Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

Abstract

Background: The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment.

Results: In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5-10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+). Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer.

Conclusion: The strong binding kinetics of the mesothelin-MUC16 interaction and the cell adhesion between ovarian tumor cells and A431-Meso+ even in the presence of peritoneal fluid strongly support the importance of these two glycoproteins in the peritoneal metastasis of ovarian tumors. The demonstration that N-linked glycans are essential for mediating mesothlein-MUC16 binding may lead to novel therapeutic targets to control the spread of ovarian carcinoma.

Figures

Figure 1

Mesothelin binds to native MUC16. (A) Meso-Fc binding to MUC16 samples from the ascites of patient #2 (100 U of CA125; lane 1) and from OVCAR-3 cells (500 U of CA125; lane 2) is shown (left panel). A control blot (right panel) with MUC16 from patient #2 (100 U of CA125; lane 1) and OVCAR-3 (500 U of CA125; lane 2) and meso-Fc (Lane 3) was overlaid with secondary antibody only. (B) For comparison, MUC16 from OVCAR-3 (100 U of CA125) was detected by VK-8 antibody. Note that since only 100 U (CA125) of OVCAR-3-derived MUC16 was loaded the banding pattern is identical to that of lane 1 of (A).

Figure 2

Mesothelin primarily binds to MUC16 on the OVCAR-3cell surface. (A)MUC16 expression on the parental OVCAR-3 and the sublines #7, #9, and #12 was determined by flow cytometry using the VK-8 antibody. (B) Meso-Fc binding to the OVCAR-3 cells and the sublines #7, #9, and #12 was determined by flow cytometry using a GAR-FITC reporter antibody. (C-E) The MB antibody was used to determine meso-Fc binding to #7 (C), #9 (D), and #12 (E) and compared to expression of native mesothelin and MUC16 on the surface of these cells.

Figure 3

Kinetics of the mesothelin-MUC16 interaction. (A) Binding of specified amounts of meso-Fc to OVCAR-3 was detected by flow cytometry. The left panel shows data for one representative experiment. Composite analysis of three independent experiments is shown in the panel on the right. Middle panel shows native expression of MUC16 on the OVCAR-3 cells used in this assay. (B) Time kinetics of meso-Fc (25 nM) binding to OVCAR-3 cells was determined by flow cytometry. After incubation with meso-Fc for the designated time intervals, the cell suspensions were diluted with 2 ml of buffer (a step that takes approximately 10 s) and analyzed. Raw flow cytometry data from one experiment is shown in panel on the left and composite analysis of two independent experiments is in the right panel. The middle panel shows expression of MUC16 on the OVCAR-3 cells used in this experiment. (C) Inhibition of meso-Fc binding to OVCAR-3 cells by soluble MUC16 was also measured by using a flow cytometry assay. Meso-Fc preincubated with designated amounts of MUC16 was added to the cells. Binding was analyzed by flow cytometry. Left panel shows raw data for one experiment and panel on right shows composite data of two independent assays. The middle panels show VK-8 binding to the OVCAR-3 cells used in this experiment in the presence (green) or absence (blue) of soluble MUC16 (100 nM).

Figure 4

Mesothelin recognizes oligosaccharides expressed on MUC16. (A) Meso-Fc binding to OVCAR-3-derived MUC16 (200 U CA125/lane) after oxidation with 1 mM (lane 2) or 10 mM (lane 4) SMP was detected in overlay experiments. Matching controls are in lanes 1 and 3, respectively. The blots were sequentially overlaid with meso-Fc, MN and a horseradish peroxidase conjugated secondary. (B) Meso-Fc binding to desailylated (lane 2) and non-desialylated (lane 1) MUC16 (150 U CA125/lane) from OVCAR-3 cells was determined in overlay experiments. (C) Meso-Fc binding to MUC16 (200 U CA125/lane) treated with PNGaseF (lane 2) or with buffer only (lane 1) was determined by Western blot overlay experiments. (D) Inhibition of bacterial mesothelin binding to OVCAR-3-derived MUC16 (200 U of CA125/blot) by ConA (blot 2), WGA (blot 3), and E-PHA (blot 4) detected in overlay experiments (D). No lectin was added in blot 1. (E) MUC16 from patient #2 (lane 1; 100 U of CA125) or from OVCAR-3 (lane 2; 500 U of CA125) were overlaid with meso-Fc in the presence (blot on left) or the absence (blot on right) of 0.5 M α-methylmannopyranoside. MN was used to detect meso-Fc binding in all experiments. Since full gel profiles have been shown in Fig. 1, only partial blots are shown here.

Figure 5

Effect of glycoconjugates on mesothelin-MUC16 interaction. (A) Binding of meso-Fc to OVCAR-3 cells was measured in the presence of 0.25 mM N-acetylglucosamine (GlcNAc) or N-acetyllactosamine (LacNAc) by flow cytometry. (B and C) Similarly the effect of ovomucoid (B) and ovotransferrin (C) on meso-Fc binding to OVCAR-3 was measured using the same technique. Meso-Fc binding was monitored by using the GAR-FITC reporter antibody.

Figure 6

MUC16 expressing ovarian tumor cells form conjugates with mesothelin positive cells. (A) CellTracker green labeled sublines #12 and #7 were coincubated with either A431-Meso+ or A431-Meso- cells in PBS containing 1% BSA. The heterotypic doublets formation was measured by flow cytometry. The #7 and #12 sublines are represented in green, the A431 cells are in blue and the heterotypic doublets are depicted in orange. The percentages of all live cells that form heterotypic doublets are shown for each plot. Cell debris is in red. (B) Heterotypic doublet formation between the sublines #12 and #7 and the A431 cells in the presence of ascites from patient#15 is shown. The sublines are represented in green and the A431 cells are in blue. Heterotypic doublets are in orange. The percentage of heterotypic doublets is shown for each plot. Cell debris is in red.

Figure 7

Soluble MUC16 has a lower affinity for mesothelin. (A) A431-Meso+ cells were incubated with purified MUC16 (50,000 U/ml). After 1 h, the amount of MUC16 on the cell surface was measured using the VK-8 antibody and a FITC labeled secondary. (B) Binding of A431-Meso+ cells to MUC16 from peritoneal fluid was measured by flow cytometry. The cells were cultured in the peritoneal fluid from patient #15 that contains (84,000 U of CA125/ml). MUC16 binding was detected using the VK-8 and the FITC-labeled secondary antibodies. (C) The A431-Meso+ cells were labeled with MN and a FITC-conjugated secondary. Mesothelin expression was measured by flow cytometry.

Figure 8

MUC16-Mesothelin binding increases spheroid formation in ovarian tumor cells. The #7 and the #12 sublines were labeled with CellTracker Green. After keeping the cells in suspension for 1 h in peritoneal fluid, the homotypic doublet formation was measured by flow cytometry. The singlet events are shown in pink and the doublets are in green. The percentage of live cells that form the homotypic doublets is shown for each plot.

Figure 9

Model for peritoneal metastasis of ovarian tumors. A model showing the importance of MUC16-mesothelin interaction in the peritoneal metastasis of ovarian tumors is shown.