The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition

• DOI: 10.1074/jbc.m700075200
• Publication Date: 2007-04-11T03:52:44Z
• Authors: Samudra K. Dissan...; Michael Wade; Carrie E. Johnson; Michael P. O'Connell; Poloko D. Leotlela; Amanda D. French; Kavita V. Shah; Kyle J. Hewitt; Devin T. Rosenthal; Fred E. Indig; Yuan Jiang; Brian J. Nickoloff; Dennis D. Taub; Jeffrey M. Trent; Randall T. Moon; Michael Bittner; Ashani T. Weeraratna
• Keywords: WNT5A; Metastasis suppressor

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) WNT5A by microarray analysis. Increasing suppressed expression several genes, which were re-expressed after small interference RNA-mediated knockdown WNT5A. Genes affected include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at protein level using both RNA recombinant (rWnt5A). Among genes up-regulated was gene vimentin, associated with an epithelial to mesenchymal transition (EMT), involves decreases E-cadherin, due up-regulation transcriptional repressor, Snail. rWnt5A treatment Snail vimentin expression, even presence dominant-negativeTCF4, suggesting this activation is independent Wnt/β-catenin signaling. Because can signal via kinase C (PKC), role PKC Wnt5A-mediated EMT also assessed inhibition studies. Treating cells expressing low levels phorbol ester increased inhibiting high decreased Furthermore, before blocked implying potentiate induction PKC-dependent manner. The molecular mechanisms govern are not well understood. prognosis for patients recurrent has no improvement over past 50 years. Many these tumors histopathologically quite similar but subclassified based upon their profiles (1Bittner M. Meltzer P. Chen Y. Jiang Seftor E. Hendrix Radmacher Simon R. Yakhini Z. Ben-Dor A. Sampas N. Dougherty Wang Marincola F. Gooden C. Lueders J. Glatfelter Pollock Carpten Gillanders Leja D. Dietrich K. Beaudry Berens Alberts Sondak V. Nature. 2000; 406: 536-540Crossref PubMed Scopus (1717) Google Scholar, 2Clark E.A. Golub T.R. Lander E.S. Hynes R.O. 532-535Crossref (1309) Scholar). In study Bittner et al. Scholar), best separated highly aggressive from less WNT5A, consistently underexpressed motile tumors. member Wnt family proteins, first identified studies development Drosophila (3McMahon A.P. Moon R.T. Development. 1989; 107: 161-167PubMed Scholar) mouse mammary virus (4Nusse van Ooyen Rijsewijk Lohuizen Schuuring van't Veer L. Proc. Soc. Lond. B. Biol. Sci. 1985; 226: 3-13Crossref (13) Unlike its members Wnt1 Wnt3A, canonical pathway, resulting nuclear translocation β-catenin, acts G-protein-coupled receptors activate (PKC) 6The abbreviations used are: PKC, C; siRNA, RNA; GFP, green fluorescent protein; PMA, 4β-phorbol-12-myristate-13-acetate; CAMKII, calmodulin-dependent II; r, recombinant; EMT, transition; NFAT, factor activating T-cells; dn, dominant negative; MMP-2, matrix-metalloproteinase-2. intracellular calcium (5Sheldahl L.C. Park Malbon C.C. Curr. 1999; 9: 695-698Abstract Full Text PDF (404) 6Sheldahl Slusarski D.C. Pandur Miller J.R. Kuhl Cell 2003; 161: 769-777Crossref (270) interplay between two understood, it does appear non-canonical pathway inhibit β-catenin stabilization vitro human HEK293 vivo zebrafish (7Westfall T.A. Brimeyer Twedt Gladon Olberding Furutani-Seiki 162: 889-898Crossref (217) 8Topol X. Choi H. Garrett-Beal Carolan P.J. Yang 899-908Crossref (629) overexpressing resulted increase (9Weeraratna A.T. Hostetter G. Rosenblatt Duray Trent J.M. Cancer Cell. 2002; 1: 279-288Abstract (790) High correlated poor outcome study. addition, many highlighted importance G-protein-mediated signaling resultant calcium, progression (10Parker Sherbet G.V. Melanoma Res. 1992; 2: 337-343Crossref (26) 11Li S. Huang Peng S.B. Int. Oncol. 2005; 27: 1329-1339PubMed 12Fink-Puches Helige Kerl Smolle Tritthart H.A. Exp. Dermatol. 1993; 17-24Crossref (14) Serial analysis samples confirmed observation, specifically, expressed libraries, including WNT10A, FRIZZLED7, junction plakoglobin (13Weeraratna Becker Carr K.M. P.H. K.P. Strausberg R.L. Riggins G.J. Wagner U. Kallioniemi O.P. Morin P.S. Oncogene. 