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Estrogen and Progestin Regulation of Thrombospondin-1 in Breast Cancer Cells
Tumor Cell Biology I
Background: Angiogenesis, or formation of new blood vessels for tissue nourishment, is an important target for inhibiting tumor growth. Recent studies have identified a naturally occurring protein, thrombospondin-1 (TSP-1), as a useful agent for arresting tumor growth. However, the role of TSP-1 in breast tissue is not clear and there are reports that TSP-1 might actually be pro-angiogenic and pro-proliferative in breast cancer cells. We recently discovered that the proliferative hormones, estradiol (E) and progesterone (P) induce TSP-1 in breast cancer cells. Therefore, it is vital that the regulation and role of hormone induced TSP-1 in breast cancer be studied further. Objective/Hypothesis: It is our hypothesis that E and P directly control the transcription and release of TSP-1 in human breast cancer cells, and that TSP-1 increases the proliferation of breast cancer cells. Specific Aims : 1. a) Define the pattern of regulation of TSP-1 expression in human breast cancer cells by E, P, and anti-hormones, and b) Determine if TSP-1 induces proliferation of breast tumor epithelial and/or endothelial cells in vitro and in vivo. 2. Determine whether steroid receptor isoforms differentially regulate TSP-1 expression via the cis-regulatory elements in the TSP-1 gene. Study Design: Northern blot and real-time PCR will be used to quantify TSP-1 mRNA in breast cancer cells. Secretion of TSP-1 protein in media will be measured by ELISA. Bromodeoxyuridine incorporation will be employed to assess proliferation of cells in response to TSP-1 treatment. Reporter constructs will be used to identify cis-acting DNA regulatory elements in the TSP-1 promoter. Putative E and P receptor binding sites will be characterized by gel-shift analysis, and by chromatin immuno-precipitation studies. Potential Outcomes/Benefits of the Research: The proposed studies could potentially identify a novel proliferative role for TSP-1 in breast cancer, as well as novel roles for E and P receptors in regulating TSP-1. Since TSP-1 is present at higher levels in malignant and invasive human breast tumors, it is our contention that elevated levels of TSP-1 will likely lead to progression of the breast disease. Such information will be important in furthering our understanding of the etiology and progression of breast cancer as it relates to TSP-1 and, furthermore, in the design of future therapeutic strategies for controlling the progression of breast cancer.
Angiogenesis, the process by which new blood vessels are formed, is an essential component of tumor growth. Controlling angiogenesis is important for arresting the progression of cancer. Recently, many studies have focused on anti-angiogenic agents and their efficacy in retarding the growth of tumors. The naturally produced protein thrombospondin-1 (TSP-1) has received great attention, due to observations which suggest that it may inhibit angiogenesis, leading to arrest of tumor growth. However, many recent studies have shown that paradoxically, TSP-1 production by breast cancer cells is associated with tumor cell proliferation, rather than regression. Thus, unexpectedly TSP-1 seems to have a pro-angiogenic role in breast cancer progression. TSP-1 is a multi-functional protein which may play a role in breast tumor progression by acting as an adhesive agent, maintaining cells ‘glued’ in place as they multiply. We have shown that estrogens and progestins increase the production of TSP-1 in hormone-dependent human breast cancer cell lines via their respective receptors. Since both classes of sex-steroid are used in contraceptives and hormone replacement therapies, it is imperative to understand the role played by these agents in regulating levels of TSP-1. If TSP-1 production is under the control of E and P via their steroid receptors then one can envision immediate design of therapeutic strategies with specific hormone receptor antagonists (anti-hormones) or antagonists of TSP-1 to curtail tumor growth. We propose therefore, to study the effects of sex steroids and anti-hormones such as tamoxifen, faslodex and mifpristone, on the regulation of TSP-1 in breast cancer cells. Such an approach is essential to fully understand the basic molecular mechanisms by which TSP-1 regulates breast cancer progression. It is vital that we ascertain whether or not TSP-1 mediates estrogen and progestin mediated breast tumor progression, since women are exposed to high levels of sex steroids from puberty through menopause and many receive hormone supplements as oral contraceptives or hormone replacement therapy . The proposed studies will provide definitive answers to the question, should tumor TSP-1 levels be manipulated in order to arrest or eradicate breast cancer?. In addition, the information gained could lead to immediate design of therapeutic strategies to inhibit the biological activity of TSP-1, and thus to control the progression of breast cancer.