Identification of RhoGEFs as novel therapeutic targets for malignant glioma

描述(申请人提供)：多形性胶质母细胞瘤是一种极具侵袭性的癌症，患者的平均生存期不到一年。这些脑肿瘤治疗中的一个关键问题是单个肿瘤细胞广泛渗透到邻近的脑组织。这些侵袭细胞对放射和化疗具有高度抵抗力，目前还没有抗侵袭疗法可用。Rho家族成员的小GTP酶在体外有助于胶质母细胞瘤细胞的侵袭。这项建议的长期目标是剖析Rho GTPase介导的信号通路，这些信号通路对胶质瘤的侵袭至关重要，并利用这些信息来识别新的药物靶点，用于脑瘤扩散的治疗干预。Rho蛋白被鸟嘌呤核苷酸交换因子(GEF)激活。这项建议的目的有两个：以全基因组的方式鉴定胶质瘤侵袭所必需的RhoGEF，并提供有关这些RhoGEF在脑瘤组织中的激活状态的临床信息。这一建议的中心假设是，胶质母细胞瘤的扩散在很大程度上是由相对较小的RhoGEF亚群的过度激活引起的。为了实现这一应用的目标，将追求以下具体目标：1)以全基因组的方式确定哪些RhoGEF是胶质瘤体外侵袭所必需的。通过筛选针对人类基因组中所有RhoGEF的小干扰RNAs(SiRNAs)文库，利用96孔格式侵袭分析，将有助于胶质瘤体外侵袭的RhoGEF被鉴定出来。将使用器官型体外脑片侵袭分析来验证Rhogef的命中率。2)使用一种新的Rhogef下拉试验确定哪些侵袭性有效的RhoGEF在恶性胶质瘤和低级别胶质瘤中是高活性的。还将使用免疫组织化学或原位杂交来确定胶质瘤进展过程中Rhogef的表达水平。此外，使用一种监测组织中特定Rho GTP酶激活状态的新方法，将检验这一假设，即至少一些Rho GTP酶在恶性和低级别胶质瘤中过度活跃。预计这项提案中概述的方法将确定相当数量的RhoGEF作为新的胶质瘤侵袭基因。预计这项研究将产生关于这些RhoGEF的重要临床信息，从而为动物模型和深入的机制研究确定这些侵袭基因的优先顺序。这项应用解决了NINDS和NCI在2000年进行的脑瘤进展审查小组的一个关键目标，即增加我们对脑瘤扩散的了解，这是脑瘤治疗中的一个关键问题。我们预计，拟议的研究将确定一些代表新的潜在治疗靶点的侵袭基因。我们还希望我们将获得重要的临床信息，这些信息将有助于验证这些侵袭基因。

DESCRIPTION (provided by applicant): Glioblastoma multiforme is an extremely aggressive cancer, with a mean patient survival time of less than one year. A critical problem in the treatment of these brain tumors is the extensive infiltration of individual tumor cells into adjacent brain tissue. These invading cells are highly resistant to radiation and chemotherapy and currently, there are no anti-invasive therapies available. Members of the Rho family of small GTPases contribute to glioblastoma cell invasion in vitro. The long-term goal of this proposal is to dissect Rho GTPase-mediated signaling pathways that are critical for glioma invasion and to use this information to identify novel drug targets for therapeutic intervention against brain tumor dispersal. Rho proteins are activated by guanine nucleotide exchange factors (GEFs). The objective of this proposal is two-fold: to identify in a genome-wide fashion RhoGEFs that are necessary for glioma invasion and to provide clinical information about the activation state of these RhoGEFs in brain tumor tissue. The central hypothesis of this proposal is that glioblastoma dispersal is caused in large part by the hyperactivation of a relatively small subset of RhoGEFs. To accomplish the goals of this application, the following specific aims will be pursued: 1) to determine in a genome-wide fashion which RhoGEFs are required for glioma invasion in vitro. RhoGEFs that contribute to glioma invasion in vitro will be identified by screening a focused library of small interfering RNAs (siRNAs) directed against all RhoGEFs in the human genome using a 96-well format invasion assay. RhoGEF hits will be validated using an organotypic ex vivo brain slice invasion assay. 2) To determine which of the invasion-validated RhoGEFs are hyperactive in malignant versus low grade glioma using a novel RhoGEF pull-down assay. RhoGEF expression levels during glioma progression will also be determined using immunohistochemistry or in situ hybridization. In addition, using a novel assay that monitors the activation state of specific Rho GTPases in tissues, the hypothesis will be tested that at least some Rho GTPases are hyperactive in malignant versus low grade glioma. It is expected that the approaches outlined in this proposal will identify a significant number of RhoGEFs as novel glioma invasion genes. It also is anticipated that this study will yield significant clinical information on these RhoGEFs that will allow prioritization of these invasion genes for animal model and in depth mechanistic studies. This application addresses a key goal of the Brain Tumor Progress Review Group, conducted in 2000 by the NINDS and NCI, namely to increase our understanding of brain tumor dispersal, which is a critical problem in the treatment of brain tumors. We anticipate that the proposed research will identify a number of invasion genes that represent novel potential therapeutic targets. We also expect that we will obtain significant clinical information that will aid in the validation of these invasion genes.