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Simultaneous Expression of Caveolin-1 and E-Cadherin in Ovarian Carcinoma Cells Stabilizes Adherens Junctions through Inhibition of src-Related Kinases

2005; Elsevier BV; Volume: 167; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)61228-x

1525-2191

Silvia Miotti, Antonella Tomassetti, Ileana Facetti, Elena Sanna, Valeria Berno, Silvana Canevari,

Wnt/β-catenin signaling in development and cancer

Cadherin-mediated adhesion plays an important role in maintaining cell-cell contacts and reducing tumor metastasis. However, neo-expression of E-cadherin in ovarian carcinoma does not prevent the release and spread of cells from the primary tumor. Because caveolin-1 is down-regulated concomitantly with E-cad expression, we investigated whether the stability of adherens junctions in ovarian carcinoma was affected by caveolin-1 expression. We used IGROV1 cells transfected with caveolin-1 (IGtC3), mock-transfected control cells (IGtM87), and SKOV3 cells that endogenously express caveolin-1. Simultaneous expression of caveolin-1 and E-cadherin favored membrane distribution of E-cadherin and its associated catenin (p120ctn), even when caveolin-1 was only focally associated with adherens junctions. Silencing of caveolin-1 induced intracellular E-cadherin redistribution in IGtC3 and SKOV3 cells. Treatment with the specific src kinase inhibitor PP1 increased E-cadherin expression in IGtM87 and SKOV3 cells and enhanced membrane localization of both E-cadherin and p120ctn. However, PP1 could not completely reverse the detrimental effects on cell-cell adhesion induced by Ca2+ depletion in IGtM87 cells. Together, our data suggest that caveolin-1 expression indirectly promotes cell-cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of src-related kinases. Thus, down-regulation or loss of caveolin-1 might contribute significantly to the spread of tumor cells from the primary tumor. Cadherin-mediated adhesion plays an important role in maintaining cell-cell contacts and reducing tumor metastasis. However, neo-expression of E-cadherin in ovarian carcinoma does not prevent the release and spread of cells from the primary tumor. Because caveolin-1 is down-regulated concomitantly with E-cad expression, we investigated whether the stability of adherens junctions in ovarian carcinoma was affected by caveolin-1 expression. We used IGROV1 cells transfected with caveolin-1 (IGtC3), mock-transfected control cells (IGtM87), and SKOV3 cells that endogenously express caveolin-1. Simultaneous expression of caveolin-1 and E-cadherin favored membrane distribution of E-cadherin and its associated catenin (p120ctn), even when caveolin-1 was only focally associated with adherens junctions. Silencing of caveolin-1 induced intracellular E-cadherin redistribution in IGtC3 and SKOV3 cells. Treatment with the specific src kinase inhibitor PP1 increased E-cadherin expression in IGtM87 and SKOV3 cells and enhanced membrane localization of both E-cadherin and p120ctn. However, PP1 could not completely reverse the detrimental effects on cell-cell adhesion induced by Ca2+ depletion in IGtM87 cells. Together, our data suggest that caveolin-1 expression indirectly promotes cell-cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of src-related kinases. Thus, down-regulation or loss of caveolin-1 might contribute significantly to the spread of tumor cells from the primary tumor. Caveolin-1 (cav-1) is a 22- to 24-kd integral membrane protein present in numerous tissue types, which localizes in membrane subdomains called caveolae. Interaction of cav-1 with a variety of protein and nonprotein molecules results in pleiotropic effects on numerous cellular events such as signal transduction, gene regulation, lipid metabolism, and vesicular traffic.1Galbiati F Razani B Lisanti MP Emerging themes in lipid rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 2Liu P Rudick M Anderson RG Multiple functions of caveolin-1.J Biol Chem. 2002; 277: 41295-41298Crossref PubMed Scopus (498) Google Scholar, 3Carver LA Schnitzer JE Caveolae: mining little caves for new cancer targets.Nat Rev Cancer. 