2004; 23: 2264-2274Crossref (61) further our previous results concerning effects on cells, as better dissect might affecting melanoma, siRNA technology knock down then assayed changes subsequently combination treatments, approach allows us identify subset specifically provides insights how mediating Lines—The line UACC1273, subclones UACC-1273EV, UACC1273-4-3, UACC12734-7, lines UACC647, M93-047, UACC-903, cultured RPMI 1640 medium, G361 McCoy's 5A medium. All media supplemented 10% fetal bovine serum (HyClone, Logan, UT), 100 units/ml penicillin G, units streptomycin. cultures incubated 37 °C 5% CO2/95% air, medium replaced every second day. Transfections Treatments—siRNA designed Qiagen online design tools, designs 21-nucleotide according Tuschl rules design. Three siRNAs described text purchased rhodamine-tagged untagged forms. Rhodamine-tagged control Qiagen. These transfected into (60-70% confluency) Lipofectamine Plus (Invitrogen). Cells allowed reach 60-70% confluency within 48 h seeding. After 6 transfection, fresh serum-containing Transfection efficiencies usually upwards 90% oligonucleotides gauged transfection siRNAs. For confocal microscopy diluted 1:3 so fluorescence would overwhelming. negative TCF4 vector (a generous gift Dr. Bert Vogelstein, Sidney Kimmel Comprehensive Center, Johns Hopkins Medical Institutes, Baltimore, MD), around 75%, simultaneous GFP transfection. Interestingly, efficiency dependent confluency, densities higher than 80% result inefficient data available authors request. obtained R&D systems, reconstituted sterile phosphate-buffered saline containing 0.1% albumin stock concentration 10 μg/ml. testing range concentrations, time points, 0.1-0.2 μg/ml 12-16 serum-free decided upon, because 24 starts lose activity media. According Systems measurements, assess ability Wnt3A activity, 5 times thus treated 0.04 same points. studies, times, inhibitors tested, ultimately, Gö6983 specific inhibitor α, β, γ, δ, ζ, μ) GF 109203X (inhibitor ϵ) attempt use different conventional concentrations 1 μm each. either pre-treated Western analysis, or 12 wound healing assays, above, continuing inhibitor, alone indicated times. ester, 4β-phorbol-12-myristate-13-acetate (PMA) 200 nm. vehicle controls, equivalent amounts Me2SO. Wound Healing Assays—Wound assays performed plating plates coated fibronectin collagen IV. attach scratch made through confluent monolayer pipette tip. Photographs invading taken triplicate, each experiment, experiment repeated three Microarray Analysis—RNA extracted culture concentrated 3.6 μg/μl Micron-100, total μg array bound anchored oligonucleotide-deoxythymidine min 75 °C, cooled 42 °C. Oligonucleotides Cy dyes added, reverse transcriptase, reaction carried out h. Samples hydrolyzed 0.5 m EDTA NaOH 15 65 neutralized equal volume HCl, probe purified running PCR purification kit, manufacturer's protocol, exception addition 35% guanidium-HCl wash prior PE buffer step. Cy-3 probes Cy-5 combined point dried lyophilizer. Dried resuspended Agilent hybridization protocol applied slides overnight. customized cDNA arrays. hybridization, washed 2 0.1× SSC, SDS room temperature final rinse 0.05× SSC temperature. Fluorescence images DNA scanner, analyzed IPLAB suite. Gene intensities background subtracted (taking mean channel median background). normalization, ratios red intensity generate multidimensional scaling diagram perform selection among classes, linear discriminant previously evaluate probability given achieving significant weight chance, random permutation test 1000 permutations empirical significance threshold comparison calculated p value gene. analyses NHGRI, NIH in-house suite web-based tools. Blotting—Sources antibody follows: Phospho-Pan-PKC (1:1,000), Phospho-CaMKII (1:500), CaMKII (1:1,000) Signaling Technology (Danvers, MA). (1:100) Biosystems (Minneapolis, MN). KISS1 (1:100), Tubulin (1:2,000) Abcam Inc. (Cambridge, E-Cadherin antibodies non-phosphorylated isoforms BD Biosciences (San Jose, CA). grown harvested ice lysis (20 mm Tris, pH 7.5, 150 NaCl, EDTA, 1% Triton X-100, 1× protease mixture (Roche Applied Science), sodium orthovanadate). Dounce-homogenized centrifuged 10,000 × g min. supernatant quantitated Pierce BCA quantitation assay. lysate run SDS-PAGE Tris-glycine NuPAGE gels transferred onto 0.2-μm nitrocellulose. membranes probed visualized ECL system (Amersham Biosciences). Phosphatase Assays—Protein lysates subjected electrophoresis polyvinylidene difluoride membrane. duplicate sets single gel, membrane cut two. One-half (one set samples) (0.1 Tris·Cl, 8.5, 0.2 EDTA) alkaline phosphatase units/ml), Maya (16Maya Oren 19: 3213-3215Crossref (34) against γ CAMKII above. Gelatin Zymography—The MMP-2 gelatin zymography. plated density 10-centimeter tissue dishes grow ∼60% they PMA (Gö6983 109203X) serum-free, phenol red-free Medium Centriplus YM-10 columns (Millipore, Billerica, MA), determined assay kit (Pierce). 