Title: Distinct Selectin Ligands on Colon Carcinoma Mucins Can Mediate Pathological Interactions among Platelets, Leukocytes, and Endothelium
Abstract: Selectins are adhesion molecules that mediate calcium-dependent cell-cell interactions among leukocytes, platelets, and endothelial cells. The naturally occurring vascular ligands for the selectins are mostly mucin-type glycoproteins. Increased expression and altered glycosylation of mucins are known to be prominent features of carcinoma progression. We have previously shown that all three selectins bind to colon carcinoma cell lines in a calcium-dependent fashion and that carcinoma growth and metastasis formation are attenuated in P-selectin-deficient mice. Here we show that the three recombinant soluble selectins recognize ligands within primary colon carcinoma tissue samples. Affinity chromatography showed that the ligands for all three selectins are O-sialoglycoprotease-sensitive mucins that are recognized in a calcium- and sialic acid-dependent manner. Furthermore, there are separate binding sites on the mucins for each selectin, allowing cross-binding of a single mucin molecule by more than one selectin. We also show that the selectin ligands on purified carcinoma mucins can mediate at least four different pathological interactions among platelets, leukocytes, and endothelial cells. These findings could explain some of the adhesive events of blood-borne tumor cells reported to occur with leukocytes, platelets, and endothelial cells, which are believed to play a part in modulating some early events in tumor metastases. Selectins are adhesion molecules that mediate calcium-dependent cell-cell interactions among leukocytes, platelets, and endothelial cells. The naturally occurring vascular ligands for the selectins are mostly mucin-type glycoproteins. Increased expression and altered glycosylation of mucins are known to be prominent features of carcinoma progression. We have previously shown that all three selectins bind to colon carcinoma cell lines in a calcium-dependent fashion and that carcinoma growth and metastasis formation are attenuated in P-selectin-deficient mice. Here we show that the three recombinant soluble selectins recognize ligands within primary colon carcinoma tissue samples. Affinity chromatography showed that the ligands for all three selectins are O-sialoglycoprotease-sensitive mucins that are recognized in a calcium- and sialic acid-dependent manner. Furthermore, there are separate binding sites on the mucins for each selectin, allowing cross-binding of a single mucin molecule by more than one selectin. We also show that the selectin ligands on purified carcinoma mucins can mediate at least four different pathological interactions among platelets, leukocytes, and endothelial cells. These findings could explain some of the adhesive events of blood-borne tumor cells reported to occur with leukocytes, platelets, and endothelial cells, which are believed to play a part in modulating some early events in tumor metastases. Many interactions that occur among leukocytes, platelets, and endothelial cells in the vasculature have been shown to be mediated by the selectin family of cell adhesion molecules.1Rosen SD Bertozzi CR The selectins and their ligands.Curr Opin Cell Biol. 1994; 6: 663-673Crossref PubMed Scopus (448) Google Scholar, 2Lasky LA Selectin-carbohydrate interactions and the initiation of the inflammatory response.Annu Rev Biochem. 1995; 64: 113-139Crossref PubMed Google Scholar, 3Ley K Tedder TF Leukocyte interactions with vascular endothelium: New insights into selectin-mediated attachment and rolling.J Immunol. 1995; 155: 525-528PubMed Google Scholar, 4Nelson RM Venot A Bevilacqua MP Linhardt RJ Stamenkovic I Carbohydrate-protein interactions in vascular biology.Annu Rev Cell Biol. 1995; 11: 601-631Crossref Scopus (101) Google Scholar, 5Tedder TF Steeber DA Chen A Engel P The selectins: vascular adhesion molecules.FASEB J. 1995; 9: 866-873Crossref PubMed Scopus (861) Google Scholar, 6Butcher EC Picker LJ Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2519) Google Scholar, 7Crocker PR Feizi T Carbohydrate recognition systems: functional triads in cell-cell interactions.Curr Opin Struct Biol. 1996; 6: 679-691Crossref PubMed Scopus (274) Google Scholar, 8Hynes RO Wagner DD Genetic manipulation of vascular adhesion molecules in mice.J Clin Invest. 1996; 98: 2193-2195Crossref PubMed Scopus (58) Google Scholar, 9Kansas GS Selectins and their ligands: current concepts and controversies.Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar, 10Lowe JB Ward PA Therapeutic inhibition of carbohydrate-protein interactions in vivo.J Clin Invest. 1997; 99: 822-826Crossref PubMed Scopus (72) Google Scholar, 11McEver RP Cummings RD Role of PSGL-1 binding to selectins in leukocyte recruitment.J Clin Invest. 1997; 100: 485-492Crossref PubMed Google Scholar, 12Varki A Selectin ligands: will the real ones please stand up?.J Clin Invest. 1997; 99: 158-162Crossref PubMed Scopus (260) Google Scholar The naturally occurring ligands for the three selectins are mostly mucin-type glycoproteins carrying sialylated, fucosylated glycans.1Rosen SD Bertozzi CR The selectins and their ligands.Curr Opin Cell Biol. 1994; 6: 663-673Crossref PubMed Scopus (448) Google Scholar, 2Lasky LA Selectin-carbohydrate interactions and the initiation of the inflammatory response.Annu Rev Biochem. 1995; 64: 113-139Crossref PubMed Google Scholar, 3Ley K Tedder TF Leukocyte interactions with vascular endothelium: New insights into selectin-mediated attachment and rolling.J Immunol. 1995; 155: 525-528PubMed Google Scholar, 7Crocker PR Feizi T Carbohydrate recognition systems: functional triads in cell-cell interactions.Curr Opin Struct Biol. 1996; 6: 679-691Crossref PubMed Scopus (274) Google Scholar, 9Kansas GS Selectins and their ligands: current concepts and controversies.Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar, 11McEver RP Cummings RD Role of PSGL-1 binding to selectins in leukocyte recruitment.J Clin Invest. 1997; 100: 485-492Crossref PubMed Google Scholar, 12Varki A Selectin ligands: will the real ones please stand up?.J Clin Invest. 1997; 99: 158-162Crossref PubMed Scopus (260) Google Scholar, 13Rosen SD Bertozzi CR Leukocyte adhesion: two selectins converge on sulphate.Curr Biol. 1996; 6: 261-264Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 14Vestweber D Ligand-specificity of the selectins.J Cell Biochem. 1996; 61: 585-591Crossref PubMed Scopus (45) Google Scholar, 15Sassetti C Tangemann K Singer MS Kershaw DB Rosen SD Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.J Exp Med. 1998; 187: 1965-1975Crossref PubMed Scopus (220) Google Scholar Recent studies suggest that selectin interactions with carcinoma cells may play pathological roles in tumor biology. Interestingly, progression and poor prognosis of carcinomas are associated with enhanced expression of sialylated, fucosylated epithelial mucins. 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Thus, our working hypothesis is that cell surface and/or secreted tumor mucins bearing selectin binding sites may interact in the bloodstream with leukocytes, platelets, and endothelial cells that are expressing selectins, and that these interactions can play roles in tumor biology. In this report, we have therefore examined primary human colon carcinoma samples for the presence of selectin ligands. We also explored the cross-binding and competition assays among the selectins for the carcinoma mucins. Finally, we showed that the carcinoma mucins can mediate a variety of pathological interactions among blood cells and endothelium. In so doing, we have provided a potential link between the selectins and the previously reported association of hematogenously borne cancer cells with leukocytes, platelets, and endothelium during the early events of tumor metastasis.45Karpatkin S Pearlstein E Role of platelets in tumor cell metastases.Ann Intern Med. 1981; 95: 636-641Crossref PubMed Scopus (232) Google Scholar, 46Gasic GJ Role of plasma, platelets, and endothelial cells in tumor metastasis.Cancer Metastasis Rev. 1984; 3: 99-114Crossref PubMed Scopus (241) Google Scholar, 47Weiss L Principles of Metastasis. Academic Press, Orlando FL1985Google Scholar, 48Fidler IJ Critical factors in the biology of human cancer metastasis: Twenty-eighth G. H. A. Clowes Memorial Award lecture.Cancer Res. 1990; 50: 6130-6138PubMed Google Scholar, 49Honn KV Tang DG Crissman JD Platelets and cancer metastasis: a causal relationship?.Cancer Metastasis Rev. 1992; 11: 325-351Crossref PubMed Scopus (283) Google Scholar, 50Toyoshima M Nakajima M Yamori T Tsuruo T Purification and characterization of the platelet-aggregating sialoglycoprotein gp44 expressed by highly metastatic variant cells of mouse colon adenocarcinoma 26.Cancer Res. 1995; 55: 767-773PubMed Google Scholar Unless specified, all chemicals and reagents were from Sigma (St. Louis, MO). Frozen sections of primary human colon carcinomas were from the University of California San Diego Cancer Center Histology Core. O-sialoglycoprotease (OSGPase) was from Cedarlane (Hornby, ON). LS180 colon carcinoma cells (ATCC CL 187) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Recombinant selectin immunoglobulin-Fc fusion proteins (selectin-Igs) were prepared and purified as reported.19Crottet P Kim YJ Varki A Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin: lack of evidence for unique oligosaccharide sequences mediating binding.Glycobiology. 