targeting of oncogenes, re-expression of the miRNAs could be an effective approach to cancer therapy, and unraveling the relationship between epigenetic alteration and miRNA dysregulation may lead to the discovery of new therapeutic targets. © 2013 Suzuki, Maruyama, Yamamoto and Kai.

DOI PubMed

Gastric cancers with microsatellite instability sharing clinical features, chemoresistance and germline MSH6 variants

Kentaro Yamashita, Yoshiaki Arimura, Mayuko Saito, Hiromu Suzuki, Tomohisa Furuhata, Koichi Hirata, Yasuhisa Shinomura

Clinical Journal of Gastroenterology ( Springer-Verlag Tokyo ) 6 ( 2 ) 122 - 126 2013 [Refereed]

Recent studies suggest that colorectal cancer with microsatellite instability (MSI) might be resistant to fluorouracil-based chemotherapy however, little is known about the clinical significance of MSI analysis for gastric cancer. We therefore conducted a prospective single center study analyzing MSI in gastric cancer. Of 123 consecutive gastric cancers analyzed, 12 (9.8 %) were MSI-positive and two patients with MSI-positive gastric cancer underwent systemic chemotherapy because of unresectable disease. Neither cases had concurrent nor past history of other malignancies but one had cancer family history meeting the revised Bethesda criteria. These two MSI-positive gastric cancers shared multiple clinical features, such as direct invasion to adjacent organs, extensive nodal metastasis but no distant metastasis, resistance to fluorouracil and death of tumor bleeding. The two cases had unmethylated MLH1 promoter, suggestive of Lynch syndrome. Germline mutation analysis demonstrated MSH6 alterations of unknown significance in both cases. © Springer 2013.

DOI PubMed

Upregulation of adenylate cyclase 3 (ADCY3) increases the tumorigenic potential of cells by activating the CREB pathway

Seung-Hyun Hong, Sung-Ho Goh, Sang Jin Lee, Jung-Ah Hwang, Jieun Lee, Il-Ju Choi, Hyehyun Seo, Jong-Hoon Park, Hiromu Suzuki, Eiichiro Yamamoto, In-Hoo Kim, Jin Sook Jeong, Mi Ha Ju, Dong-Hee Lee, Yeon-Su Lee

Oncotarget ( Impact Journals LLC ) 4 ( 10 ) 1791 - 1803 2013 [Refereed]

Adenylate cyclase 3 (ADCY3) is a widely expressed membrane-associated protein in human tissues, which catalyzes the formation of cyclic adenosine-3′,5′-monophosphate (cAMP). However, our transcriptome analysis of gastric cancer tissue samples (NCBI GEO GSE30727) revealed that ADCY3 expression was specifically altered in cancer samples. Here we investigated the tumor-promoting effects of ADCY3 overexpression and confirmed a significant correlation between the upregulation of ADCY3 and Lauren's intestinal-type gastric cancers. ADCY3 overexpression increased cell migration, invasion, proliferation, and clonogenicity in HEK293 cells conversely, silencing ADCY3 expression in SNU-216 cells reduced these phenotypes. Interestingly, ADCY3 overexpression increased both the mRNA level and activity of matrix metalloproteinase 2 (MMP2) and MMP9 by increasing the levels of cAMP and phosphorylated cAMP-responsive element-binding protein (CREB). Consistent with these findings, treatment with a protein kinase A (PKA) inhibitor decreased MMP2 and MMP9 expression levels in ADCY3-overexpressing cells. Knockdown of ADCY3 expression by stable shRNA in human gastric cancer cells suppressed tumor growth in a tumor xenograft model. Thus, ADCY3 overexpression may exert its tumor-promoting effects via the cAMP/PKA/CREB pathway. Additionally, bisulfite sequencing of the ADCY3 promoter region revealed that gene expression was reduced by hypermethylation of CpG sites, and increased by 5-Aza-2′-deoxycytidine (5-AzadC)-induced demethylation. Our study is the first to report an association of ADCY3 with gastric cancer as well as its tumorigenic potentials. In addition, we demonstrate that the expression of ADCY3 is regulated through an epigenetic mechanism. Further study on the mechanism of ADCY3 in tumorigenesis will provide the basis as a new molecular target of gastric cancer.

DOI PubMed

Analysis of LINE-1 methylation level and microRNA expression in CRC for identifying a new biomarker

Igarashi Hisayoshi, Nosho Katsuhiko, Ito Miki, Naito Takafumi, Kunimoto Hiroaki, Sukawa Yasutaka, Yoshii Shinji, Yamamoto Eiichiro, Suzuki Hiromu, Yamamoto Hiroyuki, Adachi Yasushi, Shinomura Yasuhisa

Japan Journal of Molecular Tumor Marker Research ( Japanese Society for Molecular Tumor Marker Research ) 28 62 - 63 2013

DOI CiNii

New detection method for integrating Hepatitis B Virus DNA Sequences in PLC/PRF/5 Cell Line

Hiraishi Tetsuya, Watanabe Yoshiyuki, Oikawa Ritsuko, Yamada Norie, Okuse Chiaki, Suzuki Hiromu, Yotsuyanagi Hiroshi, Suzuki Michihiro, Itoh Fumio

Japan Journal of Molecular Tumor Marker Research ( Japanese Society for Molecular Tumor Marker Research ) 28 53 - 53 2013

DOI CiNii

Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

Yasutaka Sukawa, Hiroyuki Yamamoto, Katsuhiko Nosho, Hiroaki Kunimoto, Hiromu Suzuki, Yasushi Adachi, Mayumi Nakazawa, Takayuki Nobuoka, Mariko Kawayama, Masashi Mikami, Takashi Matsuno, Tadashi Hasegawa, Koichi Hirata, Kohzoh Imai, Yasuhisa Shinomura

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBL GRP CO LTD ) 18 ( 45 ) 6577 - 6586 2012.12 [Refereed]

AIM: To investigate human epidermal growth factor receptor 2 (HER2)-phosphatidylinositol 3-kinase (PI3K)-v-Akt murine thymoma viral oncogene homolog signaling pathway. METHODS: We analyzed 231 formalin-fixed, paraffin-embedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment. The patients' age, sex, tumor location, depth of invasion, pathological type, lymph node metastasis, and pathological stage were determined by a review of the medical records. Expression of HER2 was analyzed by immunohistochemistry (IHC) using the HercepTes (TM) kit. Standard criteria for HER2 positivity (0, 1+, 2+, and 3+) were used. Tumors that scored 3+ were considered HER2-positive. Expression of phospho Akt (pAkt) was also analyzed by IHC. Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more. PI3K, catalytic, alpha polypeptide (PIK3CA) mutations in exons 1, 9 and 20 were analyzed by pyrosequencing. Epstein-Barr virus (EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA (EBER) with an EBER-RNA probe. Microsatellite instability (MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26. RESULTS: HER2 expression levels of 0, 1+, 2+ and 3+ were found in 167 (72%), 32 (14%), 12 (5%) and 20 (8.7%) samples, respectively. HER2 overexpression (IHC 3+) significantly correlated with intestinal histological type (15/20 vs 98 /205, P = 0.05). PIK3CA mutations were present in 20 cases (8.7%) and significantly correlated with MSI (10/20 vs 9/211, P < 0.01). The mutation frequency was high (21%) in T4 cancers and very low (6%) in T2 cancers. Mutations in exons 1, 9 and 20 were detected in 5 (2%), 9 (4%) and 7 (3%) cases, respectively. Two new types of PIK3CA mutation, R88Q and R108H, were found in exon1. All PIK3CA mutations were heterozygous missense single-base substitutions, the most common being H1047R (6/20, 30%) in exon20. Eighteen cancers (8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type (13/18 vs 93/198, P = 0.04). There were 7 cases of lymphoepithelioma-like carcinomas (LELC) and 6 of those cases were EBV-positive (percent/EBV: 6/18, 33%; percent/all LELC: 6/7, 86%). pAkt expression was positive in 119 (53%) cases but showed no correlation with clinicopathological characteristics. pAkt expression was significantly correlated with HER2 overexpression (16/20 vs 103/211, P < 0.01) but not with PIK3CA mutations (12/20 vs 107/211, P = 0.37) or EBV infection (8/18 vs 103/211, P = 0.69). The frequency of pAkt expression was higher in cancers with exon20 mutations (100%) than in those with exon1 (40%) or exon9 (56%) mutations. One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection. However, no cases showed both PIK3CA mutations and HER2 overexpression. One EBV-positive cancer with PIK3CA mutation (H1047R) was MSI-positive. Three of these 4 cases were positive for pAkt expression. In survival analysis, pAkt expression significantly correlated with a poor prognosis (hazard ratio 1.75; 95%CI: 1.12-2.80, P = 0.02). CONCLUSION: HER2 expression, PIK3CA mutations and EBV infection in gastric cancer were characterized. pAkt expression significantly correlates with HER2 expression and with a poor prognosis.

DOI PubMed

Genomic vulnerability to LINE-1 hypomethylation is a potential determinant of the clinicogenetic features of multiple myeloma

Yuka Aoki, Masanori Nojima, Hiromu Suzuki, Hiroshi Yasui, Reo Maruyama, Eiichiro Yamamoto, Masami Ashida, Mitsuhiro Itagaki, Hideki Asaoku, Hiroshi Ikeda, Toshiaki Hayashi, Kohzoh Imai, Mitsuru Mori, Takashi Tokino, Tadao Ishida, Minoru Toyota, Yasuhisa Shinomura

GENOME MEDICINE ( BIOMED CENTRAL LTD ) 4 ( 12 ) 101 2012.12 [Refereed]

Background: The aim of this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM). Methods: We assessed global methylation levels using four repetitive elements (long interspersed nuclear element-1 (LINE-1), Alu Ya5, Alu Yb8, and Satellite-a) in clinical samples comprising 74 MM samples and 11 benign control samples (7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPC)). We also evaluated copy-number alterations using array-based comparative genomic hybridization, and performed methyl-CpG binding domain sequencing (MBD-seq). Results: Global levels of the repetitive-element methylation declined with the degree of malignancy of plasma cells (NPC>MGUS>MM), and there was a significant inverse correlation between the degree of genomic loss and the LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA-methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also declined during the development of MM. We confirmed that levels of methylation of the 5' untranslated region of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and poorer overall survival (hazard ratio 2.8, P = 0.015). Conclusion: Global hypomethylation of LINE-1 is associated with the progression of and poorer prognosis for MM, possibly due to frequent copy-number loss.

DOI PubMed

CLCA2, a target of the p53 family, negatively regulates cancer cell migration and invasion

Yasushi Sasaki, Ryota Koyama, Reo Maruyama, Takehiro Hirano, Miyuki Tamura, Jun Sugisaka, Hiromu Suzuki, Masashi Idogawa, Yasuhisa Shinomura, Takashi Tokino

CANCER BIOLOGY & THERAPY ( LANDES BIOSCIENCE ) 13 ( 14 ) 1512 - 1521 2012.12 [Refereed]

The tumor suppressor p53 transcriptionally regulates a number of genes that are involved in cell-cycle inhibition, apoptosis and the maintenance of genetic stability. Recent studies suggest that p53 also contributes to the regulation of cell migration and invasion. Here, we show that human chloride channel accessory-2 (CLCA2) is a target gene of the p53 family (p53, p73 and p63). CLCA2 is induced by DNA damage in a p53-dependent manner. The p53 family proteins activate the CLCA2 promoter by binding directly to the conserved consensus p53-binding site present in the CLCA2 promoter. In terms of function, ectopic expression of CLCA2 inhibited cancer cell migration. In contrast, silencing CLCA2 with siRNA stimulated cancer cell migration and invasion. We also found that inactivation of CLCA2 enhanced the expression of focal adhesion kinase (FAK), as well as its promoter activation. A small-molecule FAK inhibitor reduced the effect of CLCA2 siRNA on cell migration and invasion, suggesting that CLCA2 inhibits cancer cell migration and invasion through suppression of the FAK signaling pathway. Furthermore, there was an inverse correlation between CLCA2 and FAK expression in 251 human breast cancer tissues. These results strongly suggest that CLCA2 is involved in the p53 tumor suppressor network and has a significant effect on cell migration and invasion.

DOI PubMed

DNA methylation and microRNA dysregulation in cancer

Hiromu Suzuki, Reo Maruyama, Eiichiro Yamamoto, Masahiro Kai

MOLECULAR ONCOLOGY ( ELSEVIER SCI LTD ) 6 ( 6 ) 567 - 578 2012.12 [Refereed]

DNA methylation plays a key role in the silencing of numerous cancer-related genes, thereby affecting a number of vital cellular processes, including the cell cycle checkpoint, apoptosis, signal transduction, cell adhesion and angiogenesis. Also widely altered in human malignancies is the expression of microRNAs (miRNAs), a class of small noncoding RNAs that act as posttranscriptional regulators of gene expression. Furthermore, emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer. This is in part the result of technological advances that enable more comprehensive analysis of miRNA expression profiles and the epigenome in cancer cells, which has led to the identification of a number of epigenetically regulated miRNAs. As with protein-coding genes, it appears that miRNA genes involved in regulating cancer-related pathways are silenced in association with CpG island hypermethylation. In addition, methylation in CpG island shore regions and DNA hypomethylation also appear to contribute to miRNA dysregulation in cancer. Aberrant DNA methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Moreover, re-expression of miRNAs and the replacement of tumor suppressive miRNAs using miRNA mimics or expression vectors could be effective approaches to cancer therapy. (c) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI PubMed

Model of translational cancer research in multiple myeloma

Hiroshi Yasui, Tadao Ishida, Reo Maruyama, Masanori Nojima, Hiroshi Ikeda, Hiromu Suzuki, Toshiaki Hayashi, Yasuhisa Shinomura, Kohzoh Imai

Cancer Science 103 ( 11 ) 1907 - 1912 2012.11 [Refereed]

Recently, intensive laboratory and preclinical studies have identified and validated therapeutic molecular targets in multiple myeloma (MM). The introduction of novel agents such as the proteasome inhibitor bortezomib and the immunomodulatory drugs thalidomide and lenalidomide, which were rapidly translated from preclinical studies at the Dana-Farber Cancer Institute into clinical trials, has changed the treatment paradigm and markedly extended overall survival MM has therefore become a remarkable example of translational cancer research in new drug development. In this article, with the aim of determining the key factors underlying success in translational research, we focus on our studies of MM at Dana-Farber Cancer Institute as well as at our institutes. The identification of these key factors will help to promote translational cancer research not only in MM but also in other hematologic malignancies and solid tumors, to develop novel therapies, to overcome drug resistance, and to thereby improve the prognosis of cancer patients. © 2012 Japanese Cancer Association.

DOI PubMed

Molecular Dissection of Premalignant Colorectal Lesions Reveals Early Onset of the CpG Island Methylator Phenotype

Eiichiro Yamamoto, Hiromu Suzuki, Hiro-o Yamano, Reo Maruyama, Masanori Nojima, Seiko Kamimae, Takeshi Sawada, Masami Ashida, Kenjiro Yoshikawa, Tomoaki Kimura, Ryo Takagi, Taku Harada, Ryo Suzuki, Akiko Sato, Masahiro Kai, Yasushi Sasaki, Takashi Tokino, Tamotsu Sugai, Kohzoh Imai, Yasuhisa Shinomura, Minoru Toyota

AMERICAN JOURNAL OF PATHOLOGY ( ELSEVIER SCIENCE INC ) 181 ( 5 ) 1847 - 1861 2012.11 [Refereed]

The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIR3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly; a subset of GIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway gems. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations. (Am J Pathol 2012, 181:1847-1861; http://dx.doi.org/10.1016/j.ajpath.2012.08.007)

DOI PubMed

Long noncoding RNA involvement in cancer

Reo Maruyama, Hiromu Suzuki

BMB REPORTS ( KOREAN SOCIETY BIOCHEMISTRY & MOLECULAR BIOLOGY ) 45 ( 11 ) 604 - 611 2012.11 [Refereed]

Recent advances in genome and transcriptome analysis have enabled identification of numerous members of a new class of noncoding RNA, long noncoding RNA (IncRNA). IncRNAs are broadly defined as RNA molecules greater than 200 nt in length and lacking an open reading frame. Recent studies provide evidence that IncRNAs play central roles in a wide range of cellular processes through interaction with key component proteins in the gene regulatory system, and that alteration of their cell- or tissue-specific expression and/or their primary or secondary structures is thought to promote cell proliferation, invasion and metastasis. The biological and molecular characteristics of the large majority of IncRNAs remains unknown, and it is anticipated that improved understanding of the roles played by IncRNAs in cancer will lead to the development of novel biomarkers and effective therapeutic strategies. [BMB Reports 2012; 45(11): 604-611]

DOI PubMed

Genome-wide analysis of DNA methylation identifies novel cancer-related genes in hepatocellular carcinoma

Masahiro Shitani, Shigeru Sasaki, Noriyuki Akutsu, Hideyasu Takagi, Hiromu Suzuki, Masanori Nojima, Hiroyuki Yamamoto, Takashi Tokino, Koichi Hirata, Kohzoh Imai, Minoru Toyota, Yasuhisa Shinomura

TUMOR BIOLOGY ( SPRINGER ) 33 ( 5 ) 1307 - 1317 2012.10 [Refereed]

Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n = 4; hepatitis C virus (HCV)-positive, n = 5; HBV/HCV-negative, n = 7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n = 57) and noncancerous liver tissue (n = 50) from HCC patients and in HCC cell lines (n = 10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.

