Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

Published OnlineFirst May 9, 2012; DOI: 10.1158/1541-7786.MCR-11-0616

Molecular Cancer Signaling and Regulation Research

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. 2012 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 2012 American Association for Cancer Research. Rac1 activation (7). Fn14 is a transmembrane receptor

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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 2 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, 1.5 105 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 ing that the aberrant expression of Fn14 observed in (500 mg) were precleared and immunoprecipitated from the glioblastoma may play an important role in glioblastoma lysates and then washed with lysis buffer followed by S1 progression, especially cell invasion. buffer [10 mmol/L HEPES (pH 7.4), 0.15 mol/L NaCl, 2 mmol/L EDTA, 1.5% Triton X-100, 0.5% deoxycholate, and 0.2% SDS]. Samples were then resuspended in 2 SDS Materials and Methods sample buffer and boiled in the presence of 2-mercaptoetha- Cell culture conditions nol (Sigma), separated by SDS-PAGE, transferred to nitro- Human astrocytoma cell lines T98G and U118 (Amer- cellulose for 1 hours at 4C, and proteins were detected using ican Type Culture Collection) were maintained in Dulbec- SuperSignal West Dura Chemiluminescent Substrate co's Modified Eagle's Media (DMEM; Gibco) supplemen- (Thermo Scientific). ted with 10% heat-inactivated FBS (Gibco) at 37 C with Activity assays for Rac1 and Cdc42 were done according 5% CO2. For all assays with TWEAK treatment, cells were to the protocol of the manufacturer (Pierce). Lysates were cultured in reduced serum (0.5% FBS) for 16 hours before harvested, and equal concentrations of lysates were assessed stimulation with recombinant TWEAK at 100 ng/mL in for Rac or Cdc42 activity. Affinity pull-down analyses of DMEM þ 0.1% bovine serum albumin (BSA) for the active GEFs bound to Rho GTPases were conducted using a indicated time. nucleotide-free Rac1 mutant (G15A) expressed and purified

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as described (17). Recombinant Rac1 G15A-GST protein bilaterally onto the putamen of 400-mm thick slices of freshly was produced in BL21 cells. Cells were lysed in B-PER lysis isolated 4- to 6-week-old murine brains. Glioma cell inva- buffer (Pierce) containing protease inhibitors and purified sion into the brain slices was quantified using the LSM 5 with glutathione (GSH) beads. Equal amounts of total confocal microscope, and depth of invasion (z-axis stacks) glutathione S-transferase (GST) fusion protein were then was calculated as previously described (20). incubated with total cellular protein lysate (1 mg), and precipitated lysates were resolved with SDS-PAGE. Matrix-assisted laser desorption/ionization–time-of- flight mass spectrometry siRNA transfection For matrix-assisted laser desorption/ionization–time-of- siRNA oligonucleotides specific for Rac1, GL2 luciferase flight mass spectrometry (MALDI-TOF MS), protein bands (18), Ect2, and Trio were previously described (14). siRNA were stained with SYPRO ruby protein staining kit (Bio- sequences for Cdc42 are Cdc42-1 (50-AAAGACTC- Rad) according to the manufacturer's protocol. Prominent CTTTCTTGCTT-GT) and Cdc42-2 (50-AATAACT- protein bands present in the nucleotide-free Rac1-G15A CACCACTGTCCAAA). Transient transfection of siRNA mutant lysates were visualized under UV light and isolated. was conducted using Lipofectamine 2000. Cells were plated The samples of generated peptides were dissolved in 5 mL of at 70% confluency in DMEM þ 10% FBS without anti- 0.5% trifluoroacetic acid and measured by MALDI-TOF biotics and were transfected within 8 hours of plating. The MS analysis on a Voyager reflector instrument (Applied siRNA and Lipofectamine were diluted in serum-free Biosystems) and a Q-STAR mass spectrometer (Perceptive DMEM. After 5 minutes, the mixtures were combined and Biosystems) in positive ion mode at the University of incubated for 20 minutes at room temperature to enable Arizona (Tucson, AZ) Proteomic Facility. Data searches complex formation. siRNA oligonucleotides were trans- were done using the National Center for Biotechnology fected at 50 nmol/L, and no cell toxicity was observed. Information (NCBI) protein data bank with a minimum Maximum inhibition of mRNA and protein levels was matching peptide setting of 4, a mass tolerance setting of achieved 48 to 72 hours posttransfection. 50 to 200 ppm, and a single trypsin miss cut setting.

