研究者詳細 - 時野　隆至

論文 - 時野　隆至

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Dominant inheritance in hereditary angioedema associated with carboxypeptidase N deficiency.

Tokimasa Hida, Masashi Idogawa, Masae Okura, Takashi Tokino, Hisashi Uhara

Allergology international : official journal of the Japanese Society of Allergology 2025年04月 [国際誌]

DOI PubMed

The genomic landscape of cutaneous squamous cell carcinoma in Japan.

Junji Kato, Tokimasa Hida, Masashi Idogawa, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 2024年12月 [国際誌]

Comprehensive studies of the genetic profiles of cutaneous squamous cell carcinoma (cSCC) in Japanese patients have been lacking, although an understanding of these profiles is crucial for improving treatment outcomes. Since 2019, comprehensive genomic profiling (CGP) has been covered by Japan's health insurance, and the resulting data have been compiled into a comprehensive database by the country's Center for Cancer Genomics and Advanced Therapeutics (C-CAT). In this retrospective study, we used CGP data from the C-CAT database to analyze genomic characteristics of cSCC in Japanese patients. The patients' clinical and genomic data, including the chemotherapy regimens, tumor mutational burden (TMB), and survival status, were obtained. We analyzed the cases of 152 patients, with only those evaluated by the FoundationOne® CDx included for accuracy. Among the 152 patients, the most common gene oncogenic alterations were observed in TP53 (67%), CDKN2A (54%), TERT (49%), CDKN2B (33%), and NOTCH1 (18%). TMB-high (≥10 mut/Mb) was observed in 27% (n = 41) of the patients, with a median age of 75 years for this group. TMB-low (<10 mut/Mb) was observed in 73% (n = 111) of the patients; their median age was 67 years.

DOI PubMed

Genetic Characteristics of Cutaneous, Acral, and Mucosal Melanoma in Japan.

Tokimasa Hida, Masashi Idogawa, Junji Kato, Yukiko Kiniwa, Kohei Horimoto, Sayuri Sato, Masahide Sawada, Shoichiro Tange, Masae Okura, Ryuhei Okuyama, Takashi Tokino, Hisashi Uhara

Cancer medicine 13 ( 22 ) e70360 2024年11月 [国際誌]

BACKGROUND: Acral and mucosal melanomas are more prevalent in Asians than in Caucasians, unlike cutaneous melanomas, which are predominant in Caucasians. Recent studies have suggested that non-Caucasian cutaneous melanomas responded less to immune checkpoint inhibitors, highlighting the need for genetic profiling across ethnicities. This study aimed to elucidate the genetic characteristics of Japanese melanomas, which is an under-researched topic. METHODS: Single-nucleotide variants, indels, and copy number alterations in 104 Japanese melanoma patients (37 cutaneous, 52 acral, and 15 mucosal) were analyzed using custom panel sequencing. RESULTS: Driver events were detected in 94% of the cases. Among cutaneous melanoma cases, 76% had BRAF mutations, and 8% had NRAS mutations. In acral melanoma, BRAF (9%), NRAS (17%), KRAS (8%), KIT (19%), and NF1 (7%) mutations were detected. Major driver mutations in mucosal melanoma were detected in NRAS, KRAS, NF1, PTEN, GNAQ, and KIT. The median tumor mutational burden across all melanoma types was 4.6 mutations/Mb, with no significant difference between the cutaneous and acral/mucosal types. Of the 21 patients with both primary and metastatic lesions, 11 showed distinct mutations in each. Potentially actionable mutations were detected in 58 patients in addition to BRAF V600E/K mutations in 31. CONCLUSIONS: This study highlights distinct genetic abnormalities and actionable alterations in Japanese melanoma patients. This suggests a lower tumor mutational burden in East Asian cutaneous melanoma, which may affect the efficacy of immune checkpoint inhibitors. The heterogeneity of driver mutations across and within individuals highlights the need for personalized treatment approaches.

DOI PubMed

A case of familial progressive hyperpigmentation with or without hypopigmentation presenting with hypopigmented striae along the lines of Blaschko.

Tokimasa Hida, Masashi Idogawa, Aki Ishikawa, Masae Okura, Satoru Sasaki, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 2024年09月 [国際誌]

Familial progressive hyperpigmentation with or without hypopigmentation (FPHH) is an autosomal dominant disorder characterized by widespread skin hyperpigmentation, café-au-lait spots, and hypopigmented circular macules, resulting from KITLG variants. KITLG, expressed by keratinocytes, binds to KIT on melanocytes, stimulating melanogenesis. Disturbances in the KITLG-KIT interaction result in diffuse hyperpigmentation in FPHH. However, the mechanisms behind hypopigmented macule formation remain unclear. This report presents a unique FPHH case in a patient with a novel KITLG mutation (Ser78Leu). Notably, the patient showed multiple hypopigmented macules and striae along the lines of Blaschko. Digital polymerase chain reaction analysis of the DNA from skin and blood tissues indicated a copy-neutral loss of heterozygosity at the KITLG locus, only in the hypopigmented macule. These findings suggest that the hypopigmented macules might result from revertant mosaicism. Conversely, café-au-lait spots do not follow the lines of Blaschko and can superimpose on the hypopigmented striae, indicating a distinct pathogenesis. This case contributes to the understanding of the genetic mechanisms in FPHH.

DOI PubMed

Genomic landscape of cutaneous, acral, mucosal, and uveal melanoma in Japan: analysis of clinical comprehensive genomic profiling data.

Tokimasa Hida, Junji Kato, Masashi Idogawa, Takashi Tokino, Hisashi Uhara

International journal of clinical oncology 2024年09月 [国内誌]

BACKGROUND: Cutaneous melanoma (CM) is the most common type in Caucasians, while acral melanoma (AM) and mucosal melanoma (MM), which are resistant to immunotherapies and BRAF/MEK-targeted therapies, are more common in East Asians. Genomic profiling is essential for treating melanomas, but such data are lacking in Japan. METHODS: Comprehensive genomic profiling data compiled in the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) were analyzed. RESULTS: A total of 380 melanomas was analyzed, including 136 CM, 46 AM, 168 MM, and 30 uveal melanoma (UM). MM included conjunctival, sinonasal, oral, esophageal, anorectal, and vulvovaginal melanomas. No significant difference in the median tumor mutational burden (TMB) of CM (3.39 mutations/megabase), AM (2.76), and MM (3.78) was the key finding. Microsatellite instability-high status was found in one case. BRAF V600E/K was found in only 45 patients (12%). Key driver mutations in CM were BRAF (38%), NRAS (21%), NF1 (8%), and KIT (10%), with frequent copy number alterations (CNAs) of CDKN2A, CDKN2B, and MYC. AM was characterized by altered KIT (30%), NRAS (26%), and NF1 (11%) and CDKN2A, CDKN2B, CDK4, MDM2, and CCND1 CNAs. MM was characterized by altered NRAS (24%), KIT (21%), and NF1 (17%) and MYC, KIT, and CDKN2A CNAs, with differences based on anatomical locations. UM bore GNAQ or GNA11 driver mutations (87%) and frequent mutations in SF3B1 or BAP1. CONCLUSION: The distinct genomic profiling in Japanese patients, including lower TMB, compared to Caucasians, is associated with poorer treatment outcomes. This result underscores the need for more effective therapeutic agents.

DOI PubMed

口腔がんにおける悪性化関連遺伝子群転写抑制機構の解明(Nuclear protein X represses the transcription of malignancy-related genes in oral cancer cell lines)

丹下正一朗, 後藤生子, 川島秀器, 井戸川雅史, 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 ) 83回 P - 2241 2024年09月

Mosaic SUFU mutation associated with a mild phenotype of multiple hereditary infundibulocystic basal cell carcinoma syndrome.

Marina Hamada, Tokimasa Hida, Masashi Idogawa, Shoichiro Tange, Takafumi Kamiya, Masae Okura, Toshiharu Yamashita, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 2024年08月 [国際誌]

Multiple hereditary infundibulocystic basal cell carcinoma syndrome (MHIBCC), an autosomal dominant disorder caused by variants in SUFU, is characterized by numerous infundibulocystic basal cell carcinomas (IBCCs). In this report, we present a possible case of mosaic MHIBCC. A 57-year-old woman underwent the removal of four papules on her face, which were diagnosed as IBCCs. Exome sequencing revealed a SUFU c.1022+1G>A mutation within the skin tumor. The same mutation was detected in her blood but at a lower allele frequency. TA cloning revealed that the allele frequency of the mutation in the blood was 0.07. Additionally, tumor assessment revealed loss of heterozygosity (LOH) in chromosome 10, including the SUFU locus. These results indicate the patient had mosaicism for the SUFU mutation in normal tissues, aligning with the mosaic MHIBCC diagnosis. This, combined with LOH, likely contributed to IBCC development. Mosaic MHIBCC may present with milder symptoms. However, it may still increase the risk of developing brain tumors and more aggressive basal cell carcinoma. The possibility of mosaicism should be investigated in mild MHIBCC cases, where standard genetic tests fail to detect SUFU germline variants.

DOI PubMed

LMNA::NTRK1 Fusion-positive Leiomyosarcoma: Discrepancy between DNA-based Comprehensive Genomic Profiling and RNA Sequencing.

Norito Suzuki, Masashi Idogawa, Makoto Emori, Kazuyuki Murase, Yohei Arihara, Hajime Nakamura, Makoto Usami, Tomohiro Kubo, Ichiro Kinoshita, Shintaro Sugita, Takashi Tokino, Tadashi Hasegawa, Akihiro Sakurai, Kohichi Takada

Internal medicine (Tokyo, Japan) 63 ( 15 ) 2215 - 2219 2024年08月 [国内誌]

A 26-year-old man presented with a tumor in the left soleus muscle. The tumor was diagnosed as a locally advanced leiomyosarcoma. The patient was treated with irradiation followed by wide resection. One year after surgery, the patient presented with multiple lung metastases. Despite aggressive sequential chemotherapy, systemic metastatic tumors continued to develop. To explore therapeutic options for the patient, we performed DNA-based CGP with FoundationOne® CDx (F1). F1 identified an out-of-strand rearrangement of the NOS1AP::NTRK1 gene, which has not been previously reported. In contrast, RNA sequencing revealed an in-frame LMNA::NTRK1 gene, which is an oncogenic fusion gene.

DOI PubMed

Genomic profiles of Merkel cell carcinoma in Japan.

Junji Kato, Tokimasa Hida, Masashi Idogawa, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 2024年07月 [国際誌]

DOI PubMed

クローズアップ実験法(series 370) 知っていますか?TCGAの更新状況

丹下正一朗, 井戸川雅史, 時野隆至

実験医学 ( (株)羊土社 ) 42 ( 9 ) 1414 - 1424 2024年06月

クローズアップ実験法(series 370) 知っていますか?TCGAの更新状況

丹下正一朗, 井戸川雅史, 時野隆至

実験医学 ( (株)羊土社 ) 42 ( 9 ) 1414 - 1424 2024年06月

The Clinical Validity of Urinary Pellet DNA Monitoring for the Diagnosis of Recurrent Bladder Cancer.

Masakazu Abe, Hayato Hiraki, Takashi Tsuyukubo, Sadahide Ono, Shigekatsu Maekawa, Daichi Tamura, Akiko Yashima-Abo, Renpei Kato, Hiromitsu Fujisawa, Takeshi Iwaya, Woong-Yang Park, Masashi Idogawa, Takashi Tokino, Wataru Obara, Satoshi S Nishizuka

The Journal of molecular diagnostics : JMD 26 ( 4 ) 278 - 291 2024年04月 [国際誌]

The aim of this study was to evaluate the clinical validity of monitoring urine pellet DNA (upDNA) of bladder cancer (BC) by digital PCR (dPCR) as a biomarker for early recurrence prediction, treatment efficacy evaluation, and no-recurrence corroboration. Tumor panel sequencing was first performed to select patient-unique somatic mutations to monitor both upDNA and circulating tumor DNA (ctDNA) by dPCR. For longitudinal monitoring using upDNA as well as plasma ctDNA, an average of 7.2 (range, 2 to 12) time points per case were performed with the dPCR assay for 32 previously treated and untreated patients with BC. Clinical recurrence based on imaging and urine cytology was compared using upDNA variant allele frequency (VAF) dynamics. A continuous increasing trend of upDNA VAF ≥1% was considered to indicate molecular recurrence. Most (30/32; 93.8%) cases showed at least one traceable somatic mutation. In 5 of 7 cases (71.4%) with clinical recurrence, upDNA VAF >1% was detected 7 to 15 months earlier than the imaging diagnosis. The upDNA VAF remained high after initial treatment for locally recurrent cases. The clinical validity of upDNA monitoring was confirmed with the observation that 26 of 30 cases (86.7%) were traceable. Local recurrences were not indicated by ctDNA alone. The results support the clinical validity of upDNA monitoring in the management of recurrent BC.

DOI PubMed

The frequency and pathogenicity of BRCA1 and BRCA2 variants in the general Japanese population.

Masashi Idogawa, Tasuku Mariya, Yumi Tanaka, Tsuyoshi Saito, Hiroshi Nakase, Takashi Tokino, Akihiro Sakurai

Journal of human genetics 2024年02月 [国際誌]

Hereditary breast and ovarian cancer syndrome (HBOC) resulting from pathogenic variants of BRCA1 or BRCA2 is the most common and well-documented hereditary tumor. Although founder variants have been identified in population-based surveys in various countries, the types of variants are not uniform across races and regions. Recently, the Tohoku Medical Megabank Organization (ToMMo) released whole-genome sequence data including approximately 54,000 individuals from the general population of the Tohoku area in Japan. We analyzed these data and comprehensively identified the prevalence of BRCA1/2 pathogenic and truncating variants. We believe that an accurate understanding of the unique distribution and characteristics of pathogenic BRCA1/2 variants in Japan through this analysis will enable better surveillance and intervention for HBOC patients, not only in Japan but also worldwide.

DOI PubMed

Long Noncoding RNA RP11-278A23.1, a Potential Modulator of p53 Tumor Suppression, Contributes to Colorectal Cancer Progression.

Masayo Kamikokura, Shoichiro Tange, Hiroshi Nakase, Takashi Tokino, Masashi Idogawa

Cancers 16 ( 5 ) 2024年02月 [国際誌]

Recently, many studies revealed that long noncoding RNAs (lncRNAs) play important roles in cancers. To identify lncRNAs contributing to colorectal cancers, we screened lncRNAs through expression and survival analyses in datasets from The Cancer Genome Atlas (TCGA). The screen revealed that RP11-278A23.1 expression is significantly increased in colorectal cancer tissues compared with normal tissues and that high RP11-278A23.1 expression correlates with poor prognosis. The knockdown of RP11-278A23.1 inhibited the growth of and promoted apoptosis in colorectal cancer cells. Next, to comprehensively examine differentially expressed genes after RP11-278A23.1 knockdown, RNA sequencing was performed in HCT116 cells. The expression of p21, a p53 target gene, was significantly upregulated, and the expression of several p53 target proapoptotic genes was also altered. RP11-278A23.1 knockdown increased p53 expression at the translational level but not at the transcriptional level. Interestingly, RP11-278A23.1 knockdown also altered the expression of these proapoptotic genes in DLD1 cells with mutated p53 and in p53-knockout HCT116 cells. These results suggest that RP11-278A23.1 modifies the expression of these apoptosis-related genes in p53-dependent and p53-independent manners. In summary, lncRNA RP11-278A23.1 contributes to colorectal cancer progression by promoting cell growth and inhibiting apoptosis, suggesting that this lncRNA may be a useful therapeutic target.

DOI PubMed

Effects of temozolomide on tumor mutation burden and microsatellite instability in melanoma cells.

Masahide Sawada, Tokimasa Hida, Takafumi Kamiya, Tomoyuki Minowa, Junji Kato, Masae Okura, Masashi Idogawa, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 2023年09月 [国際誌]

The efficacy of combination therapy with an immune checkpoint inhibitor (ICI) and cytotoxic chemotherapeutic agents has been investigated in cancer, including melanoma. Before ICIs were introduced, dacarbazine or temozolomide (TMZ) were used to treat melanoma. Several studies using glioma or colorectal cancer cells showed that TMZ can increase the tumor mutation burden (TMB) and induce mismatch repair (MMR) deficiency associated with microsatellite instability (MSI). These could increase immunoreactivity to an ICI, but this has not been evaluated in melanoma cells. We investigated the effects of TMZ on MSI status and TMB in melanoma cells. To evaluate the TMB, we performed whole-exome sequencing using genomic DNA from the human melanoma cell lines Mel18, A375, WM266-4, G361, and TXM18 before and after TMZ treatment. Polymerase chain reaction amplification of five mononucleotide repeat markers, BAT25, BAT26, NR21, NR24, and MONO27, was performed, and we analyzed changes in the MSI status. In all cell lines, the TMB was increased after TMZ treatment (the change amount of TMB with ≤ 5% variant allele frequency [VAF] was 18.0-38.3 mutations per megabase) even in the condition without obvious cytological damage. MSI after TMZ treatment was not observed in any cells. TMZ increased TMB but did not change MSI status in melanoma cells.

DOI PubMed

再発転移頭頸部癌におけるctDNAモニタリングによる治療効果予測(Prediction of treatment response by ctDNA monitoring in recurrent metastatic head and neck cancer)

古後龍之介, 真子知美, 開勇人, 西塚哲, 丹下正一朗, 井戸川雅史, 時野隆至, 中川尚志

日本癌学会総会記事 ( (一社)日本癌学会 ) 82回 1424 - 1424 2023年09月

予後不良の腫瘍と発現が相関する霊長類特異的遺伝子の解析(Analysis of primate-specific genes contributes to malignant cancer)

丹下正一朗, 平野朋美, 井戸川雅史, 平田英周, 井本逸勢, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 82回 1287 - 1287 2023年09月

長鎖非コードRNA IncAC1はp53標的遺伝子の発現変化を介して大腸癌進展に寄与する(Long non-coding RNA IncAC1 contributes to colorectal cancer progression via the expression change of p53-targeting genes)

井戸川雅史, 小泉昌代, 丹下正一朗, 平野朋美, 大久保陽介, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 82回 1242 - 1242 2023年09月

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年07月

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

がん遺伝子パネル検査解析支援ソフトウェア「Varporter」の開発と応用

井戸川雅史, 田中佑弥, 櫻井晃洋, 時野隆至

日本遺伝カウンセリング学会誌 ( (一社)日本遺伝カウンセリング学会 ) 44 ( 2 ) 92 - 92 2023年06月

メラノーマ細胞におけるtumor mutation burden及びmicrosatellite instabilityに対するtemozolomideの影響

澤田匡秀, 肥田時征, 神谷崇文, 箕輪智幸, 加藤潤史, 黄倉真恵, 井戸川雅史, 時野隆至, 宇原久

日本皮膚悪性腫瘍学会学術大会プログラム・抄録集 ( (一社)日本皮膚悪性腫瘍学会 ) 39回 229 - 229 2023年06月

Prospective observational study of monitoring gene alterations in plasma cell-free DNA using droplet digital PCR system during anti-EGFR antibody treatment in patients with RAS wild-type advanced colorectal cancer.

Naoki Takahashi, Keishi Nakamura, Yosuke Kito, Eiji Kubota, Masako Asayama, Yosuke Kumekawa, Hiroki Hara, Tomohiro Matsushima, Kunihiro Tsuji, Hiromi Kataoka, Akihiro Tsuyada, Yasushi Sasaki, Masashi Idogawa, Takashi Tokino, Takeshi Sawada

Journal of Clinical Oncology ( American Society of Clinical Oncology (ASCO) ) 41 ( 4_suppl ) 172 - 172 2023年02月

172 Background: Anti-EGFR antibody is recommended as one of the standard treatments in patients with RAS wild-type advanced colorectal cancer (CRC). Few reports have evaluated low-frequency subclones of alterations in EGFR signaling pathway genes in plasma cell-free DNA (cfDNA) during anti-EGFR treatment in prospective cohort of advanced CRC patients. Methods: Key inclusion criteria of this study was as follows: 20 years old and over, performance status 0 to 2, metastatic or recurrent CRC (adenocarcinoma), RAS wild-type CRC diagnosed by PCR-rSSO using tumor tissue, no prior use of anti-EGFR antibody. Plasma samples were prospectively collected at pre-treatment, at 4, 10 weeks, every 10 weeks thereafter, and at the end of the anti-EGFR treatment. With QX200 Droplet Digital PCR platform, we analyzed hotspot mutations of KRAS, NRAS, BRAF, and PIK3CA and copy number (CN) variant of HER2 and MET using plasma samples of pre- and post-treatment as well as during treatment (limited to patients with any gene alterations at pre- and post-treatment). Results: Total 50 patients were registered in this study, but 3 patients were excluded because inclusion criteria were not met. Finally, 47 patients were included in full analysis set. In 15 patients (31.9%), genetic alterations including KRAS (G12G13: 10.1%, A59Q61: 2.2%), NRAS (G12G13: 2.2%, Q61: 2.1%), BRAF (V600: 6.4%) , PIK3CA (E545: 4.3%, H1047: 4.3%) , and HER2 (amplification: 6.5%) were detected in pre-treatment samples. Analysis of pre-treatment samples showed that response rates of patients with any gene alterations (N = 13) and that without any gene alterations (N = 29) were 30.8% and 58.6%, respectively (p = 0.095). When the cut-off of variant allele frequency (VAF) was set as 0.5% in patients with any gene mutations, the response rates for cases with VAF ≥0.5% (N = 6) and those with VAF < 0.5% (N = 7) were 0% and 57.1%, respectively (p = 0.049). Among 41 patients with detectable target lesions by CT scan, there was significant difference in percentage of best tumor shrinkage between patients with gene alterations and those without gene alterations (32.1% vs 10.5%, p = 0.037), when cut-off VAF of any gene mutations was set as 0.5% and cut-off CN of HER2 or MET was set as 4 at pre-treatment. Due to dynamics of each gene alteration in cfDNA during treatment, mutations of KRAS A59Q61, NRAS Q61, and MET amplification were characterized as acquired alterations which were newly detected after the administration of anti-EGFR antibodies. Conclusions: Our study demonstrated the clinical relevance of minor mutant subclones in EGFR signaling pathway genes in plasma for predicting response to anti-EGFR treatment. Although sample size of this study was small, preferable threshold for distinguishing responders from non-responders may be 0.5% as VAF of these gene mutations. Clinical trial information: 000034923 .

DOI

抗PD-1抗体が著効したNF1変異型末端型メラノーマの1例

松田宇充, 加藤潤史, 肥田時征, 堀本浩平, 佐藤さゆり, 黄倉真恵, 宇原久, 井戸川雅史, 時野隆至

日本皮膚科学会雑誌 ( (公社)日本皮膚科学会 ) 133 ( 2 ) 271 - 272 2023年02月

MYEOV overexpression induced by demethylation of its promoter contributes to pancreatic cancer progression via activation of the folate cycle/c-Myc/mTORC1 pathway.

Shoichiro Tange, Tomomi Hirano, Masashi Idogawa, Eishu Hirata, Issei Imoto, Takashi Tokino

BMC cancer 23 ( 1 ) 85 - 85 2023年01月 [国際誌]

BACKGROUND: While molecular targeted drugs and other therapies are being developed for many tumors, pancreatic cancer is still considered to be the malignant tumor with the worst prognosis. We started this study to identify prognostic genes and therapeutic targets of pancreatic cancer. METHODS: To comprehensively identify prognostic genes in pancreatic cancer, we investigated the correlation between gene expression and cancer-specific prognosis using transcriptome and clinical information datasets from The Cancer Genome Atlas (TCGA). In addition, we examined the effects of the suppression of candidate prognostic genes in pancreatic cancer cell lines. RESULT: We found that patients with high expression levels of MYEOV, a primate-specific gene with unknown function, had significantly shorter disease-specific survival times than those with low expression levels. Cox proportional hazards analysis revealed that high expression of MYEOV was significantly associated with poor survival and was an independent prognostic factor for disease-specific survival in pancreatic cancer patients. Analysis of multiple cancer samples revealed that the MYEOV promoter region is methylated in noncancer tissues but is demethylated in tumors, causing MYEOV overexpression in tumors. Notably, the knockdown of MYEOV suppressed the expression of MTHFD2 and other folate metabolism-related enzyme genes required for the synthesis of amino acids and nucleic acids and also restored the expression of c-Myc and mTORC1 repressors. CONCLUSION: There is a significant correlation between elevated MYEOV expression and poor disease-specific survival in pancreatic cancer patients. MYEOV enhances the activation of several oncogenic pathways, resulting in the induction of pancreatic cancer cell proliferation. Overall, MYEOV acts as an oncogene in pancreatic cancer. Furthermore, MYEOV may be a prognostic biomarker and serve as an 'actionable' therapeutic target for pancreatic cancers.

DOI PubMed

抗PD-1抗体が著効したNF1-mutant acral melanoma肝転移の1例

松田宇充, 加藤潤史, 肥田時征, 堀本浩平, 佐藤さゆり, 黄倉真恵, 宇原久, 井戸川雅史, 時野隆至

日本皮膚科学会雑誌 ( (公社)日本皮膚科学会 ) 132 ( 12 ) 2699 - 2699 2022年11月

CRISPRを用いた遺伝子活性化システムによる腫瘍細胞株におけるlncRNAの網羅的機能スクリーニング(Comprehensive screening of long noncoding RNAs(lncRNAs) based on CRISPR-mediated gene activation in cancer cells)

平野朋美, 井戸川雅史, 丹下正一朗, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 81回 P - 3096 2022年09月

がん遺伝子パネル検査解析支援ソフトウェア「Varporter」の開発(The development of Varporter, a software supporting the evaluation of cancer gene panel test.)