DOI PubMed

Aberrant Methylation of RASGRF1 Is Associated with an Epigenetic Field Defect and Increased Risk of Gastric Cancer

Hiroyuki Takamaru, Eiichiro Yamamoto, Hiromu Suzuki, Masanori Nojima, Reo Maruyama, Hiro-o Yamano, Kenjiro Yoshikawa, Tomoaki Kimura, Taku Harada, Masami Ashida, Ryo Suzuki, Hiroyuki Yamamoto, Masahiro Kai, Takashi Tokino, Tamotsu Sugai, Kohzoh Imai, Minoru Toyota, Yasuhisa Shinomura

CANCER PREVENTION RESEARCH ( AMER ASSOC CANCER RESEARCH ) 5 ( 10 ) 1203 - 1212 2012.10 [Refereed]

Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P < 0.001; 8.3% vs. 19.4%, P < 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203-12. (c) 2012 AACR.

DOI PubMed

DNAメチル化の網羅的解析により同定された新規肝癌関連遺伝子

志谷真啓, 鈴木拓, 阿久津典之, 高木秀安, 佐々木茂, 野島正寛, 山本博幸, 時野隆至, 平田公一, 篠村恭久

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 109 ( 臨増大会 ) A700 - A700 2012.09

PLC/PRF/5細胞株におけるB型肝炎ウイルスDNAの新しい検出法

平石哲也, 渡邊嘉行, 及川律子, 山田典栄, 奥瀬千晃, 鈴木拓, 四柳宏, 鈴木通博, 伊東文生

日本分子腫瘍マーカー研究会プログラム・講演抄録 ( 日本分子腫瘍マーカー研究会 ) 32回 72 - 72 2012.09

多発性骨髄腫におけるトランスレーショナルリサーチの試み(A model of translational cancer research in multiple myeloma)

安井寛, 石田禎夫, 青木由佳, 丸山玲緒, 野島正寛, 池田博, 鈴木拓, 林敏昭, 篠村恭久, 今井浩三

日本癌学会総会記事 ( (一社)日本癌学会 ) 71回 301 - 301 2012.08

多発性骨髄腫の発生におけるDNAメチロームの意義 MBDシーケンシング法によるアプローチ(The significance of DNA methylome for tumor progression in multiple myeloma: An MBD-sequencing-based approach)

野島正寛, 青木由佳, 安井寛, 丸山玲緒, 麻奥英毅, 石田禎夫, 時野隆至, 森満, 鈴木拓, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 71回 385 - 385 2012.08

Cigarette smoking, alcohol consumption, and risk of systemic lupus erythematosus: A case-control study in a Japanese population

Chikako Kiyohara, Masakazu Washio, Takahiko Horiuchi, Toyoko Asami, Saburo Ide, Tatsuya Atsumi, Gen Kobashi, Yoshifumi Tada, Hiroki Takahashi, Hiroko Kodama, Koichi Akashi, Mine Harada, Hiroaki Niiro, Hiroshi Tsukamoto, Takao Hotokebuchi, Kohei Nagasawa, Osamu Ushiyama, Mitsuru Mori, Asae Oura, Yasuhisa Sinomura, Hiromu Suzuki, Motohisa Yamamoto, Tetsuya Horita, Takao Koike, Takashi Abe, Hisato Tanaka, Norihiko Nogami, Kazushi Okamoto, Naomasa Sakamoto, Satoshi Sasaki, Yoshihiro Miyake, Tetsuji Yokoyama, Yoshio Hirota, Yutaka Inaba, Masaki Nagai

Journal of Rheumatology 39 ( 7 ) 1363 - 1370 2012.07 [Refereed]

Objective. Cigarette smoking may be associated with increased risk of systemic lupus erythematosus (SLE), whereas the role of alcohol consumption is unknown. We examined the association between SLE risk and smoking or drinking. Methods. We investigated the relationship of smoking and drinking compared to SLE risk among 171 SLE cases and 492 healthy controls in female Japanese subjects. Unconditional logistic regression was used to compute OR and 95% CI, with adjustments for several covariates. Results. Compared with nonsmoking, current smoking was significantly associated with increased risk of SLE (OR 3.06, 95% CI 1.86-5.03). The higher the level of exposure to cigarette smoke, the higher the risk of SLE. Inhalation was also associated with increased SLE risk (OR 3.73, 95% CI 1.46-9.94 for moderate inhalation OR 3.06, 95% CI 1.81-5.15 for deep inhalation). In contrast, light/moderate alcohol consumption had a protective effect on SLE risk (OR 0.38, 95% CI 0.19-0.76). As for beer, the risks for non-beer drinkers and beer drinkers were similar. This also applies to alcoholic beverages other than beer. Conclusion. Our results suggest that smoking was positively associated with increased SLE risk whereas light/moderate alcohol consumption was inversely associated with SLE risk, irrespective of the type of alcoholic beverage. Additional studies are warranted to confirm these findings. The Journal of Rheumatology Copyright © 2012. All rights reserved.

DOI PubMed

Interrelationship between microsatellite instability and microRNA in gastrointestinal cancer

Hiroyuki Yamamoto, Yasushi Adachi, Hiroaki Taniguchi, Hiroaki Kunimoto, Katsuhiko Nosho, Hiromu Suzuki, Yasuhisa Shinomura

WORLD JOURNAL OF GASTROENTEROLOGY ( BAISHIDENG PUBLISHING GROUP INC ) 18 ( 22 ) 2745 - 2755 2012.06 [Refereed]

There is an increasing understanding of the roles that microsatellite instability (MSI) plays in Lynch syndrome (by mutations) and sporadic (by mainly epigenetic changes) gastrointestinal (GI) and other cancers. Deficient DNA mismatch repair (MMR) results in the strong mutator phenotype known as MSI, which is the hallmark of cancers arising within Lynch syndrome. MSI is characterized by length alterations within simple repeated sequences called microsatellites. Lynch syndrome occurs primarily because of germline mutations in one of the MMR genes, mainly MLH1 or MSH2, less frequently MSH6, and rarely PMS2. MSI is also observed in about 15% of sporadic colorectal, gastric, and endometrial cancers and in lower frequencies in a minority of other cancers where it is often associated with the hypermethylation of the MLH1 gene. miRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level and are critical in many biological processes and cellular pathways. There is accumulating evidence to support the notion that the interrelationship between MSI and miRNA plays a key role in the pathogenesis of GI cancer. As a possible new mechanism underlying MSI, overexpression of mR-155 has been shown to downreijulate expression of MLH1, MSH2, and MSH6. Thus, a subset of MSI-positive (MSI+) cancers without known MMR defects may result from miR-155 overexpression. Target genes of frameshift mutation for MSI are involved in various cellular functions, such as DNA repair, cell signaling, and apoptosis. A novel class of target genes that included not only epigenetic modifier genes, such as HDAC2, but also miRNA processing machinery genes, including TARBP2 and XPO5, were found to be mutated in MSI+ GI cancers. Thus, a subset of MSI+ colorectal cancers (CRCs) has been proposed to exhibit a mutated miRNA machinery phenotype. Genetic, epigenetic, and transcriptomic differences exist between MSI+ and MSI cancers. Molecular signatures of miRNA expression apparently have the potential to distinguish between MSI+ and MSI- CRCs. In this review, we summarize recent advances in the MSI pathogenesis of GI cancer, with the focus on its relationship with miRNA as well as on the potential to use MSI and related alterations as biomarkers and novel therapeutic targets. (C) 2012 Baishideng. All rights reserved.

DOI PubMed

Hypermethylation of Sox17 gene is useful as a molecular diagnostic application in early gastric cancer

Yoshichika Oishi, Yoshiyuki Watanabe, Yoshihito Yoshida, Yoshinori Sato, Tetsuya Hiraishi, Ritsuko Oikawa, Tadateru Maehata, Hiromu Suzuki, Minoru Toyota, Hirohumi Niwa, Michihiro Suzuki, Fumio Itoh

TUMOR BIOLOGY ( SPRINGER ) 33 ( 2 ) 383 - 393 2012.04 [Refereed]

Although minimal invasive treatment is widely accepted in the early stages of gastric cancer (GCa), we still do not have any appropriate risk markers to detect residual neoplasia and the potential for recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for the detection of gastric neoplasia. Our goal is to find and identify some candidate genes, using genome-wide DNA methylation analysis, as a treatment marker for early gastric cancer (EGC). We performed methylated CpG island amplification microarray analysis using 12 gastric washes (six each of pre- and post-endoscopic treatment in each of the same patients). We finally focused on Sox17 gene. We examined the DNA methylation status of Sox17 in a validation set consisting of 128 wash samples (pre, 64; post, 64) at EGC. We next carried out functional studies to identify Sox17. Sox17 showed significant differential methylation between pre- and post-treatments in EGC patients (Sox17, p < 0.0001). Moreover, treating GCa cells that lacked Sox17 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Additionally, the introduction of exogenous Sox17 into silenced cells suppressed colony formation. Gastric wash-based DNA methylation analysis could be useful for early detection of recurrence following endoscopic resection in EGC patients. Our data suggest that the silencing of Sox17 occurs frequently in EGC and may play a key role in the development and progression of the disease.

DOI PubMed

DNA methylation biomarker candidates for early detection of colon cancer

Joo Mi Yi, Mashaal Dhir, Angela A. Guzzetta, Christine A. Iacobuzio-Donahue, Kyu Heo, Kwang Mo Yang, Hiromu Suzuki, Minoru Toyota, Hwan-Mook Kim, Nita Ahuja

TUMOR BIOLOGY ( SPRINGER ) 33 ( 2 ) 363 - 372 2012.04 [Refereed]

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (> 80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine (TM) website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.

DOI PubMed

CHFR Protein Regulates Mitotic Checkpoint by Targeting PARP-1 Protein for Ubiquitination and Degradation

Lisa Kashima, Masashi Idogawa, Hiroaki Mita, Miki Shitashige, Tesshi Yamada, Kazuhiro Ogi, Hiromu Suzuki, Minoru Toyota, Hiroyoshi Ariga, Yasushi Sasaki, Takashi Tokino

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC ) 287 ( 16 ) 12975 - 12984 2012.04 [Refereed]

The mitotic checkpoint gene CHFR (checkpoint with fork-head-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

DOI PubMed

Emerging links between epigenetic alterations and dysregulation of noncoding RNAs in cancer

Reo Maruyama, Hiromu Suzuki, Eiichiro Yamamoto, Kohzoh Imai, Yasuhisa Shinomura

TUMOR BIOLOGY ( SPRINGER ) 33 ( 2 ) 277 - 285 2012.04 [Refereed]

Epigenetic changes, including DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, numerous studies have shown that noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs exert tumor-suppressive or oncogenic effects. In addition, it now appears that long ncRNAs may be causally related to epigenetic dysregulation of critical genes in cancer. Dissection of the relationships between ncRNAs and epigenetic alterations may lead to the development of novel approaches to the diagnosis and treatment of cancer.

DOI PubMed

p53により発現抑制される分泌性増殖因子HDGFの同定とがん組織における発現解析

佐々木泰史, 根岸秀明, 小山良太, 井戸川雅史, 鈴木拓, 今井浩三, 篠村恭久, 時野隆至

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 49回 133 - 133 2012.04

Aberrant DNA methylation associated with aggressiveness of gastrointestinal stromal tumour

Yasuyuki Okamoto, Akira Sawaki, Seiji Ito, Toshirou Nishida, Tsuyoshi Takahashi, Minoru Toyota, Hiromu Suzuki, Yasuhisa Shinomura, Ichiro Takeuchi, Keiko Shinjo, Byonggu An, Hidemi Ito, Kenji Yamao, Makiko Fujii, Hideki Murakami, Hirotaka Osada, Hiromi Kataoka, Takashi Joh, Yoshitaka Sekido, Yutaka Kondo

GUT ( B M J PUBLISHING GROUP ) 61 ( 3 ) 392 - 401 2012.03 [Refereed]

Background and aims The majority of gastrointestinal stromal tumors (GISTs) have KIT mutations; however, epigenetic abnormalities that could conceivably potentiate the aggressiveness of GISTs are largely unidentified. Our aim was to establish epigenetic profiles associated with the malignant transformation of GISTs. Methods Methylation of four tumor suppressor genes, RASSF1A, p16, CDH1, and MGMT was analyzed in GISTs. Additionally, genome-wide DNA methylation profiles were compared between small, malignant-prone, and malignant GISTs using methylated GpG island amplification microarrays (MCAM) in a training set (n=40). Relationships between the methylation status of genes identified by MCAM and clinical features of the disease were tested in a validation set (n=75). Results Methylation of RASSF1A progressively increased from small to malignant GISTs. p16 was specifically methylated in malignant-prone and malignant GISTs. MCAM analysis showed that more genes were methylated in advanced than in small GISTs (average of 473 genes vs 360 genes, respectively, P=0.012). Interestingly, the methylation profile of malignant GISTs was prominently affected by their location. Two genes, REC8 and PAX3, which were newly-identified via MCAM analysis, were differentially methylated in small and malignant GISTs in the training and validation sets. Patients with methylation of at least REC8, PAX3, or p16 had a significantly poorer prognosis (P=0.034). Conclusion Our results suggest that GIST is not, in epigenetic terms, a uniform disease and that DNA methylation in a set of genes is associated with aggressive clinical behavior and unfavorable prognosis. The genes identified may potentially serve as biomarkers for predicting aggressive GISTs with poor survivability.

DOI PubMed

Upregulation of miR-196a and HOTAIR Drive Malignant Character in Gastrointestinal Stromal Tumors

Takeshi Niinuma, Hiromu Suzuki, Masanori Nojima, Katsuhiko Nosho, Hiroyuki Yamamoto, Hiroyuki Takamaru, Eiichiro Yamamoto, Reo Maruyama, Takayuki Nobuoka, Yasuaki Miyazaki, Toshirou Nishida, Takeo Bamba, Tatsuo Kanda, Yoichi Ajioka, Takahiro Taguchi, Satoshi Okahara, Hiroaki Takahashi, Yasunori Nishida, Masao Hosokawa, Tadashi Hasegawa, Takashi Tokino, Koichi Hirata, Kohzoh Imai, Minoru Toyota, Yasuhisa Shinomura

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 72 ( 5 ) 1126 - 1136 2012.03 [Refereed]

Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXCand HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs. Cancer Res; 72(5); 1126-36. (C) 2012 AACR.

DOI PubMed

A Novel Pit Pattern Identifies the Precursor of Colorectal Cancer Derived From Sessile Serrated Adenoma

Tomoaki Kimura, Eiichiro Yamamoto, Hiro-o Yamano, Hiromu Suzuki, Seiko Kamimae, Masanori Nojima, Takeshi Sawada, Masami Ashida, Kenjiro Yoshikawa, Ryo Takagi, Ryusuke Kato, Taku Harada, Ryo Suzuki, Reo Maruyama, Masahiro Kai, Kohzoh Imai, Yasuhisa Shinomura, Tamotsu Sugai, Minoru Toyota

AMERICAN JOURNAL OF GASTROENTEROLOGY ( NATURE PUBLISHING GROUP ) 107 ( 3 ) 460 - 469 2012.03 [Refereed]

OBJECTIVES: Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic to clarify which endoscopic findings are specific to SSAs. METHODS: The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing. RESULTS: Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns. CONCLUSIONS: Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.

DOI PubMed

【消化器癌治療における新しい分子標的】GIST治療抵抗例からみた新たな分子標的治療の可能性を探る

新沼猛, 鈴木拓, 篠村恭久

分子消化器病 ( (株)先端医学社 ) 9 ( 1 ) 27 - 32 2012.03

消化管間質腫瘍(GIST)に対する薬物治療としてはイマチニブメシル酸塩(イマチニブ)、スニチニブリンゴ酸塩(スニチニブ)が用いられているが、これらの薬剤に耐性をもつ症例も出現してきている。イマチニブ耐性にはもとから効果が低いと考えられる一次耐性、新たな遺伝子変異の獲得による二次耐性がある。スニチニブ耐性の機序については完全には解明されてはいないが、活性化ループ(activation loop)の変異によるKITの自律活性化やプロモーター領域のメチル化によるPTENの発現低下とAkt経路の活性化などの報告がある。イマチニブ、スニチニブ耐性例に対しては新規のチロシンキナーゼ阻害薬であるニロチニブ塩酸塩や、mTORなどの細胞内シグナル伝達経路の阻害薬、チロシンキナーゼと細胞内シグナル伝達分子の両者を標的とするソラフェニブトシル酸塩などのマルチターゲットチロシンキナーゼなどの治療が考えられている。また熱ショック蛋白(Hsp)90などの腫瘍形成に関与する分子を標的とする薬剤などが治療標的として考えられており、臨床試験も進んでいるところである。(著者抄録)

Insulin-like growth factor receptor expression is associated with aggressive phenotypes and has therapeutic activity in biliary tract cancers

Hirokazu Ohashi, Yasushi Adachi, Hiroyuki Yamamoto, Hiroaki Taniguchi, Katsuhiko Nosho, Hiromu Suzuki, Yoshiaki Arimura, Kohzoh Imai, David P. Carbone, Yasuhisa Shinomura

Cancer Science 103 ( 2 ) 252 - 261 2012.02 [Refereed]

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeutic target have not been studied comprehensively in biliary tract carcinomas (BTC). We investigated the immunohistochemical expression of elements of the IGF axis, matrilysin, overexpression of p53 and the methylation status of the IGFBP-3 promoter in 80 surgically resected BTC. We also assessed the effect of IGF-IR blockade on signal transduction, proliferation and survival in three BTC cell lines using a new tyrosine kinase inhibitor, BMS-536924, and dominant negative IGF-IR (IGF-IR/dn). The effects of IGF-IR blockade was also studied in nude mouse xenograft models. IGF-I was expressed in 60% and IGF-II in 50% of tumors. High expression was associated with tumor size. IGF-IR was expressed in 69% of the cases and was associated with advanced stage and matrilysin expression. Hypermethylation of the IGFBP-3 promoter was detected in 41% of BTC and was inversely correlated with p53 expression. BMS-536924 blocked autophosphorylation of IGF-IR and both Akt and ERK activation by both IGF-I and insulin. BMS-536924 suppressed proliferation and tumorigenicity in vitro in a dose-dependent fashion. This inhibitor upregulated chemotherapy-induced apoptosis in a dose-dependent fashion. Moreover, IGF-IR blockade was effective against tumors in mice. IGF-IR might identify a subset of BTC with a particularly aggressive phenotype and is a candidate therapeutic target in this disease. BMS-536924 might have significant therapeutic utility. © 2011 Japanese Cancer Association.

