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erbB-2 Overexpression in Human Mammary Epithelial Cells Confers Growth Factor Independence¹

Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical School, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109-0948

Address all correspondence and requests for reprints to: Stephen P. Ethier, Ph.D., 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0948. E-mail: spethier{at}umich.edu

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.

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