-
Yuka Aoki,
Masanori Nojima,
Hiromu Suzuki,
Hiroshi Yasui,
Reo Maruyama,
Eiichiro Yamamoto,
Masami Ashida,
Mitsuhiro Itagaki,
Hideki Asaoku,
Hiroshi Ikeda,
Toshiaki Hayashi,
Kohzoh Imai,
Mitsuru Mori, Takashi Tokino,
Tadao Ishida,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Our aim in this study was to clarify the role of global hypomethylation of repetitive elements in determining the genetic and clinical features of multiple myeloma (MM). METHODS: We assessed global methylation levels using four repetitive elements (LINE-1, Alu Ya5, Alu Yb8, and Satellite-alpha) in clinical samples that included 74 cases of MM and 11 benign controls consisting of 7 cases of monoclonal gammopathy of undetermined significance (MGUS) and 4 samples of normal plasma cells (NPCs). We also evaluated copy number alterations using array-based comparative genomic hybridization (aCGH) and performed methyl-CpG binding domain sequencing (MBD-seq). RESULTS: Global levels of the repetitive element methylation declined with the degree of malignancy of plasma cells (NPC > MGUS > MM), and there was a significant inverse correlation between the degree of genomic loss and LINE-1 methylation levels. We identified 80 genomic loci as common breakpoints (CBPs) around commonly lost regions, which were significantly associated with increased LINE-1 densities. MBD-seq analysis revealed that average DNA methylation levels at the CBP loci and relative methylation levels in regions with higher LINE-1 densities also decline during the development of MM. We confirmed that levels of methylation of the 5' UTR of respective LINE-1 loci correlated strongly with global LINE-1 methylation levels. Finally, there was a significant association between LINE-1 hypomethylation and a poorer overall survival (hazard ratio = 2.8, P = 0.015). CONCLUSION: Global hypomethylation of LINE-1 is associated with the progression and a poor prognosis in MM, possibly due to frequent copy number loss.
Genome Medicine 12/2012; 4(12):101.
-
Takashi Shimizu,
Hiromu Suzuki,
Masanori Nojima,
Hiroshi Kitamura,
Eiichiro Yamamoto,
Reo Maruyama,
Masami Ashida,
Tomo Hatahira,
Masahiro Kai,
Naoya Masumori, Takashi Tokino,
Kohzoh Imai,
Taiji Tsukamoto,
Minoru Toyota
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), although the mechanism is not fully understood. OBJECTIVE: We aimed to explore the involvement of epigenetic alteration of miRNA expression in BCa. DESIGN, SETTING, AND PARTICIPANTS: Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan array (Life Technologies, Carlsbad, CA, USA). Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic performance of the miRNA gene panel. RESULTS AND LIMITATIONS: Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, miR-124-2, miR-124-3, and miR-9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137: 68.7%; miR-124-2: 50.6%; miR-124-3: 65.1%; miR-9-3: 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion. CONCLUSIONS: Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection.
European urology 11/2012; · 7.67 Impact Factor
-
Rena Morita,
Yoshihiko Hirohashi,
Hiromu Suzuki,
Akari Takahashi,
Yasuaki Tamura,
Takayuki Kanaseki,
Hiroko Asanuma,
Satoko Inoda,
Toru Kondo,
Satoshi Hashino,
Tadashi Hasegawa, Takashi Tokino,
Minoru Toyota,
Masahiro Asaka,
Toshihiko Torigoe,
Noriyuki Sato
[show abstract]
[hide abstract]
ABSTRACT: DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.
Experimental and Molecular Pathology 10/2012; · 2.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cancer biology & therapy 09/2012; 13(14). · 2.64 Impact Factor
-
Eiichiro Yamamoto,
Hiromu Suzuki,
Hiro-O Yamano,
Reo Maruyama,
Masanori Nojima,
Seiko Kamimae,
Takeshi Sawada,
Masami Ashida,
Kenjiro Yoshikawa,
Tomoaki Kimura, [......],
Taku Harada,
Ryo Suzuki,
Akiko Sato,
Masahiro Kai,
Yasushi Sasaki, Takashi Tokino,
Tamotsu Sugai,
Kohzoh Imai,
Yasuhisa Shinomura,
Minoru Toyota
[show abstract]
[hide abstract]
ABSTRACT: 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.
