Title: Galectin-3 Interaction with Thomsen-Friedenreich Disaccharide on Cancer-associated MUC1 Causes Increased Cancer Cell Endothelial Adhesion
Abstract: Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 μg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 ± 21%, mean ± S.D.) and 93% (93 ± 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galβ1,3 GalNAc-α (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-β1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression. Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 μg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 ± 21%, mean ± S.D.) and 93% (93 ± 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galβ1,3 GalNAc-α (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-β1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression. Galectin-3 is one of 15 known members of the galectin family of naturally occurring galactoside-binding lectins that are expressed intracellularly and extracellularly by many cell types (1Liu F.T. Rabinovich G.A. Nat. Rev. Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1196) Google Scholar). Galectin-3 concentrations are increased up to 5-fold in the sera of patients with breast, gastrointestinal, or lung cancer (2Iurisci I. Tinari N. Natoli C. Angelucci D. Cianchetti E. Iacobelli S. Clin. Cancer Res. 2000; 6: 1389-1393PubMed Google Scholar). Moreover, higher galectin-3 concentrations are seen in the sera of patients with metastatic disease than in the sera of patients with localized tumors (2Iurisci I. Tinari N. Natoli C. Angelucci D. Cianchetti E. Iacobelli S. Clin. Cancer Res. 2000; 6: 1389-1393PubMed Google Scholar). The source of increased circulating galectin-3 in cancer patients is not clear, but it is probably generated by tumor cells as well as by peritumoral inflammatory and stromal cells (2Iurisci I. Tinari N. Natoli C. Angelucci D. Cianchetti E. Iacobelli S. Clin. Cancer Res. 2000; 6: 1389-1393PubMed Google Scholar). It is not known whether this increased circulating galectin-3 has any functional implications for cancer progression. Cytoplasmic galectin-3 is known to be anti-apoptotic (3Yang R.Y. Hsu D.K. Liu F.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6737-6742Crossref PubMed Scopus (684) Google Scholar), whereas nuclear galectin-3 promotes pre-mRNA splicing (4Dagher S.F. Wang J.L. Patterson R.J. Proc. 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Carmi P. Raz A. Biochemistry. 1993; 32: 4455-4460Crossref PubMed Scopus (95) Google Scholar, 9Takenaka Y. Fukumori T. Raz A. Glycoconj. J. 2004; 19: 543-549Crossref PubMed Scopus (279) Google Scholar). Galectin-3 expressed on the endothelial cell surface has been shown to promote the adhesion of breast cancer cells to endothelium by interaction with cancer-associated Thomsen-Friedenreich (galactose β1,3N-acetylgalactosamine α-(TF)) 2The abbreviations used are: TF antigen, Thomsen-Friedenreich antigen (galactose β1,3N-acetylgalactosamine α-); Tn antigen, N-acetylgalactosamine α-; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular cell adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; PBS, phosphate-buffered saline; ANOVA, one way analysis of variance. antigen expressed by unknown cell surface molecules (10Glinsky V.V. Huflejt M.E. Glinsky G.V. Deutscher S.L. Quinn T.P. Cancer Res. 2000; 60: 2584-2588PubMed Google Scholar, 11Glinsky V.V. Glinsky G.V. Rittenhouse-Olson K. Huflejt M.E. Glinskii O.V. Deutscher S.L. Quinn T.P. Cancer Res. 2001; 61: 4851-4857PubMed Google Scholar, 12Glinsky V.V. Glinsky G.V. Glinskii O.V. Huxley V.H. Turk J.R. Mossine V.V. Deutscher S.L. Pienta K.J. Quinn T.P. Cancer Res. 2003; 63: 3805-3811PubMed Google Scholar, 13Khaldoyanidi S.K. Glinsky V.V. Sikora L. Glinskii A.B. Mossine V.V. Quinn T.P. Glinsky G.V. Sriramarao P. J. Biol. Chem. 2003; 278: 4127-4134Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Zou J. Glinsky V.V. Landon L.A. Matthews L. Deutscher S.L. Carcinogenesis. 