1996; 6: 191-208Crossref PubMed Scopus (50) Google Scholar, 51Norgard KE Han H Powell L Kriegler M Varki A Varki NM Enhanced interaction of L-selectin with the high endothelial venule ligand via selectively oxidized sialic acids.Proc Natl Acad Sci USA. 1993; 90: 1068-1072Crossref PubMed Scopus (66) Google Scholar For some experiments, we used selectin-Ig proteins incorporating the FLAG epitope tag (Eastman Kodak; details of these constructs will be reported elsewhere). The α2–3 linkage specific sialidase L was a kind gift from Dr. Y.-T. Lee of Tulane University (New Orleans, LA). Blocking antibodies against the selectins were kindly provided by M. Bevilacqua (H18/7, anti-E-selectin), R. McEver (C138A, anti-P-selectin), and T. Tedder (LAM 1.14, anti-L-selectin). Paraffin or frozen sections (fixed in 10% formalin/0.1 mol/L cacodylic acid for 30 minutes at room temperature) of human colon carcinomas or of LS180 cells were probed for selectin binding. For tissue sections, endogenous peroxidase was destroyed with 0.03% H2O2 for 10 minutes at room temperature, followed by a rinse in binding buffer (3% bovine serum albumin (BSA) in 20 mmol/L HEPES, 100 mmol/L NaCl, 2 mmol/L CaCl2, 2 mmol/L MgCl2, pH 7.4). Sialic acid side chains were next selectively oxidized with 2 mmol/L periodate in phosphate buffered saline (PBS) for 30 minutes in the dark at 4°C, followed by biotinylated or FLAG-epitope-tagged recombinant selectins in binding buffer for 1 hour at room temperature. FLAG-epitope-tagged selectins were detected with mouse anti-FLAG-IgG, followed by biotinylated horse anti-mouse IgG. Biotin groups were then detected with streptavidin-peroxidase conjugates (Binding Site, Birmingham, UK) and enzymatic color development performed at room temperature using VIP substrate (Vector Labs, Burlingame, CA) or 3′3′9-aminoethyl carbazole (Sigma). Slides were finally counterstained with Mayer's hematoxylin and rinsed in binding buffer without BSA. Control slides were incubated with either 5–10 mmol/L EDTA in place of calcium or without the primary probe. Each selectin-Ig (∼6 nmol, 0.5 mg protein) was incubated with 1 ml of Protein-A Sepharose resin (PAS) (Pharmacia) in pH 8.0 buffer for several hours with end-over-end rotation, and the resin then prepared as a column in a 2-ml Pasteur pipette.19Crottet P Kim YJ Varki A Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin: lack of evidence for unique oligosaccharide sequences mediating binding.Glycobiology. 1996; 6: 191-208Crossref PubMed Scopus (50) Google Scholar Secreted or detergent-extracted glycoconjugates from LS180 cells metabolically radiolabeled with [6-3H]glucosamine were enriched for labeled mucins by Jacalin-Sepharose chromatography and gel filtration on Sephacryl S200.19Crottet P Kim YJ Varki A Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin: lack of evidence for unique oligosaccharide sequences mediating binding.Glycobiology. 1996; 6: 191-208Crossref PubMed Scopus (50) Google Scholar These were dissolved in 0.5 ml binding buffer, loaded onto each of the selectin columns, and 0.5-ml fractions collected under gravity flow (∼0.05 ml/min). After ∼10 column volume washes, bound materials were eluted with 20 mmol/L MOPS, 100 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L MgCl2. Bound and run-through fractions were pooled, the latter adjusted for divalent cations, and aliquots reloaded onto each selectin column to test capacity and cross-binding. Aliquots of bound material were also treated with sialidase L in sodium acetate, pH 5.5, or with OSGPase in binding buffer, boiled, adjusted into binding buffer as needed, and reapplied to the selectin columns. Appropriate background subtraction was determined with blank vials. Samples were counted at a constant quench level in aqueous-compatible scintillation fluid for a period long enough to give a 95% confidence level. Thus, although there were low levels of radioactivity available for some analyses, the signal-to-noise ratios were acceptable. Confluent LS180 cells were released in 5 mmol/L EDTA containing medium, washed in PBS and 2 × 106 cells subcutaneously injected into the flanks of immunodeficient Rag-2 