Involvement of Large Tenascin-C Splice Variants in Breast Cancer Progression
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64320-9
ISSN1525-2191
AutoresTakatsugu Tsunoda, Hiroyasu Inada, Ilunga Kalembeyi, Kyoko Imanaka‐Yoshida, Mirei Sakakibara, Ray Okada, Koji Katsuta, Teruyo Sakakura, Yuichi Majima, Toshimichi Yoshida,
Tópico(s)Cellular Mechanics and Interactions
ResumoAlternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer. Alternative splicing of fibronectin-like type III (FNIII) repeats of tenascin-C (Tn-C) generates a number of splice variants. The distribution of large variants, typical components of provisional extracellular matrices that are up-regulated during tumor stroma remodeling, was here studied by immunoblotting and immunohistochemistry using a monoclonal antibody against the FNIII B domain (named 4C8MS) in a series of human breast cancers. Large Tn-C variants were found at only low levels in normal breast tissues, but were highly expressed at invading sites of intraductal cancers and in the stroma of invasive ductal cancers, especially at invasion fronts. There was a positive correlation between the expression of large Tn-C variants and the cell proliferation rate determined by immunolabeling of the Ki-67 antigen. Of the Tn-C recombinant fragments (all FNIII repeats or mFNIII FL, the conserved FNIII domain only, the epidermal growth factor-like domain, and the fibrinogen-like domain) which were expressed by CHO-K1 cells transfected with mouse Tn-C cDNAs, only the mFNIII FL enhanced in vitro migration and mitotic activity of mammary cancer cells derived from a Tn-C-null mouse. Addition of 4C8MS blocked the function of mFNIII FL. These findings provide strong evidence that the FNIII alternatively spliced region has important roles in tumor progression of breast cancer. During tumor progression, the cancer stroma becomes remodeled by both tumor cells and stromal cells, and protein components of the extracellular matrix (ECM) are dynamically changed by degradation and neosynthesis. Cellular interaction with the ECM strongly influences the behavior of cancer and stromal cells, resulting in modulation of cell growth, migration, differentiation, and apoptosis.1Donjacour A Cunha G Stromal regulation of epithelial function.Cancer Treat Res. 1991; 53: 335-364Crossref PubMed Scopus (143) Google Scholar, 2Liotta L Kohn E The microenvironment of the tumour-host interface.Nature. 2001; 411: 375-379Crossref PubMed Scopus (2020) Google Scholar, 3Wernert N The multiple roles of tumor stroma.Virchows Arch. 1997; 430: 433-443Crossref PubMed Scopus (170) Google Scholar Compositional change of the ECM in cancer stroma is thus a key determinant of tumor growth and cancer progression. A variety of ECM glycoproteins, such as tenascin-C (Tn-C) and fibronectin, are overexpressed in cancer stroma. 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There are eight conserved FN III repeats (designated as numbers 1–8) and, in the case of human Tn-C, up to nine alternatively spliced FN III repeats (designated as letters A-D) inserted between the conserved repeats 5 and 6. 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After balloon catheterization, smooth muscle cells express a Tn-C isoform containing only A1 and A2 repeats.30Wallner K Shah PK Sharifi BG Balloon catheterization induces arterial expression of new tenascin-C isoform.Atherosclerosis. 2002; 161: 75-83Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Transcripts for splice variants including the AD2 repeat are found in oral cancer tissues, but not in normal and pre-malignant tissues.31Mighell AJ Thompson J Hume WJ Markham AF Robinson PA Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant, and reactive oral mucosae.Int J Cancer. 1997; 72: 236-240Crossref PubMed Scopus (49) Google Scholar Immunohistochemistry using a recombinant antibody to the spliced repeat C has shown that the repeat is not detectable in normal adult tissues, not or barely detectable in malignant epithelial tumors, but abundant in glioblastomas.32Carnemolla B Castellani P Ponassi M Borsi L Urbini S Nicolo G Dorcaratto A Viale G Winter G Neri D Zardi L Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody.Am J Pathol. 