Of note, c Myc siRNA had no impact on cell viability by itself. As shown in Figure 5C, decreased c Myc e pression dimin ished cell death induced by transfection with Mcl 1 siRNA, indicating that this transcription factor contri butes to the Mcl 1 dependence of BT474 cells. Decrease of c Myc e pression upon inhibition of mTORC1 diminishes Bim e pression levels and mitigates the Mcl 1 http://www.selleckchem.com/products/CHIR-258.html dependence of BT474 cells In HER2 overe pressing cells with high Akt activity, mTORC1 downstream of Akt is e pected to actively contribute to c Myc e pression. Thus, Bim e pres sion in such cells may directly result from oncogenic signaling. To confirm this notion, we treated BT474 cells with the mTORC1 inhibitor RAD001, under condi tions that proved sufficient to prevent their growth, arrest these cells in the G1 phase of the cell cycle and prevent phosphorylation of S6K.

Importantly, this treatment by itself did not induce significant apoptosis rates in BT474 cells and had no detectable impact on Mcl 1 e pression. In contrast, this treatment lead to a decrease in c Myc e pression. Coinciden tally, RAD001 treatment significantly decreased Bim e pression in BT474 cells. Since c Myc both affects Bim e pression in BT474 cells as well as their Mcl 1 dependence, we then ana lyzed whether RAD001 treatment, which impacts on Bim e pression, also impacts on such dependence. Cells were treated with RAD001 or not prior to their transfection with control or Mcl 1 siRNA, and cell death rates were analyzed as described above.

As shown in Figure 6C, RAD001 treatment did not enhance cell death rates induced by Mcl 1 siRNA, indicating that RAD001 has no pro apoptotic effect even in Mcl 1 depleted BT474 cells. Instead, we found that RAD001 significantly prevented cell death induced by Mcl 1 siRNA. Western blot analysis showed that RAD001 treatment did not interfere with the ability of Mcl 1 siRNA to down regulate Mcl 1 and that, conver sely, RAD001 treatment was Entinostat still efficient in Mcl 1 depleted cells. Moreover, RAD001 treatment decreased Bim e pression in cells treated with a control siRNA and in Mcl 1 depleted cells. In contrast, the e pression levels of IAP, another anti apoptotic pro tein whose e pression was reported to be enhanced by mTORC1 inhibition in some cases were left unchanged by RAD001 treatment. Thus, these data reveal a genuine anti apoptotic effect e erted by RAD001 treatment in BT474 cells, which allows them to survive even when Mcl 1 is depleted and which correlates with a decrease in Bim e pression.