A Binding Domain on Mesothelin for CA125/MUC16

Abstract

Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296 -359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296 -359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.

Difference in mesothelin‐binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer

“…These results indicate that the competitive inhibitory effect of endogenous mesothelin on the CA125 meso assay is virtually negligible, and are also consistent with previous reports that CA125 much more preferentially binds to immobilized than soluble mesothelin. 10,20 An analogous sugar structure(s) on other serum mucins may competitively inhibit the CA125-mesothelin binding in the CA125 meso assay. Elevated serum levels of MUC1 have been reported in patients with EOC.…”

NCCN Working Group Report: Designing Clinical Trials in the Era of Multiple Biomarkers and Targeted Therapies

“…This interaction may be important for mediating adhesion between tumor cells migrating into the peritoneum and the mesothelial epithelium. 111 …”

The Role of MUC16 Mucin (CA125) in the Pathogenesis of Ovarian Cancer

“…The binding site for mesothelin on MUC16 is likely located in the 156 amino acid tandem repeats of the molecule (Gubbels et al, 2006). MUC16 binds primarily to the N-terminus of the extracellular domain of mesothelin (residues 296-359) (Kaneko et al, 2009). Mesothelin is a glycoprotein normally expressed on the mesothelial cells lining the peritoneal cavity (Chang et al, 1996), and by ovarian tumor cells and mesotheliomas (Rump et al, 2004).…”