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2005 Scientific Report

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During prostate cancer

During prostate cancer progression there is a shift in the expression of laminin-specific integrins: β4 integrins are lost, and there is a concomitant increase in α6β1 and α3β1, which both interact with CD82/KAI1. We predict that the loss of CD82/KAI1 alters the function of α6β1 and α3β1. Using primary prostate epithelial cells, which express high levels of CD82/KAI1, as well as several prostate tumor cell lines that do not, we are exploring the role of CD82/KAI1 in regulating α6β1- and α3β1-mediated cell adhesion, migration, and integrin signaling. We have found that overexpression of CD82/KAI1 in tumor cells suppresses laminin-specific migration and invasion; integrin-induced EGFR and c-Met receptor activation; Src and Lyn activation; and activation of the Src/Lyn substrates Cas and FAK. Furthermore, integrin activation of Src is dependent on c-Met, and laminin-mediated invasion depends on both Src and c-Met. Together these data indicate that CD82/KAI1 normally acts to regulate integrin signaling to c-Met such that upon its loss of expression in tumor cells, signaling through c-Met to Src is increased, leading to increased motility and invasion (Fig. 2). We are currently determining the mechanism by which CD82/KAI1 down-regulates c-Met signaling. In reciprocal experiments, we are inhibiting the expression of CD82/KAI1 in primary cells using siRNA and mouse models. Integrin regulation of melanoma progression by PKC The incidence of melanoma has been steadily increasing over the last 10 years. If diagnosed at an early stage, melanoma is curable, but once it has become invasive, it progresses very rapidly and is virtually untreatable. Metastasis and invasion by tumor cells require the activity of integrins, and in melanoma, expression of the αvβ3 integrin is induced during the development of invasive disease. Therefore, an understanding of how the αvβ3 integrin functions to regulate invasion will help our understanding of melanoma metastasis. We have been focusing our attention on two signaling molecules, PKCα and Src, both of which are regulated by the αvβ3 integrin and whose activities are enhanced in metastatic melanoma. Adhesion of normal melanocytes to extracellular matrix induces the formation of focal adhesion complexes and actin stress fibers. However, in a highly invasive metastatic melanoma cell line, C8161.9, these structures are absent. We have shown that the levels of Src activity and PKCα protein are elevated in these cells, and overexpression of PKCα in immortalized normal melanocytes is sufficient to confer an invasive phenotype in vitro. We have found that the activity of Rac, a small GTPase that is required for the formation of lamellipodia, is elevated in these cells and that inhibition of PKCα blocks Rac activity. The activity of the small GTPase Rho, which is involved in stress fiber and focal adhesion formation, is negatively regulated by active Src. Thus PKCα, acting to enhance Rac, and active Src, acting to inhibit Rho, together drive the formation of lamellipodia and the dissolution of stress fibers and focal adhesions, leading to increased motility (Fig. 3). We are currently exploring how Src is activated in melanoma cells, as well as the effects of blocking Src and PKCα expression with siRNA on migration and invasion. Figure 2. CD82 reexpression in prostate tumor cells inhibits invasion by blocking integrinmediated signaling to Met and Src. Figure 3. PKCa and Src cooperate to enhance migration and invasion in melanoma cells by differentially targeting Rac and Rho. 36

External Collaborators Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Recent Publications Sridhar, S.C., and C.K. Miranti. In press. Tumor metastasis suppressor KAI1/CD82 is a tetraspanin. In Contemporary Cancer Research: Metastasis, C. Rinker-Schaeffer, M. Sokoloff, and D. Yamada, eds. Totowa, N.J.: Humana Press. Bill, Heather M., Beatrice Knudsen, Sheri L. Moores, Senthil K. Muthuswamy, Vikram R. Rao, Joan S. Brugge, and Cindy K. Miranti. 2004. Epidermal growth factor receptor–dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells. Molecular and Cellular Biology 24(19): 8586–8599. Lee, Chong-Chou, Andrew J. Putnam, Cindy K. Miranti, Margaret Gustafson, Ling-Mei Wang, George F. Vande Woude, and Chong-Feng Gao. 2004. Overexpression of sprouty-2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23(30): 5193–5202. From left to right: Schulz, Edick, Miranti, Long, Freiter, Sridhar 37

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