Mucin 16 is a functional selectin ligand on pancreatic cancer cells

Abstract

Selectins promote metastasis by mediating specific interactions between selectin ligands on tumor cells and selectin-expressing host cells in the microvasculature. Using affinity chromatography in conjunction with tandem mass spectrometry and bioinformatics tools, we identified mucin 16 (MUC16) as a novel selectin ligand expressed by metastatic pancreatic cancer cells. While up-regulated in many pancreatic cancers, the biological function of sialofucosylated MUC16 has yet to be fully elucidated. To address this, we employed blot rolling and cell-free flow-based adhesion assays using MUC16 immunopurified from pancreatic cancer cells and found that it efficiently binds E- and L- but not P-selectin. The selectin-binding determinants are sialofucosylated structures displayed on O- and N-linked glycans. Silencing MUC16 expression by RNAi markedly reduces pancreatic cancer cell binding to E- and L-selectin under flow. These findings provide a novel integrated perspective on the enhanced metastatic potential associated with MUC16 overexpression and the role of selectins in metastasis.

Figure 1.

Sialofucosylated glycoproteins expressed by SW1990 pancreatic carcinoma cells support selectin-mediated adhesion. A) Western blot of SW1990 cell lysate was stained with a HECA-452 mAb. Two major bands were observed at ∼200 and >460 kDa. B) Selectin-dependent adhesion to SDS-PAGE resolved SW1990 lysate under physiological flow conditions. CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm² over the HECA-452 stained Western blots. Number of interacting cells per square millimeter was tabulated as a function of molecular weight to compile an adhesion histogram. Data represent means ± se of n = 5 experiments.

Figure 2.

Identification of MUC16 as the sialofucosylated glycoprotein > 460 kDa expressed by SW1990 cells. 1) HECA-452-reactive molecules were purified from SW1990 cell lysate by immunoaffinity chromatography using anti-sLe^a mAb immobilized on recombinant protein G agarose supports. HECA-452-reactive molecules were eluted with low-pH elution buffer. 2) Samples were separated by SDS-PAGE and stained in gel with ProQ Emerald 300 glycoprotein stain. Fluorescently labeled band >460 kDa was subsequently excised from the gel. 3) Proteins were extracted, trypsin-digested, and subjected to tandem MS. 4, 5) bioinformatics analysis of the MS data revealed the presence of MUC16 in the HECA-452-reactive band >460 kDa.

Figure 3.

MUC16 carries sialofucosylated glycans and supports selectin-mediated adhesion. A) Western blots of whole-cell lysate or IP MUC16 or sLe^a from SW1990 pancreatic carcinoma cells. HECA-452 (lanes 1, 3, 5) or anti-MUC16 (lanes 2, 4, 6) mAbs were used to stain Western blots of cell lysate (lanes 1, 2), IP MUC16 (lanes 3, 4), and IP sLe^a (lanes 5, 6) from the cells. B) Selectin-dependent adhesion to SDS-PAGE resolved immunopurified MUC16 from SW1990 lysate. CHO-P cells, lymphocytes, or CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm² over Western blots of MUC16 immunopurified from SW1990 lysate. For control experiments, CHO-P cells, lymphocytes, or CHO-E cells were pretreated with anti-P-, L-, or E-selectin function-blocking mAbs (20 μg/ml), respectively, before blot rolling assays. This concentration was maintained throughout the experiment. Data represent means ± se of n = 3 experiments. *P < 0.05 vs. untreated cells.

Figure 4.

Immunodepletion of MUC16 eliminates selectin-mediated binding >460 kDa. A) Western blots of SW1990 lysate depleted of MUC16 by sequential IP. Anti-MUC16 mAbs (X306 or X75) were used to immunodeplete MUC16 from the cell lysate by 4 rounds of IP. Anti-MUC16 mAb and HECA-452 were used to stain the untreated lysate (ctrl) and sequentially depleted lysates (depletions 1–4). B) CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm² over 2 major HECA-452-reactive bands on SDS-PAGE Western blots of SW1990 cell lysate depleted of MUC16 by sequential rounds of IP. Number of interacting cells per square millimeter was tabulated as described in Fig. 1. Data represent means ± se of n = 3 experiments. *P < 0.05; ANOVA.

