研究者詳細 - 佐々木　泰史

論文 - 佐々木　泰史

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Associations between serum insulin-like growth factor-related molecules and colorectal cancer risk by tumor location: a nested case-control study.

Yasushi Adachi, Masanori Nojima, Yingsong Lin, Yasushi Sasaki, Hiro-O Yamano, Hiroshi Nakase, Kenji Wakai, Mitsuru Mori, Akiko Tamakoshi

Digestion 1 - 19 2025年03月 [査読有り] [国際誌]

Introduction, The activity of the mitogen insulin-like growth factor (IGF) is controlled by IGF-binding protein (IGFBP). Colorectal cancers (CRCs) are heterogeneous, with left- and right-sided CRC showing different clinical and molecular characteristics. This case-control study, nested in the Japan Collaborative Cohort study, assessed associations between serum levels of IGF-related molecules and incidences of CRC by location. Methods, A baseline survey obtained serum samples from 39,242 participants. Subjects diagnosed with CRC during follow-up were regarded as cases. Conditional logistic regression modeling was used to calculate odds ratios (ORs) for cancer incidence associated with IGF-related molecules. Results, This analysis included 176 cases and 524 controls. No IGF-related molecules appeared associated with risks of overall or left-sided CRC. Both total IGFBP3 and free IGFBP3 (estimated as IGFBP3-(IGF1+IGF2)) were associated with incidence of right-sided CRC (P-for-trends=0.027 and 0.003, respectively), with the third tertile of total and free IGFBP3 showing the highest risk (OR=6.25 and 7.96, respectively). Free IGF, estimated as (IGF1+IGF2)/IGFBP3, was inversely associated with incidence of right-sided CRC (P-for-trends=0.014), with the third tertile showing the lowest risk (OR=0.18). Among subjects followed for over 3 years, association of IGF-related molecules with overall CRC was similar. Free IGFBP3 was associated with incidence of right-sided CRC (P-for-trends=0.004). Free IGF was inversely associated with incidence of right-sided CRC (P-for-trends=0.002). However, free IGFs were associated with risk of left-sided CRC (P-for-trends=0.041), with the third tertile showing the highest risk (OR=3.10). Conclusions, Serum IGF-related molecules are associated with risk of CRC. These associations might differ by tumor location.

Impact of Sensitive Circulating Tumor DNA Monitoring on CT Scan Intervals During Postoperative Colorectal Cancer Surveillance

Tomoko Sasaki, Takeshi Iwaya, Mizunori Yaegashi, Masashi Idogawa, Hayato Hiraki, Masakazu Abe, Yuka Koizumi, Noriyuki Sasaki, Akiko Yashima-Abo, Ryosuke Fujisawa, Fumitaka Endo, Shoichiro Tange, Koki Otsuka, Akira Sasaki, Mari Masuda, Masashi Fujita, Hidewaki Nakagawa, Fumiaki Takahashi, Yasushi Sasaki, Takashi Tokino, Satoshi S. Nishizuka

Annals of Surgery Open ( Ovid Technologies (Wolters Kluwer Health) ) 6 ( 1 ) e549 - e549 2025年02月 [査読有り]

Objective: This study investigated whether digital polymerase chain reaction (dPCR)-based circulating tumor DNA (ctDNA) monitoring can allow longer intervals between computed tomography (CT) scans during postoperative surveillance of colorectal cancer (CRC). Background: Practical guidelines still recommend intensive postoperative surveillance of CRC using periodical CT scans and serum carcinoembryonic antigen testing. Methods: The longitudinal dynamics of ctDNA for 52 patients with CRC as measured by dPCR using probes targeting 87 individual tumor-specific mutations (1–5 per patient) were compared with results from conventional (ie, clinical) surveillance using serum tumor markers and CT. Results: A total of 382 CT procedures were carried out for the patient cohort (3.3/year per patient) and the median lead time from ctDNA relapse to clinical relapse was 182 days (range, 0–376 days). If the CT interval was annual, potential delays in the detection of clinical relapse would have occurred for 7 of the 10 patients who experienced clinical relapse (9 of 13 events), with a median delay of 164 days (range, 0–267 days). If annual CT surveillance was performed together with ctDNA monitoring, 218 (57.1%) CTs would not have been needed to detect the first clinical relapse. In addition, the ctDNA monitoring would have provided a lead time of 339 days for detection of clinical relapse (range, 42–533 days). Conclusions: Our findings suggest that the ctDNA monitoring as part of postoperative surveillance and clinical relapse detection for patients with CRC could allow the CT interval to be lengthened. Trial Registration: This trial was registered with University Hospital Medical Information Network Clinical Trial Registry (UMIN000045114).

Targeted dynamic phospho-proteogenomic analysis of gastric cancer cells suggests host immunity provides survival benefit.

Kohei Kume, Midori Iida, Takeshi Iwaya, Akiko Yashima-Abo, Yuka Koizumi, Akari Endo, Kaitlin Wade, Hayato Hiraki, Valerie Calver, Julia Wulfkuhle, Virginia Espina, Doris R Siwak, Yiling Lu, Kazuhiro Takemoto, Yutaka Suzuki, Yasushi Sasaki, Takashi Tokino, Emanuel Petricoin, r, Lance A Liotta, Gordon B Mills, Satoshi S Nishizuka

Molecular & cellular proteomics : MCP 100870 - 100870 2024年10月 [査読有り] [国際誌]

Despite of massive emergence of molecular targeting drugs, the mainstay of advanced gastric cancer (GC) therapy is DNA-damaging drugs. Using a reverse-phase protein array-based proteogenomic analysis of a panel of eight GC cell lines, we identified genetic alterations and signaling pathways, potentially associated with resistance to DNA-damaging drugs, including 5-fluorouracil (5FU), cisplatin, and etoposide. Resistance to cisplatin and etoposide, but not 5FU, was negatively associated with global copy number loss, vimentin expression, and caspase activity, which are considered hallmarks of previously established EMT subtype. The segregation of 19,392 protein expression time courses by sensitive and resistant cell lines for the drugs tested revealed that 5FU-resistant cell lines had lower changes in global protein dynamics, suggesting their robust protein level regulation, compared to their sensitive counterparts, whereas the cell lines that are resistant to other drugs showed increased protein dynamics in response to each drug. Despite faint global protein dynamics, 5FU-resistant cell lines showed increased STAT1 phosphorylation and PD-L1 expression in response to 5FU. In publicly available cohort data, expression of STAT1 and NFκB target genes induced by proinflammatory cytokines was associated with prolonged survival in GC. In our validation cohort, total lymphocyte count (TLC), rather than PD-L1 positivity, predicted a better relapse-free survival rate in GC patients with 5FU-based adjuvant chemotherapy than those with surgery alone. Moreover, TLC+ patients who had no survival benefit from adjuvant chemotherapy were discriminated by expression of IκBα, a potent negative regulator of NFκB. Collectively, our results suggest that 5FU resistance observed in cell lines may be overcome by host immunity or by combination therapy with immune checkpoint blockade.

Synchronous cancers of the stomach and esophagus in a patient with autoimmune gastritis and pernicious anemia: a case report and review of the literature.

Toshiyuki Kubo, Yasushi Adachi, Yasushi Sasaki, Yasuyo Adachi, Yukinari Yoshida, Takao Endo, Yoshifumi Ishii, Hiroaki Takahashi, Akira Goto

International cancer conference journal 13 ( 4 ) 367 - 373 2024年10月 [査読有り] [国際誌]

Type-A gastritis/autoimmune gastritis (AIG) has gained renewed attention due to declining Helicobacter pylori infection rates and increasing eradication. AIG is associated with pernicious anemia (PA) and is prone to complicate various tumors, such as gastric cancer and neuroendocrine tumors. This report describes a case of AIG with PA in which gastric and esophageal cancers arose simultaneously. An 86-year-old woman had been diagnosed with PA and AIG 9 years earlier. As routine blood tests revealed high levels of carcinoembryonic antigen, she underwent esophagogastroduodenoscopy, which revealed a type 0-IIa lesion in the middle part of the stomach and a type 0-IIa + IIb lesion in the lower esophagus. Endoscopic submucosal dissection was performed on both lesions, since neither distant nor lymph node metastases were identified. Histological examination showed gastric tubular adenocarcinoma and esophageal squamous cell carcinoma. Immunohistology revealed that cells from neither cancer expressed gastrin, gastrin receptor, or p53. Whole-exome sequencing showed 8 gene mutations in the esophageal cancer and 6 mutations in 5 genes in the gastric cancer. The reason for the lack of p53 immunostaining was that TP53 was mutated in these cancers, although TP53 mutations differed. Thus, TP53 mutations may not be detected by immunostaining alone. When treating patients with AIG/PA, clinicians must be aware of the possibility of esophageal cancer coexisting with gastric cancer.

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

Association of serum superoxide dismutase activity and the incidence of colorectal cancer in a nested case-control study.

Yasushi Adachi, Masanori Nojima, Mitsuru Mori, Hiro-O Yamano, Yasushi Sasaki, Hiroshi Nakase, Yingsong Lin, Kenji Wakai, Akiko Tamakoshi

Cancer epidemiology 87 102455 - 102455 2023年09月 [査読有り] [国際誌]

BACKGROUND: Superoxide dismutase (SOD) is an antioxidant enzyme that degrades superoxide, a major causative factor in carcinogenesis. We assessed associations between serum SOD activities and incidence of colorectal carcinoma (CRC) in a case-control study nested in the Japan Collaborative Cohort (JACC) study. METHODS: At baseline, 39,242 subjects donated serum samples. Participants diagnosed with CRC during follow-up were regarded as cases. Odds ratios (ORs) for CRC incidence associated with SOD were evaluated with conditional logistic regression models. In the current study, 176 cases and 524 controls were analyzed. RESULTS: For the overall cohort, a decreasing trend in risk of CRC with increasing SOD was observed (P for trend=0.054) and the fourth quartile of SOD level showed the lowest risk compared to the first (OR=0.52, 95% confidence interval [CI]=0.29-0.93). This was significant in men (P for trend=0.001), with the fourth quartile of SOD level showing the lowest risk compared to the first (OR, 0.23; 95%CI, 0.09-0.60). It was also exclusively observed for rectal cancer and left-sided CRC (P for trend, 0.037 and 0.020, respectively), with the fourth quartile again showing the lowest risk compared to the first (OR, 0.28 and 0.38; 95%CI, 0.09-0.84 and 0.16-0.91, respectively). Limiting subjects to those followed-up over 2 years, all trends remained unchanged. CONCLUSIONS: Our findings suggest that serum SOD activity correlates inversely with risk of CRC, particularly in men and individuals with rectal cancer/left-sided CRC.

DOI PubMed J-GLOBAL

Esophageal intraepithelial squamous cell neoplasia with epidermalization-A case with molecular analysis.

Takao Endo, Toshiyuki Kubo, Yasushi Sasaki, Hiroaki Takahoshi, Yoshifumi Ishii, Yasushi Adachi

Pathology international 2023年06月 [査読有り] [国際誌]

Serum soluble Fas levels and incidence of liver cancer in nested case-control study.

Yasushi Adachi, Masanori Nojima, Mitsuru Mori, Toshiyuki Kubo, Noriyuki Akutsu, Yasushi Sasaki, Hiroshi Nakase, Yingsong Lin, Youichi Kurozawa, Kenji Wakai, Akiko Tamakoshi

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 82nd 2022年12月 [査読有り] [国際誌]

BACKGROUND: Soluble Fas (sFas) plays various roles in carcinogenesis and tumor dissemination by preventing apoptosis via binding to Fas ligand. We analyzed associations of serum sFas levels with the incidence of liver cancer in a prospective case-control study nested in the JACC Study. METHODS: A baseline survey was conducted from 1988, with blood samples obtained from 39,242 subjects. Patients diagnosed with liver cancer were regarded as cases. Two or three controls were selected and matched for sex, age, and geographical area. Conditional logistic regression was used to estimate odds ratios (ORs) for cancer incidence associated with sFas. RESULTS: This study contained 86 cases and 249 controls. After controlling for alcohol intake, body mass index, smoking, and hepatitis viral infection, participants with high sFas showed elevated risk of cancer (P-trend = 0.003) and the third tertile of sFas showed a higher risk compared to the first tertile (OR = 3.53, 95% confidence interval (CI) = 1.28-9.69). In hepatocellular carcinoma, high sFas was associated with elevated risk (P-trend < 0.001). In men and the elderly, subjects in the highest tertiles showed higher cancer risk. Limiting subjects to those followed for 3 years, high sFas was related to liver cancer risk (P-trend = 0.033) and the third tertile showed a higher risk compared to the first (OR = 2.94, 95%CI = 0.94-9.14). CONCLUSIONS: High serum sFas may be related to future risk of liver cancer. IMPACT: Our findings highlight this biomarker for further analysis in pooled investigations with different/larger prospective cohorts.

DOI PubMed J-GLOBAL

Genomic analysis of an aggressive case with metastatic intrahepatic mucinous cholangiocarcinoma.

Yoshiharu Masaki, Noriyuki Akutsu, Yasushi Adachi, Keisuike Ishigami, Norikazu Iwata, Takao Endo, Yoshifumi Ishii, Yasushi Sasaki, Minoru Nagayama, Yasutoshi Kimura, Hiroshi Nakase

Clinical journal of gastroenterology 15 ( 4 ) 809 - 817 2022年08月 [査読有り] [国内誌]

Intrahepatic mucinous cholangiocarcinoma (IHMC) is rare and behaves notoriously; however, the details of the clinicopathological characteristics of IHMC remain unknown. A 70-year-old man was admitted for examination of the hepatic mass in the S1 segment. He underwent extended left hepatic lobectomy. Histopathological evaluation demonstrated mixed papillary carcinoma that comprised well to moderately differentiated tubular adenocarcinoma and signet-ring cell carcinoma with large amounts of mucus lakes. Tumor was relapsed 9 months after surgery. Although he received chemotherapy with the combination of gemcitabine and cisplatin, he had renal failure and discontinued the chemotherapy. He received palliative radiotherapy for metastasis in the cervical spine. Then, the patient treated with S-1, however, he died 16 months after the initial diagnosis. The autopsy findings showed multiple nodules in the lungs, pleura, kidneys, adrenal glands, stomach, pancreas, and lymph nodes. Histological examination revealed that all nodules were IHMC. Next-generation sequencing revealed that somatic mutations in ADGRB3, TAF1L and EPHA3 may affect carcinogenesis, and those in TAF1, EPHA3, PIK3C2B, FN1, ERBB3, BRIP1, SYNE1 and TGFBR2 may affect metastasis. Molecular carcinogenesis of IHMC may be distinct from that of ordinary cholangiocarcinoma. Further studies are needed to elucidate the genetic mutations and their functions in IHMC.

Individualized circulating tumor DNA monitoring in head and neck squamous cell carcinoma.

Ryunosuke Kogo, Tomomi Manako, Takeshi Iwaya, Satoshi Nishizuka, Hayato Hiraki, Yasushi Sasaki, Masashi Idogawa, Takashi Tokino, Ayaka Koide, Noritaka Komune, Ryuji Yasumatsu, Takashi Nakagawa

Cancer medicine 2022年03月 [査読有り] [国際誌]

There is no useful biomarker to evaluate treatment response and early relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) is a promising biomarker for detecting minimal residual diseases and monitoring treatment effect. We investigated whether individualized ctDNA analysis could help monitor treatment response and relapse in HNSCC. Mutation analysis of tumor and peripheral blood mononuclear cell (PBMC) DNAs of 26 patients with HNSCC was performed using a custom squamous cell carcinoma (SCC) panel. The identified individualized mutated genes were defined as ctDNA candidates. We investigated whether frequent ctDNA monitoring via digital PCR (dPCR) is clinically valid for HNSCC patients. TP53 was the most frequently mutated gene and was detected in 14 of 24 cases (58.2%), wherein two cases were excluded owing to the absence of tumor-specific mutations in the SCC panel. Six cases were excluded because of undesignable and unusable primer-probes for dPCR. Longitudinal ctDNA was monitored in a total of 18 cases. In seven cases, ctDNA tested positive again or did not test negative, and all seven cases relapsed after initial curative treatment. In 11 cases, after initial curative treatment, ctDNA remained negative and patients were alive without recurrence. Patients who remained negative for ctDNA during follow-up after initial curative treatment (n = 11) had a significantly better prognosis than those who reverted to ctDNA positivity (n = 7; p < 0.0001; log-rank test). Individualized ctDNA monitoring using SCC panel and dPCR might be a novel and promising biomarker for HNSCC.

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 ) 51 2021年12月 [査読有り] [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.

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 [査読有り] [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.

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 [査読有り] [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.

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 [査読有り] [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.

Generation of functional liver tissue by establishing hepatobiliary connections ex vivo

Tanimizu N, Ichinohe N, Sasaki Y, Itoh T, Sudo R, Yamaguchi T, Katsuda T, Tokino T, Ochiya T, Miyajima A, Mitaka T

2020年11月

Frequent tumor burden monitoring of esophageal squamous cell carcinoma with circulating tumor DNA using individually designed digital PCR.

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

Gastroenterology 160 ( 1 ) 463 - 465 2020年10月 [査読有り] [国際誌]

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09月 [査読有り] [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.

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 [査読有り] [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.

Circulating-tumor DNA from Two Types of Blood Collection Tubes for Monitoring Tumor Burden Validated with Esophageal Squamous Cell Carcinoma Patients

Fumitaka Endo, Takeshi Iwaya, Yasushi Sasaki, Takehiro Chiba, Mizunori Yaegashi, Kohei Kume, Kei Sato, Atsuhiro Arisue, Ryoko Kawagishi, Teppei Matuo, Yuji Akiyama, Akira Sasaki, Yuji Suzuki, Takashi Tokino, Mari Masuda, Tesshi Yamada, Hidewaki Nakagawa, Satoshi S Nishizuka

( Cold Spring Harbor Laboratory ) 2020年08月 [査読有り]

Objective: To evaluate long-term whole blood (WB) storage at room temperature (RT), plasma and peripheral blood mononuclear cell (PBMC) DNA was collected simultaneously using BD Vacutainer® CPT™ Mononuclear Cell Preparation Tubes (CPT) and Streck Cell-Free DNA Blood Collection Tubes (BCT®). Methods: Plasma DNA was isolated from both types of tubes at various time points at RT. DNA from PBMCs was extracted using CPT from WB stored in BCT. The extracted DNA was used to monitor esophageal cancer treatment. Results: BCT maintained steady levels of plasma DNA for up to nine days. After transfer of BCT-stored WB to CPT, PBMC-DNA were yielded with suitable quality for up to seven days. ctDNA from esophageal cancer patients carrying TP53 mutations reflected treatment efficacy. Conclusion: BCT/CPT combinatory procedure allows storage of blood samples for up to seven days at RT for valid clinical assays using ctDNA.

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 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.

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.

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 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.

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]

担当区分：最終著者

© 2020 Nakanishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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.

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 [査読有り] [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.

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 2019年12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.

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

Fujita Y, Matsuda S, Sasaki Y, Masugi Y, Kitago M, Yagi H, Abe Y, Shinoda M, Tokino T, Sakamoto M, Kitagawa Y

Cancer science 111 ( 2 ) 739 - 748 2019年12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.

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

Suzuki N, Idogawa M, Tange S, Ohashi T, Sasaki Y, Nakase H, Tokino T

Oncogene 39 ( 10 ) 2202 - 2211 2019年12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.

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 2019年12月 [査読有り] [国際誌]

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]

担当区分：筆頭著者

© 2019 by the authors. 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.

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06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.

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 and Clinical Cancer Research 38 ( 1 ) 127 - 127 2019年03月 [査読有り] [国際誌]

© 2019 The Author(s). 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.

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]

© 2019 Masashi Idogawa et al. 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.

Corrigendum to: Prognostic Effect of Long Noncoding RNA NEAT1 Expression Depends on p53 Mutation Status in Cancer (Journal of Oncology (2019) 2019 (4368068) DOI: 10.1155/2019/4368068)

Masashi Idogawa, Hiroshi Nakase, Yasushi Sasaki, Takashi Tokino

Journal of Oncology ( Hindawi Limited ) 2019 2019年

In the article titled "Prognostic Effect of Long Noncoding RNA NEAT1 Expression Depends on p53 Mutation Status in Cancer" [1], there was an error in the Results section. In the third paragraph, the sentence "Although total RNA was treated with oligo dT to select for polyadenylated mRNAs in the RNA-seq protocol used to obtain TCGA data, the NEAT1_2 sequence includes five consecutive T repeats, with at least ten repeats (maximum: 17)" should be corrected to "Although total RNA was treated with oligo dT to select for polyadenylated mRNAs in the RNA-seq protocol used to obtain TCGA data, the NEAT1_2 sequence includes five consecutive A repeats, with at least ten repeats (maximum: 22).

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]

Malignant paraganglioma of the posterior mediastinum: A case report with genetic analysis.

Yasushi Adachi, Hiroaki Mita, Yasushi Sasaki, Ryogo Himori, Kei Onodera, Masahiro Nakamura, Takefumi Kikuchi, Kentaro Yamashita, Yukinari Yoshida, Yoshifumi Ishii, Takao Endo

Molecular and Clinical Oncology 10 ( 1 ) 10 - 16 2018年11月 [査読有り] [国際誌]

Paraganglioma and pheochromocytoma are rare neuroendocrine neoplasms that originate from chromaffin cells. In many of these tumors, several mutations are reported to occur in the genes of germline and/or somatic cells. A case of paraganglioma in the posterior mediastinum with highly malignant potential is reported. The patient had a rapid clinical course, and it was difficult to reach the final diagnosis. The initial diagnosis on fine-needle aspiration biopsy was a gastrointestinal stromal tumor (GIST) arising from the esophagus. Although radiation therapy was effective for the main tumor, the lung metastases did not respond sufficiently to several tyrosine kinase inhibitors. Autopsy and immunohistochemical examination using a battery of different markers resulted in a final diagnosis of malignant paraganglioma. Next-generation sequencing revealed several gene mutations and copy number variations, including of fumarate hydratase (FH), neurofibromatosis type-1 (NF1) and RET. Those gene alterations may contribute to the pathogenesis of this malignant phenotype to a certain extent. To confirm this, further cases and studies are required. In addition, it should be noted that histological examination of a small piece of tumor might have sampling bias and could cause misdiagnosis.

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 ( Elsevier BV ) 434 91 - 100 2018年10月 [査読有り]

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.

特発性肺線維症における肺マイクロバイオームについての検討

高橋洋平, 齋藤充史, 池田貴美之, 小林智史, 黒沼幸治, 千葉弘文, 佐々木泰史, 高橋弘毅

日本サルコイドーシス/肉芽腫性疾患学会雑誌 ( 日本サルコイドーシス ) 38 ( 1-2 ) 104 - 104 2018年10月

Prognostic nutritional index and early mortality with percutaneous endoscopic gastrostomy.

Adachi Y, Akino K, Nojima M, Himori R, Kikuchi T, Mita H, Nakamura M, Tsukuda H, Yamano HO, Sasaki Y, Yoshida Y, Kato Y, Nakase H, Endo T.

QJM: An International Journal of Medicine 111 ( 9 ) 635 - 641 2018年09月 [査読有り]

Background:Although percutaneous endoscopic gastrostomy (PEG) is a well-accepted and less invasive method of feeding tube placement in patients with swallowing difficulties, complications and early death after PEG have been reported. Aim:This study aimed to evaluate predictive factors associated with 30-day mortality after PEG, and to assess the utility of nutritional supporting period before PEG in reducing early mortality following PEG. Design:An observational study. Methods:We retrospectively analyzed 268 patients who underwent PEG at Sapporo Shirakaba-dai Hospital from 2006 to 2010, using clinical and laboratory data to analyze predictive factors associated with early death after PEG. Then, we prospectively assessed 152 consecutive patients assessed for eligibility for PEG from 2011 to 2014. We assessed the patients' nutritional condition using Onodera's prognostic nutritional index (PNI), and supported nutrition for more than 10 days before PEG in patients with a poor nutritional index (PNI < 37). Results:In both univariate and multivariate analyses in the retrospective study, Onodera's PNI of less than 37 was the only predictive factor for early mortality. In the second study, among the 115 patients who finally underwent PEG, early mortality rates improved to 1.7% from 5.2% in the first study. Conversely, 32% of patients with malnutrition who did not undergo PEG died within 30 days. Conclusion:Nutritional status might be a predictive factor for early mortality after PEG. In patients with poor nutritional status, nutritional supporting period before PEG might improve the outcomes and reduce unnecessary PEG.

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

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

Tumor Biology 40 ( 9 ) 1010428318800180 - 1010428318800180 2018年09月 [査読有り] [国際誌]

担当区分：最終著者 , 責任著者

© The Author(s) 2018. 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.

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

Nuryadi E, Sasaki Y, Hagiwara Y, Permata TBM, Sato H, Komatsu S, Yoshimoto Y, Murata K, Ando K, Kubo N, Okonogi N, Takakusagi Y, Adachi A, Iwanaga M, Nakano T

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

担当区分：筆頭著者

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.

