Cytosine-phosphoguanine methylation of estrogen receptors in endometrial cancer.

We hypothesize that estrogen receptors (ERs) are differentially expressed in endometrial cancer. To test this hypothesis, we investigated the expression profile of ERalpha (ERalpha-A, ERalpha-B, ERalpha-C) and ERbeta genes and CpG methylation status in endometrial cancer cell lines and tissues using reverse transcription-PCR and methylation-specific PCR and direct DNA sequencing. The results demonstrated that ERalpha-A, ERalpha-B, and ERbeta were normally expressed whereas ERalpha-C gene was inactivated in all endometrial cancer cell lines. We further investigated the mechanisms of ERalpha-C gene inactivation through CpG methylation pathways. The treatment with demethylating agent (5'-aza-2'-deoxycytidine) restored ERalpha-C gene expression in all endometrial cancer cell lines. We further confirmed these findings with methylation-specific PCR and direct DNA sequencing and found that only ERalpha-C was methylated on all five different CpG sites in all cell lines. We further analyzed 88 cancerous and 46 normal endometrial tissues. The results demonstrated that only ERalpha-C was inactivated and methylated in 94% of cancer tissues. In 32 pairs of cancerous and normal endometrial tissues from the same patient, ERalpha-C was methylated in 29 of 32 cancer tissues but unmethylated in all normal endometrial tissues. This is the first report that demonstrates selective ERalpha-C gene inactivation through CpG methylation pathway in uterine endometrial cancer.