(86f) Novel Ligands Are Involved in Breast Cancer Cell Adhesion to Endothelial E-Selectin

Metastasis is a process of formation of secondary tumors at a site different from primary mass. Blood borne metastasis, called hematogenous metastasis, involves a systematic series of events in which cancer cells detach from primary tumor, flow in blood to adhere to endothelial cell wall lining blood vessels. Adhering cancer cells exit the blood vessel to form a secondary tumor. Although several complex molecular mechanisms define the possibility of formation of secondary tumor, successful metastasis cannot proceed without cancer cell adhesion to endothelium. Cell adhesion at molecular level is an interaction between ligand and receptor molecules those are expressed by cancer cells and endothelial cells, respectively. E-selectin is a receptor adhesion molecule which is implicated in colon and prostate cancer metastases. Breast cancer frequently spreads to bone where E-selectin is constitutively expressed. Moreover, E-selectin levels are increased in breast cancer patients with metastases. Hence we hypothesized that endothelial E-selectin and its ligands mediate breast cancer cell adhesion to vascular endothelium in dynamic flow. To test the hypothesis MDA-MB-468 and BT-20 invasive breast cancer cell lines were perfused over human umbilical cord vein endothelial cells (HUVECs) that are activated to express E-selectin. At a physiological wall shear stress breast cancer cells adhered to HUVECs but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that the adhesion was specifically mediated by E-selectin. Efforts were made to characterize E-selectin ligands. The essential requirement for an E-selectin ligand is its decoration by appropriate carbohydrates called as glycans. Characterization of glycans expressed by breast cancer cells revealed that BT-20 cells but not MDA-MB-468 cells expressed sialyl Lewis X (sLex) and sialyl Lewis A (sLea). Hence, BT-20 may have classical while MDA-MB-468 cells may have novel glycans involved in E-selectin mediated adhesion. Nevertheless, sialidase treated breast cancer cells failed to adhere to activated HUVECs, indicating a sialylation requirement for E-selectin ligand function. To characterize the contribution of glycoproteins or glycolipids as E-selectin ligands, protease treated breast cancer cells were perfused over activated HUVECs at physiological shear stress. The number of tethering cells did not decrease after protease treatment, implying the involvement of lipids in the adhesion. The involvement of lipid ligands was confirmed when significantly large number of E-selectin transfected cells under flow adhered to immobilized gangliosides. In summary, breast cancer cells express sialylated lipids called as gangliosides as an E-selectin ligands that may be decorated with novel glycans. The structural identity breast cancer cell E-selectin reactive gangliosides may provide a new target for metastasis treatment.