Radial cell migration assay Quantification of lamellipodia formation Cell migration was quantified as previously described (19). Glioma cells were transfected with siRNA targeting lucif- Glioma cells were transfected with siRNA targeting lucifer- erase, Rac1, Cdc42, Ect2, or Trio. After 24 hours, cells were ase, Ect2, or Cdc42. After 24 hours, cells were plated onto plated onto 10-well glass slides precoated with 10 mg/mL 10-well glass slides precoated with 10 mg/mL laminin. Cells laminin. Twenty-four hours later, cells were cultured in were cultured in reduced serum (0.5% FBS) for an addi- reduced serum (0.5% FBS) for an additional 16 hours before tional 16 hours before TWEAK addition, and migration rate TWEAK stimulation for 5 minutes. Subsequently, cells were was assessed over 24 hours. fixed in 4% formaldehyde/PBS, permeabilized with 0.1% Triton-X100, dissolved in PBS, and incubated with Alexa- Lentiviral production Fluor phalloidin (Molecular Probes) to stain for F-actin. The cDNA encoding murine myc-tagged Fn14 wild-type Slides were mounted with ProLong Gold Antifade Reagent protein (pSec/Tag2/Fn14wt) or signal-deficient myc- with 40,6-diamidino-2-phenylindole (DAPI; Molecular Fn14tCT (7) was excised and ligated into the lentiviral Probes). Images were collected using a Zeiss LSM 510 transfer vector pCDH (System Biosciences) that contains microscope, equipped with a 63 objective, ZEN 2009 a second transcriptional cassette for the expression of GFP. image analysis software, and Adobe Photoshop CS3. For An empty pCDH vector expressing only GFP was used as a each experimental condition, 10 images were taken ran- control. VSV-G pseudo-typed recombinant lentiviruses domly. Lamellipodia were traced using ImageJ software. For were produced by co-transfection of 293 packaging cells each cell, the fraction of the cell perimeter that displayed with the pCDH construct and the pPACK packaging mix lamellipodia was calculated. (System Biosystems) according to the manufacturer's direc- tions. For lentiviral transduction, medium containing RCAS astrocyte-specific infection in transgenic mouse recombinant lentiviruses was harvested from the packaging models cells after 48 hours, concentrated by polyethylene glycol Immortalized DF-1 chicken fibroblasts (obtained from precipitation and centrifugation, and added to subconfluent American Type Culture Collection) were transfected by cultures of T98G and U118 cells together with 8 mg/mL calcium phosphate precipitation with plasmids in which the polybrene for 4 to 6 hours. Positively transduced cells were cDNAs for alkaline phosphatase (AP), Fn14, Ect2, and enriched by mass-sorting the GFP-positive cells on a Vantage hepatocyte growth factor (HGF) were cloned into the flow cytometer (BD Biosciences). ALV-A expression vector, RCAS-Y [a derivative of RCASBP(A); ref. 21]. Transfected cells were cultured in Organotypic brain slice invasion assay DMEM with 10% FBS and 1 penicillin/streptomycin, Preparation and culture of brain slices was carried out as and passaged 1:3 every other day to maintain maximal described previously (20) with minor modification. Glioma logarithmic growth and viral production. Viral expression cells (T98G and U118) stably expressing GFP were placed was detected by Western blotting using whole-cell lysates.

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Ect2 and Trio Regulate Fn14-Induced Glioblastoma Invasion

Mice (G-tva) expressing the TVA receptor protein under fixed in 10% neutral-buffered formalin overnight. After control of the GFAP promoter were used (22). Viral-pro- fixing, brains were dehydrated in 20% sucrose, 2% glycerol ducing DF-1 cells from a confluent T-75 flask were trypsi- in PBS. Dehydrated tissues used for alkaline phosphatase nized, centrifuged, resuspended in 100 mL of medium, and staining were cut into 60-mm sections on a vibratome. Fixed placed on ice. DF-1 cells infected with RCASBP(A)-AP and brain tissue was incubated at 65C in PBS (pH 9.5) for 1 RCASBP(A)-Fn14, -Ect2, or -HGF were combined in equal hour to remove endogenous alkaline phosphatase activity. proportions and co-injected into the newborn mouse brain. Sections were then subject to 1-Step NBT/BCIP (Thermo A single intracranial injection of 5 mL was made at the Scientific) until violet staining was visualized. intersection of the coronal and sagittal sutures using a Hamilton Gastight Syringe. The number of wild-type mice Statistical analysis t per cohort injected with virally producing DF-1 cells was as Statistical analyses were done using the 2-sample test. P < fi follows: alkaline phosphatase (n ¼ 2), Fn14 (n ¼ 15), Ect2 0.05 was considered signi cant. (n ¼ 19), and HGF (n ¼ 4). The number of transgenic (TG/ þ) mice per cohort injected with virally producing DF-1 Results cells was as follows: alkaline phosphatase (n ¼ 4), Fn14 Ect2 binds to the Fn14 receptor and regulates TWEAK- (n ¼ 14), Ect2 (n ¼ 18), and HGF (n ¼ 10). stimulated Rac1 activation Mice injected with RCAS viruses were euthanized 10 We have previously shown that TWEAK-Fn14 signaling weeks postinjection, and whole brains were removed and stimulates Rac1 activity (7). Consistent with our previous

