Defining the role and regulation of the RhoGEF Ect2 in ovarian cancer cells

描述(申请人提供)：卵巢癌是妇科癌症中最致命的一种，迫切需要更好地了解这种恶性肿瘤的发生机制。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，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家族的鸟嘌呤核苷酸交换因子，能激活Rho家族的GTP酶。Rho GTP酶作为信号节点调节许多对癌症表型很重要的属性，其中几个(RhoA/C、rac1、CDc42)在卵巢癌中过度活跃。我推测，这种过度的活动至少有一部分是由于ect2的异常活动。究竟哪些Rho GTP酶被ECC2激活是有争议的，控制特异性的机制还没有确定，但体外研究涉及到磷酸化。ECT2也是一个不寻常的Rho家族全环基金，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ECC2)可能激活了GTP酶的核池。另一方面，缺乏核定位信号的ect2的截短序列正在发生转化。因此，ECT2从细胞核到细胞质的错误定位可能是Rho GTP酶激活和细胞转化所必需的。ECT2激活Rho家族GTP酶的亚细胞定位从未被直接研究过。我假设ECT2通过激活Rho家族GTP酶对卵巢上皮性肿瘤的一种亚型的恶性变是必需的，并且它被丝氨酸/苏氨酸激酶磷酸化改变了它的亚细胞定位和底物。我提出了三个具体目标来检验我的假设。1)通过比较ect2基因敲除细胞和对照细胞的锚定非依赖性生长、迁移、侵袭和细胞增殖，进一步确定ect2转化的必要性。我还将使用我验证的一种新的Ect2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Ect2的表达/定位，并将这些数据与患者的预后相关联。2)为了了解ect2的相关下游靶点，我将对表达或缺失ect2的细胞进行GTP酶激活的下拉实验。我将使用FRET生物传感器和/或荧光 标记效应器，以确定ECT2激活在哪里导致信号传递。3)为了开始解开调控ect2的上游途径，我在两个残基上制造了可能对ect2的定位和功能重要的拟磷和缺磷突变体。我将研究这些突变体的亚细胞定位，并测试它们挽救Ect2基因敲除细胞中GTP酶活性的能力。这些研究将调查在卵巢癌中被忽视的癌基因的细胞功能，并扩大我们对Rho GTP酶调控的知识。如果像我预测的那样，ECT2依赖的转化是由激酶调节的，那么这些激酶可能是卵巢癌的可用药靶点。

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.