Defining the role and regulation of the RhoGEF Ect2 in ovarian cancer cells
定义 RhoGEF Ect2 在卵巢癌细胞中的作用和调节
基本信息
- 批准号:8256342
- 负责人:
- 金额:$ 2.94万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2012
- 资助国家:美国
- 起止时间:2012-03-16 至 2014-03-15
- 项目状态:已结题
- 来源:
- 关键词:Anchorage-Independent GrowthAntibodiesBiological AssayBiosensorC-terminalCancer cell lineCell LineCell NucleusCell ProliferationCell membraneCell physiologyCellsChromosomal DuplicationCollaborationsCytoplasmCytoskeletal ModelingDataDependenceEpithelial CellsFamilyFibroblastsFluorescence Resonance Energy TransferGuanine Nucleotide Exchange FactorsGuanosine Triphosphate PhosphohydrolasesImmunofluorescence ImmunologicIn VitroInterphaseKnowledgeLabelLocationMalignant Female Reproductive System NeoplasmMalignant NeoplasmsMalignant neoplasm of lungMalignant neoplasm of ovaryMatrigel Invasion AssayMeasuresMethodsMonitorNuclearNuclear Localization SignalOncogenesOutcomeOutputOvarianOvarian Surface Epithelial-Stromal TumorPathologistPathway interactionsPatientsPhenotypePhosphorylationPhosphorylation SitePhosphotransferasesPlasmaPropertyProtein KinaseProtein-Serine-Threonine KinasesRegulationRoleSerineSignal TransductionSiteSoft Agar AssaySpecificityStagingStaining methodStainsSubstrate SpecificitySurvival AnalysisTestingThreonineTissue MicroarrayTumor-Derivedbasecancer cellcancer therapycell motilityinhibitor/antagonistknock-downmetaplastic cell transformationmigrationmutantnoveloutcome forecastovarian neoplasmoverexpressionrhorho GTP-Binding Proteinssmall hairpin RNAtumor
项目摘要
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.
描述(由申请人提供):卵巢癌是妇科癌症中最致命的,迫切需要更好地了解驱动这种恶性肿瘤的机制。癌基因ECT2(上皮细胞转化序列2)足以驱动成纤维细胞转化,在多种肿瘤中过度表达并与不良预后相关,并且位于卵巢癌中最常见的扩增子上。然而,Ect2在卵巢癌中的作用尚未被研究。Ect2是一种Dbl家族鸟嘌呤核苷酸交换因子(GEF),可激活Rho家族gtpase。Rho gtpase作为信号节点调节许多对癌症表型重要的特性,其中一些(RhoA/C, Rac1, Cdc42)在卵巢癌中过度活跃。我假设至少有一部分过度活动是由于异常的ec2活动造成的。确切地说,哪些Rho gtpase被Ect2激活是有争议的,控制特异性的机制尚未确定,但体外研究暗示磷酸化。Ect2也是一个不寻常的Rho家族GEF,在间期定位于细胞核(图1)。Dogma认为Rho家族GTPase在质膜上被激活,然而一些Rho GTPase定位于细胞核的证据表明,罕见的核gef(如Ect2)可能激活了GTPase的核池。另一方面,缺少核定位信号的Ect2的截断序列正在发生转化。因此,Ect2从细胞核到细胞质的错误定位可能是Rho GTPase激活和细胞转化所必需的。Ect2激活Rho家族GTPases的亚细胞定位从未被直接检测过。我假设通过激活Rho家族gtpase, Ect2对于卵巢上皮肿瘤的一个亚群的恶性肿瘤是必要的,并且丝氨酸/苏氨酸激酶对其磷酸化改变了其亚细胞定位和底物。我提出三个具体目标来检验我的假设。1)我将通过比较Ect2敲低细胞和对照细胞的非锚定生长、迁移、侵袭和细胞增殖,进一步确定Ect2在转化中的必要性。我还将使用一种我验证过的新型Ect2抗体,通过对400多个卵巢肿瘤进行组织微阵列染色来检测Ect2的表达/定位,并将这些数据与患者的预后联系起来。2)为了了解Ect2的相关下游靶点,我将在表达或缺乏Ect2的细胞中进行GTPase激活的下拉实验。我将使用FRET生物传感器和/或荧光
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Lauren Parker Huff其他文献
Lauren Parker Huff的其他文献
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{{ truncateString('Lauren Parker Huff', 18)}}的其他基金
Defining the role and regulation of the RhoGEF Ect2 in ovarian cancer cells
定义 RhoGEF Ect2 在卵巢癌细胞中的作用和调节
- 批准号:
8465752 - 财政年份:2012
- 资助金额:
$ 2.94万 - 项目类别:
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