Figure 1. Ect2 binds to Fn14 and regulates TWEAK-induced Rac1 activation and glioma cell migration. A, T98G cells were cultured in reduced serum followed by treatment with 100 ng/mL TWEAK for the indicated times. Whole-cell lysates were assessed for active Rac1 via afﬁnity pull-down with the Pak1-binding domain (PBD). B, T98G cells were infected with adenoviruses expressing myc-tagged Fn14 wild-type (wt), Fn14 C-terminal–truncated (Fn14tCT), or control LacZ. Whole-cell lysates were immunoprecipitated (IP) for myc with subsequent Western blot analysis for Ect2 and myc. C, T98G cells were transfected with 2 siRNA oligonucleotides targeting Ect2 and assessed for the level of shutdown efﬁciency relative to nontransfected (NT) or ctrl siRNA transfection targeting nonmammalian luciferase (Ctrl) by Western blot analysis. D, T98G cells were cultured in reduced serum followed by treatment with TWEAK (100 ng/mL) for 10 minutes in the presence of either control (ctrl) luciferase–targeted siRNA or siRNA-targeting Ect2 and were assessed by Western blotting. E, glioma cells were transfected with siRNA targeting either control (ctrl) luciferase or 2 independent siRNA oligonucleotidesto Ect2 (Ect2-1 and Ect2-2). After 24 hours, cells were cultured in reduced serum medium (0.5% FBS) for 16 hours and were seeded onto 10-well glass slides precoated with 10 mg/mL human laminin. Cells were either left untreated or treated with TWEAK, and glioma cell migration was assessed over 24 hours. Data represent the average of 3 independent experiments (, P < 0.01).

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data, TWEAK stimulation of glioma cells results in a rapid increase of Rac1 activity (Fig. 1A). We also reported that speciﬁc Rac1 GEFs (Ect2, Trio, and Vav3) are upregulated in glioblastomas and regulate glioma migration and invasion (14). Here, we examined which GEFs are important in regulating TWEAK-induced Rac1 activation. Co-immunoprecipitation studies indicated that the Fn14 wild-type receptor, but not an Fn14 mutant with a truncated cytoplasmic tail (tCT), interacted with Ect2 (Fig. 1B) but not Trio or Vav3 (data not shown). To determine whether Ect2 is important for TWEAK-induced Rac1 activation, we obtained 2 siRNAs targeting Ect2 and analyzed whether Ect2 depletion affected TWEAK-stimulated Rac1 activation. siRNA-mediated knockdown of Ect2 expression in the glioma cell lines with 2 independent siRNA oligonucleotides was approximately 90% effective (Fig. 1C). Depletion of Ect2 suppressed TWEAK-induced Rac1 activation to levels below the untreated baseline of GTP-bound Rac1 (Fig. 1D) and also suppressed TWEAK-induced glioma cell migration to levels below baseline (Fig. 1E). These data implicate Ect2 as a nucleotide exchange factor for TWEAK-Fn14–mediated Rac1 activation and cell migration. Figure 2. TWEAK stimulation of Rac1 activation is dependent upon TWEAK regulation of Rac1 activation is dependent upon Cdc42. A, T98G cells were treated with TWEAK for the indicated times Cdc42 function and assayed for Cdc42 activity using the Pak1-binding domain (PBD) Rac1 has been shown to function downstream of Cdc42 in assay. B, T98G cells were transfected with Ect2 siRNA oligonucleotides or luciferase control siRNA (Ctrl). Cells were then cultured under reduced a number of different settings (23, 24). We therefore serum (0.5% FBS) for 24 hours, followed by treatment with TWEAK for 5 examined whether TWEAK also stimulates Cdc42 and minutes and then assessed for Cdc42 activity. C, T98G cells were either whether Cdc42, in turn, mediates TWEAK-induced Rac1 left nontransfected (NT), transfected with 2 independent siRNAs activation. We found that Cdc42 is activated by TWEAK designed against Cdc42 (Cdc42-1 and Cdc42-2), or transfected with control siRNA. Cells were then lysed, and Western blot analysis for Cdc42 with maximal activation achieved at 5 minutes (Fig. 2A). was conducted to conﬁrm protein depletion. D, cells transfected with Because Ect2 can catalyze nucleotide exchange on Cdc42 either control or Cdc42-targeting siRNA were cultured under reduced (18, 25), we also examined whether Cdc42 activation by serum for 16 hours. Cells were then either left untreated or treated with TWEAK was dependent upon Ect2. Depletion of Ect2 by TWEAK for 10 minutes and assayed for Rac1 activity. E, cells transfected siRNA oligonucleotides prevents TWEAK-induced Cdc42 with either control or Rac1-targeting siRNA were cultured under reduced serum for 16 hours. Cells were then either left untreated or treated with activation (Fig. 2B), indicating that Ect2 indeed mediates TWEAK for 5 minutes and assessed for Cdc42 activity. TWEAK-induced Cdc42 activation. To determine the epi- static relationship between Cdc42 and Rac1 in TWEAK signaling, we examined whether Cdc42 activity was neces- level of inhibition of cell migration is similar to that caused sary for Rac1 activation and vice versa. Using siRNA directed by Ect2 depletion (Fig. 1E). We next assessed whether against Cdc42 (Fig. 2C), we assessed Rac1 activity under Cdc42 or Ect2 knockdown affects Fn14-stimulated inva- depletion of Cdc42. Depletion of Cdc42 expression resulted sion using an ex vivo rat brain slice model as detailed in the suppression of TWEAK-induced Rac1 activation (Fig. previously (20). Glioma cells infected with recombinant 2D), whereas Rac1 depletion had no effect on TWEAK- lentiviruses encoding GFP alone (V), wild-type Fn14 induced Cdc42 activation (Fig. 2E). Collectively, these data (WT), or Fn14 containing a truncation of the cytoplasmic indicate that Ect2 mediates the activation of Cdc42 down- domain (tCT) were transfected with siRNA oligonucleo- stream of TWEAK-Fn14 and that, in turn, Cdc42 activates tides targeting either Cdc42 or Ect2 and subsequently Rac1. assessed for depth of invasion into the brain slices over 48 hours. As reported previously (7), glioma cells overexpres- Depletion of Cdc42 or Ect2 by siRNA suppresses sing the Fn14wt receptor have increased invasive activity, TWEAK- or Fn14-induced cell migration and invasion whereas cells expressing the Fn14tCT receptor do not Cdc42 is known to control directional responses to (Fig. 3B). siRNA-mediated knockdown of Cdc42 and extracellular cues at the front of the cell (4) and has been Ect2 in glioma cells expressing Fn14wt abrogated reported to play an important role in chemotaxis and cell Fn14-induced cell invasion relative to control nonmam- invasion (26–28). Therefore, we investigated the role of malian-targeted siRNA in Fn14wt cells (Fig. 3B). These Cdc42 function in TWEAK-induced glioma cell migration. data corroborate the cell migration data and further show TWEAK stimulation of migration was ablated in glioma cell that Ect2-Cdc42 mediates Fn14-induced glioma cell lines under Cdc42-knockdown conditions (Fig. 3A). This migration and invasion.