2003; 3: 571-581Crossref PubMed Scopus (230) Google Scholar Down-regulation of cav-1 in human tumor samples of different histological origin has been documented, suggesting that this protein has a negative regulatory role in tumor development and acts as an onco-suppressor.4Racine C Belanger M Hirabayashi H Boucher M Chakir J Couet J Reduction of caveolin 1 gene expression in lung carcinoma cell lines.Biochem Biophys Res Commun. 1999; 255: 580-586Crossref PubMed Scopus (152) Google Scholar, 5Bagnoli M Tomassetti A Figini M Flati S Dolo V Canevari S Miotti S Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression.Oncogene. 2000; 19: 4754-4763Crossref PubMed Scopus (79) Google Scholar, 6Bender FC Reymond MA Bron C Quest AF Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity.Cancer Res. 2000; 60: 5870-5878PubMed Google Scholar, 7Razani B Schlegel A Liu J Lisanti MP Caveolin-1, a putative tumour suppressor gene.Biochem Soc Trans. 2001; 29: 494-499Crossref PubMed Scopus (114) Google Scholar, 8Wiechen K Sers C Agoulnik A Arlt K Dietel M Schlag PM Schneider U Down-regulation of caveolin-1, a candidate tumor suppressor gene, in sarcomas.Am J Pathol. 2001; 158: 833-839Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 9Aldred MA Ginn-Pease ME Morrison CD Popkie AP Gimm O Hoang-Vu C Krause U Dralle H Jhiang SM Plass C Eng C Caveolin-1 and caveolin-2, together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis.Cancer Res. 2003; 63: 2864-2871PubMed Google Scholar, 10Wiechen K Diatchenko L Agoulnik A Scharff KM Schober H Arlt K Zhumabayeva B Siebert PD Dietel M Schafer R Sers C Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.Am J Pathol. 2001; 159: 1635-1643Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar Although cav-1-null mice are no more prone to tumor development than are the cav-1-expressing counterparts, the null mice reveal an increased susceptibility to dysplastic mammary11Williams TM Cheung MW Park DS Razani B Cohen AW Muller WJ Di Vizio D Chopra NG Pestell RG Lisanti MP Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice.Mol Biol Cell. 2003; 14: 1027-1042Crossref PubMed Scopus (135) Google Scholar or epidermal lesions12Capozza F Williams TM Schubert W McClain S Bouzahzah B Sotgia F Lisanti MP Absence of caveolin-1 sensitizes mouse skin to carcinogen-induced epidermal hyperplasia and tumor formation.Am J Pathol. 2003; 162: 2029-2039Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar in the presence of an initial oncogenic stimulus. However, in a few tumor histotypes, cav-1 up-regulation has been associated with tumor progression and enhanced metastatic capability.13Ho CC Huang PH Huang HY Chen YH Yang PC Hsu SM Up-regulated caveolin-1 accentuates the metastasis capability of lung adenocarcinoma by inducing filopodia formation.Am J Pathol. 2002; 161: 1647-1656Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 14Yang G Truong LD Timme TL Ren C Wheeler TM Park SH Nasu Y Bangma CH Kattan MW Scardino PT Thompson TC Elevated expression of caveolin is associated with prostate and breast cancer.Clin Cancer Res. 1998; 4: 1873-1880PubMed Google Scholar In vitro, cav-1 expression is up-regulated in confluent cells, and the protein distributes in areas of cell-cell contacts,15Volonte D Galbiati F Lisanti MP Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras: the subcellular distribution of caveolin-1 is modulated by cell-cell contact.FEBS Lett. 1999; 445: 431-439Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar in which molecules involved in intercellular cell adhesion, such as E-cadherin (E-cad), are localized. E-cad mediates cell-cell adhesion through calcium-dependent homophilic interaction of the extracellular domains forming cis-dimers on the same cell, which interact with cis-dimers on neighboring cells to generate trans-interactions. Stable cell-cell interactions require binding of catenins (cat) to the cytoplasmic domain of E-cad. Commonly, β- or γ-cat bind the carboxy-terminal sequence of E-cad via armadillo-repeats and simultaneously interact with cytoplasmic α-cat to link the entire complex to the cytoskeletal cortical actin ring.16Strumane K Van Roy F Berx G The role of E-cadherin in epithelial differentiation and cancer progression.Recent Results Dev Cell Biochem. 