Accumulation of aberrant DNA methylation in normal-appearing gastric mucosae, mostly induced by H. pylori infection, is now known to be deeply involved in predisposition to gastric cancers (epigenetic field defect), and silencing of protein-coding genes has been analyzed so far. In this study, we aimed to clarify the involvement of microRNA (miRNA) gene silencing in the field defect. First, we selected three miRNA genes as methylation-silenced after analysis of six candidate ''methylation-silenced'' tumor-suppressor miRNA genes. Methylation levels of the three genes (miR124a-1, miR-124a-2 and miR-124a-3) were quantified in 56 normal gastric mucosae of healthy volunteers (28 volunteers with H. pylori and 28 without), 45 noncancerous gastric mucosae of gastric cancer patients (29 patients with H. pylori and 16 without), and 28 gastric cancer tissues (13 intestinal and 15 diffuse types). Among the healthy volunteers, individuals with H. pylori had 7.8-13.1-fold higher methylation levels than those without (p < 0.001). Among individuals without H. pylori, noncancerous gastric mucosae of gastric cancer patients had 7.2-15.5-fold higher methylation levels than gastric mucosae of healthy volunteers (p < 0.005). Different from protein-coding genes, individuals with past H. pylori infection retained similar methylation levels to those with current infection. In cancer tissues, methylation levels were highly variable, and no difference was observed between intestinal and diffuse histological types. This strongly indicated that methylationsilencing of miRNA genes, in addition to that of protein-coding genes, contributed to the formation of a field defect for gastric cancers. ' 2008 Wiley-Liss, Inc.Key words: field for cancerization; microRNA; methylation; gastric cancer; Helicobacter pylori Metachronous occurrence of gastric cancers is becoming an important issue as localized resection of early gastric cancers by endoscopic submucosal dissection (ESD) has become common. 1 The incidence of secondary primary gastric cancers after ESD reaches as high as 2.0% per year 2 whereas the incidence of gastric cancer in the general Japanese population is 0.14% per year. 3 This indicates that noncancerous gastric mucosae are already predisposed to developing gastric cancers, forming a field defect (field for cancerization). High incidences of metachronous cancers have been known not only for gastric cancers but also for bladder, liver, and esophageal cancers 4-6 and are becoming recognized for lung, breast and colorectal cancers. [7][8][9] A molecular basis for the field defect has been considered as an accumulation of genetic and epigenetic alterations in normalappearing tissues. Traditionally, cells with a genetic alteration were considered to form a physically continuous patch, producing a genetically altered field. 10 Recently, we found that aberrant DNA methylation of specific genes can be induced in as high as several percentage of cells in noncancerous gastric mucosae (thus in multiple independent gastric glands), and the degre...

BackgroundEpigenetic alterations accumulate in normal-appearing tissues of patients with cancer, producing an epigenetic field defect. Cross-sectional studies show that the degree of the defect may be associated with risk in some types of cancer, especially cancers associated with chronic inflammation.ObjectiveTo demonstrate, by a multicentre prospective cohort study, that the risk of metachronous gastric cancer after endoscopic resection (ER) can be predicted by assessment of the epigenetic field defect using methylation levels.DesignPatients with early gastric cancer, aged 40–80 years, who planned to have, or had undergone, ER, were enrolled at least 6 months after Helicobacter pylori infection discontinued. Methylation levels of three preselected genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy, and the primary endpoint was defined as detection of a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrolment.ResultsAmong 826 patients enrolled, 782 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1–2, 2–3 and ≥3 years after the enrolment, respectively. The highest quartile of the miR-124a-3 methylation level had a significant univariate HR (95% CI) (2.17 (1.07 to 4.41); p=0.032) and a multivariate-adjusted HR (2.30 (1.03 to 5.10); p=0.042) of developing authentic metachronous gastric cancers. Similar trends were seen for EMX1 and NKX6-1.ConclusionsAssessment of the degree of an epigenetic field defect is a promising cancer risk marker that takes account of life history.