井戸川雅史, 平野朋美, 丹下正一朗, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 81回 J - 2034 2022年09月

頭頸部扁平上皮癌におけるctDNAモニタリングの特徴(The characteristics of ctDNA monitoring in head and neck squamous cell carcinoma)

古後龍之介, 真子知美, 岩谷岳, 開勇人, 西塚哲, 井戸川雅史, 時野隆至, 中川尚志

日本癌学会総会記事 ( (一社)日本癌学会 ) 81回 P - 1283 2022年09月

膵臓がんにおける新規予後予測遺伝子と1炭素代謝経路の関係(A relationship between the novel prognosticator gene in malignant pancreatic cancer and one-carbon metabolism)

丹下正一朗, 平野朋美, 井戸川雅史, 平田英周, 井本逸勢, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 81回 P - 2314 2022年09月

Multiomics identifies the link between intratumor steatosis and the exhausted tumor immune microenvironment in hepatocellular carcinoma.

Hiroki Murai, Takahiro Kodama, Kazuki Maesaka, Shoichiro Tange, Daisuke Motooka, Yutaka Suzuki, Yasuyuki Shigematsu, Kentaro Inamura, Yoshihiro Mise, Akio Saiura, Yoshihiro Ono, Yu Takahashi, Yota Kawasaki, Satoshi Iino, Shogo Kobayashi, Masashi Idogawa, Takashi Tokino, Tomomi Hashidate-Yoshida, Hideo Shindou, Masanori Miyazaki, Yasuharu Imai, Satoshi Tanaka, Eiji Mita, Kazuyoshi Ohkawa, Hayato Hikita, Ryotaro Sakamori, Tomohide Tatsumi, Hidetoshi Eguchi, Eiichi Morii, Tetsuo Takehara

Hepatology (Baltimore, Md.) 77 ( 1 ) 77 - 91 2022年05月 [国際誌]

BACKGROUND&AIMS: Immunotherapy has become the standard-of-care treatment for hepatocellular carcinoma (HCC), but its efficacy remains limited. To identify immunotherapy-susceptible HCC, we profiled the molecular abnormalities and tumor immune microenvironment (TIME) of rapidly increasing nonviral HCC. APPROACHES&RESULTS: We performed RNA-seq of tumor tissues in 113 nonviral HCC patients and cancer genome sequencing of 69 genes with recurrent genetic alterations reported in HCC. Unsupervised hierarchical clustering classified nonviral HCCs into 3 molecular classes (Class I, II, III), which stratified patient prognosis. Class I, with the poorest prognosis, was associated with TP53 mutations, whereas class III, with the best prognosis, was associated with CTNNB1 mutations. Thirty-eight percent of nonviral HCC was defined as an immune class characterized by a high frequency of intratumoral steatosis and a low frequency of CTNNB1 mutations. Steatotic HCC, which accounts for 23% of nonviral HCC cases, presented an immune-enriched but immune-exhausted TIME characterized by T-cell exhaustion, M2 macrophage and cancer-associated fibroblast (CAF) infiltration, high PD-L1 expression, and TGF-β signaling activation. Spatial transcriptome analysis suggested that M2 macrophages and CAFs may be in close proximity to exhausted CD8+ T cells in steatotic HCC. An in vitro study showed that palmitic acid-induced lipid accumulation in HCC cells upregulated PD-L1 expression and promoted immunosuppressive phenotypes of cocultured macrophages and fibroblasts. Steatotic HCC patients, confirmed by chemical-shift MR imaging, had significantly longer PFS with combined immunotherapy using anti-PD-L1 and anti-VEGF antibodies. CONCLUSIONS: Multiomics stratified nonviral HCCs according to prognosis or TIME. We identified the link between intratumoral steatosis and immune-exhausted immunotherapy-susceptible TIME.

DOI PubMed

Mutation Analysis of Radioresistant Early-Stage Cervical Cancer.

Tae Oike, Yoshihito Sekiguchi, Yuya Yoshimoto, Takahiro Oike, Ken Ando, Wenchao Gu, Yasushi Sasaki, Takashi Tokino, Akira Iwase, Tatsuya Ohno

International journal of molecular sciences 23 ( 1 ) 2021年12月 [国際誌]

Radiotherapy is a definitive treatment for early-stage cervical cancer; however, a subset of this disease recurs locally, necessitating establishment of predictive biomarkers and treatment strategies. To address this issue, we performed gene panel-based sequencing of 18 stage IB cervical cancers treated with definitive radiotherapy, including two cases of local recurrence, followed by in vitro and in silico analyses. Simultaneous mutations in KRAS and SMAD4 (KRASmt/SMAD4mt) were detected only in a local recurrence case, indicating potential association of this mutation signature with radioresistance. In isogenic cell-based experiments, a combination of activating KRAS mutation and SMAD4 deficiency led to X-ray resistance, whereas either of these factors alone did not. Analysis of genomic data from 55,308 cancers showed a significant trend toward co-occurrence of mutations in KRAS and SMAD4. Gene Set Enrichment Analysis of the Cancer Cell Line Encyclopedia dataset suggested upregulation of the pathways involved in epithelial mesenchymal transition and inflammatory responses in KRASmt/SMAD4mt cancer cells. Notably, irradiation with therapeutic carbon ions led to robust killing of X-ray-resistant KRASmt/SMAD4mt cancer cells. These data indicate that the KRASmt/SMAD4mt signature is a potential predictor of radioresistance, and that carbon ion radiotherapy is a potential option to treat early-stage cervical cancers with the KRASmt/SMAD4mt signature.

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年10月 [国際誌]

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

Early dynamics of circulating tumor DNA predict chemotherapy responses for patients with esophageal cancer.

Ryosuke Fujisawa, Takeshi Iwaya, Fumitaka Endo, Masashi Idogawa, Noriyuki Sasaki, Hayato Hiraki, Shoichiro Tange, Tomomi Hirano, Yuka Koizumi, Masakazu Abe, Tomoko Takahashi, Mizunori Yaegashi, Yuji Akiyama, Mari Masuda, Akira Sasaki, Fumiaki Takahashi, Yasushi Sasaki, Takashi Tokino, Satoshi S Nishizuka

Carcinogenesis 42 ( 10 ) 1239 - 1249 2021年09月 [国際誌]

We investigated whether early circulating tumor DNA (ctDNA) changes, measured using digital PCR (dPCR), can predict later chemotherapy responses in esophageal squamous cell cancer (ESCC). We compared the dynamics of ctDNA and tumor volumes during chemotherapy in 42 ESCC. The accuracy of predictions of later chemotherapy responses were evaluated by the ratio of the variant allele frequency (VAF) of ctDNA (post-/pre-ctDNA) and the total tumor volume (post-/pre-volume) before and after an initial chemotherapy cycle using a receiver-operating characteristic curve analysis. Total positive and negative objective responses (ORs) were defined as either >50% or ≤50% reductions, respectively, in the total tumor volume at the end of first-line chemotherapy. Mutation screening of 43 tumors from 42 patients revealed 96 mutations. The pretreatment dPCR-ctDNA data were informative in 38 patients, using 70 selected mutations (1-3 per patient). The areas under the curve (AUCs) for the post-/pre-volume and post-/pre-ctDNA levels used in predicting the total OR were 0.85 and 0.88, respectively. The optimal cutoff value of post-/pre-ctDNA was 0.13. In 20 patients with post-/pre-volume ≥50%, the total OR could be predicted by the post-/pre-ctDNA with high accuracy; the AUC by post-/pre-ctDNA was higher than that by post-/pre-volume (0.85 vs 0.76, respectively). Patients with low post-/pre-ctDNA (n = 18) had a significantly better overall survival rate than those with high post-/pre-ctDNA (n = 20; P = 0.03). Early ctDNA changes after an initial cycle of chemotherapy predict later responses to treatment with high accuracy in ESCC patients.

DOI PubMed

Dead or alive? Pitfall of survival analysis with TCGA datasets.

Masashi Idogawa, Masayo Koizumi, Tomomi Hirano, Shoichiro Tange, Hiroshi Nakase, Takashi Tokino

Cancer biology & therapy 22 ( 10-12 ) 1 - 2 2021年09月 [国際誌]

We often encounter situations in which data from the TCGA that have been analyzed in papers we read or reviewed cannot be reproduced, even when TCGA datasets are used, especially in survival analyses. Therefore, we attempted to confirm the data source for TCGA survival analysis and found that several websites used to analyze the survival data of TCGA datasets inappropriately handle the survival data, causing differences in statistical analyses. This causes the misinterpretation of results because figures of survival analysis results in several papers are sometimes exactly as generated by these sites, and the results depend on only the tools provided by these sites. We would like to make this situation widely known and raise the problem for scientific soundness.

DOI PubMed

Genetic analyses of a secondary poroma and trichoblastoma in a HRAS-mutated sebaceous nevus.

Tomoyuki Minowa, Takafumi Kamiya, Tokimasa Hida, Masae Okura, Junji Kato, Masashi Idogawa, Shoichiro Tange, Tomomi Hirano, Takashi Tokino, Hisashi Uhara

The Journal of dermatology 48 ( 8 ) 1268 - 1272 2021年08月 [国際誌]

A sebaceous nevus is a congenital skin hamartoma caused by postzygotic HRAS or KRAS mosaic mutations. With age, affected individuals may develop secondary tumors within a sebaceous nevus. RAS mutations are harbored from the onset of sebaceous nevus, and further mutations can be expected to be required in order to explain the initiation of secondary tumors. However, genetic analyses of the secondary tumors have not been conducted. Herein, we describe the rare coexistence of a poroma and a trichoblastoma arising in a sebaceous nevus. This is the first report of an investigation of multiple genes in a secondary tumor in an SN. First, HRAS c.37G>C, which is the common mutation in sebaceous nevus, was detected in all three lesions (sebaceous nevus, poroma, and trichoblastoma). Next, to elucidate the potential second-hit mutations in the secondary poroma and trichoblastoma, we applied a panel sequencing for skin cancers that was newly developed in our institution. Our comparison of the mutational profile of 95 skin cancer-related genes in each of the three lesions newly revealed TP53 p.R158P in the poroma and NOTCH2 p.G329S in the trichoblastoma. TP53 p.R158P has been determined as a pathogenic mutation in other tumors, and NOTCH2 p.G329S was a novel mutation. We identified two novel mutations that may have contributed to the pathogenesis of the secondary tumor's development. The roles of the mutations remain unclear.

DOI PubMed

Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo.

Naoki Tanimizu, Norihisa Ichinohe, Yasushi Sasaki, Tohru Itoh, Ryo Sudo, Tomoko Yamaguchi, Takeshi Katsuda, Takafumi Ninomiya, Takashi Tokino, Takahiro Ochiya, Atsushi Miyajima, Toshihiro Mitaka

Nature communications 12 ( 1 ) 3390 - 3390 2021年06月 [国際誌]

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

DOI PubMed

カスタムパネルシーケンスを用いた日本人メラノーマのドライバー遺伝子の検出

肥田時征, 井戸川雅史, 堀本浩平, 加藤潤史, 佐藤さゆり, 藤岡茉生, 丹下正一朗, 時野隆至, 宇原久

日本皮膚科学会雑誌 ( (公社)日本皮膚科学会 ) 131 ( 5 ) 1363 - 1363 2021年05月

ALK-positive atypical Spitz tumour with conspicuous rosette-like structures.

Tomoyuki Minowa, Tokimasa Hida, Kohei Horimoto, Junji Kato, Takafumi Kamiya, Shintaro Sugita, Masashi Idogawa, Toshiaki Saida, Tadashi Hasegawa, Takashi Tokino, Hisashi Uhara

European journal of dermatology : EJD 31 ( 2 ) 256 - 258 2021年04月 [国際誌]

DOI PubMed

Circulating tumor DNAの初期変動を用いた食道癌化学療法効果予測に関する検討

藤澤良介, 岩谷岳, 遠藤史隆, 佐々木教之, 二階春香, 馬場誠朗, 秋山有史, 開勇人, 小泉優香, 阿部正和, 片桐弘勝, 木村聡元, 大塚幸喜, 井戸川雅史, 新田浩幸, 佐々木章, 佐々木泰史, 時野隆至, 西塚哲

日本外科学会定期学術集会抄録集 ( (一社)日本外科学会 ) 121回 SF - 1 2021年04月

Circulating tumor DNAの初期変動を用いた食道癌化学療法効果予測に関する検討

日本外科学会定期学術集会抄録集 ( (一社)日本外科学会 ) 121回 SF - 1 2021年04月

メラノーマに特化したシーケンスパネルの開発

肥田時征, 堀本浩平, 加藤潤史, 佐藤さゆり, 藤岡茉生, 宇原久, 井戸川雅史, 丹下正一朗, 時野隆至

日本皮膚科学会雑誌 ( (公社)日本皮膚科学会 ) 131 ( 3 ) 541 - 542 2021年03月

Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction.

Takeshi Iwaya, Fumitaka Endo, Fumiaki Takahashi, Takashi Tokino, Yasushi Sasaki, Satoshi S Nishizuka

Gastroenterology 160 ( 1 ) 463 - 465 2021年01月 [国際誌]

DOI PubMed

Mutation profiling of uterine cervical cancer patients treated with definitive radiotherapy.

Yuya Yoshimoto, Yasushi Sasaki, Kazutoshi Murata, Shin-Ei Noda, Yuhei Miyasaka, Junko Hamamoto, Mio Furuya, Junko Hirato, Yoshiyuki Suzuki, Tatsuya Ohno, Takashi Tokino, Takahiro Oike, Takashi Nakano

Gynecologic oncology 159 ( 2 ) 546 - 553 2020年11月 [国際誌]

OBJECTIVE: To elucidate tumor mutation profiles associated with outcomes of uterine cervical cancer (UCC) patients treated with definitive radiotherapy. METHODS: Ninety-eight patients with newly diagnosed and pathologically confirmed UCC (82 squamous cell carcinomas, 12 adenocarcinomas, and four adenosquamous carcinomas) who were treated with definitive radiotherapy were analyzed. DNA was extracted from pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next-generation sequencer. Genetic mutations were identified and analyzed for correlations with clinical outcome. RESULTS: Recurrent mutations were observed in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes (i.e., FGFR1-4) was almost as high (24.5%) as that in PIK3CA and ARID1A, both of which are well-studied drivers of UCC. Fifty-five percent (21 of 38) of the identified FGFR mutations were located in the FGFR protein tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%, respectively; P = 0.010). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS (P = 0.005), independent of clinicopathological variables. CONCLUSIONS: FGFR mutations are associated with worse PFS in UCC patients treated with definitive radiotherapy. These results warrant further validation in prospective studies.

DOI PubMed

Neurofibromatosis type 1 without cutaneous neurofibromas: a rare genotype-phenotype correlation?

Tokimasa Hida, Masashi Idogawa, Aki Ishikawa, Miyako Mizukami, Junji Kato, Yasuyuki Sumikawa, Masae Okura, Takashi Tokino, Hisashi Uhara

European journal of dermatology : EJD 30 ( 5 ) 608 - 609 2020年10月 [国際誌]

DOI PubMed

p53標的遺伝子ARVCFはスプライシング変化によりp53の腫瘍抑制能を補助する

井戸川雅史, 鈴木菜摘, 丹下正一朗, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 79回 OJ4 - 3 2020年10月

予後不良膵臓癌のマーカー候補となる新規上皮特異的遺伝子の特定

丹下正一郎, 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 ) 79回 PJ15 - 6 2020年10月

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年10月

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年08月 [国際誌]

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

Integrated genetic and epigenetic analysis of cancer-related genes in non-ampullary duodenal adenomas and intramucosal adenocarcinomas.

Ryosuke Ota, Takeshi Sawada, Sho Tsuyama, Yasushi Sasaki, Hiromu Suzuki, Yasuharu Kaizaki, Kenkei Hasatani, Eiichiro Yamamoto, Hiroyoshi Nakanishi, Satoko Inagaki, Shigetsugu Tsuji, Naohiro Yoshida, Hisashi Doyama, Hiroshi Minato, Keishi Nakamura, Satomi Kasashima, Eiji Kubota, Hiromi Kataoka, Takashi Tokino, Takashi Yao, Toshinari Minamoto

The Journal of pathology 252 ( 3 ) 330 - 342 2020年08月 [査読有り] [国際誌]

The molecular and clinical characteristics of non-ampullary duodenal adenomas and intramucosal adenocarcinomas are not fully understood because they are rare. To clarify these characteristics, we performed genetic and epigenetic analysis of cancer-related genes in these lesions. One-hundred seven non-ampullary duodenal adenomas and intramucosal adenocarcinomas, including 100 small intestinal type tumors (90 adenomas and 10 intramucosal adenocarcinomas) and 7 gastric type tumors (2 pyloric gland adenomas and 5 intramucosal adenocarcinomas), were investigated. Using bisulfite pyrosequencing, we assessed the methylation status of CpG island methylator phenotype (CIMP) markers and MLH1. Then using next-generation sequencing, we performed targeted exome sequence analysis within 75 cancer-related genes in 102 lesions. There were significant differences in the clinicopathological and molecular variables between small intestinal and gastric type tumors, which suggests the presence of at least two separate carcinogenic pathways in non-ampullary duodenal adenocarcinomas. The prevalence of CIMP-positive lesions was higher in intramucosal adenocarcinomas than adenomas. Thus, concurrent hypermethylation of multiple CpG islands is likely associated with development of non-ampullary duodenal intramucosal adenocarcinomas. Mutation analysis showed that APC was the most frequently mutated gene in these lesions (56/102; 55%), followed by KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS (8/102; 8%), ERBB3 (7/102; 7%), and RNF43 (6/102; 6%). Additionally, the high prevalence of diffuse or focal nuclear β-catenin accumulation (87/102; 85%) as well as mutations of WNT pathway components (60/102; 59%) indicate the importance of WNT signaling to the initiation of duodenal adenomas. The higher than previously reported frequency of APC gene mutations in small bowel adenocarcinomas as well as the difference in the APC mutation distributions between small intestinal type adenomas and intramucosal adenocarcinomas may indicate the adenoma-carcinoma sequence has only limited involvement in duodenal carcinogenesis. This article is protected by copyright. All rights reserved.

DOI PubMed

Interactive Web-based Graphs of Coronavirus Disease 2019 Cases and Deaths per Population by Country.

Masashi Idogawa, Shoichiro Tange, Hiroshi Nakase, Takashi Tokino

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 71 ( 15 ) 902 - 903 2020年07月 [査読有り] [国際誌]

DOI PubMed

Combination of KRAS and SMAD4 mutations in formalin-fixed paraffin-embedded tissues as a biomarker for pancreatic cancer.

Takahiro Yokose, Minoru Kitago, Sachiko Matsuda, Yasushi Sasaki, Yohei Masugi, Yuki Nakamura, Masahiro Shinoda, Hiroshi Yagi, Yuta Abe, Go Oshima, Shutaro Hori, Fujita Yusuke, Yutaka Nakano, Yutaka Endo, Kodai Abe, Takashi Tokino, Yuko Kitagawa

Cancer science 111 ( 6 ) 2174 - 2182 2020年06月 [査読有り] [国際誌]

Formalin-fixed paraffin-embedded (FFPE) tissues used for routine pathological diagnosis are valuable for cancer genomic analysis; however, the association between mutation status derived from these specimens and prognosis in pancreatic ductal adenocarcinoma (PDAC) remains unclear. We analyzed 50 cancer-related gene mutations including driver genes in PDAC, using next-generation sequencing (NGS) to clarify the association between gene mutations and prognosis. DNA was extracted from FFPE tissues obtained from 74 patients with untreated resectable PDAC who underwent surgery at our institution between 2013 and 2018. Fifty of the 74 patients with DNA extracts from FFPE samples suitable for NGS were analyzed. The prevalence of driver gene mutations was as follows: 84% for KRAS, 62% for TP53, 32% for SMAD4, and 18% for CDKN2A. There were no cases of single SMAD4 mutations; its rate of coincidence with KRAS or TP53 mutations was 30% and 2%, respectively. The combination of KRAS and SMAD4 mutations resulted in significantly shorter relapse-free survival (RFS; median survival time [MST], 12.3 vs. 28.9 months, P = .014) and overall survival (OS; MST, 22.3 months vs. not reached, P = .048). On multivariate analysis, the combination of KRAS and SMAD4 mutations was an independent prognostic factor for RFS (hazard ratio [HR] 4.218; 95% confidence interval [CI], 1.77-10.08; P = .001) and OS (HR 6.730; 95% CI, 1.93-23.43; P = .003). The combination of KRAS and SMAD4 mutations in DNA obtained from FFPE tissues is an independent poor prognostic factor in PDAC.

DOI PubMed

Genetic analyses of mosaic neurofibromatosis type 1 with giant café-au-lait macule, plexiform neurofibroma and multiple melanocytic nevi.

Tokimasa Hida, Masashi Idogawa, Masae Okura, Shintaro Sugita, Taro Sugawara, Yasushi Sasaki, Takashi Tokino, Toshiharu Yamashita, Hisashi Uhara

The Journal of dermatology 47 ( 6 ) 658 - 662 2020年04月 [査読有り] [国際誌]

Neurofibromatosis type 1 (NF1) is a genodermatosis caused by heterozygous germ line variations in the NF1 gene. A second-hit NF1 aberration results in the formation of café-au-lait macules, cutaneous neurofibroma and plexiform neurofibroma (PNF). Mosaic NF1 (mNF1), caused by a postzygotic NF1 mutation, is characterized by localized or generalized NF1-related manifestations. Although NF1 and mNF1 are associated with pigmentary skin lesions, clinically recognizable melanocytic nevi that developed over PNF have not been reported. Here, we report the first case of multiple melanocytic nevi that developed on a giant café-au-lait macule and PNF. The PNF had biallelic NF1 deletions, a whole deletion of NF1 and a novel intragenic deletion involving exons 25-30. The deletions were not detected in the blood, which resulted in the diagnosis of mNF1. Furthermore, the nevus cells had not only biallelic NF1 deletions but also NRAS Q61R, a common mutation found in congenital melanocytic nevi. These analyses revealed the coexistence of the two different mosaic diseases, mNF1 and congenital melanocytic nevi. For a diagnosis of cases with atypical NF1-like symptoms, genetic analyses using blood and lesional tissues are useful and aid in genetic counseling.

DOI PubMed

Renal angiomyolipoma (AML) harboring a missense mutation of TSC2 with copy-neutral loss of heterozygosity (CN-LOH).

Masashi Idogawa, Tokimasa Hida, Toshiaki Tanaka, Noriaki Ohira, Shoichiro Tange, Yasushi Sasaki, Hisashi Uhara, Naoya Masumori, Takashi Tokino, Hiroshi Natori

Cancer biology & therapy 21 ( 4 ) 315 - 319 2020年04月 [査読有り] [国際誌]

Angiomyolipoma (AML) is classified as a perivascular epithelioid cell neoplasm, mostly occurring in the kidney. Twenty percent of patients with renal AML have tuberous sclerosis complex (TSC) caused by germline variation in the TSC1 or TSC2 gene. In this paper, we report the first case of renal AML harboring somatic missense mutations of the TSC2 gene and concomitant copy-neutral loss of heterozygosity (CN-LOH). The patient presented with solitary renal AML and pulmonary lymphangiomyomatosis and without other findings suggestive of TSC. Exome sequencing analysis of the renal AML, however, identified a pathogenic somatic missense mutation in the TSC2 gene (NM_000548:c.5228G>A:p. R1743Q), although no other somatic mutation was detected. Furthermore, no germline mutation in TSC1 or TSC2 was detected. Interestingly, the mutant allele ratio was too high for a somatic heterozygous mutation without loss of heterozygosity (LOH). Furthermore, no copy number variation was detected around the TSC2 locus (16p13.3). To clarify the allelic status, we analyzed heterozygous single-nucleotide polymorphisms (SNPs) in chromosome 16. In these SNPs, an unbalanced allele ratio was accumulated inside the 16p13.3 region. These results suggested copy-neutral LOH (CN-LOH). Consequently, we concluded that the missense mutation of the TSC2 gene and CN-LOH of the TSC2 locus caused renal AML.

DOI PubMed

p53-induced ARVCF modulates the splicing landscape and supports the tumor suppressive function of p53.

Natsumi Suzuki, Masashi Idogawa, Shoichiro Tange, Tomoko Ohashi, Yasushi Sasaki, Hiroshi Nakase, Takashi Tokino

Oncogene 39 ( 10 ) 2202 - 2211 2020年03月 [査読有り] [国際誌]

p53 is one of the most important tumor suppressor genes, and the exploration of p53-target genes is important for elucidation of its functional mechanisms. In this study, we identified Armadillo Repeat gene deleted in Velo-Cardio-Facial syndrome (ARVCF) as a direct target of p53 through ChIP-sequencing analysis. Activated p53 protein was found to bind to two distinct sites in the ARVCF gene, resulting in induction of ARVCF expression at both the mRNA and protein levels. We revealed that the knockdown of ARVCF inhibited p53-induced apoptosis. Interestingly, ARVCF interacted with hnRNPH2, which is involved in pre-mRNA splicing, and ARVCF knockdown induced dynamic changes in alternative splicing patterns. These results suggest that p53-induced ARVCF indirectly, but not directly, regulates p53 target selectivity through splicing alterations of specific genes. Thus, we demonstrated that the induction of ARVCF expression contributed to the tumor suppressive function of p53. Recently, it has been reported that many tumors have thousands of alternative splicing events that are not detectable in normal samples. ARVCF may play a role in alternative splicing events in cancer and may provide clues to explore novel approaches for cancer diagnosis and therapy.

DOI PubMed

Chemotherapy after progression on nivolumab is essential for responders with genetic alterations of driver gene: Review of two recurrent/metastatic oral squamous cell carcinoma patients.

Kazuhiro Ogi, Junichi Kobayashi, Takafumi Nakagaki, Junya Okamoto, Kazushige Koike, Naoki Hirokawa, Masanori Someya, Hiroki Sakamoto, Kohichi Takada, Takashi Tokino, Yasushi Sasaki, Hiroyoshi Hiratsuka, Akihiro Miyazaki

Oral oncology 102 104509 - 104509 2020年03月 [査読有り] [国際誌]

DOI PubMed

Pathogenesis of multiple pancreatic cancers involves multicentric carcinogenesis and intrapancreatic metastasis.

