Figure 1. Loss of ER activity abrogates mTORC1 inhibitor-induced feedback activation of PI3K/AKT.

A. ER+ breast cancer cells were treated with dose ranges of RAD001 and/or fulv for 5 d. Relative numbers of viable cells were measured by SRB assay (full growth curves are shown in Supplementary Figure 1). Combination Index Values (CI) at the IC₂₅, IC₅₀, and IC₇₅ for the drug combination were determined by the Median Effect method. B-D) Cell lysates were analyzed by immunoblot using the indicated antibodies. In B., cells were pretreated +/− 1 μM fulv for 24 h, then treated +/− 20 nM RAD001 for an additional 1 h or 24 h (total fulv treatment time = 48 h). In C., siRNA was used to knock down ER. Two days later, cells were treated +/− 20 nM RAD001 for 1 h or 24 h. In D., cells were pretreated with hormone-depleted medium (10% DCC-FBS) for 3 d, then treated +/− 1 nM E2 for 24 h, 20 nM RAD001 for 1 h or 24 h, or combinations (total E2/RAD001 treatment time = 24 h).