Figure 5

Fulvestrant combined with the AKT inhibitor potently suppresses GFRs downstream signaling, induces apoptosis, and accelerates tumor regression. (A and B) MCF7L-shPTEN cells were prepared as before and treated with Ful combined with single or two-agent kinase inhibitors, under -/+Dox (gray/red color) conditions. Cell growth was analyzed the same way as in Figure 4. (C) Cells were treated with Ful combined with single-kinase inhibitors (-/+Dox) for 48 hours and stained with Annexin-V-APC. Flow cytometer analysis was performed to quantify the apoptotic cells with positive staining. (D) MCF7L-shPTEN cells were prepared as before and then treated with Ful alone, or E2 or Ful in combination with mTORi (0.2 μm), AKTi (1 μm), or MEKi (1 μm). Cell lysates were harvested after 48 hours of treatment and immunoblotted with the indicated antibodies. Cells of MCF7L (E), T47D (F), and BT483 (G) -shPTEN models were prepared as before (all + Dox) and then treated with E2, Ful, or each in combination with AKTi (1 μm). Cell growth within five days was normalized to the E2 groups. (H) Kaplan-Meier plots of proportion of tumor burden without response (defined `response' as tumor size halving since randomization) within 60 days of treatment of AKTi alone in the presence of supplemented E2 pellets, or Ful combined with drug carrier (Veh) or AKTi without E2 pellets (n ≥10 in each arm). The AKTi (120 mg/kg) or drug carrier (Veh) was administered twice daily by gavage for both E2 and Ful arms. The Wilcoxon method with adjusted pairwise comparison was applied. All the pairwise comparisons of cell growth were performed by the Bonferroni post hoc test (^*P <0.05, ^**P <0.01, ^***P <0.001). AKT, protein kinase B; Dox, doxycycline; E2, β-estradiol; Ful, fulvestrant; GFRs, growth factor receptors; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog.