The Role Of ER β And Its Regulation In Breast Cancer Cells

ERβ, a new estrogen receptor, was successfully cloned from rat prostate and ovarycDNA libraries in 1996. From then on, the classical ER was named ERα. Chromosome localization, tissue distribution and ligand binding affinity appear to be different for ERpand ERa, suggesting that each may have distinct biological roles.Breast cancer is the most frequently malignant tumor in female. Endogenous estrogen is thought to play a major role in breast cancer development. Estrogen receptor blockers are the most important drugs in the treatment of breast cancer. Although approximately 50%-60% of ERa-positive women receive some degree of benefit from endocrine treatment, about 40% of patients are of no effect. ERa-negative breast cancer patients seldom respond to endocrine therapy. With the discovery of ERp, many studies have focused on the effect of ERP on breast cancer cell growth, development and progression, and on prediction of the response of endocrine treatment in breast cancer.However, the majority of studies assessed ERp at mRNA levels, which might not accurately reflect ERP protein levels. Because of limited number of tumors examined, some of the results of previous studies on ERP was contradictory. In order to elucidate direct effect of ERP on breast cancer, we first constructed an ERp eukaryotic expression vector, and then transfected the vector into MCF7 breast cancer cells. The transfected cells were selected by G418 , and the ERp stable expression breast cancer cell lines were obtained. The growth of the ERp-transfected breast cancer cell lines were measured under different treatment including E2(17beta-estradiol) and 4-OHT(4-hydroxytamoxifen). Exogenous ERβ expression neither changed the growth properties and the morphology of MCF-7 cells under normal condition nor increased the resistance to 4-OHT. However, in the presence of E2, a strong inhibition of the proliferation of the ERp-transfected breast cancer cells were observed.Our previous study revealed that COBRA 1, a novel protein, can interact with breast cancer susceptibility protein ( BRCA1 ). Like BRCA1, COBRA 1 can interact with ERα and inhibit the transcriptional activation of ERα , suggesting that COBRA 1 is one of theco-repressors of ER a .Because of high homology at ammo-acid level between ER a and ERβ, we investigated the possibility of the interaction between COBRA 1 and ERβ, in order to elucidate the molecular mechanisms of breast cancer. We constructed a GST-COBRA1 prokaryotic fusion expression vector, and obtained sufficient purified GST-COBRA1 protein. The purified GST-COBRA1 protein was injected into New Zealand white male rabbits for polyclonal antibody production, and a highly specific rabbit polyclonal anti-COBRAl antibody was developed. This antibody may be a useful tool to examine COBRA 1 protein in Western blot assay, Immunofluorescence analysis and Co-immunoprecipitation. Using the COBRA 1 antibody, COBRA 1 was shown to interact with ER 3 in vivo by Co-immunoprecipitation. COBRA 1 inhibited the transcriptional activity of ER 3 in transient transfections of an estrogen responsive reporter construct.