Elucidating the Molecular Mechanisms of Ect2 in cytokinesis and oncogenesis

描述（由申请人提供）：在过去的几年中，Ras，一种小的GT3癌基因一直是癌症研究的焦点，因为它是与所有人类癌症相关的最常见的突变基因之一（33%）。Ras是一个大的小GTP酶超家族的创始成员，最近的研究表明，Ras超家族蛋白，特别是Ras同源（Rho）GTP酶的异常活性与肿瘤发生有关。然而，与Ras不同，Rho GTP酶在癌症中不直接突变，而是其异常活性与其异常表达和/或调节有关。也许最重要的机制，已经出现，其中异常活动的Rho GTdR是异常的结果激活蛋白质称为RhoGEF（鸟嘌呤核苷酸交换因子）。我的研究集中在一个RhoGEF，Ect2（上皮细胞转化序列2）。 Ect2是RhoGEF的人Dbl家族的成员。先前的研究表明Ect 2在正常哺乳动物胞质分裂中是必需的。相反，在许多癌症中观察到Ect 2的异常过表达，最近的研究表明Ect 2在肺癌细胞系生长和致瘤性中起关键作用。我们已经发现Ect2表达在结直肠癌（CRC）患者肿瘤组织、具有自发性肠腺瘤的APCmin小鼠和人CRC细胞系中升高。有证据表明，存在于正常细胞核中的Ect 2在肺癌和脑癌中错误定位于细胞质中。这种错误定位可能导致细胞质中不适当的Rho激活。除了其RhoGEF催化结构域外，Ect 2还由多个附加结构域组成。Ect 2在癌症中如何异常激活，以及Ect 2在正常细胞和肿瘤细胞中的功能机制是否不同，目前还知之甚少，也是我研究的重点。我们假设Ect 2在胞质分裂中的功能与促进肿瘤发生的功能不同，并且错误定位可能有助于Ect 2在癌症中的激活。这些研究将要求我应用非常多样化的实验技术，促进我发展成为一名独立的研究人员，并建立我进行基础和转化癌症研究的能力。 公共卫生相关性：与Ras癌蛋白一样，异常Rho（Ras同源;例如，RhoA、Rac1和Cdc42）小GT3信号转导也与人类肿瘤发生有关。然而，Ras的直接突变导致其激活，而间接机制导致Rho激活。我们的初步研究发现，Ect2，鸟嘌呤核苷酸交换因子（RhoGEF）和Rho激活剂，在结直肠癌（CRC）的过度表达。虽然Ect 2与正常细胞胞质分裂有关，但我们推测Ect 2的异常过表达和错误定位促进了CRC的致瘤性、侵袭性和转移性生长。我们建议研究细胞培养和小鼠模型研究，以解决Ect 2在正常和肿瘤细胞功能中的作用和机制。有新兴的兴趣，RhoGEFs可能是一类重要的新的蛋白质靶点的癌症药物发现。我们的研究将提供Ect 2在CRC中的重要性的验证，并为治疗干预阐明Ect 2激活和肿瘤发生的机制。研究结直肠癌的意义：CRC是美国癌症死亡的第三大原因，虽然有许多针对CRC的治疗选择（例如，贝伐单抗和西妥昔单抗），仍然迫切需要新的和改进的分子靶向疗法。

DESCRIPTION (provided by applicant): In previous years, Ras, a small GTPase oncogene has been a focus in cancer research because it is one of the most commonly mutated genes associated with all human cancers (33%). Ras is the founding member of a large superfamily of small GTPases, and recent studies have linked the abnormal activity implicated Ras superfamily proteins, in particular, the Ras homologous (Rho) GTPases to tumorigenesis. However, unlike Ras, Rho GTPases are not mutated directly in cancer, but instead their abnormal activity has been linked to their abnormal expression and/or regulation. Perhaps the most significant mechanism that has emerged in which aberrant activity of Rho GTPase is the result of abnormalities in activating proteins called RhoGEFs (guanine nucleotide exchange factors). My studies are focused on one RhoGEF, Ect2 (Epithelial cell transforming sequence 2). Ect2 is a member of the human Dbl family of RhoGEFs. Previous studies indicate that Ect2 is essential in normal mammalian cytokinesis. In contrast, abnormal overexpression of Ect2 has been observed in many cancers, and a recent study demonstrated a critical role for Ect2 in lung carcinoma cell line growth and tumorigenicity. We have found that Ect2 expression is elevated in colorectal carcinoma (CRC) patient tumor tissue, APCmin mice with spontaneous intestinal adenomas, and human CRC cell lines. There is evidence that Ect2, which is present in the nucleus of normal cells, is mislocalized into the cytoplasm in lung and brain cancers. This mislocalization may lead to inappropriate Rho activation in the cytoplasm. In addition to its RhoGEF catalytic domain, Ect2 is comprised of multiple addition domains. How Ect2 becomes aberrantly activated in cancer and whether the mechanisms of Ect2 function in normal and neoplastic cells are distinct, are poorly understood and the focus of my studies. We hypothesize that the functions of Ect2 in cytokinesis are distinct from functions which promote oncogenesis and that mislocalization may contribute to Ect2 activation in cancer. These studies will require my application of a very diverse repertoire of experimental techniques, foster my development into an independent researcher and establish my abilities to do basic and translational cancer research. PUBLIC HEALTH RELEVANCE: Like the Ras oncoprotein, aberrant Rho (Ras homologous; e.g., RhoA, Rac1 and Cdc42) small GTPase signal transduction is also implicated in human oncogenesis. However, whereas direct mutation of Ras leads to its activation, indirect mechanisms lead to Rho activation. Our preliminary findings identified overexpression of Ect2, a guanine nucleotide exchange factor (RhoGEF) and Rho activator, in colorectal cancer (CRC). While Ect2 has been implicated in normal cell cytokinesis, we hypothesize that the aberrant overexpression and mislocalization of Ect2 promotes CRC tumorigenic, invasive and metastatic growth. We propose studies cell culture and mouse model studies to address the role and mechanism of Ect2 in normal and neoplastic cellular function. There is emerging interest that RhoGEFs may be an important new class of protein targets for cancer drug discovery. Our studies will provide validation for Ect2 importance in CRC and eludicate mechanisms of Ect2 activation and oncogenesis for therapeutic intervention. Significance of studying colorectal cancer: CRC is the 3rd leading cause of cancer deaths in the US and while there are a number of targeted therapeutic options available for CRC (e.g., bevacizumab and cetuximab) there is still a dire need for new and improved molecularly-targeted therapies.