Williams, Bart O.

Abstract Glioblastoma multiforme (GBM) is the most common primary central nervous system tumor accounting for approximately 40% of all primary malignant brain tumors. The mechanism driving the development and recurrence of GBM is still largely unknown which greatly limits the successful treatment of this disease. The tumor necrosis factor receptor superfamily member TNFRSF19 (TROY) is a type I cell surface receptor protein containing the highly conserved TNFR cysteine-rich motifs in the extracellular domain and a tumor necrosis factor-receptor-associated factor (TRAF) - binding sequence in the cytoplasmic domain. We recently demonstrated that increased expression of TROY stimulated glioma cell migration in vitro and increased cell invasion in an organotypic brain slice model. Conversely, siRNA mediated knockdown of TROY expression inhibited glioma cell migration. In addition, profiling of TROY in brain tumor samples indicated that TROY mRNA expression was significantly increased in GBM samples, directly correlated with increasing glial tumor grade, and inversely correlated with patient outcome suggesting that TROY expression may play a role in GBM invasion and is a good indicator of survival outcome. In the current study, we investigated the role of TROY in therapeutic resistance and survival signaling. We report that TROY protein expression was significantly increased in patient GBM tumor samples with TROY mRNA exhibiting increased expression in the invasive cell population. Aberrant expression of TROY in mouse astrocytes in situ using glial-specific gene transfer in transgenic mice induced astrocyte migration within the brain supporting an important role for TROY in glioma cell migration. Notably, increased TROY expression did not increase cell proliferation but increased resistance of glioma cells to both radiation and temozolomide induced apoptosis while knockdown of TROY increased temozolomide sensitivity. TROY induced resistance to TMZ was dependent upon Akt and NF-κB activation. We also report that TROY induced NF-κB phosphorylation and stimulation of migration required the membrane proximal region of the TROY cytoplasmic domain and that knockdown of TROY expression increased survival in a xenograft model. The current results further support a role for TROY in GBM and suggest that targeting TROY and its signaling pathway represents a potential approach to increase tumor vulnerability and improve the therapeutic response of glioblastoma. Citation Format: Harshil D. Dhruv, Serdar Tuncali, Jean Kloss, Zhongbo Yang, Cassie Schumacher, Bart Williams, Julianna Ross, Nhan Tran, Joseph Loftus. TNFRSF19 (TROY) promotes glioma cell survival signaling and therapeutic resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2013-2621

Abstract Of the features that characterize glioblastoma, arguably none is more clinically relevant than the propensity of malignant glioma cells to aggressively invade into the surrounding normal brain tissue. These invasive cells render complete resection impossible, confer significant resistance to chemo- and radiation-therapy, and virtually assure tumor recurrence. Expression of TROY (TNFRSF19), a member of the TNF receptor superfamily, inversely correlates with patient survival and stimulates glioblastoma cell migration and invasion in vitro. In this study, we report that TROY is overexpressed in glioblastoma tumor specimens and TROY mRNA expression is increased in the invasive cell population in vivo. In addition, inappropriate expression of TROY in mouse astrocytes in vivo using glial-specific gene transfer in transgenic mice induces astrocyte migration within the brain, validating the importance of the TROY signaling cascade in glioblastoma cell migration and invasion. Knockdown of TROY expression in primary glioblastoma xenografts significantly prolonged survival in vivo. Moreover, TROY expression significantly increased resistance of glioblastoma cells to both IR- and TMZ-induced apoptosis via activation of Akt and NF-κB. Inhibition of either Akt or NF-κB activity suppressed the survival benefits of TROY signaling in response to TMZ treatment. These findings position aberrant expression and/or signaling by TROY as a contributor to the dispersion of glioblastoma cells and therapeutic resistance. Implications: Targeting of TROY may increase tumor vulnerability and improve therapeutic response in glioblastoma. Mol Cancer Res; 11(8); 865–74. ©2013 AACR.

Abstract Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. Mol Cancer Res; 10(7); 958–68. ©2012 AACR.