Loss of estrogen receptor β isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma

Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ER beta in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ER beta isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription-polymerase chain reaction were used to evaluate methylation status and expression of ER beta isoforms. The relative abundance of exon 0N, ER beta 1, ER beta 2, and ER beta 4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ER beta 5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ER beta gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ER beta 1, ER beta 2, and ER beta 4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5-aza-2' deoxycytidine resulted in reexpression of the ER beta gene. The results of our present study suggest that ER beta is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of epithelial ovarian cancer. (Cancer Sci 2008; 99: 2365-2372).