A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease

• DOI: 10.1074/jbc.m801192200
• Publication Date: 2008-04-12T00:58:49Z
• Authors: Masahiro Yanagisawa; Deborah Huveldt; Pamela Kreinest; Christine M. Lohse; John C. Cheville; Alexander S. Parker; John A. Copland; Panos Z. Anastasi...
• Keywords: Ectopic expression

p120 catenin is a cadherin-associated protein that regulates Rho GTPases and promotes the invasiveness of E-cadherin-deficient cancer cells. Multiple isoforms are expressed in cells via alternative splicing, all them essential for HGF signaling to Rac1. However, only full-length (isoform 1) invasiveness. This selective ability isoform 1 mediated by reduced RhoA activity, both under basal conditions following treatment. All can bind vitro, central binding site. cooperative domain alternatively spliced N terminus stabilizes inhibits activity. Consistent with this, increased expression 1, when compared other isoforms, predictive renal tumor micrometastasis systemic progression, nephrectomy. Furthermore, ectopic RhoA-binding, N-terminal sufficient block inhibit promote The data indicate during progression contributes invasive phenotype cadherin-deficient carcinomas valid therapeutic target. During epithelial acquire invade surrounding tissues eventually metastasize. A number pathways have been uncovered date local metastatic spread, recent evidence argues most these converge on loss E-cadherin or function (1Berx G. Van Roy F. Breast Cancer Res. 2001; 3: 289-293Crossref PubMed Scopus (355) Google Scholar, 2Conacci-Sorrell M. Zhurinsky J. Ben-Ze'ev A. Clin. Invest. 2002; 109: 987-991Crossref (525) Scholar). main cell-cell adhesion molecule, its tumors coincides an mesenchymal transition (EMT), 2The abbreviations used are: EMT, transition; HGF, hepatocyte growth factor; ccRCC, clear cell carcinoma; shRNA, short hairpin RNA; mAb, monoclonal antibody; ANOVA, analysis variance; GEF, guanine nucleotide exchange PBS, phosphate-buffered saline; GST, glutathione S-transferase; ROCK, RhoA-Rho kinase. where shed their characteristics more phenotype. EMT associated normal development wound healing, but aberrant regulation metastasis (3Thiery J.P. Nat. 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Christofori Nature. 1998; 392: 190-193Crossref (1205) reestablishing lines reverse phenotype, suggesting acts suppressor invasion (9Vleminckx Vakaet Jr., Fiers W. 1991; 66: 107-119Abstract (1513) mechanism which suppression still unclear. adhesive function, extracellular domain, thought be involved this effect (10Wong A.S. Gumbiner B.M. 2003; 161: 1191-1203Crossref (219) intracellular interacts β-catenin (p120), separate, conserved interaction domains. Recent argue important anti-invasive properties 11Yanagisawa Anastasiadis P.Z. 2006; 174: 1087-1096Crossref (111) stabilization cadherin complexes plasma membrane thus strengthens (12Xiao Allison D.F. Buckley Kottke M.D. Vincent P.A. Faundez V. Kowalczyk A.P. 163: 535-545Crossref (347) 13Davis Ireton R.C. Reynolds A.B. 525-534Crossref (571) In addition, overexpression induces dramatic changes morphology increases motility (14Anastasiadis Curr. Opin. 13: 604-610Crossref (229) These effects apparently suppress activity (15Anastasiadis Moon S.Y. Thoreson Mariner D.J. Crawford H.C. Zheng Y. 637-644Crossref (380) 16Noren N.K. Liu B.P. Burridge Kreft B. 150: 567-580Crossref (470) Scholar) induce activities related GTPases, Rac1 Cdc42 (16Noren 17Grosheva Shtutman Elbaum Bershadsky A.D. Sci. 114: 695-707Crossref When overexpressed cells, rapidly saturates available cadherin-binding sites then accumulates cytoplasm. Endogenous also migration (11Yanagisawa blocks motility, recruitment reduces toward thereby determining balance between sessile motile states 15Anastasiadis Based observations, could play two different roles human (reviewed Ref. 18Thoreson Differentiation. 70: 583-589Crossref (135) Loss result stability promoting invasiveness, whereas mislocalize p120-mediated GTPases. Most types express multiple variants (termed therein) p120, derived splicing single gene (19Mo Y.Y. 1996; 56: 2633-2640PubMed 20Keirsebilck Bonne S. Staes Hengel Nollet Genomics. 