DNA methylation in the upstream CpG island of the GPER locus and its relationship with GPER expression in colon cancer cell lines

The seven-transmembrane G-protein coupled estrogen receptor (GPER) relays short-term non-genomic responses in target cells and tissues. It is a proposed tumor suppressor, which frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. A study by Liu and co-workers reported the loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. The present study is based on the hypothesis that GPER expression is inversely correlated with methylation in the upstream CpG island (upCpGi) in the GPER locus. Methylation in the upCpGi was analysed by bisulfite sequencing and correlated with GPER expression in a panel of colon cancer cell lines The bisulfite sequencing results show the presence of a differentially methylated region (DMR) comprising of the downstream eight CpGs of the upCpGi. Methylation in the DMR correlated inversely with GPER expression. We compared two cell lines, namely SW620 and COLO-320DM, in terms of their viability in response to varying concentrations of G1, a GPER specific agonist, which is known to induce cell cycle arrest and apoptosis in colon cancer cell lines. SW-620 cells, which had the least methylated DMR and the highest level of GPER expression, showed significant loss of viability with 1 μM G1. On the other hand, COLO-320DM, which had the most methylated DMR and the lowest level of GPER expression, did not show a significant response to 1 μM G1. At 5 μM G1, SW620 cells showed a greater reduction in viability than COLO-320DM cells. Our study demonstrates the inverse correlation between DNA methylation in the DMR and GPER expression. GPER is a non-canonical form of estrogen receptor, and estrogen is believed to exert its oncoprotective effect in the colon via GPER. DNA methylation-dependent silencing of GPER may, at least in part, the underlying reason behind the loss of estrogen’s oncoprotective effect in the colon. Future studies should explore the utility of DNA methylation in the upCpGi, particularly the DMR, in diagnosis or prognosis.