c-Src Kinase Activity Is Required for Hepatocyte Growth Factor-induced Motility and Anchorage-independent Growth of Mammary Carcinoma Cells

• DOI: 10.1074/jbc.273.50.33714
• Publication Date: 2002-07-26T14:56:47Z
• Authors: Nader Rahimi; Wesley Hung; Eric Tremblay; Ron Saulnier; Bruce Elliott
• Keywords: Mammary carcinoma

Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types human cancers, suggesting a critical role for Met development malignant cells. However, the molecular mechanism by which contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has implicated as modulator cell proliferation, spreading, migration; these functions are also regulated Met. To explore whether involved HGF-induced growth, mouse mammary carcinoma line (SP1) that co-expresses HGF nonmalignant epithelial (Mv1Lu) expresses but were used. In this study, we shown activity constitutively elevated SP1 cells induced response Mv1Lu addition, associates with following stimulation HGF. enhanced correlates its ability associate Expression dominant negative double mutant (SRC-RF), lacking both (K295R) regulatory residue (Y527F), significantly reduced strongly blocked motility colony soft agar. contrast, expression had no effect on proliferation plastic. Taken together, our data suggest association activation play anchorage-independent carcinomas further support notion presence paracrine autocrine loops transformed phenotype Evidence supports 1The abbreviations used are: HGF, factor; PI, phosphatidylinositol; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; PDGF, platelet-derived factor. receptor, product met protooncogene, normal (1Brinkmann V. Foroutan H. Sachs M. Weidner K.M. Birchmeier W. J. Cell Biol. 1995; 131: 1573-1586Crossref PubMed Scopus (296) Google Scholar, 2Niranjan B. Buluwela L. Yant Perusinghe N. Atherton A. Phippard D. Dale T. Gusterson Kamalati Development. 121: 2897-2908Crossref Scholar) (3Fixman E.D. Naujokas M.A. Rodrigues G.A. Moran M.F. Park Oncogene. 10: 237-249PubMed 4Bellusci S. Moens G. Gaudino Comoglio P. Nakamura Thiery J.P. Jouanneau 1994; 9: 1091-1099PubMed 5Maggiora Gambarotta Olivero Giordano Di Renzo P.M. Cell. 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Panayotou 77: 261-271Abstract (896) It important establish proteins regulate steps tumor progression, because different various cellular functions, mitogenic, motogenic, morphogenic signals target (18Ponzetto 19Tajima Matsumoto FEBS Lett. 1991; 291: 229-232Crossref (206) 20Rubin J.S. A.M. Bottaro D.P. Burgess W.H. Taylor W.G. Cech Hirschfield D.W. Wong Miki Finch P.W. S.A. 88: 415-419Crossref (471) 21Rosen E.M. Knesel I.D. Jin Bhargava Zitnik Wines Kelley Rockwell 57: 706-714Crossref (84) 22Schmidt Bladt Goedecke Brinkmann Zschiesche Sharpe Gherardi 373: 699-702Crossref (1231) HGF-mediated pathway complicated observation SH2-containing effectors bind single multifunctional docking site domain Met, whereas second required Grb2 Recent findings using mutational approach effects separate sites transducers (23Ponzetto Audero Basile M.L. Narsimhan 271: 14119-14123Abstract (143) 24Fixman Fournier T.M. Kamikura D.M. 13116-13122Abstract (108) 25Giordano Menard Ponzetto 94: 13868-13872Crossref (82) complementation intrans between two invasive-metastatic (25Giordano study specific function, approaches individual required. Recently, (26Rahimi Tremblay 24850-24855Abstract (54) others (27Royal 270: 27780-27787Abstract (229) PI 3-kinase mitogenic motogenic These argue activated factors (PDGF) (28Oude Weernink P.A. Rijksen 2264-2267Abstract (89) 29Courtneidge Kypta R.M. Cooper J.A. Kazlauskas Growth Differ. 