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Contribution of Nuclear Receptor Corepressor SMRT in Breast Cancer Cell Proliferation
The sex steroids, estrogens, regulate development, growth and functioning of breast tissue. The effects of estrogens (such as 17beta-estradiol or E2) are mediated by estrogen receptors (ERs), a member of a large family of ligand-dependent transcription factors. The ER¡¦s transcriptional activity is modulated by a set of coregulator proteins consisting of coactivators and corepressors. It is believed that the differential responsiveness of ER to an agonist or antagonist is mainly due to the biased recruitment of cofactors that ultimately determines the fate of transcription. In an attempt to understand the role of corpressors in gene expression, we conducted a siRNA-based SMRT depletion study, which indicated that reduction of cellular SMRT levels is associated with suppression of ER transcriptional activity, endogenous target gene expression and reduction in the rate of cell proliferation. Conversely, SMRT overexpression enhanced ER transcriptional activity. This finding led us to hypothesize that the corepressor SMRT plays a stimulatory role in ER transcriptional activity and induction of endogenous target gene expression in breast cancer cells that in turn stimulates cell proliferation. This hypothesis will be tested in specific aims which seek to investigate the effect of altered SMRT expression on anchorage dependent and independent cell proliferation, programmed cell death and regulation of gene expression in relation to cell cycle and apoptosis. I will generate, using a tet-regulated expression systems, two stable cell lines; in one SMRT expression will be increased and in the other SMRT expression will be suppressed. Using these cell lines we will determine the effects of altered SMRT expression on cell proliferation, cell cycle kinetics and apoptosis. To determine the mechanisms underlying altered growth properties in relation to SMRT modulation, pathway specific PCR-array analyses will be carried out. These studies will enable me to define SMRT regulated molecular pathways that affect breast cancer proliferation, apoptosis and ultimately breast tumorogenesis. Recent studies indicate that the diversity of estrogen receptor function stems from differential recruitment and interaction of coactivators and corepressors with this nuclear receptor. Although corepressors are believed to be a part of the transcription inactivation machinery, some reports suggest that corepressors can interact with coactivators and may play a role in receptor activation from the basal state. There are also limited reports on elevated SMRT expression in intraductal carcinoma, and in antiestrogen-resistant breast cancer, and it is clear that the biological roles of SMRT in breast are poorly understood. This study will reveal the importance of SMRT in controlling ER activity and breast cancer cell growth and survival, and will reveal whether SMRT should be evaluated as a prognostic or therapeutic target for breast cancer.
Breast cancer is one of the most common malignancies in the world and recent estimates are that one in eight American women will develop breast cancer in their lifetime (1,2). The female sex steroid estrogen is important for normal growth and development of breast, although an elevated exposure to this hormone plays a crucial role in progression of carcinogenesis. Generally, estrogen exerts its effects by binding to its specific intracellular receptors (estrogen receptor, ER) leading to recruitment of accessory molecules (e.g. coactivators and corepressors) and ultimately changes in gene expression. It is believed that binding of estrogen or antiestrogens to the ER and differential recruitment of those accessory molecules determines patterns of gene expression, cellular growth and differentiation. However, while much is known about the mechanism by which coactivators stimulate ER activity, little is known about the role of corepressors in modulation of gene expression. To understand the involvement of corepressors in gene expression, we employed a strategy in which the expression of the corepressor SMRT was down-regulated in ER-positive breast cancer cells. We expected that reduced corepressor expression would augment ER activity. Surprisingly, we found that SMRT depletion not only decreased ER activity, but also decreased cell proliferation. Moreover, the activity of antiestrogen-bound ER was unaffected. Thus, we hypothesize that in addition to its context-dependent role as a corepressor of gene expression, SMRT also is required for full activation of ER transcriptional activity and it therefore plays a critical role in breast cancer cell proliferation and survival. The goal of the studies proposed in this application is to determine the role of SMRT in breast cancer cell growth and survival. We will generate two engineered breast cancer cell lines in which SMRT expression is either inhibited or significantly enhanced. These cell lines will be used to determine the effect of altered SMRT expression on cell proliferation and/or cell death. Collectively, the proposed experiments will provide detailed information on processes that are critical for breast tumorigenesis. At the end of this study we expect to have a clearer understanding of corepressor action in breast cancer. Corepressors like SMRT/NCoR are known to be a part of the repressive machinery that is utilized by antiestrogen-bound ERs to prevent or treat breast cancer. Several recent reports indicating that SMRT/NCoR levels are increased in intraductal carcinoma and also in antiestrogen-resistant breast cancer cells, together with our preliminary results demonstrating that SMRT is required for full ER activity as well as cell proliferation indicates an additional and unexpected function of SMRT. This study will unravel these unique SMRT functions which in the future could be exploited as potential therapeutic targets for breast cancer treatment.