50: 129-146Crossref (139) events lead use four translation start site ATGs (20Keirsebilck Scholar), resulting type 2, 3, 4, respectively. Epithelial normally 3 predominantly 21Aho Levansuo Montonen O. Kari Rodeck Uitto 115: 1391-1402Crossref mesenchyme-specific (full-length p120) often turned switch overall pattern. switches cadherin-independent manner (22Ohkubo T. Ozawa 2004; 117: 1675-1685Crossref (283) physiological significance has it expected will functional differences, since they differ inclusion several motifs, including coiled-coil (N-terminal "regulatory" (lacking 4). Both domains likely mediate protein-protein interactions, either indirectly phosphorylation, occurs predominantly, exclusively, at (23Xia X. Biochemistry. 42: 9195-9204Crossref (59) 24Mariner Keilhack Bohmer F.D. Wang Chem. 276: 28006-28013Abstract (126) truncation mutant lacking entire abrogated epidermal factor-stimulated HGF-mediated scattering (25Cozzolino Stagni Spinardi Campioni N. Fiorentini Salvati Alema Salvatore A.M. 14: 1964-1977Crossref (71) receptor tyrosine kinase-induced motility. report, tested first time. Using we found depends expressed. Expression no effect, 4 correlates domain; however, required inhibition. Testing classic example (ccRCC), ratio predicts show validating potential Culture, Infections, Transfections—Culture described previously MDA-MB-231 expressing p120-specific shRNA retroviral infection (pRS vector) As indicated, vector control (pRS) p120-depleted were infected second time LZRS-neo zeo viruses, viruses murine 1A, 3A, 4A, N1 mutant, selected mg/ml G418 350 μg/ml zeocin. Amaxa electroporations performed according company's protocol. brief, 106 resuspended 100 μl solution T (Amaxa Inc.) containing 2 μg plasmid DNA. Electroporation was using program A-23. Electroporated plated 60-mm dishes incubated 24 h culture medium. Cells washed PBS another 12 serum-free Dulbecco's minimal medium prior performing assays. Constructs—LZRS-neo, LZRS-mp120 1A-neo, 3A-neo (26Ireton Davis Barnes Matrisian Bundy L.M. Sealy Gilbert 159: 465-476Crossref (439) pRS LZRS-MS-zeocin provided Dr. Al (Vanderbilt University, Nashville, TN) encodes zeocin instead neomycin resistance. pcDNA3-N1 (21Aho To generate LZRS-N1-zeo, initially digested XhoI, ends filled Klenow, fragment released additional digestion EcoRI. ligated EcoRI AfeI LZRS-MS-zeocin. 4A-neo generated subcloning EcoRI-XhoI LZRS-murine 4A-GFP respective 1A-neo vector. Finally, pcDNA3-RhoA-V14-Myc (CA-RhoA) constructs verified sequencing. Western Blotting—Western blotting procedures conducted Primary antibodies follows: 0.25 anti-p120 mAb (BD Biosciences), 5 anti-FLAG tag (M2; Sigma), anti-Myc (9E10; 0.5 anti-cadherin 11 (Zymed Laboratories Inc.), 0.6 anti-RhoA (26C4; Santa Cruz Biotechnology, Inc., Cruz, CA), 0.75 anti-Rac1 anti-actin goat polyclonal antibody (I-19; Biotechnology). Secondary peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunologicals) anti-goat (Santa Biotechnology) 1:10,000. Invasion Migration Assays—Cell measured vitro BioCoat Matrigel-coated chambers (8-μm pore size; BD Briefly, serum-starved overnight, harvested Stripper (Mediatech, density × 105 cells/ml. μl(5 104 cells) suspension added top chamber, 20 ng/ml (Peprotech, 5% fetal bovine serum lower chamber chemoattractant. allowed Matrigel migrate underside 37 °C CO2. surface removed gentle scrubbing cotton swab, stained 0.2% crystal violet counted. Control established significant differences existed assay. Data from percentage represent mean ± S.E. least three independent determinations duplicate. One asterisks p < 0.05 0.01, respectively (t test one-way ANOVA followed post hoc comparisons Newman-Keuls test). Y27632 kinase inhibitor (Calbiochem) some 0.1 0.01 μm. Rho/Rac Activity Assays—The determined MDA-231 specific pull-down assays activated forms proteins reported Rhotekin RBD PAK-1 PBD (Upstate bound glutathione-agarose beads precipitate GTP-bound lysates, Active, total RhoA/Rac1 visualized SDS-PAGE RhoA-specific Rac1-specific Biosciences). Guanine