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

Ishiguro K, Kitajima H, Niinuma T, Ishida T, Maruyama R, Ikeda H, Hayashi T, Sasaki H, Wakasugi H, Nishiyama K, Shindo T, Yamamoto E, Kai M, Suzuki H

Haematologica 104 ( 1 ) 155 - 165 2018年08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.

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, 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.

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.

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06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.

Therapeutic response monitoring using digital PCR for patient-specific circulating tumor DNA identified by an esophageal squamous cell carcinoma-specific sequencing panel.

Iwaya Takeshi, Endo Fumitaka, Sasaki Yasushi, Akiyama Yuji, Sasaki Akira, Masuda Mari

JOURNAL OF CLINICAL ONCOLOGY 36 ( 15 ) 2018年05月 [査読有り]

Impaired diversity of the lung microbiome predicts progression of idiopathic pulmonary fibrosis.

Youhei Takahashi, Atsushi Saito, Hirofumi Chiba, Koji Kuronuma, Kimiyuki Ikeda, Tomofumi Kobayashi, Shigeru Ariki, Motoko Takahashi, Yasushi Sasaki & Hiroki Takahashi

Respiratory research 19 ( 1 ) 34 - 34 2018年02月 [査読有り] [国際誌]

BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. Although IPF has not been thought to be associated with bacterial communities, recent papers reported the possible role of microbiome composition in IPF. The roles of microbiomes in respiratory functions and as clinical biomarkers for IPF remain unknown. In this study, we aim to identify the relationship between the microbial environment in the lung and clinical findings. METHODS:Thirty-four subjects diagnosed with IPF were included in this analysis. The 16S rDNA was purified from bronchoalveolar lavage fluid obtained at the time of diagnosis and analyzed using next-generation sequencing techniques to characterize the bacterial communities. Furthermore, microbiomes from mice with bleomycin-induced lung fibrosis were analyzed. RESULTS:The most prevalent lung phyla were Firmicutes, Proteobacteria and Bacteroidetes. Decreased microbial diversity was found in patients with low forced vital capacity (FVC) and early mortality. Additionally, the diversity and relative abundance of Firmicutes, Streptococcaceae, and Veillonellaceae were significantly associated with FVC, 6-min walk distance, and serum surfactant protein D. Bleomycin-induced lung fibrosis resulted in decrease of diversity and alteration of microbiota in PCoA analysis. These results support the observations in human specimens. CONCLUSIONS:This study identified relationships between specific taxa in BALF and clinical findings, which were also supported by experiments in a mouse model. Our data suggest the possibility that loss of microbial diversity is associated with disease activities of IPF.

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

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

Oncotarget 9 ( 4 ) 4707 - 4721 2018年01月 [査読有り] [国際誌]

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

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]

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 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]

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

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

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

担当区分：最終著者 , 責任著者

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. 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.

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 2017年11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.

Fledglings in p53 signaling network.

Tokino T, Idogawa M, Sasaki Y

Oncotarget 8 ( 34 ) 55768 - 55769 2017年07月 [査読有り] [国際誌]

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]

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05月 [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.

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

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

International journal of cancer 140 ( 12 ) 2785 - 2791 2017年03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.

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01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.

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

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

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

担当区分：最終著者 , 責任著者

© Nakagaki et al. 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.

TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells.

Kai M, Niinuma T, Kitajima H, Yamamoto E, Harada T, Aoki H, Maruyama R, Toyota M, Sasaki Y, Sugai T, Tokino T, Nakase H, Suzuki H

PloS one 11 ( 12 ) e0168281 2016年12月 [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.

Systemic Prophylactic Antibiotics for the Modified Introducer Method for Percutaneous Endoscopic Gastrostomy: A Prospective, Randomized, Double-Blind Study.

Yasushi Adachi, Kimishige Akino, Hiroaki Mita, Takefumi Kikuchi, Kentaro Yamashita, Yasushi Sasaki, Yoshiaki Arimura, Takao Endo

Journal of clinical gastroenterology 50 ( 9 ) 727 - 32 2016年10月 [査読有り] [国際誌]

Percutaneous endoscopic gastrostomy (PEG) is the most common method of enteral nutrition in patients who require long-term tube feeding. According to meta-analyses, administration of systemic prophylactic antibiotics for PEG reduces peristomal infection. However, with several recent developments in the procedure and instruments, the risk of infection might have been reduced. The aim of this study was to evaluate the use of systemic antibiotic prophylaxis for a modified introducer method of PEG.This prospective, randomized, double-blind trial assessed 278 patients undergoing PEG for inclusion. Ninety-one patients with an indication for PEG who gave informed consent to participate were randomized. Forty-six patients received prophylactic ampicillin and 45 patients received a placebo. A modified introducer method of PEG using a Seldinger PEG kit was performed. The primary outcome was the occurrence of clinically evident wound infection within 3 days after PEG.Wound infection within 3 days was observed in none in the prophylaxis group and in 1 patient in the control group (P=0.4945). There was no significant difference between 2 groups in the other parameters, including peristomal infection within 7 days, overall infection, white blood cell counts, C-reactive protein level, and successive rate of finishing antibiotics.For wound infection within 3 days, noninferiority of the placebo group to the antibiotics group was preliminarily suggested with our criteria, but not for peristomal infection within 7 days. More strict criteria for noninferiority should be examined in a further large sample study.

DOI DOI2 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, Sato N

Oncotarget ( Impact Journals, LLC ) 7 ( 31 ) 50043 - 50056 2016年08月 [査読有り]

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 2016年06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.

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

Yasushi Sasaki, Miyuki Tamura, Ryota Koyama, Takafumi Nakagaki, Yasushi Adachi, Takashi Tokino

World Journal of Gastroenterology 22 ( 7 ) 2284 - 2293 2016年02月 [国際誌]

担当区分：筆頭著者

© The Author(s) 2016. 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.

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

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

Oncotarget ( (一社)日本癌学会 ) 7 ( 38 ) 61426 - 61437 2016年 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

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.

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

Hiroshi Ikeda, Kazuya Ishiguro, Tetsuyuki Igarashi, Yuka Aoki, Toshiaki Hayashi, Tadao Ishida, Yasushi Sasaki, Takashi Tokino, Yasuhisa Shinomura

OncoTargets and Therapy 8 2805 - 2815 2015年10月 [査読有り] [国際誌]

担当区分：責任著者

© 2015 Ikeda et al. 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.

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, 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.

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 106 ( 8 ) 1033 - 1040 2015年08月 [査読有り] [国際誌]

担当区分：責任著者

© 2015 The Authors. 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.

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

Hiroo Yamano, Yoshihito Tanaka, Hiroo Matsushita, Kenjiro Yoshikawa, Ryo Takagi, Eiji Harada, Michiko Nakaoka, Ryogo Himori, Yuko Yoshida, Daiki Tanaka, Kentaro Sato, Yasushi Imai, Tamotsu Sugai, Makoto Eizuka, Eiichiro Yamamoto, Hironori Aoki, Hiromu Suzuki

Journal of Japanese Society of Gastroenterology ( Japanese Society of Gastroenterology ) 112 ( 4 ) 676 - 682 2015年 [査読有り]

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 33 ( 40 ) 4837 - 4846 2014年10月 [査読有り]

担当区分：責任著者

© 2014 Macmillan Publishers Limited. 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, TAp73β and TAp63γ. 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.

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.

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, Shinomura Y

International journal of cancer 135 ( 11 ) 2507 - 15 2014年04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.

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01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.

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 ( (一社)日本癌学会 ) 11 ( 12 ) 1554 - 63 2013年10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.

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

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

Tumour biology 35 ( 2 ) 973 - 85 2013年09月 [査読有り] [国際誌]

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

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

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

The American journal of pathology 181 ( 5 ) 1847 - 61 2012年09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.

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

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

Cancer gene therapy 2012年09月 [査読有り]

担当区分：筆頭著者

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09月 [査読有り] [国際誌]

担当区分：筆頭著者

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.

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07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.

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02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.

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10月 [査読有り] [国際誌]

担当区分：筆頭著者

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.

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07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.

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, 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.

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

Ikuko Yokota, Yasushi Sasaki, Lisa Kashima, Masashi Idogawa, Takashi Tokino

International Journal of Oncology 37 ( 6 ) 1407 - 1416 2010年12月 [査読有り] [国際誌]

担当区分：責任著者

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.

