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 ~9-10% 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, SNU719) and EBV lines (AGS, SNU16, MKN74) were treated with a DNA methyl-transferase inhibitor (DNMT), decitabine (DCB), for three days followed by RNA sequencing to identify EBV-specific responses. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) 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 TCGA. 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 receptor α (RARα) binding motifs, suggesting that DCB-mediated demethylation restores RA pathway accessibility and transcriptional activity. Further, inhibiting RAR signaling reduced DCB induced apoptosis. Although DCB can induce both host gene re-expression and viral lytic gene activation in EBV-positive tumors, its impact on RA signaling in EBVaGC has not been studied. Decitabine promotes extensive epigenetic reprogramming in EBVaGC, with preferential effects in CIMP-positive, EBV-infected cell lines.