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Ect2 and Trio Regulate Fn14-Induced Glioblastoma Invasion

Figure 3. Cdc42 and Ect2 regulate TWEAK-induced glioma cell migration in vitro and Fn14-induced invasion ex vivo. A, T98G and U118 glioma cells were transfected with siRNA targeting either control nonmammalian luciferase (Ctrl) or 2 independent siRNA oligonucleotides targeting Cdc42 (Cdc42-1 and Cdc42-2). After 24 hours, cells were cultured in reduced serum medium (0.5% FBS) for 16 hours and were seeded onto 10-well glass slides precoated with 10 mg/mL human laminin. Cells were either left untreated or treated with TWEAK, and glioma cell migration was assessed over 24 hours. Data represent the average of 3 independent experiments (, P < 0.01). B, T98G and U118 glioma cells were stably transduced with lentiviruses expressing GFP alone (V), the Fn14 wild-type (WT), or the cytoplasmic domain–truncated Fn14 receptor (tCT). In certain experiments, cells were either transfected with siRNA oligonucleotides against Cdc42, Ect2 or control luciferase (ctrl). Cells were implanted bilaterally onto the putamen of murine organotypic brain slices and observed at 48 hours. Depth of invasion was then calculated from z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from 6 independent experiments (, P < 0.01).

