The Insulin-like Growth Factor-I Receptor Is Required for EWS/FLI-1 Transformation of Fibroblasts

IRS1 Cell fusion
DOI: 10.1074/jbc.272.49.30822 Publication Date: 2002-07-26T15:07:12Z
ABSTRACT
Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 has been shown to act as potent chimeric transcription factor. Overexpression insulin-like growth factor-I receptor (IGF-IR) implicated in many tumor models playing role cell and tumorigenesis. In addition, blockade the IGF-IR inhibits cells. Therefore, we first studied whether presence required for transformation protein. To perform this study, used two previously fibroblast lines, R− W, derived from an knockout wild-type littermate, respectively. Neither W nor cells without formed soft agar colonies. However, clones expressing message (WF cells) colonies, whereas (R−F did not form Because transformation, chose investigate altered signaling occurs when present. WF demonstrated greater degree ligand-stimulated insulin substrate-1 phosphorylation compared with cells, suggesting expression alters pathway. sarcoma (ESFT) 1The abbreviations are: ESFT, tumors; IGF-IR, receptor; IRS, substrate; bp, base pair; PCR, polymerase chain reaction. are translocation 95% (1Delattre O. Zucman J. Melot T. Garau X.S. Zucker J.M. Lenoir G.M. Ambros P.F. Sheer D. Turc C.C. Triche T.J. Aurias A. Thomas G. N. Engl. Med. 1994; 331: 294-299Crossref PubMed Scopus (919) Google Scholar). This joins EWS gene located on chromosome 22 ets gene, either FLI-1 11, t(11;22) (2Delattre Plougastel B. Desmaze C. Peter M. Kovar H. Joubert I.deJ.P. Rouleau Nature. 1992; 359: 162-165Crossref (1562) Scholar) or ERG 21, t(21;22) (3Dunn Praissman L. Hagag Viola M.V. Cancer Genet. Cytogenet. 76: 19-22Abstract Full Text PDF (50) Scholar, 4Sorensen P.H. Lessnick S.L. Lopez T.D. Liu X.F. Denny C.T. Nat. 6: 146-151Crossref (668) generated result these translocations contains primary domains. domain transcriptional activator, highly conserved DNA binding (5May W.A. Braun B.S. Klemsz Lewis B.C. Lunsford L.B. Hromas R. Mol. Cell. Biol. 1993; 13: 7393-7398Crossref (447) 6Mao X. Miesfeldt S. Yang Leiden Thompson C.B. Chem. 269: 18216-18222Abstract 7Bailly R.A. Bosselut Cormier F. Delattre Roussel Ghysdael 14: 3230-3241Crossref (326) 8Ohno Rao V.N. Reddy E.S. Res. 53: 5859-5863PubMed EWS/ets resulting thought play both etiology promotion ESFT. appears maintaining growth. Growth inhibition three methods inhibiting protein: 1) RNA antisense (9Ouchida Ohno Fujimura Y. Oncogene. 1995; 11: 1049-1054PubMed Scholar), 2) dominant negative truncated intact (10Kovar Aryee D.N. Jug Henockl Schemper Gadner Cell Differ. 1996; 7: 429-437PubMed 3) oligodeoxynucleotides (11Tanaka K. Iwakuma Harimaya Sato Iwamoto Clin. Invest. 1997; 99: 239-247Crossref (233) 12Toretsky Connell Neckers Bhat Neuro-Oncol. 31: 9-16Crossref Each demonstrates its transcript decreased, reduced. fibroblasts, requires functional domains be (13May Gishizky M.L. Proc. Natl. Acad. Sci. U. 90: 5752-5756Crossref (489) mechanism whereby remains unknown. α2-β2 heterodimeric where α-subunit extracellular ligand β-subunit transmembrane intracellular tyrosine kinase binds IGF-I IGF-II lesser extent insulin. transduces signal via autophosphorylation residues subsequent downstream targets, including (IRS-1), IRS-2, Shc, Crk (14Rubin Baserga Lab. 73: 311-331PubMed critical physiologic functions development, growth, prevention apoptosis (15Resnicoff Burgaud J.L. Rotman H.L. Abraham 55: 3739-3741PubMed 16Resnicoff Yutanawiboonchai W. Kajstura Rubin Zoltick P. 2463-2469PubMed Embryos mice targeted disruption were 45% normal size nonviable at birth (17Liu J.P. Baker Perkins A.S. Robertson E.J. Efstratiadis 75: 59-72Abstract (2597) Blockade ligand-mediated transduction through using monoclonal antibody model systems (18Furlanetto R.W. Harwell S.E. Baggs R.B. 2522-2526PubMed 19Kalebic Tsokos Helman L.J. 54: 5531-5534PubMed Transformation overexpressed fibroblasts but only (20Kaleko Rutter W.J. Miller A.D. 1990; 10: 464-473Crossref (388) Until recently, all oncogenes investigated found require IGF-IR. constitutively active G-protein mutant Gα13 do express 2J.