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

Jennifer Aa Gubbels,Byungkook Lee,Bangalore K Sathyanarayana,Joseph Connor,Masanori Onda,Manish S Patankar,Tapan K Bera,Jennifer Belisle,Martine Migneault,Ira Pastan,Claudine Rancourt,Mitchell Ho

doi:10.1186/1476-4598-5-50

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionThe 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.

Highlights

The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors
Mesothelin binds to native MUC16 mesothelin has been previously shown in direct assays to bind a recombinant fragment of MUC16 [9], it was important to conclusively demonstrate this interaction with native forms of this mucin
MUC16 was partially purified, by size exclusion chromatography, from OVCAR-3 cell culture media and from the peritoneal fluid of epithelial ovarian cancer (EOC) patients as described in our earlier report [5]. These MUC16 samples showed strong binding to the recombinant mesothelin tagged with the Fc fragment of rabbit IgG in western blot overlay experiments (Figure 1a)

Summary

Introduction

The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The mucin MUC16 carries the peptide epitope CA125, which is a prominent molecular marker for monitoring the progression and recurrence of epithelial ovarian cancer (EOC) [1,2,3]. The peptide backbone of MUC16 is composed of the Nterminal region, an extensive tandem repeat domain, and a C-terminal region with a short cytoplasmic tail [2]. The tandem repeat, a hallmark of all mucins, is composed of 18–60 repeats, each of which contains 156 amino acids [2]. The N-terminal domain has been proposed to be made up of 13,000 amino acids [4]. In addition to the extensive protein structure, MUC16 is heavily glycosylated with both O-linked and N-linked oligosaccharides [5]

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