DOI PubMed

Insulin-like growth factor receptor expression is associated with aggressive phenotypes and has therapeutic activity in biliary tract cancers

Hirokazu Ohashi, Yasushi Adachi, Hiroyuki Yamamoto, Hiroaki Taniguchi, Katsuhiko Nosho, Hiromu Suzuki, Yoshiaki Arimura, Kohzoh Imai, David P. Carbone, Yasuhisa Shinomura

CANCER SCIENCE ( WILEY ) 103 ( 2 ) 252 - 261 2012.02 [Refereed]

DOI

Aberrant DNA methylation associated with aggressiveness of gastrointestinal stromal tumour.

Okamoto Yasuyuki, Sawaki Akira, Ito Seiji, Nishida Toshirou, Takahashi Tsuyoshi, Toyota Minoru, Suzuki Hiromu, Shinomura Yasuhisa, Takeuchi Ichiro, Shinjo Keiko, An Byonggu, Ito Hidemi, Yamao Kenji, Fujii Makiko, Murakami Hideki, Osada Hirotaka, Kataoka Hiromi, Joh Takashi, Sekido Yoshitaka, Kondo Yutaka

Gut 61 ( 3 ) 2012

BACKGROUND AND AIMS:The majority of gastrointestinal stromal tumors (GISTs) have KIT mutations; however, epigenetic abnormalities that could conceivably potentiate the aggressiveness of GISTs are largely unidentified. Our aim was to establish epigenetic profiles associated with the malignant transformation of GISTs.;METHODS:Methylation of four tumor suppressor genes, RASSF1A, p16, CDH1, and MGMT was analyzed in GISTs. Additionally, genome-wide DNA methylation profiles were compared between small, malignant-prone, and malignant GISTs using methylated GpG island amplification microarrays (MCAM) in a training set (n=40). Relationships between the methylation status of genes identified by MCAM and clinical features of the disease were tested in a validation set (n=75).;RESULTS:Methylation of RASSF1A progressively increased from small to malignant GISTs. p16 was specifically methylated in malignant-prone and malignant GISTs. MCAM analysis showed that more genes were methylated in advanced than in small GISTs (average of 473 genes vs 360 genes, respectively, P=0.012). Interestingly, the methylation profile of malignant GISTs was prominently affected by their location. Two genes, REC8 and

DOI

【大腸癌研究の新たな展開と治療戦略】大腸発癌進展におけるCa感受性受容体のエピジェネティックな不活化と治療戦略

山本博幸, 能正勝彦, 須河恭敬, 國本浩明, 五十嵐央祥, 中澤眞由美, 新沼猛, 志谷真啓, 山本英一郎, 鈴木拓, 佐々木茂, 足立靖, 篠村恭久

消化器内科 ( (有)科学評論社 ) 53 ( 6 ) 639 - 645 2011.12

p53 Negatively Regulates the Hepatoma Growth Factor HDGF

Yasushi Sasaki, Hideaki Negishi, Masashi Idogawa, Ikuko Yokota, Ryota Koyama, Masanobu Kusano, Hiromu Suzuki, Masahiro Fujita, Reo Maruyama, Minoru Toyota, Tsuyoshi Saito, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 71 ( 22 ) 7038 - 7047 2011.11 [Refereed]

Hepatoma-derived growth factor (HDGF) is a secreted heparin-binding growth factor that has been implicated in cancer development and progression. Here, we report that HDGF is a critical target for transcriptional repression by the tumor suppressor p53. Endogenous HDGF expression was decreased in cancer cells with introduction of wild-type p53, which also downregulated HDGF expression after DNA damage. In support of the likelihood that HDGF is a critical driver of cancer cell growth, addition of neutralizing HDGF antibodies to culture media was sufficient to block cell growth, migration, and invasion. Similarly, these effects were elicited by conditioned culture medium from p53-expressing cells, and they could be reversed by the addition of recombinant human HDGF. Interestingly, we found that HDGF was overexpressed also in primary gastric, breast, and lung cancer tissues harboring mutant p53 genes. Mechanistic investigations revealed that p53 repressed HDGF transcription by altering HDAC-dependent chromatin remodeling. Taken together, our results reveal a new pathway in which loss of p53 function contributes to the aggressive pathobiological potential of human cancers by elevating HDGF expression. Cancer Res; 71(22); 7038-47. (C)2011 AACR.

DOI PubMed

形質細胞異常症における血清遊離軽鎖定量法の臨床的有用性の検討

安井寛, 池田博, 青木由佳, 丸山玲緒, 野島正寛, 鈴木拓, 林敏昭, 石田禎夫, 今井浩三, 篠村恭久

生物物理化学 ( 日本電気泳動学会 ) 55 ( Suppl. ) 18 - 18 2011.11

Genome-wide Profiling of Chromatin Signatures Reveals Epigenetic Regulation of MicroRNA Genes in Colorectal Cancer

Hiromu Suzuki, Shintaro Takatsuka, Hirofumi Akashi, Eiichiro Yamamoto, Masanori Nojima, Reo Maruyama, Masahiro Kai, Hiro-o Yamano, Yasushi Sasaki, Takashi Tokino, Yasuhisa Shinomura, Kohzoh Imai, Minoru Toyota

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 71 ( 17 ) 5646 - 5658 2011.09 [Refereed]

Altered expression of microRNAs (miRNA) occurs commonly in human cancer, but the mechanisms are generally poorly understood. In this study, we examined the contribution of epigenetic mechanisms to miRNA dysregulation in colorectal cancer by carrying out high-resolution ChIP-seq. Specifically, we conducted genome-wide profiling of trimethylated histone H3 lysine 4 (H3K4me3), trimethylated histone H3 lysine 27 (H3K27me3), and dimethylated histone H3 lysine 79 (H3K79me2) in colorectal cancer cell lines. Combining miRNA expression profiles with chromatin signatures enabled us to predict the active promoters of 233 miRNAs encoded in 174 putative primary transcription units. By then comparing miRNA expression and histone modification before and after DNA demethylation, we identified 47 miRNAs encoded in 37 primary transcription units as potential targets of epigenetic silencing. The promoters of 22 transcription units were associated with CpG islands (CGI), all of which were hypermethylated in colorectal cancer cells. DNA demethylation led to increased H3K4me3 marking at silenced miRNA genes, whereas no restoration of H3K79me2 was detected in CGI-methylated miRNA genes. DNA demethylation also led to upregulation of H3K4me3 and H3K27me3 in a number of CGI-methylated miRNA genes. Among the miRNAs we found to be dysregulated, many of which are implicated in human cancer, miR-1-1 was methylated frequently in early and advanced colorectal cancer in which it may act as a tumor suppressor. Our findings offer insight into the association between chromatin signatures and miRNA dysregulation in cancer, and they also suggest that miRNA reexpression may contribute to the effects of epigenetic therapy. Cancer Res; 71(17); 5646-58. (C)2011 AACR.

DOI PubMed

Co-expression of laminin beta 3 and gamma 2 chains and epigenetic inactivation of laminin alpha 3 chain in gastric cancer

Masanori II, Hiroyuki Yamamoto, Hiroaki Taniguchi, Yasushi Adachi, Mayumi Nakazawa, Hirokazu Ohashi, Tokuma Tanuma, Yasutaka Sukawa, Hiromu Suzuki, Shigeru Sasaki, Kohzoh Imai, Yasuhisa Shinomura

INTERNATIONAL JOURNAL OF ONCOLOGY ( SPANDIDOS PUBL LTD ) 39 ( 3 ) 593 - 599 2011.09 [Refereed]

Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that regulates cell adhesion and migration. Molecular alterations of LM-332 are involved in cancer progression. The aim of this study was to clarify alterations of LM-332 in gastric carcinoma. The expression of LM-332 subunits in 10 gastric carcinoma cell lines was investigated by RT-PCR, Western blotting, and immunocytochemical/immunofluorescent analyses. The promoter methylation status of LM-332-encoding genes (LAMA3, LAMB3 and LAMC2) was analyzed by methylation-specific PCR (MSP). The relationship between cell migration and LM-332 expression was assessed by the scratch assay. The expression of LM-332 was analyzed immunohistochemically in 90 gastric cancer tissues. Co-expression of laminin beta 3 and gamma 2 chains was often observed in gastric carcinoma cell lines at mRNA and protein levels. In contrast, there was no expression of laminin alpha 3 at either the mRNA or protein levels. Extracellular secretion of laminin beta 3 and gamma 2 chains was found in 2 of the 10 cell lines. The LAMA3 gene was transcriptionally silenced by methylation of the promoter CpG islands in all of the cell lines, while the LAMB3 and LAMC2 genes were silenced in several cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored expression of the LM-332-encoding genes. Methylation frequency of LAMA3 was higher than those of the LAMB2 and LAMC2 genes in gastric cancer tissues. Migration distances were significantly correlated with cytoplasmic laminin gamma 2 chain expression. Immunohistochemistry showed frequent co-expression of laminin beta 3 and gamma 2 chains in gastric carcinoma cells, which was significantly correlated with depth of invasion and advanced tumor stage. The results suggest that the laminin beta 3 and gamma 2 chains accumulate intracellularly and play a role in gastric cancer progression, while epigenetic silencing of the laminin alpha 3 chain may lead to inability to synthesize the basement membrane and may affect cancer cell invasion. Cancer cell motility appears to be associated with the cytoplasmic laminin gamma 2 chain in vitro.

DOI PubMed

p53により発現抑制される分泌性増殖因子HDGFの同定とがんの浸潤・遊走への関与(Negative regulation of hepatoma-derived growth factor by p53)

佐々木泰史, 根岸秀明, 横田育子, 鹿島理沙, 井戸川雅史, 丸山玲緒, 鈴木拓, 豊田実, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 70回 153 - 153 2011.09

多発性骨髄腫におけるDNA繰り返し配列の低メチル化と染色体異常の関連(The relationship between DNA hypomethylation of repetitive elements and chromosomal aberrations in multiple myeloma)

青木由佳, 野島正寛, 安井寛, 丸山玲緒, 鈴木拓, 石田禎夫, 麻奥英毅, 時野隆至, 豊田実, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 70回 137 - 137 2011.09

多発性骨髄腫におけるDNAメチロームと染色体異常メチル化DNA結合蛋白を利用した次世代シーケンシングによる検討(DNA methylome and chromosomal aberrations in multiple myeloma: an integrated analysis based on MBD-sequencing)

野島正寛, 青木由佳, 安井寛, 丸山玲緒, 鈴木拓, 石田禎夫, 時野隆至, 篠村恭久, 豊田実

日本癌学会総会記事 ( (一社)日本癌学会 ) 70回 47 - 47 2011.09

Clinicopathological and Molecular Features of Colorectal Serrated Neoplasias With Different Mucosal Crypt Patterns

Yuichiro Yano, Kazuo Konishi, Toshiko Yamochi, Atsushi Katagiri, Hisako Nozawa, Hiromu Suzuki, Minoru Toyota, Yutaro Kubota, Takashi Muramoto, Yoshiya Kobayashi, Masayuki Tojo, Kenichi Konda, Reiko Makino, Kazuhiro Kaneko, Nozomi Yoshikawa, Hidekazu Ota, Michio Imawari

AMERICAN JOURNAL OF GASTROENTEROLOGY ( NATURE PUBLISHING GROUP ) 106 ( 7 ) 1351 - 1358 2011.07 [Refereed]

OBJECTIVES: Endoscopic examination shows that serrated neoplasias (SNs), such as serrated adenomas and sessile serrated adenomas, exhibit different mucosal crypt patterns. However, it remains unclear whether advanced serrated polyps with different mucosal crypt patterns have different clinicopathological or molecular features. METHODS: We classified the mucosal crypt patterns of 86 SNs into three types (hyperplastic, adenomatous, and mixed pattern) and evaluated their clinicopathological and molecular features. RESULTS: We found significant differences in the proliferative activity status between SNs with mixed/adenomatous patterns and those with the hyperplastic patterns. SNs with the hyperplastic pattern were frequently located in the proximal colon and had a macroscopically superficial appearance, whereas SNs with the adenomatous pattern were often located in the distal colon and had a protruding appearance. Furthermore, a significant difference was observed in the frequency of the CpG island methylator phenotype (CIMP), involving the methylation of two or more CIMP-related genes (MINT1, MINT2, MINT31, p16, and MLH1), between SNs with the hyperplastic pattern and those with the mixed/adenomatous patterns (18/32 (56%) vs. 8/28 (29%) or 7/26 (27%); P = 0.0309 or P = 0.0249, respectively). Moreover, the prevalence of KRAS mutations was significantly higher in SNs with the adenomatous pattern than in those with the hyperplastic pattern (7/26 (27%) vs. 1/32 (3%); P = 0.0173). In comparison with other patterns, the mixed pattern was detected more frequently in mixed serrated polyps (MSPs), which contain separate histological components. Some MSPs exhibited concordant molecular alterations among the different histological components. CONCLUSIONS: The clinicopathological and molecular features of SNs correlated strongly with their mucosal crypt patterns, which were observed using chromoendoscopy.

DOI PubMed

Epigenetic inactivation of calcium-sensing receptor in colorectal carcinogenesis

Keiichi Hizaki, Hiroyuki Yamamoto, Hiroaki Taniguchi, Yasushi Adachi, Mayumi Nakazawa, Tokuma Tanuma, Norihiro Kato, Yasutaka Sukawa, Jose V. Sanchez, Hiromu Suzuki, Shigeru Sasaki, Kohzoh Imai, Yasuhisa Shinomura

MODERN PATHOLOGY ( NATURE PUBLISHING GROUP ) 24 ( 6 ) 876 - 884 2011.06 [Refereed]

Ca(2+) is a chemopreventive agent for colon cancer. Ion transport systems are often altered in human cancer. The aim of this study was to clarify the alterations of calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed the expression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues by RT-PCR and immunostaining. In addition, we analyzed methylation of the CASR promoter by using bisulfite sequence analysis and methylation-specific PCR. CASR mRNA and protein expression was significantly downregulated in most of the cancer cell lines. CpG islands were densely methylated in cancer cell lines with reduced CASR mRNA expression. Treatment with a demethylating agent, 5-aza-2 '-deoxycytidine, and/or a histone deacetylase inhibitor, trichostatin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression in CASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca(2+) on the cytotoxic response to 5-fluorouracil. CASR expression was observed in normal colonic epithelial cells and was retained in most adenoma tissues. CASR mRNA and protein expression was significantly downregulated in cancer tissues. There was an inverse relationship between CASR expression and degree of differentiation. Immunohistochemical CASR staining was reduced more predominantly in less-differentiated cancer tissues and/or in cancer cells at the invasive front, where nuclear/cytoplasmic beta-catenin was often localized. CASR methylation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatic tissues and was significantly correlated with reduced CASR expression. CASR methylation was also detected in 32% of advanced adenoma tissues but was detected in only 9% of adenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylation seems to occur at an early stage and progress in colorectal carcinogenesis. The results suggest that epigenetic inactivation of CASR has an important role in colorectal carcinogenesis. Modern Pathology (2011) 24, 876-884; doi:10.1038/modpathol.2011.10; published online 11 February 2011

DOI PubMed

【腫瘍マーカー-その今日的解釈(理解)と応用】間葉系腫瘍の腫瘍マーカー

新沼猛, 鈴木拓, 篠村恭久

成人病と生活習慣病 ( (株)東京医学社 ) 41 ( 6 ) 735 - 737 2011.06

・消化管間葉系腫瘍の多くを占めるGISTのマーカーとしてはKITの発現が広く用いられている。・一部のKIT陰性のGISTに対してPKCθ、DOG1などの発現を検索することが有用であることがある。・GISTにおいては悪性度や予後と相関するとされるさまざまなマーカーが用いられており、細胞増殖の指標として用いられているMIB-1 labeling indexのほかにも、癌抑制遺伝子であるp16の発現低下やT細胞活性化抗原であるCD26の高発現が高リスク、予後不良と関連すると報告されている。・その他の間葉系腫瘍としては平滑筋腫瘍、神経性腫瘍、血管性腫瘍、線維芽細胞性腫瘍があり、平滑筋腫瘍においてはactinやmyosinが、神経性腫瘍においてはS100やGFAP(glial fibrillary acidic protein)、血管性腫瘍においては内皮系のマーカーであるCD31、CD34、von Willebrand因子などが用いられている。・線維芽細胞性腫瘍のうち腸間膜線維腫症においてはβ-cateninの発現上昇が認められることが多く診断の一助となる。(著者抄録)

Interferon-alpha/beta and Anti-Fibroblast Growth Factor Receptor 1 Monoclonal Antibody Suppress Hepatic Cancer Cells In Vitro and In Vivo

Shigeru Sasaki, Tadao Ishida, Minoru Toyota, Akinobu Ota, Hiromu Suzuki, Akinori Takaoka, Hiroshi Yasui, Hiroyuki Yamamoto, Hideyasu Takagi, Masahiro Maeda, Tsutomu Seito, Masayuki Tsujisaki, Yasuhisa Shinomura, Kohzoh Imai

PLOS ONE ( PUBLIC LIBRARY SCIENCE ) 6 ( 5 ) e19618 2011.05 [Refereed]

Background: Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC. Methodology/Principal Findings: We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-alpha/beta in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-alpha/beta in a murine xenograft model of human HCC. We found that interferon-alpha/beta induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-alpha, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro. Conclusions: Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-alpha/beta is a promising approach to the treatment of HCC.