Trio activates Rac1 downstream of Fn14 suggesting that Trio functions downstream of Cdc42 to Our data show that Ect2 depletion suppresses TWEAK- induce Rac1 activation. In addition, depletion of Trio induced Cdc42 and Rac1 activation. Furthermore, deple- inhibited TWEAK-induced glioma cell migration in vitro tion of Cdc42 expression by siRNA oligonucleotides (Fig. 4D). Likewise, Trio depletion also abrogated Fn14- suppresses TWEAK-induced Rac1 activation, which sug- induced cell invasion in the ex vivo rat brain slice model (Fig. gests that Ect2 is an indirect exchange factor for Rac1 and 4E), further supporting the function of Trio in mediating argues that an additional GEF(s) may activate Rac1 down- Fn14-induced cell invasion. Therefore, these data suggest stream of TWEAK-Fn14 signaling. To identify such GEFs, that Trio functions downstream of Ect2 and Cdc42 to we conducted a Rho GEF pull-down assay using a nucle- mediate TWEAK-Fn14–stimulated Rac1 activation. otide-free Rac1 GTPase mutant (G15A-Rac1) GST-fusion protein that can precipitate activated Rac1 GEFs out of cell Depletion of Rac1, Cdc42, Ect2, or Trio by siRNA lysates. T98G glioma cells were treated with TWEAK, suppresses TWEAK-induced lamellipodia formation subjected to the nucleotide-free Rac1 GEF pull-down assay, Activated Cdc42 and Rac1 are known to control actin and proteins were resolved by SDS-PAGE analysis. Prom- cytoskeleton–based dynamics and promote lamellipodia inent protein bands present in the G15A-Rac1 complex formation. To determine whether TWEAK activation of under TWEAK stimulation but absent in the untreated Cdc42 and Rac1 signaling confers changes in the actin G15A-Rac1 complex were recovered from the gel. Proteins cytoskeleton, we treated cells with TWEAK and examined were eluted, trypsin-digested, and MALDI-TOF and tan- filamentous actin using phalloidin staining. We found that dem mass spectrometric (MS-MS) analysis of the trypsin TWEAK stimulation of glioma cells strongly stimulates digests were conducted. A known Rac1-GEF, Trio, was a lamellipodia formation [Fig. 5A, (ii, arrows)] as compared candidate protein identified by MS in the TWEAK-treated with control untreated cells [Fig. 5A, (i)]. Furthermore, G15A-Rac1 complex. We therefore pursued Trio as a treatment with siRNA oligonucleotides targeting Rac1, potential Rac1 activator, using 2 siRNAs that significantly Cdc42, Ect2, or Trio significantly inhibited TWEAK-stim- reduce Trio expression (Fig. 4A). The increased activity of ulated lamellipodia formation (Fig. 5A and B). These data Rac1 seen upon TWEAK stimulation is eliminated under are in line with the migration and invasion data and are Trio-knockdown conditions (Fig. 4B), indicating that Trio consistent with the existence of a signaling cascade compris- mediates TWEAK-induced Rac1 activation. Interestingly, ing Ect2, Cdc42, Trio, and Rac1 that functions downstream depletion of Trio expression by siRNA oligonucleotides had of Fn14 to control the invasive behavior of glioblastoma no effect on TWEAK-induced Cdc42 activation (Fig. 4C), cells.

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Figure 4. Trio activates Rac1 upon TWEAK stimulation or Fn14 overexpression. A, T98G cells were transfected with 2 siRNA oligonucleotides targeting Trio and assessed for the level of shutdown efﬁciency relative to nontransfected (NT) or ctrl siRNA transfection targeting nonmammalian luciferase (ctrl) by Western blot analysis. Glioma cells transfected with Trio siRNA were cultured under reduced serum for 16 hours followed by treatment with TWEAK for 10 minutes and assessment for Rac1 activity (B) or Cdc42 activity (C). D, T98G and U118 glioma cells were transfected with siRNA targeting either control nonmammalian luciferase (ctrl) or 2 independent siRNA oligonucleotides targeting Trio (Trio-1 and Trio-2). After 24 hours, cells were cultured in reduced serum medium (0.5% FBS) for 16 hours and were seeded onto 10-well glass slides precoated with 10 mg/mL human laminin. Cells were either left untreated or treated with TWEAK, and glioma cell migration was assessed over 24 hours. Data represent the average of 3 independent experiments (, P < 0.01). E, T98G and U118 glioma cells were stably infected with lentiviruses expressing GFP and either the control vector (V), the Fn14 wild-type (WT), or the cytoplasmic domain–truncated Fn14 receptor (tCT). In certain experiments, cells were either transfected with siRNA oligonucleotides against Trio or control luciferase gene (ctrl). Cells were implanted bilaterally onto the putamen of murine organotypic brain slices and observed at 48 hours. Depth of invasion was then calculated from z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from 6 independent experiments (, P < 0.01).

We also examined focal adhesions in TWEAK-stimulated the avian cell surface receptor for subgroup A avian leukosis glioma cells using immunofluorescence staining for vinculin viruses (ALV-A), under control of the astrocyte-specific and paxillin. However, we did not observe any significant GFAP promoter. This allows for the selective infection of changes in the number or distribution of focal adhesions astrocytes with ALV-A from virus-infected and -producing upon TWEAK treatment of control cells or TWEAK stim- DF-1 chicken fibroblasts to deliver both the Fn14 or Ect2 ulation of cells depleted of Rac1, Cdc42, Ect2, or Trio (data and alkaline phosphatase genes (22, 29, 30). It is important not shown). to note that the same cells producing alkaline phosphatase– expressing virus are used to infect TVAþ mice with alkaline Fn14 and Ect2 promote proliferation and migration of phosphatase alone or in the combination of alkaline phos- astrocytes in vivo phatase and either Fn14-, Ect2-, or HGF-expressing viruses. We previously showed that activation of the Fn14 receptor Co-injection of DF-1 cells with Fn14 or Ect2 expressing and results in glioma cell migration and invasion in vitro (7); alkaline phosphatase–expressing viral vectors into the brains furthermore, Ect2 has also been implicated as a regulator of of the transgenic mice induced astrocytic cell migration glioma cell migration and invasion (14), a key feature of along tracts at the intersection of the coronal and sagittal glioblastoma tumors. To assess the role of Fn14 and Ect2 sutures (Fig. 6B and C and F and G). This effect was similar in vivo, we used transgenic mice engineered to express TVA, to that caused by co-injection of DF-1 cells infected with