1999; 154: 1345-1352Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar From quantitative analysis using antibodies against alternative spliced domains, an increased fraction of Tn-C containing A1-A4 appears related to the tumor stage of colorectal cancer, while domain D is down-regulated in metastasizing cancers.10Dueck M Riedl S Hinz U Tandara A Möller P Herfarth C Faissner A Detection of tenascin-C isoforms in colorectal mucosa, ulcerative colitis, carcinomas, and liver metastases.Int J Cancer. 1999; 82: 477-483Crossref PubMed Scopus (45) Google Scholar In breast tissues, expression of two Tn-C variants, one containing domain D and the other both B and D, was found to be associated with invasive carcinomas in one study.11Adams M Jones JL Walker RA Pringle JH Bell SC Changes in tenascin-C isoform expression in invasive and preinvasive breast disease.Cancer Res. 2002; 62: 3289-3297PubMed Google ScholarMalignant properties of tumors highly expressing Tn-C may to some extent be dependent on functions of alternative spliced domains of the FNIII repeats, since it has been reported that the alternatively spliced region, FNIII A-D, promotes migration and proliferation of endothelial cells through interaction with annexin II.33Murphy-Ullrich JE Lightner VA Aukhil I Yan YZ Erickson HP Höök M Focal adhesion integrity is down-regulated by the alternatively spliced domain of human tenascin.J Cell Biol. 1991; 115: 1127-1136Crossref PubMed Scopus (243) Google Scholar, 34Chung CY Erickson HP Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.J Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar, 35Chung CY Murphy-Ullrich JE Erickson HP Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II.Mol Biol Cell. 1996; 7: 883-892Crossref PubMed Scopus (180) Google Scholar Thus an involvement of Tn-C with the repeats in angiogenesis is possible. The spliced region is further known to support attachment of chick embryonic neurons,36Fischer D Brown-Ludi M Schulthess T Chiquet-Ehrismann R Concerted action of tenascin-C domains in cell adhesion, anti-adhesion, and promotion of neurite outgrowth.J Cell Sci. 1997; 110: 1513-1522Crossref PubMed Google Scholar to mediate neurite outgrowth and guidance,37Meiners S Mercado ML Nur-e-Kamal MS Geller HM Tenascin-C contains domains that independently regulate neurite outgrowth and neurite guidance.J Neurosci. 1999; 19: 8443-8453PubMed Google Scholar, 38Meiners S Nur-e-Kamal MS Mercado ML Identification of a neurite outgrowth-promoting motif within the alternatively spliced region of human tenascin-C.J Neurosci. 2001; 15: 7215-7225Google Scholar and to promote proliferation of hemopoietic precursor cells in bone marrow.39Seiffert M Beck SC Schermutzki F Müller CA Erickson HP Klein G Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains.Matrix Biol. 1998; 17: 47-63Crossref PubMed Scopus (44) Google Scholar In addition, the receptor protein tyrosine phosphatase β present on glial tumor cells binds to FNIII A1, 2, and 4, suggesting it to be an adhesion receptor to ECM.40Adamsky K Schilling J Garwood J Faissner A Peles E Glial tumor cell adhesion is mediated by binding of the FNIII domain of receptor protein tyrosine phosphatase β(RPTPβ) to tenascin C.Oncogene. 2001; 20: 609-618Crossref PubMed Scopus (41) Google Scholar However, it is unclear whether the alternatively spliced region of Tn-C directly enhances progressive behavior of tumor cells.The aim of this study, therefore, was to examine the expression and distribution of large Tn-C splice variants in breast tissues using a novel monoclonal antibody (mAb) specific to FNIII B. An investigation of direct effects of recombinant Tn-C fragments containing the alternatively spliced region on migration and proliferation of mammary cancer cells was also included.Materials and MethodsPurification of Tn-C and Tn-C Recombinant FragmentsTo produce a monoclonal antibody specific for large Tn-C splice variants, we first prepared a recombinant protein containing FNIII A4-D domains of human Tn-C (Figure 1A). Complementary DNA encoding the region was generated by PCR using human fetal brain Marathon-Ready cDNA (BD Biosciences Clontech, Palo Alto, CA) as