DOI PubMed

Epigenetic Alteration of DNA in Mucosal Wash Fluid Predicts Invasiveness of Colorectal Tumors

Seiko Kamimae, Eiichiro Yamamoto, Hiro-o Yamano, Masanori Nojima, Hiromu Suzuki, Masami Ashida, Tomo Hatahira, Akiko Sato, Tomoaki Kimura, Kenjiro Yoshikawa, Taku Harada, Seiko Hayashi, Hiroyuki Takamaru, Reo Maruyama, Masahiro Kai, Morie Nishiwaki, Tamotsu Sugai, Yasushi Sasaki, Takashi Tokino, Yasuhisa Shinomura, Kohzoh Imai, Minoru Toyota

CANCER PREVENTION RESEARCH ( AMER ASSOC CANCER RESEARCH ) 4 ( 5 ) 674 - 683 2011.05 [Refereed]

Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors, We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest. that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors. Cancer Prey Res; 4(5); 674-83. (c) 2011 AACR.

DOI PubMed

Molecular analysis of gastrointestinal tumor and translational research

Hiroyuki Yamamoto, Hiromu Suzuki, Yasushi Adachi, Yasuhisa Shinomura

Journal of Japanese Society of Gastroenterology 108 ( 1 ) 1 - 10 2011.01 [Refereed]

PubMed

消化器癌の分子解析とトランスレーショナルリサーチ

山本博幸, 鈴木拓, 足立靖, 篠村恭久

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 108 ( 1 ) 1 - 10 2011.01

消化器癌の分野においても、医療につながる基礎研究成果を臨床に実用化させるトランスレーショナルリサーチ(橋渡し研究)が盛んである。大規模な医薬品開発などをともなわなくとも、消化器癌の分子解析の結果を臨床にトランスレートすることが可能となってきた。本稿では、ゲノムワイドな低メチル化、CpG island methylator phenotype(CIMP)、microRNA(miRNA)、microsatellite instability(MSI)、insulin-like growth factor-I receptor(IGF-IR)に関して最近のデータを中心に紹介し、近未来的な展望についても概説する。(著者抄録)

Gastric Wash-Based Molecular Testing for Antibiotic Resistance in Helicobacter pylori

Satoshi Baba, Yoshichika Oishi, Yoshiyuki Watanabe, Ritsuko Oikawa, Ryo Morita, Yoshihito Yoshida, Tetsuya Hiraishi, Tadateru Maehata, Yoshihiko Nagase, Yasunobu Fukuda, Midori Nakazawa, Shinya Ishigouoka, Nobuhiro Hattori, Hiromu Suzuki, Minoru Toyota, Hirohumi Niwa, Michihiro Suzuki, Fumio Itoh

DIGESTION ( KARGER ) 84 ( 4 ) 299 - 305 2011 [Refereed]

Background: A number of noninvasive tests have been developed to establish the presence of Helicobacter pylori infection. However, thus far these tests have only been capable of detecting its presence. An increasing number of antibiotic-resistant H. pylori infections have been reported and they are known to be correlated with 23S rRNA single nucleotide polymorphisms (SNPs). We hypothesized that genomic analysis of H. pylori recovered from gastric washes could not only be less invasive, but also useful as a screening test and for assessing the outcome of eradication therapy. Methods: Biopsy specimens and gastric washes were collected from 100 patients during endoscopic examination. Then we analyzed 23S rRNA, ureA, and cagA genes using PCR and high-throughput pyrosequencing analysis. Results: Forty-five percent (44/97) of patients tested positive for ureA and 42.3% (41/97) tested positive by a rapid urease test. One hundred percent (35/35) of patients who tested positive by both methods were observed to have the cagA gene. Among these 35 patients, 23S rRNA SNPs were present in 34.3% (12/35). Conclusions: Gastric wash-based PCR and a pyrosequencing assay were used to rapidly detect and estimate the number of 23S rRNA SNPs in clinical isolates of H. pylori. Not only is this a less invasive technique, but it can also diagnose drug resistance. Copyright (C) 2011 S. Karger AG, Basel

DOI PubMed

Role of DNA Methylation in the Development of Diffuse-Type Gastric Cancer

Eiichiro Yamamoto, Hiromu Suzuki, Hiroyuki Takamaru, Hiroyuki Yamamoto, Minoru Toyota, Yasuhisa Shinomura

DIGESTION ( KARGER ) 83 ( 4 ) 241 - 249 2011 [Refereed]

Cancer cells exhibit two opposing methylation abnormalities: genome-wide hypomethylation and gene promoter hypermethylation. Downregulation of E-cadherin (CDH1) plays a key role in the development of diffuse-type gastric cancer, and DNA methylation is a major cause of the gene's silencing. Hereditary diffuse gastric cancer is caused by germline mutation of CDH1 gene, and DNA methylation frequently serves as the second hit completely inactivating the gene. In sporadic diffuse-type gastric cancer, methylation of CDH1 is more prevalent than mutation of the gene. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBV-associated GC) is characterized by concurrent methylation of multiple genes, and diffuse-type gastric cancer is frequently seen among EBV-associated GCs. Patients with pangastritis or enlarged-fold gastritis, which are both caused by Helicobacter pylori infection, reportedly have an increased risk for diffuse-type gastric cancer. Notably, the gastric mucosa of enlarged-fold gastritis patients exhibits CDH1 hypermethylation and genome-wide hypomethylation. These data suggest that aberrant DNA methylation is an essential promoter of carcinogenesis in individuals at high risk for diffuse-type gastric cancer. Copyright (C) 2011 S. Karger AG, Basel

DOI PubMed

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Hiromu Suzuki, Eiichiro Yamamoto, Masanori Nojima, Masahiro Kai, Hiro-o Yamano, Kenjiro Yoshikawa, Tomoaki Kimura, Toyoki Kudo, Eiji Harada, Tamotsu Sugai, Hiroyuki Takamaru, Takeshi Niinuma, Reo Maruyama, Hiroyuki Yamamoto, Takashi Tokino, Kohzoh Imai, Minoru Toyota, Yasuhisa Shinomura

CARCINOGENESIS ( OXFORD UNIV PRESS ) 31 ( 12 ) 2066 - 2073 2010.12 [Refereed]

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

DOI PubMed

Epigenetic alterations in human tumors and their clinical applications

鈴木拓, 山本博幸, 篠村恭久

札幌医学雑誌 ( 札幌医科大学医学部 ) 79 ( 1 ) 1 - 5 2010.12

Cancer is fundamentally a genetic and epigenetic disease that requires the accumulation of genomic alterations that inactivate tumor suppressors and activate proto-oncogenes. Two contradicting epigenetic events coexist in cancer: global hypomethylation, which is mainly observed in repetitive sequences within the genome, and regional hypermethylation, which is frequently associated with CpG islands within gene promoters. Hypermethylation alters the activity in a number of important signaling pathways by silencing expression of genes encoding Wnt antagonists, negative Ras effectors, and p53 targets. The list of genes aberrantly methylated in cancer is growing, and methylation of a p53 target micoRNA gene has been recently demonstrated. Global hypomethylation is strongly implicated in chromosomal instability and malignant potential of tumors. Sites of aberrant DNA methylation could be promising markers and targets for risk assessment, early detection and treatment of cancer.

DOI CiNii

A Novel Correlation between LINE-1 Hypomethylation and the Malignancy of Gastrointestinal Stromal Tumors

Shinichi Igarashi, Hiromu Suzuki, Takeshi Niinuma, Haruo Shimizu, Masanori Nojima, Hiroyuki Iwaki, Takayuki Nobuoka, Toshirou Nishida, Yasuaki Miyazaki, Hiroyuki Takamaru, Eiichiro Yamamoto, Hiroyuki Yamamoto, Takashi Tokino, Tadashi Hasegawa, Koichi Hirata, Kohzoh Imai, Minoru Toyota, Yasuhisa Shinomura

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 16 ( 21 ) 5114 - 5123 2010.11 [Refereed]

Purpose: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. Experimental Design: A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-a, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using Methy-Light. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens. Results: LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-a and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations. Conclusions: Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations. Clin Cancer Res; 16(21); 5114-23. (C)2010 AACR.

DOI PubMed

Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer

Tomoko Fujikane, Noriko Nishikawa, Minoru Toyota, Hiromu Suzuki, Masanori Nojima, Reo Maruyama, Masami Ashida, Mutsumi Ohe-Toyota, Masahiro Kai, Toshihiko Nishidate, Yasushi Sasaki, Tousei Ohmura, Koichi Hirata, Takashi Tokino

BREAST CANCER RESEARCH AND TREATMENT ( SPRINGER ) 122 ( 3 ) 699 - 710 2010.08 [Refereed]

Breast cancer arises through the accumulation of multiple genetic alterations and epigenetic changes such as methylation, which silences gene expression in a variety of cancers. In the present study, we applied genomic screening to identify genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC) in a human breast cancer cell line (MCF7). We identified 288 genes upregulated and 29 genes downregulated more than fivefold after treatment with DAC, and gene ontology analyses revealed the genes to be involved in immune responses, apoptosis, and cell differentiation. In addition, real-time PCR analysis of ten genes silenced in MCF7 cells confirmed that they are upregulated by DAC, while bisulfite-pyrosequencing analysis confirmed that nine of those genes were silenced by methylation. We also found that treating MCF7 cells with DAC restored induction of DFNA5 by p53, as well as by two other p53 family genes, p63 gamma and p73 beta. Introduction of NTN4 into MCF7 cells suppressed cell growth, indicating that NTN4 has tumor suppressive activity. In primary breast cancers, we detected cancer-specific methylation of NTN4, PGP9.5, and DKK3, suggesting that methylation of these genes could be useful markers for diagnosis of breast cancer. Thus, DNA methylation appears to be a common event in breast cancer, and the genes silenced by methylation could be useful targets for both diagnosis and therapy.

DOI PubMed

p53により発現抑制される分泌性増殖因子HDGFの同定(Negative regulation of hepatoma-derived growth factor by p53)

佐々木泰史, 根岸秀明, 横田育子, 鹿島理沙, 井戸川雅史, 鈴木拓, 豊田実, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 69回 303 - 303 2010.08

次世代シークエンサーを用いた遺伝子転写開始点予測とエピゲノム解析への応用(Prediction of transcription start sites using next-generation sequencer and its application to epigenome analysis)

豊田実, 鈴木拓, 野島正寛, 佐々木泰史, 篠村恭久, 時野隆至, 今井浩三

日本癌学会総会記事 ( (一社)日本癌学会 ) 69回 417 - 417 2010.08

肝細胞癌におけるLINE-1低メチル化とCpGアイランド高メチル化の意義(Implications of LINE-1 hypomethylation and CpG island hypermethylation in hepatocellular carcinoma)

谷口博昭, 山本博幸, 阿久津典之, 佐々木茂, 足立靖, 大橋広和, 田沼徳真, 宮本千絵, 宮本伸樹, 鈴木拓, 有村佳昭, 今井浩三, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 69回 246 - 246 2010.08

DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae

Masaki Yamashita, Minoru Toyota, Hiromu Suzuki, Masanori Nojima, Eiichiro Yamamoto, Seiko Kamimae, Yoshiyuki Watanabe, Masahiro Kai, Hirofumi Akashi, Reo Maruyama, Yasushi Sasaki, Hiroo Yamano, Tamotsu Sugai, Yasuhisa Shinomura, Kohzoh Imai, Takashi Tokino, Fumio Itoh

CANCER SCIENCE ( WILEY-BLACKWELL ) 101 ( 7 ) 1708 - 1716 2010.07 [Refereed]

Interferon regulatory factors (IRFs) are transcription factors known to play key roles in innate and adaptive immune responses, cell growth, apoptosis, and development. Their function in tumorigenesis of gastric cancer remains to be determined, however. In the present study, therefore, we examined epigenetic inactivation of IRF1-9 in a panel of gastric cancer cell lines. We found that expression of IRF4, IRF5, and IRF8 was frequently suppressed in gastric cancer cell lines; that methylation of the three genes correlated with their silencing; and that treating the cells with the demethylating agent 5-aza-2'-deoxycytidine (DAC) restored their expression. Expression of IRF5 in cancer cells was enhanced by the combination of DAC treatment and adenoviral vector-mediated expression of p53, p63, or p73. Interferon-gamma-induced expression of IRF8 was also enhanced by DAC. Moreover, treating gastric cancer cells with DAC enhanced the suppressive effects of interferon-alpha, interferon-beta, and interferon-gamma on cell growth. Among a cohort of 455 gastric cancer and noncancerous gastric tissue samples, methylation of IRF4 was frequently observed in both gastric cancer specimens and noncancerous specimens of gastric mucosa from patients with multiple gastric cancers, which suggests IRF4 methylation could be a useful molecular marker for diagnosing recurrence of gastric cancers. Our findings indicate that epigenetic IRF inactivation plays a key role in tumorigenesis of gastric cancer, and that inhibition of DNA methylation may restore the antitumor activity of interferons through up-regulation of IRFs. (Cancer Sci 2010).

DOI PubMed

多発性骨髄腫におけるDNAメチル化の網羅的解析と治療抵抗性予測への応用

安井寛, 石田禎夫, 野島正寛, 青木由佳, 丸山玲緒, 多羅澤功, 池田博, 鈴木拓, 林敏昭, 酒井基, 麻奥英毅, 今井浩三, 篠村恭久, 豊田実

生物物理化学 ( 日本電気泳動学会 ) 54 ( Suppl. ) 18 - 18 2010.07

Array-based genomic resequencing of human leukemia

Y. Yamashita, J. Yuan, I. Suetake, H. Suzuki, Y. Ishikawa, Y. L. Choi, T. Ueno, M. Soda, T. Hamada, H. Haruta, S. Takada, Y. Miyazaki, H. Kiyoi, E. Ito, T. Naoe, M. Tomonaga, M. Toyota, S. Tajima, A. Iwama, H. Mano

Oncogene 29 ( 25 ) 3723 - 3731 2010.06 [Refereed]

To identify oncogenes in leukemias, we performed large-scale resequencing of the leukemia genome using DNA sequence arrays that determine ∼9 Mbp of sequence corresponding to the exons or exon-intron boundaries of 5648 protein-coding genes. Hybridization of genomic DNA from CD34-positive blasts of acute myeloid leukemia (n=19) or myeloproliferative disorder (n=1) with the arrays identified 9148 nonsynonymous nucleotide changes. Subsequent analysis showed that most of these changes were also present in the genomic DNA of the paired controls, with 11 somatic changes identified only in the leukemic blasts. One of these latter changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in a total of 9 out of 286 cases of leukemia. Our experiments also showed a somatic change responsible for an Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which resulted in a loss of DNA methylation activity of > 50%. Our data have thus shown a unique profile of gene mutations in human leukemia. © 2010 Macmillan Publishers Limited All rights reserved.

DOI PubMed

IGFBP7 is a p53-responsive gene specifically silenced in colorectal cancer with CpG island methylator phenotype

Hiromu Suzuki, Shinichi Igarashi, Masanori Nojima, Reo Maruyama, Eiichiro Yamamoto, Masahiro Kai, Hirofumi Akashi, Yoshiyuki Watanabe, Hiroyuki Yamamoto, Yasushi Sasaki, Fumio Itoh, Kohzoh Imai, Tamotsu Sugai, Lanlan Shen, Jean-Pierre J. Issa, Yasuhisa Shinomura, Takashi Tokino, Minoru Toyota

CARCINOGENESIS ( OXFORD UNIV PRESS ) 31 ( 3 ) 342 - 349 2010.03 [Refereed]

A subset of colorectal cancers (CRCs) show simultaneous methylation of multiple genes; these tumors have the CpG island methylator phenotype (CIMP). CRCs with CIMP show a specific pattern of genetic alterations, including a high frequency of BRAF mutations and a low frequency of p53 mutations. We therefore hypothesized that genes inactivated by DNA methylation are involved in the BRAF- and p53-signaling pathways. Among those, we examined the epigenetic inactivation of insulin-like growth factor-binding protein 7 (IGFBP7) expression in CRCs. We found that in CRC cell lines, the silencing of IGFBP7 expression was correlated with high levels of DNA methylation and low levels of histone H3K4 methylation. Luciferase and chromatin immunoprecipitation assays in unmethylated cells revealed that p53 induces expression of IGFBP7 upon binding to a p53 response element within intron 1 of the gene. Treating methylated CRC cell lines with 5-aza-2'-deoxycytidine restored p53-induced IGFBP7 expression. Levels of IGFBP7 methylation were also significantly higher in primary CRC specimens than in normal colonic tissue (P < 0.001). Methylation of IGFBP7 was correlated with BRAF mutations, an absence of p53 mutations and the presence of CIMP. Thus, epigenetic inactivation of IGFBP7 appears to play a key role in tumorigenesis of CRCs with CIMP by enabling escape from p53-induced senescence.