Figure 5.

MUC16-coated microspheres interact with immobilized selectins, and the binding determinants of MUC16 are sialofucosylated structures displayed on both O- and N-linked glycans. A) Extent of interaction of microspheres (10⁶/ml) coated with nonspecific IgG or MUC16 purified from SW1990 cells with E-, L-, or P-selectin (20 μg/ml) at a wall shear stress level of 1.0 dyn/cm² for 2 min. In control experiments, MUC16-coated microspheres were incubated with anti-E-, L-, or P-selectin function-blocking mAbs (20 μg/ml) before use in flow assays. Nonspecific adhesion was assessed by adding EDTA (5 mM) to the perfusion medium. B) Extent of interaction of MUC16-coated microspheres pretreated with V. cholera sialidase, PNGase F, or OSGE with E- or L-selectin at a wall shear stress level of 1.0 dyn/cm². C) Extent of interaction of microspheres coated with MUC16 immunopurified from SW1990 cells cultured in DMJ or benzyl-GalNAc-containing medium with E- or L-selectin at a wall shear stress level of 1.0 dyn/cm². Data represent means ± se of n = 3–4 experiments. *P < 0.05 vs. control MUC16-coated microspheres.

Figure 6.

Knockdown of MUC16 decreases E- and L-selectin-mediated interaction of SW1990 cells. A) Representative flow cytometric histograms of MUC16 and CD29 surface expression by wild-type, mammalian scramble control, and 2 clones of MUC16-KD SW1990 cells. Cells were stained by indirect single-color immunofluorescence using the anti-MUC16 or CD29 mAbs and their isotype control antibodies. B) Western blot of whole-cell lysate from wild-type and MUC16-KD SW1990 cells using anti-MUC16 or anti-β-actin antibody. C) Western blot of whole-cell lysate from wild-type and MUC16-KD SW1990 cells using HECA-452 mAb. D) Selectin-dependent adhesion under physiological flow conditions to SDS-PAGE resolved wild-type and MUC16-KD SW1990 cell lysates. CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm² over the HECA-452 stained Western blots. Number of interacting cells per square millimeter was tabulated as described in Fig. 1. E) Extent of adhesion of wild-type, mammalian scramble control, and MUC16-KD SW1990 cell lines (10⁶/ml) to E-selectin (20 μg/ml) under physiological flow conditions at the wall shear stress levels of 0.5 and 1.0 dyn/cm². F) Extent of tethering of wild-type, mammalian scramble control, and MUC16-KD SW1990 cell lines (10⁶/ml) to L-selectin (20 μg/ml) under flow conditions at shear stress levels of 0.5 and 1.0 dyn/cm². Data represent means ± se of n = 3–5 experiments.*P < 0.05 vs. wild type.

Figure 7.

MUC16 is a functional E- and L-selectin ligand on metastatic Pa03C pancreatic cancer cells. A) Representative flow cytometric histograms of MUC16 and CD29 surface expression by wild-type, mammalian scramble control, and 2 clones of MUC16-KD Pa03C cells. Cells were stained by indirect single-color immunofluorescence using the anti-MUC16 or CD29 mAbs and their isotype control antibodies. B) Western blot of whole-cell lysate from wild-type and MUC16-KD Pa03C cells using anti-MUC16 or anti-β-actin antibody. C) Western blot of whole-cell lysate from wild-type and MUC16-KD Pa03C cells using HECA-452 or anti-β-actin antibody. D) Selectin-dependent adhesion under flow conditions to SDS-PAGE resolved wild-type or MUC16-KD Pa03C cell lysates. CHO-E cells were perfused at the wall shear stress level of 0.5 dyn/cm² over the anti-MUC16 mAb-stained Western blots. E) Extent of adhesion of wild-type and MUC16-KD Pa03C cell lines (10⁶/ml) to E-selectin (20 μg/ml) at the wall shear stress levels of 0.5 dyn/cm². F) Extent of tethering of wild-type and MUC16-KD Pa03C cell lines (10⁶/ml) to L-selectin (20 μg/ml) at the wall shear stress level of 0.5 dyn/cm². Data represent means ± se of n = 3 experiments. *P < 0.05 vs. wild type.