The extracellular domain of the c-erbB-2 oncoprotein is released from tumor cells by proteolytic cleavage

A molecule that is immunologically related to the c-erbB-2 oncogene product (p185HER2/neu) was detected in the conditioned culture medium from neu-overexpressing tumor cell lines and in sera of advanced-stage breast carcinoma patients. Using a sensitive (in the range of 0.5 ng ml-1) double-determinant radioimmunoassay (DDIRMA) with two monoclonal antibodies (MAbs) directed against the neu extracellular domain (ECD), soluble oncoproteins were detected in supernatants from several neu-positive tumor cell lines, independent of the levels of membrane p185HER2 expression. The molecule detected did not react with a MAb directed against an intracytoplasmic epitope of the p185HER2. Western blot analysis of the concentrated supernatant revealed a protein of approximately 110 kDa molecular mass, which closely matches the predicted size of the glycosylated p185HER2 ECD. Immunoprecipitation of culture supernatant from cell surface-radioiodinated cells confirmed the 110 kDa molecular mass of the glycosylated shed protein, which migrated to 86 kDa after deglycosylation. Proteolytic cleavage of the p185HER2 molecule was demonstrated in release assays carried out with protease inhibitors. The combined use of leupeptin and EDTA completely inhibited release of the molecule. Analysis of sera from breast carcinoma patients and healthy donors by DDIRMA revealed the presence of soluble neu in 15% of pathologic sera but none of the normal sera. A good correlation was found between neu-overexpression in the primary tumor and the soluble marker in serum of patients with advanced disease; sera of early-stage patients were always negative, independent of neu-overexpression in the tumor. These results suggest the usefulness of soluble neu as an indicator of tumor aggressiveness but not as a diagnostic marker of breast cancer.