DOI PubMed

Polymorphisms in estrogen related genes may modify the protective effect of isoflavones against prostate cancer risk in Japanese men

Tomoko Sonoda, Hiromu Suzuki, Mitsuru Mori, Taiji Tsukamoto, Akira Yokomizo, Seiji Naito, Kiyohide Fujimoto, Yoshihiko Hirao, Naoto Miyanaga, Hideyuki Akaza

EUROPEAN JOURNAL OF CANCER PREVENTION ( LIPPINCOTT WILLIAMS & WILKINS ) 19 ( 2 ) 131 - 137 2010.03 [Refereed]

Soy isoflavones and estrogen related genes may play a major role in the etiology of prostate cancer. This study examined whether the genetic polymorphisms of estrogen receptors (ESR-alpha and ESR-beta) and cytochrome P450 19A1 (CYP19A1) modified the protective effect of isoflavones against prostate cancer. One hundred and eighty cases and 177 controls were selected from three geographic areas of Japan. The odds ratio for more than or equal to 60 versus less than 60 mg/day of the intake of isoflavones was 0.63 (95% confidence interval = 0.41-0.96). The TTTA long repeat was significantly associated with an increased risk (odds ratio = 1.76, 95% confidence interval = 1.15-5.70). The interaction between the polymorphisms and the intake of isoflavones on prostate cancer risk was analyzed by the multifactor dimensionality reduction method. The combination of the TTTA long repeats and the minor alleles of rs10046 in CYP19A1 and rs2077647 in ESR-alpha was a high risk for prostate cancer despite greater than or equal to 60 mg isoflavones/day. The combination of the TTTA short repeat and those homozygous for the major allele of rs10046 in CYP19A1 was low risk despite less than 60 mg isoflavones/day. In conclusion, the findings of this case-control study suggest that the protective effect of isoflavones may differ between the genotypes of estrogen related genes. European Journal of Cancer Prevention 19: 131-137 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

DOI PubMed

Immune response against tumor antigens expressed on human cancer stem-like cells/tumor-initiating cells

Yoshihiko Hirohashi, Toshihiko Torigoe, Satoko Inoda, Akari Takahashi, Rena Morita, Satoshi Nishizawa, Yasuaki Tamura, Hiromu Suzuki, Minoru Toyota, Noriyuki Sato

IMMUNOTHERAPY ( FUTURE MEDICINE LTD ) 2 ( 2 ) 201 - 211 2010.03 [Refereed]

Cancer stem-like cells (CSCs)/tumor-initiating cells (TICs) are a small population of cancer cells that have the properties of tumor-initiating ability, self-renewal and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence and distant metastasis. Thus, elimination of CSCs/TICs is essential to cure malignant diseases. However, there are several studies reporting that CSCs/TICs are more resistant to standard cancer therapies, including chemotherapy and radiotherapy, than nonCSC/TIC populations. How then, can we eliminate CSCs/TICs? Immunotherapy might be the possible answer. In recent analysis, innate immunity (natural killer cells and gamma delta T cells) and also adaptive immunity (cytotoxic T lymphocyte-based cellular immunity and antibody-based humoral immunity) can recognize CSCs/TICs in vitro efficiently. Furthermore, CSC/TIC-specific monoclonal antibody therapies are also efficient in vivo. In this article, we describe the potency, possibilities and problems of CSC/TIC-targeting immunotherapy.

DOI PubMed

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/beta-Catenin Pathway In Vivo

Michael Buchert, Dimitris Athineos, Helen E. Abud, Zoe D. Burke, Maree C. Faux, Michael S. Samuel, Andrew G. Jarnicki, Catherine E. Winbanks, Ian P. Newton, Valerie S. Meniel, Hiromu Suzuki, Steven A. Stacker, Inke S. Nathke, David Tosh, Joerg Huelsken, Alan R. Clarke, Joan K. Heath, Owen J. Sansom, Matthias Ernst

PLOS GENETICS ( PUBLIC LIBRARY SCIENCE ) 6 ( 1 ) e1000816 2010.01 [Refereed]

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

DOI PubMed

Molecular analysis of single isolated glands in gastric cancers and their surrounding gastric intestinal metaplastic mucosa

Tamotsu Sugai, Wataru Habano, Yu-Fei Jiao, Minoru Toyota, Hiromu Suzuki, Mitsunori Tsukahara, Hitohiko Koizuka, Risaburo Akasaka, Keisuke Koeda, Go Wakabayashi, Kazuyuki Suzuki

ONCOLOGY REPORTS ( SPANDIDOS PUBL LTD ) 23 ( 1 ) 25 - 33 2010.01 [Refereed]

The biological properties and underlying genetics of gastric cancer and gastric intestinal metaplasia evolve with neoplastic progression from the genetics of the original gland cell. PCR assay with crypt isolation was used in tumors from 20 patients to examine microsatellite alterations (allelic imbalance at 17p, 5q, 18q, 3p, 4p, and 9p, and microsatellite instability) in glands from each tumor and from intestinal metaplastic lesions. Tumor specimens were processed as either pooled-gland samples or single-gland samples. Pooled gland sample was composed of 10-20 tumor glands, intestinal metaplastic glands, or nonmetaplastic glands. Single gland sample was 10 tumor glands from tumor and single gland sample was 5 gastric intestinal metaplastic and 5 nonmetaplastic glands from its surrounding metaplastic mucosa. Multiple genetic alterations were found in individual tumor glands, with various subclonal expansions seen within the same tumor. Although microsatellite instability was found in 2 of 20 tumor single-gland samples, none was detected in metaplastic single-gland samples. Most cancers appear to have a heterogeneous composition. On the other hand, microsatellite alterations were also detected within the non-metaplastic as well as intestinal metaplastic single-gland samples. In conclusion, the present data on tumor and corresponding intestinal metaplastic and nonmetaplastic glands suggest that genetic alterations already occur within the surrounding of the noncancerous mucosa.

DOI PubMed

Epigenetic Drivers of Genetic Alterations

Minoru Toyota, Hiromu Suzuki

EPIGENETICS AND CANCER, PT A ( ELSEVIER ACADEMIC PRESS INC ) 70 309 - 323 2010 [Refereed]

DNA methylation plays a key role in the silencing of cancer-related genes, thereby affecting numerous cellular processes, including the cell cycle checkpoint, apoptosis, signal transduction, cell adhesion, and angiogenesis. DNA methylation also affects the expression of genes involved in maintaining the integrity of the genome through DNA repair and detoxification of reactive oxygen species. Here, we discuss how epigenetic changes lead to genetic alterations, including microsatellite instability and nucleotide and chromosomal alterations. Epigenetic inactivation of hMLH1 is a major cause of microsatellite instability in sporadic colorectal cancers, and germline epimutation of hMLH1 and hMSH2 is a cause of hereditary nonpolyposis colorectal cancers, which do not show mutation of mismatch repair genes. Epigenetic inactivation of MGMT is often associated with G:C-to-A:T mutations in K-ras and p53, while epigenetic inactivation of BRCA1, WRN, FANCF, and CHFR impairs the machinery involved in maintaining genomic integrity. Epigenetic alteration of the genes involved in the induction of senescence is often associated with cancers showing mutations in the Ras signaling pathway. In addition to regional hypermethylation, global hypomethylation is also a common feature of tumors. Hypomethylation of short and long interspersed repetitive elements has been reported, and hypomethylation affecting the integrity of the genome has been observed in ICF syndrome and various cancers. Dissection of the epigenetic drivers of genetic instability may be important for the development of novel approaches to the treatment of cancer. (C) 2010, Elsevier Inc.

DOI PubMed

Molecular mechanisms involved in epigenetic alterations in cancer

Minoru Toyota, Hiromu Suzuki, Takahiro Nishizaka, Akiko Sato, Toshiharu Yamashita

Japanese Journal of Cancer and Chemotherapy ( Japanese Journal of Cancer and Chemotherapy Publishers Inc. ) 37 ( 9 ) 1650 - 1653 2010 [Refereed]

Epigenetic alterations such as DNA methylation and histone modification play a role in gene silencing in tumorigenesis. DNA methylation affects the expression of genes involved in cell cycle checkpoint, apoptosis, and DNA repair. Recently, epigenetic alterations are shown to play a role in silencing microRNA. Mutations of DNA methyltransferase and histone modification enzymes such as DNMT3A, UTX and EZH2 have been shown in various types of tumors. Genes involved in epigenetic regulation may be novel targets of cancer therapy in the near future.

PubMed

Integrated analysis of genetic and epigenetic alterations in cancer

Minoru Toyota, Hiromu Suzuki, Eichiro Yamamoto, Hiroo Yamano, Kohzoh Imai, Yasuhisa Shinomura

EPIGENOMICS ( FUTURE MEDICINE LTD ) 1 ( 2 ) 291 - 299 2009.12 [Refereed]

A proposed genetic model describing the transition from normal colonic epithelium to malignant cancer involves mutation of a number of key oncogenes and tumor suppressor genes. However, only subsets of colorectal cancers contain such mutations. Moreover, the heterogeneous pattern of tumor mutations suggests there are multiple alternative pathways leading to colonic tumorigenesis. These alternative pathways involve epigenetic alterations such as the methylation of multiple CpG islands, termed the CpG island methylator phenotype, and cancers with CpG island methylator phenotype show distinct genetic and clinicopathological features. The causes of these epigenetic alterations are still not fully understood, but exogenous pathogens such as Helicobacter pylori and Epstein Barr virus, and the chromosomal translocations seen in leukemia, have all been shown to induce epigenetic alterations of genes.

DOI PubMed

Epigenetic Profiles Distinguish Malignant Pleural Mesothelioma from Lung Adenocarcinoma

Yasuhiro Goto, Keiko Shinjo, Yutaka Kondo, Lanlan Shen, Minoru Toyota, Hiromu Suzuki, Wentao Gao, Byonggu An, Makiko Fujii, Hideki Murakami, Hirotaka Osada, Tetsuo Taniguchi, Noriyasu Usami, Masashi Kondo, Yoshinori Hasegawa, Kaoru Shimokata, Keitaro Matsuo, Toyoaki Hida, Nobukazu Fujimoto, Takumi Kishimoto, Jean-Pierre J. Issa, Yoshitaka Sekido

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 69 ( 23 ) 9073 - 9082 2009.12 [Refereed]

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets. [Cancer Res 2009;69(23):9073-82]

DOI PubMed

Elevated Dnmt3a Activity Promotes Polyposis in Apc(Min) Mice by Relaxing Extracellular Restraints on Wnt Signaling

Michael S. Samuel, Hiromu Suzuki, Michael Buchert, Tracy L. Putoczki, Niall C. Tebbutt, Therese Lundgren-May, Aliki Christou, Melissa Inglese, Minoru Toyota, Joan K. Heath, Robyn L. Ward, Paul M. Waring, Matthias Ernst

GASTROENTEROLOGY ( W B SAUNDERS CO-ELSEVIER INC ) 137 ( 3 ) 902 - 913 2009.09 [Refereed]

BACKGROUND & AIMS: Aberrant DNA methylation is a common early event in neoplasia, but it is unclear how this relates to dysregulation of DNA (cytosine-5) methyltransferases (Dnmts). Here we use knock-in transgenic mice to investigate the consequences of intestinal epithelium-specific overexpression of de novo Dnmt3a. METHODS: A novel gene targeting strategy, based on the intestinal epithelium-specific, uniform expression of the A33 glycoprotein, is employed to restrict Dnmt3a overexpression in homozygous A33(Dnmt3a) mutant mice. RESULTS: A33(Dnmt3a) mice infrequently develop spontaneous intestinal polyps. However, when genetically challenged, tumor multiplicity in A33(Dnmt3a);Apc(Min) compound mice is 3-fold higher than in Apc(Min) mice. Although we observe a requirement for spontaneous loss of heterozygosity of the adenomatous polyposis coli (Apc) gene to trigger tumorigenesis in Apc(Min) mice, lesions in A33(Dnmt3a); Apc(Min) mice frequently retain the wild-type Apc allele. However, epithelia from normal mucosa and polyps of A33(Dnmt3a);Apc(Min) mice show hypermethylation-mediated transcriptional silencing of the Writ antagonists Sfrp5, and to a lesser extent, Sfrp1 and increased nuclear beta-catenin alongside activation of the Wnt-target gene Axin2/Conductin. Conversely, enforced Sfrp5 expression suppresses canonical Writ-signaling more effectively in wildtype than in Apc(Min) cells. CONCLUSIONS: Aberrant activation of the canonical Wnt pathway, either by mono-allelic Apc loss or transcriptional silencing of Sfrp5 is largely insufficient to promote polyposis, but epistatic interactions between these genetic and epigenetic events enables initiation and promotion of disease. This mechanism is likely to play a role in human colorectal cancer, because we also show that elevated DNMT3A expression coincides with repressed SFRP5 and enhanced AXIN2/CONDUCTIN expression in paired patient biopsies.

DOI PubMed

DNAメチル化の網羅的解析によって解明された、多発性骨髄腫におけるRASD1遺伝子不活化とデキサメサゾン耐性の相関(Genomic Screening by DNA Methylation Revealed an Association between RASD 1 Inactivation and Dex Resistance in MM)

野島正寛, 丸山玲緒, 安井寛, 鈴木拓, 佐々木泰史, 麻奥英毅, 酒井基, 石田禎夫, 森満, 今井浩三, 時野隆至, 豊田実, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 68回 224 - 224 2009.08

p53誘導性膜貫通蛋白質hCLCA2は癌細胞移動/接着を制御する(hCLCA2, a p53 inducible transmembrane protein regulates cancer cell migration and adhesion)

佐々木泰史, 安保直樹, 鹿島理沙, 井戸川雅史, 鈴木拓, 豊田実, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 68回 140 - 140 2009.08

潜在的腫瘍抑制因子CHFRはNFκB抑制を介してインターロイキン-8を下方制御する(CHFR, a potential tumor suppressor, downregulates interleukin-8 via inhibition of NF-kappaB)

鹿島理沙, 豊田実, 見田裕章, 鈴木拓, 井戸川雅史, 荻和弘, 佐々木泰史, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 68回 155 - 155 2009.08

p53応答配列のゲノム網羅的in silico解析によるp53誘導性non-coding RNAの同定(Genome wide in silico analysis of p53 response elements identified non-coding RNA regulated by p53)

高丸博之, 丸山玲緒, 豊田実, 鈴木拓, 佐々木泰史, 野島正寛, 明石浩史, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 68回 141 - 141 2009.08

CHFR, a potential tumor suppressor, downregulates interleukin-8 through the inhibition of NF-κB

L. Kashima, M. Toyota, M. Toyota, H. Mita, H. Suzuki, H. Suzuki, M. Idogawa, K. Ogi, Y. Sasaki, T. Tokino

Oncogene 28 2643 - 2653 2009.07

The mitotic checkpoint gene CHFR (checkpoint with forkhead and ring finger domains) is silenced in various human cancers by promoter hypermethylation, suggesting that CHFR is a tumor suppressor. Here, we show that CHFR functions as a negative regulator of the nuclear factor-κB (NF-κB) pathway. Expression of CHFR inhibited NF-κB reporter activity, whereas knockdown of CHFR activated reporter activity. These activities are independent of its RING finger domain. Furthermore, we found that CHFR physically interacts with p65 in cells. Electrophoretic mobility shift assays (EMSAs) and ELISA-based NF-κB-binding assays showed that CHFR negatively regulated transcriptional activity of p65. In addition, our data show that interleukin (IL)-8 is significantly downregulated by CHFR, and that the migration of human endothelial cells is suppressed in culture medium conditioned from CHFR-expressing cancer cells. Using a xenograft model, we show that neovascularization is suppressed by adenovirus-mediated transfer of CHFR. These results indicate that expression of CHFR markedly reduces the expression of IL-8 through the inhibition of NF-κB. As the NF-κB signaling pathway plays a critical role in the development and progression of cancer, our findings show the functional relationship between epigenetic alteration and inflammation/angiogenesis in human cancer cells, thereby showing several potential targets for therapeutic intervention. © 2009 Macmillan Publishers Limited All rights reserved.

DOI PubMed

Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma

Masanori Nojima, Reo Maruyama, Hiroshi Yasui, Hiromu Suzuki, Yumiko Maruyama, Isao Tarasawa, Yasushi Sasaki, Hideki Asaoku, Hajime Sakai, Toshiaki Hayashi, Mitsuru Mori, Kohzoh Imai, Takashi Tokino, Tadao Ishida, Minoru Toyota, Yasuhisa Shinomura

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 15 ( 13 ) 4356 - 4364 2009.07 [Refereed]

Purpose: Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone. Experimental Design: Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples. WST-8 assays were used to assess cell viability after treatment with 5-aza-2'-deoxycytidine and/or dexamethasone. Results: Microarray analysis was done to screen for genes up-regulated by 5-aza-2'-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5' regions, including RASD1, was confirmed. Methylation of RASD1 was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2'-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2'-deoxycytidine plus dexamethasone. Conclusion: Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.

DOI PubMed

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

Hiroaki Mita, Minoru Toyota, Fumio Aoki, Hirofumi Akashi, Reo Maruyama, Yasushi Sasaki, Hiromu Suzuki, Masashi Idogawa, Lisa Kashima, Kazuyoshi Yanagihara, Masahiro Fujita, Masao Hosokawa, Masanobu Kusano, Sorin Vasile Sabau, Haruyuki Tatsumi, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

BMC CANCER ( BIOMED CENTRAL LTD ) 9 198 2009.06 [Refereed]

Background: Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods: DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results: DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8-18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8-50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Conclusion: Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.

DOI PubMed

A Single Recombinant Adenovirus Expressing p53 and p21-targeting Artificial microRNAs Efficiently Induces Apoptosis in Human Cancer Cells

Masashi Idogawa, Yasushi Sasaki, Hiromu Suzuki, Hiroaki Mita, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 15 ( 11 ) 3725 - 3732 2009.06 [Refereed]

Purpose: Gene transfer involving p53 is viewed as a potentially effective cancer therapy, but does not result in a good therapeutic response in all human cancers. The activation of p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest in response to p53 activation is mediated primarily through the induction of the cyclin-dependent kinase inhibitor p21. Because p21 also has an inhibitory effect on p53-mediated apoptosis, the suppression of p53-induced p21 expression would be expected to result in the preferential induction of apoptosis. However, p21 also has tumor-suppressive properties. In this study, we developed an adenovirus vector that expresses p53 and suppresses p21 simultaneously to enhance p53-mediated apoptosis. Experimental Design: We constructed a replication-deficient recombinant adenovirus (Ad-p53/miR-p21) that enabled cocistronic expression of the p53 protein and artificial microRNAs that targeted p21, and examined the therapeutic effectiveness of this vector in vitro and in vivo. Results: The levels of p21 were significantly attenuated following infection with Ad-p53/miR-p21. In colorectal and hepatocellular carcinoma cells, infection with Ad-p53/miR-p21 augmented apoptosis as compared with an adenovirus that expressed p53 alone (Ad-p53/miR-control). Ad-p53/miR-p21 also significantly increased the chemosensitivity of cancer cells to adriamycin (doxorubicin). In a xenograft tumor model in nude mice, tumor volume was significantly decreased following the direct injection of Ad-p53/miR-p21 into the tumor, as compared with the injection of Ad-p53/miR-control. Conclusion: These results suggest that adenovirus-mediated transduction of p53 and p21-specific microRNAs may be useful for gene therapy of human cancers.