2005; 6: 309-318Google Scholar). TF antigen is the core I structure of mucin-type O-linked glycans, but in its simplest nonsialylated, nonextended form it acts as an oncofetal antigen, and its presence/expression is increased in malignant and premalignant epithelia (15Campbell B.J. Finnie I.A. Hounsell E.F. Rhodes J.M. J. Clin. Investig. 1995; 95: 571-576Crossref PubMed Google Scholar). MUC1 (also known as episialin and DF3) is a large (Mr > 250,000) transmembrane mucin protein expressed on the apical surface of most normal secretory epithelia including those in the mammary gland, and the gastrointestinal, respiratory, urinary, and reproductive tracts. The MUC1 extracellular domain consists of variable numbers of 20-amino acid tandem repeat peptides (VNTR) that are rich in serines, threonines, and prolines. These tandem repeat domains are heavily glycosylated with complex O-glycans (16Taylor-Papadimitriou J. Burchell J. Miles D.W. Dalziel M. Biochim. Biophys. Acta. 1999; 1455: 301-313Crossref PubMed Scopus (428) Google Scholar). There are several splice variants of MUC1, and no functional differences between these MUC1 variants are known (17Ligtenberg M.J. Gennissen A.M. Vos H.L. Hilkens J. Nucleic Acids Res. 1991; 19: 297-301Crossref PubMed Scopus (59) Google Scholar, 18Ligtenberg M.J. Vos H.L. Gennissen A.M. Hilkens J. J. Biol. 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Cancer. 1984; 34: 197-206Crossref PubMed Scopus (406) Google Scholar, 26Wesseling J. wan der walk S.W. Vos H.J. Sonnenberg A. Hilkens J. J. Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (450) Google Scholar). MUC1 has been shown to interact via its cytoplasmic domain with important intracellular proteins including β-catenin (27Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) and p53 (28Wei X. Xu H. Kufe D. Cancer Cell. 2005; 7: 167-178Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and is therefore involved in signal transduction and regulation of apoptosis. Because of its massive size and length, ∼250 nm in comparison with ∼28 nm for typical cell surface adhesion molecules like liver cell adhesion molecule (29Becker J.W. Erickson H.P. Hoffman S. Cunningham B.A. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1088-1092Crossref PubMed Scopus (89) Google Scholar), cell surface MUC1 is also believed to function as an anti-adhesion molecule by masking cell surface adhesion molecules (30Ligtenberg M.J. Buijs F. Vos H.L. Hilkens J. Cancer Res. 1992; 52: 2318-2324PubMed Google Scholar). Thus, overexpression of MUC1 inhibits integrin-mediated adhesion of human breast epithelial cells to extracellular matrix proteins in vitro (31Wesseling J. van der Valk S.W. Hilkens J. Mol. Biol. Cell. 1996; 7: 565-577Crossref PubMed Scopus (361) Google Scholar), and down-regulation of MUC1 by antisense oligonucleotide increases E-cadherin-mediated cell-cell aggregation of breast cancer cells (32Kondo K. Kohno N. Yokoyama A. Hiwada K. Cancer Res. 1998; 58: 2014-2019PubMed Google Scholar). Capping of MUC1 on the cell surface of human breast cancer cells as a result of the addition of a cross-linking anti-MUC1 antibody exposes cell adhesion molecules and increases adherence of these cells to the extracellular matrix (30Ligtenberg M.J. Buijs F. Vos H.L. Hilkens J. Cancer Res. 1992; 52: 2318-2324PubMed Google Scholar). In this study we show that MUC1 is a novel and natural ligand of endogenous galectin-3 in human colon cancer cells and that recombinant galectin-3, at concentrations similar to those found in the blood of cancer patients, causes a significant increase in adhesion of epithelial cancer cells to endothelium as a consequence of its interaction with TF expressed on MUC1. Materials—Anti-MUC1 (B27.29) and anti-STn (B195.3) (33Reddish M.A. Jackson L. Koganty R.R. Qiu D. Hong W. Longenecker B.M. Glycoconj. J. 1997; 14: 549-560Crossref PubMed Scopus (72) Google Scholar) antibodies were kindly provided by Dr. Mark Reddish (Biomira Inc., Edmonton, Canada). Mouse anti-galectin-3 antibody was obtained from Novocastra (Newcastle upon Tyne, United Kingdom). Human anti-TF antibody (TF5) was kindly provided by Dr. Bo Jansson (BioInvent Therapeutic, Lund, Sweden) (34Yu L.G. Jansson B. Fernig D.G. Milton J.D. Smith J.A. Gerasimenko O.V. Jones M. Rhodes J.M. Int. J. Cancer. 