McDonough, Wendy S.

Abstract Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5128.

Abstract Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and significant technical advances in surgical and radiation treatment, the impact on clinical outcome for patients with malignant gliomas is disappointing. We have previously shown that tumor necrosis factor–like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell survival via binding to the Fn14 receptor, activation of the NF-κB pathway, and upregulation of BCL-XL gene expression. Here, we show that TWEAK treatment of glioma cells leads to phosphorylation of Akt and BAD. TWEAK stimulation results in the phosphorylation of both Akt1 and Akt2. However, small interfering RNA (siRNA)–mediated depletion of either Akt1 or Akt2 showed that BAD serine 136 phosphorylation is dependent specifically on Akt2 function. Depletion of Akt2 expression by siRNA also abrogates TWEAK-stimulated glioma cell survival, whereas no effect on glioma cell survival was observed after siRNA-mediated depletion of Akt1 expression. Surprisingly, although siRNA-mediated depletion of BAD in glioma cells abrogates cytotoxic- and chemotherapy-induced apoptosis, TWEAK still displays a strong protective effect, suggesting that BAD serine 136 phosphorylation plays a minor role in TWEAK-Akt2–induced glioma cell survival. We also report here that AKT2 gene expression levels increased with glioma grade and inversely correlate with patient survival. Additionally, immunohistochemical analysis showed that Akt2 expression positively correlates with Fn14 expression in glioblastoma multiforme specimens. We hypothesize that the TWEAK-Fn14 signaling axis functions, in part, to enhance glioblastoma cell survival by activation of the Akt2 serine/threonine protein kinase. (Mol Cancer Res 2009;7(11):1871–81)

Abstract Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-κB (NF-κB) activity. The Fn14 promoter region contains NF-κB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior. (Cancer Res 2006; 66(19): 9535-42)