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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA180769-06
Application #
8692720
Study Section
Intercellular Interactions (ICI)
Program Officer
Ault, Grace S
Project Start
2009-06-01
Project End
2018-04-30
Budget Start
2014-05-29
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$303,562
Indirect Cost
$107,716
Name
University of California San Diego
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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
Miller, Nichol L G; Lawson, Christine; Kleinschmidt, Elizabeth G et al. (2013) A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation. J Cell Sci 126:5074-85
Lawson, Christine; Schlaepfer, David D (2013) pHocal adhesion kinase regulation is on a FERM foundation. J Cell Biol 202:833-6