2003; 1: 33-37Google Scholar, 17Cavallaro U Christofori G Cell adhesion and signalling by cadherins and Ig-CAMs in cancer.Nat Rev Cancer. 2004; 4: 118-132Crossref PubMed Scopus (1062) Google Scholar Colocalization of cav-1 with E-cad/β-cat complexes at the cell junctions, co-fractionation in caveolae, and reciprocal co-immunoprecipitation of all of these molecules has been demonstrated in normal epithelial MDCKII cells.18Galbiati F Volonte D Brown AM Weinstein DE Ben-Ze'ev A Pestell RG Lisanti MP Caveolin-1 expression inhibits Wnt/beta-catenin/Lef-1 signaling by recruiting beta-catenin to caveolae membrane domains.J Biol Chem. 2000; 275: 23368-23377Crossref PubMed Scopus (166) Google Scholar E-cad is frequently down-regulated in epithelial tumors, with different mechanisms contributing at the transcriptional and posttranscriptional level to this down-regulation.16Strumane K Van Roy F Berx G The role of E-cadherin in epithelial differentiation and cancer progression.Recent Results Dev Cell Biochem. 2003; 1: 33-37Google Scholar, 17Cavallaro U Christofori G Cell adhesion and signalling by cadherins and Ig-CAMs in cancer.Nat Rev Cancer. 2004; 4: 118-132Crossref PubMed Scopus (1062) Google Scholar In cav-1-positive/E-cad-positive A431 carcinoma cells, continuous stimulation by epidermal growth factor (EGF) was shown to down-regulate the expression of both E-cad and cav-1 first at the membrane level through caveolae-mediated E-cad internalization and then at the transcriptional level with consequent activation of β-cat signaling.19Lu Z Ghosh S Wang Z Hunter T Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion.Cancer Cell. 2003; 4: 499-515Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar In addition, NIH-3T3 cells co-transfected with cav-1 and β-cat showed inhibition of β-cat signaling.18Galbiati F Volonte D Brown AM Weinstein DE Ben-Ze'ev A Pestell RG Lisanti MP Caveolin-1 expression inhibits Wnt/beta-catenin/Lef-1 signaling by recruiting beta-catenin to caveolae membrane domains.J Biol Chem. 2000; 275: 23368-23377Crossref PubMed Scopus (166) Google Scholar Among the mechanisms involved in remodeling of the adherens junctions, tyrosine phosphorylation of adherens junction components mediated by both receptor and nonreceptor tyrosine kinases plays an important role.20Daniel JM Reynolds AB Tyrosine phosphorylation and cadherin/catenin function.BioEssays. 1997; 19: 883-891Crossref PubMed Scopus (285) Google Scholar In particular, tyrosine phosphorylation of β-cat and of p120ctn, the latter catenin binding to the E-cad cytoplasmic juxtamembrane region, reduces their respective affinity for the E-cad cytoplasmic domain21Hu P O'Keefe EJ Rubenstein DS Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin reversibly regulates their binding to E-cadherin and alpha-catenin.J Invest Dermatol. 2001; 117: 1059-1067Crossref PubMed Google Scholar, 22Ozawa M Ohkubo T Tyrosine phosphorylation of p120(ctn) in v-Src transfected L cells depends on its association with E-cadherin and reduces adhesion activity.J Cell Sci. 2001; 114: 503-512PubMed Google Scholar and enhances the disassembly of junctional complexes. In the human normal ovary, the surface epithelium (OSE) is negative for E-cad expression23Sundfeldt K Piontkewitz Y Ivarsson K Nilsson O Hellberg P Brännström M Janson PO Enerbäck S Hedin L E-cadherin expression in human epithelial ovarian cancer and normal ovary.Int J Cancer. 1997; 74: 275-280Crossref PubMed Scopus (149) Google Scholar and displays a baso-lateral distribution of cav-1.5Bagnoli M Tomassetti A Figini M Flati S Dolo V Canevari S Miotti S Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression.Oncogene. 2000; 19: 4754-4763Crossref PubMed Scopus (79) Google Scholar, 10Wiechen K Diatchenko L Agoulnik A Scharff KM Schober H Arlt K Zhumabayeva B Siebert PD Dietel M Schafer R Sers C Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.Am J Pathol. 