Role of p190RhoGEF (Rgnef) in cell motility and tumor progression

Summary

Principal Investigator: Nichol L G Miller
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2011-07-01 - 2014-06-30
more information: NIH RePORT

Top Publications

  1. pmc Rgnef (p190RhoGEF) knockout inhibits RhoA activity, focal adhesion establishment, and cell motility downstream of integrins
    Nichol L G Miller
    Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 7:e37830. 2012
  2. pmc Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression
    Ssang Taek Lim
    Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, La Jolla, CA 92093, USA
    J Cell Biol 197:907-19. 2012
  3. pmc Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression
    Kristy K Ward
    Department of Reproductive Medicine, Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093, USA
    Clin Exp Metastasis 30:579-94. 2013
  4. pmc A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation
    Nichol L G Miller
    Moores UCSD Cancer Center, La Jolla, CA 92093, USA
    J Cell Sci 126:5074-85. 2013
  5. pmc Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function
    Christine Jean
    Department of Reproductive Medicine and 2 Department of Pathology, Moores University of California, San Diego Cancer Center, La Jolla, CA 92093
    J Cell Biol 204:247-63. 2014
  6. pmc RhoGEFs in cell motility: novel links between Rgnef and focal adhesion kinase
    N L G Miller
    University of California San Diego, Moores Cancer Center, Department of Reproductive Medicine, MC 0803, 3855 Health Sciences Dr, La Jolla, CA 92093 USA
    Curr Mol Med 14:221-34. 2014

Research Grants

  1. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
  2. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
  3. Digitalis-Induced Signaling by Cardiac Na+/K+-ATPase
    Amir Askari; Fiscal Year: 2013
  4. Role of 11q23 Chromosome Abnormalities in the Causation of Acute Leukemia
    Carlo M Croce; Fiscal Year: 2013
  5. CSHL CANCER CENTER SUPPORT GRANT
    BRUCE W STILLMAN; Fiscal Year: 2013

Detail Information

Publications6

  1. pmc Rgnef (p190RhoGEF) knockout inhibits RhoA activity, focal adhesion establishment, and cell motility downstream of integrins
    Nichol L G Miller
    Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 7:e37830. 2012
    ..Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility...
  2. pmc Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression
    Ssang Taek Lim
    Department of Reproductive Medicine, University of California San Diego, Moores Cancer Center, La Jolla, CA 92093, USA
    J Cell Biol 197:907-19. 2012
    ..These studies reveal new developmental and anti-inflammatory roles for kinase-inhibited FAK in limiting VCAM-1 production via nuclear localization and promotion of GATA4 turnover...
  3. pmc Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression
    Kristy K Ward
    Department of Reproductive Medicine, Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093, USA
    Clin Exp Metastasis 30:579-94. 2013
    ..These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer...
  4. pmc A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation
    Nichol L G Miller
    Moores UCSD Cancer Center, La Jolla, CA 92093, USA
    J Cell Sci 126:5074-85. 2013
    ..These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner...
  5. pmc Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function
    Christine Jean
    Department of Reproductive Medicine and 2 Department of Pathology, Moores University of California, San Diego Cancer Center, La Jolla, CA 92093
    J Cell Biol 204:247-63. 2014
    ....
  6. pmc RhoGEFs in cell motility: novel links between Rgnef and focal adhesion kinase
    N L G Miller
    University of California San Diego, Moores Cancer Center, Department of Reproductive Medicine, MC 0803, 3855 Health Sciences Dr, La Jolla, CA 92093 USA
    Curr Mol Med 14:221-34. 2014
    ..As revealed by ongoing Rgnef-FAK investigations, exploring GEF roles in cancer will yield fundamental new information on the molecular mechanisms promoting tumor spread and metastasis. ..

Research Grants30

  1. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
    ....
  2. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  3. Digitalis-Induced Signaling by Cardiac Na+/K+-ATPase
    Amir Askari; Fiscal Year: 2013
    ..abstract_text> ..
  4. Role of 11q23 Chromosome Abnormalities in the Causation of Acute Leukemia
    Carlo M Croce; Fiscal Year: 2013
    ..abstract_text> ..
  5. CSHL CANCER CENTER SUPPORT GRANT
    BRUCE W STILLMAN; Fiscal Year: 2013
    ....