-L. LeRoith, personal communication. transform their IGF-IR-dependent pathways currently being elucidated. aberrant factor, yet there no how expressed ESFT lines; signaling, blocking stimulation, reduced (21Scotlandi Benini Sarti Serra Lollini P.L. Maurici Picci Manara M.C. Baldini 56: 4570-4574PubMed 22Yee Favoni R.E. Lebovic G.S. Lombana Powell D.R. Reynolds C.P. Rosen 86: 1806-1814Crossref (164) investigators have other transforming proteins, SV-40 large T antigen (23Sell Rubini 11217-11221Crossref (542) sought determine receptors was activity respectively (24Sell Dumenil Deveaud Miura Coppola DeAngelis 3604-3612Crossref (505) Both lines transfected, individual selected, evaluated anchorage-independent We show here also demonstrate leads generous gift (Philadelphia, PA), R+ transfected overexpress All grown Dulbecco's modified Eagles medium + 10% fetal bovine serum. TC-32, CHP-100, RD-ES contain (25Whang P.J. Knutsen Miser Kao S.S. Tsai Israel M.A. 1986; 21: 185-208Abstract (277) 26Dockhorn D.B. Schafer K.L. Dantcheva Blasius Winkelmann Strehl Burdach van Valen Jurgens Bocker Virchows Arch. Pathol. 425: 107-112Crossref (51) RPMI 1640 maintained humidified, 6% CO2 atmosphere 37 °C. type I cDNA (Los Angeles, CA). Lyophilized αIR−3 (IGF-IR antibody) purchased Oncogene Science Inc. (Cambridge, MA), IgG1 isotype-matched MOPC 21 Sigma. Santa Cruz Biotechnology, (Santa Cruz, Phosphotyrosine antibodies 4G10 PY-20 Upstate Biotechnology (Lake Placid, NY) Transduction Laboratories (Lexington, KY), IRS-1 antibodies, pleckstrin carboxyl domains, Grb2 Laboratories. obtained Ci77ba-Geigy. Immunoblot analysis performed R−, stimulated 20 nm IGF-I. Lysates prepared Tris/sodium chloride buffer Nonidet P-40, vanadate, protease inhibitors (12Toretsky 500 μg immunoprecipitated αIR−3. Separation polyacrylamide gel followed transfer nitrocellulose blotting anti-phosphotyrosine (4G10), chemiluminescent detection, below. Solution hybridization assays reported (27Lowe Roberts Lasky LeRoith 1987; 84: 8946-8950Crossref (304) 28Werner Woloschak Adamo Shen-Orr Z. Jr., 1989; 7451-7455Crossref (327) [32P]UTP probe exon 3 40 total Soft 5000 cells/well 0.3% agarose top 0.6% feeder layer. Colonies than 0.12 mm scored counted (29D'Ambrosio Hongo Li 12: 371-378PubMed 30Fei Z.L. D'Ambrosio Surmacz E. 15: 4232-4239Crossref A 1400-bp HindIII/XbaI fragment containing transferred shuttle vector, pSP72 (Promega, Madison, WI), NotI restriction sites each end multiple cloning region. TheNotI bidirectionally cloned into pCI.Neo vector (Promega), orientation determined mapping. vitro translation yielded full-length Plasmids purified ion exchange chromatography (Qiagen, Chatsworth, CA) according instructions manufacturer. introduced electroporation serum-free 0.4-cm gap chamber 350 V 1600 microfarad. Cells then prewarmed medium. 