Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells

BACKGROUND: Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection. MATERIALS AND METHODS: Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction. RESULTS: GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively). CONCLUSIONS: This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.     

Virulence of infecting Helicobacter pyloristrains and intensity of mononuclear cell infiltration are associated with levels of DNA hypermethylation in gastric mucosae

DNA methylation changes are known to occur in gastric cancers and in premalignant lesions of the gastric mucosae. In order to examine variables associated with methylation levels, we quantitatively evaluated DNA methylation in tumors, non-tumor gastric mucosae, and in gastric biopsies at promoters of 5 genes with methylation alterations that discriminate gastric cancers from non-tumor epithelia (EN1, PCDH10, RSPO2, ZIC1, and ZNF610). Among Colombian subjects at high and low risk for gastric cancer, biopsies from subjects from the high-risk region had significantly higher levels of methylation at these 5 genes than samples from subjects in the low risk region (p ≤ 0.003). When results were stratified by Helicobacter pylori infection status, infection with a cagA positive, vacA s1m1 strain was significantly associated with highest methylation levels, compared with other strains (p = 0.024 to 0.001). More severe gastric inflammation and more advanced precancerous lesions were also associated with higher levels of DNA methylation (p ≤ 0.001). In a multivariate model, location of residence of the subject and the presence of cagA and vacA s1m1 in the H. pylori strain were independent variables associated with higher methylation in all 5 genes. High levels of mononuclear cell infiltration were significantly related to methylation in PCDH10, RSPO2, and ZIC1 genes. These results indicate that for these genes, levels of methylation in precancerous lesions are related to H. pylori virulence, geographic region and measures of chronic inflammation. These genes seem predisposed to sustain significant quantitative changes in DNA methylation at early stages of the gastric precancerous process.     

Estimation of the Fraction of Cancer Cells in a Tumor DNA Sample Using DNA Methylation

Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.     

Connections between epigenetic gene silencing and human disease

Alterations in epigenetic gene regulation are associated with human disease. Here, we discuss connections between DNA methylation and histone methylation, providing examples in which defects in these processes are linked with disease. Mutations in genes encoding DNA methyltransferases and proteins that bind methylated cytosine residues cause changes in gene expression and alterations in the patterns of DNA methylation. These changes are associated with cancer and congenital diseases due to defects in imprinting. Gene expression is also controlled through histone methylation. Altered levels of methyltransferases that modify lysine 27 of histone H3 (K27H3) and lysine 9 of histone H3 (K9H3) correlate with changes in Rb signaling and disruption of the cell cycle in cancer cells. The K27H3 mark recruits a Polycomb complex involved in regulating stem cell pluripotency, silencing of developmentally regulated genes, and controlling cancer progression. The K9H3 methyl mark recruits HP1, a structural protein that plays a role in heterochromatin formation, gene silencing, and viral latency. Cells exhibiting altered levels of HP1 are predicted to show a loss of silencing at genes regulating cancer progression. Gene silencing through K27H3 and K9H3 can involve histone deacetylation and DNA methylation, suggesting cross talk between epigenetic silencing systems through direct interactions among the various players. The reversible nature of these epigenetic modifications offers therapeutic possibilities for a wide spectrum of disease.     

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.     

The role of epigenetics in environmental and occupational carcinogenesis

Over the last few years there has been an increasing effort in identifying environmental and occupational carcinogenic agents and linking them to the incidence of a variety of human cancers. The carcinogenic process itself is multistage and rather complex involving several different mechanisms by which various carcinogenic agents exert their effect. Amongst them are epigenetic mechanisms often involving silencing of tumor suppressor genes and/or activation of proto-oncogenes, respectively. These alterations in gene expression are considered critical during carcinogenesis and have been observed in many environmental- and occupational-induced human cancers. Some of the underlying mechanisms proposed to account for such differential gene expression include alterations in DNA methylation and/or histone modifications. Throughout this article, we aim to provide a current account of our understanding on how the epigenetic pathway is involved in contributing to an altered gene expression profile during human carcinogenesis that ultimately will allow us for better cancer diagnostics and therapeutic strategies.     

Genetic and epigenetic alterations of tumor suppressor and tumor-related genes in gastric cancer

Both genetic and epigenetic alterations of tumor suppressor and tumor-related genes involved in the pathogenesis of gastric cancer are reviewed here, and molecular pathways of gastric carcinogenesis are proposed. Gastric carcinomas are believed to evolve from native gastric mucosa or intestinal metaplastic mucosa that undergoes genetic and epigenetic alterations involving either the suppressor pathway (defects in tumor suppressor genes) or mutator pathway (defects in DNA mismatch repair genes). Methylation of E-cadherin in native gastric mucosa results in undifferentiated carcinomas (suppressor pathway), while methylation of hMLHI results in differentiated foveolar-type carcinomas (mutator pathway). The majority of differentiated gastric carcinomas however, arise from intestinal metaplastic mucosa and exhibit structural alterations of tumor suppressor genes, especially p53. They appear to be related to chronic injury, perhaps due to Helicobacter pylori infection. Approximately 20% of differentiated carcinomas (ordinary-type) have evidence of mutator pathway tumorigenesis. Mutations of E-cadherin are mainly involved in the progression of differentiated carcinomas to undifferentiated tumors. The molecular pathways of gastric carcinogenesis depend on the histological background, and gastric carcinomas show distinct biological behaviors as a result of discernible cellular genetic and epigenetic alterations.     