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]

Myogenic lineage differentiated mesenchymal stem cells enhance recovery from dextran sulfate sodium-induced colitis in the rat.

Hiroki Tanaka, Yoshiaki Arimura, Takashi Yabana, Akira Goto, Masayo Hosokawa, Kanna Nagaishi, Kentaro Yamashita, Hiroyuki Yamamoto, Yasushi Sasaki, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

Journal of gastroenterology 46 ( 2 ) 143 - 52 2010年09月 [査読有り] [国内誌]

Although mounting evidence implicates mesenchymal stem cells (MSCs) in intestinal tissue repair, uncertainty remains concerning the distribution, function, and fate of repopulating MSCs in recipient colonic tissues. Therefore, we investigated the role of transplanted MSCs in the repair phase of DSS colitis.LacZ-labeled rat MSCs were transplanted into rats with colitis induced by 4% DSS on day 2. Regular water replaced the DSS solution on day 6. Therapeutic effect was evaluated on day 9 by clinicopathologic and growth factor/cytokine expression profiles. We analyzed the Notch signaling pathway by Western blotting and characterized immunofluorescence of lacZ-labeled MSCs with confocal laser microscopy. In vivo differentiation of MSC was confirmed by transmission electron microscopy (TEM).Recovery of colitis was modestly but significantly promoted by MSC transplantation due to proceeding cell cycle and inhibiting apoptosis in the epithelia. Tgfa mRNA expression increased significantly, while Notch signaling was inhibited in the colonic tissues with MSC transplantation. β-Galactosidase-positive cells, which expressed α-SMA, desmin, and vimentin, were infrequently detected in the lamina propria stroma. DSS exposure in vitro proved to be the most potent inducer for α-SMA in MSCs where TEM demonstrated myogenic lineage differentiation.We found that MSCs transplantation modestly promoted the repair of DSS colitis. The donor-derived MSCs were likely reprogrammed to differentiate to myogenic lineage cells by cues from the micro milieu. Further characterization of these cells is warranted as a basis for applying cell-based therapy for inflammatory bowel disease.

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, Itoh F

Cancer Science 101 ( 7 ) 1708 - 16 2010年04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.

幽門狭窄を来した進行胃癌に対して経皮内視鏡的胃瘻造設術（PEG）の腸瘻化（PEG-J）が奏効した１例

山下真幸, 足立靖, 田中浩紀, 安達靖代, 明石浩史, 佐々木泰史, 加藤康夫, 伊東文生, 篠村恭久, 遠藤高夫

日本消化器内視鏡学会雑誌 ( (一社)日本消化器内視鏡学会 ) 52 ( 5 ) 1415 - 1420 2010年 [査読有り]

幽門狭窄をきたした切除不能進行胃癌の90歳代男性症例について検討した。減圧と栄養・投薬を目的に小腸留置型ダブルルーメンチューブを用いた経皮内視鏡的胃瘻造設術(PEG)の腸瘻化(PEG-J)を二期的に行った。胃瘻造設から7日目に経胃瘻的内視鏡(TGE)を用いてチューブを十二指腸水平脚へ誘導した。TGEは軽い抵抗はあったものの、容易に狭窄部を通過でき、有用であった。術後、経鼻胃管留置および中心静脈栄養を中止することができ、退院可能となった。PEG-Jは、幽門狭窄胃癌に対する緩和医療として、選択しうる治療法であると思われた。

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 2009年10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.

Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity.

Takashi Yabana, Yoshiaki Arimura, Hiroki Tanaka, Akira Goto, Masayo Hosokawa, Kanna Nagaishi, Kentaro Yamashita, Hiroyuki Yamamoto, Yasushi Adachi, Yasushi Sasaki, Masaharu Isobe, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

The Journal of pathology 218 ( 3 ) 350 - 9 2009年07月 [査読有り] [国際誌]

The cellular origin, in vivo function and fate of donor bone marrow-derived cells residing in the recipient intestinal epithelial cells, pericryptal myofibroblasts or endothelial cells remain obscure. Although 'immunoprivileged' mesenchymal stem cells (MSCs) are prime candidates for cell- and gene-based therapy, their precise role in colitis remains largely undetermined. Using a dextran sulphate sodium (DSS) colitis with busulphan (BU)-induced hypoplastic marrow model, we examined the therapeutic effects of MSC transplantation, focusing on the role of MSCs as both cell providers and immunomodulators. Donor-derived MSCs were detected by eGFP immunofluorescence and fluorescence in situ hybridization for Y-chromosome (Y-FISH) analysis. Western blot analysis of apical-most tight junction proteins was performed with antibodies against claudin-2, -7, -8, -12, -13, -15 and ZO-1. Cytokine and cell cycle profiles were analysed by semi-quantitative RT-PCR and flow cytometry. Susceptibility to DSS colitis was significantly increased by co-existing BU-induced bone marrow hypoplasia and this increase was significantly reduced by enhancing epithelial engraftment of MSCs, an effect depending on restoring epithelial barrier integrity rather than inhibiting host immune responses. We provide evidence that implicates MSCs in maintaining epithelial barrier function by reassembling apical-most tight junction proteins, claudins. The therapeutic efficacy of extrinsic MSCs depends on enhancing epithelial engraftment in damaged crypts by busulphan conditioning. Such a role for the MSC-derived intestinal cells in colitis therapy merits further examination and may offer a promising new treatment for inflammatory bowel disease (IBD).

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, Toyota M

Carcinogenesis 31 ( 3 ) 342 - 9 2009年07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.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: Involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

Hiroaki Mita, Minoru Toyota, Fumio Aoki, Hirofumi Akashi, Reo Maruyama, Yasushi Sasaki, Hiromu Suzuki, Masashi Idogawa, Lisa Kashima, Kazuyoshi Yanagihara, Masahiro Fujita, Masao Hosokawa, Masanobu Kusano, Sorin Vasile Sabau, Haruyuki Tatsumi, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

BMC Cancer 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. © 2009 Mita et al; licensee BioMed Central Ltd.

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 15 ( 13 ) 4356 - 64 2009年06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.

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

CHFR, a potential tumor suppressor, downregulates interleukin-8 through the inhibition of NF-kappaB.

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

Oncogene 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.

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

Clinical cancer research 15 ( 11 ) 3725 - 32 2009年05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.

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

The Journal of investigative dermatology 129 ( 9 ) 2233 - 41 2009年03月 [査読有り] [国際誌]

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.

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.

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 2008年11月 [査読有り] [国際誌]

担当区分：筆頭著者

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.

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, Shinomura Y

Cancer epidemiology, biomarkers & prevention 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.

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.

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06月 [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.

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.

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]

担当区分：筆頭著者

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.

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02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.

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 6 ( 3 ) 395 - 406 2008年02月 [査読有り] [国際誌]

担当区分：筆頭著者

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.

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, Tokino T

Carcinogenesis 29 ( 7 ) 1312 - 8 2008年02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.

Gene amplification and overexpression of PRDM14 in breast cancers.

Noriko Nishikawa, Minoru Toyota, Hiromu Suzuki, Toshio Honma, Tomoko Fujikane, Tousei Ohmura, Toshihiko Nishidate, Mutsumi Ohe-Toyota, Reo Maruyama, Tomoko Sonoda, Yasushi Sasaki, Takeshi Urano, Kohzoh Imai, Koichi Hirata, Takashi Tokino

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 DOI2 PubMed

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

Hironobu Sato, Hiromu Suzuki, Minoru Toyota, Masanori Nojima, Reo Maruyama, Shigeru Sasaki, Hideyasu Takagi, Yohei Sogabe, Yasushi Sasaki, Masashi Idogawa, Tomoko Sonoda, Mitsuru Mori, Kohzoh Imai, Takashi Tokino, Yasuhisa Shinomura

Carcinogenesis 28 ( 12 ) 2459 - 66 2007年08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.

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

Yoshiyuki Watanabe, Minoru Toyota, Yutaka Kondo, Hiromu Suzuki, Takashi Imai, Mutsumi Ohe-Toyota, Reo Maruyama, Masanori Nojima, Yasushi Sasaki, Yoshitaka Sekido, Hiroyoshi Hiratsuka, Yasuhisa Shinomura, Kohzoh Imai, Fumio Itoh, Takashi Tokino

Clinical cancer research 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 DOI2 PubMed

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

Yuichiro Oshima, Yasushi Sasaki, Hideaki Negishi, Masashi Idogawa, Minoru Toyota, Toshiharu Yamashita, Takuro Wada, Satoshi Nagoya, Satoshi Kawaguchi, Toshihiko Yamashita, Takashi Tokino

Cancer Biology and Therapy 6 ( 7 ) 1058 - 1066 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 p63γ induced apoptosis in OS cells that are resistant to p53-mediated apoptosis, while less effect was observed following transduction of p73α or p63α. Interestingly, the apoptotic effects of p63γ 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 p63γ significantly suppressed tumor growth compared with p53. In addition, exogenous p73β and p63γ 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. ©2007 Landes Bioscience.