DOI PubMed

Cancer epigenomics: implications of DNA methylation in personalized cancer therapy.

Minoru Toyota, Hiromu Suzuki, Toshiharu Yamashita, Koichi Hirata, Kohzoh Imai, Takashi Tokino, Yasuhisa Shinomura

Cancer science 100 ( 5 ) 787 - 91 2009.05 [Refereed] [International journal]

Genetic alterations in cancer can provide information for predicting a tumor's sensitivity to chemotherapeutic drugs. But although such information is certainly useful, the relatively low frequency of mutations seen in many cancers limits the utility of pharmacogenomics in large numbers of cancer patients, necessitating consideration of other approaches. Epigenetic changes such as DNA methylation are a hallmark of human cancers. Methylation of genes involved in DNA repair and maintaining genome integrity (e.g. MGMT, hMLH1, WRN, and FANCF), and cell-cycle checkpoint genes (e.g. CHFR and 14-3-3 sigma, CDK10, and p73), all reportedly influence the sensitivity to chemotherapeutic drugs, suggesting that DNA methylation could serve as a molecular marker for predicting the responsiveness of tumors to chemotherapy. However, the comprehensive study of pharmacoepigenomics awaits the advent of genome-wide analysis of DNA methylation using microarrays and next-generation sequencers.

DOI PubMed

p53により発現抑制される分泌性増殖因子HDGFの同定胃癌組織における発現解析

佐々木泰史, 井戸川雅史, 鈴木拓, 豊田実, 今井浩三, 篠村恭久

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 106 ( 臨増総会 ) A234 - A234 2009.03

DNAメチル化により引き起こされるSda糖鎖発現抑制の炎症発癌における意義

河村由紀, 豊田実, 河村裕, 小西文雄, 斉藤幸夫, 鈴木拓, 今井浩三, 土肥多惠子

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 106 ( 臨増総会 ) A309 - A309 2009.03

Inflammation-related aberrant patterns of DNA methylation: detection and role in epigenetic deregulation of cancer cell transcriptome.

Suzuki H, Toyota M, Kondo Y, Shinomura Y

Methods in molecular biology (Clifton, N.J.) 512 55 - 69 2009 [Refereed]

DOI PubMed

Epigenetic profiles distinguish malignant pleural mesothelioma from lung adenocarcinoma.

Goto Yasuhiro, Shinjo Keiko, Kondo Yutaka, Shen Lanlan, Toyota Minoru, Suzuki Hiromu, Gao Wentao, An Byonggu, Fujii Makiko, Murakami Hideki, Osada Hirotaka, Taniguchi Tetsuo, Usami Noriyasu, Kondo Masashi, Hasegawa Yoshinori, Shimokata Kaoru, Matsuo Keitaro, Hida Toyoaki, Fujimoto Nobukazu, Kishimoto Takumi, Issa Jean-Pierre J, Sekido Yoshitaka

Cancer research 69 ( 23 ) 2009

:Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B,

DOI

Cigarette smoking, N-acetyltransferase 2 polymorphisms and systemic lupus erythematosus in a Japanese population

Kiyohara C, Washio M, Horiuchi T, Tada Y, Asami T, Ide S, Takahashi H, Kobashi G, Kodama H, Washio M, Akashi K, Harada M, Horiuchi T, Kiyohara C, Tsukamoto H, Asami T, Hotokebuchi T, Nagasawa K, Tada Y, Ushiyama O, Mori M, Oura A, Sinomura Y, Suzuki H, Takahashi H, Yamamoto M, Kobashi G, Abe T, Tanaka H, Nogami N, Okamoto K, Sakamoto N, Sasaki S, Miyake Y, Yokoyama T, Inaba Y, Nagai M

Lupus 18 ( 7 ) 630 - 638 2009 [Refereed]

DOI

Cigarette smoking, STAT4 and TNFRSF1B polymorphisms, and systemic lupus erythematosus in a Japanese population

Kiyohara C, Washio M, Horiuchi T, Tada Y, Asami T, Ide S, Atsumi T, Kobashi G, Takahashi H, Kodama H, Akashi K, Harada M, Tsukamoto H, Hotokebuchi T, Nagasawa K, Ushiyama O, Mori M, Oura A, Sinomura Y, Suzuki H, Yamamoto M, Horita T, Koike T, Abe T, Tanaka H, Nogami N, Okamoto K, Sakamoto N, Sasaki S, Miyake Y, Yokoyama T, Hirota Y, Inaba Y, Nagai M

Journal of Rheumatology 36 ( 10 ) 2195 - 2203 2009 [Refereed]

DOI

Prediction of p53 target genes based on integrative analysis of chromatin- immunoprecipitated and sequenced tags,by using Galaxy,a web-based interactive platform for large-scale genome analysis

Mita H, Sasaki Y, Suzuki H, Idogawa M, Kashima L, Anbo N, Akashi H, Tatsumi H, Toyota M, Tokino T

Tumor Research ( Sapporo Medical University ) 43 1 - 23 2008.12 [Refereed]

Chromatin immunoprecipitation (ChIP) followed by sequencing of immunoprecipitated DNA fragments is the high-throughput method for identifying transcription factor binding sites. In one such method, ChIP-PET, paired-end ditags (PETs) derived from both ends of the immunoprecipitated DNA fragments are sequenced and mapped to the genome. We report here the prediction of p53 target genes by metaanalyzing tags of p53 ChIP-PET and by combining with other genomic annotations, using Galaxy, a web-based platform for large-scale genome analysis. We found 327 of p53 binding sites on the genome of 5-fluorouracil (5-FU)- treated HCT116 colon cancer cells by searching the total 65,509 PETs for PET clusters. The search for p53 target gene, which focused on PET clusters with computationally-predicted p53 binding motif, identified 20 of putative p53 target genes as well as 11 of known p53 targets. Another search for p53 target genes, which focused on PET clusters located within 50-kb flanking regions of transcription start sites of genes, identified 278 of Refseq genes, 79 of noncoding RNAs and 5 of microRNAs as p53 targets which included lots of known validated targets. Our results indicate that sequencing-based ChIP analysis combined with the existing genome annotation is effective method to predict p53 binding loci and target genes, and also show that the Galaxy platform is well-suited for multiple-type analyses and visualization of ChIP data, leading to functional annotation of transcription factor binding sites.

DOI CiNii

DNA methylation and cancer pathways in gastrointestinal tumors

Hiromu Suzuki, Takashi Tokino, Yasuhisa Shinomura, Kohzoh Imai, Minoru Toyota

PHARMACOGENOMICS ( FUTURE MEDICINE LTD ) 9 ( 12 ) 1917 - 1928 2008.12 [Refereed]

Cancer is fundamentally a genetic and epigenetic disease that requires the accumulation of genomic alterations that inactivate tumor suppressors and activate proto-oncogenes. In addition to genetic mutation or allelic loss, epigenetic gene silencing associated with DNA methylation is now recognized as an alternative mechanism by which tumor suppressor genes are inactivated. In gastrointestinal cancers, for example, DNA methylation frequently alters the activity in a number of important signaling pathways by silencing expression of genes encoding Wnt antagonists, negative Ras effectors and p53 targets. Indeed, the list of genes aberrantly methylated in cancer is growing, and methylation of a p53 target micoRNA gene has recently been demonstrated. Sites of DNA methylation could be promising markers and targets for risk assessment, early detection and treatment of cancer.

DOI PubMed

LINE-1 hypomethylation is associated with increased CpG island methylation in Helicobacter pylori - Related enlarged-fold gastritis

Eiichiro Yamamoto, Minoru Toyota, Hiromu Suzuki, Yutaka Kondo, Tamana Sanomura, Yoko Murayama, Mutsumi Ohe-Toyota, Reo Maruyama, Masanori Nojima, Masami Ashida, Kyoko Fujii, Yasushi Sasaki, Norio Hayashi, Mitsuru Mori, Kohzoh Imai, Takashi Tokino, Yasuhisa Shinomura

CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION ( AMER ASSOC CANCER RESEARCH ) 17 ( 10 ) 2555 - 2564 2008.10 [Refereed]

Background: The molecular mechanism by which Helicobacter pylori infection leads to gastric cancer is not fully understood. Similarly, patients with enlarged-fold (EF+) gastritis, one cause of which is H. pylori infection, have an increased risk for gastric cancer, although again molecular mechanism is unclear. In the present study, we analyzed the methylation status of long interspersed nucleotide elements (LINE-1) and three cancer-related genes in a panel of gastric mucosae, with or without EF+ gastritis. Methods: We used bisulfite pyrosequencing to assess the levels of LINE-1, CDH1, CDH13, and PGP9.5 methylation in 78 gastric mucosa specimens from 48 patients. Results: Levels of LINE-1 methylation were significantly reduced in mucosae from patients with EF+ gastritis. This hypomethylation of LINE-1 was associated with increased methylation of the 5' CpG islands of the genes, which suggests that, in EF+ gastritis, the methylation of the promoter regions of certain genes is accompanied by global demethylation of repetitive sequences. Conclusions: Our results indicate that genomewide hypomethylation and regional hypermethylation occur in EF+ gastritis and may contribute to the tumorigenesis of diffuse-type gastric cancers.

DOI PubMed

Epigenetic inactivation of secreted Frizzled-related proteins in acute myeloid leukaemia

E. Jost, J. Schmid, S. Wilop, C. Schubert, H. Suzuki, J. G. Herman, R. Osieka, O. Galm

British Journal of Haematology 142 ( 5 ) 745 - 753 2008.09 [Refereed]

The Wnt signalling pathway has a key function in stem cell maintenance and differentiation of haematopoietic progenitors. Secreted Frizzled-related protein genes (SFRPs), functioning as Wnt signalling antagonists, have been found to be downregulated by promoter hypermethylation in many tumours. To analyse epigenetic dysregulation of SFRPs in acute myeloid leukaemia (AML), we examined the promoter methylation status of SFRP1, -2, -4 and -5 in AML cell lines by methylation-specific polymerase chain reaction (MSP). Aberrant CpG island methylation was found for all four SFRP genes. By real-time reverse transcription-PCR, corresponding transcriptional silencing for SFRP1 and -2 was demonstrated and treatment of cell lines with 5-aza-2′-deoxycytidine resulted in re-expression. The methylation status of the SFRP genes was analysed in 100 specimens obtained from AML patients at diagnosis. The frequencies of aberrant methylation among the patient samples were 29% for SFRP1, 19% for SFRP2, 0% for SFRP4 and 9% for SFRP5. For SFRP2, a correlation between promoter hypermethylation and transcriptional downregulation was found in primary AML samples. Among AML cases with a favourable karyotype, hypermethylation of SFRP genes was restricted to patients with core binding factor (CBF) leukaemia, and aberrant methylation of the SFRP2 promoter was an adverse risk factor for survival in CBF leukaemia. © 2008 The Authors.

DOI PubMed

胃癌におけるKRAS遺伝子増幅と細胞内シグナルの活性化(Gene amplification of KRAS and activation of intracellular signaling pathways in gastric cancer)

見田裕章, 豊田実, 青木文夫, 明石浩史, 丸山玲緒, 佐々木泰史, 鈴木拓, 井戸川雅史, 鹿島理沙, サバウ・ソリン, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 163 - 163 2008.09

CHFRはNF-kappaBを抑制する(CHFR, a potential tumor suppressor, inhibits the transcriptional activity of NF-kappaB)

鹿島理沙, 見田裕章, 豊田実, 佐々木泰史, 鈴木拓, 井戸川雅史, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 410 - 410 2008.09

p53による肝癌由来増殖因子(HDGF)の抑制機構(Negative regulation of hepatoma-derived growth factor by p53)

安保直樹, 佐々木泰史, 根岸秀明, 井戸川雅史, 小山良太, 豊田実, 鈴木拓, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 136 - 136 2008.09

大腸癌におけるmicroRNA-34b/cおよびBTG4のCpGアイランドメチル化によるエピジェネティックな不活化(Epigenetic silencing of microRNA-34b/c and BTG4 is associated with CpG island hypermethylation in colorectal cancer)

鈴木拓, 豊田実, 野島正寛, 佐々木泰史, 時野隆至, 今井浩三, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 71 - 71 2008.09

口腔扁平上皮癌におけるSFRP遺伝子のエピジェネティックな不活化(Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma)

曽我部陽平, 鈴木拓, 豊田実, 今井崇, 野島正寛, 佐々木泰史, 時野隆至, 平塚博義

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 174 - 174 2008.09

DNA hypermethylation contributes to incomplete synthesis of carbohydrate determinants in gastrointestinal cancer

Yuki I. Kawamura, Minoru Toyota, Rei Kawashima, Teruki Hagiwara, Hiromu Suzuki, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino, Reiji Kannagi, Taeko Dohi

GASTROENTEROLOGY ( W B SAUNDERS CO-ELSEVIER INC ) 135 ( 1 ) 142 - 151 2008.07 [Refereed]

Background & Aims: it has long been known that malignant transformation is associated with abnormal expression of carbohydrate determinants. The aim of this study was to clarify the cause of cancer-associated abnormal glycosylation in gastrointestinal (GI) cancers. Methods: We compared the expression levels of "glyco-genes," including glycosyltransferases and glycosidases, in normal GI mucosa and in gastric and colorectal cancer cells. To examine the possibility that DNA hypermethylation contributed to the down-regulation of these genes, we treated GI cancer cells with 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferase. Results: The silencing of some of these glyco-genes, but not up-regulation of certain molecules, was observed. The Sd(a) carbohydrate was abundantly expressed in the normal GI mucosa, but its expression was significantly decreased in cancer tissues. When human colon and gastric cancer cells were treated with 5-aza-dC, cell surface expression of Sda and the transcription of B4GALNT2, which catalyzes the synthesis of the Sda, were induced. The promoter region of the human B4GALNT2 gene was heavily hypermethylated in many of the GI cancer cell lines examined as well as in gastric cancer tissues (39 out of 78 cases). In addition, aberrant methylation of the B4GALNT2 gene was strongly correlated with Epstein-Barr virus-associated gastric carcinomas and occurred coincidentally with hypermethylation of the ST3GAL6 gene. Conclusions: Epigenetic changes in a group of glycosyltransferases including B4GALNT2 and ST3GAL6 represent a malignant phenotype of gastric cancer caused by silencing of the activity of these enzymes, which action may eventually induce aberrant glycosylation and expression of cancer-associated carbohydrate antigens.

DOI PubMed

Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer

Reo Maruyama, Kimishige Akino, Minoru Toyota, Hiromu Suzuki, Takashi Imai, Mutsumi Ohe-Toyota, Eiichiro Yamamoto, Masanori Nojima, Tomoko Fujikane, Yasushi Sasaki, Toshiharu Yamashita, Yoshiyuki Watanabe, Hiroyoshi Hiratsuka, Koichi Hirata, Fumio Itoh, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CARCINOGENESIS ( OXFORD UNIV PRESS ) 29 ( 7 ) 1312 - 1318 2008.07 [Refereed]

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2'-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein-Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

DOI PubMed

Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma

Yohei Sogabe, Hiromu Suzuki, Minoru Toyota, Kazuhiro Ogi, Takashi Imai, Masanori Nojima, Yasushi Sasaki, Hiroyoshi Hiratsuka, Takashi Tokino

INTERNATIONAL JOURNAL OF ONCOLOGY ( PROFESSOR D A SPANDIDOS ) 32 ( 6 ) 1253 - 1261 2008.06 [Refereed]

Although mutations of APC, CTNNB1 (beta-catenin) and AXIN1 are rare in oral squamous cell carcinoma (OSCC), activation of the Wnt signaling pathway is thought to play an important role in oral carcinogenesis. In the present study, we examined the relationship between Wnt signaling and epigenetic alteration of the secreted frizzled-related protein (SFRP) genes in OSCC. We frequently detected loss of membrane localization of beta-catenin and its cytoplasmic or nuclear accumulation in OSCC cell lines, although these cell lines showed no APC or CTNNB1 (beta-catenin) mutations and no methylation of CDH1 (E-cadherin). By contrast, we frequently detected methylation of SFRP1 (7/17, 41%) SFRP2 (16/17, 94%) and SFRP5 (14/17, 82%) in a panel of OSCC cell lines, as well as in specimens of primary tumors collected from 44 OSCC patients (SFRP1, 10/42, 24%; SFRP2, 16/44, 36%; SFRP5, 7/43, 16%). We also observed that OSCC cell lines express various Wnt ligands, and that ectopic expression of SFRPs inhibited cancer cell proliferation. Our results confirm the frequent methylation and silencing of SFRP genes in OSCC, and suggest that their loss of function contributes to activation of Wnt signaling that leads to cell proliferation during oral carcinogenesis.

DOI PubMed

Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer

Minoru Toyota, Hiromu Suzuki, Yasushi Sasaki, Reo Maruyama, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 68 ( 11 ) 4123 - 4132 2008.06 [Refereed]

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2'-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC.