Yusuke Fujita, Sachiko Matsuda, Yasushi Sasaki, Yohei Masugi, Minoru Kitago, Hiroshi Yagi, Yuta Abe, Masahiro Shinoda, Takashi Tokino, Michiie Sakamoto, Yuko Kitagawa

Cancer science 111 ( 2 ) 739 - 748 2020年02月 [査読有り] [国際誌]

There are increased opportunities in oncology clinics to identify multiple pancreatic ductal adenocarcinomas (PDAC) that co-occur simultaneously or arise metachronously in the pancreatic parenchyma, yet their pathogenesis remains elusive. We hypothesized that two potential pathways, multicentric carcinogenesis and intrapancreatic metastasis, might contribute to forming multiple PDAC. Among 241 resected cases, we identified 20 cancer nodules from nine patients with multiple PDAC (six with synchronous PDAC, one with metachronous PDAC, and two with both synchronous and metachronous PDAC). Integrated clinical, pathological, and mutational analyses, using TP53 and SMAD4 immunostaining and targeted next-generation sequencing of 50 cancer-related genes, were conducted to examine the intertumor relationships. Four of the nine patients were assessed as having undergone multicentric carcinogenesis because of heterogeneity of immunohistochemical and/or mutation characteristics. In contrast, tumors in the other five patients showed intertumor molecular relatedness. Two of these five patients, available for matched sequencing data, showed two or more shared mutations. Moreover, all the smaller nodules in these five patients showed identical TP53 and SMAD4 expression patterns to the corresponding main tumors. Consequently, these five patients were considered to have undergone intrapancreatic metastasis. None of the five smaller nodules arising from intrapancreatic metastasis was accompanied by pancreatic intraepithelial neoplasia, and three of them were tiny (≤1mm). Patients whose tumors resulted from intrapancreatic metastasis appeared to have higher disease stages and worse outcome than those with tumors from multicentric carcinogenesis. Our results provide insight into pancreatic carcinogenesis, showing that the development of multiple PDAC involves distinct evolutionary paths that potentially affect patient prognosis.

DOI PubMed

神経線維腫を伴わない神経線維腫症1型2家系の遺伝子解析

肥田時征, 井戸川雅史, 石川亜貴, 水上都, 加藤潤史, 澄川靖之, 時野隆至, 宇原久

日本レックリングハウゼン病学会学術大会プログラム・抄録集 ( 日本レックリングハウゼン病学会 ) 11回 15 - 15 2020年02月

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年 [査読有り] [国際誌]

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

Whole-exome sequencing of 79 xenografts as a potential approach for the identification of genetic variants associated with sensitivity to cytotoxic anticancer drugs.

Chihiro Udagawa, Yasushi Sasaki, Yasuhiro Tanizawa, Hiroshi Suemizu, Yasuyuki Ohnishi, Yasukazu Nakamura, Takashi Tokino, Hitoshi Zembutsu

PloS one 15 ( 9 ) e0239614 2020年 [国際誌]

Chemotherapy response remains unpredictable in most patients with cancer. In this study, we performed whole-exome sequencing of 79 cancer xenografts derived from human cancer tissues to identify genetic predictors of chemosensitivity to nine cytotoxic anticancer drugs. Xenografts were harvested from 12 organs with cancer and implanted into nude mice. The mice were exposed to one of nine cytotoxic anticancer drugs (5-fluorouracil, nimustine, adriamycin, cyclophosphamide, cisplatin, mitomycin C, methotrexate, vincristine, and vinblastine) to assess the correlation between chemosensitivity response and variant allele frequency. We found 162 candidate variants that were possibly associated with chemosensitivity to one or more of the nine anticancer drugs (P < 0.01). In a subgroup analysis of breast and gastric cancer xenografts, 78 and 67 variants, respectively, were possibly associated with chemosensitivity. This approach may help to contribute to the development of personalized treatments that may allow for the prescription of optimal chemotherapy regimens among patients with cancer.

DOI PubMed

Genomic characterization for familial cases with urothelial carcinoma.

Tetsuya Shindo, Megumi Hirobe, Yasushi Adachi, Yasushi Sasaki, Takashi Tokino, Naoya Masumori

International cancer conference journal 8 ( 4 ) 185 - 189 2019年10月 [査読有り] [国際誌]

In this report, we present familial cases of urothelial carcinoma. To investigate the possibility of hereditary urothelial cancer, we performed semiconductor-based next-generation DNA sequencing. A woman in her 80s who had bladder and left ureteral cancer was hospitalized in Sapporo Shirakaba-dai Hospital due to consciousness disturbance. Radiographic evaluation revealed multiple liver metastases and she died 38 days later. Needle necropsy was done for a left ureteral tumor that continued to her bladder tumor and for liver metastases. At the same time, her son in his 60s, who also had muscle-invasive bladder cancer, was admitted to Sapporo Medical University Hospital and underwent neoadjuvant chemotherapy followed by laparoscopic radical cystectomy. DNA was isolated from both cancers and normal controls in each case and analyzed by massive parallel sequencing of 409 cancer-related genes using a targeted, multiplex PCR approach followed by semiconductor sequencing. Somatic mutations of KMT2C and KMT2D were detected in the mother's tumor. Copy number gains of FGFR1, IkBKB, NFkB2, FGFR2, and FLT3 and copy number losses of IGF2R and TP53 were also found in her cancer. In her son's tumor, somatic mutations of FGFR3 and EP300 were identified. Copy number gains of IkBKE/MAPK1/PARP1, EGFR, BRAF, IRS2, MAPK2K1, IGF1R, and ERBB2 and copy number loss of TP53 were also found in his cancer. There were no germline gene mutations related to familial urothelial carcinoma. Although somatic mutation of TP53 was a common feature, these cases with urothelial carcinoma might not be the result of a heredity syndrome.

DOI PubMed

FGFR Signaling as a Candidate Therapeutic Target for Cancers Resistant to Carbon Ion Radiotherapy.

Narisa Dewi Maulany Darwis, Ankita Nachankar, Yasushi Sasaki, Toshiaki Matsui, Shin-Ei Noda, Kazutoshi Murata, Tomoaki Tamaki, Ken Ando, Noriyuki Okonogi, Shintaro Shiba, Daisuke Irie, Takuya Kaminuma, Takuya Kumazawa, Mai Anakura, Souichi Yamashita, Takashi Hirakawa, Sangeeta Kakoti, Yuka Hirota, Takashi Tokino, Akira Iwase, Tatsuya Ohno, Atsushi Shibata, Takahiro Oike, Takashi Nakano

International journal of molecular sciences 20 ( 18 ) 2019年09月 [査読有り] [国際誌]

Radiotherapy is an essential component of cancer therapy. Carbon ion radiotherapy (CIRT) promises to improve outcomes compared with standard of care in many cancers. Nevertheless, clinicians often observe in-field recurrence after CIRT. This indicates the presence of a subset of cancers that harbor intrinsic resistance to CIRT. Thus, the development of methods to identify and sensitize CIRT-resistant cancers is needed. To address this issue, we analyzed a unique donor-matched pair of clinical specimens: a treatment-naïve tumor, and the tumor that recurred locally after CIRT in the same patient. Exon sequencing of 409 cancer-related genes identified enrichment of somatic mutations in FGFR3 and FGFR4 in the recurrent tumor compared with the treatment-naïve tumor, indicating a pivotal role for FGFR signaling in cancer cell survival through CIRT. Inhibition of FGFR using the clinically available pan-FGFR inhibitor LY2874455 sensitized multiple cancer cell lines to carbon ions at 3 Gy (RBE: relative biological effectiveness), the daily dose prescribed to the patient. The sensitizer enhancement ratio was 1.66 ± 0.17, 1.27 ± 0.09, and 1.20 ± 0.18 in A549, H1299, and H1703 cells, respectively. Our data indicate the potential usefulness of the analytical pipeline employed in this pilot study to identify targetable mutations associated with resistance to CIRT, and of LY21874455 as a sensitizer for CIRT-resistant cancers. The results warrant validation in larger cohorts.

DOI PubMed

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年09月

変異TP53遺伝子との相関が確認された新規遺伝子群の発見(Identification of novel TP53 mutation-associated genes through pan-cancer analysis)

丹下正一郎, 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 ) 78回 P - 1192 2019年09月

新規p53標的遺伝子ARVCFはスプライシング変化を誘導し腫瘍抑制に寄与する(ARVCF, a novel p53 target, modulates the splicing landscape and supports the tumor suppressive function of p53)

井戸川雅史, 鈴木菜摘, 丹下正一郎, 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 ) 78回 J - 3018 2019年09月

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年09月

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年05月 [査読有り] [国際誌]

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

A subset of diffuse-type gastric cancer is susceptible to mTOR inhibitors and checkpoint inhibitors.

Hiroshi Fukamachi, Seon-Kyu Kim, Jiwon Koh, Hye Seung Lee, Yasushi Sasaki, Kentaro Yamashita, Taketo Nishikawaji, Shu Shimada, Yoshimitsu Akiyama, Sun-Ju Byeon, Dong-Hyuck Bae, Keisuke Okuno, Masatoshi Nakagawa, Toshiro Tanioka, Mikito Inokuchi, Hiroshi Kawachi, Kiichiro Tsuchiya, Kazuyuki Kojima, Takashi Tokino, Yoshinobu Eishi, Yong Sung Kim, Woo Ho Kim, Yasuhito Yuasa, Shinji Tanaka

Journal of experimental & clinical cancer research : CR 38 ( 1 ) 127 - 127 2019年03月 [査読有り] [国際誌]

BACKGROUND: Mechanistic target of rapamycin (mTOR) pathway is essential for the growth of gastric cancer (GC), but mTOR inhibitor everolimus was not effective for the treatment of GCs. The Cancer Genome Atlas (TCGA) researchers reported that most diffuse-type GCs were genomically stable (GS). Pathological analysis suggested that some diffuse-type GCs developed from intestinal-type GCs. METHODS: We established patient-derived xenograft (PDX) lines from diffuse-type GCs, and searched for drugs that suppressed their growth. Diffuse-type GCs were classified into subtypes by their gene expression profiles. RESULTS: mTOR inhibitor temsirolimus strongly suppressed the growth of PDX-derived diffuse-type GC-initiating cells, which was regulated via Wnt-mTOR axis. These cells were microsatellite unstable (MSI) or chromosomally unstable (CIN), inconsistent with TCGA report. Diffuse-type GCs in TCGA cohort could be classified into two clusters, and GS subtype was major in cluster I while CIN and MSI subtypes were predominant in cluster II where PDX-derived diffuse-type GC cells were included. We estimated that about 9 and 55% of the diffuse-type GCs in cluster II were responders to mTOR inhibitors and checkpoint inhibitors, respectively, by identifying PIK3CA mutations and MSI condition in TCGA cohort. These ratios were far greater than those of diffuse-type GCs in cluster I or intestinal-type GCs. Further analysis suggested that diffuse-type GCs in cluster II developed from intestinal-type GCs while those in cluster I from normal gastric epithelial cells. CONCLUSION: mTOR inhibitors and checkpoint inhibitors might be useful for the treatment of a subset of diffuse-type GCs which may develop from intestinal-type GCs.

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年01月 [査読有り] [国際誌]

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

Prognostic Effect of Long Noncoding RNA NEAT1 Expression Depends on p53 Mutation Status in Cancer.

Masashi Idogawa, Hiroshi Nakase, Yasushi Sasaki, Takashi Tokino

Journal of oncology 2019 4368068 - 4368068 2019年 [査読有り] [国際誌]

Recently, many studies have revealed that long noncoding RNAs (lncRNAs) play important roles in various biological and pathological processes. Our previous study reported that lncRNA NEAT1 is a direct transcriptional target of p53. NEAT1 is an essential component of paraspeckles, which have recently been identified as a novel type of nuclear compartment. Although our previous findings indicate that NEAT1 induction contributes to the tumor-suppressor function of p53, the role of NEAT1 in cancer is still controversial. In this study, we comprehensively analyzed the relationship between NEAT1 expression and p53 mutation status. Interestingly, survival analysis based on NEAT1 expression in several cancer tissues revealed that the p53 wild-type group with high NEAT1 expression had a good prognosis, while poor prognosis or no correlation between NEAT1 expression and survival was observed in the p53-mutated group. These results demonstrate that the tumor-suppressive effect of NEAT1 depends on p53 function and is consistent with our previous report showing that NEAT1 supports the tumor-suppressive function of p53. Specifically, NEAT1 seems to play a tumor-suppressive role only in the presence of wild-type p53. These results provide important clues to the roles of NEAT1 in cancer.

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年12月 [査読有り] [国内誌]

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

Exome sequence analysis of carcinomatous and sarcomatous elements of an esophageal carcinosarcoma

Sasaki Noriyuki, Iwaya Takeshi, Endo Fumitaka, Konosu Masafumi, Akiyama Yuji, Sasaki Yasushi, Tokino Takashi, Nishizuka Satoshi

CANCER SCIENCE 109 216 2018年12月 [査読有り]

Patient-specific circulating tumor DNA monitoring using digital PCR in esophageal squamous cell cancer patients

Iwaya Takeshi, Endo Fumitaka, Sasaki Yasushi, Yaegashi Mizunori, Sato Kei, Sasaki Noriyuki, Akiyama Yuji, Sasaki Akira, Masuda Mari, Tokino Takashi, Nishizuka Satoshi

CANCER SCIENCE 109 708 2018年12月 [査読有り]

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年10月 [査読有り] [国際誌]

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

Targeted next-generation sequencing of 50 cancer-related genes in Japanese patients with oral squamous cell carcinoma.

Nakagaki T, Tamura M, Kobashi K, Omori A, Koyama R, Idogawa M, Ogi K, Hiratsuka H, Tokino T, Sasaki Y

Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 40 ( 9 ) 1010428318800180 - 1010428318800180 2018年09月 [査読有り] [国際誌]

Somatic mutation analysis is a standard of practice for human cancers to identify therapeutic sensitization and resistance mutations. We performed a multigene sequencing screen to explore mutational hotspots in cancer-related genes using a semiconductor-based sequencer. DNA from oral squamous cell carcinoma samples was used as a template to amplify 207 regions from 50 cancer-related genes. Of the 80 oral squamous cell carcinoma specimens from Japanese patients, including formalin-fixed paraffin-embedded samples, 56 specimens presented at least one somatic mutation among the 50 investigated genes, and 17 of these samples showed multiple gene somatic mutations. TP53 was the most commonly mutated gene (50.0%), followed by CDKN2A (16.3%), PIK3CA (7.5%), HRAS (5.0%), MET (2.5%), and STK11 (2.5%). In total, 32 cases (40.0%) were human papillomavirus positive and they were significantly less likely to have a TP53, mutation than human papillomavirus-negative oral squamous cell carcinomas (8/32, 25.0% vs 32/48, 66.7%, p = 0.00026). We also detected copy number variations, in which segments of the genome could be duplicated or deleted from the sequencing data. We detected the tumor-specific TP53 mutation in the plasma cell-free DNA from two oral squamous cell carcinoma patients, and after surgery, the test for these mutations became negative. Our approach facilitates the simultaneous high-throughput detection of somatic mutations and copy number variations in oral squamous cell carcinoma samples.

DOI PubMed

中枢神経系原発悪性リンパ腫におけるIon Reporterエクソームシーケンシングによる分子マーカー候補の同定(Identification of molecular marker candidate by Ion Reporter exome sequencing in primary central nervous system lymphoma)

高島康郎, 佐々木泰史, 早野あづさ, 本間順平, 深井順也, 岩立康男, 梶原浩司, 石澤伸, 本道洋昭, 時野隆至, 山中龍也

日本癌学会総会記事 ( 日本癌学会 ) 77回 2381 - 2381 2018年09月 [査読有り]

Digital PCRを用いた食道癌患者における症例特異的血漿中遊離DNAモニタリング(Patient-specific circulating tumor DNA monitoring using digital PCR in esophageal squamous cell cancer patients)

岩谷岳, 遠藤史隆, 佐々木泰史, 八重樫瑞典, 佐藤慧, 佐々木教之, 秋山有史, 佐々木章, 増田万里, 時野隆至, 西塚哲

日本癌学会総会記事 ( 日本癌学会 ) 77回 930 - 930 2018年09月 [査読有り]

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年09月

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年09月

日本人口腔扁平上皮癌における癌関連50遺伝子を標的とした次世代シークエンサーによる解析(Targeted next-generation sequencing of 50 cancer-related genes in Japanese patients with oral squamous cell carcinoma)

荻和弘, 中垣貴文, 井戸川雅史, 宮崎晃亘, 時野隆至, 佐々木泰史

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 784 - 784 2018年09月

新規p53標的遺伝子として同定されたp120カテニンファミリー蛋白の癌における役割(The roles of p120 catenin family protein as a novel p53 target in cancer)

鈴木菜摘, 井戸川雅史, 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 ) 77回 1301 - 1301 2018年09月

大腸癌における発現ネットワーク解析によるハブ長鎖非コードRNA(hub-lncRNA)の同定と機能解析(Identification of hub-long non-coding RNAs(lncRNAs) by the network analysis of lncRNA expression in colorectal cancers)

井戸川雅史, 鈴木菜摘, 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 ) 77回 463 - 463 2018年09月

食道癌肉腫の遺伝子変異解析(Exome sequence analysis of carcinomatous and sarcomatous elements of an esophageal carcinosarcoma)

佐々木教之, 岩谷岳, 遠藤史隆, 鴻巣正史, 秋山有史, 佐々木泰史, 時野隆至, 西塚哲

日本癌学会総会記事 ( (一社)日本癌学会 ) 77回 326 - 326 2018年09月

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年09月

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年09月

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年09月

Mutational analysis of uterine cervical cancer that survived multiple rounds of radiotherapy.

Endang Nuryadi, Yasushi Sasaki, Yoshihiko Hagiwara, Tiara Bunga Mayang Permata, Hiro Sato, Shuichiro Komatsu, Yuya Yoshimoto, Kazutoshi Murata, Ken Ando, Nobuteru Kubo, Noriyuki Okonogi, Yosuke Takakusagi, Akiko Adachi, Mototaro Iwanaga, Keisuke Tsuchida, Tomoaki Tamaki, Shin-Ei Noda, Yuka Hirota, Atsushi Shibata, Tatsuya Ohno, Takashi Tokino, Takahiro Oike, Takashi Nakano

Oncotarget 9 ( 66 ) 32642 - 32652 2018年08月 [査読有り] [国際誌]

Radiotherapy is an essential component of cancer therapy. Despite advances in cancer genomics, the mutation signatures of radioresistant tumors have not yet been fully elucidated. To address this issue, we analyzed a unique set of clinical specimens from a uterine cervical cancer that repeatedly locally recurred after multiple rounds of radiotherapy. Exon sequencing of 409 cancer-related genes in the treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy identified (i) activating mutations in PIK3CA and KRAS, and putative inactivating mutations in SMAD4, as trunk mutation signatures that persisted over the clinical course; and (ii) mutations in KMT2A, TET1, and NLRP1 as acquired mutation signatures observed only in recurrent tumors after radiotherapy. Comprehensive mining of published in vitro genomics data pertaining to radiosensitivity revealed that simultaneous mutations in KRAS and SMAD4, which have not been described previously in uterine cervical cancer, are associated with cancer cell radioresistance. The association between this mutation signature and radioresistance was validated by isogenic cell-based experiments. These results provide proof-of-principle for the analytical pipeline employed in this study, which explores clinically relevant mutation signatures for radioresistance, and demonstrate that this approach is worth pursuing with larger cohorts in the future.

DOI PubMed

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

Nishiyama K, Maruyama R, Niinuma T, Kai M, Kitajima H, Toyota M, Hatanaka Y, Igarashi T, Kobayashi JI, Ogi K, Dehari H, Miyazaki A, Yorozu A, Yamamoto E, Idogawa M, Sasaki Y, Sugai T, Tokino T, Hiratsuka H, Suzuki H

Cell death & disease 9 ( 8 ) 826 - 826 2018年08月 [査読有り] [国際誌]

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

Target amplicon exome-sequencing identifies promising diagnosis and prognostic markers involved in RTK-RAS and PI3K-AKT signaling as central oncopathways in primary central nervous system lymphoma.

Takashima Y, Sasaki Y, Hayano A, Homma J, Fukai J, Iwadate Y, Kajiwara K, Ishizawa S, Hondoh H, Tokino T, Yamanaka R

Oncotarget 9 ( 44 ) 27471 - 27486 2018年06月 [査読有り] [国際誌]

Exome-sequencing for somatic mutation detection and copy number variation analysis are effective and valid methods for evaluating human cancers in current molecular medicine. We conducted target amplicon exome-sequencing analyses using PCR target enrichment and next-generation sequencing on Ion Proton semiconductor sequencers. Twenty-seven primary central nervous system lymphoma (PCNSL) specimens and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverages of the entire coding regions of 409 cancer-related genes. The average of the total numbers of somatic mutations including single-nucleotide variations and insertion/deletion mutations in each specimen was 13.3. Of these, the average of the ratios of nonsynonymous substitutions in each specimen was 74.8%. The most frequent mutations in 27 specimens were in PIM1, MYD88, CD79B, DST, IRF4, ERBB3, MYH11, DCC, and KMT2D. Furthermore, somatic mutations of MYH11 were related to poor prognoses in PCNSL patients. Copy number variations were also duplicated and/or deleted from deep-sequencing in segmental genomic islands. In addition to these prognostic marker candidates, analysis of RTK-RAS-MAPK signaling and the PTEN-PI3K-AKT proapoptotic pathway showed that somatic activations and aberrations, respectively, may be involved in a promising central oncopathway harboring mTOR, c-Myc, FOXO1, and p53. This study provides a foundation for molecular targeted therapies based on genome diagnostics and prognosis in PCNSL.

DOI PubMed

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

Shindo T, Niinuma T, Nishiyama N, Shinkai N, Kitajima H, Kai M, Maruyama R, Tokino T, Masumori N, Suzuki H

Oncotarget 9 ( 36 ) 24457 - 24469 2018年05月 [査読有り] [国際誌]

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

Targeted sequencing reveals genetic variants associated with sensitivity of 79 human cancer xenografts to anticancer drugs.

Udagawa C, Sasaki Y, Suemizu H, Ohnishi Y, Ohnishi H, Tokino T, Zembutsu H

Experimental and therapeutic medicine 15 ( 2 ) 1339 - 1359 2018年02月 [査読有り] [国際誌]

Although there has been progress moving from a 'one-size-fits-all' cytotoxic approach to personalized molecular medicine, the majority of patients with cancer receive chemotherapy using cytotoxic anticancer drugs. The sequencing analysis of 409 genes associated with cancer was conducted in the present study using 59 DNA sequences extracted from human cancer xenografts implanted into nude mice, of which sensitivity to 9 cytotoxic anticancer drugs [5-fluorouracil, nimustine, adriamycin, cyclophosphamide, cisplatin, mitomycin C (MMC), methotrexate, vincristine (VCR), and vinblastine] was examined. The present study investigated the association between the sensitivities of the xenografts to the 9 anticancer drugs and the frequency of single nucleotide variants (SNV). The correlation between the expression level of the genes and sensitivities to the 9 drugs in the above xenografts was also estimated. In the screening study using 59 xenografts, 3 SNVs (rs1805321, rs62456182 in PMS1 Homolog 2, Mismatch Repair System Component and rs13382825 in LDL Receptor Related Protein 1B), were associated with sensitivity to VCR and MMC, respectively (P<0.001). A replication study of 596 SNVs was subsequently performed, which indicated P<0.05 in the screening study using independent samples of 20 xenografts. A combined result of the screening and replication studies indicated that 35 SNVs were potentially associated with sensitivities to one or more of the nine anticancer drugs (Pcombined=0.0011-0.035). Of the 35 SNVs, rs16903989 and rs201432181 in Leukemia Inhibitory Factor Receptor α and Adhesion G Protein-Coupled Receptor A2 were commonly associated with sensitivity to 2 or 4 anticancer drugs, respectively. These findings provide novel insights which may benefit the development of personalized anticancer therapy for patients with cancer in the future.

DOI PubMed

Exploitation of a liquid biopsy system using circulating tumor DNA in esophageal cancer patients.

Iwaya Takeshi, Endo Fuinitaka, Sasaki Yasushi, Yaegashi Mizunori, Chiba Takehiro, Akiyama Yuji, Masuda Mar, Yamada Tesshi, Tokino Takashi, Nishizuka Satoshi

CANCER SCIENCE 109 953 2018年01月 [査読有り]

Identification of genomic alterations in oral squamous cell carcinomas using amplicon-based whole-exome sequencing

Sasaki Yasushi, Tamura Miyuki, Nakagaki Takafumi, Koyama Ryota, Idogawa Masashi, Ogi Kazuhiro, Hiratsuka Hiroyoshi, Nakase Hiroshi, Tokino Takashi

CANCER SCIENCE 109 990 2018年01月 [査読有り]

Exploration of p53-targeting long non-coding RNAs (lncRNAs) and network analysis of lncRNA expression in cancers

Idogawa Masashi, Ohashi Tomoko, Sasaki Yasushi, Tokino Takashi

CANCER SCIENCE 109 82 2018年01月 [査読有り]

Profiling cancer-related gene mutations in oral squamous cell carcinoma from Japanese patients by targeted sequencing

Nakagaki Takafumi, Sasaki Yasushi, Idogawa Masashi, Koyama Ryouta, Tamura Miyuki, Fukushima Hisayo, Ogi Kazuhiro, Hiratsuka Hiroyoshi, Tokino Takashi

CANCER SCIENCE 109 57 2018年01月 [査読有り]

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年12月 [査読有り]

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

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

Koyama R, Tamura M, Nakagaki T, Ohashi T, Idogawa M, Suzuki H, Tokino T, Sasaki Y

Cancer science 108 ( 12 ) 2413 - 2421 2017年12月 [査読有り] [国際誌]

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

新規p53標的である蛋白コード遺伝子LIMA1と長鎖非コードRNA(IncRNA)NEAT1のp53機能における役割

鈴木菜摘, 井戸川雅史, 大箸智子, 佐々木泰史, 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 [1P - 0990] 2017年12月

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年12月

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 14 ( 5 ) 5703 - 5710 2017年11月 [査読有り] [国際誌]

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

癌におけるp53標的長鎖非コードRNA(lncRNA)の探索と発現ネットワーク解析

井戸川雅史, 大箸智子, 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 ) 76回 J - 1077 2017年09月

次世代シークエンサーによる日本人口腔扁平上皮癌におけるがん関連409遺伝子の変異解析

中垣貴文, 佐々木泰史, 井戸川雅史, 小山良太, 田村みゆき, 福島久代, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 76回 J - 1063 2017年09月

口腔扁平上皮癌の全エクソン解析

佐々木泰史, 田村みゆき, 中垣貴文, 小山良太, 井戸川雅史, 荻和弘, 平塚博義, 仲瀬裕志, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 76回 P - 3044 2017年09月

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年09月

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年09月

Profiling cancer-related gene mutations in oral squamous cell carcinoma from Japanese patients by targeted amplicon sequencing.

