The assembly of the fibronectin matrix is a dynamic, cell dependent process which impacts on adhesion based signaling pathways important in the control of cell migration, growth and survival. There is now accumulating evidence that the fibronectin matrix plays a significant role in metastatic progression of certain tumors. The effects of fibronectin matrix on cancer progression are varied and include a role in the release of tumors from a dormant state, resistance of tumors to chemotherapeutics and the establishment of premetastatic niches required for seeding of metastases. During the previous funding period, we discovered a novel role for the urokinase-type plasminogen activator receptor (uPAR) in the regulation of fibronectin matrix assembly in connective tissue cells. uPAR ligation results in a Src-dependent transactivation of the epidermal growth factor receptor (EGFR) which results in the formation of EGFR/a5?1 integrin complexes, integrin activation and increased fibronectin matrix. During the next funding period, we intend to dissect the molecular mechanisms which control this pathway. Experiments in Aim 1 will identify the mechanism by which uPAR regulates Src and EGFR activation. The role of lipid rafts and caveolin in the uPAR activation of Src/EGFR will be assessed biochemically through the identification of raft associated uPAR containing complexes. The roles of uPAR co-receptors in the activation of Src/EGFR (integrins and FPRL1) will be addressed using a combination of pharmacological inhibitors, gene knockdowns and receptor down regulation. Mutational analysis of uPAR will be done to determine the domains of uPAR which are required , for Src/EGFR activation.
Aim 2 will define the mechanism by which EGFR transactivation activates the oc5p1 integrin. Experiments will test the hypothesis that uPAR mediated EGFR transactivation does not result in EGFR internalization, but in the lateral movement of EGFR to focal adhesions, where it forms complexes with the a5?1 integrin. EGFR sequestration in response to uPAR or EGF will be analyzed by FLOW cytometry and ELISA. Complex formation between EGFR and a5?1 will be evaluated by coimmunoprecipitation and FRET. A role for EGFR/a5?1 complex formation in integrin activation and matrix assembly will be demonstrated using dominant/negative EGFR. These experiments will provide novel information on the nature of uPAR signaling complexes and their role in the regulation of fibronectin deposition. Therapies directed at controlling this pathway may be useful for the treatment of metastatic disease. a ?