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Ect2 and Trio Regulate Fn14-Induced Glioblastoma Invasion

Figure 5. TWEAK-induced lamellipodia formation in glioma cells requires the function of Rac1, Cdc42, Ect2, and Trio. T98G glioma cells were transfected with siRNA targeting either control nonmammalian luciferase (ctrl), Rac1, Cdc42, Ect2, or Trio. After 24 hours, cells were seeded onto 10-well glass slides precoated with 10 mg/mL human laminin. Cells were further grown for 24 additional hours and then cultured in reduced serum medium (0.5% FBS) for 16 hours. Cells were either left untreated or treated with TWEAK (5 minutes) and stained for filamentous actin using AlexaFluor-phalloidin. A, representative images of control transfected nontreated (i) or TWEAK-treated (ii) cells, as well as TWEAK-treated cells after depletion of Rac1 (iii), Cdc42 (iv), Ect2 (v), or Trio (vi) are shown. Arrows indicate lamellipodia. Bar, 10 mm. B, quantification of lamellipodia formation. Data represent the average of 10 cells per condition (, P < 0.001). a.u., arbitrary units. avian leucosis viruses expressing alkaline phosphatase and a positive cells in the Fn14 or Ect2 and AP co-injection known robust inducer of cell motility, HGF (ref. 31; Fig. 6D conditions was clearly increased relative to conditions in and H). The injection of DF-1 cells producing alkaline which alkaline phosphatase was injected alone, indicating a phosphatase–expressing viruses alone did not stimulate cell role for Fn14 and Ect2 in astrocyte proliferation in addition migration (Fig. 6A and E). Given the limited number of cells to migration. Co-injection of HGF- and alkaline phospha- injected and the lack of selective proliferation or survival tase–producing DF-1 cells also resulted in increased advantage under alkaline phosphatase expression alone, it is numbers of alkaline phosphatase–staining cells. Moreover, technically difficult to find these cells after necropsy and analysis of virally infected cells in culture suggests no staining and thus the injection sites are labeled with a box evidence of toxicity associated with alkaline phosphatase (Fig. 6). Furthermore, the number of alkaline phosphatase– expression. This is consistent with previous studies using

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Figure 6. Proliferation and migration of RCAS-Fn14 and RCAS-Ect2–infected astrocytic cells in vivo. DF-1 chicken ﬁbroblasts producing subgroup A avian leukosis viruses (ALV-A) carrying the RCAS-AP plasmid along with either the RCAS-Fn14 (B and F), RCAS-Ect2 (C and G) or RCAS-HGF (D and H) plasmids were injected at the intersection of the coronal and sagittal sutures of transgenic mice expressing the TVA receptor for ALV-A under control of the GFAP promoter. In certain animals, chicken DF-1 cells producing RCAS-AP ALV-A viruses were injected alone as a negative control (A and E). Rectangles indicate area of injection. Arrows indicate alkaline phosphatase staining 10 weeks postinfection. Images (A–D) and (E–H) represent magniﬁcation at 2 and 4, respectively.

