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O-Glycosylated Cd44 Variant Isoforms Are the Major Functional P-Selectin Ligands on Colon Carcinoma Cells

Susan L. Napier, Zachary Healy, and Konstantinos Konstantopoulos. Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Maryland Hall Room 223, Baltimore, MD 21218

The selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium can regulate their hematogenous spread into distant tissues. We recently demonstrated that O-linked sialofucosylated glycans on CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here, we extend our findings by showing that T84 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan sulfate and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion of T84 cells sorted based on their CD44-expression levels, and LS174T cells transfected with CD44 short hairpin (sh)-RNA to purified selectin substrates under shear. High versus low CD44-expressing T84 cells (Mean Fluorescence Intensity: 621 versus 304) bound more efficiently to P- and L-, but not E-, selectin, and rolled more slowly on P- and E-selectin. CD44 shRNA transfection of LS174T cells markedly diminished CD44 expression, and inhibited by >50% their binding to P- and L-, but not E-, selectin. Interestingly, this genetic intervention did not affect the rolling velocity of LS174T cells relative to scramble-transfected cells on E-selectin. Immunoblot analysis reveals the enhanced HECA-452 reactivity of alternative glycosylation acceptors in CD44-knockdown cells, which can mediate E-selectin binding. Our finding that CD44v is a functional P- and L-selectin ligand on colon carcinomas provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v over-expression, and the critical role of selectins in regulating metastasis.