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

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

Oncogene 2007年02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.

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

Kimishige Akino, Minoru Toyota, Hiromu Suzuki, Takashi Imai, Reo Maruyama, Masanobu Kusano, Noriko Nishikawa, Yoshiyuki Watanabe, Yasushi Sasaki, Tamaki Abe, Eiichiro Yamamoto, Isao Tarasawa, Tomoko Sonoda, Mitsuru Mori, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

Cancer Science 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.

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.

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 noncanonical 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.

Activation of the ribosomal protein L13 gene in human gastrointestinal cancer

Toshihisa Kobayashi, Yasushi Sasaki, Yuichiro Oshima, Hiroyuki Yamamoto, Hiroaki Mita, Hiromu Suzuki, Minoru Toyota, Takashi Tokino, Fumio Itoh, Kohzoh Imai, Yasuhisa Shinomura

International Journal of Molecular Medicine 18 ( 1 ) 161 - 170 2006年07月 [査読有り] [国際誌]

担当区分：責任著者

Although ribosomal proteins are major components of ribosomes, recent data have shown them to have extra-ribosomal functions apart from ribosome and protein biosynthesis. In our earlier study, we showed that ribosomal protein L13 mRNA was up-regulated in response to DNA damage in hamster cells. We report here that L13 expression is up-regulated in human gastrointestinal cancers. We also examined the biological role of L13 on human cancer cells. Knocking down L13 expression using small interfering RNA (siRNA) resulted in drastic attenuation of cancer cell growth with significant G1 and G2/M arrest of the cell cycle. Moreover, L13 siRNA significantly enhanced the cellular sensitivity to certain DNA damaging agents and, concordantly, L13-overexpressing cells demonstrated greater chemoresistance compared to parent cells, suggesting an inverse correlation between L13 expression and chemosensitivity. By using semi-quantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of 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.

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

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

Reo Maruyama, Fumio Aoki, Minoru Toyota, Yasushi Sasaki, Hirofumi Akashi, Hiroaki Mita, Hiromu Suzuki, Kimishige Akino, Mutsumi Ohe-Toyota, Yumiko Maruyama, Haruyuki Tatsumi, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

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 DOI2 PubMed

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

Hiroshi Ikeda, Yasushi Sasaki, Toshihisa Kobayashi, Hiromu Suzuki, Hiroaki Mita, Minoru Toyota, Fumio Itoh, Yasuhisa Shinomura, Takashi Tokino, Kohzoh Imai

International Journal of Oncology 27 ( 4 ) 933 - 940 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 Tfimbrin 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.

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

Kazuhiro Ogi, Minoru Toyota, Hiroaki Mita, Ayumi Satoh, Lisa Kashima, Yasushi Sasaki, Hiromu Suzuki, Kimishige Akino, Noriko Nishikawa, Makoto Noguchi, Yasuhisa Shinomura, Kohzoh Imai, Hiroyoshi Hiratsuka, Takashi Tokino

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.

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

Kimishige Akino, Minoru Toyota, Hiromu Suzuki, Hiroaki Mita, Yasushi Sasaki, Mutsumi Ohe-Toyota, Jean-Pierre J Issa, Yuji Hinoda, Kohzoh Imai, Takashi Tokino

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.

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]

担当区分：筆頭著者

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.

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.

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

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

British Journal of Cancer 92 ( 6 ) 1165 - 1172 2005年03月 [査読有り]

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.

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 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.

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.

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.

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]

担当区分：筆頭著者

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.

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 [査読有り]

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.

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]

担当区分：筆頭著者

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.

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 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.

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.

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.

Analysis of lung tumorigenesis in chimeric mice indicates the Pulmonary adenoma resistance 2 (Par2) locus to operate in the tumor-initiation stage in a cell-autonomous manner: detection of polymorphisms in the Poli gene as a candidate for Par2.

Gang-Hong Lee, Hiroyuki Nishimori, Yasushi Sasaki, Hiroshi Matsushita, Tomoyuki Kitagawa, Takashi Tokino

Oncogene 22 ( 15 ) 2374 - 82 2003年04月 [査読有り] [国際誌]

The Pulmonary adenoma resistance 2 (Par2) locus of the BALB/cByJ mouse, located within 0.5 cM of chromosome 18, is responsible for reducing the mean multiplicity of urethane-induced lung tumors relative to those in C57BL/6J, A/J and C3H/HeJ mice. Thus, BALB/B6-Par2 congenic strain genetically identical to BALB/cByJ except carrying C57BL/6J Par2 alleles develops seven times more tumors than BALB/cByJ. To gain clues for identification of Par2 candidate genes, we analysed lung tumorigenesis in BALB/cByJ<-->BALB.B6-Par2 chimeric animals. Of 100 tumors induced by urethane in 16 chimeras, 82 originated from BALB.B6-Par2 cells, indicating the Par2 phenotype to be cell-autonomous. In addition, the BALB.B6-Par2- and BALB/cByJ-derived tumors were similar in mean size, implying that the phenotype is primarily expressed during initiation rather than in the promotion stage of carcinogenesis. Given these results, we surveyed a comprehensive mouse genome database and physically mapped Par2 within a 2.3 Mbp segment containing three known genes, Poli, Mbd2 and Dcc. Among those, the Poli seemed to be the most reasonable Par2 candidate, since it encodes an extremely error-prone DNA polymerase preferentially incorporating G or T opposite template T in vitro, reminiscent of the Kras2 activation because of an A to G or T point mutation within codon 61 with which most urethane-induced lung tumors are initiated. Indeed, our sequencing of Poli cDNAs from BALB/cByJ, C57BL/6J, A/J and C3H/HeJ lungs revealed 21 BALB/cByJ-specific single-nucleotide polymorphisms in the coding region accompanied by seven amino-acid substitutions and an elevated frequency of alternative splicing, while no polymorphisms associated with tumor susceptibility were found for either Mbd2 or Dcc. Notably, we obtained evidence that BALB/cByJ Par2 alleles may selectively decrease the frequency of Kras2-mutated tumors compared with C57BL/6J alleles. Consequently, the Poli is an intriguing Par2 candidate clearly deserving further evaluation.

Induction of tenascin-C by tumor-specific EWS-ETS fusion genes.

Goichi Watanabe, Hiroyuki Nishimori, Hideto Irifune, Yasushi Sasaki, Setsuko Ishida, Hitoshi Zembutsu, Toshihiro Tanaka, Satoshi Kawaguchi, Takuro Wada, Jun-ichi Hata, Moriaki Kusakabe, Koichi Yoshida, Yusuke Nakamura, Takashi Tokino

Genes, chromosomes & cancer 36 ( 3 ) 224 - 32 2003年03月 [査読有り] [国際誌]

Ewing sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNETs) 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 (most commonly FLI1 or ERG). Although previous reports suggested that these chimera 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 Tenascin-C (TNC) is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Furthermore, through use of an antisense cDNA expression vector we show that expression of endogenous TNC mRNA and protein were reduced coordinately with attenuation of EWS-FLI1 fusion protein expression. A chromatin immunoprecipitation assay showed direct interaction between the TNC promoter and the EWS-FLI1 fusion protein in vivo. In addition, a luciferase reporter assay revealed that EWS-ETSs upregulated the TNC gene through four ETS binding sites in the TNC promoter. High levels of TNC expression were observed in a subset of ES cell lines (3 of 6) and primary tumors (4 of 6). Together with previous studies showing that TNC expression 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 upregulating of TNC expression.

The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors.

