論文 - 時野　隆至

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

井戸川 雅史, 大箸 智子, 佐々木 泰史, 時野 隆至

日本癌学会総会記事 ( (一社)日本癌学会 )  74回   J - 1294  2015年10月

薬剤耐性を示した多発性骨髄腫患者の次世代シークエンサーを用いた遺伝子解析

池田 博, 石黒 一也, 青木 由佳, 石田 禎夫, 佐々木 泰史, 時野 隆至

日本癌学会総会記事 ( (一社)日本癌学会 )  74回   P - 3191  2015年10月

消化器癌における変異kRAS遺伝子の影響の解析およびIGF-1R阻害

松永 康孝, 足立 靖, 佐々木 泰史, 能正 勝彦, 高木 秀安, 山本 博幸, 佐々木 茂, 有村 佳昭, 遠藤 高夫, 加藤 康夫, 時野 隆至, 篠村 恭久

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

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

栄永 直樹, 山口 裕美, 中山 裕貴, 川路 美子, 佐々木 泰史, 時野 隆至, 江角 眞理子

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

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

DOI

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

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

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

DOI

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

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

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

　概要を見る

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

DOI PubMed

CRKL oncogene is downregulated by p53 through miR-200s

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

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

　概要を見る

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

DOI

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

　概要を見る

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

DOI PubMed

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

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

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

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

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

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

　概要を見る

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

DOI PubMed

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

Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki

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

DOI

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

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

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

　概要を見る

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

DOI PubMed

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

佐々木 泰史, 中垣 貴文, 田村 みゆき, 竹田 康佑, 井戸川 雅史, 大箸 智子, 鈴木 拓, 篠村 恭久, 時野 隆至

日本癌学会総会記事 ( (一社)日本癌学会 )  73回   P - 2146  2014年09月

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

中垣 貴文, 佐々木 泰史, 井戸川 雅史, 竹田 康佑, 田村 みゆき, 大箸 智子, 荻 和弘, 平塚 博義, 時野 隆至

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