Aberrant CpG methylations play important roles in cancer development and progression. In this study, aberrant methylations in human breast cancer were searched for using methylation-sensitive representational difference analysis (MS-RDA). A CpG island (CGI) in the 5 0 region of the heparan sulfate d-glucosaminyl 3-Osulfotransferase-2 (3-OST-2) gene was found to be hypermethylated, while its exon 2 was hypomethylated. In seven breast cancer cell lines, hypermethylation of the 5 0 region and loss of 3-OST-2 expression were observed. Treatment with a demethylating agent, 5-aza-2 0 -deoxycytidine, removed the methylation of the CGI in the 5 0 region and restored its expression, demonstrating silencing of the 3-OST-2 gene. Methylation-specific PCR (MSP) analysis in 85 primary breast cancers showed that the hypermethylation of the CGI in the 5 0 region was present in 75 (88%) of them. Quantitative reverse transcriptase-PCR (RT-PCR) analysis in 37 primary breast cancers showed that the average expression level was decreased in them. Further, MSP analysis in primary colon, lung and pancreatic cancers showed that hypermethylation of the CGI in the 5 0 region was present in the colon (8/10, 80%), lung (7/10, 70%) and pancreatic (10/10, 100%) cancers. These results showed that silencing of 3-OST-2 was present in a wide range of human cancers. The 3-OST-2 gene encodes an enzyme involved in the final modification step of heparan sulfate proteoglycans (HSPGs), and its silencing is expected to result in abnormal modification of HSPGs and abnormal signal transduction. From the high incidence, silencing of the 3-OST-2 gene is expected to have high diagnostic, and potentially therapeutic, values.

Nonalcoholic steatohepatitis (NASH) may cause fibrosis, cirrhosis, and hepatocellular carcinoma (HCC); however, the exact mechanism of disease progression is not fully understood. Angiogenesis has been shown to play an important role in the progression of chronic liver disease. The aim of this study was to elucidate the role of angiogenesis in the development of liver fibrosis and hepatocarcinogenesis in NASH. Zucker rats, which naturally develop leptin receptor mutations, and their lean littermate rats were fed a choline-deficient, amino acid-defined diet. Both Zucker and littermate rats showed marked steatohepatitis and elevation of oxidative stress markers (e.g., thiobarbital acid reactive substances and 8-hydroxydeoxyguanosine). In sharp contrast, liver fibrosis, glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions, and HCC developed in littermate rats but not in Zucker rats. Hepatic neovascularization and the expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, only increased in littermate rats, almost in parallel with fibrogenesis and carcinogenesis. The CD31-immunopositive neovessels were mainly localized either along the fibrotic septa or in the GST-P-positive lesions. Our in vitro study revealed that leptin exerted a proangiogenic activity in the presence of VEGF. In conclusion, these results suggest that leptin-mediated neovascularization coordinated with VEGF plays an important role in the development of liver fibrosis and hepatocarcinogenesis in NASH. Supplementary material for this article can be found on the HEPATOLOGY website

Background A previous multicenter prospective randomized study from Japan showed that Helicobacter pylori eradication reduced the development of metachronous gastric cancer (MGC) after endoscopic resection for early gastric cancer. MGC risk, however, is not eliminated; yet few studies have evaluated its long-term incidence and risk factors. In this study, we investigated the incidence of and risk factors for MGC in patients who underwent endoscopic resection for early gastric cancer with successful H. pylori eradication. Methods A total of 594 patients who underwent endoscopic resection for early gastric cancer and successful H. pylori eradication at three institutions (National Cancer Center Hospital, University of Tokyo Hospital, and Wakayama Medical University Hospital) were analyzed retrospectively. Annual endoscopic surveillance was performed after initial endoscopic resection. MGC was defined as a gastric cancer newly detected at least 1 year after successful H. pylori eradication. Results Ninety-four MGCs were detected in 79 patients during the 4.5-year median follow-up period. KaplanMeier analysis showed the cumulative incidence of MGC 5 years after successful H. pylori eradication was 15.0 %; the incidence of MGC calculated by use of the person-year method was 29.9 cases per 1000 person-years. Multivariate analysis using the Cox proportional hazards model revealed that male sex, severe gastric mucosal atrophy, and multiple gastric cancers before successful H. pylori eradication were independent risk factors for MGC. Eleven percent of MGCs (10 of 94) were detected more than 5 years after successful H. pylori eradication. Conclusion Surveillance endoscopy for MGC in patients who have undergone endoscopic resection for early gastric cancer should be performed even after successful H. pylori eradication.

