Expression of c-Met and Heparan-Sulfate Proteoglycan Forms of CD44 in Colorectal Cancer
2000; Elsevier BV; Volume: 157; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64793-1
ISSN1525-2191
AutoresVera J.M. Wielenga, Robbert van der Voort, Taher E.I. Taher, Lia Smit, Esther A. Beuling, C. van Krimpen, Marcel Spaargaren, Steven T. Pals,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoIn colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis. In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis. 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Mouse monoclonal antibodies (mAbs) used were: 3G5 (anti-CD44v3, IgG2b; R&D Systems, Abington, UK); 3G10 (anti-desaturated uronate from heparitinase-treated HS; HS stub, IgG2b)45David G Bai XM Van Der Schueren B Cassiman JJ Van Den Berghe H Developmental changes in heparan sulfate expression: in situ detection with mAbs.J Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (413) Google Scholar; PY-20 (anti-phosphotyrosine, IgG2b; Affiniti, Nottingham, UK); anti-human c-Met (IgG2a; R&D Systems); anti-human HGF/SF (IgG1; R&D Systems). Polyclonal antibodies used were (rabbit anti-c-Met, IgG, C12; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-phospho-p44/42 MAP kinase (Thr202/Tyr204; New England Biolabs, Beverly, MA); rabbit anti-ERK 1 (C-16; Santa Cruz Biotechnology); horseradish-peroxidase (HRP)-conjugated rabbit anti-mouse (DAKO, Glostrup, Denmark); HRP-conjugated goat anti-rabbit (DAKO); HRP-conjugated swine anti-rabbit (DAKO). biotin-conjugated rabbit anti-mouse (DAKO). In addition we used phycoerythrin- conjugated streptavidin (DAKO). The colon carcinoma cell lines SW480, SW620, colo 201, colo 205, colo 320, and HT-29, were purchased from the American Type Culture Collection (ATCC, Rockville, MD). HT-29 cells were cultured in modified McCoy's 5A medium (Gibco BRL/Life Technologies, Paisley, UK), whereas SW480 and SW620 were cultured in L-15 (Leibovitz) medium (Gibco BRL/Life Technologies). The other cell lines were cultured in RPMI 1640 (Gibco BRL/Life Technologies). All media were supplemented with 10. heat-inactivated fetal calf serum, 2 mmol/L l-glutamine, 100 IU/ml penicillin, and 100 IU/ml streptomycin (all from Gibco BRL/Life Technologies). The construction of pVL1393 vectors (Pharmingen, San Diego, CA. containing wild-type or mutant HGF/SF (HP1) cDNA was described elsewhere.46Hartmann G Prospero T Brinkmann V Ozcelik O Winter G Hepple J Batley S Bladt F Sachs M Birchmeier C Engineered mutants of HGF/SF with reduced binding to heparan sulfate proteoglycans, decreased clearance and enhanced activity in vivo.Curr Biol. 1998; 8: 125-134Abstract Full Text Full Text PDF PubMed Google Scholar HGF/SF (wild type and HP1) was produced in a Baculovirus system as described previously.47Hartmann G Naldini L Weidner KM Sachs M Vigna E Comoglio PM Birchmeier W A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis.Proc Natl Acad Sci USA. 1992; 89: 11574-11578Crossref PubMed Scopus (192) Google Scholar In brief, sf 9 insect cells were transduced with an amplified virus stock and after 3 days media were pooled and analyzed for scattering activity in the Madin-Darby canine kidney dissociation assay.48Weidner KM Behrens J Vandekerckhove J Birchmeier W Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.J Cell Biol. 1990; 111: 2097-2108Crossref PubMed Scopus (581) Google Scholar Then, HGF/SF was purified with Ni-NTA-resin from the QIA expressionist system (Qiagen, Hilden, Germany). HGF/SF concentrations were measured by enzyme-linked immunosorbent assay as described previously,49van der Voort R Taher TEI Keehnen RMJ Smit L Groenink M Pals ST Paracrine regulation of germinal center B cell adhesion through the c-Met-hepatocyte growth factor/scatter factor pathway.J Exp Med. 1997; 185: 2121-2131Crossref PubMed Scopus (108) Google Scholar and in addition, HGF/SF (wild type and mutant) was analyzed by Western blotting using anti-goat-HGF/SF. For enzymatic cleavage of GAGs, cells were treated with either heparitinase (Flafobacterium heparinum, EC 4.2.2.8. ICN Biomedicals, Aurora, OH) or chondroitinase avidin-biotin-peroxidase complex (Proteusvulgaris, EC 4.2.2.4; Boehringer Mannheim, Almere, The Netherlands) in phosphate-buffered saline (PBS) at 37°C for the periods indicated. Enzyme treatments were followed by immunoprecipitation. Immunoprecipitation was performed as described.49van der Voort R Taher TEI Keehnen RMJ Smit L Groenink M Pals ST Paracrine regulation of germinal center B cell