American Journal Of Pathology 09/2012; 181(5):1847-61. · 4.89 Impact Factor
-
Hiroyuki Takamaru,
Eiichiro Yamamoto,
Hiromu Suzuki,
Masanori Nojima,
Reo Maruyama,
Hiro-O Yamano,
Kenjiro Yoshikawa,
Tomoaki Kimura,
Taku Harada,
Masami Ashida,
Ryo Suzuki,
Hiroyuki Yamamoto,
Masahiro Kai, Takashi Tokino,
Tamotsu Sugai,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P < 0.001; 8.3% vs. 19.4%, P < 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203-12. ©2012 AACR.
Cancer Prevention Research 09/2012; 5(10):1203-12. · 4.91 Impact Factor
-
Akari Takahashi,
Toshihiko Torigoe,
Yasuaki Tamura,
Takayuki Kanaseki,
Tomohide Tsukahara,
Yasushi Sasaki,
Hidekazu Kameshima,
Tetsuhiro Tsuruma,
Koichi Hirata, Takashi Tokino,
Yoshihiko Hirohashi,
Noriyuki Sato
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cell Stress and Chaperones 07/2012; 17(6):757-63. · 3.01 Impact Factor
-
Masahiro Shitani,
Shigeru Sasaki,
Noriyuki Akutsu,
Hideyasu Takagi,
Hiromu Suzuki,
Masanori Nojima,
Hiroyuki Yamamoto, Takashi Tokino,
Koichi Hirata,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n = 4; hepatitis C virus (HCV)-positive, n = 5; HBV/HCV-negative, n = 7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n = 57) and noncancerous liver tissue (n = 50) from HCC patients and in HCC cell lines (n = 10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.
Tumor Biology 03/2012; 33(5):1307-17. · 1.94 Impact Factor
-
Takeshi Niinuma,
Hiromu Suzuki,
Masanori Nojima,
Katsuhiko Nosho,
Hiroyuki Yamamoto,
Hiroyuki Takamaru,
Eiichiro Yamamoto,
Reo Maruyama,
Takayuki Nobuoka,
Yasuaki Miyazaki, [......],
Satoshi Okahara,
Hiroaki Takahashi,
Yasunori Nishida,
Masao Hosokawa,
Tadashi Hasegawa, Takashi Tokino,
Koichi Hirata,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs.
Cancer Research 03/2012; 72(5):1126-36. · 7.86 Impact Factor
-
Lisa Kashima,
Masashi Idogawa,
Hiroaki Mita,
Miki Shitashige,
Tesshi Yamada,
Kazuhiro Ogi,
Hiromu Suzuki,
Minoru Toyota,
Hiroyoshi Ariga,
Yasushi Sasaki, Takashi Tokino
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 02/2012; 287(16):12975-84. · 4.77 Impact Factor
-
Yasushi Sasaki,
Hideaki Negishi,
Masashi Idogawa,
Ikuko Yokota,
Ryota Koyama,
Masanobu Kusano,
Hiromu Suzuki,
Masahiro Fujita,
Reo Maruyama,
Minoru Toyota,
Tsuyoshi Saito, Takashi Tokino
[show abstract]
[hide abstract]
ABSTRACT: Hepatoma-derived growth factor (HDGF) is a secreted heparin-binding growth factor that has been implicated in cancer development and progression. Here, we report that HDGF is a critical target for transcriptional repression by the tumor suppressor p53. Endogenous HDGF expression was decreased in cancer cells with introduction of wild-type p53, which also downregulated HDGF expression after DNA damage. In support of the likelihood that HDGF is a critical driver of cancer cell growth, addition of neutralizing HDGF antibodies to culture media was sufficient to block cell growth, migration, and invasion. Similarly, these effects were elicited by conditioned culture medium from p53-expressing cells, and they could be reversed by the addition of recombinant human HDGF. Interestingly, we found that HDGF was overexpressed also in primary gastric, breast, and lung cancer tissues harboring mutant p53 genes. Mechanistic investigations revealed that p53 repressed HDGF transcription by altering HDAC-dependent chromatin remodeling. Taken together, our results reveal a new pathway in which loss of p53 function contributes to the aggressive pathobiological potential of human cancers by elevating HDGF expression.
