Mac-2 Binding Protein Is a Novel E-Selectin Ligand Expressed by Breast Cancer Cells

<div><p>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.</p> </div

Identification of a novel protein, Mac-2BP, as an E-selectin ligand.

<p>(<b>A</b>) The steps followed for identification of the E-selectin reactive protein. Biotinylated ZR-75-1 cells (5 × 10⁶) were lysed and immunoprecipitated using E-Ig chimera. Immunoprecipitates were resolved by SDS-PAGE and subsequently blotted with streptavidin-AP. The intensity histogram was obtained by analysis of digitalized image using Image Lab software. (<b>B</b>) Lysate from ZR-75-1 cells (2 × 10⁶) was subjected to Western blotting with anti-Mac-2BP pAb. Immunoprecipitates from ZR-75-1 cell lysate (5 × 10⁶ cells) by anti-Mac-2BP mAb were Western blotted with E-Ig chimera or HECA-452 mAb.</p

Immunostaining of ZR-75-1 cells shows co-localization of signals for Mac-2BP and E-selectin ligand activity.

<p>(<b>A</b>) ZR-75-1 cells were surface labeled with anti-Mac-2BP pAb and analyzed by flow cytometry. Open curve shows isotype, and filled curve shows specific antibody. (<b>B</b>) ZR-75-1 cells were dually surface labeled with anti-Mac-2BP pAb (green) and E-Ig chimera (red). Images of slices, 0.5 µm apart, were obtained in epifluorescence microscopy, and projected to obtain a composite image. The composite image was deconvoluted using AutoQuant X software. Co-localization of two molecules is shown in the overlapped image (orange). Scale bar indicates 10 µm.</p

Several breast cancer cell lines express Mac-2BP.

<p>(<b>A</b>) Lysates of 2×10⁶ cells obtained from a variety of breast cancer cell lines were subjected to Western blot analysis with anti-Mac-2BP pAb. β-actin staining was used as the loading control. (<b>B</b>) Breast cancer cells were surface labeled with anti-Mac-2BP pAb and analyzed by flow cytometry. Open curve shows isotype, and filled curve shows specific antibody. (<b>C</b>) Immunoprecipitates from ZR-75-1 or BT-20 cell lysate (5 × 10⁶ cells) by anti-Mac-2BP mAb were subjected to Western blotting with E-Ig chimera.</p

Hypothesized model for the role of Mac-2BP in metastasis.

<p>Three possible scenarios for Mac-2BP expressing breast cancer cells. (1) Breast cancer cells expressing high levels of Mac-2BP bind to Gal-1 yet possess enough free epitopes for E-selectin binding. Thus, these cells are more likely to form metastatic lesions than (2) cells expressing low levels of Mac-2BP, which may not bind to endothelium due to blockade of E-selectin ligand function by Gal-1. (3) Absence of Gal-1 may lead to detection and deletion of cancer cells by immune cells.</p

ZR-75-1 cell adhesion to IL-1β activated HUVECs is E-selectin mediated.

<p>(<b>A</b>) ZR-75-1 cells (10⁶/ml) were perfused over activated (left bar) or anti-CD62E mAb treated activated HUVECs (middle bar), or sialidase (neuraminidase; 1% at 37°C for 30 min) treated ZR-75-1 cells were perfused over activated HUVECs (right bar) for 2 min at a wall shear rate of 80 s⁻¹. Data are mean ± SE for n = 3–6 independent experiments. *P<0.05 with respect to untreated cells control. (<b>B</b>) ZR-75-1 cells were surface labeled with anti-sLe^x (CSLEX-1) or anti-sLe^a (KM-231) mAbs and analyzed by flow cytometry. Open curves show isotype and filled curves show specific mAb. (<b>C</b>) Untreated or protease treated (1% bromelain at 37°C for 1 hr) ZR-75-1 cells were surface labeled with HECA-452 mAb and analyzed by flow cytometry. Open curves show isotype, and filled curves show specific mAb.</p

Mac-2BP silencing of ZR-75-1 cells reduces their adhesion to E-selectin.

<p>(<b>A</b>) Vector or Mac-2BP silenced cells were surface labeled with anti-Mac-2BP pAb and analyzed by flow cytometry. The data are represented as mean fluorescence intensity ± SE for n = 5 independent experiments. *P<0.05 with respect to vector. (<b>B</b>) Vector or Mac-2BP silenced cells (10⁶/ml) were perfused over CHO-E cells for 4 min at a wall shear rate of 80 s⁻¹ and the number of adhering cancer cells were counted. Data are mean ± SE for n = 5 independent experiments. *P<0.05 with respect to vector. (<b>C</b>) The rolling velocity of vector and silenced cells over CHO-E cells was determined at a wall shear rate of 80 s⁻¹. Data are mean ± SE for n = 10 cells. *P<0.05 with respect to vector. (<b>D</b>) Vector or silenced cells (10⁶/ml) were perfused over CHO-E cells for 4 min at a wall shear rate of 80 s⁻¹ and then the shear rate was doubled in 30 s time intervals. Data are mean ± SE for n = 5 independent experiments. *P<0.05 with respect to vector.</p

Fluid shear regulates the kinetics and molecular mechanisms of activation-dependent platelet binding to colon carcinoma cells.

This study was undertaken to investigate the kinetics and molecular requirements of platelet binding to tumor cells in bulk suspensions subjected to a uniform linear shear field, using a human colon adenocarcinoma cell line (LS174T) as a model. The effects of shear rate (20-1000 s(-1)), shear exposure time (30-300 s), shear stress (at constant shear rate by adjusting the viscosity of the medium from 1.3-2.6 cP), cell concentration, and platelet activation on platelet-LS174T heteroaggregation were assessed. The results indicate that hydrodynamic shear-induced collisions augment platelet-LS174T binding, which is further potentiated by thrombin/GPRP-NH(2). Peak adhesion efficiency occurs at low shear and decreases with increasing shear. Intercellular contact duration is the predominant factor limiting heteroaggregation at shear rates up to 200 s(-1), whereas these interactions become shear stress-sensitive at > or = 400 s(-1). Heteroaggregation increases with platelet concentration due to an elevation of the intercellular collision frequency, whereas adhesion efficiency remains nearly constant. Moreover, hydrodynamic shear affects the receptor specificity of activation-dependent platelet binding to LS174T cells, as evidenced by the transition from a P-selectin-independent/Arg-Gly-Asp (RGD)-dependent process at 100 s(-1) to a P-selectin/alpha(IIb)beta(3)-dependent interaction at 800 s(-1). This study demonstrates that platelet activation and a fluid-mechanical environment representative of the vasculature affect platelet-tumor cell adhesive interactions pertinent to the process of blood-borne metastasis