Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers

Abstract

Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (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 previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (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. Finally, 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 showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.

Transcriptional Regulation of the Human Tumor Suppressor DOK1 by E2F1

“…Because 5=-aza appears to affect both DNA methylation and chromatin remodeling in cells, the two key events in the regulation of gene expression (28,35), we investigated whether histone modifications could also play a role in 5=-aza-induced E2F1 recruitment to the DOK1 promoter in HNC cells. To this end, we evaluated the levels of different epigenetic markers on the DOK1 promoter of HNC cells, including H3K9 acetylation and H3K4 trimethylation (epigenetic markers for transcriptional activation) and H3K27 trimethylation, a key marker for epigenetic repression that often precedes DNA methylation (26).…”

Comprehensive Study on Associations Between Nine SNPs and Glioma Risk

“…Previous studies have assessed the several SNPs in EGFR gene for the association with the risk of several cancers, such as lung cancer, gastric cancer, breast cancer, prostate cancer and esophageal cancer (Wong et al, 1992;Tokunaga et al, 1995;Kharrat et al, 2007;Upadhyay et al, 2008;Perez et al, 2010;Han et al, 2011;Yun et al, 2012). Somatic alterations of the EGFR gene are common in glioma and influence several mechanisms of malignant transformation (Wong et al, 1992).…”

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition

“…27 Aberrant methylation, which is thought to be one of the causes of cancer, performed mostly through the induction of transcriptional silencing of tumor suppressor genes. 28 Deactivation of tumor suppressor genes by hypermethylation of promoters have been observed in various stages of colon cancer. 27 While there are different theories about the mechanism underlying this abnormal methylation in tumor cells, none of them has been proved.…”