Estrogen regulation of anti-apoptotic Bcl-2 family member Mcl-1 expression in breast cancer

INTRODUCTION: Estrogen is implicated as an important factor in stimulating breast cancer cell proliferation, and presence of estrogen receptor (ER) is an indication of a good prognosis in breast cancer patients. Mcl-1 is an anti-apoptotic Bcl-2 family member that is often overexpressed in breast tumors, correlating with poor survival. Estrogen has been previously shown to regulate Bcl-2 family members, leading to an evasion of apoptosis, however the role of estrogen in regulating Mcl-1 expression is unclear. I hypothesize that estrogen increases the expression of anti-apoptotic gene Mcl-1 through binding of ERalpha to a half estrogen response element (ERE) site within the promoter of Mcl-1 gene. This leads to increased Mcl-1 expression in breast cancer cells, ultimately contributing to an evasion of apoptosis.;RESULTS: In ERalpha positive cell lines, estrogen treatment increased Mcl-1 expression at both the protein and mRNA level. In two ERalpha negative cell lines, SK-BR-3 and MDA-MB-231, estrogen failed to increase in Mcl-1 protein expression. ERalpha antagonists decreased estrogen mediated Mcl-1 expression at both the protein and message level. Upon knockdown of ERalpha, Mcl-1 mRNA expression after estrogen treatment was also decreased. ChIP showed an enrichment of ERalpha to the Mcl-1 promoter at a region 3683 bp upstream of the translation start site containing a half ERE site. Streptavidin-pull down assay showed both ERalpha and transcription factor Sp1 bind to this region and mutation of the half ERE site eliminated this binding.;CONCLUSIONS These results suggest that estrogen is involved in regulating Mcl-1 expression specifically through a mechanism involving ERalpha. Ultimately, a better understanding of the role of estrogen in regulating Mcl-1 expression will determine whether Mcl-1 is a valid molecular target for breast cancer therapy.;METHODS: Four distinct breast cancer cell lines: MCF-7 and ZR-75, which both express ERalpha, and SKB-BR-3 and MDA-MB-231, which do not express ERalpha, to investigate the role of estrogen plays in regulating Mcl-1 expression. Cells were grown in serum-starved white media with charcoal-stripped FBS for five days prior to treatment with estrogen. Cells were treated with ERalpha antagonists Tamoxifen and Fulvestrant in combination with estrogen. Also, siRNA knockdown of ERalpha was performed and mRNA expression was evaluated. Chromatin immunoprecipitation (ChIP) was used to investigate if ERalpha binds to a specific ERE half-site within the Mcl-1 promoter. To further validate this data, a streptavidin pull-down assay was performed using a biotin-labeled probe specific to this region.