Expression of estrogen receptor β isoforms in normal breast epithelial cells and breast cancer: Regulation by methylation

Two novel estrogen receptor β (ERβ) mRNA isoforms that diverge in their 5′-untranslated regions, ERβ mRNA (0K-1) and ERβ mRNA (0N-1), have recently been identified. This indicates that transcription of the human ERβ gene occurs from at least two different promoters, named promoter 0K and promoter 0N. The aim of this study was to investigate the expression of ERβ isoforms in primary cultures of normal breast epithelial cells, a panel of breast cancer cell lines and in normal breast and breast cancer tissues; and to examine whether methylation of the two ERβ promoters is involved in regulation of ERβ gene expression. Using quantitative real-time PCR techniques, we found that ERβ mRNA levels were significantly lower in breast cancer cell lines than in primary cultures of normal breast epithelial cells. Bisulfite genomic sequencing analysis revealed that two promoters of the ERβ gene exhibit distinct methylation patterns. Promoter 0N was unmethylated in normal breast epithelial cells, but extensively methylated in breast cancer cell lines. In contrast, promoter 0K was unmethylated in both normal and malignant breast epithelial cells. We demonstrated a significant correlation between promoter 0N hypermethylation and loss of ERβ mRNA expression in breast cancer cell lines. Treatment of breast cancer cells with demethylating agent effectively reactivated the expression of ERβ mRNA. The observations from the cell lines were also reflected in primary breast cancer tumors. Thus, expression of ERβ mRNA in breast tumors was found to be inversely associated with the degree of methylation of promoter 0N. Our results suggest that a decreased level of ERβ mRNA may be associated with breast tumorigenesis, and that DNA methylation is an important mechanism for ERβ gene silencing in breast cancer.