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.
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 21 MT-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 p185erb-2 measured in the absence of exogenous growth factors in culture. Normal mammary epithelial cells require both insulin-like growth factor (IGF)-l (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 c-erbB-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.
Ram
T G
TG
Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor 48105-0582, USA.
Dilts
C A
CA
Dziubinski
M L
ML
Pierce
L J
LJ
Ethier
S P
SP
D001483:Base Sequence; D001940:Breast / Q000378:metabolism / Q000502:physiology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D004815:Epidermal Growth Factor / Q000502:physiology*; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D008969:Molecular Sequence Data; D020880:Neuregulins; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D004815:Epidermal Growth Factor; D006023:Glycoproteins; D007334:Insulin-Like Growth Factor I; D020880:Neuregulins; D018719:Erb-b2 Receptor Tyrosine Kinases; D014443:Tyrosine