DOI PubMed

Epigenetic inactivation of RASSF2 in oral squamous cell carcinoma

Takashi Imai, Minoru Toyota, Hiromu Suzuki, Kimishige Akino, Kazuhiro Ogi, Yohei Sogabe, Lisa Kashima, Reo Maruyama, Masanori Nojima, Hiroaki Mita, Yasushi Sasaki, Fumio Itoh, Kohzoh Imai, Yasuhisa Shinomura, Hiroyoshi Hiratsuka, Takashi Tokino

CANCER SCIENCE ( BLACKWELL PUBLISHING ) 99 ( 5 ) 958 - 966 2008.05 [Refereed]

Genetic and epigenetic alterations in tumor-suppressor genes play important roles in human neoplasia. Ras signaling is often activated in oral squamous cell carcinoma (OSCC), although Ras mutations are rarely detected in Japanese OSCC patients, and the mechanisms underlying the gene's activation remain unclear. Here, we examined the expression of Ras association family (RASSF) genes in a panel of OSCC cell lines and found that RASSF2 is often downregulated by DNA methylation in OSCC cells. In addition, aberrant methylation of RASSF2 was detected in 12 of 46 (26%) primary OSCC, and 18 (39%) of those OSCC showed methylation of at least one RASSF gene. Ectopic expression of RASSF2 in OSCC cells suppressed cell growth and induced apoptosis. A RASSF2 deletion mutant lacking the Ras-association domain, which was therefore unable to interact with Ras, exhibited less pro-apoptotic activity than the full-length protein, indicating that the pro-apoptotic activity of RASSF2 is related to its association with Ras. Genomic screening of genes regulated by RASSF2 showed that genes involved in immune responses, angiogenesis, and metastasis are suppressed by RASSF2. Our results suggest that epigenetic inactivation of RASSF2 plays an important role in OSCC tumorigenesis, and that RASSF2 may be a useful molecular target for the diagnosis and treatment of OSCC.

DOI PubMed

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models

Yasushi Sasaki, Hideaki Negishi, Masashi Idogawa, Hiromu Suzuki, Hiroaki Mita, Minoru Toyota, Yasuhisa Shinornura, Kohzoh Imai, Takashi Tokino

MOLECULAR CANCER THERAPEUTICS ( AMER ASSOC CANCER RESEARCH ) 7 ( 4 ) 779 - 787 2008.04 [Refereed]

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.

DOI PubMed

A requirement for DICER to maintain full promoter CpG island hypermethylation in human cancer cells

Angela H. Ting, Hiromu Suzuki, Leslie Cope, Kornel E. Schuebel, Byron H. Lee, Minoru Toyota, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino, Stephen B. Baylin

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 68 ( 8 ) 2570 - 2575 2008.04 [Refereed]

Promoter hypermethylation is a prevalent phenomenon, found in virtually all cancer types studied thus far, and accounts for tumor suppressor gene silencing in the absence of genetic mutations. The mechanism behind the establishment and maintenance of such aberrant hypermethylation has been under intense study. Here, we have uncovered a link between aberrant gene silencing associated with promoter CpG island DNA methylation and the siRNA/miRNA processing enzyme, DICER, in human cancer cells. By comparing demethylated HCT116 colon cancer cells with HCT116 cells genetically rendered hypomorphic for DICER, we identified a group of epigenetically silenced genes that became reactivated in the absence of functional DICER. This reactivation is associated with a dramatic loss of localized promoter DNA hypermethylation. Thus, intact DICER is required to maintain full promoter DNA hypermethylation of select epigenetically silenced loci in human cancer cells.

DOI PubMed

Histone Deacetylase Inhibitor FK228 Enhances Adenovirus-mediated p53 Family Gene Therapy in Cancer Models

Sasaki Yasushi, Idogawa Masashi, Suzuki Hiromu, Mita Hiroaki, Toyota Minoru, Shinomura Yasuhisa, Imai Kohzoh, Tokino Takashi

Tumor Biology 29 ( 4 ) 24 - 787 2008.04 [Refereed]

DOI PubMed

今井崇, 秋野公臣, 豊田実, 鈴木拓, 佐々木泰史, 見田裕章, 井戸川雅史, 鹿島理沙, 荻和弘, 曽我部陽平, 今井浩三, 平塚博義, 時野隆至

日本口腔科学会雑誌 ( (NPO)日本口腔科学会 ) 57 ( 1 ) 160 - 160 2008.01

Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma

Hideyasu Takagi, Shigeru Sasaki, Hiromu Suzuki, Minoru Toyota, Reo Maruyama, Masanori Nojima, Hiroyuki Yamamoto, Masao Omata, Takashi Tokino, Kohzoh Imai, Yasuhisa Shinomura

JOURNAL OF GASTROENTEROLOGY ( SPRINGER TOKYO ) 43 ( 5 ) 378 - 389 2008 [Refereed]

Background. Activation of the Wnt signaling pathway is frequently observed in hepatocellular carcinoma (HCC), though mutation of three of its components, CTNNB1, AXIN1, and AXIN2, is observed substantially less often. Methods. We examined the relationship between Wnt signaling and epigenetic alteration of secreted frizzled-related protein (SFRP) genes in HCC. Results. We frequently detected the active form of beta-catenin and accumulation of nuclear beta-catenin in liver cancer cell lines. We detected methylation of SFRP family genes in liver cancer cell lines (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) and primary HCCs (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42 %), though methylation of SFRP4 was not found in primary HCCs. SFRP methylation also was detected in hepatitis B or C virus-associated chronic hepatitis (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) and liver cirrhosis (SFRP1, 10/28,36%; SFRP2,9128,32%; SFRP5, 3/28, 11%), suggesting that methylation of these genes is an early event in liver carcinogenesis. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity in liver cancer cells, while overexpression of a P-catenin mutant and depletion of SFRP1 using siRNA synergistically upregulated TCF/LEF transcriptional activity. Conclusions. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in HCC, and suggest that their loss of function contributes to activation of Wnt signaling during hepatocarcinogenesis.

DOI PubMed

【消化器疾患とエピジェネティクス】大腸癌でのDNAメチル化異常

野島正寛, 豊田実, 鈴木拓, 森満, 篠村恭久

分子消化器病 5 364 - 369 2008 [Refereed]

Genomic Mapping of 12p Amplifications in Gastric Cancer Reveal a 0.5-Mb Target Region Including KRAS Locus

Mita Hiroaki, Aoki Fumio, Akashi Hirofumi, Maruyama Reo, Sasaki Yasushi, Suzuki Hiromu, Idogawa Masashi, Kashima Lisa, Toyota Minoru, Imai Kohzoh, Shinomura Yasuhisa, Tokino Takashi

Tumor Biology 29 83 2008 [Refereed]

Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer

Suzuki H, Toyota M, Carraway H, Gabrielson E, Ohmura T, Fujikane T, Nishikawa N, Sogabe Y, Nojima M, Sonoda T, Mori M, Hirata K, Imai K, Shinomura Y, Baylin SB, Tokino T

Br J Cancer 98 1147 - 1156 2008 [Refereed]

DOI

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors

Suzuki Hiromu, Toyota Minoru, Sato Hironobu, Nojima Masanori, Imai Kohzoh, Shinomura Yasuhisa

Japan Journal of Molecular Tumor Marker Research ( Japanese Society for Molecular Tumor Marker Research ) 23 37 - 38 2008

DOI CiNii

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors

Hironobu Sato, Hiromu Suzuki, Minoru Toyota, Masanori Nojima, Reo Maruyama, Shigeru Sasaki, Hideyasu Takagi, Yohei Sogabe, Yasushi Sasaki, Masashi Idogawa, Tomoko Sonoda, Mitsuru Mori, Kohzoh Imai, Takashi Tokino, Yasuhisa Shinomura

CARCINOGENESIS ( OXFORD UNIV PRESS ) 28 ( 12 ) 2459 - 2466 2007.12 [Refereed]

Activation of Wnt signaling has been implicated in tumorigenesis, and epigenetic silencing of Wnt antagonist genes has been detected in various cancers. In the present study, we examined the expression and methylation of DICKKOPF (DKK) family genes in gastrointestinal cancer cell lines. We found that all known DKK genes were frequently silenced in colorectal cancer (CRC) cells (DKK1, 3/9, 33%; DKK2, 8/9, 89%; DKK3, 5/9, 56% and DKK4, 5/9, 56%), but not in normal colon mucosa. DKK1, -2 and -3 have 5' CpG islands, and show an inverse relation between expression and methylation. DKK methylation also was frequently observed in gastric cancer (GC) cell lines (DKK1, 6/16, 38%; DKK2, 15/16, 94% and DKK3, 10/16, 63%), but was seen less frequently in hepatocellular carcinoma and pancreatic cancer cell lines. DKKs also were frequently methylated in primary CRCs (DKK1, 7/58, 12%; DKK2, 45/58, 78% and DKK3, 12/58, 21%) and GCs (DKK1, 15/31, 48%; DKK2, 26/31, 84% and DKK3, 12/31, 39%). Against a background of CTNNB1 or APC mutations, Dickkopfs (Dkks) were less effective inhibitors of Wnt signaling than secreted frizzled-related proteins, though over-expression of Dkks suppressed colony formation of CRC cells with such mutations. Our results demonstrate that DKKs are frequent targets of epigenetic silencing in gastrointestinal tumors, and that loss of DKKs may facilitate tumorigenesis through beta-catenin/T-cell factor-independent mechanisms.

DOI PubMed

Gene amplification and overexpression of PRDM14 in breast cancers

Noriko Nishikawa, Minoru Toyota, Hiromu Suzuki, Toshio Honma, Tomoko Fujikane, Tousei Ohmura, Toshihiko Nishidate, Mutsmui Ohe-Toyota, Reo Maruyama, Tomoko Sonoda, Yasushi Sasaki, Takeshi Urano, Kohzoh Imai, Koichi Hirata, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 67 ( 20 ) 9649 - 9657 2007.10 [Refereed]

Several genes that encode PR (PRDI-BF1 and RIZ) domain proteins (PRDM) have been linked to human cancers. To explore the role of the PR domain family genes in breast carcinogenesis, we examined the expression profiles of 16 members of the PRDM gene family in a panel of breast cancer cell lines and primary breast cancer specimens using semiquantitative real-time PCR. We found that PRDM14 mRNA is overexpressed in about two thirds of breast cancers; moreover, immunohistochemical analysis showed that expression of PRDM14 protein is also up-regulated. Analysis of the gene copy number revealed that PRDM14 is a target of gene amplification on chromosome 8q13, which is a region where gene amplification has frequently been detected in various human tumors. Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 by siRNA induced apoptosis in breast cancer cells and increased their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 may play an important role in the proliferation of breast cancer cells. That little or no expression of PRDM14 is seen in noncancerous tissues suggests that PRDM14 could be an ideal therapeutic target for the treatment of breast cancer.

DOI PubMed

Epigenetic silencing of CDX2 is a feature of squamous esophageal cancer

MingZhou Guo, Michael G. House, Hiromu Suzuki, Ying Ye, Malcolm V. Brock, Fengmin Lu, Zhihua Liu, Anil K. Rustgi, James G. Herman

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 121 ( 6 ) 1219 - 1226 2007.09 [Refereed]

CDX2, a mammalian homologue of the homeobox gene 'caudal,' is expressed in gut epithelia and plays an important role in establishing the intestinal phenotype during development. Mice heterozygously disrupted for CDX2 develop disorganized polypoid hamartomas with glandular epithelium and stratified squamous metaplasia resembling foregut mucosa. Since no genetic disruptions of CDX2 have been reported to explain loss of gene function, we examined whether epigenetic mechanisms altered CDX2 expression. Eleven of 17 squamous esophageal cancer cell lines lacked expression of CDX2 that was restored following treatment with 5-aza-2'-deoxycytidine, while all colorectal cancer cell lines expressed CDX2. Loss of expression was associated with DNA methylation in the 5' region of CDX2 determined by methylation specific PCR and bisulfite sequencing. Methylation of CDX2 was rare in primary colorectal (I of 44 tumors, 2%) and esophageal adenocarcinoma neoplasms (2 of 43 tumors, 5%), but was common in esophageal squamous carcinoma (24 of 45 tumors, 49%). No CDX2 methylation was found in normal tissues. Using semi-quantitative RT-PCR, expression of CDX2 was found in low level in normal esophagus, at higher levels in primary adenocarcinoma of the esophagus, but not in primary squamous cancers of the esophagus. Restoration of CDX2 in silenced cell lines resulted in expression of the CDX2 target gene MUC2, a gene important in glandular differentiation. Our results suggest that the inactivation of l in esophageal cancer associated with DNA methylation may be an important determinant of the squamous or non-adenomatous phenotype. (c) 2007 Wiley-Liss, Inc.

DOI PubMed

Comparing the DNA hypermethylome with gene mutations in human colorectal cancer

Kornel E. Schuebel, Wei Chen, Leslie Cope, Sabine C. Glöckner, Hiromu Suzuki, Joo-Mi Yi, Timothy A. Chan, Leander Van Neste, Wim Van Criekinge, Sandra Van Den Bosch, Manon Van Engeland, Angela H. Ting, Kamwing Jair, Wayne Yu, Minoru Toyota, Kohzoh Imai, Nita Ahuja, James G. Herman, Stephen B. Baylin

PLoS Genetics 3 ( 9 ) 1709 - 1723 2007.09 [Refereed]

We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumorspecific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken. © 2007 Schuebel et al.

DOI PubMed

Comparing the DNA hypermethylome with gene mutations in human colorectal cancer

Kornel E. Schuebel, Wei Chen, Leslie Cope, Sabine C. Gloeckner, Hiromu Suzuki, Joo-Mi Yi, Timothy A. Chan, Leander Van Neste, Wim Van Criekinge, Sandra van den Bosch, Manon van Engeland, Angela H. Ting, Kamwing Jair, Wayne Yu, Minoru Toyota, Kohzoh Imai, Nita Ahuja, James G. Herman, Stephen B. Baylin

PLOS GENETICS ( PUBLIC LIBRARY SCIENCE ) 3 ( 9 ) 1709 - 1723 2007.09 [Refereed]

We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

DOI

PRDM5 identified as a target of epigenetic silencing in colorectal and gastric cancer

Yoshiyuki Watanabe, Minoru Toyota, Yutaka Kondo, Hiromu Suzuki, Takashi Imai, Mutsumi Ohe-Toyota, Reo Maruyama, Masanori Nojima, Yasushi Sasaki, Yoshitaka Sekido, Hiroyoshi Hiratsuka, Yasuhisa Shinomura, Kohzoh Imai, Fumio Itoh, Takashi Tokino

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 13 ( 16 ) 4786 - 4794 2007.08 [Refereed]

Purpose: PR (PRDI-BF1 and RIZ) domain proteins (PRDM) are a subfamily of the kruppel-like zinc finger gene products that play key roles during cell differentiation and malignant transformation. The aim of the present study was to begin to examine the involvement of epigenetic alteration of PRDM expression in gastric and colorectal cancer. Experimental Design: We used real-time PCR to assess expression of PRDM1-17. In addition, we used bisulfite PCR to assess DNA methylation and chromatin immunoprecipitation to assess histone modification in colorectal and gastric cancer cell lines lacking PRDM5 expression. Results: Among the 17 PRDM family genes tested, we found that PRDM5 is the most frequently silenced in colorectal and gastric cancer cell lines. Silencing of PRDM5 was mediated by either DNA methylation or trimethylation of Lys(27) of histone H3. Introduction of PRDM5 into cancer cells suppressed cell growth, suggesting that it acts as a tumor suppressor in gastrointestinal cancers. Methylation of PRDM5 was detected in 6.6% (4 of 61) of primary colorectal and 50.0% (39 of 78) of primary gastric cancers but not in noncancerous tissue samples collected from areas adjacent to the tumors. Conclusions: Our data suggest that epigenetic alteration of PRDM5 (e.g., methylation of its 5'-CpG island or trimethylation of Lys(27) of histone H3) likely plays a key role in the progression of gastrointestinal cancers and may be a useful molecular marker.

DOI PubMed

口腔癌におけるRAS関連遺伝子の異常について(Alterations of RAS signaling pathway in oral squamous cell carcinoma)

今井崇, 秋野公臣, 豊田実, 鈴木拓, 佐々木泰史, 見田裕章, 井戸川雅史, 鹿島理沙, 曽我部陽平, 渡邊嘉行, 今井浩三, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 314 - 314 2007.08

デジタルゲノムスキャンにより同定された胃癌におけるK-rasゲノム増幅(Genomic amplification of K-ras in gastric cancer identified by digital genome scanning)

見田裕章, 豊田実, 青木文夫, 明石浩史, 丸山玲緒, 佐々木泰史, 鈴木拓, 井戸川雅史, 鹿島理沙, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 231 - 231 2007.08

アデノウイルス介在性p53ファミリー遺伝子療法効果のヒストンデアセチラーゼ阻害剤FK228による増大(The Histone Deacetylase Inhibitor FK228 Increases the Efficacy of Adenovirus-mediated p53 Family Gene Therapy)

佐々木泰史, 根岸秀明, 井戸川雅史, 鈴木拓, 見田裕章, 豊田実, 丸山玲緒, 明石浩史, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 129 - 129 2007.08

CHFR有糸分裂チェックポイントタンパク質はNFκBの転写活性を抑制する(CHFR mitotic checkpoint protein inhibits the transcriptional activity of NF-κB)

鹿島理沙, 見田裕章, 豊田実, 佐々木泰史, 鈴木拓, 井戸川雅史, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 482 - 482 2007.08

多発性骨髄腫においてDNAメチル化により不活化される遺伝子の網羅的解析(Genomic Screening for Genes Silenced by DNA Methylation in Multiple Myeloma)

丸山玲緒, 豊田実, 鈴木拓, 安井寛, 林敏昭, 酒井基, 石田禎夫, 今井浩三, 時野隆至, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 458 - 458 2007.08

Identification of DFNA5 as a target of epigenetic inactivation in gastric cancer

Kimishige Akino, Minoru Toyota, Hiromu Suzuki, Takashi Imai, Reo Maruyama, Masanobu Kusano, Noriko Nishikawa, Yoshiyuki Watanabe, Yasushi Sasaki, Tamaki Abe, Eiichiro Yamamoto, Isao Tarasawa, Tomoko Sonoda, Mitsuru Mori, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CANCER SCIENCE ( BLACKWELL PUBLISHING ) 98 ( 1 ) 88 - 95 2007.01 [Refereed]

Epigenetic gene inactivation plays a key role in the development of various types of cancer. Using methylated CpG island amplification coupled with representational difference analysis to identify genes inactivated by DNA methylation in gastric cancer, we identified seven DNA fragments corresponding to the 5' CpG islands of the affected genes. One of the clones recovered was identical to the 5' flanking region of DFNA5, a gene previously shown to be associated with deafness and induced by DNA damage. Further analysis revealed that DFNA5 is expressed in normal tissues but is downregulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Treating gastric cancer cells that lacked DFNA5 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Methylation of DFNA5 was detected in 50% of primary gastric tumors, and was correlated with positivity for Epstein-Barr virus and the absence of metastasis. Moreover, introduction of exogenous DFNA5 into silenced cells suppressed colony formation. Taken together, these data suggest that the silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the disease.

