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，患者平均生存时间不到一年。治疗这些脑肿瘤的一个关键问题是单个肿瘤细胞广泛浸润到邻近脑组织。这些入侵的细胞对放疗和化疗有很强的抵抗力，目前还没有抗侵入的治疗方法。小gtpase的Rho家族成员在体外参与胶质母细胞瘤细胞的侵袭。本提案的长期目标是解剖Rho gtpase介导的对胶质瘤侵袭至关重要的信号通路，并利用这些信息确定新的药物靶点，用于治疗干预脑肿瘤扩散。Rho蛋白被鸟嘌呤核苷酸交换因子（gef）激活。该提案的目的有两个方面：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在脑肿瘤组织中激活状态的临床信息。该提议的中心假设是，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体外胶质瘤侵袭所需的rhogef。通过96孔格式的入侵实验，筛选针对人类基因组中所有rhogef的小干扰rna （sirna）集中文库，鉴定有助于体外胶质瘤侵袭的rhogef。RhoGEF命中将使用器官型离体脑切片侵袭试验进行验证。2)使用一种新的RhoGEF下拉试验来确定哪些侵袭验证的RhoGEF在恶性胶质瘤和低级别胶质瘤中是过度活跃的。在胶质瘤进展过程中，RhoGEF的表达水平也将通过免疫组织化学或原位杂交来确定。此外，使用一种监测组织中特定Rho gtpase激活状态的新方法，将验证至少一些Rho gtpase在恶性胶质瘤和低级别胶质瘤中过度活跃的假设。预计本提案中概述的方法将确定大量rhogef作为新型胶质瘤侵袭基因。预计这项研究将产生关于这些rhogef的重要临床信息，这将允许在动物模型和深入的机制研究中优先考虑这些入侵基因。该应用程序解决了由NINDS和NCI于2000年开展的脑肿瘤进展审查小组的一个关键目标，即增加我们对脑肿瘤扩散的理解，这是脑肿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入侵基因，代表新的潜在治疗靶点。我们也期望我们将获得重要的临床信息，这将有助于验证这些入侵基因。

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.