Identification of RhoGEFs as novel therapeutic targets for malignant glioma

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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.