adhesion through the c-Met-hepatocyte growth factor/scatter factor pathway.J Exp Med. 1997; 185: 2121-2131Crossref PubMed Scopus (108) Google Scholar The only modification were that, for precipitation of CD44, cells were lysed in lysis buffer containing 50 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 1% Nonidet P-40, 10 μg/ml aprotinin (Sigma), 10 μg/ml leupeptin (Sigma), 1 mmol/L sodium orthovanadate (Sigma), 2 mmol/L ethylenediaminetetraacetic acid, and 5 mmol/L NaF. For precipitation of c-Met, cells were lysed in 10 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 10% glycerol, 1% Nonidet P-40, 10 μg/ml aprotinin (Sigma), 10 μg/ml leupeptin (Sigma), 2 mmol/L sodium orthovanadate (Sigma), 5 mmol/L ethylenediaminetetraacetic acid, and 5 mmol/L NaF. Western blotting of immunoprecipitates and total cell lysates was essentially performed as described previously,50Taher TEI Smit L Griffioen W Schilder-Tol EJM Borst J Pals ST Signaling through CD44 is mediated by tyrosine kinases. Association with p56lck in T lymphocytes.J Biol Chem. 1996; 271: 2863-2867Crossref PubMed Scopus (150) Google Scholar with the modification that, for analysis of phosphorylated proteins, membranes were blocked and stained in 2% bovine serum albumin, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5, and 0.05% Tween-20 (Sigma). Films were scanned with an Eagle Eye II video system (Stratagene, La Jolla, CA) and band intensities were determined with ONE-Dscan software (Stratagene). c-Met phosphorylation was expressed as the ratio of phosphorylated c-Met to precipitated c-Met. Activation of the MAP kinases ERK 1 and 2 was analyzed by immunoblotting of total cell lysates with the phospho-specific p44/42 MAP kinase antibody. The study set consisted of 54 primary colorectal carcinomas,20Mulder JWR Kruyt PM Sewnath M Oosting J Seldenrijk CA Weidema WF Offerhaus GJA Pals ST Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins.Lancet. 1994; 344: 1470-1472Abstract PubMed Scopus (245) Google Scholar removed at operation between January 1, 1983 and January 1, 1986 at the Department of Surgery, Reinier de Graaf Hospital, Delft, The Netherlands, of which snap-frozen tissue and follow-up till June 1, 1992 (6.5 to 9.5 years) was available. The mean age of the patients at diagnosis was 69.7 (range, 39 to 92) and the male-to-female ratio was 28/40. Colorectal tissue samples of six adenomas and six normal controls, removed at operation between January 1, 1992 and January 1, 1999 were obtained from the tissue bank of the Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Frozen tissue sections were tested for the expression of CD44v3, c-Met, and HGF/SF by immunohistochemistry as described previously.18Koopman G Heider KH Horst E Adolf GR van den Berg F Ponta H Herrlich P Pals ST Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44.J Exp Med. 1993; 177: 897-904Crossref PubMed Scopus (318) Google Scholar, 19Heider K-H Hofmann M Horst E Van den Berg F Ponta H Herrlich P Pals ST A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps.J Cell Biol. 1993; 120: 227-233Crossref PubMed Scopus (368) Google Scholar A single modification was that HRP-conjugated rabbit anti-mouse and HRP-conjugated swine anti-rabbit were used as secondary and tertiary antibodies, respectively. All slides were read by two independent observers, and discrepancies were solved by consensus. The tumor samples were scored as described previously:20Mulder JWR Kruyt PM Sewnath M Oosting J Seldenrijk CA Weidema WF Offerhaus GJA Pals ST Colorectal cancer prognosis and expression of exon-v6-containing CD44 proteins.Lancet. 1994; 344: 1470-1472Abstract PubMed Scopus (245) Google Scholar, 23Wielenga VJM van der Voort R Mulder JWR Kruyt PM Weidema WF Oosting J Selderijk CA van Krimpen C Offerhaus GJA Pals ST CD44 splice variants as prognostic markers in colorectal cancer.Scan J Gastroenterol. 1998; 33: 82-87Crossref PubMed Scopus (57) Google Scholar 0 (low/negative) = <10% of the cells positive; 1 (intermediate) = 10 to 50% of the cells positive; 2 (high) = >50% of the cells positive. Survival functions were estimated by the Kaplan-Meier method and comparison of survival functions between groups was performed by the log-rank test. RNA isolation and first-strand cDNA synthesis were performed as described previously.49van der Voort R Taher TEI Keehnen RMJ Smit L Groenink M Pals ST Paracrine regulation of germinal center B cell adhesion through the c-Met-hepatocyte
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