Stratification of Gastric Cancer Based on Methylation Status of Enhancer Regions and Effects on Transcriptional Regulation

Epstein-Barr virus-positive gastric cancer (EBV(+) GC) shows extensive DNA hypermethylation in gene promoter regions. DNA methylation induced by EBV infection contributes to carcinogenesis through tumor suppressor gene inactivation. Whereas aberrant DNA methylation in enhancer regions might reportedly contribute to carcinogenesis, its impact has not been fully elucidated. Therefore, we here performed DNA methylome analysis on clinical samples, including GC and normal gastric mucosa, using the Infinium MethylationEPIC BeadChip array to evaluate the epigenome in the enhancer regions. We stratified GC cases into three subclusters using hierarchical clustering analysis based on the methylation status of enhancer regions; EBV(+) GC samples were classified into the most extensively methylated subcluster. We identified genes with promoters unmethylated but enhancers aberrantly methylated in EBV(+) GC. Such methylated genes included Inositol polyphosphate-5-phosphatase A (INPP5A), which is associated with the phosphoinositide 3-kinase/Akt signaling pathway and cell proliferation and survival, and showed lower expression levels in EBV(+) GC compared with normal mucosa and other GC samples. We infected GES1, an immortalized gastric epithelial cell line, with EBV in vitro, and demonstrated the induction of aberrant DNA methylation in the INPP5A enhancer region and the downregulation of its expression. These findings indicate that EBV(+) GC exhibits a specific DNA hypermethylation phenotype in enhancer regions, and EBV infection in gastric cells could induce aberrant DNA methylation in enhancer regions, involving dysregulation of cancer-related gene expression.