2001; 159: 1635-1643Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar Cav-1 expression is frequently lost or down-regulated in malignant ovarian carcinomas,5Bagnoli M Tomassetti A Figini M Flati S Dolo V Canevari S Miotti S Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression.Oncogene. 2000; 19: 4754-4763Crossref PubMed Scopus (79) Google Scholar, 10Wiechen K Diatchenko L Agoulnik A Scharff KM Schober H Arlt K Zhumabayeva B Siebert PD Dietel M Schafer R Sers C Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.Am J Pathol. 2001; 159: 1635-1643Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar whereas unlike in other carcinomas, E-cad is expressed de novo at least during early stages of tumor progression.23Sundfeldt K Piontkewitz Y Ivarsson K Nilsson O Hellberg P Brännström M Janson PO Enerbäck S Hedin L E-cadherin expression in human epithelial ovarian cancer and normal ovary.Int J Cancer. 1997; 74: 275-280Crossref PubMed Scopus (149) Google Scholar, 24Davies BR Worsley SD Ponder BA Expression of E-cadherin, alpha-catenin and beta-catenin in normal ovarian surface epithelium and epithelial ovarian cancers.Histopathology. 1998; 32: 69-80Crossref PubMed Scopus (118) Google Scholar, 25Marques FR Fonsechi-Carvasan GA Angelo Andrade LA Bottcher-Luiz F Immunohistochemical patterns for alpha- and beta-catenin, E- and N-cadherin expression in ovarian epithelial tumors.Gynecol Oncol. 2004; 94: 16-24Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar In the present study, we investigated the effect of the contemporaneous expression of cav-1 and E-cad on β-cat signaling, on subcellular distribution of cav-1 and cell-cell adhesion molecules, and on the stability of adherens junctions in ovarian carcinoma cells. Our overall results indicate that cav-1 does not contribute physically to the formation of adherens junctions, but promotes their stability by modulating the level and/or the subcellular distribution of E-cad and p120ctn through inhibition of src kinase activity. Phenylmethylsulfonylfluoride, 3,3′-diaminobenzidine, Triton X-100 (TX-100), 2-(N-morpholino) ethanesulfonic acid (MES), Na3VO4, dimethylsulfoxide (DMSO), and aprotinin were from Sigma-Aldrich Fine Chemicals (St. Louis, MO); geneticin sulfate (G418) was from GIBCO-BRL (Paisley, Scotland); protease inhibitory cocktail and octyl-β-glucoside (OG) were from Boehringer-Mannheim (Germany); the src inhibitor PP1 was from BIOMOL Research Laboratories (Plymouth Meeting, PA); and Tween 20 was from Merck (Hohenbrunn, Germany). The following primary antibodies were used at the dilution recommended by the manufacturer: anti-cav 1 (rabbit), anti-lamin A/C Mab (mouse), anti-E-cad (rabbit), (Santa Cruz Biotechnology, Santa Cruz, CA); anti-E-cad Mab (mouse), anti-β-cat Mab (mouse) (Chemicon International, Temecula, CA); anti-tubulin-α Mab (mouse) (NeoMarkers, Inc., Fremont, CA); anti-β-cat (rabbit), anti-actin (rabbit) (Sigma); anti-phosphotyrosine Mab (mouse) (Cell Signaling Technology, New England BioLabs, Beverly, MA), anti-p120ctn Mab (mouse), anti-p120ctn (pY228) phospho-specific Mab (mouse) (Transduction Laboratories, BD Biosciences Pharmingen, Palo Alto, CA); anti-E-cad clone HECD1 Mab (mouse) (Zymed Laboratories, Inc., San Francisco, CA); anti-src (pY418) (rabbit) (Biosource International, Camarillo, CA). Fluorochrome-conjugated Alexa Fluor 488 and Alexa Fluor 546 secondary antibodies were from Molecular Probes (Eugene, OR). The ovarian carcinoma cell lines IGtC3 and IGtM87, obtained by cav-1 cDNA or mock transfection, respectively, of E-cad-positive IGROV1 ovarian carcinoma cells5Bagnoli M Tomassetti A Figini M Flati S Dolo V Canevari S Miotti S Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression.Oncogene. 