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alkaline phosphatase expression as a glial cell marker with no in glioblastoma cell invasion have not been investigated. evidence of deleterious effects on glial cell proliferation or Here, we show that Cdc42 plays a critical role in glioblas- survival in vitro (32) or in vivo (33) These data suggest that toma cell migration and invasion stimulated by TNF family the abnormal expression of Fn14 or Ect2 can be an inducer member TWEAK. Cdc42 is best known for its key role in of migration in situ. Thus, increased expression of Fn14 or the control of cell polarity (36), but it also has been Ect2 observed in glioblastoma may play a critical role in the implicated in the regulation of cell migration and invasion malignant behavior of glioblastoma. in a variety of different cell types (26, 28). Our data also place Cdc42 upstream of Rac1 in TWEAK- Discussion stimulated cell migration and invasion. Importantly, deple- In this article, we describe a signaling cascade comprising tion of Cdc42 completely inhibits TWEAK-stimulated Ect2, Cdc42, Trio, and Rac1 that mediates TWEAK-stim- Rac1 activation, indicating that the Ect2-Cdc42 module ulated glioblastoma cell invasion via the TNFRSF member mediates the main, if not the only, signaling pathway that Fn14 (Fig. 7). In addition, we report that ectopic expression controls Rac1 activation downstream of TWEAK. We note of either Fn14 or Ect2 in the astrocytic lineage induces that Cdc42 has been shown to function upstream of Rac1 in aberrant proliferation and migration of glial cells, under- the regulation of a variety of different biologic functions, lining the importance of Fn14 and Ect2 in the malignant including cell polarity, cell migration, and glucose-stimu- behavior of glioblastoma. lated insulin secretion (37, 38), although several Rac1- Over the past several years, a number of Rho GEFs that independent functions of Cdc42 also have been delineated contribute to the invasive behavior of glioblastoma cells have (23, 24). been identiﬁed: Ect2, Vav3, Trio, SWAP-70, and Dock180 We also showed that depletion of the GEF Trio strongly (14–16). All these GEFs can act on the Rac1 GTPase in vitro inhibits TWEAK-stimulated activation of Rac1, but not of (6, 25, 34, 35). In addition, siRNA-mediated depletion of Cdc42, suggesting that Trio relays Cdc42 activation to that these GEFs has been shown to diminish Rac1 activation in of Rac1. We note however that we cannot exclude the glioblastoma cells (14–16). Several of these GEFs can also act possibility that additional GEFs contribute to the activation on Cdc42 and RhoA, but thus far, the roles of these GTPases of Rac1 by Cdc42. Indeed, in other systems, other GEFs have been shown to mediate Cdc42-induced Rac1 activation, including Cool-2/aPix and Tiam1 (37, 39), suggesting that Cdc42 may couple to Rac1 via different GEFs, depending on the biologic setting. Trio contains 2 GEF domains, TWEAK which act on Rac1 and RhoA, respectively, but not Cdc42 (40). The role of RhoA in glioblastoma cell invasion remains to be elucidated however. Notably, we previously showed Fn14 Extracellular that stimulation of glioblastoma cells with TWEAK leads to PM a decrease in RhoA activation (7), making it unlikely that Cytoplasmic RhoA activity contributes to TWEAK-stimulated glioblastoma invasion. Our data showing that depletion of Ect2 completely inhibits TWEAK-stimulated Cdc42 activation also indicate that Ect2 is the sole GEF that is responsible for Cdc42 GDP activation in this setting. It is interesting to note that Ect2 Ect2 Cdc42 can also act as an exchange factor for Rac1, both in vitro (25) GTP and in cells (41). However, even though we observed that depletion of Ect2 strongly inhibits TWEAK-induced Rac1 activation, it is unlikely that Ect2 directly acts on Rac1 in GDP TWEAK-stimulated glioblastoma cells, as depletion of Cdc42 completely inhibits TWEAK-stimulated Rac1 acti- Trio Rac1 vation. Thus, our data support a model in which TWEAK- GTP Fn14 in glioblastoma cells signals via Ect2, leading to Cdc42 and subsequently to Rac1 activation (Fig. 7). In addition, although our results suggest that Trio is a critical mediator of Rac1 activation downstream of TWEAK and Cdc42, in the Cell migration/invasion absence of biochemical data showing that Trio activity is regulated by Cdc42, we cannot exclude the possibility that Trio may be activated via cross-talk with other TWEAK- Figure 7. Schematic model of TWEAK-Fn14 signaling via Cdc42 and Rac1 stimulated signaling events. Interestingly, in lung cancer to drive glioma migration/invasion. We propose that TWEAK engagement of the Fn14 receptor results in the activation of Cdc42 by the Ect2 GEF. cells, Ect2 has been shown to mediate the activation of Rac1, Ect2-Cdc42 signaling results in the activation of Rac1 by the Trio GEF to but not Cdc42 (41). Moreover, during mitosis, Ect2 acti- drive glioma cell migration and invasion. PM, plasma membrane. vates Cdc42 during metaphase but regulates RhoA during

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Ect2 and Trio Regulate Fn14-Induced Glioblastoma Invasion

