title: erbB-2 overexpression in human mammary epithelial cells confers growth factor independence.
abstract: Previously, we demonstrated that human breast cancer cells with progressively elevated levels of constitutively tyrosine phosphorylated erbB-2 are independent of growth factors required by normal human mammary epithelial (HME) cells for proliferation in serum-free medium. To determine whether erbB-2 overexpression alone is sufficient to confer the growth factor-independence phenotype in HME cells, the spontaneously immortalized MCF-10A cell line and the HPV-16-immortalized H16N2 cell line were infected with the bicistronic retroviral vector pTPerbB-2 and tested for their ability to grow in the absence of specific factors. Selection of infected cells in G418-containing medium resulted in moderate levels of erbB-2 overexpression in approximately 40% of cells. The subpopulation of erbB-2 overexpressing cells could be selected for by culturing the cells in medium devoid of insulin. When MCF-10A or H16N2 cells were infected with pTPerbB-2 and directly selected in growth factor-deficient medium over long periods of time, populations of both cell lines emerged that expressed levels of erbB-2 protein equivalent to levels expressed by breast cancer cells with an erbB-2 gene amplification. Furthermore, overexpressed p185(erbB-2) was constitutively tyrosine phosphorylated in these cells. The levels of tyrosine phosphorylated p185(erbB-2) differed in the two recipient lines, with H16N2-erbB-2 cells having higher levels of activated receptor than MCF-10AerbB-2 cells. Furthermore, only the H16N2-erbB-2 cells were independent of both insulin and epidermal growth factor for growth in serum-free medium. Overexpression of erbB-2 also resulted in progressively increasing levels of tyrosine-phorphorylated erbB-3, without any significant changes in p180(erbB-3) levels. These studies demonstrate a direct relationship between the level of expression and activation of p185(erbB-2) and the requirements of HME cells for insulin-like and epidermal growth factor-like growth factors. The results also suggest that genetic alterations present in breast cancer cells, or mediated by HPV-16-induced alterations in pRb and p53, can influence the expression level and activation status of erbB-2 as well as erbB-3 and, in turn, their degree of growth factor independence.
authors.affiliation: Department of Radiation Oncology, University of Michigan Medical School, University of Michigan Comprehensive Cancer Center, Ann Arbor 48109-0948, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Ignatoski
authors.forename: A J
authors.initials: AJ
authors.lastname: Lapointe
authors.forename: E H
authors.initials: EH
authors.lastname: Radany
authors.forename: S P
authors.initials: SP
authors.lastname: Ethier
mesh_terms: D000818:Animals; D001943:Breast Neoplasms; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D018734:Genes, erbB-2; D005822:Genetic Vectors; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000235:genetics*; D020893:Receptor, ErbB-3; D012190:Retroviridae; D014407:Tumor Cells, Cultured
chemical_list: D066246:ErbB Receptors; D006133:Growth Substances; D007328:Insulin; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3