1997; 73: 424-431Crossref PubMed Scopus (30) Google Scholar). Antibodies against E-Selectin, ICAM-1, VCAM-1, integrin β1, and CD44H were from R & D Systems Europe Ltd (Abingdon, United Kingdom). Peroxidase-conjugated peanut lectin (PNA) and mushroom lectin (ABL), biotin-conjugated jacalin (JAC), Maackia amurensis (MAL-II), and Griffonia simplicifolia lectin (GSL) were purchased from Vector Laboratories Ltd. (Peterborough, United Kingdom). Arthrobacter ureafaciens sialidase (EC 3.2.1.18), Streptococcus pneumoniae endo-N-acetyl-galactosaminidase (EC 3.2.1.97), O-glycanase, and recombinant N-glycosidase (peptide-N-glycosidase F; EC 3.2.2.18) were obtained from Glyko Inc., (Oxford, United Kingdom). The non-enzymatic cell dissociation solution was from Sigma. The Vybrant DIO and DiI cell labeling solutions were from Molecular Probes (Eugene, OR). Cell Lines—The HT-29 human colon cancer cell line was obtained from the European Cell Culture Collection via the Public Health Laboratory Service (Porton Down, Wiltshire, United Kingdom). HT29-5F7 cells, kindly provided by Dr. Thecla Lesuffleur (INSERM U560, Lille, France), are enterocyte-like subpopulations of HT29 cells that express mainly MUC1 and MUC5B and were isolated as a consequence of their resistance to 5-fluorouracil (35Leteurtre E. Gouyer V. Rousseau K. Moreau O. Barbat A. Swallow D. Huet G. Lesuffleur T. Biol. Cell. 2004; 96: 145-151Crossref PubMed Scopus (64) Google Scholar). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 4 mm glutamine at 37 °C in a humidified atmosphere of 5% CO2. ZR-75-1 human breast cancer cells were kindly provided by Professor David Fernig, School of Biological Science, University of Liverpool and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.1 μg/ml estradiol, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 4 mm glutamine. Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection and were cultured in F12K medium supplemented with 0.1 mg/ml heparin, 0.03 mg/ml endothelial cell growth supplement (Sigma), and 10% fetal calf serum at 37 °C. MUC1 transfection of HBL-100 human breast epithelial cells with fulllength cDNA encoding MUC1 and the subsequent selection of the MUC1 positive transfectant HCA1.7+ and the negative revertant HCA1.7-cells were as previously described (26Wesseling J. wan der walk S.W. Vos H.J. Sonnenberg A. Hilkens J. J. Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (450) Google Scholar). MUC1 transfection of Ras-transformed Madin-Darby canine kidney epithelial-like MDCK-Ras-e cells with full-length cDNA encoding MUC1 and the subsequent selection of the MUC1 positive transfectant MDE9.2+ and the negative revertant MDE9.2-were also as previously described (26Wesseling J. wan der walk S.W. Vos H.J. Sonnenberg A. Hilkens J. J. Cell Biol. 1995; 129: 255-265Crossref PubMed Scopus (450) Google Scholar). Production of Human Recombinant Galectin-3—Recombinant human galectin-3 was produced in Escherichia coli using pET21a expression vector, which was ligated with a cDNA sequence encoding for human galectin-3, and affinity-purified using asialofetuin-Sepharose 4B as previously described (36Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (829) Google Scholar). Immunoprecipitation and Immunoblotting—Subconfluent HT-29 cells were released from the culture plates using a cell scraper (Coster) and lysed on ice in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, plus 2% aprotinin, and 