Insulin/Insulin-like growth factor-I and estrogen cooperate to stimulate cyclin E-Cdk2 activation and cell cycle progression in MCF-7 breast cancer cells through differential regulation of cyclin E and p21(WAF1/Cip1)

Estrogens and insulin/insulin-like growth factor-I (IGF-I) are potent mitog ens for breast epithelial cells and, when co-administered, induce synergist ic stimulation of cell proliferation. To investigate the molecular basis of this effect, a MCF-7 breast cancer cell model was established where serum deprivation and concurrent treatment with the pure estrogen antagonist, ICI 182780, inhibited growth factor and estrogen action and arrested cells in G(0)/G(1) phase. Subsequent stimulation with insulin or IGF-I alone failed to induce significant S-phase entry. However, these treatments increased cy . clin D1, cyclin E, and p21 gene expression and induced the formation of a ctive Cdk4 complexes but resulted in only minor increases in cyclin E-Cdk2 activity, likely due to recruitment of the cyclin-dependent kinase (CDK) in hibitor p21(WAF1/Cip1) into these complexes. Treatment with estradiol alone resulted in a greater increase in cyclin D1 gene expression but markedly d ecreased p21 expression, with a concurrent increase in Cdk4 and Cdk2 activi ty and subsequent synchronous entry of cells into S phase. Go-administratio n of insulin/ IGF-I and estrogen induced synergistic stimulation of S-phase entry coincident with synergistic: activation of high molecular mass (simi lar to 350 kDa) cyclin E-Cdk2 complexes lacking p21, To determine if the ab ility of estrogen to deplete p21 was central to these effects, cells stimul ated with insulin and estradiol were infected with an adenovirus expressing p21, Induction of p21 to levels equivalent to those following treatment wi th insulin alone markedly inhibited the synergism between estradiol and ins ulin on S-phase entry. Thus the ability of estradiol to antagonize the insu lin-induced increase in p21 gene expression, with consequent activation of cy. clin E-Cdk2, is a central component of the synergistic stimulation of b reast epithelial cell proliferation induced by simultaneous activation of t he estrogen and insulin/IGF-I signaling pathways.