Project Funding Details


Title
Defining the role and regulation of the RhoGEF Ect2 in ovarian cancer cells
Alt. Award Code
1F31CA165844-01
Funding Organization
National Cancer Institute
Budget Dates
2012-03-16 to 2013-03-15
Principal Investigator
Parker, Lauren
Institution
University of North Carolina at Chapel Hill
Region
North America
Location
Chapel Hill, NC, US

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Technical Abstract

DESCRIPTION (provided by applicant): Ovarian cancer is the deadliest of gynecological cancers, and a better understanding of the mechanisms that drive this malignancy is urgently needed. The oncogene ECT2 (epithelial cell transforming sequence 2) is sufficient to drive fibroblast transformation, is overexpressed and associated with poor outcome in a variety of tumors, and is located on the most frequent amplicon in ovarian cancer. Yet, the role of Ect2 in ovarian cancer has not been investigated. Ect2 is a Dbl family guanine nucleotide exchange factor (GEF) that activates Rho family GTPases. Rho GTPases act as signaling nodes to regulate many properties important for cancer phenotypes, and several (RhoA/C, Rac1, Cdc42) are overactive in ovarian cancer. I hypothesize that at least some of this excessive activity is due to aberrant Ect2 activity. Exactly which Rho GTPases are activated by Ect2 is controversial, and the mechanisms controlling specificity have not been defined but in vitro studies implicate phosphorylation. Ect2 is also an unusual Rho family GEF, localized to the nucleus during interphase (Fig. 1). Dogma states that Rho family GTPases are activated at the plasma membrane, yet evidence of some Rho GTPase localization to the nucleus suggests that the rare nuclear GEFs (such as Ect2) may be activating nuclear pools of GTPases. On the other hand, truncated sequences of Ect2 that lack their nuclear localization signals are transforming. Thus, mislocalization of Ect2 from the nucleus to the cytoplasm may be necessary for Rho GTPase activation and cellular transformation. The subcellular localization of where Ect2 activates Rho family GTPases has never been directly examined. I hypothesize that Ect2 is necessary for malignancy of a subset of epithelial ovarian tumors through activation of Rho family GTPases, and that its phosphorylation by serine/threonine kinases alters its subcellular localization and substrates. I propose three specific aims to test my hypotheses. 1) I will further determine the necessity of Ect2 for transformation by comparing anchorage- independent growth, migration, invasion, and cell proliferation in Ect2 knockdown and control cells. I will also examine Ect2 expression/localization by staining a tissue microarray with over 400 ovarian tumors, using a novel Ect2 antibody I validated, and I will correlate this data with patient outcome. 2) To understand the relevant downstream targets of Ect2, I will perform pulldown assays for GTPase activation in cells expressing or lacking Ect2. I will utilize FRET biosensors and/or fluorescently labeled effectors to determine where Ect2 activation is causing signaling. 3) To start to unravel upstream pathways that regulate Ect2, I have made phosphomimetic and phosphodeficient mutants at two residues that may be important for Ect2 localization and function. I will look at subcellular localization of these mutants and test their ability to rescue GTPase activity in Ect2 knockdown cells. These studies will investigate the cellular functions of an oncogene overlooked in ovarian cancer and expand our knowledge of Rho GTPase regulation. If Ect2-dependent transformation is regulated by kinases as I predict, then these kinases may be druggable targets for ovarian cancer.

Cancer Types

  • Ovarian Cancer

Common Scientific Outline (CSO) Research Areas

  • 1.3 Biology Cancer Initiation: Oncogenes and Tumor Suppressor Genes