2: 483-486PubMed 30Kypta Ulug E.T. Courtneidge 1990; 62: 481-492Abstract (480) fibroblast (31Zhan X. Plourde Hu Friesel Maciag 20221-20224Abstract epidermal (32Muthuswamy S.K. Muller W.J. 11: 271-279PubMed known modulate (33Barone M.V. 378: 509-512Crossref (283) 34Broome Hunter 16798-16806Crossref (117) spreading (35Kaplan K.B. Swedlow J.R. D.O. Varmus H.E. Genes Dev. 1505-1517Crossref (295) 36Rodier J-M. Vallés Denoyelle Boyer 761-773Crossref (72) migration (36Rodier 37Hansen Johnell Siegbahn Rorsman Engstrom Wernstedt Heldin C.H. Ronnstrand EMBO 15: 5299-5313Crossref (107) 38Hall C.L. Lange L.A. Prober D.A. Zhang Turley E.A. 13: 2213-2224PubMed types; (19Tajima 23Ponzetto 4-fold cancer (39Rosen Bolen J.B. Schwartz Cohen DeSeau Israel 1986; 261: 13754-13759Abstract 40Ottenhoff-Kalff A.E. G.A.U. Hennipman Michels A.A. Staal G.E. 52: 4773-4778PubMed Neu-induced transgenic mice (41Muthuswamy Siegel Dankort D.L. Webster 14: 735-743Crossref 42Guy C.T. Muthuswamy Cardiff R.D. Soriano 8: 23-32Crossref (187) Activation induces hyperplasias required, sufficient, induction tumors polyoma virus middle T-transgenic (42Guy 43Webster 92: 7849-7853Crossref (71) Altogether, plays clearly analyze line, SP1, tyrosine-phosphorylated thereby generating (44Rahimi McAdam Schwall & 7: 263-270PubMed Our current results demonstrate cells, compared We therefore examined expressing form significant characteristics phenotype. Rabbit anti-sheep IgG conjugated horseradish peroxidase was from Jackson ImmunoResearch Laboratories (Westgrove, PN). Mouse anti-phosphotyrosine (PY20) monoclonal antibody purchased Transduction (Lexington, KT). anti-c-Src IgG, anti-Met (mouse) anti-PLC-γ1 obtained Santa Cruz Biotechnology (San Diego, CA). members mink lung ATCC (Rockville, MA). Maintenance medium Dulbecco's modified Eagle's (Life Technologies, Inc.) supplemented 10% FBS. derived spontaneous poorly metastatic murine intraductal adenocarcinoma described elsewhere (45Rahimi Saulnier DNA 1189-1197Crossref (98) Scholar,46Elliott Tam S.P. Dexter Chen Z.Q. 51: 416-424Crossref (80) RPMI 1640 7% FBS Inc.). cDNAs encoding wild typec-src (SRC) ofc-src (SRC-RF) loss-of-function mutations (Y527F) ligated into pRc/CMV plasmid (Invitrogen, San CA) carrying neomycin resistance marker Dr. Brugge (47Mukhoupadhyay Tsiokas Zhou X.M. Foster Sukhatme V.P. 375: 577-581Crossref (539) type established stable transfection LipofectAMINE method (48Seth G.N. Katayose Gress Pastan Cowan 56: 1346-1351PubMed Briefly, grown 80% confluence. (1 μg) mixed reagent (9 μl) 200 μl serum-free incubated 15 min at room temperature. Before transfection, washed once 2 ml medium. For each combined 0.8 medium, mixture. After 5 h incubation, equal volume RPMI/14% added incubation proceeded additional 24 h. most experiments, pooled transfected selected G418 (450 μg/ml) one experiment, SRC-RF SRC, clones isolated tested Src forming efficiency. carried out plated 104cells/well 24-well plates under conditions indicated. synthesis measured adding 0.2 μCi [3H]thymidine (Amersham Pharmacia Biotech, Oakville, ON, Canada) h, harvested trypsin/EDTA. Aliquots placed 96-well microtiter transferred filters Titertek harvester (ICN, Costa Mesa, CA), incorporation scintillation counter (Beckman, Mississauga, Canada). Results expressed mean cpm/well ± S.D. triplicates. Colony