30 non-reducing conditions zymogram gel electrophoresis, zymogram-renaturing remove followed 24- 48-h incubation developing Gels stained 30% methanol, glacial acetic acid, 0.25% Coomassie Blue destained 4 methanol/10% acid. Clear bands Coomassie-stained blue areas gelatinolytic activity. Experiments least four Real-time Analysis—Primers PerkinElmer Life Sciences 5700 system. Primers products bp, efficient listed forward 5′-ATTCCACTTTGCGTTCAAGG-3′; 5′-CTTCAGAGAGAGGAAGCCGA-3′; SNAIL, 5′-AGGTTGGAGCGGTCAGC-3′, 5′-CCTTCTCTAGGCCCTGGCT-3′. transcribed RNA, (Applied Biosystems, Foster City, CA), ng real-time transcription-PCR reaction. normalized 18 S gene, universal primers (Ambion, Austin, TX) ratio S, -fold melanocyte controls calculation 2-ΔΔCT (PerkinElmer Sciences). Immunofluorescence—Cells glass (note transfections 60% mentioned before). They fixed 95% methanol saline. Nuclei permeabilized incubating X-100 (pH 7.4, magnesium free) Slides immunofluorescence blocking (0.2% 3% albumin, 0.2% casein, gelatin, 0.02% azide) h, overnight incubation. Primary include: (1:50, Inc.), (1:100, E-cadherin Biosciences), CD44 Systems). again Alexafluor 488 594 secondary (Molecular Probes, Eugene, OR), washed, mounted Prolong gold anti-fade Probes), examined under imaged Zeiss LSM 510 Meta microscope (Thornwood, NY). experiments light (Zeiss Axiovert 200). sequences BLAST base ensure target regions synthesized (Fig. 1A). efficacy tested endogenously line, M93-047 1B). most sequence, A2, decrease had been stably (UACC1273-4-7), UACC1273-4-7 protein, immunofluorescent detection 1c). Finally, Wnt5A-A2 Both UACC903 (with levels), whether untreated, able close respectively. However, yet closed time, confirming 1D). phosphorylation all phosphorylation. predominantly PKCα,-β, -γ status Pan-PO4-PKC (Cell Signaling). independently empty vector-transfected (UACC1273EV), (Wnt5Alow) parental (UACC1273-4-7). A2 sequence transfectants (Wnt5Atfx), without Wnt5Alow dramatically 2A). (Wnt5Ahigh) showed amount phosphorylated when siRNA-A2 ascertain deactivation investigated translocated active site cell, Wnt5A. PKCβII. transiently GFP-tagged PKCβII microscopy. 2B). Some did exhibit cytoplasmic staining, accounted only ∼20% When GFP-PKCβII (Wnt5A-A2Rh) 2c), moved cytoplasm. Although vast majority percentage (in case 10%) staining. Levels 2D, all) unaffected isoform (BD mix observed -γ), whereas decreased. observation calmodulindependent II (CAMKII), known activated Ca2+ release binding (14Kuhl Sheldahl Chem. 275: 12701-12711Abstract (402) Activation Thr286 residue (15Chin Means A.R. Biochemistry. 41: 14001-14009Crossref (20) phospho-Thr286 significantly 2E). As antibodies, unclear inability recognize form, if truly represented pool CAMKII. determine this, employed dephosphorylate proteins already Upon phosphatase, detected lanes -γ, contrary what un-phosphatased PKCα,β,γ+PPase). appeared affect blot, making harder definitively determine, possibly sensitive, untreated samples, something 2E, CAMKII+PPase). indicates phosphorylation-sensitive epitopes. Regardless, corresponds demonstrated activation, motility, migration Recently manipulating other namely claudin-1 (17Leotlela P.D. Wade M.S. Rhode M.J. Brown H.F. Rosenthal D.T. Dissanayake S.K. Earley Indig F.E. Nickoloff B.J. Taub D.D. Weeraratna 2006; press)PubMed turn migratory capacity Here provide additional pieces demonstrate itself act inhibited Wnt5Ahigh measured PO4-PKC 3A). situation knockdown, where there inversely along PO4-PKC, treating inhibitors, working PKC. others (18Bartolome R.A. Molina-Ortiz I. Samaniego Sanchez-Mateos Bustelo X.R. Teixido 66: 248-258Crossref (107) critical show here secretion zymography 3A, white dark Additionally, inflicting marked 3B). Next, looked An interesting should addressed here: long term depletion It experience that, (200 nm), fluctuate increasing, decreasing, finally stabilizing 4-12 remain elevated up depending 3C), requires replenishment looking found too steadily (see Fig. 5C). implies like some depletion. another manner visually PKC-GFPβII them nm) slides, 3D). caused early points such min, late much still remained present cytoplasm Increases mimicked 3E) imaging cell-associated 3F). Accordingly, rWnt5A, dramatic invade 3G, rWnt5A). PMA). Inhibiting