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2002 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 from 1995 to 1997 in the laboratory of Joan Brugge at ARIAD Pharmaceuticals, Cambridge, Massachusetts, and from 1997 to 2000 in the Department of Cell Biology at Harvard Medical School. 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. Staff Suganthi Chinnaswamy, Ph.D. Andrew Putnam, Ph.D. Veronique Schultz Patacsil, B.S. Laboratory Members Students Heather Bill, B.S. Andrea Pearson Research Projects Our laboratory is interested in understanding the mechanisms by which integrin receptors interacting with the extracellular matrix regulate cell function in normal and tumorigenic processes. Alterations in integrin receptors and their downstream signaling targets are common events in tumorigenesis, leading to a disruption of normal cell function. Using tissueculture models, biochemistry, molecular genetics, and ultimately mouse models, we are defining the signaling pathways and molecular events involved in integrin-dependent adhesion and migration that are important for tumorigenesis in general and specifically for melanoma and prostate cancer. Role of integrins in tumorigenesis Integrins are a class of heterodimeric transmembrane receptors for which there are currently 24 known family members: 15 alpha and 9 beta subunits. Each subunit contains a short cytoplasmic region with no known enzymatic activity, but through protein-protein interactions, subunits are able to interact with actin-containing microfilaments and important signaling molecules. Thus, the engagement of the integrin receptor by extracellular matrix components induces changes in actin structures, as well as the induction of several signal transduction pathways. Both the loss and gain of different integrins contribute to tumorigenesis and metastasis in many tumor types. In addition to changes in integrin expression, other contributors to tumorigenesis are alterations in integrin ligands, altered regulation of integrin function, or alterations in integrin-dependent signal transduction pathways. How integrins activate growth factor receptors Recent work in our laboratory has focused on characterizing the interactions between integrins and receptor tyrosine kinase. Adhesion of epithelial cells to several different extracellular matrices induces ligand-independent activation of the epidermal growth factor receptor (EGFR) and the Met receptor. Overexpression or mutation of EGFR family members or the Met receptor are common events in many epithelial tumors. We have shown that by recruiting EGFR, integrins are able to activate a subset of integrin-induced signaling pathways (Figure 1). In the absence of EGFR activation, the ability of the cells to induce the Ras/Erk signaling pathway and Akt is severely impaired. However, not all Figure 1. Integrin-induced activation of EGFR is required for a subset of integrin-regulated signaling pathways 33

integrin signaling pathways are dependent on EGFR (e.g., FAK, Src, and PKC). We further have demonstrated that integrinmediated adhesion of epithelial cells, including primary prostate epithelial cells, is sufficient to induce several G1 cell cycle events, including increases in cyclin D1, p21, cdk4 kinase activity, and Rb phosphorylation. This is dependent on integrin activation of EGFR, Erk, and PI-3K (Figure 2). However, adhesion alone was not Figure 2. Integrin-induced activation of EGFR, and subsequently Erk and PI-3K, is required for a entry into G1 of the cell cycle, but is not sufficient for entry into S phase sufficient for induction of DNA synthesis, indicating that additional signals are required. We are currently attempting to define what steps in G1 are blocked. Interestingly, HGF-mediated induction of DNA synthesis through the Met receptor was also dependent on integrin activation of EGFR. These data indicate that integrin regulation of EGFR activation is a critical mediator of cell cycle regulation. Integrin-mediated regulation of EGFR may be one mechanism that tumor cells use to regulate cell growth in the absence of exogenous growth factor. We are also exploring the mechanisms by which integrins activate EGFR and how integrins cooperate with the Met receptor to regulate cellular signaling. Integrin signaling in prostate cancer The development of metastatic prostate cancer is slow and is accompanied by the loss of androgen sensitivity. In normal epithelial cells, the α6 integrin is usually found in association with β4 integrin (α6β4) and is specifically localized to desmosomal junctions. However, in prostate carcinoma, β4 integrins are often lost, and there is a concomitant increase in the α6β1 and α3b1 integrins and a loss of typical epithelial structures. We are interested in understanding how α6β1 and α3β1 integrins contribute to latestage prostate carcinoma and how androgen may regulate this process. The integrins α6β1 and α3β1 are known interact with an integrin-associated protein called CD82 (or alternatively, KAI1). Loss of expression of CD82 correlates with prostate metastasis, and the loss of CD82 would be predicted to alter the function of α6β1 and α3β1 integrins. Using primary prostate epithelial cells, which express high levels of CD82, as well as several prostate tumor cell lines that do not, we are exploring the role of CD82 in regulating α6β1- and α3β1- mediated cell adhesion, migration, and cell signaling. We are using molecular genetic approaches such as mutagenesis, siRNA, and mouse models to alter CD82 expression in prostate cells. Integrin regulation of melanoma progression The incidence of melanoma has been steadily increasing in the last 10 years. If caught at an early stage it is usually curable, but once it has become invasive, metastatic melanoma is virtually untreatable and progresses very rapidly. Induced expression of the αvβ3 integrin correlates with increased invasive capacity of melanomas, yet the mechanisms underlying this shift in expression and increased invasiveness are unknown. We have initiated studies to determine how expression of the αvβ3 integrin in normal melanocytes alters cell function and the integrindependent signaling pathways involved. The serine/threonine protein kinase family, PKC, is a family of 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 of these kinases is largely unknown. In normal melanocytes, PKC is required for cell growth and survival; in tumor cells, however, 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 expression of different PKC isoforms can regulate melanoma proliferation, migration, and invasion. 34

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