The efficacy of treating malignant glioblastoma multiforme (GB) with adjuvant therapies remains largely unsuccessful due to the inability to effectively target invading cells. Fn14 expression level is elevated in advanced glial tumors. Fn14-induced glioma invasion is mediated by the small GTPase Rac1; deletion of the TRAF binding region of Fn14 failed to activate Rac1 protein. We found that Ect2, a guanine nucleotide exchange factor (GEF), is overexpressed in GBM, and that overexpression of both Ect2 and Fn14 is correlated directly with tumor grade and inversely with patient outcome. Ect2 can associate with the Fn14 cytoplasmic complex and regulate Rac1 activation. Rac1 is a ubiquitous in many normal tissues, however the central position of Rac1 as a downstream player in many signaling pathologies operating in malignant states, suggest that targeting key regulators of Rac1 should be exploited to discover key vulnerabilities for invading glioma cells. We HYPOTHESIZE that Fn14 operates via Rac1 to enhance malignant glioblastoma cell invasion and survival.
The specific aims are as follow: 1) to define the proximal mechanism(s) by which the Fn14 activates Rac1 in glioma cells. We will query a specific biochemical model of the signaling protein(s) involved in Fn14- mediated Rac1 activation. We will first validate the role of Ect2 in this Fn14 induced Rac1 activation and determine whether Ect2 is a direct exchange factor for Rac1 or indirect exchange factor for Rac1 via Cdc42 activation downstream of Fn14. Secondly, we will determine whether Ect2 binds directly or indirectly to the TRAF site of the Fn14's cytoplasmic tail. 2) to demonstrate the impact of the Fn14-Rac1 signaling axis on glioma migration, invasion and therapy response. Biological validation using siRNAs directed to suppress the expression levels of the Fn14 signaling proteins (Ect2, TRAFs) will be performed to assess the effects of these proteins on glioma cell migration in vitro, cell invasion using ex vivo rat brain slices, and vulnerability of invasive glioma cells to cytotoxic therapy in vitro. Additionally, validation of the abundance and activation of Fn14 signaling proteins will be determined using immunohistochemical (IHC) techniques on glioma invasion TMAs. 3) to determine whether inhibition of Fn14 or its signaling protein(s) in glioblastoma xenografts leads to reduced tumor growth and cell invasion and facilitated activation of cell death in vivo. Inhibition and manipulation of Fn14 signaling components using blocking monoclonal antibodies to human Fn14 or shRNA directed against Fn14 signaling protein(s) (Ect2) will be used to assess the suitability of Fn14 and its signaling components as targeted therapies for invasive primary human malignant glioblastoma xenografts. Orthotopic models of GBMs will be used to assess effects of these biological therapies on induction of cell death by the cytotoxic agent temozolomide (Temador). Median cohort survival (Kaplan-Meier) as well as histological assessment of tumor size, invasion pattern and cell death in addition to examination of downstream signaling targets (Rac1 and NF:B activation) by ELISA and IHC techniques will be employed to measure the effectiveness of the biological therapies. ? PROJECT NARRATIVE: A hallmark of glioblastoma multiforme (GBM) is the ability of tumor cells to invade into surrounding normal brain. These invading cells are missed by surgery, escape focused radiation, and hide from chemotherapy. Preliminary studies demonstrate that the transmembrane receptor, Fn14, is upregulated with glioma progression; Fn14 expression correlates inversely with poor patient outcome. The activation of Fn14 mediates cell invasion and survival phenotypes of these malignant cells. Thus, the use of therapy against the transmembrane receptor Fn14 or its signaling proteins as an adjuvant to surgical extirpation may specifically target invasive glioma cells and improve the outcome of this devastating cancer. ? ? ?
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