2000; 19: 4754-4763Crossref PubMed Scopus (79) Google Scholar were used as a model to investigate how cav-1 expression affects E-cad/β-cat in ovarian carcinoma. IGtM87 and IGtC3 cells were maintained in folate-deficient RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (FCS) (Sigma), 2 mmol/L l-glutamine (Sigma), and 400 μg/ml G418. Ovarian carcinoma cell line SKOV3 was from ATCC (Manassas, VA). MDCKII cells were kindly provided by Dr. R. Sitia (Vita-Salute University-San Raffaele, DiBiT, San Raffaele Scientific Institute, Milan, Italy). Both SKOV3 and MDCKII cells were grown in standard RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum and 2 mmol/L l-glutamine. Short-term cultured normal OSE cells were derived from scrapings at the time of the surgery for gynecological diseases other than ovarian carcinoma. Cells were cultured in 199-MCDB105 medium (Sigma) supplemented with 15% FCS, 2 mmol/L l-glutamine, and 25 mg/ml gentamicin as described.26Kruk PA Maines-Bandiera SL Auersperg N A simplified method to culture human ovarian surface epithelium.Lab Invest. 1990; 63: 132-136PubMed Google Scholar, 27De Cecco L Marchionni L Gariboldi M Reid JF Lagonigro MS Caramuta S Ferrario C Bussani E Mezzanzanica D Turatti F Delia D Daidone MG Oggioni M Bertuletti N Ditto A Raspagliesi F Pilotti S Pierotti MA Canevari S Schneider C Gene expression profiling of advanced ovarian cancer: characterization of molecular signature involving the fibroblast growth factor 2.Oncogene. 2004; 23: 8171-8183Crossref PubMed Scopus (77) Google Scholar All clinical specimens used in this study were obtained with Institutional Review Board approval and informed consent to use excess biological material for investigative purposes from all patients participating in the study. Tissue samples were obtained at the time of initial surgery, immediately frozen in liquid nitrogen, and stored at −80°C until use. For immunohistochemical analysis, frozen 5-μm serial sections were fixed in cold acetone:methanol (3:2) for 10 minutes and stained as described previously, with minor modifications.5Bagnoli M Tomassetti A Figini M Flati S Dolo V Canevari S Miotti S Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression.Oncogene. 2000; 19: 4754-4763Crossref PubMed Scopus (79) Google Scholar Briefly, samples were preincubated for 20 minutes in phosphate-buffered saline containing 0.9 mmol/L CaCl2 (PBS+), 1% bovine serum albumin (BSA), and 0.1% Tween 20, followed by incubation for 30 minutes at room temperature with primary antibody in PBS+ containing 1% BSA. PBS+/BSA alone was used as a negative control. Incubation with secondary biotinylated antibody in the same buffer was performed for 30 minutes at room temperature. Endogenous peroxidase activity was inhibited by incubation with 0.4% H2O2 in PBS+ for 40 minutes. Bound antibodies were detected by using the avidin-biotin complex method (Vectastain kit; Vector Laboratories, Inc., Burlingame, CA) and 3,3′-diaminobenzidine as substrate. Cells (3 × 103/sample) seeded on glass coverslips were grown for 24 hours or 72 to 96 hours to obtain compact cell monolayers and fixed for 10 minutes in 10% cold methanol. After saturation in PBS+/BSA and 0.1% Tween 20, cells were incubated with primary antibodies, diluted in PBS+/BSA for 30 minutes at room temperature. After three washes in PBS+, secondary fluorochrome-conjugated (Alexa Fluor 488 and Alexa Fluor 546) antibodies in the same buffer were applied for an additional 30 minutes. Controls were exposed to relevant secondary antibodies alone and showed no significant degree of labeling. Coverslips were mounted on glass slides using MOWIOL-DABCO solution and analyzed by confocal microscopy (Microradiance 2000; Bio-Rad Laboratories, Inc., Hercules, CA) equipped with Ar (488 nm) and HeNe (543 nm) lasers. Images (1024 × 1024 pixels) were obtained using a 60× oil immersion lens and analyzed using Lasersharp 2000 software. Reported images represent a single Z-section of the samples. Confluent cells were washed in PBS+ containing 0.1 mmol/L Na3VO4, detached by scraping in hypotonic buffer HB-7 (5 mmol/L Tris, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 11% sucrose, pH 7.4), and passed through a 0.3-mm-gauge needle. The cell lysate was centrifuged for 10 minutes at 3000 rpm in a SS34 rotor (Sorvall) at 4°C. The pellet was resuspended twice in the same buffer and pelleted under the same conditions. The last pellet was considered the nuclear fraction (Nu). Supernatants derived from nuclear pellet washings were pooled and centrifuged at 14,000 rpm in the