20 μg/ml leupeptin) for 10 min before a brief sonication (30 s). The supernatants of the cell lysates were collected after centrifugation at 100,000 × g for 1 h. After dilution to 1 mg of protein/ml with lysis buffer, 1-ml supernatants were precleaned with 50 μl of protein A-agarose for 20 min at 4 °C before incubation with either 5 μl (20 μg) anti-MUC1 antibody (B27.29) or 20 μl of anti-galectin-3 antibody for 2 h at 4°C followed by the addition of 50 μl of protein A-agarose for a further hour. After washing, the immunoprecipitates were retrieved by mixing the beads with 40 μl of SDS sample buffer and boiling for 10 min before separation on either a 4% (for MUC1 analysis) or 12% (for galectin-3 analysis) SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membranes and probed with either anti-MUC1 or anti-galectin-3 primary antibodies. After application of the peroxidase-conjugated secondary antibodies, the blots were developed with SuperSignal West Dura Extended During Substrate (Pierce) and visualized using a Flu- or-S Imager (Bio-Rad). Desialylation and Deglycosylation—MUC1 immunoprecipitates prepared as described above were divided into six equal aliquots and incubated with or without 0.02 unit/ml N-glycanase, 0.02 unit/ml A. ureafaciens sialidase, which cleaves 2-3, 2-6, and 2-8-linked sialic acid, 0.02 unit/ml S. pneumoniae O-glycanase, which is highly specific for unsubstituted O-linked Galβ1,3 GalNAcα-, or 0.02 unit/ml sialidase plus 0.02 unit/ml O-glycanase for 16 h at 37 °C (37Yu L.G. Andrews N. Weldon M. Gerasimenko O.V. Campbell B.J. Singh R. Grierson I. Petersen O.H. Rhodes J.M. J. Biol. Chem. 2002; 277: 24538-24545Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 38Bhavanandan V.P. Umemoto J. Davidson E.A. Biochem. Biophys. Res. Commun. 1976; 70: 738-745Crossref PubMed Scopus (51) Google Scholar). The immunoprecipitates were separated on SDS-PAGE (4% running gel and 3.75% stack gel), transferred to nitrocellulose membranes, and probed with 1 μg/ml recombinant galectin-3 and then anti-galectin-3 antibody followed by peroxidase-conjugated secondary antibody. In some experiments, 30 μg of cell lysates of MUC1-transfected HCA1.7+/- or MDE9.2+/-cells were separated on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then incubated with A. ureafaciens sialidase (0.02 unit/ml) for 16 h at 37 °C before probing with anti-TF5 antibody. In other experiments, subconfluent MDE9.2+, MDE9.2-, HCA1.7+, or HCA1.7-cells were incubated with or without A. ureafaciens sialidase (0.02 unit/ml), or O-glycanase (0.02 unit/ml) for 1-3 h at 37 °C before lysis of the cells and followed by blotting with anti-MUC1, anti-TF5 antibody, or TF-binding peanut lectin (PNA). Cell Adhesion to HUVEC—Subconfluent epithelial cancer (ZR-75-1, HT29-5F7, or HT29) cells or MUC1 transfectant/revertant cells (HCA1.7+/- or MDE9.2+/-) cultured in 24-well plates were washed with PBS and labeled with 5 μg/ml DIO fluorescent cell labeling solution in serum-free Dulbecco's modified Eagle's medium for 30 min at 37 °C. The cells were washed with PBS and treated with a nonenzymatic cell dissociation solution (Sigma) that releases the cells from the culture plates while keeping the cell membrane proteins intact. After washing, 5 × 104 cells were incubated with or without recombinant galectin-3 in the presence or absence of 50 mm lactose for 30 min at 37 °C before application for 1 h at 37 °C to a HUVEC monolayer cultured on chamber slides. The chamber slides were then gently washed with PBS and inverted for 10 min at room temperature. The slides were mounted, and the fluorescent-labeled cells were counted between three and ten randomly chosen low power fields using an Olympus B51 fluorescent microscope with a 20× objective (200× magnifications). Effect of Antibodies against Adhesion Molecules on Cell Adhesion to HUVEC—A range of monoclonal antibodies against