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
• 负责人: Lauren Parker Huff
• 金额: $ 2.39万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-16 至 2013-12-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465752
• 关键词: Anchorage-Independent Growth; Antibodies; Biological Assay; Biosensor; C-terminal; Cancer cell line; Cell Line; Cell Nucleus; Cell Proliferation; Cell membrane; Cell physiology; Cells; Chromosomal Duplication; Collaborations; Cytoplasm; Cytoskeletal Modeling; Data; Dependence; Epithelial Cells; Family; Fibroblasts; Fluorescence Resonance Energy Transfer; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Immunofluorescence Immunologic; In Vitro; Interphase; Knowledge; Label; Location; Malignant Female Reproductive System Neoplasm; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Matrigel Invasion Assay; Measures; Methods; Monitor; Nuclear; Nuclear Localization Signal; Oncogenes; Outcome; Output; Ovarian; Ovarian Surface Epithelial-Stromal Tumor; Pathologist; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Plasma; Property; Protein Kinase; Protein-Serine-Threonine Kinases; Regulation; Role; Serine; Signal Transduction; Site; Soft Agar Assay; Specificity; Staging; Staining method; Stains; Substrate Specificity; Survival Analysis; Testing; Threonine; Tissue Microarray; Tumor-Derived; base; cancer cell; cancer therapy; cell motility; inhibitor/antagonist; knock-down; metaplastic cell transformation; migration; mutant; novel; outcome forecast; ovarian neoplasm; overexpression; rho; rho GTP-Binding Proteins; small hairpin RNA; tumor

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）：卵巢癌是最致命的妇科癌症，迫切需要更好地了解这种恶性肿瘤的机制。癌基因ECT 2（上皮细胞转化序列2）足以驱动成纤维细胞转化，在多种肿瘤中过表达并与不良结局相关，并且位于卵巢癌中最常见的扩增子上。然而，Ect 2在卵巢癌中的作用尚未研究。Ect 2是激活Rho家族GTP酶的Dbl家族鸟嘌呤核苷酸交换因子（GEF）。Rho GTP酶作为信号节点调节许多对癌症表型重要的特性，并且几种（RhoA/C，Rac 1，Cdc 42）在卵巢癌中过度活跃。我推测，至少有一些这种过度的活动是由于异常的Ect 2活动。确切地说，哪些Rho GTP酶被Ect 2激活是有争议的，并且控制特异性的机制尚未确定，但体外研究涉及磷酸化。Ect 2也是一种不寻常的Rho家族GEF，在间期定位于细胞核（图1）。Dogma指出Rho家族GTP酶在质膜上被激活，但一些Rho GTP酶定位于细胞核的证据表明，罕见的核GEF（如Ect 2）可能激活GTP酶的核库。另一方面，缺乏核定位信号的Ect 2截短序列正在转化。因此，Ect 2从细胞核到细胞质的错误定位可能是Rho GT3激活和细胞转化所必需的。Ect 2激活Rho家族GTP酶的亚细胞定位从未被直接研究过。我假设Ect 2是必要的恶性上皮性卵巢肿瘤的一个子集，通过激活Rho家族GTP酶，其磷酸化丝氨酸/苏氨酸激酶改变其亚细胞定位和基板。我提出了三个具体目标来检验我的假设。1)我将通过比较Ect 2敲低和对照细胞中的锚定非依赖性生长、迁移、侵袭和细胞增殖来进一步确定Ect 2用于转化的必要性。我还将使用我验证过的新型Ect 2抗体，通过对400多个卵巢肿瘤的组织微阵列进行染色来检查Ect 2表达/定位，并将此数据与患者结果相关联。2)为了了解Ect 2的相关下游靶点，我将在表达或缺乏Ect 2的细胞中进行GTdR激活的下拉测定。我将利用FRET生物传感器和/或荧光 标记的效应物，以确定Ect 2激活引起信号传导的位置。3)为了开始解开调控Ect 2的上游途径，我在两个残基上制备了磷酸模拟物和磷酸缺陷突变体，这两个残基可能对Ect 2的定位和功能很重要。我将研究这些突变体的亚细胞定位，并测试它们在Ect 2敲低细胞中拯救GTdR活性的能力。这些研究将调查卵巢癌中被忽视的癌基因的细胞功能，并扩大我们对Rho GT3调节的了解。如果Ect 2依赖性转化如我预测的那样受激酶调节，那么这些激酶可能是卵巢癌的药物靶点。