Additive effects of c‐erbB‐2, c‐Ha‐ras, and transforming growth factor‐α genes on in vitro transformation of human mammary epithelial cells

Abstract

MCF‐10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point‐mutated c‐Ha‐ras proto‐oncogene, the rat c‐neu (c‐erbB‐2) proto‐oncogene, or the human transforming growth factor‐α (TGF‐α) gene in MCF‐10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF‐10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF‐10A Ha‐ras and MCF‐10A TGF‐α cells with a recombinant retroviral vector containing the human c‐erbB‐2 proto‐oncogene and the hygromycin‐resistance gene. Ten MCF‐10A TGF‐α/c‐ethB‐2 (MCF‐10A TE) and 10 MCF‐10A Ha‐ras/c‐erbB‐2 (MCF‐10A HE) hygromycin‐resistant clones were randomly selected and expanded into cell lines. MCF‐10A TE and MCF‐10A HE clones expressed a 10‐fold to 40‐fold increase in p185 erbB‐2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum‐free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF‐α compared with MCF‐10A cells. Moreover, both MCF‐10A TE and MCF‐10A HE clones exhibited a fivefold to 20‐fold higher cloning efficiency in soft agar than MCF‐10A Ha‐ras, MCF‐10A c‐erbB‐2, or MCF‐10A TGF‐α clones. However, neither MCF‐10A TE nor MCF‐10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c‐Ha‐ras, c‐erbB‐2, and TGF‐α genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto‐oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.