potentially relevant adhesion molecules (E-Selectin, CD44H, Integrin-β1, ICAM-1, and VCAM-1) was incubated with HUVEC cells at 25 μg/ml for 30 min at 37 °C and remained present during the subsequent 1-h cell adhesion assay as described above. Effect of Galectin-3 on MUC1 Cell Surface Localization—Subconfluent HCA1.7+/-cells were released from the culture plates using the nonenzymatic cell dissociation solution. After washing, 104 cells were incubated with or without 0.5-1 μg/ml recombinant galectin-3 for 1 h at 37°C. The cell suspensions were then applied to polylysine-coated slides for 30 min at room temperature. After gentle washing, the cells were fixed with 2% paraformaldehyde, blocked with 5% normal goat serum/PBS, and probed with anti-MUC1 antibody, followed by fluorescent-labeled secondary antibody. MUC1 localization was visualized using an Olympus B51 fluorescent microscope. Focal rearrangement of cell surface MUC1 expression was scored by two observers blinded to the cell treatment who counted the percentage of cells lacking a continuous rim of MUC1 in eight randomly selected low power fields. Laser Scanning Confocal Microscopy of Cell Adhesion to HUVEC—Samples of epithelial cells adherent to HUVEC were prepared as described above. Before introduction of recombinant galectin-3-treated HCA1.7+ cells to the HUVEC monolayer, the HUVEC cells were prelabeled with Dil (5 μl/well) cell labeling solution for 30 min at 37 °C. After interaction of the HCA1.7+/-HUVEC cells at 37 °C for 30 min, the cells were washed with PBS, fixed in 2% paraformaldehyde/PBS for 20 min, treated with 0.1% Triton X-100 for 5 min, and then treated with 5% normal goat serum/PBS for 30 min. B27.29 anti-MUC1 antibody at 0.5 μg/ml in 1% bovine serum albumin/PBS was introduced followed by fluorescein isothiocyanate-labeled secondary antibody. The laser scanning confocal microscopy was performed with Leica SP2 laser scanning confocal microscope (63× water immersion objective). Sequential line by line scanning with 476- and 543-nm lasers were used to separate fluorescence of dyes. z-Stacks were prepared by obtaining serial sections with 0.5-μm increments and analyzed in orthogonal projections (x-z sections) using Leica software. Statistical Analysis—The statistical analyses were performed using unpaired t test, Fisher's exact test, or one-way analysis of variance (ANOVA) followed by Newman and Keuls multiple comparisons (StatsDirect for Windows, StatsDirect Ltd., Sale, United Kingdom), where appropriate. The differences were considered significant when p < 0.05. Co-immunoprecipitation of Endogenous Galectin-3 and MUC1 in HT29 Human Colon Cancer Cells—MUC1 immunoprecipitation of HT29 cell lysates using B27.29 anti-MUC1 antibody followed by immunoblotting with anti-galectin-3 antibody shows the presence of endogenous galectin-3 in MUC1 immunoprecipitates but not in the control Bcl-2 immunoprecipitates (Fig. 1A). In the reciprocal experiment, galectin-3 immunoprecipitation followed by MUC1 immunoblotting also shows the presence of MUC1 in galectin-3 immunoprecipitates but not in the control immunoglobulin immunoprecipitates (Fig. 1B). These results suggest a probable interaction between galectin-3 and MUC1 in colon cancer cells. Direct Binding of Recombinant Galectin-3 to MUC1 through the Oncofetal TF Carbohydrate Antigen on MUC1—Recombinant galectin-3 probing of MUC1 immunoprecipitates from HT29 cell lysates showed that recombinant galectin-3 bound predominantly to the higher molecular weight allelic form of MUC1 (17Ligtenberg M.J. Gennissen A.M. Vos H.L. Hilkens J. Nucleic Acids Res. 1991; 19: 297-301Crossref PubMed Scopus (59) Google Scholar) (Fig. 1, C and D). This binding was not affected if MUC1 immunoprecipitates were pretreated with N-glycanase, but binding was markedly reduced if the immunoprecipitates were pretreated with Streptococcal endo-N-acetylgalactosaminidase (O-glycanase), which is highly specific for liberating unsubstituted TF from serine or threonine residues (38Bhavanandan V.P. Umemoto J. Davidson E.A. Biochem. Biophys. Res. Commun. 