The long-term goal of this application is to investigate the role of maspin in prostate cancer. Maspin is a novel serine protease inhibitor (serpin), initially identified in human mammary epithelial cells (Mammary serpin). Maspin has been shown to have a tumor suppressive activity in breast cancer, acting at the level of invasion and metastasis. Recent functional evidence demonstrates that maspin inhibits prostate tumor invasion in vitro, and metastasis in nude mice. These data suggest a potential application of maspin in prostate cancer intervention. However, the development of maspin-based anti-invasive strategies is hampered by the lack of understanding of its molecular mechanism. A central issue concerns whether maspin inhibits tumor invasion by blocking the pericellular proteolysis since maspin has a novel reactive site loop (RSL) sequence and does not inhibit soluble serine proteases. Our preliminary evidence demonstrates that maspin is down-regulated in human prostate carcinoma tissues as well as in established human prostate carcinoma cell lines. Functional studies demonstrate a critical role of the RSL of maspin in its inhibition of prostate tumor cell detachment, motility and invasion in vitro. Furthermore, in vitro evidence shows that maspin is a potent inhibitor of cell surface-bound uPA. Cellular distribution studies revealed a specific co-localization of maspin with cell surface. Upon detachment, both uPA and maspin are internalized. These findings suggest that maspin may regulate the availability of uPA on the cell surface and thus controls pericellular proteolysis. Based on these preliminary results, we propose that maspin exerts its anti-invasive effect by inhibiting the cell surface uPA-mediated detachment. To test this hypothesis, we plan to focus on the following three specific aims: 1) To investigate the molecular mechanism by which maspin inhibits cell surface-associated uPA (Hypothesis: Maspin inhibits the uPAR-bound uPA); 2) To investigate the role of maspin in uPA-dependent cell detachment (Hypothesis: Maspin inhibits prostate tumor cell detachment by inhibiting the multifunctional uPA complex on the cell surface); 3) To establish the structural basis of the anti-invasive activity of maspin (Hypothesis: Three domains of maspin implicated by biological evidence coordinate the novel cell membrane-dependent anti-invasive activity of maspin). These studies will address a new paradigm in the regulation of uPA cellular effect by the novel serpin maspin, and provide a better understanding for future drug intervention aimed at interfering with tumor invasion.
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