(74h) Distinct Glycosylations On the Hcellv Isoform of CD44 Mediate Breast Cancer Cell Adhesion to E-Selectin

Authors: 
Burdick, M. M., Ohio University
Shirure, V. S., Ohio University
Liu, T., Ohio University


Metastasis is the culmination of an elaborate cascade of events in which cancer cells separate from the primary mass, intravasate through blood vessel walls into the vasculature, traverse throughout the body, and finally seed new colonies in distant organs. E-selectin expressed by vascular endothelium plays a significant role in the cascade, mediating the tethering and rolling of circulating tumor cells (CTCs) that initiate the recruitment process in the secondary organ.  We have previously shown that sialylated glycolipids on breast cancer cells are E-selectin ligands.  Glycoprotein E-selectin ligands are also expressed by breast cancer cells, but it is not known whether O-linked and/or N-linked glycans confer ligand activity. Therefore, we sought to define the type(s) of glycosylation(s) leading to E-selectin ligand function of cell surface proteins expressed by breast cancer cells.  When BT-20 breast cancer cells were perfused over IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) at 0.8 dyn/cm2 in the parallel plate flow chamber adhesion assay, the breast cancer cells attached in an endothelial E-selectin-dependent manner.  Pretreatment of the BT-20 cells with Vibrio cholerae neuraminidase, which enzymatically cleaves terminal sialic acid residues, essentially abrogated attachment to HUVECs, consistent with the sialylation requirement of E-selectin ligands.  To more specifically characterize the glycans expressed by glycoprotein E-selectin ligands, BT-20 cells were treated with deoxymannojirimycin (DMJ) to prevent N-linked glycosylation of glycoproteins, whereas cells were treated with benzyl-N-acetyl-α-galactosaminide (Bzl-GalNAc) to prevent O-linked glycosylation of glycoproteins.  Immunoprecipitation, Western blotting, flow cytometry, and parallel plate flow chamber adhesion assays revealed that an N-glycosylated high molecular weight variant of CD44 (~150 kDa; CD44v) was a major contributor to E-selectin-mediated adhesion.  O-glycans also contributed to E-selectin ligand activity of CD44v (~150 kDa), albeit to a lesser extent than N-glycans.  However, no E-selectin ligand activity was attributed to CD44s (~100 kDa).  The breast cancer glyco-isoforms of CD44 possessing E-selectin ligand activity, termed HCELLv, thereby demonstrate similarities to both N-glycosylated HCELLs and HCELLv predominantly expressed by normal hematopoietic stem cells and leukemic cells, as well as O-glycosylated HCELLv expressed by colon cancer cells.  With increased understanding of the role that glycosylation plays in E-selectin ligand function of breast cancer cells, vital improvements in the diagnosis, prognosis, and treatment of breast cancer can be made.