the template and the primers shown in Table 1. PCR products were cloned into the BamHI and HindIII sites of the pQE30 expression vector (Qiagen, Hilden, Germany) and the sequence confirmed. For protein expression, single colonies of E. coli JM109 cells transformed with the construct were grown in LB medium. Expression of the recombinant protein was induced by addition of 1 mmol/L IPTG. The bacteria were collected by centrifugation at 7,000 × g for 15 minutes, and resuspended in lysis buffer (phosphate buffer pH 7.4, 20 mmol/L imidazole, 1% Tween 20, 6 mol/L urea) with protease inhibitor cocktail tablets (Roche Diagnostic, Basel, Switzerland). The mixture was stirred for 30 minutes at room temperature and then centrifuged at 15,000 × g for 30 minutes at 4°C, the supernatant being collected and applied to a HisTrap column (Amersham Pharmacia, Buckinghamshire, UK). Recombinant proteins were eluted with elution buffer (PB pH 7.4, 0.5 mol/L imidazole, 1% Tween-20, 6 mol/L urea). Recombinant hFNIII A4-C, A4-B, and A4 fragments were also prepared by the same procedures using the reverse primers shown in Table 1.Table 1PCR Primers Used for the Generation of Recombinant Proteins Issued from the Alternatively Spliced Domain of Human and Mouse Tn-CRecombinant proteinPrimers (5′-3′)hFNIII A4-DF:CTGGGATCCCAGGTGCAGGAGGTCAACAAAGTGR:CCCAAGCTTGGGGGCAAGTAGGGTTATTTCCAGhFNIII A4-CR:TACAAGCTTGTAACAATCTCAGCCCTCAAGhFNIII A4-BR:GACAAGCTTGTCGTGGCTGTGGCACTGATGhFNIII A4R:CACAAGCTTGTGGAGGCCTCAGCAGAGTACTGmFNIII FLF:AAGGAATTCCTTCTGAGGTGTCCCCTCCCAAAR:AACTCGAGGGAATGGGTACAGGAGTCCAATmFNIII SOF(1):AAGGAATTCCTTCTGAGGTGTCCCCTCCCAAAR(5):AACCTCGAGGCCTTCACACGTGCAGGCTTGCF(6):ACGAATTCTGTCGACGGCCATGGGTTCTCCGAAGGR(8):AACTCGAGGGAATGGGTACAGGAGTCCAATmEGFF:AACGAATTCGCTGTGTCTGTGAACCAGGCTGGR:TCCTCGAGAGCAGTCTATCCCTGTGTAACCmFBGF:TTGGAATTCCCAGGGACTGCTCTCAAGCAATGR:CGGGCAGCGGCCGCTGCCCGCTTACGCCTGCCT Open table in a new tab To analyze functions of Tn-C domains, we also prepared recombinant fragments of mouse Tn-C using a cell expression system. Complementary DNAs encoding EGF-like repeats (mEGF), FNIII repeats (mFNIIII FL and SO), and the FBG-like domain (mFBG) of mouse Tn-C (Figure 1B) were generated by PCR using mouse Tn-C cDNA as a template and the primers shown in Table 1. The complete cDNA of mouse Tn-C was cloned from a cDNA library of a mammary cancer cell line.21Saga Y Tsukamoto T Jing N Kusakabe M Sakakura T Murine tenascin: cDNA cloning, structure, and temporal expression of isoforms.Gene. 1991; 104: 177-185Crossref PubMed Scopus (90) Google Scholar Restriction sites were included in the primers to facilitate directional cloning and PCR products were cloned into the pSecTag2A expression vector having 6xHis and myc tags (Invitrogen, Carlsbad, CA), and the sequences confirmed. Since the template Tn-C cDNA includes five alternatively spliced repeats of the FNIII domain, repeats of FNIII 1–5, A1, A2, A4, B, D, and 6–8 comprised cDNA for mFNIII FL. The plasmids containing FNIII 1–5 were then restricted with XhoI, and those containing FNIII 6–8 were restricted with SalI and XhoI. These constructs were ligated again, and then the plasmids for mFNIII SO were completed. For transfection, CHO-K1 cells were used and grown in Iscove's modified Dulbecco's medium (IMDM, Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Grand Island, NY) under 95% air/5% CO2 atmosphere at 37°C. The constructs were transfected into CHO-K1 cells with Lipofectamin plus reagent (Invitrogen). Forty-eight hours later, the cells were cultured in medium containing Zeocin (Invitrogen) to select stable transfectants. The clones producing the recombinant proteins were chosen by Western blotting using anti-myc-tag antibody (MBL, Nagoya, Japan), and subcloned three times. Conditioned media containing the proteins were collected. The recombinant proteins were precipitated with 50% ammonium sulfate, and purified using a HisTrap column (Amersham Pharmacia). Purified proteins of Tn-C domains from conditioned media of stable transfectants are shown in Figure 2. Human Tn-C was purified from conditioned media of a glioma cell line, U251MG, by a previously described method.15Yoshida T Yoshimura H 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 