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2005 Scientific Report

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Laboratory of Integrin

Laboratory of Integrin Signaling and Tumorigenesis Cindy K. Miranti, Ph.D. Dr. Miranti received her M.S. in microbiology from Colorado State University in 1982 and her Ph.D. in biochemistry from Harvard Medical School in 1995. She was a postdoctoral fellow in the laboratory of Dr. Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Mass., from 1995 to 1997 and in the Department of Cell Biology at Harvard Medical School from 1997 to 2000. Dr. Miranti joined VARI as a Scientific Investigator in January 2000. She is also an Adjunct Assistant Professor in the Department of Physiology at Michigan State University. Laboratory Members Staff Suganthi Chinnaswamy, Ph.D. Mathew Edick, Ph.D. Robert Long, B.A. Veronique V. Schulz, B.S. Student Erik Freiter Research Interests Our laboratory is interested in understanding the mechanisms by which integrin receptors, interacting with the extracellular matrix, regulate cell processes involved in the development of cancer. Using tissue culture models, biochemistry, molecular genetics, and mouse models, we are defining the cellular and molecular events of integrindependent adhesion and downstream signaling that are important in melanoma and prostate tumorigenesis and metastasis. Integrins are transmembrane proteins that serve as receptors for extracellular matrix (ECM) proteins. By interacting with the ECM, integrins stimulate intracellular signaling transduction pathways that regulate cell shape, proliferation, migration, survival, gene expression, and differentiation. Integrins do not act autonomously; they are involved in “crosstalk” with receptor tyrosine kinases (RTKs) to regulate many cellular processes. Studies in our lab, for example, indicate that integrin-mediated adhesion to ECM proteins activates the epidermal growth factor receptors EGFR and ErbB2 and the HGF/SF receptor Met. Integrin-mediated activation of these RTKs is ligand-independent and is required for activation of a subset of intracellular signaling molecules in response to cell adhesion. The prostate gland and cancer Tumors that develop in cells of epithelial origin, i.e., carcinomas, represent the largest tumor burden in the United States. Prostate cancer is the most frequently diagnosed cancer in U.S. men and the second leading cause of cancer death in men. Eighty percent of human prostate tumors arise in the peripheral zone of the gland and are primarily confined to the intermediate basal and secretory epithelial cells. Patients who at the time of diagnosis have androgen-dependent and organ-confined prostate cancer are relatively easy to cure through radical prostatectomy or localized radiotherapy. However, patients with aggressive and metastatic disease have fewer options. Androgen ablation can significantly reduce the tumor burden in these patients, but the potential for relapse and the development of androgen-independent cancer is high. Currently there are no effective treatments for patients who reach this stage of disease. In the human prostate secretory glands, basal epithelial cells form a contiguous layer adjacent to the basement membrane. Upon them rests a layer of secretory luminal cells, forming a stratified epithelium. The basal cells express a broad repertoire of integrins, including α2, α3, α6, β1, and β4. The secretory cells express primarily α6 and β1, with some α2. In vivo, basal cells secrete and organize a laminin 5–containing basement membrane that also contains collagens IV and VII and laminin 10. The basal cells bind to laminin 5 and collegen VII through α6β4 to form hemidesmosomal complexes at the basal surface. Basal cells adhering to this matrix respond to growth factors secreted by the surrounding stroma, including EGF and HGF. They proliferate and give rise to the nonproliferating secretory cells through 34

the generation of an intermediate, transiently amplifying cell population that has traits of both cell types. In primary prostate tumors, α6β4 integrin and its ligands, laminin 5 and collagen VII, are lost. The tumor cells, unlike normal secretory cells, develop the ability to adhere, via α2β1 and α6β1, to an altered basement membrane consisting of collagen IV and laminin 10. Thus, tumor cells have characteristics of both basal and secretory cells, but not all the properties of either. Whether the tumor cells are derived from the transient differentiating population of basal cells or from differentiated secretory cells has not been unequivocally determined, but it is clear that the way in which these cells interact with the ECM has been changed. If tumor cells are derived from basal cells, they are now interacting with an altered matrix and using different integrins to engage it. If they are derived from the secretory cells, the tumor cells are now engaging a matrix, which they did not do previously. A fundamental question in our lab is whether the changes in integrin/matrix interactions that occur in tumor cells are required for or help to drive the survival of tumor cells. The role of integrins and RTKs in prostate epithelial cell survival Increased cell survival due to resistance to cell death is a prerequisite for tumorigenesis. Several reports have suggested that the signaling pathways that regulate cell survival in normal prostate epithelial cells are different from those in prostate tumor cells. How integrin engagement of different ECMs regulates survival pathways is not known. We have recently found that integrin-induced activation of both EGFR and c-Met in primary prostate epithelial cells is required for cell survival in the absence of growth factors. Our goal is to determine how integrin activation of EGFR and c-Met regulate cell survival. We have previously shown that integrin activation of EGFR is required for integrinmediated induction of the Ras/Erk and PI3K/Akt signaling pathways. Recent studies indicate that integrin activation of Src depends on c-Met. Interestingly, inhibition of either Src or Ras/Erk signaling—but not PI3K/Akt signaling—induces cell death in primary prostate cells even when they are still adherent to matrix. Together these data indicate that Src signals generated by c-Met, as well as Ras/Erk signals generated by EGFR, are driving cell survival in primary cells (Fig. 1). We are currently exploring the downstream events that Src and Erk regulate to maintain cell survival on ECM. Figure 1. Met and EGFR both act independently to regulate integrin-mediated survival of primary prostate epithelial cells. Activation of Src and Erk through Met and EGFR, respectively, are proposed to be involved in integrin-mediated survival. We are also exploring the mechanisms by which integrins activate EGFR and ErbB2. This activation is ligand independent, requires only the cytoplasmic domain of EGFR, and stimulates the phosphorylation of only a subset of sites on EGFR. ErbB2 activation depends 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; we are currently investigating the role of several phosphatases. Integrin and RTK crosstalk in prostate cancer metastasis Death from prostate cancer is due to the development of metastatic disease, which is difficult to control. The mechanisms involved in progression to metastatic disease are not understood. Our approach is to characterize genes that are specifically associated with metastatic prostate cancer. CD82/KAI1 is a metastasis suppressor gene whose expression is specifically lost in metastatic cancer but not in primary tumors. CD82/KAI1 is known to associate with both integrins and RTKs. Our goal has been to determine how loss of CD82/KAI1 expression promotes metastasis by studying the role of CD82/KAI1 in integrin and RTK crosstalk. 35

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