Epidermal growth factor-dependent cell cycle progression is altered in mammary epithelial cells that overexpress c-myc.

Amplification and overexpression of the c-myc gene are common in primary human breast cancers and have been correlated with highly proliferative tumors. Components of the epidermal growth factor (EGF) receptor signaling pathway are also often overexpressed and/or activated in human breast tumors, and transgenic mouse models have demonstrated that c-myc and transforming growth factor alpha (a member of the EGF family) strongly synergize to induce mammary tumors. These bitransgenic mammary tumors exhibit a higher proliferation rate than do tumors arising in single transgenics. We, therefore, chose to investigate EGF-dependent cell cycle progression in mouse and human mammary epithelial cells with constitutive c-myc expression. In both species, c-myc overexpression decreased the doubling time of mammary epithelial cells by approximately 6 h, compared to parental lines. The faster growth rate was not due to increased sensitivity to EGF but rather to a shortening of the G1 phase of the cell cycle following EGF-induced proliferation. In cells with exogenous c-myc expression, retinoblastoma (Rb) was constitutively hyperphosphorylated, regardless of whether the cells were growth-arrested by EGF withdrawal or were traversing the cell cycle following EGF stimulation. In contrast, the parental cells exhibited a typical Rb phosphorylation shift during G1 progression in response to EGF. The abnormal phosphorylation status of Rb in c-myc-overexpressing cells was associated with premature activation of cdk2 kinase activity, reduced p27 expression, and early onset of cyclin E expression. These results provide one explanation for the strong tumorigenic synergism between deregulated c-myc expression and EGF receptor signal transduction in the mammary tissue of transgenic mice. In addition, they suggest a possible tumorigenic mechanism for c-myc deregulation in human breast cancer.