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Tenascin C Induces Epithelial-Mesenchymal Transition–Like Change Accompanied by SRC Activation and Focal Adhesion Kinase Phosphorylation in Human Breast Cancer Cells

2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2010.10.015

ISSN

1525-2191

Autores

Keiki Nagaharu, Xinhui Zhang, Toshimichi Yoshida, Daisuke Katoh, Noriko Hanamura, Yuji Kozuka, Tomoko Ogawa, Taizo Shiraishi, Kyoko Imanaka‐Yoshida,

Tópico(s)

Wnt/β-catenin signaling in development and cancer

Resumo

Tenascin C (TNC) is an extracellular matrix glycoprotein up-regulated in solid tumors. Higher TNC expression is shown in invading fronts of breast cancer, which correlates with poorer patient outcome. We examined whether TNC induces epithelial-mesenchymal transition (EMT) in breast cancer. Immunohistochemical analysis of invasive ductal carcinomas showed that TNC deposition was frequent in stroma with scattered cancer cells in peripheral margins of tumors. The addition of TNC to the medium of the MCF-7 breast cancer cells caused EMT-like change and delocalization of E-cadherin and β-catenin from cell-cell contact. Although amounts of E-cadherin and β-catenin were not changed after EMT in total lysates, they were increased in the Triton X-100-soluble fractions, indicating movement from the membrane into the cytosol. In wound healing assay, cells were scattered from wound edges and showed faster migration after TNC treatment. The EMT phenotype was correlated with SRC activation through phosphorylation at Y418 and phosphorylation of focal adhesion kinase (FAK) at Y861 and Y925 of SRC substrate sites. These phosphorylated proteins colocalized with αv integrin-positive adhesion plaques. A neutralizing antibody against αv or a SRC kinase inhibitor blocked EMT. TNC could induce EMT-like change showing loss of intercellular adhesion and enhanced migration in breast cancer cells, associated with FAK phosphorylation by SRC; this may be responsible for the observed promotion of TNC in breast cancer invasion. Tenascin C (TNC) is an extracellular matrix glycoprotein up-regulated in solid tumors. Higher TNC expression is shown in invading fronts of breast cancer, which correlates with poorer patient outcome. We examined whether TNC induces epithelial-mesenchymal transition (EMT) in breast cancer. Immunohistochemical analysis of invasive ductal carcinomas showed that TNC deposition was frequent in stroma with scattered cancer cells in peripheral margins of tumors. The addition of TNC to the medium of the MCF-7 breast cancer cells caused EMT-like change and delocalization of E-cadherin and β-catenin from cell-cell contact. Although amounts of E-cadherin and β-catenin were not changed after EMT in total lysates, they were increased in the Triton X-100-soluble fractions, indicating movement from the membrane into the cytosol. In wound healing assay, cells were scattered from wound edges and showed faster migration after TNC treatment. The EMT phenotype was correlated with SRC activation through phosphorylation at Y418 and phosphorylation of focal adhesion kinase (FAK) at Y861 and Y925 of SRC substrate sites. These phosphorylated proteins colocalized with αv integrin-positive adhesion plaques. A neutralizing antibody against αv or a SRC kinase inhibitor blocked EMT. TNC could induce EMT-like change showing loss of intercellular adhesion and enhanced migration in breast cancer cells, associated with FAK phosphorylation by SRC; this may be responsible for the observed promotion of TNC in breast cancer invasion. Epithelial cells are polarized and tightly interconnected by cellular junctions, whereas mesenchymal cells never form stable intercellular contacts in adult tissues. Epithelial-mesenchymal transition (EMT) is a process whereby polarized epithelial cells are converted into mesenchymal cells during embryogenesis and in diseased tissues.1Savagner P. Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition.Bioessays. 2001; 23: 912-923Crossref PubMed Scopus (603) Google Scholar, 2Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7270) Google Scholar, 3Baum B. Settleman J. 