Nakagaki T, Tamura M, Kobashi K, Koyama R, Fukushima H, Ohashi T, Idogawa M, Ogi K, Hiratsuka H, Tokino T, Sasaki Y

Oncotarget 8 ( 35 ) 59113 - 59122 2017年08月 [査読有り] [国際誌]

Somatic mutation analysis is a standard practice in the study of human cancers to identify mutations that cause therapeutic sensitization and resistance. We performed comprehensive genomic analyses that used PCR target enrichment and next-generation sequencing on Ion Proton semiconductor sequencers. Forty-seven oral squamous cell carcinoma (OSCC) samples and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverage of the entire coding regions of 409 cancer-related genes (covered regions: 95.4% of total, 1.69 megabases of target sequence). The number of somatic mutations in 47 patients with OSCC ranged from 1 to 20 with a mean of 7.60. The most frequent mutations were in TP53 (61.7%), NOTCH1 (25.5%), CDKN2A (19.1%), SYNE1 (14.9%), PIK3CA (10.6%), ROS1 (10.6%), and TAF1L (10.6%). We also detected copy number variations (CNVs) in the segments of the genome that could be duplicated or deleted from deep sequencing data. Pathway assessment showed that the somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation and RTK-MAPK-PI3K. This study may enable better selection of therapies and deliver improved outcomes for OSCC patients when combined with clinical diagnostics.

DOI PubMed

Fledglings in p53 signaling network.

Tokino T, Idogawa M, Sasaki Y

Oncotarget 8 ( 34 ) 55768 - 55769 2017年08月 [査読有り] [国際誌]

DOI PubMed

Mutation detection by target sequence analyses using tissue-specific panels in esophageal squamous cell carcinoma

Iwaya Takeshi, Endo Fumitaka, Kume Kohei, Sasaki Yasushi, Tokino Takashi, Nishizuka Satoshi

CANCER RESEARCH 77 2017年07月 [査読有り]

DOI

Long non-coding RNA NEAT1 is a transcriptional target of p53 and modulates p53-induced transactivation and tumor-suppressor function.

Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Hiroshi Nakase, Takashi Tokino

International journal of cancer 140 ( 12 ) 2785 - 2791 2017年06月 [査読有り] [国際誌]

p53 is one of the most important tumor suppressor genes, and the direct transcriptional targets of p53 must be explored to elucidate its functional mechanisms. Thus far, the p53 targets that have been primarily studied are protein-coding genes. Our previous study revealed that several long non-coding RNAs (lncRNAs) are direct transcriptional targets of p53, and knockdown of specific lncRNAs modulates p53-induced apoptosis. In this study, analysis of next-generation chromatin immunoprecipitation-sequencing (ChIP-seq) data for p53 revealed that the lncRNA NEAT1 is a direct transcriptional target of p53. The suppression of NEAT1 induction by p53 attenuates the inhibitory effect of p53 on cancer cell growth and also modulates gene transactivation, including that of many lncRNAs. Furthermore, low expression of NEAT1 is related to poor prognosis in several cancers. These results indicate that the induction of NEAT1 expression contributes to the tumor-suppressor function of p53 and suggest that p53 and NEAT1 constitute a transcriptional network contributing to various biological functions and tumor suppression.

DOI PubMed

p53 mediates the suppression of cancer cell invasion by inducing LIMA1/EPLIN.

Ohashi T, Idogawa M, Sasaki Y, Tokino T

Cancer letters 390 58 - 66 2017年04月 [査読有り] [国際誌]

The tumor suppressor gene p53 is frequently mutated in human cancer. p53 executes various functions, such as apoptosis induction and cell cycle arrest, by modulating transcriptional regulation. In this study, LIM domain and Actin-binding protein 1 (LIMA1) was identified as a target of the p53 family using a cDNA microarray. We also evaluated genome-wide occupancy of the p53 protein by performing chromatin immunoprecipitation-sequencing (ChIP-seq) and identified two p53 response elements in the LIMA1 gene. LIMA1 protein levels were increased by treatment with nutlin-3a, a small molecule that activates endogenous p53. In addition, LIMA1 expression was significantly downregulated in cancers compared with normal tissues. Knockdown of LIMA1 significantly enhanced cancer cell invasion and partially inhibited p53-induced suppression of cell invasion. Furthermore, low expression of LIMA1 in cancer patients correlated with decreased survival and poor prognosis. Thus, p53-induced LIMA1 inhibits cell invasion, and the downregulation of LIMA1 caused by p53 mutation results in decreased survival in cancer patients. Collectively, this study reveals the molecular mechanism of LIMA1 downregulation in various cancers and suggests that LIMA1 may be a novel prognostic predictor and a therapeutic target for cancer.

DOI PubMed

The effect of forced expression of mutated K-RAS gene on gastrointestinal cancer cell lines and the IGF-1R targeting therapy.

Matsunaga Y, Adachi Y, Sasaki Y, Koide H, Motoya M, Nosho K, Takagi H, Yamamoto H, Sasaki S, Arimura Y, Tokino T, Carbone DP, Imai K, Shinomura Y

Molecular carcinogenesis 56 ( 2 ) 515 - 526 2017年02月 [査読有り] [国際誌]

Mutation in K-RAS (K-RAS-MT) plays important roles in both cancer progression and resistance to anti-epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin-like growth factor-1 receptor (IGF-1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K-RAS mutation using an anti-IGF-1R monoclonal antibody. In this study, we sought to evaluate effects of forced K-RAS-MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti-EGFR therapy and IGF-1R-targeted therapy for these transfectants. We made stable transfectants of K-RAS-MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC-3. We assessed the effect of forced expression of K-RAS-MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti-tumor effects of dominant negative IGF-1R (IGF-1R/dn) and an IGF-1R inhibitor, picropodophyllin, on the K-RAS-MT transfectants. Overexpression of K-RAS-MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF-1R blockade suppressed cell growth, colony formation, migration, and invasion, and up-regulated chemotherapy-induced apoptosis of gastrointestinal cancer cells, even when K-RAS-MT was over-expressed. IGF-1R blockade inhibited the Akt pathway more than the extracellular signal-regulated kinase (ERK) pathway in the K-RAS-MT transfectants. IGF-1R/dn, moreover, inhibited the growth of murine xenografts expressing K-RAS-MT. Thus, K-RAS-MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF-1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K-RAS is mutated. © 2016 Wiley Periodicals, Inc.

DOI PubMed

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.

Einaga N, Yoshida A, Noda H, Suemitsu M, Nakayama Y, Sakurada A, Kawaji Y, Yamaguchi H, Sasaki Y, Tokino T, Esumi M

PloS one 12 ( 5 ) e0176280 2017年 [査読有り] [国際誌]

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

DOI PubMed

p53ファミリー遺伝子の標的としてのBRMS1L(breast cancer metastasis suppressor 1 like、BRMS1-like)の同定と機能解析

小山良太, 佐々木泰史, 田村みゆき, 中垣貴文, 大箸智子, 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 ) 75回 P - 2059 2016年10月

新規p53標的遺伝子LIMA1は癌細胞浸潤を抑制し低発現が予後不良と相関する

大箸智子, 井戸川雅史, 田村みゆき, 中垣貴文, 小山良太, 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 ) 75回 J - 3027 2016年10月

半導体シーケンサーを用いた口腔扁平上皮癌の全エクソームシーケンス

田村みゆき, 佐々木泰史, 中垣貴文, 小山良太, 大箸智子, 井戸川雅史, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 P - 1033 2016年10月

口腔扁平上皮癌におけるがん関連50遺伝子の変異解析

佐々木泰史, 中垣貴文, 田村みゆき, 小山良太, 福島久代, 大箸智子, 井戸川雅史, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 P - 1032 2016年10月

癌におけるp53および長鎖非コードRNA(IncRNA)による転写ネットワーク解析

井戸川雅史, 大箸智子, 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 ) 75回 J - 3026 2016年10月

がん関連遺伝子target sequenceによる食道扁平上皮癌の解析

岩谷岳, 遠藤史隆, 久米浩平, 佐々木泰史, 時野隆至, 澤田元太, 新井田厚司, 三森功士, 西塚哲

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 J - 2024 2016年10月

次世代半導体シーケンサーを用いた口腔扁平上皮癌におけるがん関連遺伝子の網羅的解析

中垣貴文, 佐々木泰史, 井戸川雅史, 小山良太, 田村みゆき, 大箸智子, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 P - 2297 2016年10月

次世代シークエンサーを用いた多発性骨髄腫における遺伝子変異とコピー数異常の解析

福島久代, 佐々木泰史, 田村みゆき, 池田博, 石黒一也, 櫻井晃洋, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 P - 1024 2016年10月

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年10月

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年10月

がん関連遺伝子のエクソーム解析による細胞傷害性抗がん剤感受性マーカーの探索

宇田川智野, 時野隆至, 佐々木泰史, 塩谷文章, 大西保行, 末水洋志, 前佛均

日本癌学会総会記事 ( (一社)日本癌学会 ) 75回 J - 3043 2016年10月

Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 target.

Sasaki Y, Tamura M, Takeda K, Ogi K, Nakagaki T, Koyama R, Idogawa M, Hiratsuka H, Tokino T

Oncotarget 7 ( 38 ) 61426 - 61437 2016年09月 [査読有り] [国際誌]

The p53 tumor suppressor inhibits cell growth through the activation of both cell cycle arrest and apoptosis, which maintain genome stability and prevent cancer development. Here, we report that intercellular adhesion molecule-2 (ICAM2) is transcriptionally activated by p53. Specifically, ICAM2 is induced by the p53 family and DNA damage in a p53-dependent manner. We identified a p53 binding sequence located within the ICAM2 gene that is responsive to wild-type p53, TAp73, and TAp63. In terms of function, we found that the ectopic expression of ICAM2 inhibited cancer cell migration and invasion. In addition, we demonstrated that silencing endogenous ICAM2 in cancer cells caused a marked increase in extracellular signal-regulated kinase (ERK) phosphorylation levels, suggesting that ICAM2 inhibits migration and invasion of cancer cells by suppressing ERK signaling. Moreover, ICAM2 is underexpressed in human cancer tissues containing mutant p53 as compared to those with wild-type p53. Notably, the decreased expression of ICAM2 is associated with poor survival in patients with various cancers. Our findings demonstrate that ICAM2 induction by p53 has a key role in inhibiting migration and invasion.

DOI PubMed

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

Saijo H, Hirohashi Y, Torigoe T, Horibe R, Takaya A, Murai A, Kubo T, Kajiwara T, Tanaka T, Shionoya Y, Yamamoto E, Maruyama R, Nakatsugawa M, Kanaseki T, Tsukahara T, Tamura Y, Sasaki Y, Tokino T, Suzuki H, Kondo T, Takahashi H, Sato N

Oncotarget 7 ( 31 ) 50043 - 50056 2016年08月 [査読有り] [国際誌]

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

p53 downregulates CRKL oncogene through miR-200

Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 76 2016年07月 [査読有り]

DOI

The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data

Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 76 2016年07月 [査読有り]

DOI

Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma

Takafumi Nakagaki, Yasushi Sasaki, Masashi Idogawa, Ryota Koyama, Kenta Kobashi, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Hiroyoshi Hiratsuka, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 76 2016年07月 [査読有り]

DOI

Targeted sequencing of 409 cancer related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers

Yasushi Sasaki, Ryota Koyama, Takafumi Nakagaki, Miyuki Tamura, Masashi Idogawa, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 76 2016年07月 [査読有り]

DOI

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

Kumegawa K, Maruyama R, Yamamoto E, Ashida M, Kitajima H, Tsuyada A, Niinuma T, Kai M, Yamano HO, Sugai T, Tokino T, Shinomura Y, Imai K, Suzuki H

Scientific reports 6 26699 - 26699 2016年05月 [査読有り] [国際誌]

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

Low-grade mucoepidermoid carcinoma with regional lymph node metastasis: A case report and genetic review of criteria for grading

Takafumi Nakagaki, Kazuhiro Ogi, Masato Abe, Hironari Dehari, Akihiro Miyazaki, Tadashi Hasegawa, Takashi Tokino, Hiroyoshi Hiratsuka

Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology ( Elsevier Ltd ) 28 ( 2 ) 189 - 192 2016年03月 [査読有り]

Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. It accounts for 5% of all salivary gland tumors and 20% of salivary malignancies and has various clinical outcomes. Its prognosis is related to the tumor grade in the WHO classification of head and neck tumors. MEC is divided into high, intermediate and low grades. In general, low-grade MEC with cervical lymph node metastasis is rare, and primary MEC that occurs in a minor salivary gland with cervical lymph node metastasis is very rare. Low-grade lesions have quite different behavior from high-grade ones, leading to differences in the treatment course required for disease control. MEC is associated with recurring chromosomal translocation t(11 19)(q21 p13). CRTC1-MAML2 gene fusion was identified from the recurring translocation, which is often the sole cytogenetic alteration in this disease. This fusion transcript is detected frequently in MEC and is involved in the transformation of epithelial cells. Detection of the CRTC1-MAML2 fusion transcript is associated with a less advanced clinical stage and low-grade tumor histology. The presence of the transcript is associated with longer disease-free and overall survival periods.Here, we report a rare case of low-grade MEC with cervical lymph node metastasis. The findings for this patient show that the CRTC1-MAML2 fusion transcript may be specific to MEC, and associated with a distinct MEC subset that exhibits favorable clinicopathologic features.

DOI

Genomic characterization of esophageal squamous cell carcinoma: Insights from next-generation sequencing.

Sasaki Y, Tamura M, Koyama R, Nakagaki T, Adachi Y, Tokino T

World journal of gastroenterology 22 ( 7 ) 2284 - 93 2016年02月 [査読有り] [国際誌]

Two major types of cancer occur in the esophagus: squamous cell carcinoma, which is associated with chronic smoking and alcohol consumption, and adenocarcinoma, which typically arises in gastric reflux-associated Barrett's esophagus. Although there is increasing incidence of esophageal adenocarcinoma in Western counties, esophageal squamous cell carcinoma (ESCC) accounts for most esophageal malignancies in East Asia, including China and Japan. Technological advances allowing for massively parallel, high-throughput next-generation sequencing (NGS) of DNA have enabled comprehensive characterization of somatic mutations in large numbers of tumor samples. Recently, several studies were published in which whole exome or whole genome sequencing was performed in ESCC tumors and compared with matched normal DNA. Mutations were validated in several genes, including in TP53, CDKN2A, FAT1, NOTCH1, PIK3CA, KMT2D and NFE2L2, which had been previously implicated in ESCC. Several new recurrent alterations have also been identified in ESCC. Combining the clinicopathological characteristics of patients with information obtained from NGS studies may lead to the development of effective diagnostic and therapeutic approaches for ESCC. As this research becomes more prominent, it is important that gastroenterologist become familiar with the various NGS technologies and the results generated using these methods. In the present study, we describe recent research approaches using NGS in ESCC.

DOI PubMed

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 11 ( 12 ) e0168281 2016年 [査読有り] [国際誌]

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

癌細胞におけるゲノムワイドRNAiスクリーニングを用いたp53誘導アポトーシス抵抗性を解除するshRNAの探索

梅田璃子, 井戸川雅史, 大箸智子, 佐々木泰史, 時野隆至

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 88回・38回 [2LBA040] - [2LBA040] 2015年12月

新規p53標的遺伝子LIMA1の発現誘導は癌細胞浸潤の抑制に必要である

大箸智子, 井戸川雅史, 梅田璃子, 佐々木泰史, 時野隆至

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 88回・38回 [4T18 - 03(3P1065)] 2015年12月

次世代シーケンサーを用いたがん関連409遺伝子のターゲットシーケンス解析

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

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 88回・38回 [3LBA004] - [3LBA004] 2015年12月

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

Kamimae S, Yamamoto E, Kai M, Niinuma T, Yamano HO, Nojima M, Yoshikawa K, Kimura T, Takagi R, Harada E, Harada T, Maruyama R, Sasaki Y, Tokino T, Shinomura Y, Sugai T, Imai K, Suzuki H

Oncotarget 6 ( 30 ) 29975 - 90 2015年10月 [査読有り] [国際誌]

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

半導体シーケンサーを用いたがん関連遺伝子のターゲットシーケンス解析

佐々木泰史, 中垣貴文, 田村みゆき, 竹田康佑, 大箸智子, 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 ) 74回 P - 1156 2015年10月

p53による癌細胞浸潤の抑制には新規標的遺伝子LIMA1の発現誘導が必要である

大箸智子, 井戸川雅史, 田村みゆき, 竹田康佑, 中垣貴文, 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 ) 74回 J - 1280 2015年10月

CRKLはmiR-200ファミリーを介してp53により発現抑制される

田村みゆき, 佐々木泰史, 竹田康佑, 中垣貴文, 大箸智子, 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 ) 74回 P - 3013 2015年10月

次世代半導体シーケンサーを用いた口腔扁平上皮癌におけるがん関連遺伝子の網羅的解析

中垣貴文, 佐々木泰史, 井戸川雅史, 竹田康佑, 田村みゆき, 大箸智子, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 74回 J - 1330 2015年10月

RNA-seqとChIP-seqの複合解析による非コードRNAを含むp53標的遺伝子の同定

井戸川雅史, 大箸智子, 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 ) 74回 J - 1294 2015年10月

薬剤耐性を示した多発性骨髄腫患者の次世代シークエンサーを用いた遺伝子解析

池田博, 石黒一也, 青木由佳, 石田禎夫, 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 ) 74回 P - 3191 2015年10月

消化器癌における変異kRAS遺伝子の影響の解析およびIGF-1R阻害

松永康孝, 足立靖, 佐々木泰史, 能正勝彦, 高木秀安, 山本博幸, 佐々木茂, 有村佳昭, 遠藤高夫, 加藤康夫, 時野隆至, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 74回 J - 1204 2015年10月

ホルマリン固定パラフィン包埋(FFPE)組織DNAのゲノム解析に問題はないか?

栄永直樹, 山口裕美, 中山裕貴, 川路美子, 佐々木泰史, 時野隆至, 江角眞理子

日本癌学会総会記事 ( (一社)日本癌学会 ) 74回 P - 1157 2015年10月

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

Mitsuhashi K, Yamamoto I, Kurihara H, Kanno S, Ito M, Igarashi H, Ishigami K, Sukawa Y, Tachibana M, Takahashi H, Tokino T, Maruyama R, Suzuki H, Imai K, Shinomura Y, Yamamoto H, Nosho K

Oncotarget 6 ( 26 ) 22114 - 25 2015年09月 [査読有り] [国際誌]

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

One-Dimensional Sliding of p53 Along DNA Is Accelerated in the Presence of Ca(2+) or Mg(2+) at Millimolar Concentrations.

Murata A, Ito Y, Kashima R, Kanbayashi S, Nanatani K, Igarashi C, Okumura M, Inaba K, Tokino T, Takahashi S, Kamagata K

Journal of molecular biology 427 ( 16 ) 2663 - 78 2015年08月 [査読有り] [国際誌]

One-dimensional (1D) sliding of the tumor suppressor p53 along DNA is an essential dynamics required for its efficient search for the binding sites in the genome. To address how the search process of p53 is affected by the changes in the concentration of Mg(2+) and Ca(2+) after the cell damages, we investigated its sliding dynamics at different concentrations of the divalent cations. The 1D sliding trajectories of p53 along the stretched DNA were measured by using single-molecule fluorescence microscopy. The averaged diffusion coefficient calculated from the mean square displacement of p53 on DNA increased significantly at the higher concentration of Mg(2+) or Ca(2+), indicating that the divalent cations accelerate the sliding likely by weakening the DNA-p53 interaction. In addition, two distributions were identified in the displacement of the observed trajectories of p53, demonstrating the presence of the fast and slow sliding modes having large and small diffusion coefficients, respectively. A coreless mutant of p53, in which the core domain was deleted, showed only a single mode whose diffusion coefficient is about twice that of the fast mode for the full-length p53. Thus, the two modes are likely the result of the tight and loose interactions between the core domain of p53 and DNA. These results demonstrated clearly that the 1D sliding dynamics of p53 is strongly dependent on the concentration of Mg(2+) and Ca(2+), which maintains the search distance of p53 along DNA in cells that lost homeostatic control of the divalent cations.

DOI PubMed

Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing

Takafumi Nakagaki, Yasushi Sasaki, Kenta Kobashi, Kousuke Takeda, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Masashi Idogawa, Hiroyoshi Hiratsuka, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 75 2015年08月 [査読有り]

DOI

p53 downregulates CRKL proto-oncogene protein through miR-200 family

Yasushi Sasaki, Miyuki Tamura, Kenta Kobashi, Kousuke Takeda, Takafumi Nakagaki, Masashi Idogawa, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 75 2015年08月 [査読有り]

DOI

CRKL oncogene is downregulated by p53 through miR-200s.

Tamura M, Sasaki Y, Kobashi K, Takeda K, Nakagaki T, Idogawa M, Tokino T

Cancer science 106 ( 8 ) 1033 - 40 2015年08月 [査読有り] [国際誌]

Tumor suppressive miRNAs that target oncogenes are frequently downregulated in cancers, and this downregulation leads to oncogene pathway activation. Thus, tumor suppressive miRNAs and their target oncogenes have been proposed as useful targets in cancer treatment. miR-200 family downregulation has been reported in cancer progression and metastasis. The miR-200 family consists of two gene clusters, miR-200b/200a/429 and miR-200c/141, which are located on human chromosomes 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of the miR-200 family and confirmed that miR-200s are transcriptional targets of the p53 family. In silico analyses of miRNA targets established the CRKL oncogene as a potential target for miR-200b/200c/429. Moreover, miR-200b/200c/429 inhibited CRKL mRNA and protein expression by directly targeting its 3'-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells through the introduction of p53 family and endogenous p53 activation. Moreover, the downregulation of CRKL by siRNA inhibited cancer cell growth. The Oncomine database demonstrates that CRKL is overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that the p53 target miR-200b/200c/429 miRNAs are negative regulators of the CRKL oncogene.

DOI PubMed

CRKL oncogene is downregulated by p53 through miR-200s

Miyuki Tamura, Yasushi Sasaki, Kenta Kobashi, Kousuke Takeda, Takafumi Nakagaki, Masashi Idogawa, Takashi Tokino

CANCER SCIENCE ( WILEY ) 106 ( 8 ) 1033 - 1040 2015年08月 [査読有り]

Tumor suppressive miRNAs that target oncogenes are frequently downregulated in cancers, and this downregulation leads to oncogene pathway activation. Thus, tumor suppressive miRNAs and their target oncogenes have been proposed as useful targets in cancer treatment. miR-200 family downregulation has been reported in cancer progression and metastasis. The miR-200 family consists of two gene clusters, miR-200b/200a/429 and miR-200c/141, which are located on human chromosomes 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of the miR-200 family and confirmed that miR-200s are transcriptional targets of the p53 family. Insilico analyses of miRNA targets established the CRKL oncogene as a potential target for miR-200b/200c/429. Moreover, miR-200b/200c/429 inhibited CRKL mRNA and protein expression by directly targeting its 3-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells through the introduction of p53 family and endogenous p53 activation. Moreover, the downregulation of CRKL by siRNA inhibited cancer cell growth. The Oncomine database demonstrates that CRKL is overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that the p53 target miR-200b/200c/429 miRNAs are negative regulators of the CRKL oncogene.

DOI

Relation between Ku80 and microRNA-99a expression and late rectal bleeding after radiotherapy for prostate cancer.

Someya M, Yamamoto H, Nojima M, Hori M, Tateoka K, Nakata K, Takagi M, Saito M, Hirokawa N, Tokino T, Sakata K

Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 115 ( 2 ) 235 - 9 2015年05月 [査読有り] [国際誌]

BACKGROUND AND PURPOSE: Late rectal bleeding is one of the severe adverse events after radiotherapy for prostate cancer. New biomarkers are needed to allow a personalized treatment. MATERIALS AND METHODS: Four patients each with grade 0-1 or grade 2-3 rectal bleeding were randomly selected for miRNA array to examine miRNA expression in peripheral blood lymphocytes (PBLs). Based on results of miRNA array, 1 of 348 miRNAs was selected for microRNA assays. Then, expression of DNA-dependent protein kinase mRNA and miR-99a was analyzed in the PBLs of 97 patients. PBLs were exposed to 4Gy of X-ray ex-vivo. RESULTS: In the discovery cohort, grade 2-3 rectal bleeding was significantly higher in the Ku80 <1.09 expression group compared with ⩾1.09 group (P=0.011). In radiation-induced expression of miR-99a, grade 2-3 rectal bleeding was significantly higher in the miR-99a IR(+)/IR(-) >0.93 group compared with ⩽0.93 group (P=0.013). Most patients with grade 2-3 rectal bleeding were in the group with low Ku80 and high miR-99a expression. In the validation cohort, similar results were obtained. CONCLUSION: A combination of low Ku80 expression and highly-induced miR-99a expression could be a promising marker for predicting rectal bleeding after radiotherapy.

DOI PubMed

A Screen for Epigenetically Silenced microRNA Genes in Gastrointestinal Stromal Tumors.

Isosaka M, Niinuma T, Nojima M, Kai M, Yamamoto E, Maruyama R, Nobuoka T, Nishida T, Kanda T, Taguchi T, Hasegawa T, Tokino T, Hirata K, Suzuki H, Shinomura Y

PloS one 10 ( 7 ) e0133754 2015年 [査読有り] [国際誌]

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

Molecular diagnostics of a single drug-resistant multiple myeloma case using targeted next-generation sequencing.

Ikeda H, Ishiguro K, Igarashi T, Aoki Y, Hayashi T, Ishida T, Sasaki Y, Tokino T, Shinomura Y

OncoTargets and therapy 8 2805 - 15 2015年 [査読有り] [国際誌]

A 69-year-old man was diagnosed with IgG λ-type multiple myeloma (MM), Stage II in October 2010. He was treated with one cycle of high-dose dexamethasone. After three cycles of bortezomib, the patient exhibited slow elevations in the free light-chain levels and developed a significant new increase of serum M protein. Bone marrow cytogenetic analysis revealed a complex karyotype characteristic of malignant plasma cells. To better understand the molecular pathogenesis of this patient, we sequenced for mutations in the entire coding regions of 409 cancer-related genes using a semiconductor-based sequencing platform. Sequencing analysis revealed eight nonsynonymous somatic mutations in addition to several copy number variants, including CCND1 and RB1. These alterations may play roles in the pathobiology of this disease. This targeted next-generation sequencing can allow for the prediction of drug resistance and facilitate improvements in the treatment of MM patients.

DOI PubMed

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

Ito M, Mitsuhashi K, Igarashi H, Nosho K, Naito T, Yoshii S, Takahashi H, Fujita M, Sukawa Y, Yamamoto E, Takahashi T, Adachi Y, Nojima M, Sasaki Y, Tokino T, Baba Y, Maruyama R, Suzuki H, Imai K, Yamamoto H, Shinomura Y

International journal of cancer 135 ( 11 ) 2507 - 15 2014年12月 [査読有り] [国際誌]

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

口腔扁平上皮癌における50の癌関連遺伝子についての次世代シークエンス分析(A Next-Generation Sequencing for 50 Cancer-Related Genes in Oral Squamous Carcinoma)

Nakagaki Takafumi, Sasaki Yasushi, Idogawa Masashi, Takeda Kousuke, Tamura Miyuki, Ohashi Tomoko, Ogi Kazuhiro, Hiratsuka Hiroyoshi, Tokino Takashi

Personalized Medicine Universe. Japanese Edition ( (一社)国際個別化医療学会 ) 3 ( Suppl.2 ) 61 - 61 2014年11月

Forkhead transcription factor FOXF1 is a novel target gene of the p53 family and regulates cancer cell migration and invasiveness

M. Tamura, Y. Sasaki, R. Koyama, K. Takeda, M. Idogawa, T. Tokino

ONCOGENE ( NATURE PUBLISHING GROUP ) 33 ( 40 ) 4837 - 4846 2014年10月

p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of the p53 family because FOXF1 is upregulated by p53, TAp73 and TAp63. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73 beta and TAp63 gamma. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin (CDH1) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to have an important role in cancer cell invasion and migration.

DOI PubMed

Forkhead transcription factor FOXF1 is a novel p53 target gene and regulates cancer cell migration and invasion

Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 74 ( 19 ) 2014年10月 [査読有り]

DOI

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

Idogawa M, Ohashi T, Sugisaka J, Sasaki Y, Suzuki H, Tokino T

Oncotarget 5 ( 17 ) 7540 - 8 2014年09月 [査読有り] [国際誌]

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年09月

次世代半導体シーケンサーを用いた口腔扁平上皮癌におけるがん関連遺伝子の変異解析(A Next-Generation Sequencing Screen for Mutational Hotspots in 50 Cancer-Related Genes using Semiconductor Sequencer)

中垣貴文, 佐々木泰史, 井戸川雅史, 竹田康佑, 田村みゆき, 大箸智子, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 73回 P - 3270 2014年09月

p53標的遺伝子AKR1B10は大腸癌で発現減少し予後不良と相関する(AKR1B10, a Transcriptional Target of p53, is Downregulated in Colorectal Cancers Associated with Poor Prognosis)

大箸智子, 井戸川雅史, 田村みゆき, 竹田康佑, 中垣貴文, 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 ) 73回 P - 2072 2014年09月

p53のmiR-200ファミリーを介したCRKLの発現抑制(Proto-oncogene CRKL is downregulated by p53 family through miR-200 family)

田村みゆき, 佐々木泰史, 竹田康佑, 中垣貴文, 大箸智子, 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 ) 73回 P - 2065 2014年09月

ゲノム網羅的p53結合領域解析とマイクロアレイを組み合わせたp53ファミリー標的長鎖非コードRNA(lincRNA)の同定と解析(Identification of p53 target long non-coding RNAs (lincRNAs) through ChIP-seq, in silico motif analysis and microarray)

井戸川雅史, 大箸智子, 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 ) 73回 P - 2079 2014年09月

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年09月

多発性骨髄腫における単球と骨髄ストローマ細胞の関連性(The interaction between monocyte and bone marrow stroma cell in the pathogenesis of myeloma)

池田博, 五十嵐哲祥, 青木由佳, 林敏昭, 石田禎夫, 佐々木泰史, 時野隆至, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 73回 P - 2277 2014年09月

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

Idogawa M, Ohashi T, Sasaki Y, Maruyama R, Kashima L, Suzuki H, Tokino T

Human molecular genetics 23 ( 11 ) 2847 - 57 2014年06月 [査読有り] [国際誌]

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

AKR1B10, a transcriptional target of p53, is downregulated in colorectal cancers associated with poor prognosis.

Ohashi T, Idogawa M, Sasaki Y, Suzuki H, Tokino T

Molecular cancer research : MCR 11 ( 12 ) 1554 - 63 2013年12月 [査読有り] [国際誌]

UNLABELLED: 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.

DOI PubMed

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年10月

p53ファミリーの新規標的ICAM2の同定がん細胞の接着・遊走能への関与(Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 family target)

竹田康佑, 佐々木泰史, 田村みゆき, 井戸川雅史, 荻和弘, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 72回 253 - 253 2013年10月

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年10月

AKR1B10はp53ファミリーの直接転写標的でありp53誘導アポトーシスを促進する(AKR1B10 is a direct transcriptional target of p53 family members and promotes p53-induced apoptosis)

大箸智子, 井戸川雅史, 田村みゆき, 竹田康佑, 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 ) 72回 253 - 253 2013年10月

p53はmiR-200ファミリーを介してCRKLの発現を抑制する(p53 family members activate the miR-200 family leading to downregulation of the CRKL proto-oncogene protein)

田村みゆき, 佐々木泰史, 竹田康佑, 大箸智子, 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 ) 72回 253 - 253 2013年10月

Methylation of a panel of microRNA genes is a novel biomarker for detection of bladder cancer.

Shimizu T, Suzuki H, Nojima M, Kitamura H, Yamamoto E, Maruyama R, Ashida M, Hatahira T, Kai M, Masumori N, Tokino T, Imai K, Tsukamoto T, Toyota M

European urology 63 ( 6 ) 1091 - 100 2013年06月 [査読有り] [国際誌]

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.

DOI PubMed

DNA methyltransferase 1 is essential for initiation of the colon cancers.

Morita R, Hirohashi Y, Suzuki H, Takahashi A, Tamura Y, Kanaseki T, Asanuma H, Inoda S, Kondo T, Hashino S, Hasegawa T, Tokino T, Toyota M, Asaka M, Torigoe T, Sato N

Experimental and molecular pathology 94 ( 2 ) 322 - 9 2013年04月 [査読有り] [国際誌]

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.

DOI PubMed

2P026 Direct observation of the multiple sliding modes of a tumor suppressor p53(01B. Protein: Structure & Function,Poster)

Murata Agato, Kashima Risa, Itoh Yuji, Tokino Takashi, Takahashi Satoshi, Kamagata Kiyoto

生物物理 ( 一般社団法人日本生物物理学会 ) 53 ( 1 ) S163 2013年

DOI CiNii

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

Sasaki Y, Koyama R, Maruyama R, Hirano T, Tamura M, Sugisaka J, Suzuki H, Idogawa M, Shinomura Y, Tokino T

Cancer biology & therapy 13 ( 14 ) 1512 - 21 2012年12月 [査読有り] [国際誌]

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

A novel approach to cancer treatment using structural hybrids of the p53 gene family

Y. Sasaki, Y. Oshima, R. Koyama, M. Tamura, L. Kashima, M. Idogawa, T. Yamashita, M. Toyota, K. Imai, Y. Shinomura, T. Tokino

CANCER GENE THERAPY ( NATURE PUBLISHING GROUP ) 19 ( 11 ) 749 - 756 2012年11月

The p53 tumor suppressor belongs to a gene family that includes two other structurally and functionally related members: p73 and p63. The regulation of p53 activity differs significantly from that of p73 and p63. To enhance the tumor suppressive activity of p53, we constructed six recombinant adenoviruses that encode hybrid proteins with three functional domains derived from either p53 or TAp63 gamma. The potency of these hybrid molecules in suppressing tumorigenesis was evaluated using in vitro and in vivo models. Of the hybrid molecules tested, one hybrid named p63-53O was the most potent activator of apoptosis in human cancer cells. The p63-53O hybrid is composed of the transcriptional activation domain and DNA-binding domain of TAp63 gamma and the oligomerization domain of p53. The p63-53O hybrid efficiently transactivated p53AIP1. Moreover, silencing of p53AIP1 partially abolished the apoptotic response to p63-53O in human cancer cells. The p53-p63 hybrid molecule is a novel potent anti-proliferative agent for the treatment of cancer.

DOI PubMed

Molecular dissection of premalignant colorectal lesions reveals early onset of the CpG island methylator phenotype.

Yamamoto E, Suzuki H, Yamano HO, Maruyama R, Nojima M, Kamimae S, Sawada T, Ashida M, Yoshikawa K, Kimura T, Takagi R, Harada T, Suzuki R, Sato A, Kai M, Sasaki Y, Tokino T, Sugai T, Imai K, Shinomura Y, Toyota M

The American journal of pathology 181 ( 5 ) 1847 - 61 2012年11月 [査読有り] [国際誌]

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 PIK3CA 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 CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. 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.

DOI PubMed

Heat shock enhances the expression of cytotoxic granule proteins and augments the activities of tumor-associated antigen-specific cytotoxic T lymphocytes.

Takahashi A, Torigoe T, Tamura Y, Kanaseki T, Tsukahara T, Sasaki Y, Kameshima H, Tsuruma T, Hirata K, Tokino T, Hirohashi Y, Sato N

Cell stress & chaperones 17 ( 6 ) 757 - 63 2012年11月 [査読有り] [国際誌]

Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients' peripheral blood, and the CTL activities were investigated in several temperature conditions (37-41 °C). Cytotoxicity and IFN-γ secretion of CTL were greatest under 39 °C condition, whereas they were minimum under 41 °C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCRαβ by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 °C. On the other hand, CTL cell death was induced under 41 °C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 °C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

DOI PubMed

Aberrant methylation of RASGRF1 is associated with an epigenetic field defect and increased risk of gastric cancer.

Takamaru H, Yamamoto E, Suzuki H, Nojima M, Maruyama R, Yamano HO, Yoshikawa K, Kimura T, Harada T, Ashida M, Suzuki R, Yamamoto H, Kai M, Tokino T, Sugai T, Imai K, Toyota M, Shinomura Y

Cancer prevention research (Philadelphia, Pa.) 5 ( 10 ) 1203 - 12 2012年10月 [査読有り] [国際誌]

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.

DOI PubMed

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

Shitani M, Sasaki S, Akutsu N, Takagi H, Suzuki H, Nojima M, Yamamoto H, Tokino T, Hirata K, Imai K, Toyota M, Shinomura Y

Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 33 ( 5 ) 1307 - 17 2012年10月 [査読有り] [国際誌]

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

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年09月

p53ファミリーの新規標的FOXF1の同定と機能解析がん細胞の浸潤・遊走能への関与(Forkhead box F1 is a novel target gene of the p53 family and regulates cancer cell migration and invasiveness)

田村みゆき, 佐々木泰史, 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 ) 71回 175 - 176 2012年08月

p53ファミリーとがん p53ファミリーネットワークと腫瘍抑制(p53 family and cancer research p53 family network and tumor suppression)

佐々木泰史, 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 ) 71回 41 - 41 2012年08月

ゲノム網羅的p53応答配列解析とクロマチン免疫沈降シーケンシング(ChIP-seq)を組み合わせた新規p53標的遺伝子の同定(Identification of novel p53 target genes by ChIP-seq combined with genome-wide p53-binding motif analysis in silico)

井戸川雅史, 大箸智子, 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 ) 71回 257 - 258 2012年08月

ゲノム網羅的shRNAライブラリースクリーニングによるp53誘導アポトーシス阻害遺伝子の同定(The identification of target genes inhibiting p53-induced apoptosis by the screening of genome-wide shRNA library)

大箸智子, 井戸川雅史, 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 ) 71回 176 - 176 2012年08月

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年08月

CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

Kashima L, Idogawa M, Mita H, Shitashige M, Yamada T, Ogi K, Suzuki H, Toyota M, Ariga H, Sasaki Y, Tokino T

The Journal of biological chemistry 287 ( 16 ) 12975 - 84 2012年04月 [査読有り] [国際誌]

The mitotic checkpoint gene CHFR (checkpoint with forkhead-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

Identification of p53 target genes by ChIP-Seq and transcriptome analysis combined with genome-wide p53-binding motif analysis in silico

Masashi Idogawa, Yasushi Sasaki, Yasuhisa Shinomura, Kohzoh Imai, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 72 2012年04月 [査読有り]

DOI

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年04月

Upregulation of miR-196a and HOTAIR drive malignant character in gastrointestinal stromal tumors.

Niinuma T, Suzuki H, Nojima M, Nosho K, Yamamoto H, Takamaru H, Yamamoto E, Maruyama R, Nobuoka T, Miyazaki Y, Nishida T, Bamba T, Kanda T, Ajioka Y, Taguchi T, Okahara S, Takahashi H, Nishida Y, Hosokawa M, Hasegawa T, Tokino T, Hirata K, Imai K, Toyota M, Shinomura Y

Cancer research 72 ( 5 ) 1126 - 36 2012年03月 [査読有り] [国際誌]

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 HOXC and 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.

DOI PubMed

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

Aoki Y, Nojima M, Suzuki H, Yasui H, Maruyama R, Yamamoto E, Ashida M, Itagaki M, Asaoku H, Ikeda H, Hayashi T, Imai K, Mori M, Tokino T, Ishida T, Toyota M, Shinomura Y

Genome medicine 4 ( 12 ) 101 - 101 2012年 [査読有り] [国際誌]

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-α) 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

2E1412 走査型プローブ顕微鏡を用いた癌抑制因子p53の一分子構造解析(蛋白質-物性II,口頭発表,日本生物物理学会第50回年会(2012年度))

Takahashi Seiya, Sainoo Yasuyuki, Kashima Risa, Tokino Takashi, Komeda Tadahiro, Takahashi Satoshi, Kamagata Kiyoto

生物物理 ( 一般社団法人日本生物物理学会 ) 52 S45 - S46 2012年

DOI CiNii

Erratum

Tomita M, Toyota M, Ishikawa C, Nakazato T, Okudaira T, Matsuda T, Uchihara J. N, Taira N, Ohshiro K, Senba M, Tanaka Y, Ohshima K, Saya H, Tokino T, Mori N

International Journal of Cancer 129 ( 11 ) 2763 2011年12月

DOI

p53 negatively regulates the hepatoma growth factor HDGF.

Sasaki Y, Negishi H, Idogawa M, Yokota I, Koyama R, Kusano M, Suzuki H, Fujita M, Maruyama R, Toyota M, Saito T, Tokino T

Cancer research 71 ( 22 ) 7038 - 47 2011年11月 [査読有り] [国際誌]

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.

DOI PubMed

Genome-wide profiling of chromatin signatures reveals epigenetic regulation of MicroRNA genes in colorectal cancer.

Suzuki H, Takatsuka S, Akashi H, Yamamoto E, Nojima M, Maruyama R, Kai M, Yamano HO, Sasaki Y, Tokino T, Shinomura Y, Imai K, Toyota M

Cancer research 71 ( 17 ) 5646 - 58 2011年09月 [査読有り] [国際誌]

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.

DOI PubMed

p53ファミリーの新規標的遺伝子FOXF1の同定と機能解析(Forkhead transcription factor FOXF1 is a novel target gene of the p53 family and regulates cancer cell invasiveness)

田村みゆき, 佐々木泰史, 横田育子, 鹿島理沙, 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 ) 70回 153 - 153 2011年09月

ゲノム網羅的shRNAライブラリースクリーニングに基づく人工miRNA/p53共発現による癌治療(Cancer therapy by the transduction of p53 and artificial miRNAs based on the screening of genome-wide shRNA library)

井戸川雅史, 佐々木泰史, 鹿島理沙, 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 ) 70回 52 - 52 2011年09月

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年09月

p53ファミリーの新規標的ICAM2の同定がん細胞の接着・遊走能への関与(Identification and characterizatio n of the intercellular adhesion molecule-2 gene as a novel p53 family target)

横田育子, 佐々木泰史, 田村みゆき, 鹿島理沙, 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 ) 70回 153 - 153 2011年09月

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年09月

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年09月

Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis.

Cheung AK, Ko JM, Lung HL, Chan KW, Stanbridge EJ, Zabarovsky E, Tokino T, Kashima L, Suzuki T, Kwong DL, Chua D, Tsao SW, Lung ML

Proceedings of the National Academy of Sciences of the United States of America 108 ( 20 ) 8390 - 5 2011年05月 [査読有り] [国際誌]

Chromosome 14 was transferred into tumorigenic nasopharyngeal carcinoma and esophageal carcinoma cell lines by a microcell-mediated chromosome transfer approach. Functional complementation of defects present in the cancer cells suppressed tumor formation. A candidate tumor-suppressor gene, cysteine-rich intestinal protein 2 (CRIP2), located in the hot spot for chromosomal loss at 14q32.3, was identified as an important candidate gene capable of functionally suppressing tumor formation. Previous studies have shown that CRIP2 is associated with development. To date, no report has provided functional evidence supporting a role for CRIP2 in tumor development. The present study provides unequivocal evidence that CRIP2 can functionally suppress tumorigenesis. CRIP2 is significantly down-regulated in nasopharyngeal carcinoma cell lines and tumors. CRIP2 reexpression functionally suppresses in vivo tumorigenesis and angiogenesis; these effects are induced by its transcription-repressor capability. It interacts with the NF-κB/p65 to inhibit its DNA-binding ability to the promoter regions of the major proangiogenesis cytokines critical for tumor progression, including IL6, IL8, and VEGF. In conclusion, we provide compelling evidence that CRIP2 acts as a transcription repressor of the NF-κB-mediated proangiogenic cytokine expression and thus functionally inhibits tumor formation and angiogenesis.

DOI PubMed

Epigenetic alteration of DNA in mucosal wash fluid predicts invasiveness of colorectal tumors.

Kamimae S, Yamamoto E, Yamano HO, Nojima M, Suzuki H, Ashida M, Hatahira T, Sato A, Kimura T, Yoshikawa K, Harada T, Hayashi S, Takamaru H, Maruyama R, Kai M, Nishiwaki M, Sugai T, Sasaki Y, Tokino T, Shinomura Y, Imai K, Toyota M

Cancer prevention research (Philadelphia, Pa.) 4 ( 5 ) 674 - 83 2011年05月 [査読有り] [国際誌]

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.

DOI PubMed

Apoptosis induction in cancer cells by the simultaneous transduction of p53 and artificial miRNAs based on the screening of genome-wide shRNA library

Masashi Idogawa, Yasushi Sasaki, Lisa Kashima, Yasuhisa Shinomura, Kohzoh Imai, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 71 2011年04月 [査読有り]

DOI

The functional relationship between CHFR and PARP-1 controls the antephase checkpoint and tumor development

Lisa Kashima, Hiroaki Mita, Masashi Idogawa, Minoru Toyota, Yasushi Sasaki, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 71 2011年04月 [査読有り]

DOI

Identification and characterization of early growth response 2, a zinc-finger transcription factor, as a p53-regulated proapoptotic gene.

Yokota I, Sasaki Y, Kashima L, Idogawa M, Tokino T

International journal of oncology 37 ( 6 ) 1407 - 16 2010年12月 [査読有り] [国際誌]

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. In recent years, many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the apoptotic effect. Using microarray analysis, we have identified the transcription factor early growth response 2 (EGR2) as a target of the p53 family, specifically p53, p63 and p73. EGR2 expression was up-regulated by DNA damage-induced p53 activity, as well as by overexpression of p53 family genes. Furthermore, we identified a responsive element to p53, TAp63, and TAp73 within the EGR2 gene. This response element is highly conserved between human and rodents. We also found that overexpression of EGR2 induced apoptosis when combined with anticancer agents. Conversely, inactivation of EGR2 attenuated p53-mediated apoptosis. The results presented here suggest that EGR2 is a direct transcriptional target of p53 family that can in part mediate the p53-dependent apoptotic pathway.

DOI PubMed

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect.

Suzuki H, Yamamoto E, Nojima M, Kai M, Yamano HO, Yoshikawa K, Kimura T, Kudo T, Harada E, Sugai T, Takamaru H, Niinuma T, Maruyama R, Yamamoto H, Tokino T, Imai K, Toyota M, Shinomura Y

Carcinogenesis 31 ( 12 ) 2066 - 73 2010年12月 [査読有り] [国際誌]

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

p53 family network and human cancer

Sasaki Y, Idogawa M, Yokota I, Kashima L, Tokino T

Tumor Research 45 33 - 44 2010年12月 [査読有り]

CHFRとPARP-1の機能的関連が有糸分裂チェックポイントおよび腫瘍発生をコントロールする(The functional relationship between CHFR and PARP-1 controls the mitotic checkpoint and tumor development)

鹿島理沙, 見田裕章, 井戸川雅史, 豊田実, 佐々木泰史, 時野隆至

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 83回・33回 3T4 - 4 2010年12月

アデノウイルスを用いたアポトーシス阻害的p53標的遺伝子に対する人工miRNAとp53の導入による癌細胞でのアポトーシス誘導(Apoptosis induction in cancer cells by the adenovirus-mediated transduction of p53 and artificial miRNAs targeting anti-apoptotic p53 target genes)

杉坂淳, 井戸川雅史, 佐々木泰史, 鹿島理沙, 篠村恭久, 今井浩三, 時野隆至

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 83回・33回 4P - 0967 2010年12月

平野雄大, 佐々木泰史, 横田育子, 杉坂淳, 鹿島理紗, 井戸川雅史, 時野隆至

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 ( (公社)日本生化学会 ) 83回・33回 3P - 0982 2010年12月

A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors.

Igarashi S, Suzuki H, Niinuma T, Shimizu H, Nojima M, Iwaki H, Nobuoka T, Nishida T, Miyazaki Y, Takamaru H, Yamamoto E, Yamamoto H, Tokino T, Hasegawa T, Hirata K, Imai K, Toyota M, Shinomura Y

Clinical cancer research : an official journal of the American Association for Cancer Research 16 ( 21 ) 5114 - 23 2010年11月 [査読有り] [国際誌]

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-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. 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-α 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.

DOI PubMed

Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer.

Fujikane T, Nishikawa N, Toyota M, Suzuki H, Nojima M, Maruyama R, Ashida M, Ohe-Toyota M, Kai M, Nishidate T, Sasaki Y, Ohmura T, Hirata K, Tokino T

Breast cancer research and treatment 122 ( 3 ) 699 - 710 2010年08月 [査読有り] [国際誌]

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, p63gamma and p73beta. 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ファミリーの標的遺伝子EGR2の同定と機能解析(Identification and characterization of early growth response gene-2 as a p53-regulated proapoptotic gene)

横田育子, 佐々木泰史, 安保直樹, 鹿島理沙, 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 ) 69回 303 - 304 2010年08月

がん抑制分子CHFRおよびPARP-1相互作用による細胞周期制御(Functional association between CHFR and PARP-1 controls the mitotic checkpoint)

鹿島理沙, 見田裕章, 井戸川雅史, 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 ) 69回 312 - 312 2010年08月

p53とアポトーシス阻害的標的に対するmiRNAの単一アデノウイルス発現による癌治療(Cancer Therapy by single adenovirus-mediated expression of p53 and microRNAs targeting anti-apoptotic p53 target genes)

井戸川雅史, 佐々木泰史, 鹿島理沙, 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 ) 69回 322 - 322 2010年08月

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年08月

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年08月

DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae.

Yamashita M, Toyota M, Suzuki H, Nojima M, Yamamoto E, Kamimae S, Watanabe Y, Kai M, Akashi H, Maruyama R, Sasaki Y, Yamano H, Sugai T, Shinomura Y, Imai K, Tokino T, Itoh F

Cancer science 101 ( 7 ) 1708 - 16 2010年07月 [査読有り] [国際誌]

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.

DOI PubMed

IGFBP7 is a p53-responsive gene specifically silenced in colorectal cancer with CpG island methylator phenotype.

Suzuki H, Igarashi S, Nojima M, Maruyama R, Yamamoto E, Kai M, Akashi H, Watanabe Y, Yamamoto H, Sasaki Y, Itoh F, Imai K, Sugai T, Shen L, Issa JP, Shinomura Y, Tokino T, Toyota M

Carcinogenesis 31 ( 3 ) 342 - 9 2010年03月 [査読有り] [国際誌]

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

N-Propionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells

Makito Sato, Toshiharu Yamashita, Masae Ohkura, Yasue Osai, Akiko Sato, Tomoaki Takada, Hidenobu Matsusaka, Ichiro Ono, Yasuaki Tamura, Noriyuki Sato, Yasushi Sasaki, Akira Ito, Hiroyuki Honda, Kazumasa Wakamatsu, Shosuke Ito, Kowichi Jimbow

JOURNAL OF INVESTIGATIVE DERMATOLOGY ( NATURE PUBLISHING GROUP ) 129 ( 9 ) 2233 - 2241 2009年09月 [査読有り]

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43 degrees C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF.

