Expression of herstatin, an alternative her-2/neu product, in cells that express either p185her-2 or the egf receptor inhibits receptor activity and cell growth

An alternative HER-2/neu product, herstatin, consists of subdomains I and II from the ectodomain of p185HER-2 and a unique 79 amino acid C-terminus encoded by intron 8. Recombinant herstatin added to cells was found to bind to and inhibit p185HER-2. The effects of ectopic expression of herstatin in combination with either p185HER-2 or with its homolog, the EGF receptor, in several cell lines was studied. Cotransfection of herstatin with HER-2 inhibited p185HER-2 levels and caused an approximate 8-fold reduction in p185 tyrosine phosphorylation. Inhibition of p185HER-2 tyrosine phosphorylation corresponded to a dramatic decline in colony formation by cells that coexpressed p185HER-2 and herstatin. Herstatin also interferred with EGF activation of the EGF receptor in cotransfected cells as demonstrated by impaired receptor tyrosine phosphorylation, reduced receptor down-regulation, and growth suppression. For both p185HER-2 and the EGF receptor, the extent of inhibition was affected by the expression levels of herstatin relative to the receptor. Herstatin is an autoinhibitor of p185HER-2 and expands its inhibitory activity to another member of the group I family of receptor tyrosine kinases, the EGF receptor. Herstatin blocked the activated Akt-mediated EGF survival signal, as well as transforming growth factor alpha (TGFα)-mediated EGF receptor activation, survival signal and proliferation signal. Purified recombinant herstatin specifically inhibited human carcinoma cells that over-express HER-2, and was effectively absorbed into the blood of intraperitoneally injected mice, where it was not proteolytically degraded and was present for between one and three hours.