same rotor as above for 45 minutes at 4°C. The pellet, washed once in the same buffer, represents the crude membrane fraction (M), whereas the pooled supernatants constitute the cytosolic fraction (Cy). The Nu and M pellets were each resuspended in the same buffer volume. Protein concentration was determined by BCA protein assay (Pierce, Rockford, IL), according to the manufacturer's protocol. Possible contamination of each fraction with proteins of other subcellular compartments was monitored using Western blot analysis for the presence of α-tubulin and lamin A/C as markers of cytosol and nuclei, respectively. Cells (1.2 × 105) were seeded in 12-well plates and transfected the following day using positively charged liposomes (kindly provided by Silvia Arpicco [University of Turin, Italy]) in the absence of FCS. TOP- or FOP-FLASH constructs (2.5 μg) (Upstate Biotechnology, Lake Placid, NY) containing three copies of the wild-type or mutated T cell factor (TCF) DNA-binding site, respectively, were co-transfected with the pRL-SV40 vector (0.01 μg; Promega, Madison, WI) as a control for transfection efficiency. Firefly and Renilla Luciferase activities were evaluated on cell lysates using the DUAL-luciferase Reporter Assay (Promega), essentially as suggested by the manufacturer. Cells were grown to confluence in T75 flasks to enhance localization of cav-1 at the cell-cell contacts. After three washes in cold PBS+, cells were lysed in 1% TX-100 containing buffer (25 mmol/L MES, pH 6.5, and 150 mmol/L NaCl), and the lysate was mixed 1:1 with 80% sucrose solution. Gradient fractionation (5–30%) and protein separation were carried out as described previously.28Sargiacomo M Sudol M Tang Z Lisanti MP Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells.J Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 29Miotti S Bagnoli M Tomassetti A Colnaghi MI Canevari S Interaction of folate receptor with signaling molecules lyn and Ga13 in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.J Cell Sci. 2000; 113: 349-357PubMed Google Scholar After ultracentrifugation for 17 hours in SW41 Beckman rotor, twelve 1-ml fractions were collected from the top of the gradient, and the distribution of relevant proteins was analyzed by SDS-PAGE30Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar and Western blotting. The glycosyl phosphatidyl inositol (GPI)-anchored folate receptor29Miotti S Bagnoli M Tomassetti A Colnaghi MI Canevari S Interaction of folate receptor with signaling molecules lyn and Ga13 in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.J Cell Sci. 2000; 113: 349-357PubMed Google Scholar was used as a marker of lipid rafts. Confluent cells in T75 flasks were rinsed twice with cold PBS+ containing 0.1 mmol/L Na3VO4 and treated in flasks for 10 minutes at 4°C with lysis buffer (25 mmol/L MES, pH 6.5, 150 mmol/L NaCl, 3 mmol/L MgCl2, protease inhibitor cocktail) containing 0.5% TX-100 and 1 mmol/L Na3VO4. After recovery of TX-100 solubilized proteins (TX-sol), cells in flasks were further treated for 30 minutes at 4°C with the same volume of the above buffer containing 1.1% OG (OG-sol). Both treatments were applied with gentle agitation. The solubilized fractions (TX-sol and OG-sol) were clarified by centrifugation (15,000 × g for 15 minutes) at 4°C, and the supernatant was recovered. Immunoprecipitation of the bottom fractions (pool 9–12) of the sucrose gradient and from TX-sol, OG-sol fractions was carried out using magnetic beads conjugated with goat anti-mouse and sheep anti-rabbit antibodies (Dynabeads; Dynal ASA, Oslo, Norway) as described.29Miotti S Bagnoli M Tomassetti A Colnaghi MI Canevari S Interaction of folate receptor with signaling molecules lyn and Ga13 in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.J Cell Sci. 2000; 113: 349-357PubMed Google Scholar Briefly, 1 to 2 μg of each antibody was used for 25 μl of beads. Normal mouse and rabbit sera were used as negative controls. Beads were incubated with the cell lysate for 3 hours or overnight at 4°C with rotation. After incubation, the beads were washed once with cold lysis buffer, twice with