Abstract
BackgroundEndothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown.Methodology/Principal FindingsBy assessing migration of various breast cancer cells across TNF-α pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLex) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a ∼170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLex moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA.Conclusions/SignificanceWe demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin–dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis.
Highlights
Metastatic invasion is the primary cause of breast cancer mortality
human umbilical vein endothelial cells (HUVECs) monolayers cultured on collagen-coated permeable Transwell filters were used as a model for endothelial cell monolayers to assess the transmigration of breast cancer cells
CD44 variant 4 (CD44v4) serves as a major E-selectin ligand in mediating breast cancer cell transendothelial metastasis To characterize the role of CD44v4 in mediating breast cancer cell transendothelial migration, we examined the effects of CD44v4 knockdown as well as removal of tumor cell surface sialyl Lewis x moieties (sLex) moieties on tumor cell adhesion to and migration across TNF-a pre-activated HUVEC monolayers
Summary
Metastatic invasion is the primary cause of breast cancer mortality. A key step in the metastasis process is migration of tumor cells across the blood vessel-lining endothelial monolayers. It has been widely reported that endothelial cell E-selectin plays a pivotal role in mediating cell–cell interactions between tumor cells and endothelial monolayers during tumor metastasis [1,2,3]. The major ligand of endothelial E-selectin on the tumor cell surface has been identified as a sialylated glycan determinant, such as sialyl Lewis x moieties (sLex), which decorate the terminal extensions of O-linked or N-linked carbohydrates [4]. Interaction of tumor cell surface sLex moieties and sLex-decorated glycoproteins with endothelium E-selectin is a major component of cancer invasion and metastasis. Endothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. The counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown
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