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SGEF Is Regulated Via TWEAK/Fn14/NF-Kb Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide Shannon P

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SGEF Is Regulated Via TWEAK/Fn14/NF-Kb Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide Shannon P Published OnlineFirst January 13, 2016; DOI: 10.1158/1541-7786.MCR-15-0183 Signal Transduction Molecular Cancer Research SGEF Is Regulated via TWEAK/Fn14/NF-kB Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide Shannon P. Fortin Ensign1,2, Alison Roos1, Ian T. Mathews1, Harshil D. Dhruv1, Serdar Tuncali1, Jann N. Sarkaria3, Marc H. Symons4, Joseph C. Loftus5, Michael E. Berens1, and Nhan L.Tran1 Abstract Glioblastoma (GB) is the highest grade and most common ated reduction of SGEF expression sensitizes glioma cells to form of primary adult brain tumors. Despite surgical removal temozolomide-induced apoptosis and suppresses colony for- followed by concomitant radiation and chemotherapy with the mation following temozolomide treatment. Nuclear SGEF is alkylating agent temozolomide, GB tumors develop treatment activated following temozolomide exposure and complexes resistance and ultimately recur. Impaired response to treatment with the DNA damage repair (DDR) protein BRCA1. Moreover, occurs rapidly, conferring a median survival of just fifteen months. BRCA1 phosphorylation in response to temozolomide treat- Thus, it is necessary to identify the genetic and signaling mechan- ment is hindered by SGEF knockdown. The role of SGEF in isms that promote tumor resistance to develop targeted therapies promoting chemotherapeutic resistance highlights a heretofore to combat this refractory disease. Previous observations indicated unappreciated driver, and suggests its candidacy for develop- that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide ment of novel targeted therapeutics for temozolomide-refrac- exchange factor (GEF), is overexpressed in GB tumors and plays tory, invasive GB cells. a role in promoting TWEAK-Fn14–mediated glioma invasion. Here, further investigation revealed an important role for SGEF Implication: SGEF, as a dual process modulator of cell survival in glioma cell survival. SGEF expression is upregulated by and invasion, represents a novel target for treatment refractory TWEAK-Fn14 signaling via NF-kB activity while shRNA-medi- glioblastoma. Mol Cancer Res; 14(3); 302–12. Ó2016 AACR. Introduction of invasion has been shown to increase chemotherapeutic sensi- tivity (5, 6). Glioblastoma (GB) is the most common form of primary adult An increased capacity for cell survival results from the brain tumors characterized by a poorly delineated tumor mass multifaceted regulation of pathways involved in promoting resulting from highly invasive cells. The problem of resistance to cell growth, replication and spread, and preventing apoptosis the standard antiproliferative treatment of concomitant radio- in response to cytotoxic insult (7). Treatment strategies of therapy with chemotherapy using the alkylating agent temozo- tumor irradiation and temozolomide administration in glio- lomide is common, and actively invading cells survive the current blastoma lead to the formation of DNA double strand breaks therapeutic regimens. Glioma cells with the increased capacity for (DSB), either directly, or via mismatch repair conversion of migration have a decreased expression of proapoptotic genes and O(6)-methylguanine adducts into DSBs, respectively (8). DSBs are less sensitive to cytotoxic therapy–induced apoptosis (1-4); are primarily repaired through two mechanisms, homologous the knockdown of several proinvasive gene candidates in GB recombination (HR) and nonhomologous end-joining decreases glioma cell migration rate and sensitizes the cells to (NHEJ). HR repair makes use of a non-damaged homologous cytotoxic therapy and importantly, therapy directed at mediators DNA template, and thus is characterized as an error-free mechanism, while NHEJ has no homologous strand for tem- 1Cancer and Cell Biology Division, The Translational Genomics plate use resulting in sequence errors near the break point (9). Research Institute, Phoenix, Arizona. 2Cancer Biology Graduate Inter- DNA repair is initiated via sensing of DSBs by three kinases: 3 disciplinary Program, University of Arizona,Tucson, Arizona. Depart- ataxia telangiectasia mutated (ATM), ataxia telangiectasia and ment of Radiation Oncology, Mayo Clinic, Rochester, Minnesota. 4Cen- ter for Oncology and Cell Biology, The Feinstein Institute for Medical Rad3 related (ATR), and Chk2. Subsequently, the early phosphor- Research at North Shore-LIJ, Manhasset, New York. 5Department of ylation of histone H2A.X (gH2A.X) by ATM occurs at damaged Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, DNA foci and leads to the phosphorylation of mediator of DNA Arizona. damage checkpoint protein 1 (MDC1), with subsequent chro- Note: Supplementary data for this article are available at Molecular Cancer matin remodeling and recruitment of DNA repair proteins (10). Research Online (http://mcr.aacrjournals.org/). BRCA1 is one such key mediator of HR and NHEJ repair; after Corresponding Author: Nhan L. Tran, The Translational Genomics Research exposure to DNA-damaging agents BRCA1 is rapidly phosphor- Institute, 445 N. 5th St., Suite 400, Phoenix, AZ 85004. Phone: 602-343-8771; ylated by ATM, ATR, and Chk2, and relocated to sites of replica- Fax: 602-343-8717; E-mail: [email protected] tion forks with gH2A.X foci, where it recruits further proteins doi: 10.1158/1541-7786.MCR-15-0183 including BRCA2 and Rad51 to mediate strand exchange toward Ó2016 American Association for Cancer Research. DNA repair and cell survival (9). 