Abstract Epiregulin (EREG), a ligand of the epidermal growth factor receptor (EGFR), induces cell growth by binding to the EGFR. Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study performed by our group revealed that EREG is methylated in gastric cancer cells. In the present study, we found that EREG gene expression was low in seven out of 11 gastric cancer cells and this down-regulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated the CpG sites in the EREG promoter. Compared to DNMT1, knockdown of DNMT3b significantly increased the expression of EREG mRNA and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates the CpG methylation and silencing of EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues. In addition to DNA methylation, results from a ChIP assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone markers (H3K4me3 and AcH3) and of a repressive marker (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Lastly, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study demonstrated for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4017. doi:1538-7445.AM2012-4017