Effect of Helicobacter pylori Eradication on Incidence of Gastric Cancer in Human and Animal Models: Underlying Biochemical and Molecular Events

Background:  Gastric cancer remains one of the most common cancers worldwide. A strong association exists between Helicobacter pylori infection and the risk of developing noncardia gastric cancer. H. pylori eradication by antibiotic treatment is regarded as a primary chemoprevention strategy to reduce gastric cancer incidence.
Aim:  To analyze the efficacy of H. pylori eradication in preventing gastric cancer in human and animal models, and to discuss whether biochemical, genetic, and epigenetic changes associated with H. pylori infection are reversible after curing the infection.
Results:  Several intervention trials have indicated that in some patients, H. pylori eradication leads to regression and prevents the progression of precancerous lesions. The eradication therapy reduces gastric cancer incidence in patients without any precancerous lesions at the baseline and is most effective before the development of atrophic gastritis. A few recent intervention studies in Japan have demonstrated significant prophylactic effects of eradication therapy on the development of gastric cancer, suggesting the use of eradication therapy in high-risk populations as a gastric cancer reduction strategy. However, gastric cancer may still develop despite successful eradication therapy. Studies in animal models have confirmed the use of eradication therapy at an early point of infection to prevent gastric cancer development.
Conclusion:  H. pylori eradication may not completely abolish the risk of gastric cancer. However, eradication therapy may be used in high-risk populations to reduce gastric cancer incidence. It can reverse many biochemical, genetic, and epigenetic changes that H. pylori infection induces in the stomach.     

High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array

DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region (∼1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5′ regulatory regions and the 3′ regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.Key words: DNA methylation, upper aerodigestive tract, cancer, risk factors, biomarkers     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.     

E-cadherin gene promoter hypermethylation in H. pylori-induced enlarged fold gastritis

BACKGROUND: Promoter hypermethylation of E-cadherin plays an important role on gastric carcinogenesis. We have previously reported that the odds ratio for gastric carcinoma and the prevalence of diffuse-type early gastric carcinoma in Helicobacter pylori-induced enlarged fold gastritis increased with increasing fold width. Thus, we examined E-cadherin methylation in gastric mucosa from H. pylori-induced enlarged fold gastritis before and after H. pylori eradication. Moreover, we analyzed the mechanism of H. pylori infection-induced E-cadherin hypermethylation. MATERIALS AND METHODS: Twenty-three H. pylori-positive patients with enlarged folds, 18 H. pylori-positive and seven H. pylori-negative patients without enlarged folds, were involved in the study. E-cadherin promoter methylation was studied using quantitative methylation-specific polymerase chain reaction. We investigated methylation percentage and DNA methyltransferase activity in gastric cancer cell lines treated with EGF, TNFalpha, and MG132. RESULTS: E-cadherin methylation percentage of the gastric antral and body mucosa in H. pylori-positive patients with enlarged folds was much greater than that in both H. pylori-positive and -negative patients without enlarged folds. After H. pylori eradication, the methylation percentage in six patients with enlarged fold gastritis decreased significantly from 15.6 +/- 3.9 to 8.8 +/- 2.2 (p < .05). Moreover, the methylation was induced by TNFalpha, MG132, and EGF treatment, and DNA methyltransferase activity was induced by EGF treatment in MKN-1 cells. CONCLUSIONS: Our findings suggest that the hypermethylation of E-cadherin promoter might be involved in the process of gastric carcinoma through the specialized factors in H. pylori-induced enlarged fold gastritis.     

Aging, methylation and cancer

Alterations in methylation are widespread in cancers. DNA methylation of promoter-associated CpG islands is an alternate mechanism to mutation in silencing gene function, and affects tumor-suppressor genes such as p16 and RBI, growth and differentiation controlling genes such as ER and many others. Evidence is now accumulating that some of these methylation changes may initiate in subpopulations of normal cells as a function of age and progressively increase during carcinogenesis. Age-related methylation appears to be widespread and is one of the earliest changes marking the risk for neoplasia. In colon cancer, we have shown a pattern of age-related methylation for several genes, including ER, IGF2, N33 and MyoD, which progresses to full methylation in adenomas and neoplasms. Hypermethylation of these genes is associated with gene silencing. Age-related methylation involves at least 50% of the genes which are hypermethylated in colon cancer, and we propose that such age-related methylation may partly account for the fact that most cancers occur as a function of old age. Age-related methylation, then, may be a fundamental mark of the field defect in patients with neoplasia. The causes of age-related methylation are still unknown at this point, but evidence points to an interplay between local predisposing factors in DNA (methylation centers), levels of gene expression and environmental exposure. The concept that age-related methylation is a predisposing factor for neoplasia implies that it may serve as a diagnostic risk marker in cancer, and as a novel target for chemoprevention. Studies in animal models support this hypothesis and should lead to novel approaches to risk-assessment and chemoprevention in humans.     

Recent patents of DNA methylation biomarkers in gastrointestinal oncology

Gastrointestinal malignancies are among the most common malignancies worldwide. Advances in technology and treatment have improved diagnosis and monitoring of these tumors. As a consequence, identification of new biomarkers that can be applied at different levels of disease is urgently needed. DNA methylation is a process in which cytosines acquire a methyl group in 5' position only if they are followed by a guanine. An emerging catalog of specific genes inactivated by DNA methylation in gastrointestinal tumors has been established. In this review we will give a brief overview of the main sources of DNA used to investigate methylation biomarkers and several related patents. One of these is related to multiple genes that predict the risk of development of esophageal adenocarcinoma. Another evaluated methylation status of 24 genes to find one frequently methylated in primary tumors as well as plasma samples from gastric cancer patients. Others patented the epigenetic silencing of miR-342 as a promissory biomarker for colorectal carcinoma. Thus the new field of DNA methylation biomarkers holds the promise of better methods for screening, early detection, disease progression and outcome predictor of therapy response in gastrointestinal oncology.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.