(77i) Mucin-16 and Podocalyxin Are Functional Selectin Ligands Expressed by Pancreatic Cancer Cells

Metastatic dissemination of circulating tumor cells requires a multi-step cascade initiated by the interactions between selectins (E-, P-, and L-selectin) expressed by host cells and the selectin ligands displayed by cancerous cells. Following our recent identification of CD44 variant isoforms, carcinoembryonic antigen (CEA), and podocalyxin (PODXL) as functional selectin ligands in LS174T colon cancer cells we discovered ~200 and >460 kDa sialofucosylated glycoproteins that mediated selectin binding in SW1990 pancreatic carcinoma cells. Utilizing immunoaffinity chromatography and tandem mass spectrometry we identified Mucin-16 (MUC16) and PODXL as putative selectin ligands. Blot rolling and cell-free flow-based assays confirmed that MUC16 and PODXL expressed by SW1990 cells possess E- and L-selectin binding activity. In addition, the selectin binding capacity of SW1990 cells was dramatically decreased after sequential immunodepletion of MUC16 and PODXL. To assess and further investigate the functional role of MUC16 in selectin-mediated binding, we generated stable MUC16-konckdown SW1990 and E3LZ10.7 pancreatic cancer cells using MUC16 short hairpin RNA. Knocking down of MUC16 expression markedly reduced the cell binding to E- and L-selectin relative to control-transfected cells. Moreover, by treatment of glycosidases and carbohydrate metabolic inhibitors, the selectin-binding determinants on MUC16 are found as sialofucosylated epitopes displayed on both O- and N-linked glycans. Taken together, these findings offer new insight on the observed metastatic potential associated with MUC16 overexpression as well as the role of selectins in metastasis. Targeted therapies against MUC16-expressing cells could address multiple aspects of the metastatic cascade, including the invasive phenotype of metastasizing cells as well as blood-borne tumor/host cell interactions.