Abstract
Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.
Highlights
The five-year survival rate for breast cancer patients is almost 98% if the disease is detected in early stages
The breast cancer cells attached to activated Human umbilical vein endothelial cells (HUVECs), but the binding was completely diminished when the HUVECs were treated with E-selectin function blocking monoclonal antibody (mAb) (Figure 1A), demonstrating that the adhesion of ZR-75-1 cells to HUVECs is mediated by E-selectin
When sialidase treated ZR-75-1 cells were perfused over activated HUVECs, the adhesion of treated ZR-75-1 cells was significantly reduced as compared to the adhesion of untreated cells (Figure 1A), showing the need for sialylated glycans for optimal adhesion of cancer cells
Summary
The five-year survival rate for breast cancer patients is almost 98% if the disease is detected in early stages. If the primary growth has metastasized to distant organs, the survival rate decreases drastically to 27% [1]. This bleak statistic emphasizes a need for greater understanding and better interventions for the prevention of metastasis. It is believed that the attachment of circulating tumor cells to endothelium occurs through a mechanism that is similar to the recruitment of leukocytes to inflamed tissue. According to this model, flowing leukocytes form initial contacts (capture), which lead to continuous but transient interactions (rolling), and arrest of the cells on endothelium (firm adhesion). Understanding of E-selectin ligands expressed on cancer cells may be critical in devising new prognostic and therapeutic strategies against cancer metastasis
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