Regulation of cell adhesion and actin cytoskeletal structures play important roles in normal and tumor cell movement. Rho GTPases, such as RhoA, are molecular switches that act to control cell adhesion, motility and have been implicated in tumor progression. The activation of Rho proteins is mediated by specific guanine- nucleotide exchange factors or RhoGEFs, which catalyze the exchange of GDP for GTP. As the number of RhoGEFs outnumber their target GTPases by a factor of 3, it is hypothesized that the spatial and temporal regulation of RhoGEF activity is a key factor in controlling RhoA function within cells. Rgnef is a 190 kDa RhoA-specific GEF (also termed p190RhoGEF) involved in the regulation of integrin-initiated RhoA activation, focal adhesion (FA) formation, and fibroblast cell motility. A novel feature of Rgnef is that it contains a binding site for the integrin-activated focal adhesion kinase (FAK). What remains unknown is how Rgnef is regulated within cells. This proposal will test the hypothesis that Rgnef (via FAK association and tyrosine phosphorylation) regulates RhoA activity, cell adhesion and motility, ultimately influencing ovarian carcinoma tumor progression. Experiments will use gain-of-function assays via reconstitution of mouse embryo fibroblasts (MEFs) obtained from Rgnef-null mice or rescue of knockdown phenotypes in human ovarian carcinoma cells.
Three aims are proposed:
Aim 1 will determine the necessity of Rgnef in FA formation and cell motility. Experiments will include RhoA activity assays, fluorescence microscopy monitoring of FA formation, and real-time imaging to evaluate Rgnef localization and the enhancement of cell motility.
Aim 2 will test the role of FAK binding and tyrosine phosphorylation in Rgnef function. Two sites within Rgnef have been identified by mass spectrometry as phosphorylated by FAK. These sites will be mutated and re-expression of these mutants in Rgnef-null MEFs will be evaluated for effects on intrinsic Rgnef activity, integrin-associated RhoA activation, FA formation, and cell motility.
Aim 3 will evaluate the role of Rgnef in human ovarian carcinoma tumor progression. Recent studies indicate that Rgnef expression is elevated as function of colon carcinoma tumor progression and cellular studies show that Rgnef-FAK interactions contribute to an invasive cell phenotype. Rgnef-specific antibody tissue array staining reveals enhanced expression in advanced stage ovarian carcinoma and preliminary data indicate that Rgnef knockdown inhibits ovarian carcinoma progression. Lentiviral-mediated Rgnef knockdown and re-expression of Rgnef mutants in two human ovarian carcinoma cell lines will be evaluated in cell culture and upon orthotopic tumor growth in nude mice for effects on FA formation-invasion and tumor progression, respectively. Together, these experiments will provide advanced training in cellular signaling, molecular mechanisms of cell motility, and cancer biology model systems. The results will enhance our current understanding of intracellular signaling pathways promoting ovarian carcinoma tumor progression.
Ovarian cancer is a leading cause of mortality in gynecological cancer and approximately 1 in 60 women are diagnosed in the United States annually. The research proposed in this application will analyze the role of Rgnef, a protein implicated in cellular adhesion and motility, both critical factors in tumor cell progression and metastasis. These projects ultimately aim to add to the current understanding of intracellular signaling pathways that contribute to ovarian carcinoma tumor progression.