DOI PubMed

Epigenetics in gastroenterological cancer and prospects for translational research

Hiromu Suzuki, Minoru Toyota, Yasuhisa Shinomura, Kohzoh Imai

Japanese Journal of Gastroenterology 104 ( 9 ) 1319 - 1328 2007 [Refereed]

DOI PubMed

Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer

Nojima M, Suzuki H, Toyota M, Watanabe Y, Maruyama R, Sasaki S, Sasaki Y, Mita H, Nishikawa N, Yamaguchi K, Hirata K, Itoh F, Tokino T, Mori M, Imai K, Shinomura Y

Oncogene 26 4699 - 4713 2007 [Refereed]

DOI

Genomic screening for genes silenced by DNA methylation in multiple myeloma

Yasui, Hiroshi, Toyota, Minoru, Maruyama, Reo, Tarasawa, Isao, Suzuki, Hiromu, Hayashi, Toshiaki, Sakai, Hajime, Ishida, Tadao, Asaoku, Hideki, Tokino, Takashi, Imai, Kohzoh, Shinomura, Yasuhisa

TUMOR BIOLOGY ( KARGER ) 28 78 - 78 2007 [Refereed]

Proteins interacting with CHFR, mitotic-checkpoint ubiquitin ligase

Mita H, Toyota M, Sasaki Y, Suzuki H, Idogawa M, Kashima L, Tokino T

Tumor Research ( Sapporo Medical University ) 41 23 - 41 2006.12 [Refereed]

Cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome. CHFR which contains a RING domain and has ubiquitin ligase activity, a novel mitotic checkpoint gene, delays chromosome condensation in cells treated with microtubule poisons. CHFR is inactivated by promoter methylation and point mutations in various human tumors, and cancer cells lacking CHFR are sensitive to microtubule inhibitors. However, few reports are available on the molecular mechanism that accounts for the link between the sensitivity of cancer cells to microtubule inhibitors and the physiological function of CHFR. In the present study, we isolated cellular proteins capable of interacting with CHFR using yeast two-hybrid method to clarify the function of CHFR. As a result of the screening, we isolated canonical and noncanonical E2 ubiquitin conjugating enzymes as CHFR interacting proteins, which are involved in proteolytic and non-proteolytic ubiquitination respectively. This raises the possibility that CHFR is switching canonical and non-canonical ubiquitination depending on the situation of cells. On the other hand, we isolated gadd34 which interacted with the FHA domain of CHFR by two-hybrid screen. Coexpression in mammalian cells showed that gadd34 interacted with the FHA domain of CHFR, but gadd34 is not the substrate for CHFR, rather it promoted autoubiquitination of CHFR. Furthermore, CHFR moved, in part, from nucleus to cytoplasm in the presence of microtubule inhibitor docetaxel, which enabled colocalization of CHFR and gadd34 in cytoplasm. This colocalization was followed by cell death. These findings suggest that gadd34 and CHFR cooperate to mediate cell death in response to mitotic stress.

DOI CiNii

インシリコ解析による新規p53標的遺伝子群の網羅的な同定

丸山玲緒, 豊田実, 明石浩史, 佐々木泰史, 青木文夫, 鈴木拓, 見田裕章, 井戸川雅史, 今井浩三, 篠村泰久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 79 - 79 2006.09

短いDNA断片の塩基配列情報を用いた定量的ゲノム解析法の開発と癌研究への応用

見田裕章, 豊田実, 丸山玲緒, 明石浩史, 青木文夫, 佐々木泰史, 鹿島理沙, 鈴木拓, 井戸川雅史, 苗代康可, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 182 - 182 2006.09

CHFR(checkpoint with FHA and RING domain)によるNF-kBシグナリング制御

鹿島理沙, 見田裕章, 豊田実, 佐々木泰史, 井戸川雅史, 鈴木拓, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 162 - 162 2006.09

口腔扁平上皮癌におけるRAS関連遺伝子のジェネティックおよびエピジェネティックな異常の解析

今井崇, 秋野公臣, 豊田実, 鈴木拓, 佐々木泰史, 見田裕章, 井戸川雅史, 荻和弘, 鹿島理沙, 曽我部陽平, 今井浩三, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 199 - 199 2006.09

大腸癌におけるエピジェネティックな変化を指標とした診断への比較検討

渡邊嘉行, 豊田実, 鈴木拓, 岡本賢, 時野隆至, 伊東文生

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 52 - 52 2006.09

Activation of the ribosomal protein L13 gene in human gastrointestinal cancer

T Kobayashi, Y Sasaki, Y Oshima, H Yamamoto, H Mita, H Suzuki, M Toyota, T Tokino, F Itoh, K Imai, Y Shinomura

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE ( PROFESSOR D A SPANDIDOS ) 18 ( 1 ) 161 - 170 2006.07 [Refereed]

Although ribosomal proteins are major components of ribosomes, recent data have shown them to have extraribosomal functions apart from ribosome and protein biosynthesis. In our earlier study, we showed that ribosomal protein L13 mRNA was up-regulated in response to DNA damage in hamster cells. We report here that L13 expression is up-regulated in human gastrointestinal cancers. We also examined the biological role of L13 on human cancer cells. Knocking down L13 expression using small interfering RNA (siRNA) resulted in drastic attenuation of cancer cell growth with significant G1 and G2/M arrest of the cell cycle. Moreover, L13 siRNA significantly enhanced the cellular sensitivity to certain DNA damaging agents and, concordantly, L13-overexpressing cells demonstrated greater chemoresistance compared to parent cells, suggesting an inverse correlation between L13 expression and chemosensitivity. By using semi-quantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of L 13 mRNA expression was observed in 10 (28%) of 36 gastric, 19 (41%) of 46 colorectal and 5 (20%) of 25 liver cancer tissue samples compared to adjacent normal tissue samples. We also found that increased expression of the L13 gene correlated with clinical staging in gastric cancers. The results of this study suggest that L13 plays an essential role in the progression of some gastrointestinal malignancies.

PubMed

DNA断片の塩基配列情報に基づいたゲノムコピー数解析法の開発

見田裕章, 豊田実, 丸山玲緒, 青木文夫, 明石浩史, 鹿島理沙, 佐々木泰史, 鈴木拓, 井戸川雅史, 藤井恭子, 篠村恭久, 今井浩三, 時野隆至

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 43回 63 - 63 2006.07

がん抑制遺伝子p53により誘導される新規分子の網羅的同定

丸山玲緒, 豊田実, 青木文夫, 佐々木泰史, 明石浩史, 鈴木拓, 見田裕章, 時野隆至, 今井浩三, 篠村恭久

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 43回 64 - 64 2006.07

Mammary gland tissue targeted overexpression of human protease-activated receptor 1 reveals a novel link to beta-catenin stabilization

YJ Yin, Katz, V, Z Salah, M Maoz, Cohen, I, B Uziely, H Turm, S Grisaru-Granovsky, H Suzuki, R Bar-Shavit

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 66 ( 10 ) 5224 - 5233 2006.05 [Refereed]

Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking beta-catenin stabilization. Nuclear localization of beta-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PARI induces beta-catenin nuclear localization also in established epithelial tumor cell lines of intact beta-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PARI-mediated beta-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced beta-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced beta-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface (forming a neutralizing complex of "Receptors-SFRP-Wnt"). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PARI-induced Wnt-4. The striking stabilization of beta-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar-1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated beta-catenin stabilization. This link between PAR1 and beta-catenin may bear substantial implications both in developmental and tumor progression processes.

DOI PubMed

Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation

R Maruyama, F Aoki, M Toyota, Y Sasaki, H Akashi, H Mita, H Suzuki, K Akino, M Ohe-Toyota, Y Maruyama, H Tatsumi, K Imai, Y Shinomura, T Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 66 ( 9 ) 4574 - 4583 2006.05 [Refereed]

p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico, approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.

DOI PubMed

Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus

M Kusano, M Toyota, H Suzuki, K Akino, F Aoki, M Fujita, M Hosokawa, Y Shinomura, K Imai, T Tokino

CANCER ( JOHN WILEY & SONS INC ) 106 ( 7 ) 1467 - 1479 2006.04 [Refereed]

BACKGROUND. The CpG island methylator phenotype (CIMP), which is characterized by simultaneous methylation of the CpG islands of multiple genes, has been recognized as one of the important mechanisms in gastrointestinal carcinogenesis. METHODS. Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes in 78 primary gastric carcinomas was examined using combined-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors bisulfite were detected using real-time polymerase chain reaction analysis followed by all evaluation of the correlations between CIMP status, EBV-association, and genetic alteration of p53 and K-ras. The authors compared the clinicopathologic features of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N). RESULTS. The methylation profiles of 12 genes showed non-random methylation, Supporting the presence of CIMP in gastric carcinoma. No p53 mutations were detected among CIMP-H tumors, and no EBV association was detected in tumors that showed mutation of p53 and K-ras. In a multiple logistic regression model with CIMP-H as the dependent variable, proximal location (P =.011), diffuse type (P =.019), and less advanced pathologic TNM status (P =.043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly worse Survival than patients who had CIMP-H tumors (P =.004) or CIMP-L tumors (P =.012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation. CONCLUSIONS. CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that gastric carcinomas arose through different molecular pathways may affect not only tumor characteristics but also patient prognosis.

DOI PubMed

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines.

Terasawa K, Toyota M, Sagae S, Ogi K, Suzuki H, Sonoda T, Akino K, Maruyama R, Nishikawa N, Imai K, Shinomura Y, Saito T, Tokino T

British journal of cancer ( 6 ) 94 914 - 921 2006.03 [Refereed]

DOI PubMed

Smoking, drinking, sleeping habits, and other lifestyle factors and the risk of systemic lupus erythematosus in Japanese females: Findings from the KYSS study

Masakazu Washio, Takahiko Horiuchi, Chikako Kiyohara, Hiroko Kodama, Yoshifumi Tada, Toyoko Asami, Hiroki Takahashi, Gen Kobashi, Takashi Abe, Hisato Tanaka, Norihiko Nogami, Mine Harada, Hiroshi Tsukamoto, Saburo Ide, Kohei Nagasawa, Osamu Ushiyama, Takao Hotokebuchi, Kazushi Okamoto, Naomasa Sakamoto, Satoshi Sasaki, Yoshihiro Miyake, Tetsuji Yokoyama, Mitsuru Mori, Asae Oura, Yasuhisa Sinomura, Hiromu Suzuki, Motohisa Yamamoto, Yutaka Inaba, Masaki Nagai

Modern Rheumatology 16 ( 3 ) 143 - 150 2006.03 [Refereed]

Many risk factors have been proposed for systemic lupus erythematosus (SLE). However, there is little information about the relationship between lifestyles and SLE in Japan. Two case control studies were conducted in Kyushu, southern Japan, and in Hokkaido, northern Japan, to examine the relationship between lifestyles and development of SLE in females. The participants were 78 patients and 329 controls in Kyushu and 35 patients and 188 controls in Hokkaido. Smoking was associated with an increased risk of SLE after adjusting for age in both regions. However, in Hokkaido, this association between smoking and SLE did not reach statistical significance after adjusting for alcohol drinking. The present study suggests that smoking may increase the risk of SLE among Japanese females. © Japan College of Rheumatology 2006.

DOI PubMed

De novo CpG island methylation in human cancer cells

KW Jair, KE Bachman, H Suzuki, AH Ting, Rhee, I, RWC Yen, SB Baylin, KE Schuebel

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 66 ( 2 ) 682 - 692 2006.01 [Refereed]

A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.

DOI PubMed

Roles and causes of abnormal DNA methylation in gastrointestinal cancers

Hiromu Suzuki, Minoru Toyota, Hironobu Sato, Tomoko Sonoda, Fumio Sakauchi, Mitsuru Mori

Asian Pacific Journal of Cancer Prevention ( Asian Pacific Organization for Cancer Prevention ) 7 ( 2 ) 177 - 185 2006 [Refereed]

Evidence now suggests that epigenetic abnormalities, particularly altered DNA methylation, play a crucial role in the development and progression of human gastrointestinal malignancies. Two distinct DNA methylation abnormalities are observed together in cancer. One is an overall genome-wide reduction in DNA methylation (global hypomethylation) and the other is regional hypermethylation within the CpG islands of specific gene promoters. Global hypomethylation is believed to induce proto-oncogene activation and chromosomal instability, whereas regional hypermethylation is strongly associated with transcriptional silencing of tumor suppressor genes. To date, genes involved in regulation of the cell cycle, DNA repair, growth signaling, angiogenesis, and apoptosis, are all known to be inactivated by hypermethylation. Recently developed techniques for detecting changes in DNA methylation have dramatically enhanced our understanding of the patterns of methylation that occur as cancers progress. One of the key contributors to aberrant methylation is aging, but other patterns of methylation are cancer-specific and detected only in a subset of tumors exhibiting the CpG island methylator phenotype (CIMP). Although the cause of altered patterns of DNA methylation in cancer remsains unknown, it is believed that epidemiological factors, notably dietary folate intake, might strongly influence DNA methylation patterns. Recent studies further suggest that polymorphisms of genes involved in folate metabolism are causally related to the development of cancer. Identifying epidemiological factors responsible for epigenetic changes should provide clues for cancer prevention in the future.

PubMed

Application of methylation changes in a novel colon tumor suppressor gene, RASSF2, to tumor marker

Toyota Minoru, Akino Kimishige, Suzuki Hiromu, Tokino akashi, Shinomura Y, Imai Kohzoh

Japan Journal of Molecular Tumor Marker Research ( Japanese Society for Molecular Tumor Marker Research ) 21 5 - 6 2006

DOI CiNii

分裂期チェックポイント分子CHFRの結合分子探索と機能解析

見田裕章, 豊田実, 鹿島理沙, 佐々木泰史, 秋野公臣, 鈴木拓, 井戸川雅史, 下重美紀, 山田哲司, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 327 - 327 2005.09

RASエフェクター,RASSF2遺伝子は大腸がんにおける新規がん抑制遺伝子である(The Ras Effector RASSF2 is a Novel Tumor Suppressor Gene in Human Colorectal Cancer)

秋野公臣, 豊田実, 鈴木拓, 見田裕章, 佐々木泰史, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 136 - 137 2005.09

DNAの異常メチル化を指標とした大腸癌アベレージリスク群の設定と検診への応用

渡邊嘉行, 豊田実, 秋野公臣, 鈴木拓, 前畑忠照, 岡本賢, 奥瀬千晃, 今井浩三, 篠村恭久, 伊東文生, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 540 - 540 2005.09

p53によるビタミンD受容体の発現誘導と癌治療への応用

丸山玲緒, 豊田実, 青木文夫, 佐々木泰史, 明石浩史, 見田裕章, 鈴木拓, 時野隆至, 今井浩三, 篠村恭久

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 42回 112 - 112 2005.07

Aberrant methylation and silencing of the BNIP3 gene in colorectal and gastric cancer

M Murai, M Toyota, H Suzuki, A Satoh, Y Sasaki, K Akino, M Ueno, F Takahashi, M Kusano, H Mita, K Yanagihara, T Endo, Y Hinoda, T Tokino, K Imai

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 11 ( 3 ) 1021 - 1027 2005.02 [Refereed]

BNIP3 protein is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. To examine its role in the progression of gastrointestinal cancer, we examined the expression and DNA methylation status of BNIP3 gene in a panel of colorectal and gastric cancer cell lines. BNIP3 was not expressed in 14 of the 24 cell lines tested, and its absence was not caused by gene mutation or by altered expression of hypoxia inducible factor-1, a key transcription factor that regulates BNIP3 expression. On the other hand, methylation of the 5' CpG island of BNIP3 was closely correlated with silencing the gene. Moreover, treating methylated cells with the methyltransferase inhibitor 5-aza2'-deoxycytidine restored hypoxia-induced expression of BNIP3 mRNA and protein, which in turn led to cell death. Aberrant methylation of BNIP3 was also detected in 66% of primary colorectal and 49% of primary gastric cancers, but not in normal tissue samples collected from areas adjacent to the tumors. Apparently, epigenetic alteration of BNIP3 is a frequent and cancer-specific event, which suggests that inactivation of BNIP3 likely plays a key role in the progression of some gastrointestinal cancers and that it may be a useful molecular target for therapy.

PubMed

Epigenetic inactivation of SFRP's complements genetic alterations to allow constitutive Wnt pathway signaling in human colorectal cancer

S. H., T. M., T. T., M. M., I. K.

Japan Journal of Molecular Tumor Marker Research ( Japanese Society for Molecular Tumor Marker Research ) 20 73 - 74 2005

DOI CiNii

【消化器疾患における転写制御メカニズムを解明する】消化器癌とエピジェネティクスの新たな展開

鈴木拓, 公衆衛生学講座, 野島正寛, 森満, 今井浩三

分子消化器病 1 327 - 335 2004 [Refereed]

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