THE EXTRACELLULAR DOMAIN OF THE C-ERBB-2 ONCOPROTEIN IS RELEASED FROMTUMOR-CELLS BY PROTEOLYTIC CLEAVAGE

A molecule that is immunologically related to the c-erbB-2 oncogene pr oduct (p185HER2/neu) was detected in the conditioned culture medium fr om neu-overexpressing tumor cell lines and in sera of advanced-stage b reast carcinoma patients. Using a sensitive (in the range of 0.5 ng ml -1) double-determinant radioimmunoassay (DDIRMA) with two monoclonal a ntibodies (MAbs) directed against the neu extracellular domain (ECD), soluble oncoproteins were detected in supernatants from several neu-po sitive tumor cell lines, independent of the levels of membrane p185HER 2 expression. The molecule detected did not react with a MAb directed against an intracytoplasmic epitope of the p185HER2. Western blot anal ysis of the concentrated supernatant revealed a protein of approximate ly 110 kDa molecular mass, which closely matches the predicted size of the glycosylated p185HER2 ECD. Immunoprecipitation of culture superna tant from cell surface-radioiodinated cells confirmed the 110 kDa mole cular mass of the glycosylated shed protein, which migrated to 86 kDa after deglycosylation. Proteolytic cleavage of the p185HER2 molecule w as 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 h ealthy donors by DDIRMA revealed the presence of soluble neu in 15% of pathologic sera but none of the normal sera. A good correlation was f ound between neu-overexpression in the primary tumor and the soluble m arker in serum of patients with advanced disease; sera of early-stage patients were always negative, independent of neu-overexpression in th e tumor. These results suggest the usefulness of soluble neu as an ind icator of tumor aggressiveness but not as a diagnostic marker of breas t cancer.