Estradiol abrogates apoptosis in breast cancer cells through inactivation of BAD: Ras-dependent nongenomic pathways requiring signaling through ERK and Akt

Estrogens such as 17-beta estradiol (E-2) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E-2 abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-alpha, H2O2, and serum starvation in causing apoptosis. Furthermore, the ability of E-2 to prevent tumor necrosis factor-alpha-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90(RSK1) and Akt, was not phosphorylated in response to E, in vitro. E-2 treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90(RSK1) to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90(RSK1) activation, E-2 also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E-2. Dominant negative Ras blocked E-2-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E-2-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E,induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E2 prevents apoptosis.