Sayaka Funata

DNA methylation in gastric cancer, related to Helicobacter pylori and Epstein-Barr virus

Gastric cancer is a leading cause of cancer death worldwide, and significant effort has been focused on clarifying the pathology of gastric cancer. In particular, the development of genome-wide analysis tools has enabled the detection of genetic and epigenetic alterations in gastric cancer; for example, aberrant DNA methylation in gene promoter regions is thought to play a crucial role in gastric carcinogenesis. The etiological viewpoint is also essential for the study of gastric cancers, and two distinct pathogens, Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV), are known to participate in gastric carcinogenesis. Chronic inflammation of the gastric epithelium due to H. pylori infection induces aberrant polyclonal methylation that may lead to an increased risk of gastric cancer. In addition, EBV infection is known to cause extensive methylation, and EBV-positive gastric cancers display a high methylation epigenotype, in which aberrant methylation extends to not only Polycomb repressive complex (PRC)-target genes in embryonic stem cells but also non-PRC-target genes. Here, we review aberrant DNA methylation in gastric cancer and the association between methylation and infection with H. pylori and EBV.

Host SHP1 phosphatase antagonizes Helicobacter pylori CagA and can be downregulated by Epstein-Barr virus.

Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains1–4. Approximately 10% of gastric cancers also harbour Epstein–Barr virus (EBV) in the cancer cells5,6. Following delivery into gastric epithelial cells via type IV secretion7,8, the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu–Pro–Ile–Tyr–Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl9,10. Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity11,12, which has been considered to play an important role in gastric carcinogenesis13. Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer.

DNA methylation accumulation and its predetermination of future cancer phenotypes

Aberant DNA methylation is a common epigenomic alteration in carcinogenesis. Comprehensive analyses of DNA methylation have stratified gastrointestinal cancer into several subgroups according to specific DNA methylation accumulation. In gastric cancer, Helicobacter pylori infection is a cause of methylation accumulation in apparently normal mucosa. Epstein-Barr virus infection is another methylation inducer that causes more genome-wide methylation, resulting in the formation of unique epigenotype with extensive methylation. In colorectal carcinogenesis, accumulation of high levels of methylation in combination with BRAF mutation is characteristic of the serrated pathway, but not of the adenoma-carcinoma sequence through conventional adenoma. In a de novo pathway, laterally spreading tumours generate intermediate- and low-methylation epigenotypes, accompanied by different genetic features and different macroscopic morphologies. These methylation epigenotypes, with specific genomic aberrations, are mostly completed by the adenoma stage, and additional molecular aberration, such as TP53 mutation, is suggested to lead to cancer development with the corresponding epigenotype. Accumulation of DNA methylation and formation of the epigenotype is suggested to occur during the early stages of carcinogenesis and predetermines the future cancer type.

TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5′ regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

Epstein–Barr virus infection induces genome-wide de novo DNA methylation in non-neoplastic gastric epithelial cells

Epstein–Barr virus (EBV)‐positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome‐wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low‐methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non‐neoplastic epithelial cells remains unclear. Here we performed post‐infection time‐series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome‐wide analysis using Illuminas Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post‐infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC‐specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV‐positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well‐ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower‐CpG peripheral regions and extending to higher‐CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non‐neoplastic cells within a month in a spatiotemporally well‐ordered manner. Copyright

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Regulation of tumour related genes by dynamic epigenetic alteration at enhancer regions in gastric epithelial cells infected by Epstein-Barr virus

Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infection-mediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.