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Biomarkers for epithelial-mesenchymal transitions.J Clin Invest. 2009; 119: 1429-1437Crossref PubMed Scopus (1664) Google Scholar Tenascin C (TNC) is a large hexameric extracellular matrix glycoprotein that exhibits de-adhesive effects on cell-matrix interaction, enhancing cell proliferation and motility in most cell types.13Orend G. Chiquet-Ehrismann R. Tenascin-C induced signaling in cancer.Cancer Lett. 2006; 244: 143-163Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar TNC is highly expressed in remodeling tissues during embryonic development and under pathological conditions in adults. In development, its expression is known to be associated with classic EMT events including gastrulation14Crossin K.L. Hoffman S. Grumet M. Thiery J.P. Edelman G.M. Site-restricted expression of cytotactin during development of the chicken embryo.J Cell Biol. 1986; 102: 1917-1930Crossref PubMed Scopus (210) Google Scholar and formation of the neural crest,15Tucker R.P. McKay S.E. The expression of tenascin by neural crest cells and glia.Development. 1991; 112: 1031-1039PubMed Google Scholar endocardial cushion,16Sugi Y. Markwald R.R. Formation and early morphogenesis of endocardial endothelial precursor cells and the role of endoderm.Dev Biol. 1996; 175: 66-83Crossref PubMed Scopus (103) Google Scholar, 17Boyer A.S. Erickson C.P. Runyan R.B. Epithelial-mesenchymal transformation in the embryonic heart is mediated through distinct pertussis toxin-sensitive and TGFbeta signal transduction mechanisms.Dev Dyn. 1999; 214: 81-91Crossref PubMed Scopus (54) Google Scholar and secondary palate.18Ferguson M.W. Palate development.Development. 1988; 103: 41-60PubMed Google Scholar In normal mammary gland, TNC expression is limited, but elevation occurs in breast cancer tissues with production by both tumor and stromal cells.19Kalembey I. Yoshida T. Iriyama K. Sakakura T. Analysis of tenascin mRNA expression in the murine mammary gland from embryogenesis to carcinogenesis: an in situ hybridization study.Int J Dev Biol. 1997; 41: 569-573PubMed Google Scholar, 20Yoshida T. Matsumoto E. Hanamura N. Kalembeyi I. Katsuta K. Ishihara A. Sakakura T. Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign lesions and ductal carcinomas in the human breast.J Pathol. 1997; 182: 421-428Crossref PubMed Scopus (58) Google Scholar Immunohistochemical studies of invasive ductal carcinoma cases have demonstrated that expression is indicative of a poorer patient outcome.21Ishihara A. Yoshida T. Tamaki H. Sakakura T. Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance.Clin Cancer Res. 1995; 1: 1035-1041PubMed Google Scholar It has been reported that TNC promotes proliferation and migratory activity of cancer cells22Yoshida T. Yoshimura E. Numata H. Sakakura Y. Sakakura T. Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells.Virchows Arch. 1999; 435: 496-500Crossref PubMed Scopus (75) Google Scholar, 23Tsunoda T. Inada H. Kalembeyi I. Imanaka-Yoshida K. Sakakibara M. Okada R. Katsuta K. Sakakura T. Majima Y. Yoshida T. Involvement of large tenascin-C splice variants in breast cancer progression.Am J Pathol. 2003; 162: 1857-1867Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 24Hancox R.A. Allen M.D. Holliday D.L. Edwards D.R. Pennington C.J. Guttery D.S. Shaw J.A. Walker R.A. Pringle J.H. Jones J.L. Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.Breast Cancer Res. 2009; 11: R24Crossref PubMed Scopus (91) Google Scholar, 25Calvo A. Catena R. Noble M.S. Carbott D. Gil-Bazo I. Gonzalez-Moreno O. Huh J.I. Sharp R. Qiu T.H. Anver M.R. Merlino G. Dickson R.B. Johnson M.D. Green J.E. Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis.Oncogene. 2008; 27: 5373-5384Crossref PubMed Scopus (66) Google Scholar and up-regulates the expression of matrix metalloproteinases by breast cancer cells.24Hancox R.A. Allen M.D. Holliday D.L. Edwards D.R. Pennington C.J. Guttery D.S. Shaw J.A. Walker R.A. Pringle J.H. Jones J.L. Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.Breast Cancer Res. 2009; 11: R24Crossref PubMed Scopus (91) Google Scholar, 26Kalembeyi I. Inada H. Nishiura R. Imanaka-Yoshida K. Sakakura T. Yoshida T. Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: direct and synergistic effects with transforming growth factor beta1.Int J Cancer. 2003; 105: 53-60Crossref PubMed Scopus (85) Google Scholar Furthermore, it is important to note that TNC expression is frequently observed in invasion borders of cancer tissues and in microinvasive foci around intraductal carcinomas, where cells may undergo EMT.23Tsunoda T. Inada H. Kalembeyi I. Imanaka-Yoshida K. Sakakibara M. Okada R. Katsuta K. Sakakura T. Majima Y. Yoshida T. Involvement of large tenascin-C splice variants in breast cancer progression.Am J Pathol. 2003; 162: 1857-1867Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27Jahkola T. Toivonen T. von Smitten K. Blomqvist C. Virtanen I. Expression of tenascin in invasion border of early breast cancer correlates with higher risk of distant metastasis.Int J Cancer. 1996; 69: 445-447Crossref PubMed Scopus (65) Google Scholar, 28Jahkola T. Toivonen T. Virtanen I. von Smitten K. Nordling S. von Boguslawski K. Haglund C. Nevanlinna H. Blomqvist C. Tenascin-C expression in invasion border of early breast cancer: a predictor of local and distant recurrence.Br J Cancer. 1998; 78: 1507-1513Crossref PubMed Scopus (106) Google Scholar, 29Jahkola T. Toivonen T. Nordling S. von Smitten K. Virtanen I. Expression of tenascin-C in intraductal carcinoma of human breast: relationship to invasion.Eur J Cancer. 1998; 34: 1687-1692Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar TNC immunostaining in invasive fronts is correlated with higher risk of distant metastasis and local recurrence.27Jahkola T. Toivonen T. von Smitten K. Blomqvist C. Virtanen I. Expression of tenascin in invasion border of early breast cancer correlates with higher risk of distant metastasis.Int J Cancer. 1996; 69: 445-447Crossref PubMed Scopus (65) Google Scholar, 28Jahkola T. Toivonen T. Virtanen I. von Smitten K. Nordling S. von Boguslawski K. Haglund C. Nevanlinna H. Blomqvist C. Tenascin-C expression in invasion border of early breast cancer: a predictor of local and distant recurrence.Br J Cancer. 1998; 78: 1507-1513Crossref PubMed Scopus (106) Google Scholar In mammary epithelial cell differentiation, TNC expression is inversely correlated with the polarized epithelial phenotype and the addition of TNC disturbs dome formation in vitro, indicating TNC interference with induction and maintenance of cytodifferentiation.30Wirl G. Hermann M. Ekblom P. Fässler R. Mammary epithelial cell differentiation in vitro is regulated by an interplay of EGF action and tenascin-C downregulation.J Cell Sci. 1995; 108: 2445-2456PubMed Google Scholar These observations support the hypothesis that TNC is an extracellular trigger of EMT in breast cancer progression, although there is still no direct evidence to confirm this. Therefore, in the present study, we investigated the expression of TNC in different morphological types of breast cancer, solid and scattered, by immunohistochemistry. In in vitro studies using MCF-7 cells—a breast cancer cell line that shows a typical epithelial character with tight intercellular contacts and does not produce TNC under typical culture conditions24Hancox R.A. Allen M.D. Holliday D.L. Edwards D.R. Pennington C.J. Guttery D.S. Shaw J.A. Walker R.A. Pringle J.H. Jones J.L. Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.Breast Cancer Res. 2009; 11: R24Crossref PubMed Scopus (91) Google Scholar, 31Kawakatsu H. Shiurba R. Obara M. Hiraiwa H. Kusakabe M. Sakakura T. Human carcinoma cells synthesize and secrete tenascin in vitro.Jpn J Cancer Res. 1992; 83: 1073-1080Crossref PubMed Scopus (47) Google Scholar—we examined the effects of exogenous TNC on morphology and internalization of E-cadherin/β-catenin. The molecular mechanisms that underlie EMT induced by TNC were explored also. Immunohistochemical analysis was performed on 35 cases of invasive ductal carcinoma of the breast using archival samples that had been fixed in formalin and routinely processed for embedding in paraffin. Use of the samples was approved by written informed consent from the patients under a protocol authorized by the ethical committee of Mie University School of Medicine. All sections were cut at a thickness of 4 μm, placed on silane-coated glass slides, and incubated in 0.3% H2O2 in methanol for 15 minutes to block endogenous peroxidase activity. Antigen retrieval was performed using an autoclave (121°C for 1 minute). Sections were then treated with Super Block solution (Scytek Laboratories, Logan, UT) before incubation with anti-TNC antibody (4F10TT,23Tsunoda T. Inada H. Kalembeyi I. Imanaka-Yoshida K. Sakakibara M. Okada R. Katsuta K. Sakakura T. Majima Y. Yoshida T. Involvement of large tenascin-C splice variants in breast cancer progression.Am J Pathol. 