DOI

アデノウイルスを用いたp53蛋白とp21発現抑制人工miRNAの共発現による癌治療効果の改善(A single adenovirus expressing p53 protein and p21-targeting artificial microRNAs improves therapeutic outcome in cancer)

井戸川雅史, 佐々木泰史, 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 492 - 492 2009年08月

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年08月

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年08月

p53ファミリーの標的遺伝子ICAM2の同定と機能解析(Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 family target)

石田勢津子, 安保直樹, 鹿島理沙, 井戸川雅史, 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 141 - 141 2009年08月

p53ファミリーの標的遺伝子EGR2の同定と機能解析(Identification and characterization of early growth response gene-2 as a p53-regulated proapoptotic gene)

横田育子, 佐々木泰史, 安保直樹, 鹿島理沙, 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 ) 68回 141 - 141 2009年08月

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年08月

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年08月

Genomic screening for genes silenced by DNA methylation revealed an association between RASD1 inactivation and dexamethasone resistance in multiple myeloma.

Nojima M, Maruyama R, Yasui H, Suzuki H, Maruyama Y, Tarasawa I, Sasaki Y, Asaoku H, Sakai H, Hayashi T, Mori M, Imai K, Tokino T, Ishida T, Toyota M, Shinomura Y

Clinical cancer research : an official journal of the American Association for Cancer Research 15 ( 13 ) 4356 - 64 2009年07月 [査読有り] [国際誌]

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

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 ( NATURE PUBLISHING GROUP ) 28 ( 29 ) 2643 - 2653 2009年07月

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

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.

Mita H, Toyota M, Aoki F, Akashi H, Maruyama R, Sasaki Y, Suzuki H, Idogawa M, Kashima L, Yanagihara K, Fujita M, Hosokawa M, Kusano M, Sabau SV, Tatsumi H, Imai K, Shinomura Y, Tokino T

BMC cancer 9 198 - 198 2009年06月 [査読有り] [国際誌]

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

Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-kappaB activity.

Tomita M, Toyota M, Ishikawa C, Nakazato T, Okudaira T, Matsuda T, Uchihara JN, Taira N, Ohshiro K, Senba M, Tanaka Y, Ohshima K, Saya H, Tokino T, Mori N

International journal of cancer 124 ( 11 ) 2607 - 15 2009年06月 [査読有り] [国際誌]

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

DOI PubMed

A single recombinant adenovirus expressing p53 and p21-targeting artificial microRNAs efficiently induces apoptosis in human cancer cells.

Idogawa M, Sasaki Y, Suzuki H, Mita H, Imai K, Shinomura Y, Tokino T

Clinical cancer research : an official journal of the American Association for Cancer Research 15 ( 11 ) 3725 - 32 2009年06月 [査読有り] [国際誌]

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

A novel biomarker for cervical cancer.

Tokino T

Cancer biology & therapy 8 ( 12 ) 1154 - 5 2009年06月 [査読有り] [国際誌]

PubMed

miRNAによるp21発現誘導抑制能を付加したp53発現ベクターによる癌治療効果の増強

井戸川雅史, 佐々木泰史, 篠村恭久, 今井浩三, 時野隆至

Drug Delivery System ( 日本DDS学会 ) 24 ( 3 ) 332 - 332 2009年06月

ヒストン脱アセチル化酵素阻害剤の併用によるp53遺伝子治療の作用増強

佐々木泰史, 今井浩三, 篠村恭久, 時野隆至

Drug Delivery System ( 日本DDS学会 ) 24 ( 3 ) 319 - 319 2009年06月

Cancer epigenomics: implications of DNA methylation in personalized cancer therapy.

Toyota M, Suzuki H, Yamashita T, Hirata K, Imai K, Tokino T, Shinomura Y

Cancer science 100 ( 5 ) 787 - 91 2009年05月 [査読有り] [国際誌]

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 family members regulate the expression of the apolipoprotein D gene

Yasushi Sasaki, Naoki Anbo, Masashi Idogawa, Hiroaki Mita, Minoru Toyota, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 69 2009年05月 [査読有り]

A single recombinant adenovirus expressing p53 and p21-targeting artificial microRNAs efficiently induces apoptosis in human cancer cells.

Masashi Idogawa, Yasushi Sasaki, Kohzoh Mai, Yasuhisa Shinomura, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 69 2009年05月 [査読有り]

DOI

p53 family members regulate the expression of the apolipoprotein D gene.

Sasaki Y, Negishi H, Koyama R, Anbo N, Ohori K, Idogawa M, Mita H, Toyota M, Imai K, Shinomura Y, Tokino T

The Journal of biological chemistry 284 ( 2 ) 872 - 83 2009年01月 [査読有り] [国際誌]

p73 and p63 are members of the p53 gene family that play an important role in development and homeostasis, mainly by regulating transcription of a variety of genes. We report here that apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid transport proteins, is a direct transcriptional target of the p53 family member genes. We found that the expression of apoD was specifically up-regulated by either TAp73 or TAp63 but not significantly by p53. In addition, apoD transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abolishes induction of apoD transcription following cisplatin treatment. We also identified a p73/p63-binding site in the promoter of the apoD gene that is responsive to the p53 family members. The ectopic expression of TAp73 as well as the addition of recombinant human apoD to culture medium induced the osteoblastic differentiation of the human osteosarcoma cell line Saos-2, as assessed by alkaline phosphatase activity. Importantly, apoD knockdown abrogated p73-mediated alkaline phosphatase induction. Moreover, TAp73-mediated apoD expression was able to induce morphological differentiation, as well as expression of neuronal markers, in the human neuroblastoma cell line SH-SY5Y. These results suggest that apoD induction may mediate the activity of p73 in normal development.

DOI PubMed

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年12月 [査読有り]

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.

Suzuki H, Tokino T, Shinomura Y, Imai K, Toyota M

Pharmacogenomics 9 ( 12 ) 1917 - 28 2008年12月 [査読有り] [国際誌]

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.

Yamamoto E, Toyota M, Suzuki H, Kondo Y, Sanomura T, Murayama Y, Ohe-Toyota M, Maruyama R, Nojima M, Ashida M, Fujii K, Sasaki Y, Hayashi N, Mori M, Imai K, Tokino T, Shinomura Y

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 17 ( 10 ) 2555 - 64 2008年10月 [査読有り] [国際誌]

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

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年09月

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年09月

p53ファミリー(p73,p63)の標的遺伝子apolipoprotein Dの同定(p53 family members regulate the expression of the apolipoprotein D gene)

佐々木泰史, 安保直樹, 根岸秀明, 井戸川雅史, 見田裕章, 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 ) 67回 60 - 60 2008年09月

miRNAによるp21誘導抑制能をもつp53発現アデノウイルスベクターによる抗腫瘍効果の増強(Enhancement of antitumor effect by single adenovirus vector expressing p53 and suppressing p21 induction by miRNA)

井戸川雅史, 佐々木泰史, 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 ) 67回 237 - 237 2008年09月

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年09月

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年09月

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年09月

DNA hypermethylation contributes to incomplete synthesis of carbohydrate determinants in gastrointestinal cancer.

Kawamura YI, Toyota M, Kawashima R, Hagiwara T, Suzuki H, Imai K, Shinomura Y, Tokino T, Kannagi R, Dohi T

Gastroenterology 135 ( 1 ) 142 - 151 2008年07月 [査読有り] [国際誌]

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 Sd(a) and the transcription of B4GALNT2, which catalyzes the synthesis of the Sd(a), 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.

Maruyama R, Akino K, Toyota M, Suzuki H, Imai T, Ohe-Toyota M, Yamamoto E, Nojima M, Fujikane T, Sasaki Y, Yamashita T, Watanabe Y, Hiratsuka H, Hirata K, Itoh F, Imai K, Shinomura Y, Tokino T

Carcinogenesis 29 ( 7 ) 1312 - 8 2008年07月 [査読有り] [国際誌]

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 silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer.

Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K, Shinomura Y, Tokino T

Cancer research 68 ( 11 ) 4123 - 32 2008年06月 [査読有り] [国際誌]

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 SFRP genes in oral squamous cell carcinoma.

Sogabe Y, Suzuki H, Toyota M, Ogi K, Imai T, Nojima M, Sasaki Y, Hiratsuka H, Tokino T

International journal of oncology 32 ( 6 ) 1253 - 61 2008年06月 [査読有り] [国際誌]

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 inactivation of RASSF2 in oral squamous cell carcinoma.

Imai T, Toyota M, Suzuki H, Akino K, Ogi K, Sogabe Y, Kashima L, Maruyama R, Nojima M, Mita H, Sasaki Y, Itoh F, Imai K, Shinomura Y, Hiratsuka H, Tokino T

Cancer science 99 ( 5 ) 958 - 66 2008年05月 [査読有り] [国際誌]

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

A requirement for DICER to maintain full promoter CpG island hypermethylation in human cancer cells.

Ting AH, Suzuki H, Cope L, Schuebel KE, Lee BH, Toyota M, Imai K, Shinomura Y, Tokino T, Baylin SB

Cancer research 68 ( 8 ) 2570 - 5 2008年04月 [査読有り] [国際誌]

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 Y, Negishi H, Idogawa M, Suzuki H, Mita H, Toyota M, Shinomura Y, Imai K, Tokino T

Molecular cancer therapeutics 7 ( 4 ) 779 - 87 2008年04月 [査読有り] [国際誌]

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

Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer

H. Suzuki, M. Toyota, H. Caraway, E. Gabrielson, T. Ohmura, T. Fujikane, N. Nishikawa, Y. Sogabe, M. Nojima, T. Sonoda, M. Mori, K. Hirata, K. Imai, Y. Shinomura, S. B. Baylin, T. Tokino

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 98 ( 6 ) 1147 - 1156 2008年03月 [査読有り]

Although mutation of APC or CTNNB1 ( beta- catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled- related protein ( SFRP) and Dickkopf ( DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines ( SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours ( SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines ( 3 out of 11, 27%) and primary tumours ( 15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a beta- catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T- cell factor/ lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis.

DOI

Identification of flotillin-2, a major protein on lipid rafts, as a novel target of p53 family members.

Sasaki Y, Oshima Y, Koyama R, Maruyama R, Akashi H, Mita H, Toyota M, Shinomura Y, Imai K, Tokino T

Molecular cancer research : MCR 6 ( 3 ) 395 - 406 2008年03月 [査読有り] [国際誌]

p73 and p63 are members of the p53 gene family and have been shown to play an important role in development and homeostasis mainly by regulating the transcription of a variety of genes. A subset of these genes encodes secreted proteins and receptors that may be involved in the communication between adjacent cells. We report here that flotillin-2, a major hydrophobic protein on biomembrane microdomain lipid rafts, is a direct transcriptional target of the p53 family member genes. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. We found that the expression of flotillin-2 was specifically up-regulated by either TAp73beta or TAp63gamma, but not significantly by p53. In addition, flotillin-2 transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we showed that silencing endogenous p73 abolishes the induction of flotillin-2 transcription following cisplatin treatment. Furthermore, we identified a p73/p63-binding site located upstream of the flotillin-2 gene that is responsive to the p53 family members. This response element is highly conserved between humans and rodents. We also found that ectopic expression of TAp73 as well as TAp63 enhances signal transduction by assessing the interleukin-6-mediated phosphorylation of signal transducers and activators of transcription 3. Thus, in addition to direct transactivation, p53 family member genes enhance a set of cellular processes via lipid rafts.

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年01月

Histone Deacetylase Inhibitor FK228 Enhances Adenovirus-mediated p53 Family Gene Therapy in Cancer Models

Yasushi Sasaki, Masashi Idogawa, Hiromu Suzuki, Hiroaki Mita, Minoru Toyota, Yasuhisa Shinomura, Kohzoh Imai, Takashi Tokino

TUMOR BIOLOGY ( KARGER ) 29 ( 4 ) 24 - 24 2008年 [査読有り]

DOI PubMed

In vitro transformation of adult rat hepatic progenitor cells into pancreatic endocrine hormone-producing cells.

Kawasaki H, Mizuguchi T, Kikkawa Y, Oshima H, Sasaki Y, Tokino T, Kokai Y, Miyazaki J, Katsuramaki T, Mitaka T, Hirata K

Journal of hepato-biliary-pancreatic surgery 15 ( 3 ) 310 - 7 2008年 [査読有り] [国内誌]

BACKGROUND/PURPOSE: Pancreatic and duodenal homeobox factor 1 (Pdx-1) plays an important role in initiating differentiation toward pancreatic endocrine cells. The transdifferentiation or transformation of hepatocytes into pancreatic endocrine cells could be feasible, due to their similar cellular origins. Our goal in this study was to see if small hepatocytes (SHs) could give rise to pancreatic endocrine cells via exogenous Pdx-1 gene expression. METHODS: SHs were cultured for 10 days before adenovirus (Adt)-mediated Pdx-1 gene transfection. We performed western blot analysis for pancreatic transcription factors in the nuclei and reverse-transcription polymerase chain reaction (RT-PCR) for the gene expression of pancreatic endocrine hormones. Confocal laser microscanning analysis was used to observe the transformation of SHs. RESULTS: Pancreatic transcription factors such as Pdx-1, Ngn3, NeuroD, Nkx2.2, and Pax6 were induced after Adt-Pdx-1 gene transfection. The mRNA expression of pancreatic endocrine hormones (insulin, glucagon, and somatostatin) was induced after the gene transfection. Pdx-1 was expressed in the nucleus, where the cells were positive for one or more of the hormones and cytokeratin (CK) 8. Some cells were positive for multiple hormones. The insulin level increased while the glucagon level decreased after the glucose loading test, depending on the glucose concentration. CONCLUSIONS: SHs are transformed into functional pancreatic endocrine cells after Pdx-1 gene transfection.

DOI PubMed

Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma.

Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, Nojima M, Yamamoto H, Omata M, Tokino T, Imai K, Shinomura Y

Journal of gastroenterology 43 ( 5 ) 378 - 89 2008年 [査読有り] [国内誌]

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, 9/28, 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 beta-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

p53 Expression with p21 Suppression by Single Vector Enhances Antitumor Effect

Masashi Idogawa, Yasushi Sasaki, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

TUMOR BIOLOGY ( KARGER ) 29 73 - 73 2008年 [査読有り]

Genomic Mapping of 12p Amplifications in Gastric Cancer Reveal a 0.5-Mb Target Region Including KRAS Locus

Hiroaki Mita, Fumio Aoki, Hirofumi Akashi, Reo Maruyama, Yasushi Sasaki, Hiromu Suzuki, Masashi Idogawa, Lisa Kashima, Minoru Toyota, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

TUMOR BIOLOGY ( KARGER ) 29 83 - 83 2008年 [査読有り]

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors.

Sato H, Suzuki H, Toyota M, Nojima M, Maruyama R, Sasaki S, Takagi H, Sogabe Y, Sasaki Y, Idogawa M, Sonoda T, Mori M, Imai K, Tokino T, Shinomura Y

Carcinogenesis 28 ( 12 ) 2459 - 66 2007年12月 [査読有り] [国際誌]

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.

Nishikawa N, Toyota M, Suzuki H, Honma T, Fujikane T, Ohmura T, Nishidate T, Ohe-Toyota M, Maruyama R, Sonoda T, Sasaki Y, Urano T, Imai K, Hirata K, Tokino T

Cancer research 67 ( 20 ) 9649 - 57 2007年10月 [査読有り] [国際誌]

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

PRDM5 identified as a target of epigenetic silencing in colorectal and gastric cancer.

Watanabe Y, Toyota M, Kondo Y, Suzuki H, Imai T, Ohe-Toyota M, Maruyama R, Nojima M, Sasaki Y, Sekido Y, Hiratsuka H, Shinomura Y, Imai K, Itoh F, Tokino T

Clinical cancer research : an official journal of the American Association for Cancer Research 13 ( 16 ) 4786 - 94 2007年08月 [査読有り] [国際誌]

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

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年08月

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年08月

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年08月

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年08月

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年08月

Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer

M. Nojima, H. Suzuki, M. Toyota, Y. Watanabe, R. Maruyama, S. Sasaki, Y. Sasaki, H. Mita, N. Nishikawa, K. Yamaguchi, K. Hirata, F. Itoh, T. Tokino, M. Mori, K. Imai, Y. Shinomura

ONCOGENE ( NATURE PUBLISHING GROUP ) 26 ( 32 ) 4699 - 4713 2007年07月 [査読有り]

Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear beta-catenin accumulation (13/15; 87%) and detected the active form of beta-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%),and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.

DOI

Antitumor effect of adenovirus-mediated p53 family gene transfer on osteosarcoma cell lines.

Oshima Y, Sasaki Y, Negishi H, Idogawa M, Toyota M, Yamashita T, Wada T, Nagoya S, Kawaguchi S, Yamashita T, Tokino T

Cancer biology & therapy 6 ( 7 ) 1058 - 66 2007年07月 [査読有り] [国際誌]

Osteosarcoma (OS) is one of the most common malignancies of the bone. Although prognosis of OS has improved significantly during the past several years due to more intensive chemotherapy and radiotherapy regimens, new therapeutic approaches are needed for recurrent and inoperable cases, p73 and p63, like their homologue, the tumor suppressor p53, are able to induce apoptosis in several cell types. Here, we evaluated the antitumor effects of p73 and p63 on eleven different human OS cell lines. In vitro, adenovirus-mediated transduction of p63gamma induced apoptosis in OS cells that are resistant to p53-mediated apoptosis, while less effect was observed following transduction of p73alpha or p63alpha. Interestingly, the apoptotic effects of p63gamma were greater than those of wild-type p53 in OS cells carrying MDM2-amplification. We then determined the in vivo therapeutic effect of intratumoral injection of adenovirus-vector expressing p53 family members on xenografts derived from Saos-2 cells implanted in nude mice, and showed that infection with p63y significantly suppressed tumor growth compared with p53. In addition, exogenous p73beta and p63gamma significantly increased the chemosensitivity of OS cells to doxorubicin and cisplatin, chemotherapeutic agents commonly used in the treatment of OS. Our results suggest that adenovirus-mediated transduction of p53 family members may have utility in gene therapy for OS, particularly in combination with chemotherapeutic agents.

PubMed

Ku70 and poly(ADP-ribose) polymerase-1 competitively regulate beta-catenin and T-cell factor-4-mediated gene transactivation: possible linkage of DNA damage recognition and Wnt signaling.

Idogawa M, Masutani M, Shitashige M, Honda K, Tokino T, Shinomura Y, Imai K, Hirohashi S, Yamada T

Cancer research 67 ( 3 ) 911 - 8 2007年02月 [査読有り] [国際誌]

Formation of the T-cell factor-4 (TCF-4) and beta-catenin nuclear complex is considered crucial to embryonic development and colorectal carcinogenesis. We previously reported that poly(ADP-ribose) polymerase-1 (PARP-1) interacts with the TCF-4 and beta-catenin complex and enhances its transcriptional activity. However, its biological significance remains unexplained. Using immunoprecipitation and mass spectrometry, we found that two Ku proteins, Ku70 and Ku80, were also associated with the complex. Knockdown of Ku70 by RNA interference increased the amount of beta-catenin associated with TCF-4 and enhanced the transcriptional activity. PARP-1 competed with Ku70 for binding to TCF-4. Treatment with bleomycin, a DNA-damaging alkylating agent, induced polyADP-ribosylation of PARP-1 protein and inhibited its interaction with TCF-4. Bleomycin conversely increased the amounts of Ku70 coimmunoprecipitated with TCF-4 and removed beta-catenin from TCF-4. We propose a working model in which the transcriptional activity of TCF-4 is regulated by the relative amount of Ku70, PARP-1, and beta-catenin proteins binding to TCF-4. Identification of the functional interaction of Ku70 as well as PARP-1 with the TCF-4 and beta-catenin transcriptional complex may provide insights into a novel linkage between DNA damage recognition/repair and Wnt signaling.

DOI PubMed

Identification of DFNA5 as a target of epigenetic inactivation in gastric cancer.

Akino K, Toyota M, Suzuki H, Imai T, Maruyama R, Kusano M, Nishikawa N, Watanabe Y, Sasaki Y, Abe T, Yamamoto E, Tarasawa I, Sonoda T, Mori M, Imai K, Shinomura Y, Tokino T

Cancer science 98 ( 1 ) 88 - 95 2007年01月 [査読有り] [国際誌]

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 down-regulated 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

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年 [査読有り]

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年12月 [査読有り]

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

Sequence motif discovery with computational genome-wide analysis

Akashi H, Aoki F, Toyota M, Maruyama R, Sasaki Y, Mita H, Tokura H, Imai K, Tatsumi H, Tokino T

Tumor Research ( Sapporo Medical University ) 41 59 - 69 2006年12月 [査読有り]

As a result of the human genome project and advancements in DNA sequencing technology, we can utilize a huge amount of nucleotide sequence data and can search DNA sequence motifs in whole human genome. However, searching motifs with the naked eye is an enormous task and searching throughout the whole genome is absolutely impossible. Therefore, we have developed a computational genome-wide analyzing system for detecting DNA sequence motifs with biological significance. We used a multi-parallel network computing system as a powerful computing engine. Furthermore, we improved the system to work as a background engine for web-based applications. The multi-parallel computing engine consists of a head processor, which issues control commands to data processing nodes for various kinds of jobs, such as retrieving arbitrary sequences, generating mapping images, and loading data sections from genome databases. We constructed the system to function as a flexible Client/Server structure connected over the network, and this system could be adapted to cope with increases in sequence data and to deal with algorithms for new investigation needs by slightly changing the control procedures and increasing the number of the processor node. We developed two additional tools to annotate the genome sequences. The first was the cDNA Reverse Splicing Tool, which divided cDNA sequences into exons and mapped them on the genomic sequence, and the second was DNA-Protein Translation Tool which showed open reading frames (ORFs) of whole genome. In order to examine the availability and efficiency of our system, we searched and identified p53RE (p53 response element) as a representative sequence motif on genomic sequences of chromosome 21 and 22. As a result, we detected 50,000 p53REs on fifty mega base genomic DNA sequences within 27 seconds.

DOI CiNii

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年09月

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年09月

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年09月

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年09月

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年09月

Activation of the ribosomal protein L13 gene in human gastrointestinal cancer.

Kobayashi T, Sasaki Y, Oshima Y, Yamamoto H, Mita H, Suzuki H, Toyota M, Tokino T, Itoh F, Imai K, Shinomura Y

International journal of molecular medicine 18 ( 1 ) 161 - 70 2006年07月 [査読有り] [国際誌]

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 semiquantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of L13 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年07月

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年07月

p73によるIL-4シグナルの活性化と抗腫瘍活性

佐々木泰史, 見田裕章, 豊田実, 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 85 - 85 2006年07月

Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation.

Maruyama R, Aoki F, Toyota M, Sasaki Y, Akashi H, Mita H, Suzuki H, Akino K, Ohe-Toyota M, Maruyama Y, Tatsumi H, Imai K, Shinomura Y, Tokino T

Cancer research 66 ( 9 ) 4574 - 83 2006年05月 [査読有り] [国際誌]

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.

Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M, Hosokawa M, Shinomura Y, Imai K, Tokino T

Cancer 106 ( 7 ) 1467 - 79 2006年04月 [査読有り] [国際誌]

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 bisulfite-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors were detected using real-time polymerase chain reaction analysis followed by an 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 nonrandom 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 the candidate tumor suppressor gene HOXB13 in human renal cell carcinoma

H. Okuda, M. Toyota, W. Ishida, M. Furihata, M. Tsuchiya, M. Kamada, T. Tokino, T. Shuin

Oncogene 25 ( 12 ) 1733 - 1742 2006年03月 [査読有り]

Epigenetic alterations like DNA methylation and the resulting inactivation of cancer-related genes often contribute to the development of various cancers. To identify the genes that are silenced by aberrant methylation in renal cell carcinoma (RCC), we subjected two RCC lines to methylated CpG island amplification/representational difference analysis. This identified 27 CpG islands. Combined bisulfite restriction analysis of these CpG islands in primary RCC cases revealed that four were methylated in a tumor-specific manner. One of these was identified as the human homeo-box gene B13 (HOXB13) gene, but the remaining three CpG islands were not associated with known genes. The methylation frequencies of HOXB13 in primary RCC samples and lines were 30 and 73%, respectively. The methylation status of HOXB13 correlated with the loss of its expression both in RCC lines and primary tumors, and methyltransferase inhibitor treatment induced the recovery of its expression. Exogenous expression of HOXB13 in RCC cells that lacked endogenous HOXB13 expression suppressed colony formation and induced apoptotic features. Furthermore, HOXB13 methylation correlated positively with tumor grade and microvessel invasion. These results suggest that HOXB13 is a novel candidate tumor suppressor gene in RCC and that its inactivation may play an important role in both RCC tumorigenesis and progression. © 2006 Nature Publishing Group All rights reserved.

DOI PubMed

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

K Terasawa, M Toyota, S Sagae, K Ogi, H Suzuki, T Sonoda, K Akino, R Maruyama, N Nishikawa, K Imai, Y Shinomura, T Saito, T Tokino

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 94 ( 6 ) 914 - 921 2006年03月 [査読有り]

Transcription factor 2 gene ( TCF2) encodes hepatocyte nuclear factor 1 beta ( HNF1 beta), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the gene's expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5- aza- 2'deoxycitidine ( 5- aza- dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5- aza- dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4 alpha, a transcriptional target of HNF1 beta, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment.

DOI PubMed

Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis

KE King, RM Ponnamperuma, MJ Gerdes, T Tokino, T Yamashita, CC Baker, WC Weinberg

CARCINOGENESIS ( OXFORD UNIV PRESS ) 27 ( 1 ) 53 - 63 2006年01月 [査読有り]

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and Delta N. Delta Np63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, Delta N- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. Delta Np63 alpha declines with Ca2+-induced differentiation, while a smaller Delta N-form, Delta Np63(s), persists, suggesting unique functions of the two Delta N-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, Delta Np63 alpha, Delta Np63(p40), TAp63 alpha, TAp63 gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of Delta Np63(p40), which lacks the entire alpha-tail present in Delta Np63 alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length Delta Np63 alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of Delta Np63 alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the Delta N domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The Delta N-terminus is sufficient to maintain keratinocytes in a proliferative state, as both Delta N forms block Ca2+-mediated p21(WAF1) induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63 alpha or TAp63 gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

DOI

新規大腸癌抑制遺伝子RASSF2異常メチル化の腫瘍マーカーへの応用

豊田実, 秋野公臣, 鈴木拓, 時野隆至, 篠村恭久, 今井浩三

日本分子腫瘍マーカー研究会誌 ( 日本分子腫瘍マーカー研究会 ) 21 5 - 6 2006年

DOI CiNii

The role of T-fimbrin in the response to DNA damage: silencing of T-fimbrin by small interfering RNA sensitizes human liver cancer cells to DNA-damaging agents.