It is referred to as intact Tn-C in this paper.Figure 2Purified recombinant proteins of mouse Tn-C domains. Recombinant proteins were purified from the conditioned media using His-trap column, electrophoresed in sodium dodecyl sulfate polyacrylamide gel (2 to 15%), and stained with Coomassie blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Monoclonal Antibodies Against Tn-CTo obtain higher efficiencies of antibody production, we immunized congenic Tn-C-null mice obtained by back-crossing Tn-C-null mice originally generated by Saga et al41Saga Y Yagi T Ikawa Y Sakakura T Aizawa S Mice develop normally without tenascin.Genes Dev. 1992; 6: 1821-1831Crossref PubMed Scopus (529) Google Scholar with BALB/c mice. They received 100 μg of the recombinant FNIII A4-D protein mixed with Freund's complete adjuvant, and after 2 weeks, the antigen mixture with Freund's incomplete adjuvant. Three days before cell fusion, the mice were hyperimmunized with the antigen by i.v. injection. Hybridomas were prepared by fusion of immunized mouse spleen cells with myeloma cells (Sp2/0) in polyethylene glycol. Positive hybridoma clones were detected by enzyme immunoassay using plates coated with native human Tn-C and peroxidase-conjugated anti-mouse IgG antibodies (Biorad, Hercules, CA). After being cloned three times, they were transferred to serum-free medium (Wako, Osaka, Japan) with 20 units/ml IL-6 (Roche Diagnostic) and grown. The conditioned media were collected by centrifugation, and then the antibodies were purified by 50% ammonium sulfate fractionation, followed by affinity-purification using a HiTrap protein A column (Amersham Pharmacia). The isotype of the monoclonal antibodies was determined with the mouse monoclonal antibody isotype kit (Amersham Lifescience, Piscataway, NJ) and was IgG1 κ.ImmunoblottingFour breast tissue samples were obtained from specimens taken at therapeutic surgeries for breast cancers. Small pieces of normal parts and cancer tissues were isolated by gross examination and stored at −80°C. Aliquots (100 μg) were then homogenized in 1 ml of 10 mmol/L Tris-buffered saline (TBS, pH 7.6) containing 0.2% Nonidet P-40 and protease inhibitor cocktail tablets (Complete mini, Roche, Manheim, Germany), and centrifuged at 15,000 × g for 10 minutes. The pellets were resuspended in TBS, and washed twice with centrifugation, then resuspended in 1 ml of TBS containing 2 mol/L urea, and the slurries were gently shaken for 1 hour on ice. They were centrifuged at 15,000 × g for 15 minutes, and the supernatants were collected. The samples were applied to polyacrylamide gels and electrophoresed by the method of Laemmli. The electrophoresed proteins were blotted onto Immobilon membranes (Millipore Japan, Tokyo, Japan), blocked with a blocking buffer (TBS, pH 7.6 and 0.5% skimmed milk), and incubated with monoclonal anti-Tn-C antibodies (4F10TT or 4C8MS, 1 μg/ml) at 4°C overnight. The monoclonal antibody 4F10TT against the EGF-like repeats of Tn-C was as previously reported.42Imanaka-Yoshida K Hiroe M Yasutomi Y Toyozaki T Tsuchiya T Noda N Maki T Nishikawa T Sakakura T Yoshida T Tenascin-C is a useful marker for disease activity in myocarditis.J Pathol. 2002; 197: 388-394Crossref PubMed Scopus (116) Google Scholar The membranes were then washed in the blocking buffer and successively treated with peroxidase-labeled goat anti-mouse IgG Fab' (1:400, MBL, Nagoya, Japan) for 1 hour at room temperature. The reactive bands were developed with diaminobenzidine (DAB)/H2O2 solution.ImmunohistochemistryImmunohistochemical analysis was performed on 10 fiboroadenomas and 22 invasive ductal carcinomas using archival samples which were fixed with formalin and routinely processed for embedding in paraffin. All sections were cut at 4 μm and placed on silane-coated glass slides (DAKO Japan, Kyoto, Japan). For 4F10TT, the sections were incubated in 0.4% pepsin (1:60,000, Sigma) in 0.01N HCl for 20 minutes at 37°C to retrieve the antigens. The sections were incubated in 0.3% H2O2 in methanol for 15 minutes to block endogenous peroxidase activity. This proteinase treatment conversely abolished the positive reaction with the 4C8MS