The elucidation of mechanisms driving cell movement is critical to our understanding of normal development and pathological processes such as tumor invasion. Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions, sites of matrix-integrin attachment, tension generation, and cell survival signals. A complex of kinases and structural proteins are localized to adhesions and this proposal will focus on the molecular interactions of focal adhesion kinase (FAK) and a guanine nucleotide exchange factor (GEF) for Rho-family GTPases termed ARHGEF28 (alternative names are p190RhoGEF or Rgnef). GEFs are proteins that activate Rho-family GTPases and function in the regulation of focal adhesion formation and turnover. Canonically, GEFs are thought to function far downstream of matrix-integrin activating signals. An exception to this model is Rgnef, which binds directly to FAK and localizes to focal adhesions. In the previous funding period, we showed that blocking the interaction between Rgnef and FAK via a dominant-negative approach inhibited colon carcinoma motility, matrix degradation, and tumor progression. We also created an Rgnef knockout mouse and analyses of Rgnef-/- fibroblasts established the importance of Rgnef in RhoA regulation, focal adhesion formation, and cell migration downstream of integrins. Here we extend these findings by Rgnef-/- reconstitution studies and show that Rgnef phosphoinositide lipid binding as an adaptor protein (GEF independent) is required for early FAK recruitment and activation at focal adhesions. We hypothesize that subsequently, within an Rgnef-FAK complex, FAK-mediated tyrosine phosphorylation leads to Rgnef activation and promotes RhoA/C GTPase activation (GEF dependent). These events will be tested as drivers of ovarian carcinoma tumor progression as Rgnef and FAK expression are elevated as a function of tumor stage and high FAK levels in serous ovarian cancer are associated with decreased overall patient survival. We will test whether the formation of an Rgnef-FAK signaling complex promotes FAK activation independently, or in conjunction with downstream RhoA/C GTPase activation pushing ovarian carcinoma cells toward an epithelial to mesenchymal transition and invasive phenotype. Our proposed experiments will combine molecular and mechanistic signaling studies in cell culture with mouse tumor models of ovarian cancer.
Aim -1 will identify phosphorylation sites and domains of Rgnef that contribute to FAK activation, connections to Rho GTPases, cell motility, and an invasive cell phenotype through gain-of-function cell reconstitution assays using Rgnef-/- fibroblasts and ovarian tumor cells.
Aim -2 will expand the analysis of Rgnef and FAK in human tumor samples and will test the role of Rgnef in mouse ovarian orthotopic and genetic tumor models. This multi-faceted approach will yield a comprehensive understanding of Rgnef-FAK signaling axis within fibroblasts and ovarian cancer cells and provide new insights into pathways driving disease progression.
Signaling Connections Controlling Cell Motility and Invasion Project Narrative Ovarian cancer is a leading cause of cancer deaths among women and despite chemotherapy advances, the five year survival rate is less than 50% due to tumor recurrence and spread. Knowledge gained from these molecular studies will yield important information on drug- targetable intracellular signaling pathways driving motility, invasion, and tumor progression.
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|MasiÃ -BalaguÃ©, Miriam; Izquierdo, Ismael; Garrido, Georgina et al. (2015) Gastrin-stimulated GÎ±13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells. J Biol Chem 290:15197-209|
|Tancioni, Isabelle; Miller, Nichol L G; Uryu, Sean et al. (2015) FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth. Breast Cancer Res 17:47|
|Jean, Christine; Chen, Xiao Lei; Nam, Ju-Ock et al. (2014) Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function. J Cell Biol 204:247-63|
|Sulzmaier, Florian J; Jean, Christine; Schlaepfer, David D (2014) FAK in cancer: mechanistic findings and clinical applications. Nat Rev Cancer 14:598-610|
|Miller, N L G; Kleinschmidt, E G; Schlaepfer, D D (2014) RhoGEFs in cell motility: novel links between Rgnef and focal adhesion kinase. Curr Mol Med 14:221-34|
|Shah, Nina R; Tancioni, Isabelle; Ward, Kristy K et al. (2014) Analyses of merlin/NF2 connection to FAK inhibitor responsiveness in serous ovarian cancer. Gynecol Oncol 134:104-11|
|Tancioni, Isabelle; Uryu, Sean; Sulzmaier, Florian J et al. (2014) FAK Inhibition disrupts a Î²5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth. Mol Cancer Ther 13:2050-61|
|Lawson, Christine; Schlaepfer, David D (2013) pHocal adhesion kinase regulation is on a FERM foundation. J Cell Biol 202:833-6|
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