Abstract C19: C2-O-sLeX glycans on human carcinoma cells are high-affinity selectin ligands that mediate tumor adhesion to vascular endothelium and invasion
Authors: Catherine A. St. Hill, Katherine Krieser, Mariya Farooqui
Journal: Cancer Research (2009)

Abstract The family of selectin adhesion molecules, CD62-E and P-selectin expressed on vascular endothelium, and L-selectin expressed on leukocytes, bind sialyl-Lewis X (sLeX)-related glycans and are critical for leukocyte trafficking to lymphoid tissues and sites of inflammation. The sLeX-modified core 2 O-glycans (C2-O-sLeX) on leukocytes promote significantly higher affinity selectin binding than sLeX. Our objectives were to determine whether C2-O-sLeX glycans conferred carcinoma cells with high binding activity to the selectins and participated in metastasis. We investigated C2-O-sLeX expression in human colorectal adenocarcinomas and metastatic liver tumors by immunohistochemistry and RT-PCR. C2-O-sLeX was abundantly expressed in adenocarcinomas and mRNA expression of C2GnT1, the enzyme responsible for C2-O-sLeX synthesis, was greater than 15-fold upregulated. C2-O-sLeX was also present in metastatic liver tumors but not normal colorectal or liver tissues. We evaluated human lung (H1299), pancreatic (AsPC-1), colon (LS174T), and hepatic (HepG2) carcinoma cell lines for their expression of C2-O-sLeX or sLeX glycans and ability to bind to selectins. We also examined mRNA expression of genes coding for the enzymes alpha-2,3 fucosyltransferases (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1). FucT-III is responsible for sLeX synthesis and the formation of the core 2 branched O-glycan structures is dependent on C2GnT1. The activity of both enzymes is necessary for C2-O-sLeX synthesis. All four carcinoma cell lines expressed similar levels of C2GnT1 mRNA but FucT-III mRNA levels were lower in H1299 and AsPC-1 cells by RT-PCR. LS174T and HepG2 cell lines were 70 to 90% positive for cell surface expression of C2-O-sLeX and between 70 to 97% positive for sLeX by flow cytometry. C2-O-sLeX and sLeX were also detected in these cells by Western blotting. H1299 and AsPC-1 cells did not express these glycans. H1299 and AsPC-1 cells stably transfected with FucT-III were 43% and 7% positive for C2-O-sLeX respectively. Transfected (H1299/FucT-III and AsPC-1/FucTIII) and LS174T and HepG2 cell lines bound avidly to E-selection but not P- and L-selectin. We used shRNA vectors to silence gene expression of C2GnT1 in H1299/FucT-III and LS174T cells. We observed a 40–90% decrease in C2-O-sLeX but not sLeX expression and a 30–50% decrease in binding to E-selectin on cells containing C2GnT1 shRNA compared to unmanipulated cells. Migration of H1299/FucT-III cells across a reconstituted basement membrane was significantly increased compared to untransfected cells using a matrigel invasion assay. In contrast, the migration of H1299 cells containing C2GnT1 shRNA was reduced. Similarly, LS174T cells transfected with C2GnT1 shRNA exhibited significantly reduced migration and proliferation. Our findings demonstrate that C2-O-sLeX glycans are tumor-associated antigens expressed on carcinoma cells. Importantly, C2-O-sLeX glycans are high affinity ligands for E-selectin and play a role in the adhesion of carcinoma cells to endothelium and tumor invasiveness. These findings are relevant to the development of novel biomarkers and allow further insight into the role of sLeX-containing glycans in carcinoma metastasis. C2-O-sLeX may prove useful to predict patient prognosis and survival. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. Citation Information: Cancer Res 2009;69(23 Suppl):C19.