Ikeda H, Sasaki Y, Kobayashi T, Suzuki H, Mita H, Toyota M, Itoh F, Shinomura Y, Tokino T, Imai K

International journal of oncology 27 ( 4 ) 933 - 40 2005年10月 [査読有り] [国際誌]

Fimbrins (also known as plastins) are actin-binding proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. We previously reported that the up-regulation of T-fimbrin, a fimbrin isoform, in association with G2 arrest following DNA damage and that a lack of T-fimbrin expression shortens the radiation-induced G2 arrest in Chinese hamster ovarian cells. In this study, we further investigated the role of T-fimbrin in DNA-damage response using a panel of human liver cancer cell lines and small interfering RNA technology. T-fimbrin was differentially expressed in human liver cancer cell lines. Colony formation assays revealed that cell lines lacking T-fimbrin expression were highly sensitive to DNA damage compared to cell lines that express T-fimbrin. Using siRNA designed to target T-fimbrin, we demonstrated that silencing endogenous T-fimbrin causes a marked increase in the cellular sensitivity to VP-16 and UV irradiation. Moreover, T-fimbrin deletion abrogated UV-mediated cell cycle checkpoint, and consequently led to increased apoptotic cell death in resistant cells. These findings suggest that the status of T-fimbrin expression may be a useful molecular marker for predicting the responsiveness of cancer cells to treatment with chemotherapeutic drugs.

PubMed

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年09月

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年09月

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年09月

Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation.

Simbulan-Rosenthal CM, Trabosh V, Velarde A, Chou FP, Daher A, Tenzin F, Tokino T, Rosenthal DS

Oncogene 24 ( 35 ) 5443 - 58 2005年08月 [査読有り] [国際誌]

Solar ultraviolet B (UVB) acts as both an initiator and promoter in models of multistage skin carcinogenesis. We found that, whereas UVB induces apoptosis in human papillomavirus-16 E6/7-immortalized keratinocytes, it inhibits markers of differentiation in human foreskin keratinocytes (HFK). Potential mechanisms for this differential response were examined by DNA microarray, which revealed that UVB alters the expression of three of the four human inhibitor of differentiation/DNA binding (Id) proteins that comprise a class of helix-loop-helix family of transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. These results were verified by RT-PCR and immunoblot analysis of control and UVB-irradiated primary and immortalized keratinocytes. Whereas Id1 was downregulated in both cell types, Id2 expression was upregulated in primary HFK, but not immortalized cells. In contrast, Id3 expression was significantly increased only in immortalized cells. The differential expression pattern of Id2 in response to UVB was recapitulated in reporter constructs containing the 5' regulatory regions of this gene. Id2 promoter activity increased in response to UVB in HFK, but not in immortalized cells. To identify the regulatory elements in the Id2 promoter that mediate transcriptional activation by UVB in HFK, promoter deletion/mutation analysis was performed. Deletion analysis revealed that transactivation involves a 166 bp region immediately upstream to the Id2 transcriptional start site and is independent of c-Myc. The consensus E twenty-six (ETS) binding site at -120 appears to mediate UVB transcriptional activation of Id2 because point mutations at this site completely abrogated this response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays verified that the Id2 promoter interacts with known Id2 promoter (ETS) binding factors Erg1/2 and Fli1, but not with c-Myc; and this interaction is enhanced after UVB exposure. Similar to the effects of UVB exposure, ectopic expression of Id2 protein in primary HFK resulted in inhibition of differentiation, as shown by decreased levels of the terminal differentiation marker keratin K1 and inhibition of involucrin crosslinking. Reduction of Id2 expression by small interfering RNAs attenuated the UVB-induced inhibition of differentiation in these cells. These results suggest that UVB-induced inhibition of differentiation of primary HFK is at least, in part, due to the upregulation of Id2, and that upregulation of Id2 by UVB might predispose keratinocytes to carcinogenesis by preventing their normal differentiation program.

DOI PubMed

Identification of pigment epithelium-derived factor as a direct target of the p53 family member genes.

Yasushi Sasaki, Yasuyoshi Naishiro, Yuichiro Oshima, Kohzoh Imai, Yusuke Nakamura, Takashi Tokino

Oncogene 24 ( 32 ) 5131 - 6 2005年07月 [査読有り] [国際誌]

p63 and p73 show a high degree of structural homology to p53 and are members of a family of transcriptional factors that can activate transcription of p53-responsive genes. p53 is mutated in more than 50% of human cancers, whereas p63 and p73 are rarely mutated. Studies of knockout mice also revealed an unexpected functional diversity among the p53 family. To determine how p63 and p73 are involved in tumorigenesis and normal development, we used cDNA microarray to examine 9216 genes in human colorectal cancer cells. We discovered that the expression of pigment epithelium-derived factor (PEDF) was specifically induced by either p63 or p73, but not by p53. We also report here that the PEDF gene contains a response element specific for p63 and p73 in its promoter region and is a direct target of p63 and p73. Collectively, p63 and p73 may be involved in cell fate by inducing PEDF expression.

PubMed

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

Akino K, Toyota M, Suzuki H, Mita H, Sasaki Y, Ohe-Toyota M, Issa JP, Hinoda Y, Imai K, Tokino T

Gastroenterology 129 ( 1 ) 156 - 69 2005年07月 [査読有り] [国際誌]

BACKGROUND & AIMS: Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. METHODS: The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. RESULTS: Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. CONCLUSIONS: RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

DOI PubMed

Small interfering RNA-induced CHFR silencing sensitizes oral squamous cell cancer cells to microtubule inhibitors.

Ogi K, Toyota M, Mita H, Satoh A, Kashima L, Sasaki Y, Suzuki H, Akino K, Nishikawa N, Noguchi M, Shinomura Y, Imai K, Hiratsuka H, Tokino T

Cancer biology & therapy 4 ( 7 ) 773 - 80 2005年07月 [査読有り] [国際誌]

Alterations in the function of cell cycle checkpoints are frequently detected in oral squamous cell carcinomas (OSCCs), and are often associated with the sensitivity of the cancer cells to chemotherapeutic drugs. Recently, a mitotic checkpoint gene, Chfr, was shown to be inactivated by promoter methylation and point mutations in various human tumors. Here we show that the absence of its product, CHFR, is associated with mitotic checkpoint dysfunction, and that cancer cells lacking CHFR are sensitive to microtubule inhibitors. Checkpoint impairment appears to be caused by a prophase defect in this case, as OSCC cells lacking CHFR showed phosphorylation of histone H3 on Ser10 and translocation of cyclin B1 to the nucleus. When CHFR-deficient OSCC cells were treated with a microtubule inhibitor (docetaxel or paclitaxel), significant numbers of apoptotic cells were observed. Moreover, disruption of CHFR using small interfering RNA (siRNA) impaired the mitotic checkpoint, thereby reducing the ability of OSCC cells to arrest at G2/M phase and making them more sensitive to microtubule inhibitors. Our results suggest that CHFR could be a useful molecular target for chemotherapy.

PubMed

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年07月

Upregulation of BNIP3 by 5-aza-2'-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death.

Abe T, Toyota M, Suzuki H, Murai M, Akino K, Ueno M, Nojima M, Yawata A, Miyakawa H, Suga T, Ito H, Endo T, Tokino T, Hinoda Y, Imai K

Journal of gastroenterology 40 ( 5 ) 504 - 10 2005年05月 [査読有り] [国内誌]

BACKGROUND: Pancreatic cancer cells often show resistance to hypoxia-mediated apoptosis, but the molecular mechanism underlying that resistance remains unknown. The purpose of the present study, therefore, was to examine the role of epigenetic gene alteration in the resistance to hypoxia-mediated apoptosis among pancreatic cancer cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of five genes associated with hypoxia-mediated apoptosis (PUMA, Caspase-8 [CASP8], APAF-1, BNIP3, and BNIP3L) in a panel of pancreatic cancer cell lines. Protein expression was examined by Western blot analysis, using lysates from cells incubated under normoxic or hypoxic conditions. The methylation status of the genes was determined using bisulfite-PCR and sequencing. The percentages of cells that were apoptotic were determined using flow cytometry. RESULTS: Under normoxic conditions, the expression of the BNIP3 gene varied among the 12 pancreatic cancer cell lines tested, with 50% of them showing no BNIP3 expression at all, whereas expression of the other four genes was readily detected in all 12 cell lines. DNA methylation of BNIP3's CpG island in the region around the transcription start site of the gene was closely associated with its silencing. The expression of BNIP3 was restored by the methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), as was the hypoxia-mediated pancreatic cancer cell death. CONCLUSIONS: BNIP3 expression is silenced in some pancreatic cancer cells by the methylation of its CpG island. Demethylation of BNIP3, using a methyltransferase inhibitor, restores the gene's expression and induces hypoxia-mediated cell death. BNIP3 may thus be a useful target for new therapies aimed at treating pancreatic cancer.

DOI PubMed

Aberrant laminin beta3 isoforms downstream of EWS-ETS fusion genes in Ewing family tumors.

Hideto Irifune, Hiroyuki Nishimori, Goichi Watanabe, Kouichi Yoshida, Tatsuru Ikeda, Chihiro Matsui, Masaaki Morohashi, Satoshi Kawaguchi, Satoshi Nagoya, Takuro Wada, Toshihiko Yamashita, Yusuke Nakamura, Takashi Tokino

Cancer biology & therapy 4 ( 4 ) 449 - 55 2005年04月 [査読有り] [国際誌]

Ewing family tumors (EFTs) are associated with a chromosomal translocation resulting in a fusion of the amino-terminus of EWS with the DNA-binding domain of an ETS transcription factor. Although previous reports suggested that these chimeric proteins would act as aberrant transcription factors, their downstream targets have not been fully elucidated. To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line, HT-1080, by retroviral transduction. Here we report that the LAMB3 gene encoding the beta3 chain of basement membrane protein laminin-5 is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Additionally through use of an antisense oligonucleotide for EWS-ERG in the W-ES EFT cell line, laminin beta3 protein was reduced coordinately with EWS-ERG fusion protein expression. Furthermore, we found small mRNAs were preferentially transcribed from the LAMB3 gene in EFT cell lines. Molecular cloning of the entire coding region shows that the alternative transcripts from different promoter(s) located within the intron 14, which encode small proteins, likely are major products of the LAMB3 gene in EFT cells. We show that the small isoforms conferred increased anchorage-independent proliferation to NIH3T3 cells. Together with previous studies showing that laminin-5 is involved in the invasive and malignant phenotype of several tumor types, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by upregulation of LAMB3 expression.

PubMed

Abnormal beta-catenin expression in oral cancer with no gene mutation: correlation with expression of cyclin D1 and epidermal growth factor receptor, Ki-67 labeling index, and clinicopathological features.

Tetsuyo Odajima, Yasushi Sasaki, Nobuyuki Tanaka, Yuko Kato-Mori, Hiroko Asanuma, Tatsuru Ikeda, Masaaki Satoh, Hiroyoshi Hiratsuka, Takashi Tokino, Norimasa Sawada

Human pathology 36 ( 3 ) 234 - 41 2005年03月 [査読有り] [国際誌]

Beta-Catenin not only acts as a regulator of E-cadherin-mediated cell-cell adhesion but also plays an important role in Wnt signaling. To assess the prevalence of Wnt signaling, we examined beta-catenin mutation and its immunohistochemical protein expression in oral cancers. The results were linked with expression of cyclin D1, one of the target genes of Wnt signaling, expression of epidermal growth factor receptor (EGFR) relevant to beta-catenin tyrosine phosphorylation, Ki-67 labeling index, clinicopathological features, and survival. In the analysis based on membranous expression of beta-catenin, 75 (68.2%) of 110 cases showed a reduced membranous pattern, and the remaining 35 (31.8%) had a preserved membranous pattern similar to that in oral epithelium. In the analysis of another category of beta-catenin expression, a cytoplasmic/nuclear pattern was observed in 21 (19.1%) of the 110 tumors. Most (19/21, 90.5%) of these tumors had a concomitant reduction of membranous expression of beta-catenin. The reduced membranous or cytoplasmic/nuclear pattern of beta-catenin was significantly associated with an invasive growth pattern, EGFR expression, an increased Ki-67 labeling index, and shorter survival but not with cyclin D1 expression. Mutational analyses of beta-catenin were performed for 39 cases, including the 21 tumors with a cytoplasmic/nuclear pattern, but no mutations in the beta-catenin gene exon 3 were detected in these samples. Our data indicate that altered expression of beta-catenin may play an important role in tumor progression through increased proliferation and invasiveness under EGFR activation. However, mutations of beta-catenin do not appear to be responsible for tumor development and abnormal expression of beta-catenin in oral cancers.

DOI PubMed

Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

M Murai, M Toyota, A Satoh, H Suzuki, K Akino, H Mita, Y Sasaki, T Ishida, L Shen, G Garcia-Manero, JPJ Issa, Y Hinoda, T Tokino, K Imai

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 92 ( 6 ) 1165 - 1172 2005年03月

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2 '-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5 ' region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5 ' CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

DOI

Aberrant methylation and silencing of the BNIP3 gene in colorectal and gastric cancer.

Masafumi Murai, Minoru Toyota, Hiromu Suzuki, Ayumi Satoh, Yasushi Sasaki, Kimishige Akino, Masako Ueno, Fumihiko Takahashi, Masanobu Kusano, Hiroaki Mita, Kazuyoshi Yanagihara, Takao Endo, Yuji Hinoda, Takashi Tokino, Kohzoh Imai

Clinical cancer research : an official journal of the American Association for Cancer Research 11 ( 3 ) 1021 - 7 2005年02月 [査読有り] [国際誌]

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-aza-2'-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

Enhanced Bax in oral SCC in relation to antitumor effects of chemotherapy.

Kanako Takemura, Makoto Noguchi, Kazuhiro Ogi, Takashi Tokino, Hiromi Kubota, Akihiro Miyazaki, Geniku Kohama, Hiroyoshi Hiratsuka

Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 34 ( 2 ) 93 - 9 2005年02月 [査読有り] [国際誌]

BACKGROUND: Antitumor effects of chemotherapeutic agents are commonly associated with the induction of apoptosis. Bax belongs to the Bcl-2 family and induces apoptosis. The present study was conducted to investigate the relationship between enhanced Bax expression in oral squamous cell carcinoma (SCC; cell lines and clinical cases) and the antitumor effects of chemotherapy. METHODS: In three oral SCC cell lines, Bax expression before and after treatment with chemotherapeutic agents [docetaxel (TXT), cisplatin and 5-fluorouracil] was examined by reverse transcriptase-polymerase chain reaction and immunoblotting. The effects of treatment were assessed by counting the number of viable cells and determining sub-G1 cells. Tissue samples (both biopsy specimens before chemotherapy and surgically excised specimens after chemotherapy) from nine patients with oral SCC who underwent neoadjuvant chemotherapy were immunostained for Bax. The relationship between enhancement of Bax expression and chemotherapeutic effects was established. RESULTS: Two of three cell lines did not express Bax mRNA or protein before treatment. After treatment, Bax expression was enhanced only by TXT in one cell line, but by all chemotherapeutic agents in the other two cell lines. In three of nine patients, Bax expression was not found before chemotherapy. Two of these three patients showed enhanced Bax expression after chemotherapy including TXT, but one still failed to express Bax. Both in cell lines and clinical cases, enhancement of Bax after chemotherapy was associated with antitumor effects. CONCLUSION: Certain chemotherapeutic agents enhance Bax expression in oral SCC, and it is suggested that this contributes to the antitumor effects of chemotherapy.

DOI PubMed

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis.

Masaki Murata, Takashi Kojima, Toshinobu Yamamoto, Mitsuru Go, Ken-ichi Takano, Hideki Chiba, Takashi Tokino, Norimasa Sawada

Cell and tissue research 319 ( 2 ) 341 - 7 2005年02月 [査読有り] [国際誌]

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40-51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18beta-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.

DOI PubMed

Identification of a specific target sequence for p53 family in the Jagged2 gene

Yuichiro Oshima, Setsuko Ishida, Reo Maruyama, Minoru Toyota, Toshihiko Yamashita, Takashi Tokino, Yasushi Sasaki

Tumor Research 40 13 - 24 2005年 [査読有り]

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. The p53 family members, p73 and p63, have remarkable structural similarities with p53 and can activate transcription of p53-responsive genes. In contrast to p53 however, p73 and p63 are rarely mutated in human cancers. Mice that lack p53 are developmentally normal, while p73 and p63 appear to play critical roles in normal development. In our earlier study, we showed that both p73 and p63 regulate the ligands for the Notch receptor, Jagged1 (JAG1) and Jagged2 (JAG2). We also identified a p63-binding site in the second intron of the human JAG1 gene. Here, we have analyzed the responsive element within the human JAG2 gene for p73/p63-dependent induction of gene expression. Using a chromatin immunoprecipitation assay, we identified a p73/p63-binding site located in the second intron of the JAG2 gene. A heterologous reporter assay revealed that this binding site is a functional response element and is relatively specific for p73 and p63 among the p53 family. This binding site consists of four copies of 10-bp consensus p53-binding motif and is highly conserved between human and rodents. Furthermore, both JAG1 and JAG2 mRNA were specifically up-regulated by either p63 or p73, but not by p53 in mouse embryo fibroblasts. Our findings demonstrate that JAG2 is indeed a direct and evolutionarily conserved transcriptional target of p73 and p63, and suggest a potential molecular mechanism for the involvement of the p53 family genes in normal development.

SFRP遺伝子のepigeneticな不活化は大腸癌におけるWnt経路の異常活性化を補完する

鈴木拓, 豊田実, 時野隆至, 森満, 今井浩三

日本分子腫瘍マーカー研究会誌 ( Japanese Society for Molecular Tumor Marker Research ) 20 73 - 74 2005年

DOI CiNii

Dual role of p53 in DNA binding.

Takashi Tokino

Cancer biology & therapy 3 ( 12 ) 1322 - 3 2004年12月 [査読有り] [国際誌]

PubMed

Erratum: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells (British Journal of Cancer (2004) 90 (844-852) doi:10.1038/sj.bjc.6601602)

Y. Morimoto, M. Toyota, H. Ikeda, T. Tokino, K. Imai

British Journal of Cancer 91 ( 11 ) 1978 2004年11月 [査読有り]

DOI

Epigenetic inactivation of class II transactivator (CIITA) is associated with the absence of interferon-gamma-induced HLA-DR expression in colorectal and gastric cancer cells.

Ayumi Satoh, Minoru Toyota, Hideyuki Ikeda, Yoshikazu Morimoto, Kimishige Akino, Hiroaki Mita, Hiromu Suzuki, Yasushi Sasaki, Takayuki Kanaseki, Yukio Takamura, Hidenobu Soejima, Takeshi Urano, Kazuyoshi Yanagihara, Takao Endo, Yuji Hinoda, Masahiro Fujita, Masao Hosokawa, Noriyuki Sato, Takashi Tokino, Kohzoh Imai

Oncogene 23 ( 55 ) 8876 - 86 2004年11月 [査読有り] [国際誌]

Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4+ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-gamma induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5' CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.

DOI PubMed

Identification of SCN3B as a novel p53-inducible proapoptotic gene.

Katsuya Adachi, Minoru Toyota, Yasushi Sasaki, Toshiharu Yamashita, Setsuko Ishida, Mutsumi Ohe-Toyota, Reo Maruyama, Yuji Hinoda, Tsuyoshi Saito, Kohzoh Imai, Ryuichi Kudo, Takashi Tokino

Oncogene 23 ( 47 ) 7791 - 8 2004年10月 [査読有り] [国際誌]

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. To identify novel p53-inducible genes, we compared the expression of genes in normal mouse embryo fibroblasts (MEFs) to p53-null cells by cDNA representational difference analysis. We report here that expression of endogenous sodium channel subunit beta 3 (SCN3B) is upregulated in mouse embryonic fibroblasts by DNA damage in a p53-dependent manner. In addition, we found that SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of p53, but not significantly by p63 or p73. Furthermore, we identified two putative p53-binding sites upstream of the first exon (RE1) and in the third intron (RE2). The p53 protein can directly interact with the putative p53-binding sites in vivo, as assessed by chromatin immunoprecipitation. A reporter gene assay revealed that these two p53-binding sites are functional response elements. The SCN3B protein appears to be localized to the endoplasmic reticulum (ER). Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation. Furthermore, we found that adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. The results presented here suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs.

DOI PubMed

T-fimbrin遺伝子のG2/M期チェックポイントへの関与と抗がん剤感受性

池田博, 佐々木泰史, 見田裕章, 豊田実, 時野隆至, 今井浩三

日本癌学会総会記事 ( (一社)日本癌学会 ) 63回 471 - 471 2004年09月

Reply: CIITA methylation and decreased levels of HLA-DR in tumour progression

Y Morimoto, M Toyota, H Ikeda, T Tokino, K Imai

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 91 ( 4 ) 814 - 815 2004年08月

DOI

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.

Hiromu Suzuki, D Neil Watkins, Kam-Wing Jair, Kornel E Schuebel, Sanford D Markowitz, Wei Dong Chen, Theresa P Pretlow, Bin Yang, Yoshimitsu Akiyama, Manon Van Engeland, Minoru Toyota, Takashi Tokino, Yuji Hinoda, Kohzoh Imai, James G Herman, Stephen B Baylin

Nature genetics 36 ( 4 ) 417 - 22 2004年04月 [査読有り] [国際誌]

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

DOI PubMed CiNii

Epigenetic inactivation of TMS1/ASC in ovarian cancer.

Katsuhiko Terasawa, Satoru Sagae, Minoru Toyota, Kuniko Tsukada, Kazuhiro Ogi, Ayumi Satoh, Hiroaki Mita, Kohzoh Imai, Takashi Tokino, Ryuichi Kudo

Clinical cancer research : an official journal of the American Association for Cancer Research 10 ( 6 ) 2000 - 6 2004年03月 [査読有り] [国際誌]

PURPOSE: The purpose of this work was to explore the role of epigenetic inactivation of apoptotic pathways in ovarian cancer by examining the DNA methylation and expression status of four proapoptotic genes in primary ovarian cancers and cancer cell lines and to correlate those findings with the clinicopathological features of ovarian cancer patients. EXPERIMENTAL DESIGN: Genomic DNA was isolated from 15 ovarian cancer cell lines, 80 primary ovarian cancer specimens, and 4 normal ovary specimens using phenol-chloroform extraction. The methylation status of the DNA was evaluated using combined bisulfite restriction analysis, gene expression was evaluated using reverse transcription-PCR, and histone acetylation was evaluated using chromatin immunoprecipitation. RESULTS: Of the four proapoptotic genes studied, expression of TMS1/ASC was absent in six ovarian cancer cell lines. Dense methylation of the 5' region of TMS1/ASC was detected in cells not expressing TMS1/ASC. Treating methylated cells with 5-aza-deoxycytidine restored gene expression, confirming the role of methylation in silencing the gene. Chromatin immunoprecipitation revealed histone to be deacetylated in cells not expressing TMS1/ASC, indicating that histone deacetylation is also involved in silencing TMS1/ASC. Aberrant methylation of TMS1/ASC was detected in 15 of 80 ovarian cancer tissues (19%) but in none of the normal ovary specimens. Aberrant methylation of TMS1/ASC was observed significantly more often in clear cell-type ovarian cancers than in other tumor types (P < 0.0001). CONCLUSIONS: Methylation-mediated silencing of TMS1/ASC confers a survival advantage to tumor cells by enabling them to escape apoptosis. The role for aberrant methylation in human ovarian tumorigenesis may be particularly important for ovarian cancers with the clear cell phenotype.

DOI PubMed

Regulation of MHC class II expression in glioma cells by class II transactivator (CIITA).

Yukio Takamura, Hideyuki Ikeda, Takayuki Kanaseki, Minoru Toyota, Takashi Tokino, Kohzoh Imai, Kiyohiro Houkin, Noriyuki Sato

Glia 45 ( 4 ) 392 - 405 2004年03月 [査読有り] [国際誌]

We first classified 12 malignant glioma cell lines into three different groups (types 1-3) with respect to major histocompatibility complex (MHC) class II expression and analyzed each group based on the different expression status of the class II transactivator (CIITA) gene. Glioma type 1 (2 of 12) showed constitutive expression of all class II molecules that might be mediated by activation of B cell-specific CIITA promoter III. Glioma type 2 represented the major phenotype (66.7 %) of malignant glioma cell lines, and MHC class II expression was induced by interferon-gamma (IFN-gamma) in this phenotype. Analysis of glioma tissue samples revealed that CIITA promoter IV was detected in 9 of 11 patients (81.8%); however, promoter III was only in two (18.2%). Moreover, cultured glioma cells obtained from a fresh tumor sample upregulated expression of CIITA and class II molecules in the presence of IFN-gamma, strongly suggesting that glioma type 2 might be predominant in glioma tissues. Glioma type 3 (2 of 12) showed CIITA transcripts but loss of MHC class II expression even in the presence of IFN-gamma. In addition, we determined that the constitutive MHC class II expression in the glioma cell lines (type 1) was the result of transcriptional activation of the CIITA gene. This phenomenon was mediated by global histone acetylation over 6 kb upstream from the transcriptional start site of CIITA promoter III. Moreover, stable transfection of CIITA promoter IV as well as promoter III into MHC class II inducible cell lines restored the constitutive expression of all class II molecules. These studies lay the foundation to understand the molecular basis for the expression of class II molecules in gliomas.