24 h after electroporation, 400 μg/ml G418 added. Clones isolated rings macroscopic continued G418. frozen regular intervals, experiments similar passage clones. initially screened 105 K Buffer consisting 50 KCl, 10 mmTris, pH 8, 2.5 MgCl2, 0.5% Tween 20, 100 proteinase amplified sense primer within distal cytomegalovirus promoter, CI.758 (5′-GCTTTATTGCGGTAGTTTATCACAG-3′) complimentary 3′ cDNA, FLI-C (5′-CCACGCGGATCCAGCTTCTAGTAGTAGCTGCCTAAGTG-3′). PCR kit manufacturer (Perkin-Elmer, Branchberg, NJ) cycles. Cycle parameters were: annealing 65 °C 2 min, extension 72 denaturing 95 1 min. products separated 2% gels. Those 2000-bp sequence one-third promoter entire further reverse transcriptase PCR. extracted RNAzol (Tel-Test Inc., Friendswood, TX) digested DNase (Boehringer Mannheim) parts. Reverse added one part water other. synthesis random hexamer priming (Perkin-Elmer). Expression primers EWS-1 (5′-ATGGCGTCCACGGATTACAGTACC-3′) FLI-A (5′-GACTGCTGGTCGGGCCCAGG-3′). integrity confirmed β-actin (5′-GCTGCGCTGGTCGTCG-3′) B (5′-ATCCTGTCAGCAATGCCTGG-3′). cycle above, Nomenclature is: designated WF, R−F respectively, clone number (i.e. WF3, R−F17), control lack insert W5, R−6). Doubling times clonal plating equal numbers duplicate 96-well plates evaluating daily 7 days. Cultures analyzed viable 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma). Absorbance 540 nm, standardized curve created software Kaleidagraph (Synergy Software, Reading, PA). doubling time calculated maximal slope curve. Clonally above. Near-confluent monolayers washed placed 12 prior stimulation (20 nm) min (31Beitner-Johnson 270: 5187-5190Abstract (109) concentration lysate manufacturers instruction (BCA, Pierce) resolved directly 8% fresh lysates. Immunoprecipitated proteins removed A-Sepharose beads gel. Proteins semi-dry apparatus. Membranes blocked 5% nonfat milk (Carnation, Glendale, blotted Antibodies Tris-sodium-EDTA-Tween 1% serum albumin (Miles Kankakee, IL) 2.5% dry milk. Chemiluminescent detection following incubation secondary anti-mouse anti-rabbit conjugated horseradish peroxidase (Amersham Corp.) Renaissance (Dupont) exposure X-Omat film (Eastman Kodak Co.). Blots stripped β-mercaptoethanol (2% sodium dodecyl sulfate, β-mercaptoethanol, 63 6.8) 70 C, reblocked, probed various Films band density digitizing films CCD camera Macintosh computer, checking threshold bands, analyzing NIH Image version 1.61 software. Phosphorylation levels corrected based upon immunoblot. phosphotyrosine divided band, IRS-1. Fold dividing unstimulated band. equation Table II. Statistics Microsoft Excell 5.0 software.Table IISummary densitometric evaluation immunoblotsIGF-I-induced (fold stimulation)Fig. 4Fig. 6IGF-IRSDt testIRS-1SDttestWF clones9.68.7p < 0.256.04.1p 0.05W clones6.25.41.50.14Films Figs. 4 6 digitized camera, densities These average values four Values equation. stimulation=(stimulated PY/stimulated IGF­IR IRS­1)(unstimulated PY/unstimulated IRS­1where PY indicates phosphotyrosine. Open table new tab could detected surface fluorescent automated sorting analysis, (data shown). confirm solution assay. Only hybridizes protects 300-bp (Fig.1, lane 3), does (Fig. 1, 4). An internal 18S ribosomal hybridizations. pCI.Neo.EWS/FLI-1 electroporated selected 5′ CI.758, FLI-C. Fig.2 isolation six typical clones, five stably product (lanes B–F). screening identify message, so initial screening, expanded extraction RNA. Fig. shows example this, residual eliminated digestion. took place parallel tubes, contained acted avoid amplification genomic (incorporated plasmid) sequences. panelof WF6, R−F2, R−F17, WF12. W5 empty transcribe message. bands themiddle panel digestion successfully DNA. Therefore any seen treated true amplification. bottom mRNA samples. Liquid culture distinguish assay marker transformation. tested utilizing cells/35-mm2 dish. colony defined cluster larger μm diameter 14 days culture. summary TableI. our produce (expressing IGF-IRs lacking EWS/FLI-1), (lacking EWS/FLI-1) produced colonies.Table ISoft clo-genic assayCloneFusion proteinIGF-IRNumber experimentsAverageWF6++763WF22++48WF3++6138WF7++532WF36++225R − F2+−20R F17+−20R 10−−20R 4−−20R 