Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells
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Published OnlineFirst May 9, 2012; DOI: 10.1158/1541-7786.MCR-11-0616 Molecular Cancer Signaling and Regulation Research Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells Shannon P. Fortin1,2, Matthew J. Ennis1, Cassie A. Schumacher3, Cassandra R. Zylstra-Diegel3, Bart O. Williams3, Julianna T.D. Ross1, Jeffrey A. Winkles4, Joseph C. Loftus5, Marc H. Symons6, and Nhan L. Tran1 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. Ó2012 AACR. Introduction therapeutic strategies, the mechanisms of which are complex Glioblastomas are the most malignant and common and remain to be fully characterized (2). primary brain tumor in adults. Glioblastomas are highly Glioma cell invasion requires adhesion to extracellular infiltrative, leading to a poorly defined tumor mass. As a matrix, subsequent degradation, and remodeling of this result, complete resection of the tumor is not feasible matrix, as well as signaling initiated by promigratory growth factors, and Rho GTPase–mediated organization and remo- without compromising neurologic function, and despite fi adjuvant chemo- and radiation therapy, the 5-year survival deling of the actin cytoskeleton (3). Speci cally, the Rho rate is just under 10% (1). The invasive nature of glioblas- GTPase family members RhoA, Rac1, and Cdc42 are key toma correlates to an increased resistance to current regulators of cell migration and have been implicated in the formation of stress fibers, induction of lamellipodia, and filopodia protrusion (4). The regulation of Rho GTPase Authors' Affiliations: 1Cancer and Cell Biology Division, The Translational Genomics Research Institute, Phoenix; 2Cancer Biology Graduate Inter- activation is mediated by 3 classes of proteins: guanine disciplinary Program, University of Arizona, Tucson, Arizona; 3Van Andel nucleotide exchange factors (GEF), which are responsible for Research Institute, Grand Rapids, Michigan; 4University of Maryland School of Medicine, Baltimore, Maryland; 5Mayo Clinic, Scottsdale, Ari- activating Rho GTPases to their GTP-bound state; GTPase- zona; and 6Center for Oncology and Cell Biology, The Feinstein Institute for activating proteins (GAP), which enact phosphate bond Medical Research at North Shore-LIJ, Manhasset, New York hydrolysis thus inactivating Rho GTPases to a GDP-bound Corresponding Authors: Nhan L. Tran, The Translational Genomics state; and GDP dissociation inhibitors (GDI) which bind to Research Institute, 445 N Fifth Street, Suite 400, Phoenix, AZ 85004. and stabilize Rho GTPases in their inactive GDP-bound form Phone: 602-343-8771; Fax: 602-343-8717; E-mail: [email protected]; and Marc H. Symons, The Feinstein Institute, 350 Community Dr., Manhasset, (5). To date, 22 Rho GTPases and 80 Rho GEFs belonging NY 11030. Phone: 516-562-1193; Fax: 516-562-1022; E-mail: to either the Dbl or DOCK families have been identified (6). [email protected] Previous studies have shown that the fibroblast growth doi: 10.1158/1541-7786.MCR-11-0616 factor inducible–14 (Fn14) receptor can signal to induce Ó2012 American Association for Cancer Research. Rac1 activation (7). Fn14 is a transmembrane receptor 958 Mol Cancer Res; 10(7) July 2012 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst May 9, 2012; DOI: 10.1158/1541-7786.MCR-11-0616 Ect2 and Trio Regulate Fn14-Induced Glioblastoma Invasion belonging to the TNF receptor superfamily (TNFRSF) and Antibodies, reagents, and Western blot analysis serves as the receptor for the multifunctional cytokine A monoclonal Cdc42 antibody was purchased from Santa TWEAK (8). The Fn14 cytoplasmic tail lacks a death Cruz Biotechnology. Anti-myc was purchased from Cell domain but contains TNFR-associated factor (TRAF) bind- Signaling Technologies. A polyclonal Ect2 antibody and a ing sites specific for TRAF1, 2, 3, and 5 (9). Fn14 expression monoclonal antibody to tubulin were purchased from Milli- is minimal to absent in normal brain tissue but correlates pore. Human recombinant TWEAK was purchased from with tumor grade in glioblastoma (10). Activation of Fn14 PeproTech, and laminin from human placenta was obtained by TWEAK promotes glioma cell migration, invasion, and from Sigma. Lipofectamine 2000 was purchased from survival (7, 10, 11). The TWEAK-Fn14 signaling axis Invitrogen. mediates glioma migration and invasion via the Rac1 For immunoblotting, cells were lysed in 2Â SDS sample GTPase and fosters a self-promoting feedback loop whereby buffer (0.25 mol/L Tris-HCl, pH 6.8, 10% SDS, 25% Rac1-mediated TWEAK-Fn14 signaling induces Fn14 gene glycerol) containing 10 mg/mL aprotinin, 10 mg/mL leu- expression via the NF-kB pathway (7). While Rac1 is peptin, 20 mmol/L NaF, 2 mmol/L sodium orthovanadate, ubiquitously expressed among tissue types (12, 13), the and 1 mmol/L phenylmethylsulfonylfluoride. Protein con- levels of Rac1 protein expression in astrocytomas directly centrations were determined using the BCA Assay (Pierce) correlate with tumor grade in tissue microarray analysis. with BSA as a standard. Thirty micrograms of total cellular Furthermore, in glioblastoma, but not in lower grade astro- protein was loaded per lane and separated by SDS-PAGE. cytomas, prominent plasma membrane staining of Rac1 is After transfer at 4C, the nitrocellulose (Invitrogen) was observed. These observations indicate that Rac1 is consti- blocked with either 5% nonfat milk or 5% BSA in TBS, pH tutively active in glioblastomas, underlining the importance 8.0, containing 0.1% Tween-20 (TBST) before addition of of Rac1 in these tumors (14). To date, 5 GEFs that can primary antibodies and followed with peroxidase-conjugated activate Rac1 (Ect2, Vav3, Trio, SWAP-70, and Dock180- anti-mouse IgG or anti-rabbit IgG. Protein bands were ELMO1) have been shown to contribute to the invasive detected using SuperSignal West Dura Chemiluminescent behavior of glioblastoma (14–16). Four of these GEFs have Substrate (Thermo Scientific) with a UVP BioSpectrum 500 been shown to be overexpressed in glioblastoma versus non- Imaging System. neoplastic brain (Ect2, Vav3, Trio, and SWAP-70; refs. 14, 16), and expression of Dock180 is higher in the Preparation of recombinant adenoviruses and infection tumor rim than in the tumor core. Adenoviruses expressing myc-tagged Fn14 wild-type pro- In this study, we describe a role for TWEAK-Fn14 tein and the cytoplasmic domain truncation mutant myc- signaling through a multi-GEF, multi-Rho GTPase sig- Fn14tCT protein were previously described (7). Cells were naling pathway that includes Ect2, Trio, Cdc42, and infected at matched multiplicity of infections ranging from 5 Rac1. We show that Rac1 activation via TWEAK-Fn14 to 20. For adenoviral infection, 1.5 Â 105 cells were plated signaling is dependent upon Cdc42 function. We also into a 6-well plate and cultured for 24 hours before infection. report that Ect2 mediates Cdc42 activation, whereas Trio mediates Rac1 activation following TWEAK stimu- Immunoprecipitation, Rac and Cdc42 activity assays, lation. Depletion of Ect2, Trio, or Cdc42 by siRNA and nucleotide-free GEF pulldowns oligonucleotides suppresses TWEAK-Fn14–induced Rac1 For immunoprecipitation, cells were lysed on ice for activation and subsequently glioma cell migration and 10 minutes in a buffer containing 10 mmol/L Tris- invasion. Finally, we show that the inappropriate expres- HCl (pH 7.4), 0.5% Nonidet P-40, 150 mmol/L NaCl, sion of either Fn14 or Ect2 in the astrocyte population of 1 mmol/L phenylmethylsulfonylfluoride, 1 mmol/L EDTA, glial fibrillary acidic protein (GFAP)-tva transgenic mice 2 mmol/L sodium orthovanadate, 10 mg/mL aprotinin, and induces astrocyte cell motility and proliferation, suggest- 10 mg/mL leupeptin (Sigma). Equivalent amounts of protein