title: 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.
abstract: Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function 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 dominant negative form of HER-3 in which most of the cytoplasmic domain was removed 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 carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary 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 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.
authors.affiliation: School of Biological Science, Washington State University, Pullman 99164-4236, USA. tram@mail.wsu.edu
authors.forename: T G
authors.initials: TG
authors.lastname: Ram
authors.forename: M E
authors.initials: ME
authors.lastname: Schelling
authors.forename: H L
authors.initials: HL
authors.lastname: Hosick
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D005786:Gene Expression Regulation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008934:Mitogens / Q000494:pharmacology; D020890:Neuregulin-1 / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
chemical_list: D008934:Mitogens; D020890:Neuregulin-1; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3