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 pre-metastatic 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/1521 integrin complexes, integrin activation and increased fibronectin matrix. During the next funding period, we intend to dissect the molecular mechanisms which control this pathway in fibroblasts, osteosarcoma cells and MDA-MB-231 breast cancer cells. Experiments in Aim 1 will identify the mechanism by which uPAR regulates Src and EGFR activation. A tumor-fibroblast co-culture system will be used to evaluate the contribution of uPAR expression levels as well as intercellular uPAR/integrin complex formation in the regulation of Src-mediated EGFR transactivation. The roles of uPAR co-receptors in the activation of Src/EGFR (integrins, growth factor receptors 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 1521 integrin. Experiments will evaluate whether uPAR-mediated EGFR transactivation reroutes the trafficking of EGFR from lysosomes and into focal adhesions where it forms complexes with the 1521 integrin. EGFR dimerization, ubiquitination, sequestration and degradation in response to uPAR or EGFR ligands will be analyzed by Western blot, FLOW cytometry and confocal microscopy. The role of EGFR kinase activity and phosphorylated residues in the regulation of complex formation between EGFR and 1521 will be evaluated in fibroblasts using dominant/negative EGFR constructs and by expressing EGFR mutated in specific tyrosine residues in EGFR null cells. 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.
Fibronectin matrix in the tumor microenvironment is thought to contribute to cancer progression by promoting tumor metastasis and acquired resistance of tumors to chemotherapeutics. During the past funding period, we discovered a novel signaling pathway for fibronectin assembly which is initiated by the receptor for plasminogen activator (uPAR). This receptor is highly expressed in many tumors and is a negative prognostic indicator. Our studies, which are designed to understand the molecular basis underlying the uPAR regulation of fibronectin deposition, should provide novel targets for therapies directed at the control of both tumor metastasis and acquired resistance to chemotherapeutics.
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