antibody. Treatment with 0.1% sapponin in distilled water for 20 minutes or heating in an autoclave was effective for the retrieval of antigens. Sections were treated with superblock solution (Scytek Laboratories, Logan, UT) or 10% normal goat serum before incubation with anti-Tn-C antibodies (1 to 10 μg/ml) overnight at 4°C. After being washed, sections were treated with a commercially available LSAB kit (Scytek) or peroxidase-conjugated anti-mouse IgG Fab' (MBL), followed by color development with DAB/H2O2 solution. Light counterstaining with hematoxylin was performed to aid orientation. Various tissue samples from autopsy cases were also examined.For double immunohistochemistry with 4C8MS and Ki-67 antibodies, the sections were treated with 4C8MS, followed by the incubation with peroxidase-conjugated anti-mouse IgG Fab', and color development by a DAB/H2O2 solution. The immune complexes were then removed by treatment with 0.01N HCl solution. Next the sections were exposed to the Ki-67 antibody (clone MIB-1, Dako Japan), and again treated with peroxidase-conjugated anti-mouse IgG Fab'. The second coloring reaction was achieved with a Ni/DAB/H2O2 solution. Therefore, the 4C8MS immunoreactivity was brown in color, whereas the Ki-67 immunoreactivity was blue. Finally the sections were lightly counterstained with methylgreen solution.To examine whether expression of large Tn-C variants was related to cell proliferation in human cancer tissues, 12 cases having sufficient areas for analysis were chosen from fully invasive carcinomas. When the 4C8MS-staining of cancer stroma was equivalent to or stronger than that of the muscular arteries in the same tissue, the immunolabeling was considered as positive. Labeling indices for Ki-67-positive nuclei in three fields (1 mm2 each) positive for 4C8MS was compared with those for three negative fields close to the positive fields.Migration AssayThe mouse mammalian cancer cell line, GHOM5E, was established from a spontaneously developing tumor in a congenic Tn-C-null mouse of the GRS/A strain. By transwell migration assay using cell culture inserts (8-μm pore size, Becton Dickinson Labware, Franklin Lakes, NJ), we examined effects of intact Tn-C or its recombinant fragments on cancer cell migration. Into the inner chamber, 5 × 104 cells were plated in 0.5% bovine serum albumin (BSA)/serum-free IMDM, and the medium containing 0.5% BSA and 5% FCS as a chemoattractant was poured in the outer chamber (Falcon 24-well plate, Becton Dickinson Labware). After addition of the proteins tested into the medium of the upper chamber, the cells were allowed to migrate to the lower membrane surface for 18 hours. After the cells on the upper surface were wiped off, the inserts were fixed and stained with 0.1% crystal violet (Sigma) in 10% ethanol. Stained cells on the lower surface in three fields of 1 mm2 were counted under a 10× objective lens.BrdU Incorporation AssayThe cells were grown on coverslips in IMDM/10% FCS for 16 hours, washed with serum-free medium, and then incubated in the medium with 0.1% FCS. Twenty-four hours thereafter, Tn-C or the fragments were added into the medium at final concentration of 1 to 10 μg/ml. After incubation for 12 hours, the cells were labeled with 8-bromodeoxyuridine (BrdU 10 μg/ml) for 2 hours, fixed with 70% ethanol at −20°C for 30 minutes, and treated with 1N HCl solution at room temperature for 20 minutes. Labeled nuclei were detected with monoclonal anti-BrdU antibody (DAKO Japan) and anti-mouse IgG conjugated with peroxidase (MBL). The percentage of the labeled nuclei was determined.Antibody BlockingThree μg of mouse Tn-C fragments were incubated with 15 μg of the 4C8MS Fab' fragment for 30 minutes at room temperature and then added into the medium for BrdU incorporation and migration assays. As a control antibody, an isotype-matched monoclonal antibody against V protein of parainfluenza virus type II (clone 53–1) (kindly provided by the Department of Microbiology, Mie University School of Medicine, Mie, Japan) was used. The experiment was performed in triplicate.Statistical AnalysisFor quantitative analysis the arithmetical mean
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