8−−20W5−+60W6−+20W8−+21W13−+205000 plated 35-mm2 dishes incubated >0.12 inverted microscope. Results colonies/well. induced direct activation overnight synchronize achieve basal Identical cultures lysed before 3-min stimulate Equal amounts antibody, electrophoresis, Fig.4 (upper panel). blot 4,lower Densitometry blots. fold increases phosphorylation, correction levels, averaged (Table II). absolute differ significantly 9.6-fold 6.2-fold (Student's t test,p 0.25). Total lysates immunoprecipitation immunoblot showed neither if IGF-I-stimulated differed between Paired stimulated/unstimulated 5 panel) protein, alternate lanes most striking difference accentuated occurring approximately 180 kDa. 180-kDa stripping membrane specific anti-IRS-1 5, lower pair samples indicating represents increased rather higher levels. revealed trend shown), focus study phosphorylation. preparing manner legend immunoprecipitating homology (Fig.6). cut 43-kDa standard blocking. residues, middle co-precipitant Grb2. WF3 WF36, prestimulation W17, relatively comparison density, performed, 4. IGF-I-induced 6.0-fold 1.5-fold II, Student'st test, p 0.05). Grb2, adapter molecule phosphorylated IRS-1, will co-precipitate correspond state IRS-1,i.e. WF36 W17 6, reprobing carboxyl-terminal antibody. 32Zoubek Dockhorn-Dworniczak Christiansen Niggli Gatterer-Menz I. Smith T.L. Oncol. 1245-1251Crossref (273) reduces 13May combination ubiquitous nature properties reduction plays significant biology development 24Sell Scholar,33Baker 73-82Abstract (2072) Ligand generates mitogenic signal, overexpressed, can independently results decreased findings suggest pathway important developed evaluate effect absence would (not able transformed protein), 3C. Denny, so, they littermate animals Most thus far activated ras SV40 29D'Ambrosio does, however, cells.2This recent finding fact events grow fashion, same not. Thus answer question some way series ligand-induced changes When averaged, slight clones; significant. docking IRS-I, known bind apparent hypophosphorylation preceding stimulation. specifically analysis. observed increase response therefore due (lower) state. corroborated coprecipitation SH2 domain-containing studies, co-precipitated Grb-2 corroborating stimulated, coprecipitated rose tandem rise Our likely reflection communication data indicate target genes activity, may represent unique interaction turn out characteristic novel factors function precise One hypothesis testing Syp another phosphatase Syp, example, SHPTP2 (34Seely B.L. Reichart Staubs P.A. Jhun B.H. Hsu Maegawa Milarski Saltiel A.R. Olefsky 19151-19157Abstract (73) (35Xiao Rose D.W. Sasaoka Burke T.R. Roller P.P. Shoelson 21244-21248Abstract Additional studies effective (36Yamauchi Ribon V. Pessin J.E. 17716-17722Abstract (52) 37Yamauchi 92: 664-668Crossref (268) recognized mutations resistant (38Kuhne M.R. Zhao Rowles Lavan B.E. Shen S.H. Fischer E.H. Lienhard G.E. 15833-15837Abstract regulatory such platelet-derived factor (39Li Nishimura Kashishian Batzer A.G. Kim Cooper J.A. Schlessinger 509-517Crossref epidermal (40Bennett A.M. Hausdorff S.F. O'Reilly Freeman R.M. Neel B.G. 16: 1189-1202Crossref (226) albeit activator explain key It possible although system, what really target, like Combinations transfections oncogene, antigen, plus (adapter kinases, IGF-IR), capable combining Ag, overexpression targets potentially converging allow bypass conclude had those Based presume pathway; described. Further investigation needed elucidate alterations aberrent EWS-FLI-1 during thank Dr. Chris discussions reagents Renato lines. acknowledge Shili Zhan assistance protocol Chari Bachman immunoblotting.
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