Cancer Research 11/2011; 71(22):7038-47. · 7.86 Impact Factor
-
Hiromu Suzuki,
Shintaro Takatsuka,
Hirofumi Akashi,
Eiichiro Yamamoto,
Masanori Nojima,
Reo Maruyama,
Masahiro Kai,
Hiro-O Yamano,
Yasushi Sasaki, Takashi Tokino,
Yasuhisa Shinomura,
Kohzoh Imai,
Minoru Toyota
[show abstract]
[hide abstract]
ABSTRACT: Altered expression of microRNAs (miRNA) occurs commonly in human cancer, but the mechanisms are generally poorly understood. In this study, we examined the contribution of epigenetic mechanisms to miRNA dysregulation in colorectal cancer by carrying out high-resolution ChIP-seq. Specifically, we conducted genome-wide profiling of trimethylated histone H3 lysine 4 (H3K4me3), trimethylated histone H3 lysine 27 (H3K27me3), and dimethylated histone H3 lysine 79 (H3K79me2) in colorectal cancer cell lines. Combining miRNA expression profiles with chromatin signatures enabled us to predict the active promoters of 233 miRNAs encoded in 174 putative primary transcription units. By then comparing miRNA expression and histone modification before and after DNA demethylation, we identified 47 miRNAs encoded in 37 primary transcription units as potential targets of epigenetic silencing. The promoters of 22 transcription units were associated with CpG islands (CGI), all of which were hypermethylated in colorectal cancer cells. DNA demethylation led to increased H3K4me3 marking at silenced miRNA genes, whereas no restoration of H3K79me2 was detected in CGI-methylated miRNA genes. DNA demethylation also led to upregulation of H3K4me3 and H3K27me3 in a number of CGI-methylated miRNA genes. Among the miRNAs we found to be dysregulated, many of which are implicated in human cancer, miR-1-1 was methylated frequently in early and advanced colorectal cancer in which it may act as a tumor suppressor. Our findings offer insight into the association between chromatin signatures and miRNA dysregulation in cancer, and they also suggest that miRNA reexpression may contribute to the effects of epigenetic therapy.
Cancer Research 07/2011; 71(17):5646-58. · 7.86 Impact Factor
-
Arthur Kwok Leung Cheung,
Josephine M Y Ko,
Hong Lok Lung,
Kwok Wah Chan,
Eric J Stanbridge,
Eugene Zabarovsky, Takashi Tokino,
Lisa Kashima,
Toshiharu Suzuki,
Dora Lai-Wan Kwong,
Daniel Chua,
Sai Wah Tsao,
Maria Li Lung
[show abstract]
[hide abstract]
ABSTRACT: Chromosome 14 was transferred into tumorigenic nasopharyngeal carcinoma and esophageal carcinoma cell lines by a microcell-mediated chromosome transfer approach. Functional complementation of defects present in the cancer cells suppressed tumor formation. A candidate tumor-suppressor gene, cysteine-rich intestinal protein 2 (CRIP2), located in the hot spot for chromosomal loss at 14q32.3, was identified as an important candidate gene capable of functionally suppressing tumor formation. Previous studies have shown that CRIP2 is associated with development. To date, no report has provided functional evidence supporting a role for CRIP2 in tumor development. The present study provides unequivocal evidence that CRIP2 can functionally suppress tumorigenesis. CRIP2 is significantly down-regulated in nasopharyngeal carcinoma cell lines and tumors. CRIP2 reexpression functionally suppresses in vivo tumorigenesis and angiogenesis; these effects are induced by its transcription-repressor capability. It interacts with the NF-κB/p65 to inhibit its DNA-binding ability to the promoter regions of the major proangiogenesis cytokines critical for tumor progression, including IL6, IL8, and VEGF. In conclusion, we provide compelling evidence that CRIP2 acts as a transcription repressor of the NF-κB-mediated proangiogenic cytokine expression and thus functionally inhibits tumor formation and angiogenesis.
Proceedings of the National Academy of Sciences 05/2011; 108(20):8390-5. · 9.68 Impact Factor
-
Seiko Kamimae,
Eiichiro Yamamoto,
Hiro-o Yamano,
Masanori Nojima,
Hiromu Suzuki,
Masami Ashida,
Tomo Hatahira,
Akiko Sato,
Tomoaki Kimura,
Kenjiro Yoshikawa, [......],
Hiroyuki Takamaru,
Reo Maruyama,
Masahiro Kai,
Morie Nishiwaki,
Tamotsu Sugai,
Yasushi Sasaki, Takashi Tokino,
Yasuhisa Shinomura,
Kohzoh Imai,
Minoru Toyota
[show abstract]
[hide abstract]
ABSTRACT: Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors.
