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2004 Scientific Report

D1, p21, cdk4 kinase

D1, p21, cdk4 kinase activity, and Rb phosphorylation. The induction of these cell cycle events is dependent on integrin activation of EGFR, Erk, and PI-3K. However, adhesion alone is not sufficient to induce DNA synthesis; while some G1 cell cycle events were activated by integrin-dependent EGFR activation, not all were. Adhesion failed to induce p27 degradation; c-myc, cdk2, or ATF-2 activation; or cyclin A synthesis. All are required for entry into S phase. Treatment of cells with EGF—the EGFR ligand—or overexpression of EGFR (as is observed in many tumors) was sufficient to restore these additional G1 events and induce DNA synthesis. These data indicate that integrin-mediated activation of EGFR is a critical regulator of the cell cycle. Furthermore, integrinmediated regulation of EGFR, especially in EGFR-overexpressing tumors, may be one mechanism by which tumor cells induce cell growth. We have also found that integrin-induced activation of EGFR in primary prostate epithelial cells is required for their survival in the absence of growth factors. Increased cell survival and resistance to cell death is often a prerequisite for tumorigenesis. Overexpression of growth factor receptors in tumor cells may help promote integrin-mediated cell survival. We are exploring the mechanism by which EGFR mediates integrindependent survival of prostate cells. We are also exploring the mechanisms by which integrins activate EGFR and ErbB2. Integrin activation of EGFR and ErbB2 is ligand independent, requires only the cytoplasmic domain of EGFR, and stimulates the phosphorylation of only a subset of sites on EGFR. Erb2 activation is dependent on EGFR, suggesting that integrins induce the formation of an EGFR/ErbB2 heterodimer. Integrin activation of Src or FAK is not required, but integrin activation of a phosphatase may be involved. Integrin signaling in prostate cancer Prostate cancer is the second leading cause of cancer death in U.S. men. Prostate cancer deaths are due to the development of metastatic disease, which is difficult to control. 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. α6β1 and α3β1 both interact with an integrin-associated protein called CD82 or KAI1. CD82/KAI1 is a metastasis suppressor gene; loss of its expression correlates with prostate cancer metastasis. 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 in tumor cells suppresses laminin-specific migration (Fig. 2) and invasion; integrin-induced EGFR and Met receptor activation; and Src and Lyn activation. We are currently determining how EGFR, Met, Src, or Lyn contributes to integrin-mediated migration and invasion. In reciprocal experiments, we are inhibiting the expression of CD82 in primary cells using siRNA and mouse models. Figure 2. CD82 reexpression in tumor cells inhibits cell migration on laminin. Migration of CD82-expressing tumor cells was completely inhibited when cells were attached to laminin. Migration on collagen was partially inhibited. Integrin regulation of melanoma progression via PKC The incidence of melanoma has been steadily increasing in the last 10 years. If diagnosed at an early stage it is usually curable, but once it has become invasive, metastatic melanoma is virtually untreatable and progresses very rapidly. Metastasis and invasion by tumor cells require the activity of integrins. Therefore, an understanding of how integrins mediate metastasis and 36

invasion will help our understanding of melanoma progression. The serine/threonine protein kinase family, PKC, comprises 11 related kinases that can be separated into three major classes: classical, novel, and atypical. This kinase family has been implicated in differentiation, growth regulation, cell survival, cell adhesion, cell migration, and tumorigenesis, but the exact role of each kinase in the various cell functions is largely unknown. In normal melanocytes, PKC is required for cell growth and survival, while in tumor cells, stimulation of PKC activity can result in growth arrest and cell death. In addition, PKC plays an important role in regulating cell adhesion and migration. We are interested in understanding how changes in the expression of different PKC isoforms can regulate melanoma proliferation, migration, and invasion. Adhesion of normal melanocytes to the extracellular matrix induces the formation of focal adhesion complexes and actin stress fibers. However, in a highly invasive, metastatic melanoma cell line, these structures are absent. The levels of PKCα are elevated in these cells. We have found that the activity of Rac, a small GTPase that regulates actin structure, is elevated and that inhibition of PKC blocks Rac activity. Overexpression of PKCα in immortalized normal melanocytes is sufficient to confer an invasive phenotype in vitro. We are exploring the blocking of PKCα expression with siRNA and the resulting effects on actin structures, migration, and invasion. External Collaborators Joan Brugge, Harvard Medical School, Boston, Massachusetts Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Senthil Muthuswamy, Cold Spring Harbor Laboratory, New York Recent Publications Lee, C.C., A.J. Putnam, C.K. Miranti, M. Gustafson, L.M. Wang, G.F. Vande Woude, and C.F. Gao. In press. Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration and cytokinesis. Oncogene. Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914–4916. Left to right: Chinnaswamy, Patacsil, Illian, Long, Miranti, Edick 37

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