null mice (Taconic, Germantown, NY).52Shinkai Y Rathbun G Lam KP Oltz EM Stewart V Mendelsohn M Charron J Datta M Young F Stall AM RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.Cell. 1992; 68: 855-867Abstract Full Text PDF PubMed Scopus (2199) Google Scholar After several weeks, the tumors (∼2 cm in diameter) were excised, any necrotic regions discarded, and homogenized in 0.5 × PBS with 40 μg/ml aprotonin, 1 μg/ml leupeptin, 0.7 μg/ml, pepstatin, and 20 μg/ml phenylmethylsulfonyl fluoride. The insoluble debris was removed at 100,000 × g for 1 hour at 4°C and the viscous supernatant fractionated on a Superose CL-4B column run at ∼1 ml/minute flow in PBS with the protease inhibitor cocktail detailed above. High molecular weight fractions (void volume) were pooled and further enriched for mucins using a CsCl2 gradient. Fractions with a density of 1.45 or greater were pooled, dialyzed, lyophilized, and further enriched for O-linked glycoproteins using Jacalin affinity chromatography. Purification of the mucins was monitored using bovine submaxillary mucin as a standard, with A280 readings for protein, and the 2-thiobarbituric acid (TBA) or 1,2-diamino-4,5-methylene dioxybenzene (DMB) assays for sialic acids. Typically, ∼5 g of tissue yielded ∼1 mg of carcinoma mucin. For some studies, mucin samples were also extracted with chloroform:methanol (1:1, 10 volumes) or treated with heparin lyase II to exclude contamination by glycolipids or heparan sulfate, respectively. Selectin-Igs were biotinylated using 0.5 mg sulfo-NHS-biotin (Pierce, Rockford, IL) dissolved in DMSO, and mixed with 100 μg of selectin-Igs in PBS at 4°C for 1 hour. Reactions were quenched with 100 mmol/L glycine and the samples extensively dialyzed against binding buffer. Biotin content per molecule was estimated by Western blot analysis in comparison to standards. Preservation of selectin activity after biotinylation was confirmed using an enzyme-linked immunosorbent assay (ELISA) method involving binding to polyacrylamide-sLex absorbed on microtiter plates.53Koenig A Jain R Vig R Norgard-Sumnicht KE Matta KL Varki A Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1.Glycobiology. 1997; 7: 79-93Crossref PubMed Scopus (94) Google Scholar Purified mucins from tumors were absorbed onto 96-well Xeno-bind plates in sodium carbonate buffer, pH 9.5, at 5 μg/ml concentration. (Xeno-bind plates aid covalent attachment of the highly glycosylated mucins.) Absorption of mucins was confirmed using tracer amounts of [3H]mucin from LS180 cells and by detection with wheat germ agglutinin (WGA). Plates were blocked with 1% BSA in binding buffer, and incubated with various mixtures of biotinylated and nonbiotinylated selectins at 4°C for 3 hours. After washing, Neutravidin-alkaline phosphatase conjugate was used to detect the biotinylated selectin binding. Cleavage of 4-methylumbelliferyl phosphate by the bound phosphatase was detected after 4 hours (product release was linear to this point) using a 96-well fluorescent reader (CytoFluor II, Perseptive Biosystems, Bedford, MA). Corning ELISA plates were coated with 200 ng of E-, P-, or L-selectin-Ig by overnight incubation at 4°C in 100 μl of 50 mmol/L sodium carbonate/bicarbonate buffer, pH 9.5. Plates were blocked with 0.1% BSA in Hanks' balanced salt solution (HBSS, Sigma) for 30 minutes at room temperature. Purified carcinoma mucin in HBSS was added (1 μg per well) and incubated for 1 hour at room temperature. During the mucin incubation, 200 ng of selectin Ig chimeras with the FLAG epitope tag were precomplexed with monoclonal anti-FLAG M2 antibody (Sigma) at 1:300 dilution. Controls including 30 mmol/L EDTA in this step were used. Plates were washed twice with HBSS and the precomplexes added to plates for 1 hour at room temperature. Plates were washed with HBSS and incubated with alkaline phosphatase-conjugated goat-anti-mouse antibody (Biorad) at concentration 1:1000 for 1 hour at room temperature. Plates were again washed four times with HBSS, developed with p-nitrophenyl phosphate (Sigma), quenched by addition of 40 μl of 3 mol/L NaOH and the absorption at 405 nm read on a microplate reader (Molecular Devices). Blood from normal individuals who had not recently consumed caffeine or medications was collected into acid citrate dextrose, and platelet-rich plasma prepared by centrifugation. Platelets were rewashed into 10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L CaCl2, 0.5% BSA, and 20 nmol/L PGE1 added for stabilization. Aggregation was monitored in a standard platelet aggregome