Cancer Prevention Research 05/2011; 4(5):674-83. · 4.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
International Journal of Oncology 12/2010; 37(6):1407-16. · 2.40 Impact Factor
-
Shinichi Igarashi,
Hiromu Suzuki,
Takeshi Niinuma,
Haruo Shimizu,
Masanori Nojima,
Hiroyuki Iwaki,
Takayuki Nobuoka,
Toshirou Nishida,
Yasuaki Miyazaki,
Hiroyuki Takamaru,
Eiichiro Yamamoto,
Hiroyuki Yamamoto, Takashi Tokino,
Tadashi Hasegawa,
Koichi Hirata,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy.
A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens.
LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations.
Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations.
Clinical Cancer Research 10/2010; 16(21):5114-23. · 7.74 Impact Factor
-
Hiromu Suzuki,
Eiichiro Yamamoto,
Masanori Nojima,
Masahiro Kai,
Hiro-O Yamano,
Kenjiro Yoshikawa,
Tomoaki Kimura,
Toyoki Kudo,
Eiji Harada,
Tamotsu Sugai,
Hiroyuki Takamaru,
Takeshi Niinuma,
Reo Maruyama,
Hiroyuki Yamamoto, Takashi Tokino,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
[show abstract]
[hide abstract]
ABSTRACT: Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.
Carcinogenesis 10/2010; 31(12):2066-73. · 5.70 Impact Factor
-
Masaki Yamashita,
Minoru Toyota,
Hiromu Suzuki,
Masanori Nojima,
Eiichiro Yamamoto,
Seiko Kamimae,
Yoshiyuki Watanabe,
Masahiro Kai,
Hirofumi Akashi,
Reo Maruyama,
Yasushi Sasaki,
Hiroo Yamano,
Tamotsu Sugai,
Yasuhisa Shinomura,
Kohzoh Imai, Takashi Tokino,
Fumio Itoh
[show abstract]
[hide abstract]
ABSTRACT: Interferon regulatory factors (IRFs) are transcription factors known to play key roles in innate and adaptive immune responses, cell growth, apoptosis, and development. Their function in tumorigenesis of gastric cancer remains to be determined, however. In the present study, therefore, we examined epigenetic inactivation of IRF1-9 in a panel of gastric cancer cell lines. We found that expression of IRF4, IRF5, and IRF8 was frequently suppressed in gastric cancer cell lines; that methylation of the three genes correlated with their silencing; and that treating the cells with the demethylating agent 5-aza-2'-deoxycytidine (DAC) restored their expression. Expression of IRF5 in cancer cells was enhanced by the combination of DAC treatment and adenoviral vector-mediated expression of p53, p63, or p73. Interferon-gamma-induced expression of IRF8 was also enhanced by DAC. Moreover, treating gastric cancer cells with DAC enhanced the suppressive effects of interferon-alpha, interferon-beta, and interferon-gamma on cell growth. Among a cohort of 455 gastric cancer and noncancerous gastric tissue samples, methylation of IRF4 was frequently observed in both gastric cancer specimens and noncancerous specimens of gastric mucosa from patients with multiple gastric cancers, which suggests IRF4 methylation could be a useful molecular marker for diagnosing recurrence of gastric cancers. Our findings indicate that epigenetic IRF inactivation plays a key role in tumorigenesis of gastric cancer, and that inhibition of DNA methylation may restore the antitumor activity of interferons through up-regulation of IRFs.
Cancer Science 07/2010; 101(7):1708-16. · 3.33 Impact Factor
-
Tomoko Fujikane,
Noriko Nishikawa,
Minoru Toyota,
Hiromu Suzuki,
Masanori Nojima,
Reo Maruyama,
Masami Ashida,
Mutsumi Ohe-Toyota,
Masahiro Kai,
Toshihiko Nishidate,
Yasushi Sasaki,
Tousei Ohmura,
Koichi Hirata, Takashi Tokino
[show abstract]
[hide abstract]
ABSTRACT: 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.
Breast Cancer Research and Treatment 10/2009; 122(3):699-710. · 4.43 Impact Factor
-
Takashi Tokino
Cancer biology & therapy 07/2009; 8(12):1154-5. · 2.64 Impact Factor