Aberrant DNA methylation is known as an important cause of human cancers, along with mutations. Although aberrant methylation was initially speculated to be similar to mutations, it is now recognized that methylation is quite unlike mutations. Whereas the number of mutations in individual cancer cells is estimated to be 80, that of aberrant methylation of promoter CpG islands reaches several hundred to 1000. Although mutations of a specific gene are very few in non-cancerous (thus polyclonal) tissues (usually at 1 · 10 )5 ⁄ cell), aberrant methylation of a specific gene can be present up to several 10% of cells. Mutagenic chemicals and radiation are well-known inducers of mutations, whereas chronic inflammation is deeply involved in methylation induction. Although mutations are induced in mostly random genes, methylation is induced in specific genes depending on tissues and inducers. Methylation is potentially reversible, unlike mutations. These characteristics of methylation are opening up new fields of application and research. (Cancer Sci 2010; 101: 300-305) A berrant DNA methylation is deeply involved in human carcinogenesis, (1)(2)(3) and is often described as ''genomeoverall hypomethylation and regional hypermethylation''. Genome-overall hypomethylation was discovered in the early 1980s (4,5) and has been shown to induce genomic instability and promote carcinogenesis.(6-8) Regional hypermethylation denotes methylation of normally unmethylated CpG islands (CGI) and, in particular, methylation of a promoter CGI is known to silence its downstream gene by multiple mechanisms, including aberrant nucleosome formation.(9,10) Inactivation of a tumor-suppressor gene was first discovered for RB in 1993, (5,11) and now a wide variety of tumor-suppressor genes, including CDKN2A (p16), MLH1, and CDH1 (E-cadherin), are known to be inactivated by aberrant methylation.(2) In many types of cancers, aberrant promoter methylation is frequently observed and in some types of cancers, such as gastric cancers, aberrant methylation is more frequent than mutations in inactivating mechanisms of specific tumor-suppressor genes. (12) In the 1990s, investigators found that tumor-suppressor genes can be inactivated by aberrant methylation of promoter CGI, and that most CGI analyzed by conventional methods were kept unmethylated, even in cancers. This made them think that genes with aberrant methylation of promoter CGI were tumor-suppressor genes. Some investigators were inspired that they could identify tumor-suppressor genes if they could identify aberrant methylation by genome-wide screening methods.(13-16) Actually, these methods contributed to the identification of important CGI in diagnostic purposes and isolation of tumor-suppressor genes.(3) In addition, the fact that aberrant methylation of promoter CGI is an alternative to a mutation for inactivation of tumor-suppressor genes made many investigators think that epigenetic alterations would share similar features with mutations in other aspects, such as their frequencies in c...

Alteration in the methylation status of a gene is often associated with its altered expression. Based on a genome scanning technique for differences in CpG methylations, methylation-sensitive representational difference analysis, DNA fragments hypermethylated in a human breast cancer were isolated. A DNA fragment was isolated from intron 1 of guanine-nucleotide-binding protein α-11 (GNA11). mRNA expression of GNA11 was shown to be decreased in 10 of 16 breast cancers by RT-PCR analysis, and the immunoreactivity of the GNA11 product, Gα11 subunit of heterotrimeric G-protein, was observed to be reduced in 14 of the 16 cancers by immunohistochemistry. Methylation of a CpG island (CGI) in the 5′ region of GNA11 or that of intron 1 did not show a clear correlation with its decreased expression. Another DNA fragment was isolated from a CGI in the 5′ upstream region of monocarboxylate transporter 1 (MCT1), and was methylated in 4 of 20 breast cancers. The CGI was also methylated in a human breast cancer cell line, MDA-MB-231, and quantitative RT-PCR showed that its expression was almost lost in the cell line. By treatment of the cells with a demethylating agent, 5-aza-2′-deoxycytidine, the methylation was removed and the expression was restored. GNA11 is involved in signalling of gonadotropin-releasing hormone receptor, which negatively regulates cell growth. MCT1 is involved in cellular transportation of butyrate, which induces cellular differentiation. Downregulation of these two genes was suggested to be involved in human breast cancers.

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