Nucleotide Exchange Assay—The [3H]GDP/GTP 25 mg [3H]GDP-bound ∼4 GST-p120-1A, GST-p120-4A, GST-p120-4A-ΔRho His-TrioC (a Rho-specific GEF) ambient temperature GEF assay buffer (27Li R. 1997; 272: 4671-4679Abstract (60) reactions terminated indicated times nitrocellulose filter binding, amount [3H]GDP remaining radionucleotide 0. Purification Recombinant Proteins—Escherichia coli (BL21-DE3) His-RhoA cultured overnight ml 2× YT 50 ampicillin. expanded 8-fold fresh (40-ml final volume) 90 min °C. induced incubating bacteria mm isopropyl 1-thio-β-d-galactopyranoside Bacteria (6000 g 10 °C) lysed 1.5 lysis (PBS 0.5% Nonidet P-40, imidazole, proteinase inhibitors). Following brief sonication, lysates cleared insoluble debris (14,000 min), 80-100 nickel affinity gel (Sigma) supernatants. After rotation °C, wash (50 H2NaPO4, pH 8.0, 0.3 m NaCl, imidazole) twice GDP (20 Tris, 7.5, MgCl2, dithiothreitol, 0.1% Triton X-100). 500 μm (G7127; Sigma) rotate room temperature. step, GDP-bound eluted 170 elution 250 imidazole). GST fusion (GST-p120 mutants) purified BL21(DE3) coli, induction generation bacterial (in P-40 inhibitors), clarified supernatants (G4510; Sigma). agarose Tris-HCl, 7.3, 150 EDTA, Vitro Binding Assays—GST (GST, GST-p120 1N, 1A-ΔRho, 4A-ΔRho, GST-N-terminal mutants X-100) mixed 30 (above) 750 buffer. temperature, five boiled Laemmli sample remove proteins. Human Renal Carcinoma Tumor Processing Analysis—Hematoxylin/eosin-stained sections matched analyzed pathologist confirm diagnosis, staging, integrity tissue samples. Samples eligible consenting individuals treated nephrectomy newly diagnosed, histologically confirmed, localized ccRCC Mayo Clinic January 2000 July 30, 2004. Forty deidentified cases progressed 3-6-year follow-up did progress) frozen chosen extraction processed approval Institutional Review Board. Ten 10-μm-thick slices cut each block. Protein isolated direct solubilization Analysis Tissue—Protein samples quantified nitric acid-mediated nitration (28Bible K.C. Boerner S.A. Kaufmann S.H. Anal. Biochem. 1999; 267: 217-221Crossref (26) Equal amounts (∼30 μg) resolved 7% SDS-polyacrylamide gels, transferred polyvinylidene difluoride (Immobilin-P; Millipore, Billerica, MA), subjected immunoblot appropriate (mAb HECD-1 E-cadherin, 15D2 recognizing C-28 c-Met) ECL Plus detection (Amersham (29Zhang Aubrey Thompson Fields 279: 22118-22123Abstract (125) Images obtained X-Omat AR film, antigens fluorescence Typhoon 9410 variable mode imager. fluorescent signal ImageQuant 5.2 software Immunohistochemistry done paraffin-embedded primary tissues. deparaffinized placing slides xylene rehydrated graded ethanol series. rinsed water antigen retrieval citrate (pH 6.0) manufacturer (DAKO, Carpinteria, CA). Slides 3% H2O2 reduce endogenous peroxidase (w/v) Tween 20. c-Met detected 1:1000, 1:400, 1:200, dilution, respectively, PBS/Tween Envision Dual Labeled Polymer Kit manufacturer's instructions (DAKO). captured ImagePro software. Differential Effect Isoforms Invasiveness—Previously, depletion significantly reexpression rescues hypothesis differentially affect migration, GTPase signaling, amphotropic retroviruses (Fig. 1A). Polyclonal (shRNA-neo), 4A after days incubation G418. selection, than 95% given isoform. Invasiveness gradient ng/ml) transwell filters "Experimental Procedures." chemoattractant, previous studies shown blocking prevents nude (30Jiang W.G. Grimshaw Martin T.A. Davies Parr Watkins Lane Abounader Laterra Mansel R.E. 9: 4274-4281PubMed 1A promoted shRNA-neo 1B). Interestingly, 3A had blocked Identical results NIH3T3 fibroblasts, chemoattractant 1C). Therefore, regulatory consistent role maintaining predominant Activities—To differential due levels GTP-bound, active rhotekin PAK assays, Initial focused exhibited biggest First, studied conditions, overnight. seen Fig. 2A, constitutive (shRNA-neo) decreased rescued activation. Reexpression able activation comparable those 2A). characterize further course state Serum-starved (up 40 min) HGF. split two, (active) same (see 'Experimental Procedures"). densitometric GTP-b