telophase (42, 43). Thus, these findings suggest that Ect2 recent development of an immunoconjugate containing a acts on different GTPases depending on the cellular context. highly specific anti-Fn14 monoclonal antibody conjugated The molecular mechanisms that underlie the substrate to recombinant gelonin (rGel), a cytotoxic ribosome-inacti- specificity dynamics of Ect2 largely remain to be elucidated vating N-glycosidase, that can kill Fn14-expressing cells in and may involve the interaction of Ect2 with different vitro and induce long-term tumor growth suppression in binding partners, such as MgcRacGAP and the PKC-Par6 nude mice bearing T-24 human bladder cancer cell xeno- complex (41–44). grafts supports the proposal that Fn14-targeted agents may The Fn14-Rac1 axis is highly deregulated in glioblastoma have antitumor effects (48). Additional therapeutic strategies tumors. Expression levels of Fn14, Ect2, and Trio increase targeting the TWEAK-Fn14 axis, including small-molecule with astrocytoma grade and correlate with poor patient antagonists of TWEAK trimerization or TWEAK-Fn14– outcome (7, 14, 45). We also have noted an increase in the binding and Fn14-Fc fusion proteins, have been recently degree of cytoplasmic localization of Ect2 in glioblastoma reviewed (8). There is also an increasing body of literature versus low-grade astrocytoma (14). Ect2 localizes to the that supports the feasibility of targeting GTPases and their nucleus in nontransformed cells, and aberrant localization cognate GEFs as promising anti-cancer therapies (49, 50). of Ect2 to the cytoplasm correlates with its oncogenic activity Thus, targeting the TWEAK-Fn14 signaling axis presents a (46, 47). Rac1 expression levels also increase with astrocy- unique therapeutic strategy for invasive glioblastoma cells. toma grade (14). In addition, prominent Rac1 plasma fi membrane localization in a signi cant subset of glioblastoma Disclosure of Potential Conflicts of Interest tumors indicates that this GTPase is hyperactive in these No potential conflicts of interests were disclosed. tumors (14). We also note that the Fn14-Rac1-NF-kB axis contributes to the malignant behavior of glioblastoma cells Authors' Contributions Conception and design: S.P. Fortin, B.O. Williams, J.T.D. Ross, M.H. Symons, N. by promoting cell survival (7, 11). L. Tran On the basis of their role in glioma cell migration, we Development of methodology: C.R. Zylstra-Diegel, B.O. Williams, J.C. Loftus, M. assessed the ability of Fn14 and Ect2 to promote astrocyte H. Symons, N.L. Tran Acquisition of data (provided animals, acquired and managed patients, provided migration and proliferation when inappropriately expressed facilities, etc.): S.P. Fortin, M.J. Ennis, C.A. Schumacher, J.T.D. Ross, J.A. Winkles in glial cells of transgenic mice. Previous work from Holland Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- and Varmus showed that basic fibroblast growth factor tational analysis): S.P. Fortin, M.J. Ennis, C.R. Zylstra-Diegel, J.T.D. Ross, M.H. Symons, N.L. Tran production in astrocytes induces the proliferation and migra- Writing, review, and/or revision of the manuscript: S.P. Fortin, M.J. Ennis, B.O. tion of glial cells, whereas injection of cells producing Williams, J.A. Winkles, J.C. Loftus, M.H. Symons, N.L. Tran – Administrative, technical, or material support (i.e., reporting or organizing data, alkaline phosphatase expressing virus alone results in only constructing databases): C.A. Schumacher, C.R. Zylstra-Diegel, J.T.D. Ross a small number of infected cells near the site of injection that Study supervision: N.L. Tran do not migrate (22). Here, we report the ability of both Fn14 and Ect2 to induce cell proliferation and motility with no Acknowledgments evidence of toxicity to cells when introduced into the murine The authors thank Dr. Keith Burridge (University of North Carolina, Chapel Hill, astrocyte population. NC) for a gift of G15A-Rac1-GST plasmid and Dr. Eric Holland (Memorial Sloan- Kettering Cancer Center, New York, NY) for a gift of the transgenic mice. Thus, the functional observations described in this article and our previous work (7, 11), together with the observations that Fn14 and Ect2 are overexpressed in patient Grant Support The work was supported by NIH grants R01 CA130940 (N.L. Tran), R21 glioblastoma tumors in comparison with non-neoplastic NS060023 (M.H. Symons), R01 NS055126 (J.A. Winkles), R01 CA103956 (J.C. tissue (10, 14), strongly suggest that blocking Fn14 signaling Loftus), a VARI-TGen Integration Grant (N.L. Tran and B.O Williams), and the ARCS Foundation Eller Scholarship and Science Foundation Arizona Fellowship (S.P. may slow the invasion and progression of the promigratory Fortin). population of glioblastoma tumors. Furthermore, our find- The costs of publication of this article were defrayed in part by the payment of page ing that Ect2, Cdc42, and Trio are essential signaling charges. This article must therefore be hereby marked advertisement in accordance with components that mediate TWEAK-stimulated activation 18 U.S.C. Section 1734 solely to indicate this fact. of Rac1 suggests novel avenues for therapeutic intervention Received December 27, 2011; revised April 17, 2012; accepted May 2, 2012; in TWEAK-Fn14–dependent glioblastoma tumors. The published OnlineFirst May 9, 2012.

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968 Mol Cancer Res; 10(7) July 2012 Molecular Cancer Research

Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

Shannon P. Fortin, Matthew J. Ennis, Cassie A. Schumacher, et al.

Mol Cancer Res 2012;10:958-968. Published OnlineFirst May 9, 2012.

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