302 Mol Cancer Res; 14(3) March 2016 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 13, 2016; DOI: 10.1158/1541-7786.MCR-15-0183 SGEF Promotes Glioblastoma Cell Survival One key driver in GB that has been characterized to promote sulfonyl fluoride. Protein concentrations were determined using both cell invasion and cell survival is the transmembrane receptor the BCA assay (Pierce) with bovine serum albumin as a standard. fibroblast growth factor inducible-14 (Fn14). Fn14 is a member of Thirty micrograms of total protein were loaded per lane and the TNF receptor superfamily with one known ligand, the TNF- separated by SDS PAGE. After 4C transfer, the nitrocellulose like weak inducer of apoptosis (TWEAK). Signaling through Fn14 (Invitrogen) was blocked with either 5% nonfat milk or 5% BSA in by its cytokine ligand TWEAK activates Rac1, Akt, and NF-kB Tris-buffered saline, pH 8.0, containing 0.1% Tween 20 (TBST) pathways, and has been shown to promote increased cell invasion prior to addition of primary antibodies and followed with per- and resistance to cytotoxic therapy-induced apoptosis (3, 4, 11). oxidase-conjugated anti-mouse IgG or anti-rabbit IgG. Protein Here we show that the src-homology 3 domain containing GEF was detected using SuperSignal West Dura Chemiluminescent (SGEF) promotes cell survival in response to temozolomide Substrate (Thermo Scientific) with a UVP BioSpectrum 500 Imag- treatment. In GB tumors, SGEF has been shown to be significantly ing System. Densitometry was calculated via ImageJ software. overexpressed, to be correlated with poor patient outcome, and to promote glioma cell migration (12). We report that SGEF expres- RNA isolation and quantitative reverse transcriptase-PCR sion is increased in a subset of temozolomide-resistant (TMZ-R) Total RNA was isolated as previously described (1). cDNA was derived primary GB xenografts and is upregulated by TWEAK- synthesized from 500 ng of total RNA in a 20 mL reaction volume Fn14 signaling via NF-kB activity. Moreover, levels of SGEF and using the SuperScript III First-Strand Synthesis SuperMix Kit Fn14 mRNA are positively correlated in GB tumor specimens. (Invitrogen) for 50 minutes at 50C, followed by 85C for 5 Depletion of SGEF impairs colony formation following temozo- minutes. qPCR analysis of SGEF (sense: 50-TGC TGA AAG GAC lomide treatment and increases cell susceptibility to temozolo- AAG GAA CA-30; antisense: 50-GTA GTT TTG ATA CAG GAC AGC mide-induced apoptosis. temozolomide treatment of glioma cells ATT-30) and histone H3.3 (sense: 50- CCA CTG AAC TTC TGA TTC both leads to increased nuclear activation of SGEF and the SGEF GC-30; antisense: 50-GCG TGC TAG CTG GAT GTC TT-30) mRNA dependent phosphorylation of the DNA damage repair protein levels was conducted using SYBR green (Roche) fluorescence for BRCA1. SGEF and BRCA1 are found in a complex upon temo- detection of amplification after each cycle with LightCycler anal- zolomide treatment. SGEF may thus be an important mediator of ysis software and quantified as described previously (3). pro-survival signaling in response to temozolomide-induced DNA damage. Biotinylated electrophoretic mobility shift assay T98G glioma cells were plated at a density of 3 Â 106 in 100 Materials and Methods mm2 tissue culture dishes in normal growth medium. After 12 Cell culture conditions hours, cells were cultured under reduced serum (0.5% FBS) for an Human astrocytoma cell lines U87, U118, and T98G (ATCC), additional 16 hours before TWEAK (100 ng/mL) addition for 2 as well as primary glioblastoma xenograft cells (GBM and GBM hours. Isolation of cell nuclear protein was carried out using the TMZ-R lines) were maintained in DMEM (Gibco) supplemented NE-PER kit (Pierce) according to the protocol of the manufacturer. with 10% heat-inactivated FBS (Gibco) at 37 C with 5% CO2. For Protein–DNA complexes were detected using biotin end–labeled all assays with TWEAK treatment, cells were cultured in reduced double-stranded DNA 23-mer probes containing the NF-kB– serum (0.5% FBS) for 16 hours before stimulation with recom- binding sites within the SGEF promoter (NF-kB-SGEF wt target binant TWEAK at 100 ng/mL in DMEM þ 0.1% bovine serum sequence: 50- GTC TAG GAG GCA AAT CCC AGA AA -30; NF-kB- albumin for the indicated times. SGEF mt target sequence: 50- GTC TAG GAG CCA GAT CGC AGA AA -30). The binding reactions were done using the LightShift kit (Pierce) according to the manufacturer's protocol. Where indi- Antibodies, plasmids, reagents, and Western blot analysis cated, 200-fold molar excess of unlabeled NF-kB-SGEF wt oligo- A polyclonal SGEF antibody was purchased from Sigma. A nucleotides or anti-p65 antibody was included.
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