1976; 70: 738-745Crossref PubMed Scopus (51) Google Scholar). Pretreatment of MUC1 immunoprecipitates with A. ureafaciens sialidase, which cleaves nonreducing terminal α2-3, 2-6, and 2-8-linked sialic acid from galactose, N-acetylgalactosamine and N-acetylglucosamine residues, showed the characteristic reduced mobility in SDS-PAGE that results from the removal of the negatively charged sialic acids (31Wesseling J. van der Valk S.W. Hilkens J. Mol. Biol. Cell. 1996; 7: 565-577Crossref PubMed Scopus (361) Google Scholar). Desialylation of MUC1 greatly enhanced galectin-3 binding to MUC1, which was then markedly reduced following additional O-glycanase treatment. Together, these results suggest that galectin-3 interacts directly with MUC1 and that this interaction is mediated, to a large extent, by binding of galectin-3 to the unsubstituted TF disaccharide on MUC1. Recombinant Galectin-3 Enhances Epithelial Cancer Cell Adhesion to HUVEC—We next investigated the functional significance of the interaction of galectin-3 with MUC1. It had been reported previously that endothelial cell-associated galectin-3 mediates heterotypic adhesion of cancer cells to endothelium via binding to cancer-associated TF antigen expressed by unknown cell surface molecules (10Glinsky V.V. Huflejt M.E. Glinsky G.V. Deutscher S.L. Quinn T.P. Cancer Res. 2000; 60: 2584-2588PubMed Google Scholar, 11Glinsky V.V. Glinsky G.V. Rittenhouse-Olson K. Huflejt M.E. Glinskii O.V. Deutscher S.L. Quinn T.P. Cancer Res. 2001; 61: 4851-4857PubMed Google Scholar, 12Glinsky V.V. Glinsky G.V. Glinskii O.V. Huxley V.H. Turk J.R. Mossine V.V. Deutscher S.L. Pienta K.J. Quinn T.P. Cancer Res. 2003; 63: 3805-3811PubMed Google Scholar, 13Khaldoyanidi S.K. Glinsky V.V. Sikora L. Glinskii A.B. Mossine V.V. Quinn T.P. Glinsky G.V. Sriramarao P. J. Biol. Chem. 2003; 278: 4127-4134Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Zou J. Glinsky V.V. Landon L.A. Matthews L. Deutscher S.L. Carcinogenesis. 2005; 6: 309-318Google Scholar). This together with the presence of increased circulating galectin-3 concentrations in cancer patients and the overexpression in cancer of MUC1 bearing increased copy numbers of unsubstituted TF, prompted us to investigate the role of the interaction between galectin-3 and cancer-associated MUC1 in cancer cell adhesion to endothelium. We therefore preincubated MUC1-expressing epithelial cancer cells with recombinant galectin-3 at various concentrations and subsequently tested the adhesion of the cells to HUVECs. It was found that galectin-3 at concentrations (0.2-1.0 μg/ml) similar to those found in patients with metastatic breast or colon cancer induces a significant increase of cancer cell adhesion of human breast cancer cells to the HUVEC monolayer. At 1 μg/ml, recombinant galectin-3 increased adhesion of ZR-75-1 human breast cancer cells to HUVEC by 111% (111 ± 21%, mean ± S.D., p < 0.001, ANOVA) (Fig. 2). At similar concentration, recombinant galectin-3 caused little change of the adhesion of parental (standard) HT29 cells (data not shown) but 93% (93 ± 17%, p < 0.001, ANOVA) increased adhesion of HT29-5F7, a subpopulation of HT29 cells that have greater MUC1 expression than the parental HT29 cells (35Leteurtre E. Gouyer V. Rousseau K. Moreau O. Barbat A. Swallow D. Huet G. Lesuffleur T. Biol. Cell. 2004; 96: 145-151Crossref PubMed Scopus (64) Google Scholar, 39Truant S. Bruyneel E. Gouyer V. De Wever O. Pruvot F.R. Mareel M. Huet G. Int. J. Cancer. 2003; 104: 683-694Crossref PubMed Scopus (44) Google Scholar). In the absence of galectin-3, an average of 35 (35 ± 5) ZR-75-1 cells and 41 (41 ± 5) HT29-5F7 cells were adherent per randomly selected low power field. The increased adhesion