Mac-2 binding protein is a novel E-selectin ligand expressed by breast cancer cells

• Published in: PloS one 2012-09, Vol.7 (9), p.e44529-e44529
• Main Authors: Shirure, Venktesh S, Reynolds, Nathan M, Burdick, Monica M
• Format: Article
• Language: English
• Subjects: Adhesion; Adhesion tests; Analysis; Antigens; Antigens, Neoplasm - metabolism; Base Sequence; Biology; Blotting, Western; Breast cancer; Breast carcinoma; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cancer; Cancer cells; Cancer metastasis; Cell Adhesion; Cell adhesion & migration; Cell Line, Tumor; Colorectal cancer; Cytometry; E-selectin; E-Selectin - metabolism; Endothelial cells; Endothelium; Engineering schools; Epitopes; Female; Flow; Flow chambers; Flow Cytometry; Fluorescence; Fluorescence microscopy; Gene expression; Glycoproteins; Humans; Immunoglobulins; Immunoprecipitation; Invasiveness; Ligands; Mass spectrometry; Mass spectroscopy; Medical prognosis; Medical research; Medicine; Membrane Glycoproteins - metabolism; Metastases; Metastasis; Microscopy; Microscopy, Fluorescence; Monoclonal antibodies; Parallel plate flow cells; Pathology; Penicillin; Protein binding; Proteins; RNA Interference; Shear rate; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Therapeutic applications; Tumor cell lines; Tumor cells; Tumors; Umbilical vein; Wall shear rate; Western blotting
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0044529

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.