Integrated transcriptomic and methylome analysis reveals retinoic acid pathway activation after decitabine treatment in EBV-associated gastric cancer

Epstein-Barr virus-associated gastric cancer (EBVaGC) accounts for ~9% to 10% of gastric cancers worldwide and is defined by a distinctive molecular profile, including extreme hypermethylation of the DNA. Targeting this aberrant methylation may be a potential therapeutic strategy. EBV⁺ gastric cancer cell lines (YCCEL1 and SNU719) and EBV^- lines (AGS, SNU16, and MKN74) were treated with a DNA methyltransferase inhibitor, decitabine (5-aza-2'-deoxycytidine [DCB]), for 3 days followed by RNA sequencing to identify Epstein-Barr virus (EBV)-specific responses. DNA methylation profiling by reduced representation bisulfite sequencing was performed in EBV⁺ cell lines and integrated with expression data to identify epigenetically regulated networks. While DCB induced broad transcriptional changes across all lines, EBV⁺ cells exhibited the strongest transcriptional response, sharing many upregulated genes. Many of these EBV⁺-specific genes were expressed at lower baseline levels in EBV⁺ tumors from The Cancer Genome Atlas Stomach Adenocarcinoma. DCB predominantly reduced methylation at highly methylated intergenic CpGs, with a subset of promoters undergoing significant demethylation. Integrated analysis revealed a strong inverse correlation between promoter demethylation and gene expression, implicating multiple cancer-relevant pathways. Upstream regulator analysis and motif enrichment indicated that regions losing methylation were enriched for retinoic acid (RA) receptor α binding motifs, suggesting that DCB-mediated demethylation restores RA pathway accessibility and transcriptional activity. Furthermore, inhibiting retinoic acid receptor signaling reduced DCB-induced apoptosis. Although DCB can induce both host gene re-expression and viral lytic gene activation in EBV⁺ tumors, its impact on RA signaling in EBVaGC has not been studied. Decitabine promotes extensive epigenetic reprogramming in EBVaGC, with preferential effects in CpG island methylator phenotype-positive, EBV-infected cell lines.IMPORTANCEEBV⁺ gastric cancer contains hypermethylated DNA, and despite this distinct molecular phenotype, there are currently no Epstein-Barr virus (EBV)-specific treatments available. Using an FDA-approved inhibitor to target hypermethylated DNA and multiomics approach to study the cellular response, we uncovered epigenetically altered transcriptional networks that may be further exploited to improve potential therapy. Among the pathways disrupted, retinoic acid signaling is of particular interest, as retinoid receptors such as retinoic acid receptor α and RARβ are frequently hypermethylated and repressed in EBV-associated gastric cancer. Our findings indicate that DNA methyltransferase inhibition can partially reverse all-trans retinoic acid (ATRA) receptor silencing, supporting further investigation of DNA methyltransferase inhibitor-ATRA combination strategies as a novel therapy for EBV⁺ gastric cancer.