DOI PubMed

Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-gamma in haematopoietic tumour cells

Y Morimoto, M Toyota, A Satoh, M Murai, H Mita, H Suzuki, Y Takamura, H Ikeda, T Ishida, N Sato, T Tokino, K Imai

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 90 ( 4 ) 844 - 852 2004年02月

By presenting immunogenic peptides at the cell surface, major histocompatibility complex (MHC) class II molecules play a key role in the control of adaptive immune responses. Whether expressed constitutively or induced by interferon-gamma, expression of MHC class II molecules is regulated via coactivator class II transactivator (CIITA); moreover, suppression of their expression is one mechanism by which cancer cells escape host immunity. In this study, we surveyed the relationship between the expression of one MHC class II antigen, HLA-DR, and its coactivators in a group of haematopoietic cell lines, and explored the role of the aberrant DNA methylation in silencing HLA-DR expression. Among 26 cell lines studied, HLA-DR expression was lost from eight T-cell and two myeloid leukaemia cell lines, and this loss was closely associated with suppression of CIITA-PIV expression. Notably, nine of the 10 cell lines that lost CIITA-PIV expression showed methylation of the gene's 5' CpG island. Thus, DNA methylation is believed to inhibit the expression of MHC class II molecules in haematopoietic tumour cells by silencing its coactivator, CIITA-PIV. Furthermore, methylation of CIITA-PIV was detected in seven of 32 primary acute myeloid leukaemia specimens, indicating that epigenetic alteration is not a cell line-specific phenomenon. Collectively, these data suggest that, by suppressing expression of MHC class II molecules, epigenetic inactivation of CIITA provides a survival advantage to a subset of haematopoietic tumours.

DOI

Aberrant methylation and histone deacetylation associated with silencing of SLC5A8 in gastric cancer

M Ueno, M Toyota, K Akino, H Suzuki, M Kusano, A Satoh, H Mita, Y Sasaki, M Nojima, K Yanagihara, Y Hinoda, T Tokino, K Imai

TUMOR BIOLOGY ( KARGER ) 25 ( 3 ) 134 - 140 2004年

Aberrant methylation of a sodium co-transporter, solute carrier family 5 member 8 gene (SLC5A8), has been detected in a subset of colorectal cancers, suggesting SLC5A8 may also serve as a tumor suppressor. To further investigate the role of epigenetic inactivation of SLC5A8 expression in gastric cancer, we determined the methylation status of the SLC5A8 5' CpG island (CGI) in a panel of gastric cancer cell lines and primary gastric cancers. We detected methylation of the 5' CGI in ten of twelve gastric cancer cell lines, and five of those showed dense methylation, which correlated with the absence of SLC5A8 transcription. Aberrant methylation of SLC5A8 was also detected in 23 of 71 (30%) primary gastric cancers, indicating that epigenetic inactivation of SLC5A8 is not a cell-line-specific phenomenon. SLC5A8 expression was restored in methylated cell lines by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, chromatin immunoprecipitation assays showed that acetylation of histone H3 in the 5' region of the gene correlated directly with SLC5A8 expression and inversely with DNA methylation. It thus appears that aberrant methylation of its 5' CGI and histone deacetylation play key roles in silencing SLC5A8 expression in gastric cancers. Copyright (C) 2004 S. Karger AG, Basel.

DOI

Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer.

Ayumi Satoh, Minoru Toyota, Fumio Itoh, Yasushi Sasaki, Hiromu Suzuki, Kazuhiro Ogi, Takefumi Kikuchi, Hiroaki Mita, Toshiharu Yamashita, Takashi Kojima, Masanobu Kusano, Masahiro Fujita, Masao Hosokawa, Takao Endo, Takashi Tokino, Kohzoh Imai

Cancer research 63 ( 24 ) 8606 - 13 2003年12月 [査読有り] [国際誌]

Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.

PubMed

Identification of HRK as a target of epigenetic inactivation in colorectal and gastric cancer.

Toshiro Obata, Minoru Toyota, Ayumi Satoh, Yasushi Sasaki, Kazuhiro Ogi, Kimishige Akino, Hiromu Suzuki, Masafumi Murai, Takefumi Kikuchi, Hiroaki Mita, Fumio Itoh, Jean-Pierre J Issa, Takashi Tokino, Kohzoh Imai

Clinical cancer research : an official journal of the American Association for Cancer Research 9 ( 17 ) 6410 - 8 2003年12月 [査読有り] [国際誌]

PURPOSE: Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression. EXPERIMENTAL DESIGN: Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis. RESULTS: The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53. CONCLUSION: HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.

PubMed

Identification of the interleukin 4 receptor alpha gene as a direct target for p73.

Yasushi Sasaki, Hiroaki Mita, Minoru Toyota, Setsuko Ishida, Ichiro Morimoto, Toshiharu Yamashita, Toshihiro Tanaka, Kohzoh Imai, Yusuke Nakamura, Takashi Tokino

Cancer research 63 ( 23 ) 8145 - 52 2003年12月 [査読有り] [国際誌]

p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.

PubMed

口腔扁平上皮癌におけるM期チェックポイント遺伝子の発現異常とmicrotubule inhibitor感受性

荻和弘, 豊田実, 田中信幸, 野口誠, 佐藤亜由美, 佐々木泰史, 見田裕章, 鈴木拓, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 62回 222 - 222 2003年08月

MCA-RDA法を用いた消化器癌における新規メチル化標的遺伝子の同定

秋野公臣, 豊田実, 荻和弘, 小畑俊郎, 石井卓, 佐藤亜由美, 村井政史, 丸山玲緒, 見田裕章, 佐々木泰史, 遠藤高夫, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 62回 286 - 286 2003年08月

Epigenetic inactivation of CHFR in human tumors.

Minoru Toyota, Yasushi Sasaki, Ayumi Satoh, Kazuhiro Ogi, Takefumi Kikuchi, Hiromu Suzuki, Hiroaki Mita, Nobuyuki Tanaka, Fumio Itoh, Jean-Pierre J Issa, Kam-Wing Jair, Kornel E Schuebel, Kohzoh Imai, Takashi Tokino

Proceedings of the National Academy of Sciences of the United States of America 100 ( 13 ) 7818 - 23 2003年06月 [査読有り] [国際誌]

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

DOI PubMed CiNii

deltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes.

King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF, Weinberg WC

Oncogene 22 ( 23 ) 3635 - 44 2003年06月 [査読有り] [国際誌]

deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

DOI PubMed

ヒト腫瘍におけるM期チェックポイント遺伝子CHFRの異常メチル化と発現抑制

佐藤亜由美, 豊田実, 佐々木泰史, 荻和弘, 小畑俊郎, 菊地剛, 鈴木拓, 伊東文生, 時野隆至, 今井浩三

日本癌学会総会記事 ( (一社)日本癌学会 ) 61回 88 - 88 2002年10月

紫外線(UVB)によって転写誘導又は抑制されるヒトメラノサイト遺伝子の解析

金海英, 山下利春, 佐々木泰史, 時野隆至, 神保孝一

日本研究皮膚科学会年次学術大会・総会プログラム ( (一社)日本研究皮膚科学会 ) 27回 189 - 189 2002年08月

p53ファミリー遺伝子の機能解析ー遺伝子治療をめざしてー

佐々木泰史, 時野隆至

札幌医学雑誌 ( 札幌医科大学 ) 71 ( 1〜2 ) 1 - 6 2002年04月 [査読有り]

p53 gene therapy is being tested clinically for the treatment of human cancer, however some cancer models (in vivo and in vitro) are resistant to p53. To explore the potential use of two p53 homologues, p73 and p51/p63, in cancer gene therapy, we introduced p53, p73 and p51/p63 into colorectal cancer cell lines via adenoviral vectors, and compared their effects on cell growth. Among ten cell lines tested, six cell lines displayed a similar response following transduction of p53, p73 beta or p51A/p63 gamma, two lines underwent cell-cycle arrest, three lines exhibited apoptosis and one line showed no-effect following transduction. The effect on cell-cycle progression was variable in the other four cell lines. Interestingly, three cell lines were resistant to p53-mediated apoptosis, including two lines having endogenous wildtype p53 alleles, but underwent apoptosis after transduction of p73 beta or p51A/p63 gamma. Similar to p53, transduction of p51A/p63 gamma induced extensive apoptosis when combined with adriamycin or X-radiation in SW480 cells, which are normally resistant to apoptosis. Transduction of p73 beta and p51A/p63 gamma also reduced the tumorigenicity of two colorectal cancer cells in vivo. These results suggest that adenovirus-mediated p73 beta and p51A/p63 gamma transfer are potential novel approaches for the treatment of human cancers, particularly for tumors that are resistant to p53 gene therapy.

DOI CiNii

Methylated CpG Island Amplification of Representational Difference Analysis for Oral Cancer <Review>

Ogi Kazuhiro, Toyota Minoru, Kohama Geniku, Tokino Takashi

Tumor research : experimental and clinical ( 札幌医科大学 ) 37 9 - 15 2002年

CpG islands are G+C-rich regions approximately 1 kb long that are free of methylation and contain the promoters of many mammalian genes. It has been demonstrated that aberrant methylation of CpG islands is associated with gene silencing of tumor suppressor genes. To examine DNA methylation changes in oral squamous cell carcinoma (OSCC), we have applied a novel genome screening technique, methylated CpG island amplification coupled with representational difference analysis. Using DNA from an OSCC cell line as tester and DNA from normal tongue tissue as driver, DNA sequences aberrantly methylated in OSCC can be obtained. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

DOI CiNii

EWS-ETS family融合遺伝子の標的遺伝子の発現解析

渡邊吾一, 西森博幸, 入船秀仁, 佐々木泰史, 前仏均, 田中敏博, 吉田幸一, 石井清一, 中村祐輔, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 60回 334 - 334 2001年09月

Dried Feces Spots as an Alternative DNA Source: Detection of K-ras Mutations in Colorectal Cancer Screening

Yamaguchi Akihiro, Hashimoto Naoko, Tsutae Wataru, Seino Kuniyoshi, Osanai Hiroyuki, Yamamoto Naoya, Tokino Takashi, Sato Noriyuki, Kikuchi Kokichi

Tumor research : experimental and clinical ( 札幌医科大学 ) 36 7 - 13 2001年

Detection of genetic alterations in exfoliated intestinal cells into feces is expected to be a novel approach to colorectal cancer screening. We aimed here to detect cancer-related gene mutations in routine fecal specimen; dried feces spots (DFS) on a filter paper especially designated for an automated fecal occult blood (FOB) test. DNA was simply extracted from a 3 mm disc of DFS (including around 10 mg feces) with a boiling procedure followed by a cationic surfactant extraction. K-ras common mutations, as the typical cancer-related gene alterations, were detected with a nested allele specific PCR with microfluorometry (ASP/MFL). Adequate DNA was recovered from 363/451 (80.5%) DFS specimens tested. The K-ras mutations were detected in 2/24 (8.3%) from patients with colorectal cancer and 13/315 (4.1%) from screening participants with positive FOB. No mutations were detected in 24 DFS specimens with negative FOB. The results of K-ras mutation frequency were not encouraging to achieve practically a specific dreliable method for colorectal cancer screening. However this report, identifying for the first time a cancer related gene alteration in feces using identical samples with routine FOB test, will be a step to realize a molecular genetic testing for the cancer screening.

DOI CiNii

ヒト大腸癌由来細胞株におけるp53類似遺伝子p73,p51によるアポトーシス誘導と抗癌剤との併用効果

石田勢津子, 佐々木泰史, 森本一郎, 中谷宇一郎, 山下利春, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 59回 324 - 324 2000年09月

頭頸部癌におけるβ-catenin遺伝子の蛋白発現とmutation,増殖因子受容体の発現

小田島哲世, 佐々木泰史, 山口晃, 田中信幸, 池田健, 佐藤昌明, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 59回 162 - 162 2000年09月

p53類似遺伝子p73,p51の抗腫瘍効果についての検討

佐々木泰史, 森本一郎, 中谷宇一郎, 石田勢津子, 山下利春, 今井浩三, 鬼原史, 田中敏博, 中村祐輔, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 59回 55 - 55 2000年09月

Ewing肉腫特異的なEWS-ETS融合転写因子の標的遺伝子

吉田幸一, 佐々木泰史, 梅沢明弘, 秦順一, 鬼原史, 田中敏博, 中村祐輔, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 59回 53 - 53 2000年09月

子宮頸部,内膜細胞診,組織診とヒトパピローマウイルス(HPV)型,K-rasとの関係

梶野由里, 山口昭弘, 新家瑠奈, 清野邦義, 日野順子, 沓澤武, 時野隆至, 佐藤昇志, 小野江和則, 菊地浩吉

日本病理学会会誌 ( (一社)日本病理学会 ) 89 ( 1 ) 238 - 238 2000年03月

Apoptotic signaling cascade is regulated by p53 status in X-ray- irradiated cells

D. Kobayashi, T. Tokino, N. Watanabe

Biotherapy 14 ( 5 ) 482 - 484 2000年

Objectives: In the present study, we investigated the effect of the p53 status on the signal transduction pathway of X-ray-induced apoptosis using temperature sensitive p53-transduced rat embryonic fibroblast (REF 112) cells. Materials and Methods: (1) The cytotoxicity and apoptosis were examined by WST-8 reagent, DNA fragmentation assay and Hoechst 33258 staining. (2) The p53 and p21/WAF-1 expression was determined using indirect fluorescein-labeled antibody method and analyzed with Spectrafluor (SLT Labinstruments). (3) Caspase-3 activity was assayed by fluorometric real-time assay using Spectrafluor. Results: (1) There was 50% cell death by 5 Gy of X- ray irradiation in cells at mt p53 status, and this cytotoxicity was abrogated almost completely with specific caspase-3 inhibitor Ac-DMQD-CHO. In addition, caspase-3 activity increased approximately 5 times at 24 h after treatment, and this increased activity was almost completely inhibited by the administration of antioxidant N-acetyl-L-cysteine (NAC). This NAC treatment also abrogated X-ray induced cytotoxicity. However, there was no induction of p53 and p21/WAF-1. (2) In contrast, the cells in wt p53 status were more resistant to X-ray compared to cells in mt p53 status. X-ray induced cytotoxicity was not inhibited by Ac-DMQD-CHO, and caspase-3 activity was not elevated, though there was an apparent induction of p53 and p21/WAF-1. Conclusion: These findings suggest that caspase-3 activation downstream of reactive oxygen species occurs during radiation-induced apoptosis in the cells carrying mutant p53, while p21/WAF-1 induction by p53 has a crucial role at wild p53 status.

Nineteenth International Symposium on Cancer, Sapporo, 7-9 July 1999: Cancer genomics and molecular diagnosis - The Nineteenth International Symposium of Sapporo Cancer Seminar

M. Perucho, T. Tokino, Y. Nakamura

Japanese Journal of Cancer Research 90 ( 11 ) 1273 - 1276 1999年 [査読有り]

PubMed

著しい骨髄の線維化と全身性の骨転移を伴った節外性非Hodgkinリンパ腫

笠原薫, 高橋徹, 岡俊州, 山本元久, 白田智洋, 井戸川雅史, 苗代康可, 林敏昭, 安達正晃, 日野田祐治, 今井浩三

臨床血液 39 ( 12 ) 1180 - 1184 1998年12月

A 68-year-old woman was admitted in March 1997 because of lumbago, fever, vomiting, and general malaise. Laboratory data disclosed anemia and severe hypercalcemia (7.7 mEq/l). Multiple osteolytic lesions were detected in the patient's vertebra, pelvis, and bilateral tibia by x-ray films and 99mTc bone scintigrams. Bone marrow aspiration sample was not obtained due to dry tap. Marked myelofibrosis and proliferation of lymphoid cells were revealed by a bone marrow biopsy specimen. Immunohistochemical analysis showed that cells in the biopsy specimen were positive for L-26 and LCA, but not for UCHL-1. Gastrointestinal endoscopic examination found multiple polypoid lesions in the stomach; biopsy specimens of the lesion tissue disclosed invasion by B lymphoid cells. A diagnosis of diffuse large B cell lymphoma was thus made. THP-COP chemotherapy was performed, but only minimal response was obtained. Lymphoma cells subsequently invaded the brain stem, and the patient eventually died of respiratory failure.

DOI PubMed CiNii

GML遺伝子導入によってもたらされる細胞増殖抑制機構の解析

上田和毅, 三好康雄, 木村康利, 時野隆至, 綿谷正弘, 安富正幸, 中村祐輔

日本癌学会総会記事 ( 日本癌学会 ) 57回 160 - 160 1998年08月 [査読有り]

p53により発現が誘導される新規遺伝子の単離

井阪茂之, 三好康雄, 時野隆至, 東千尋, 村田雄二, 中村祐輔

日本癌学会総会記事 ( (一社)日本癌学会 ) 57回 440 - 440 1998年08月

Cloning and characterization of BAI2 and BAI3, novel genes homologous to brain-specific angiogenesis inhibitor 1 (BAI1)

T. Shiratsuchi, H. Nishimori, H. Ichise, Y. Nakamura, T. Tokino

Cytogenetic and Genome Research 79 ( 1-2 ) 103 - 108 1997年01月 [査読有り]

We have identified two novel human genes homologous to BAH (brain-specific angiogenesis inhibitor 1), an angiogenesis inhibitor that is a candidate for involvement in development of glioblastoma. Like BAH, these two genes, designated BAI2 and BAD, were specifically expressed in brain, and are likely to be expressed in the same type of cells. However, in spite of similar tissue specificity among the three BAI genes, only BAH is transcriptionally regulated by p53. BAD expression was absent in two of nine glioblastoma cell lines examined and was significantly reduced in three of the remaining seven. These data suggest that members of this novel gene family may play important roles in suppression of glioblastoma. BAH, BAI2 and BAI3 were mapped to 8q24, 1p35 and 6q12, respectively. © 1997 S. Karger AG, Basel.

DOI

Cloning, expression and mapping of a novel human zinc-finger gene TCF17 homologous to rodent Kid1

Y. Omori, H. Kyushiki, S. Takeda, M. Suzuki, A. Kawai, T. Fujiwara, E. Takahashi, Y. Nakamura

Cytogenetic and Genome Research 78 ( 3-4 ) 285 - 288 1997年01月

We isolated a novel human zinc-finger gene TCF17 homologous to rat Kid1, a zinc-finger gene of the Kr�ppel type expressed predominantly in kidney. In the rat this gene seems to be a transcription factor expressed in response to renal injury and ischemia. The 2435-bp human cDNA contained an open reading frame encoding 605 amino acids. The deduced amino acid sequence showed 86.0% and 87.2% identity (91.6% and 92.8% similarity) with the rat Kid1 and mouse Tcf17 respectively. In contrast with rat Kid1, human TCF17 was expressed in all human tissues examined, including kidney. This gene was mapped by FISH to chromosome 5q35.3, where cytogenetic and molecular abnormalities have been reported often in renal cell carcinomas. © 1997 S. Karger AG, Basel.

DOI PubMed

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

T. Katagiri, K. Ozaki, T. Fujiwara, F. Shimizu, A. Kawai, S. Okuno, M. Suzuki, Y. Nakamura, E. Takahashi, Y. Hirai

Cytogenetic and Genome Research 74 ( 1-2 ) 90 - 95 1996年01月 [査読有り]

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2→q24.3 by fluorescence in situ hybridization (FISH). © 1996 S. Karger AG, Basel.

DOI PubMed

Isolation, characterization and chromosomal assignment of the human WNT7A gene

S. Ikegawa, Y. Kumano, K. Okui, T. Fujiwara, E. Takahashi, Y. Nakamura

Cytogenetic and Genome Research ( S. Karger AG ) 74 ( 1-2 ) 149 - 152 1996年

DOI

Fifty novel sequence-tagged sites (STSs) on human chromosome 11q13.4→q25 identified from microclones generated by microdissection

H. Soejima, K. Yoshiura, T. Tamura, T. Tokino, Y. Nakamura, N. Niikawa, Y. Jinno

Cytogenetic and Genome Research 70 ( 1-2 ) 108 - 111 1995年 [査読有り]

Fifty sequence-tagged sites (STSs) from human chromosome region, 11q13.4→q25 were identified in micro-dissection-generated microclones. From a total of 124 single-copy microclones obtained from regions 11q14→q22 and 11q23→q25, 59 were sequenced, and a PCR primer pair was designed for each of them. Fifty of the 59 clones were mapped to regions ranging from 11q1 3.4 to 1 1q25 by means of PCR on a somatic hybrid cell panel with various deletions of the long arm of human chromosome 11. These STSs will contribute to the construction of physical cosmid/YAC maps representing the chromosome regions. They are also useful for analysis of chromosome aberrations, such as translocations, inversions or marker chromosomes. © 1995 S. Karger AG, Basel.

DOI PubMed

Identification and characterization of a cdna, which is highly homologous to the ribonucleoprotein gene, from a locus (D10s102) closely linked to men2 (multiple endocrine neoplasia type 2)

S. Takiguchi, T. Tokino, T. Imai, A. Tanigami, K. Koyama, Y. Nakamura

Cytogenetic and Genome Research 64 ( 2 ) 128 - 130 1993年 [査読有り]

We previously reported a nearly 1-Mb contig of cosmid and YAC clones at D10S102, a locus that is tightly linked to the gene responsible for multiple endocrine neoplasia type 2 (MEN2A). Subclones of 159 genomic EcoRI fragments in the contig were tested on “zoo blot” to detect DNA sequences conserved in various species. Nine fragments found to contain conserved sequences were used as probes for screen ing cDNA libraries. One of these probes, y300-pL50, recognized a cDNA from a human fetal brain library. The cDNA encoded a protein consisting of 270 amino acids, whose predicted sequence has a high degree of homology to heterogeneous nuclear ribonuclear protein. This expressed sequence may be a candidate for the gene responsible for MEN2A. © 1993 S. Karger AG, Basel.

DOI PubMed

A 14-MB PHYSICAL MAP OF THE REGION AT CHROMOSOME-11Q13 HARBORING THE MEN1 LOCUS AND THE TUMOR AMPLICON REGION

A TANIGAMI, T TOKINO, K TAKITA, S TAKIGUCHI, Y NAKAMURA

GENOMICS ( ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS ) 13 ( 1 ) 16 - 20 1992年05月 [査読有り]

Different type of hepatitis B virus (HBV) DNA integrants that may reflect the integration process

Kousaku Okubo, Takaaki Nakamura, Takashi Tokino, Kenichi Matsubara

Gastroenterologia Japonica ( Springer-Verlag ) 25 ( 2 ) 23 - 30 1990年09月 [査読有り]

Through analyses of HBV DNA integratns in human cellular DNA, we identified three different integrant types, each of which may reflect the process of primary integrant formation by the viral DNA. The first type, which we call "simple type" consists of integrants found in some hepatocellular carcinomas (HCC's). The structure of the viral genome is simple, and part of it is deleted. The viral cohesive end sequence appears at one of the viral-cellular DNA junctions, and integration has elicited a microdeletion in the target cellular DNA sequence. This structure suggests viral DNA replication intermediates as substrates for integration. Judging from its frequency in HCC, this type may represent the most preferred one, if not all, among the primary integration products. The second type, which we call "complex type" is essentially the same as the first type, except tht the viral genome structure is complex. We considered the possibility that they may have been produced via the same process, using preformed complex viral genomes such as "novel form DNA's" (Rogler and Summers, 1982) as substrates. In cultured fetal hepatocytes, integration of HBV DNA can occur only a few days after infection. Among such integrants, we found a third type integrant, having a simple viral genome, but having a larger cellular DNA deletion. We propose that different forms of viral DNA may be used a ssubstrates in the integration process, and the process is characterized by its eliciting of deletions of different size in the target cellular DNA. The most preferred substrate may be the one producing the simple type integrants, and the most frequently occurring deletion in the target DNA may be the microdeletion. The structure of one of the early integrants is different from any of the above, and its formation seems to have a deleterious effect on the chromosome in which the integration occurs, and hence to the host cell. © 1990 The Japanese Society of Gastroenlerology.

DOI PubMed

Regional localization of the LCA oncogene to human chromosome region 2q14→q21

T. Tokino, H. Satoh, M. C. Yoshida, T. Ochiya, K. Matsubara

Cytogenetic and Genome Research 48 ( 1 ) 63 - 64 1988年 [査読有り]

DOI PubMed

CARRIER DETECTION IN JAPANESE HEMOPHILIA A FAMILIES USING FACTOR-VIII GENE PROBE (F8A) AND THE GENE-LINKED ST 14-1 PROBE

M NISHINO, T NISHIMURA, H NAKA, S MIKAMI, T TOKINO, T MUROTSU

JAPANESE JOURNAL OF HUMAN GENETICS ( TOKYO MEDICAL DENTAL UNIV ) 32 ( 3 ) 237 - 245 1987年09月 [査読有り]

Carrier detection in Japanese haemophilia a families using factor VIII gene probe (F8A) and the gene-linked ST 14-1 probe

Masato Nishino, Takuya Nishimura, Hiroyuki Naka, Sadaaki Mikami, Takashi Tokino, Tomoaki Murotsu

The Japanese Journal of Human Genetics ( Springer-Verlag ) 32 ( 3 ) 237 - 245 1987年09月 [査読有り]

Carriers of haemophilia A were detected in 20 Japanese families with using the factor VIII gene probe (F8A) and the gene-linked ST 14-1 probe. Polymorphism by the use of the BclI and F8A probe detected the smaller allele (0.9 kb) in 86% and the larger allele (1.2 kb) in 14% of the 65 normal X chromosomes. The frequency of BclI polymorphism was 30% in 30 normal fameles. In six out of 20 haemophilia A families, the haemophilia gene was identified by BclI allele polymorphism. The use of TaqI and the ST 14-1 probe detected 11 carriers in 20 haemophilia A families. In 14 families (70%), either the BclI polymorphism in the factor VIII locus or TaqI polymorphisms in the ST 14 locus was useful for carrier detection. © 1987 The Japan Society of Human Genetics.

DOI PubMed

Replication of mini-F plasmid in vitro promoted by purified E protein

Kanae Muraiso, Takashi Tokino, Tomoaki Murotsu, Kenichi Matsubara

MGG Molecular & General Genetics ( Springer-Verlag ) 206 ( 3 ) 519 - 521 1987年03月 [査読有り]

An in vitro system for replication of mini-F plasmid DNA was constructed. This system consists of an ammonium sulfate fraction II (Fuller et al. 1981) from Escherichia coli extract, exogeneously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form. Experiments with this system showed that the 217 bp DNA region which contains the A+T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication. © 1987 Springer-Verlag.

DOI PubMed

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