assays performed previously (49Saulnier Bhardwaj Klassen Leopold Rahimi Mosher Exp. 222: 360-369Crossref (32) solution 1.2% Bactoagar (Difco Lab) (1:1) 2× 1640, final concentrations 7 1% alone indicated, layered onto 60 × 15-mm tissue culture plates. (103/2.5 ml) 0.36% prepared similar way (2.5 ml/plate) top 0.6% layer. Plates 37 °C 5% CO2 8–10 days. Colonies fixed methanol, stained Giemsa, counted manually. colonies per dish quadruplicates. measure motility, Transwell inserts (8-μm pore size) (Costar, Toronto, coated uniformly gelatin (0.25% w/v, Sigma, sides temperature (50Boyer Roche 16: 5904-5913Crossref (124) Membranes twice inserted plate 1 containing 0.5 mg/ml serum albumin Cells 50% confluence, serum-starved overnight, mm EDTA. (2 104/100 insert 6–8 °C. Following excess removed, paraformaldehyde (Sigma) hematoxylin (Fisher, upper side membrane removed wiping cotton. inverted microscope phase contrast illumination. migrating well. parallel wounding assay performed, monolayers "wounded" scraping Eppendorf yellow tip, washed, varying times. Migration assessed visually close wounded area. confluence rinsed cold phosphate-buffered saline three times lysed lysis buffer 50 Tris-HCl, pH 7.5, 150 NaCl, Nonidet P-40, Na3VO4, NaF, EGTA, μg/ml aprotinin, leupeptin, phenylmethylsulfonyl fluoride. Lysates centrifuged 10 14,000 rpm IEC/Micromax centrifuge 4 Protein concentration supernatants determined bicinchoninic acid protein (Pierce). Equal amounts lysate indicated antibodies overnight. Immunoprecipitates collected A-Sepharose Biotech), buffer, separated SDS-PAGE, nitrocellulose membrane. 3% skimmed milk TBST (10 8.0, 0.1% Tween 20), probed antibodies. peroxidase-labeled secondary anti-rabbit anti-mouse min, time. Immune complexes ECL (Amersham). anin vitro enolase substrate (51Atfi Drobetsky Boissonneault Chapdelaine Chevalier 30688-30693Abstract lysates prepared, immunoprecipitated (Santa Biotechnology) above. amount pre-determined over protein, indicating (data shown). One-half immunoprecipitate subjected SDS-PAGE nonreducing Western blot analysis confirm present. half assayed activity, incubating reaction (20 7.0, MnCl2, μmNa3VO4), freshly acid-denatured (5 μg + HCl 30 then neutralized m 7.0), [γ-32P]ATP. °C, reactions terminated addition SDS sample samples 8% SDS-PAGE. Serine threonine phosphorylations hydrolyzed acrylamide 1m KOH 45 followed fixing 45% MeOH acetic drying 80 vacuum. Autoradiograms produced quantitated Storm PhosphorImager (Molecular Dynamics, Sunnyvale, some experiments (see Fig. 3), according Cheng et al. (52Cheng Nishio Hatase Ralph Wang 267: 9248-9256Abstract family-specific cdc2 peptide substrate. Anti-c-Src immunoprecipitates above 40 (100 0.4 Mg(OAc)2), Inc., 250 μm/assay), ATP (25 μm), 2.5 control consisting immunoprecipitation more stringent RIPA (150 1.0% 0.5% deoxycholate, SDS, Tris, 8.0) where would co-precipitated out. 20 40% trichloroacetic min. subsequently blotted p81 paper (Whatman, Fisher, Ottawa, min/wash) 0.75% phosphoric acetone temperature, radiolabeled liquid counter. μmNa3VO4). [γ-32P]ATP stopped β-mercaptoethanol. Samples boiled 3 Dynamics). express consistent test possibility Met-induced pathways, HGF-sensitive Mv1Lu. capacity phosphorylate substrate, enolase. showed pronounced only slightly treatment exogenous (Fig. 1). highly dependent levels observed correlated constitutive phosphorylation shown) (Ref. 26Rahimi Scholar conceivable could resulted interaction due