Distinct mechanisms of loss of estrogen receptor α gene expression in human breast cancer: methylation of the gene and alteration of trans-acting factors

We have previously shown that the distal promoter (promoter B) of the estrogen receptor α (ERα) gene is responsible for the enhanced expression of the ERα gene seen in human breast cancer and that a novel trans-acting factor, estrogen receptor promoter B associated factor 1 (ERBF-1), is required for transcription from promoter B in breast cancer cells. In development of breast cancer, loss of ERα gene expression is one of the most important steps in acquiring hormone resistance, though the mechanisms are poorly understood. Recent studies have reported that methylation of the ERα gene promoter A and exon 1 was inversely associated with ERα gene expression in human breast cancer and cell lines. The methylation status of the promoter B region, which is responsible for overexpression of ERα protein in cancer tissue, has not been investigated. In this report, we found that the methylation status of promoter B, as well as that of promoter A, was inversely associated with ERα gene expression in human breast cancer and cell lines. Specific methylation of ERα gene promoters in vitro directly decreased transcription of the ERα gene in a reporter assay. Demethylating treatment induced transcription of ERα mRNA from promoter B in ZR-75-1 cells, which showed no transcription from promoter B, despite weak ERBF-1 expression, but not in ERα-negative MDA-MB-231 and BT-20 cells, which lack ERBF-1. ZR-75-1 cells showed promoter activity equal to that of MCF-7 cells in a reporter assay. Our results indicate that methylation of promoter B of the ERα gene is important for loss of ERα gene expression in human breast cancer, and methylation of the promoters can directly modulate ERα gene expression. However, loss of critical transcriptional factors such as ERBF-1 may also be involved in some ERα-negative cases.