PBS+/BSA and protease inhibitors, and once with PBS+ plus inhibitor (10 minutes/wash). Bound material, eluted from beads by boiling for 5 minutes at 95°C in final sample buffer containing 0.5% SDS, was analyzed by SDS-PAGE to 10% slab gels or in 4 to 12% precasted mini gels (Invitrogen, Paisley, UK). Separated proteins were transferred on nitrocellulose sheets (Amersham Bioscience-GE Healthcare, Italy) for 2 hours at constant 200 mA. Staining with red ponceau (Sigma) was used to confirm homogeneous protein loading and transfer. After blocking in Blotto (5% nonfat dry milk; Merck), sheets were immunoreacted with the relevant primary antibodies and peroxidase-conjugated secondary antibodies (Amersham Bioscience-GE Healthcare) as described previously.29Miotti S Bagnoli M Tomassetti A Colnaghi MI Canevari S Interaction of folate receptor with signaling molecules lyn and Ga13 in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.J Cell Sci. 2000; 113: 349-357PubMed Google Scholar Reactivity was usually revealed by the ECL method (Amersham Bioscience-GE Healthcare). ECL plus was used to detect the phosphorylated forms of p120ctn. The Benchmark prestained protein ladder, used as a molecular mass standard, was from Invitrogen. Cells were grown for 2 to 3 days to 50 to 60% confluence, and for another 2 days in the presence of the src family inhibitor PP1. PP1 stock solution, dissolved in 100% DMSO, was diluted at least 1000-fold into culture medium to final concentrations of 20, 4, and 0.8 μmol/L. Control cells were treated with culture medium containing DMSO at the same dilution. For immunofluorescence analysis, cells grown for 2 days on glass coverslips were treated with 4 μmol/L PP1 and stained with relevant antibodies as described above. For analysis of protein expression, cells grown in flasks were sequentially solubilized as described above. Solubilized proteins were analyzed by SDS-PAGE and Western blotting as single extracts (TX-sol or OG-sol) or as a pool obtained by mixing the same volume of each. IGM87 and IGtC3 cells were seeded in 24-well plates (5 × 104 cells/well) in RPMI 1640 medium, incubated at 37°C for 1 or 2 hours. After three vigorous PBS washings, adherent cells were fixed in cold methanol for 10 minutes, stained with 0.5% crystal violet in 20% methanol, and assessed for eluted stain at 550 nm. Background staining of wells without cells was subtracted from specific readings. Cells were grown, as described, for 2 days on glass coverslips and treated with 4 μmol/L PP1 or DMSO-containing control medium and allowed to grow for another 2 days. After removal of culture medium, cells were incubated for 90 minutes at 37°C in the presence of PBS containing high (1 mmol/L) or low (0.030 mmol/L) Ca2+ concentrations, fixed in cold methanol, and immunostained for E-cad expression. Cells (5 × 104/sample) seeded on glass coverslips were grown for 24 hours at 37°C, washed in medium without FCS, and then transfected with 20 or 40 pmol (IGtC3 and SKOV3, respectively) of siRNA oligonucleotides specific for cav-1 or with unrelated scramble (SCRB) sequence oligonucleotides (Dharmacon, Lafayette, CO) using lipofectamine 2000 (Invitrogen) (1 μl/20 pmol oligonucleotides). After 7 hours, transfection reagents were removed and replaced with RPMI 1640 medium containing 10% FCS. Sixty-four hours later, cells were processed for immunofluorescence staining or solubilized for Western blot evaluation of cav-1 silencing. Frozen sections from a panel of 11 human ovarian carcinoma, including 9 serous and 2 endometrial histotypes, were tested by immunoperoxidase staining with antibodies directed against cav-1, E-cad, and β-cat (Figure 1A). Cav-1 staining of tumor cells was absent in four cases and in five cases was only faint and focal. In the remaining two cases, marked basal reactivity of anti-cav-1 antibody on tumor cells was observed. This pattern of reactivity was associated with the distribution of both E-cad and β-cat at the membrane level in correspondence with cell-cell contacts (sample a is representative of this pattern). All samples were E-cad positive, but a heterogeneous cellular