Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification

Amplification and overexpression of the HER-2 (neu/erbB-2) gene in human br east cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patie nts. Heterodimer interactions between HER-P and HER-3 (erbB-3) are activate d by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodime rs are constitutively activated in breast cancer cells with HER-2 gene ampl ification. This indicates that inhibition of HER-2/HER-3 heterodimer functi on may be an especially effective and unique strategy for blocking the HER- 2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominan t negative form of HER-3 in which most of the cytoplasmic domain was remove d for introduction into cells. By using a bicistronic retroviral Vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast car cinoma cells with HER-P gene amplification (21 MT-1 cells) and normal mamma ry epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/HER-3 rec eptor tyrosine phosphorylation, p85(PI) (3-kinase) and SHC protein activati on, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activ ation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as t he constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. R esponses to exogenous HRG were strongly inhibited by dominant negative HER- 3, In wcontrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth f actor-independent proliferation and transformed growth of 21 MT-1 cells wer e also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRC-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by d ominant negative HER-3, whereas the epidermal growth factor-induced prolife ration of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.