Hiroyuki Nishimori, Yasushi Sasaki, Koichi Yoshida, Hideto Irifune, Hitoshi Zembutsu, Toshihiro Tanaka, Tomoki Aoyama, Taisuke Hosaka, Satoshi Kawaguchi, Takuro Wada, Jun-Ichi Hata, Junya Toguchida, Yusuke Nakamura, Takashi Tokino

Oncogene 21 ( 54 ) 8302 - 9 2002年11月 [査読有り] [国際誌]

We report here that the Id2 (inhibitor of DNA binding 2) gene is a novel target of transcriptional activation by EWS-FLI1 and EWS-ERG, two fusion proteins that characterize Ewing family tumors (EFTs). To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line by retroviral transduction. cDNA microarray analysis revealed that Id2 expression was up-regulated by introducing the EWS-ETS fusion gene but not by the normal full-length ETS gene. An Id2 promoter-luciferase reporter assay showed that transactivation by EWS-ETS involves the minimal Id2 promoter and may function in cooperation with c-Myc within the full-length regulatory region. A chromatin immunoprecipitation assay revealed direct interaction between the Id2 promoter and EWS-FLI1 fusion protein in vivo. Significantly higher expression of Id2 and c-Myc was observed in all of the six EFT cell lines examined compared to six other sarcoma cell lines. Moreover, high levels of Id2 expression were also observed in five of the six primary tumors examined. Id2 is generally thought to affect the balance between cell differentiation and proliferation in development and is highly expressed in several cancer types. Considering these previous studies, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by up-regulating Id2 expression. doi:10.1038/sj.onc.1206025

DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers.

Satoh A, Toyota M, Itoh F, Kikuchi T, Obata T, Sasaki Y, Suzuki H, Yawata A, Kusano M, Fujita M, Hosokawa M, Yanagihara K, Tokino T, Imai K

British Journal of Cancer 86 ( 11 ) 1817 - 1823 2002年06月 [査読有り]

Death-associated protein kinase is a positive regulator of programmed cell death induced by interferon gamma. To investigate the role of epigenetic inactivation of death-associated protein kinase in gastrointestinal cancer, we examined the methylation status of the 5' CpG island of the death-associated protein kinase gene. Methylation of the 5' CpG island was detected in 3 of 9 colorectal and 3 of 17 gastric cancer cell lines, while among primary tumours, it was detected in 4 of 28 (14%) colorectal and 4 of 27 (15%) gastric cancers. By contrast, methylation of the edge of the CpG island was detected in virtually every sample examined. Death-associated protein kinase expression was diminished in four cell lines that showed dense methylation of the 5' CpG island, and treatment with 5-aza-2'-deoxycitidine, a methyltransferase inhibitor, restored gene expression. Acetylation of histones H3 and H4 in the 5' region of the gene was assessed by chromatin immunoprecipitation and was found to correlate directly with gene expression and inversely with DNA methylation. Thus, aberrant DNA methylation and histone deacetylation of the 5' CpG island, but not the edge of the CpG island, appears to play a key role in silencing death-associated protein kinase expression in gastrointestinal malignancies.

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]

担当区分：筆頭著者

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.

Identification of the osteopontin gene as a direct target of TP53.

Ichiro Morimoto, Yasushi Sasaki, Setsuko Ishida, Kohzoh Imai, Takashi Tokino

Genes, chromosomes & cancer 33 ( 3 ) 270 - 8 2002年03月 [査読有り] [国際誌]

The TP53 tumor suppressor gene regulates a number of genes that are involved in cell-cycle inhibition, apoptosis, and maintaining genetic stability. Recently, two genes that have a role in immunosurveillance were identified as downstream targets of TP53. These genes, TAP1 and fractalkine, may contribute to suppress tumor growth through host immunosurveillance. It has been reported that the mouse secreted phosphoprotein osteopontin (Opn) is one of the key cytokines for type 1 immune responses mediated by macrophages. It also was reported that Opn may play a role in suppressing tumor growth in vivo. Here we identified Opn as a Tp53-target gene using mRNA differential display analysis of embryonic fibroblasts from Tp53-deficient mice. Furthermore, we found that Opn expression was upregulated by DNA damage-induced Tp53 activity and by adenovirus-mediated transfer of the human TP53 gene. In addition, a luciferase assay showed that the Opn gene has a functional Tp53-responsive element in its promoter region, and a chromatin immunoprecipitation assay confirmed interaction between the Opn promoter and Tp53 protein in vivo. These results suggest that OPN is a direct transcriptional target of TP53. The TP53-directed regulation of OPN expression suggests a novel model of TP53 participation in immunosurveillance, involving interaction with the host immune system to prevent damaged cells from undergoing malignant transformation.

Increased expression of T-fimbrin gene after DNA damage in CHO cells and inactivation of T-fimbrin by CpG methylation in human colorectal cancer cells.

Yasushi Sasaki, Fumio Itoh, Toshihisa Kobayashi, Takefumi Kikuchi, Hiromu Suzuki, Minoru Toyota, Kohzoh Imai

International journal of cancer 97 ( 2 ) 211 - 6 2002年01月 [査読有り] [国際誌]

担当区分：筆頭著者

When DNA damage is induced by unprogrammed extrinsic events, activating-cell-cycle checkpoints delay cell-cycle progression in the G1 or G2 phases and allow repair of a damaged template. In this study, we evaluated changes in gene expression upon radiation-induced G2 cell-cycle arrest using Chinese hamster ovary (CHO) cells. T-fimbrin, an actin-binding protein, was overexpressed in CHO cells in which G2 arrest had been induced by X-radiation. Northern blot analysis revealed that T-fimbrin gene expression was induced not only by X-radiation but also by a topoisomerase II inhibitor, etoposide. Transfection of CHO cells with a vector encoding T-fimbrin antisense RNA demonstrated that reduced T-fimbrin expression induced alterations in cell-cycle control; radiation-induced G2 arrest was short and decreased in cells transfected with antisense T-fimbrin. Additionally, T-fimbrin gene expression was suppressed in a human colorectal cancer cell line, SW948, because of promoter-specific DNA methylation. These results suggest that downregulation of T-fimbrin may be involved in cancer development through G2/M cell-cycle control in mammalian cells.

M期チェックポイント遺伝子の異常メチル化とMicrotubule Inhibitor感受性に対する分子マーカーとしての応用

佐藤亜由美, 豊田実, 佐々木泰史, 伊東文生, 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 ) 18 52 - 53 2002年

The p53 family member genes are involved in the Notch signal pathway.

Yasushi Sasaki, Setsuko Ishida, Ichiro Morimoto, Toshiharu Yamashita, Takashi Kojima, Chikashi Kihara, Toshihiro Tanaka, Kohzoh Imai, Yusuke Nakamura, Takashi Tokino

The Journal of biological chemistry 277 ( 1 ) 719 - 24 2001年10月 [査読有り] [国際誌]

担当区分：筆頭著者

The p53 tumor suppressor is a transcription factor that regulates cell growth and death in response to environmental stimuli such as DNA damage. p63/p51 and p73 were recently identified as members of the p53 gene family. In contrast to p53 however, p63 and p73 are rarely mutated in human cancers. Mice that lack p53 are developmentally normal, while p63 and p73 appear to play critical roles in normal development. To determine how p63 and p73 are involved in normal development, we attempted to identify target genes that are specifically regulated by p63 and/or p73 but not by p53. We found that the Jagged1 (JAG1) and Jagged2 (JAG2) genes, encoding ligands for the Notch receptors, are up-regulated by p63 and p73. Furthermore, we identified a p63-binding site in the second intron of the JAG1 gene, which can directly interact with the p63 protein in vivo, as assessed by a chromatin immunoprecipitation assay. A heterologous reporter assay revealed that this p63-binding site is a functional response element and is specific for p63. We also found a target of Notch signaling, HES-1 was up-regulated in Jurkat cells, in which Notch1 is highly expressed, when co-cultured with p63-transfected cells, suggesting that p63 can trigger the Notch signal pathway in neighboring cells. Our findings show an association between the p53 family genes and Notch signaling and suggest a potential molecular mechanism for the involvement of the p53 family genes in normal development.

DOI DOI2 PubMed

Adenovirus-mediated transfer of the p53 family genes, p73 and p51/p63 induces cell cycle arrest and apoptosis in colorectal cancer cell lines: potential application to gene therapy of colorectal cancer.

Sasaki Y, Morimoto I, Ishida S, Yamashita T, Imai K, Tokino T

Gene therapy 8 ( 18 ) 1401 - 1408 2001年09月 [査読有り]

担当区分：筆頭著者

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 10 cell lines tested, six cell lines displayed a similar response following transduction of p53, p73beta or p51A/p63gamma; 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 wild-type p53 alleles, but underwent apoptosis after transduction of p73beta or p51A/p63gamma. Similar to p53, transduction of p51A/p63gamma induced extensive apoptosis when combined with adriamycin or X-radiation in SW480 cells, which are normally resistant to apoptosis. Transduction of p73beta and p51A/p63gamma also reduced the tumorigenicity of two colorectal cancer cells in vivo. These results suggest that adenovirus-mediated p73beta and p51A/p63gamma transfer are potential novel approaches for the treatment of human cancers, particularly for tumors that are resistant to p53 gene therapy.

