Insulin-like growth factor and epidermal growth factor independence in human mammary carcinoma cells with c-erbB-2 gene amplification and progressively elevated levels of tyrosine-phosphorylated p185erbB-2

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Abstract
Growth factor‐independent proliferation is an essential aspect of the transformation process. To study the influence of c‐erbB‐2 overexpression on the autonomous growth of human mammary cancer cells, we used a series of non‐neoplastic and neoplastic human mammary epithelial cell lines isolated from a patient with intraductal and invasive ductal carcinoma of the breast. The non‐neoplastic cell line, H16N‐2, which expresses a normal level (single gene copy) of c‐erbB‐2, was used for comparison with the neoplastic cell lines. Both the metastatic tumor cell lines, 21MT‐1 and 21MT‐2, showed equivalent amplification of the c‐erbB‐2 gene; however, 21MT‐1 cells showed a higher level of c‐erbB‐2 overexpression. Therefore, the H16N‐2, 21MT‐2, and 21MT‐1 cell series forms a distinct gradient of progressively increasing c‐erbB‐2 gene expression. Furthermore, the overexpression of c‐erbB‐2 in the 21MT cell lines was concordant with increases in the constitutive tyrosine kinase activity of p185erbB‐2 measured in the absence of exogenous growth factors in culture. Normal mammary epithelial cells require both insulin‐like growth factor (IGF)‐1 (or supraphysiological concentrations of insulin) and epidermal growth factor (EGF) to proliferate under serum‐free conditions in culture. By contrast, 21MT‐2 cells showed a reduced requirement for IGF but still required EGF to proliferate. 21MT‐1 cells did not require either insulin or EGF to proliferate. Therefore, the progressive increases in constitutive p185erbB‐2 tyrosine kinase activity in the 21MT‐2 and 21MT‐1 cell lines was directly correlated with IGF independence and combined IGF and EGF independence under defined conditions in culture. Experiments using conditioned media and anti‐IGF‐1 receptor and anti‐EGF receptor neutralizing antibodies showed that the growth‐factor independence of the tumor cells did not involve detectable IGF‐ or EGF‐like autocrine activity expressed by the 21MT cells. Furthermore, neu differentiation factor/heregulin, a ligand that indirectly activates p185erbB‐2 by direct binding to erbB‐3 receptors, potently stimulated the proliferation of the growth factor‐dependent H16N‐2 cells (which expressed cerbB‐2 and c‐erbB‐3 but not c‐erbB‐4) in the absence of both IGF and EGF. Thus, HRG‐induced mitogenesis mimicked the autonomous growth seen in the 21MT cells that have the highest level of constitutive p185erbB‐2 activation. These data support the hypothesis that the constitutive activation of p185erbB‐2 in human mammary carcinoma cells causes growth‐factor independence by directly activating multiple signal‐transduction pathways that substitute for both IGF and EGF during proliferation.