2003; 162: 1857-1867Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar 1 μg/ml; IBL Japan, Takasaki, Japan) overnight at 4°C. After being washed, they were treated with a commercially available LSAB kit (Scytek), followed by color development with 3,3′-diaminobenzidine 4HCl (DAB)/H2O2 solution. Light counterstaining with hematoxylin was performed to aid orientation. The invasive patterns of ductal carcinomas at the peripheral margins of the tumors were classified into two groups: solid and scattered. A maximum of three representative areas of each type were selected in each breast cancer specimen, and TNC immunoreactivity was assessed in 63 solid- and 84 scattered-type areas. TNC labeling around or between cancer nests in the peripheral margins was defined as positive. MCF-7 cells were obtained from the Health Science Research Resources Bank of the Japan Health Sciences Foundation (Tokyo, Japan) and routinely cultured in Dulbecco's modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS), 1% nonessential amino acid solution (Invitrogen, Carlsbad, CA), and 0.01 mg/ml bovine insulin (Sigma-Aldrich, St. Louis, MO). Cells were trypsinized, collected by brief centrifugation, and resuspended in DMEM with 5% FBS. They were seeded at 5 × 104 cells per well (1 ml) in 12-well plates (BD Falcon, Franklin Lakes, NJ), then 10 μg/ml TNC was added. The TNC had been purified from conditioned medium of human glioma cell line U251MG as previously described.22Yoshida T. Yoshimura E. Numata H. Sakakura Y. Sakakura T. Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells.Virchows Arch. 1999; 435: 496-500Crossref PubMed Scopus (75) Google Scholar After 16 hours of incubation, 5 ng/ml TGF-β1 (Roche Diagnostics, Mannheim, Germany) was added and the cells were cultured for another 48 hours. Neutralizing antibodies for the αv integrin subunit (AV1, 1:40; Millipore Corporation, Billerica, MA) or β1 (P4C10, 5 μg/ml; Millipore) and the SRC kinase inhibitor (pp2, 10 μmol/L, Calbiochem, La Jolla, CA) were added to the medium when the cells were plated. A neutralizing antibody for TGF-β (50 μg/ml, 1D11; R&D Systems, Minneapolis, MN) was used also. As a negative control, TNC solution (10 μg) was reacted with 13 μg of rabbit polyclonal affinity-purified TNC antibody23Tsunoda T. Inada H. Kalembeyi I. Imanaka-Yoshida K. Sakakibara M. Okada R. Katsuta K. Sakakura T. Majima Y. Yoshida T. Involvement of large tenascin-C splice variants in breast cancer progression.Am J Pathol. 2003; 162: 1857-1867Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar for 6 hours, followed by precipitation with protein A beads (10 μL) for 16 hours, and the supernatant was added to the medium. Another breast cancer cell line, T-47D, was purchased from the American Type Culture Collection (Manassas, VA) and grown in RPMI-1640 containing 10% FBS and 0.2 U/ml bovine insulin. This cell line also shows an epithelial phenotype and does not synthesize TNC.24Hancox R.A. Allen M.D. Holliday D.L. Edwards D.R. Pennington C.J. Guttery D.S. Shaw J.A. Walker R.A. Pringle J.H. Jones J.L. Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.Breast Cancer Res. 2009; 11: R24Crossref PubMed Scopus (91) Google Scholar, 31Kawakatsu H. Shiurba R. Obara M. Hiraiwa H. Kusakabe M. Sakakura T. Human carcinoma cells synthesize and secrete tenascin in vitro.Jpn J Cancer Res. 1992; 83: 1073-1080Crossref PubMed Scopus (47) Google Scholar To examine EMT change, the bottom surfaces of the wells were incubated with 80 μL of 250 μg/ml TNC solution for 1 hour, then the cell suspension in DMEM/5% FBS medium was poured into the wells. TGF-β1 was added after 16 hours of incubation. MCF-7 or T-47D cells were grown on circular cover glasses (18-mm diameter) in 12-well plates, fixed in 2% paraformaldehyde/phosphate-buffered saline (PBS) for 10 minutes and then permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich). After treatment with 10% normal goat serum, they were exposed to primary antibodies (200-fold diluted) specific for E-cadherin (BD Bioscience, San Jose, CA), β-catenin (BD Bioscience), phosphorylated sites of FAK [pY397 (Biosource, Camarillo, CA), pY861 (Biosource), pY925 (Cell Signaling Technology, Danvers, MA)], and a phosphorylated form of SRC at Y418 (Y416 for chicken; Cell Signaling Technology). Subsequently, the cells were treated with appropriate secondary antibodies, fluorescein-labeled goat anti-mouse IgG or anti-rabbit IgG (200-fold diluted; MBL, Nagoya, Japan). For double immunofluorescence, rhodamine-labeled goat anti-mouse IgG (200-fold diluted; Tago, Burlingame, CA) was used instead of the fluorescein-labeled antibody. Observation was performed under an epifluorescence microscope with appropriate filter sets and a 40× or 60× objective lens (Olympus, Tokyo, Japan), and photographs were taken using a cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). To examine expression levels of cell adhesion molecules, FAK, and SRC, cells cultured on 6-well plates (1.4 × 105 cells/well) underwent lysis under denaturing conditions using a warmed buffer [1% sodium dodecyl sulfate (SDS), 10 mmol/L Tris-HCl buffer, pH 6.8, with 1 mmol/L sodium orthovanadate]. The amount of protein in each extract was evaluated using the BCA (bicinchoninic acid) assay (Pierce, Rockford, IL). Equal amounts of total protein were mixed with Laemmli's sample buffer and underwent electrophoresis on 5% to 20% polyacrylamide gradient gels. The proteins were electrically transferred to PVDF (polyvinylidene fluoride) membranes, and immunoblotting was performed with antibodies specific for E-cadherin, β-catenin, and α-tubulin (Cederlane, Burlington, Ontario, Canada) as an intrinsic control, followed by peroxidase-labeled anti-mouse IgG (3000–5000×; GE Healthcare, Waukesha, WI) or anti-rabbit IgG (3000×; Sigma-Aldrich) and ECL or ECL Plus detection (GE Healthcare). First antibodies were usually used at 2000- to 3000-fold dilution. Intensities of the bands were quantified using Image J software and the values were normalized to intensities of α-tubulin bands. Total FAK and phosphorylated FAK were detected using antibodies against FAK (Cell Signaling Technology) and the phosphorylated sites, Y397 (Cell Signaling Technology), Y861 (Abcam, Cambridge, MA) and Y925. Total SRC (Cell Signaling Technology) and the phosphorylation at Y418 also were examined. The values were normalized to intensities of total FAK or SRC bands. Antibodies against N-cadherin (3B9; Zymed Laboratories, Carlsbad, CA), vimentin (Vim 13.2; Sigma-Aldrich), and TNC (0.1 μg/ml) also were used. Triton X-100 fractionation was further conducted to examine intracellular localization of E-cadherin and β-catenin. The cells were extracted at 37°C with 200 μL of 0.5% Triton X-100, 2.5 mmol/L EGTA (ethylene glycol tetraacetic acid), 5 mmol/L MgCl2 (magnesium chloride), and 50 mmol/L PIPES (1,4-piperazinediethanesulfonic acid), pH 6.2, for 2 minutes.32Sadot E. Simcha I. Shtutman M. Ben-Ze'ev A. Geiger B. Inhibition of beta-catenin-mediated transactivation by cadherin derivatives.Proc Natl Acad Sci USA. 1998; 95: 15339-15344Crossref PubMed Scopus (193) Google Scholar, 33Shtutman M. Levina E. Ohouo P. Baig M. Roninson I.B. Cell adhesion molecule L1 disrupts E-cadherin-containing adherens junctions and increases scattering and motility of MCF7 breast carcinoma cells.Cancer Res. 2006; 66: 11370-11380Crossref PubMed Scopus (111) Google Scholar The Triton X-100-soluble fraction was collected and the plates were washed twice with the same buffer. The insoluble fraction was extracted with 200 μL of the buffer with 1% SDS. All quantitative experiments were performed at least in triplicate. Migration activity was determined using wound healing assay.22Yoshida T. Yoshimura E. Numata H. Sakakura Y. Sakakura T. Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells.Virchows Arch. 1999; 435: 496-500Crossref PubMed Scopus (75) Google Scholar The wells of 12-well plates were incubated with 20 μg of TNC per well for 1 hour. The suspension of MCF-7 cells (1 × 106 cells/well) was poured into a well and grown until subconfluence and further incubated with or without 5 ng/ml TGF-β1 for 24 hours. The confluent cell layer was scratched with a pipette chip, followed by medium change. Photographs (4 fields/well) were taken and TNC and/or TGF-β1 were added at the same concentration. After 24 hours, cells were fixed and stained with 0.1% crystal violet/20% ethanol/1% formaldehyde. The fields previously recorded were photographed, and the areas newly covered by the migrating cells were measured using Image J software. Ten wells per condition were examined. Differences in TNC staining with reference to histological patterns were assessed by Fisher's exact test. With quantitative data, group means were compared using one-way analysis of variance with the Bonferroni means comparison test. P values less than 0.05 were considered to be statistically significant. First, we investigated the expression of TNC in 35 cases of invasive ductal carcinomas. Dense TNC deposition was observed often in areas showing the scattered pattern (ie, the invading or EMT phenotype) of human breast cancer cells (Figure 1A), whereas solid-type large nests were negative for TNC (Figure 1B). TNC staining was dominantly positive in the stroma around and between the small cancer nests. The difference in positivity of TNC staining between solid and scattered types was highly significant (P < 0.0001; Figure 1C). It has been reported that TGF-β1 induces EMT change in some breast cancer cell lines.34Brown K.A. Aakre M.E. Gorska A.E. Price J.O. Eltom S.E. Pietenpol J.A. Moses H.L. Induction by transforming growth factor-beta1 of epithelial to mesenchymal transition is a rare event in vitro.Breast Cancer Res. 2004; 6: R215-R231Crossref PubMed Scopus (153) Google Scholar Here we investigated whether extrinsic addition of TNC and TGF-β1 to culture medium affects MCF-7 morphology (Figure 2A, top). MCF-7 cells normally showed tightly packed clusters, characteristic of epithelial cells. Although TGF-β1 (5 ng/ml) treatment did not affect the morphology, the cells treated with TNC (10 μg/ml) for 64 hours appeared to be flattened and actively spreading, and they tended to lose their cell contacts. On costimulation with TNC and TGF-β1, most cells showed apparent loss of cell-cell adhesion and a migratory phenotype with production of lamellipodia and filopodia. This phenotypic change of MCF-7 cells was reversible because the morphology was restored to the epithelial phenotype within 3 days of replacement of the normal medium (Figure 2B). As a negative control, adding a supernatant of TNC solution after immunoprecipitation by TNC antibody and protein A beads did not cause the phenotypic change either without (Figure 2C) or with (Figure 2D) TGF-β1. Because we considered the possibility that intrinsic TGF-β produced by the cells may regulate EMT change induced by TNC alone, adding a neutralizing antibody to TGF-β (1D11, 50 μg/ml) was examined, but this did not impede the EMT change (Figure 2E). These results indicate a direct effect of TNC on EMT. Immunofluorescence of E-cadherin and β-catenin (Figure 2A, middle and bottom, respectively) demonstrated membrane staining at cell contacts in control cells and TGF-β1–treated cells. After treatment with TNC or both TGF-β1 and TNC, the membranous staining was diminished, whereas cytoplasmic staining was increased and nuclear staining of β-catenin was observed. TNC deposition on the cell surfaces and substrata was detected in TNC-treated groups by immunoblot analysis, although MCF-7 cells did not synthesize TNC even after treatment by TGF-β1 alone (Figure 2F). Breast cancer cell line T-47D also showed EMT-like change by TNC treatment of 5 to 6 days. The EMT was exhibited at the margins of cell clusters, to a lesser extent than that for MCF-7 cells (Figure 3, left). Immunofluorescence demonstrated diminished β-catenin staining of intercellular contacts in the cells migrating outside the c

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