Pancreatic cancer is a devastating disease with an exceptionally poor prognosis. Tumors of ductular origin comprise 95% of pancreatic neoplasms, have a strong tendency to invade into surrounding parenchyma, and metastasize to distal organs. Currently, the molecular mechanisms that support these critical processes are poorly defined. It is known that the actin cytoskeleton plays an important role and responds dynamically to stimulation by the epidermal growth factor receptor (EGF-R), which is significantly upregulated in many pancreatic tumors. This is a proposal to study the mechanisms by which reorganization of the actin cytoskeleton mediates loss of pancreatic ductular integrity and cell polarity leading to activation of cell migration, invasion, and metastasis. We have identified a key regulatory cytoskeletal complex in these cells comprised of a large mechanochemical GTPase called dynamin (Dyn2), the actin crosslinking protein cortactin, and the transforming kinase c-Src that is activated by the EGF-R and binds and phosphorylates both Dyn2 and cortactin. This is a resubmitted proposal that received a favorable first review. In response, we have conducted many new experiments to address the reviewers' helpful suggestions. These new data, and that presented in the original proposal, support the CENTRAL HYPOTHESIS of this study that the Dyn2-cortactin-c-Src complex is activated by EGF stimulation to mediate actin-plasma membrane dynamics in pancreatic ductular cells that promote invasion and metastasis. This proposal will test the participation of this protein complex toward the neoplastic process in 3 different human pancreatic tumor cell lines in vitro, using biochemical and microscopic approaches, and in vivo, via injection into nude mice. We will test the Hypotheses that: 1. The Dyn2-cortactin-c-Src complex mediates the EGF-stimulated disassembly of pancreatic ductular cell-cell-adherens junctions before metastasis. 2. This complex in pancreatic ductular cells is activated by the EGF-R to mediate the formation of unique dorsal """"""""wave"""""""" structures that specifically sequester receptor and ligand while reorganizing the actin cytoskeleton. 3. EGF stimulation of pancreatic ductular tumor cells activates the Dyn2-cortactin-c-Src complex leading to actin membrane reorganization, lamellipod extension, migration, and metastasis. This study will provide novel information toward understanding the molecular mechanisms that make pancreatic tumors aggressive, migratory, and lethal.
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