Mutational analysis of the beta-catenin gene in gastric carcinomas.

Sasaki Y, Morimoto I, Kusano M, Hosokawa M, Itoh F, Yanagihara K, Imai K, Tokino T

Tumour biology 22 ( 2 ) 123 - 130 2001年03月 [査読有り]

担当区分：筆頭著者

Previous studies reported that mutation of the adenomatous polyposis coli (APC) gene was not observed in the majority of gastric cancers. To evaluate the role of the APC/beta-catenin/Tcf pathway, we analyzed mutations in the beta-catenin gene and the accumulation of beta-catenin protein in gastric carcinomas. An interstitial deletion spanning exon 3 of the beta-catenin gene was observed in 1 of 13 gastric cancer cell lines. No missense mutation was found in these 13 cell lines. Nuclear and/or cytoplasmic localization of beta-catenin was observed in 16 of 70 primary gastric carcinomas by immunohistochemistry, while we found no mutations in exon 3 in 35 carcinoma tissues available for PCR amplification. Our findings suggest that somatic mutations of the beta-catenin gene are rare in human gastric carcinomas and that accumulation of normal beta-catenin protein in a subset of gastric cancers may be due to other mechanisms of its activation.

Jejunal carcinoid tumor mimicking leiomyosarcoma: preoperative diagnosis by endoscopic biopsy

Yukinari Yoshida, Takao Endo, Yasushi Sasaki, Fumio Itoh, Shigeru Sasaki, Yoshiaki Arimura, Masayuki Arashi, Masanori Ohara, Masahiro Fujita, Masao Hosokawa, Kohzoh Imai

Journal of Gastroenterology ( Springer Science and Business Media LLC ) 36 ( 1 ) 39 - 43 2001年01月 [査読有り] [国際誌]

情報システムのポストゲノムプロジェクトへの応用

辰巳治之, 青木文夫, 明石浩史, 宮司正道, 豊田実, 佐々木泰史, 西森博幸, 時野隆至, 伊東文生, 今井浩三

新医療 11 64 - 69 2001年

アデノウイルスベクターを用いたp53ファミリー遺伝子導入は大腸癌由来細胞株に細胞周期停止とアポトーシスを誘導する大腸癌の遺伝子治療への応用

佐々木泰史, 時野隆至

放射線生物研究 ( 放射線生物研究会 ) 3 ( 3 ) 279 - 290 2001年 [査読有り] [招待有り]

担当区分：筆頭著者

ポストゲノム・プロジェクトのアプリケーション: p53遺伝子機能解析とマルチパラレルコンピューティング

青木文夫, 辰巳治之, 明石浩史, 宮司正道, 豊田実, 佐々木泰史, 西森博幸, 時野隆至

医療とコンピュータ 12 31 - 40 2001年

Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis.

Sasaki Y, Itoh F, Suzuki H, Kobayashi T, Kakiuchi H, Hareyama M, Imai K

Journal of clinical laboratory analysis 14 ( 6 ) 314 - 319 2000年01月 [査読有り]

担当区分：筆頭著者

Abnormal cell cycle regulation is believed to be an important step in tumorigenesis. In mammalian cells, DNA damage commonly leads to cell cycle arrest in G2; however, little is known about the detailed biochemical mechanisms underlying the DNA damage-induced G2 arrest. In order to identify genes differentially expressed in association with G2 arrest, differential display analysis was performed between exponentially growing Chinese hamster ovary (CHO) cells and G2-arrested CHO cells induced by etoposide, SN-38, or X-radiation. We identified five cDNA clones whose expression was up-regulated in G2-arrested CHO cells. Sequence analysis revealed that three clones were homologous to known genes: isogene I of translation initiation factor eIF-4A, ribosomal protein L13, and translation repressor NAT1. The remaining two clones showed no homology to known genes. These results indicate that DNA damage can alter the expression of multiple genes, including translational regulators.

医学用語解説：Caretaker遺伝子・染色体の不安定性

小林寿久, 佐々木泰史, 伊東文生

G.I.Research 7 70 - 73 1999年 [査読有り] [招待有り]

The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma

S Ishida, K Yoshida, Y Kaneko, Y Tanaka, Y Sasaki, F Urano, A Umezawa, J Hata, K Fujinaga

CYTOGENETICS AND CELL GENETICS ( KARGER ) 82 ( 3-4 ) 278 - 283 1998年 [査読有り]

Chromosome translocation creates a fusion between the EWSR1 gene and an ETS family gene. The fusion between these two genes is a characteristic feature of Ewing sarcoma. We previously identified a fourth translocation, t(17;22)(q12;q12), in genomic DNA isolated from cells of patients affected with Ewing sarcoma. The discovery of this translocation suggested that there might be a novel EWSR1-ETV4 fusion gene. In the present study, we determined the genomic breakpoint and characterized the chimeric transcript of the EWSR1-ETV4 fusion gene in two t(17;22) Ewing sarcomas. Reverse transcriptase-PCR assay showed an in-frame fusion between the 5'-terminal region of EWSR1 and the 3' end of ETV4 (alias E1AF, PEA3); the chimeric transcript could thus serve as a template for expression of a protein composed of the N-terminal portion of EWSR1 fused to the DNA-binding domain of ETV4. Long PCR and sequence analysis of genomic DNA revealed that either exon 8 or intron 7 of EWSR 1 is fused to the same intron of ETV4 in both tumors. Several palindromic oligomer sequences were found close to the breakpoints in both genes. The 159-bp Alu-like sequence was repealed in the breakpoint region of the ETV4 gene. These observations suggest a mechanism of EWSR1-ETV4 gene fusion.

Subcapsular hematoma of the liver and pylethrombosis in the setting of cholestatic liver injury

Hirofumi Sakamoto, Mitsuo Suga, Itaru Ozeki, Toshihisa Kobayashi, Toshiaki Sugaya, Yasushi Sasaki, Naoki Azuma, Fumio Itoh, Shin-ichi Sakamoto, Akira Yachi, Kohzoh Imai

Journal of Gastroenterology ( Springer Science and Business Media LLC ) 31 ( 6 ) 880 - 884 1996年11月 [国際誌]

蛋白分解酵素阻害剤メシル酸ナファモスタットの大量療法奏効した重症急性膵炎と考えられた２症例

川崎君王, 有村佳昭, 平田博巳, 鈴木隆, 角本芳隆, 村上里絵子, 金戸宏行, 佐々木泰史, 今井浩三

医薬の門 35 12 - 15 1995年 [査読有り]

大腸癌の腫瘍マーカー

佐々木泰史, 伊東文生, 日野田裕治, 今井浩三

癌の臨床 40 697 - 703 1994年 [査読有り] [招待有り]

肝内胆管粘表皮癌の一例

佐々木泰史, 日野田裕治, 高山義一, 仲川尚明, 今井浩三, 矢花剛, 谷内昭, 榎本克彦

日本消化器病学会雑誌 88 1110 - 1115 1991年04月 [査読有り]

担当区分：筆頭著者

再生不良性貧血を伴う全身性エリテマトーデスと考えられる一症例

佐々木泰史, 杉山敏郎, 石田禎夫, 嶋中昭二, 細川寿和, 今井浩三, 谷内昭

日本臨床免疫学会雑誌 12 ( 4 ) 422 - 428 1989年 [査読有り]

担当区分：筆頭著者

A case of serologically highly suspected systemic lupus erythematosus (SLE) with pancytopenia because of hypoplastic bone marrow was reported. A 38 year-old female was admitted to Sapporo Medical College Hospital because of general malaise and body weight loss. On admission, laboratory examinations revealed severe pancytopenia, positive antinuclear antibody, positive anti-DNA antibodies, high titer of anti-Sm antibodies and anti-RNP antibodies, hypocomplementemia, and severe hypoplastic bone marrow. The diagnosis of SLE (three items of the 1982 revised criteria was present in this case) with aplastic anemia was highly suggested. Since a complement-dependent serum inhibitor of the growth of the patient's bone marrow cells in vitro was detected in patient's desease phase serum, it was suggested that the marrow aplasia was mediated by autoimmune mechanisms. © 1989, The Japan Society for Clinical Immunology. All rights reserved.

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