Figure 6. A FOXO3a-BIM cascade mediates sensitivity to PARP and MEK inhibition.

(A) OVCAR8 were treated with 10 nM control siRNA, BIM siRNA, or FOXO3a siRNA. The next day, cells were treated with increasing doses of BMN673 (left panel) or combination with increasing doses of BMN673 and 5 µM MEKi (AZD6244) (right panel). Cell viability was assessed with Prestoblue after 96 h.

(B) OVCAR8 were transfected with BIM siRNA or control siRNA. Western blotting for BIM demonstrated effective BIM knockdown by siRNA (upper panel). OVCAR8 were transfected with FOXO3a siRNA or control siRNA. Western blotting for FOXO3a demonstrated effective FOXO3a knockdown by siRNA. BIM is downregulated by FOXO3a knockdown (lower panel). ERK2 was used as loading control.

(C) BIM and FOXO3a were expressed in OVCAR8 and then treated as indicated. Cell viability was assessed with Prestoblue after 96 h. In the combination, cells were treated with increasing doses of BMN673 and a constant 5 µM MEKi (AZD6244).

(D) OVCAR8 were transfected with BIM or control plasmid. Western blotting for BIM demonstrated effective BIM overexpression (upper panel). OVCAR8 were transfected with FOXO3a or control plasmid. Western blotting for FOXO3a demonstrated effective FOXO3a overexpression. BIM is upregulated by FOXO3a overexpression (lower panel). ERK2 was used as loading control.

(E) OVCAR8 were treated with BMN673 (1 µM), AZD6244 (5 µM), or combination therapy with BMN673 (1 µM), and AZD6244 (5 µM) for 96 hours and subjected to Annexin V-FITC/PI apoptosis analysis. Each value represents mean±SEM from three independent experiments.

(F) A pan-caspase inhibitor (Z-VAD-FMK) (50 µM) was added to OVCAR8. Cells were treated as indicated. Prestoblue assay was done after 96 h of combination therapy with BMN673 dosed as indicated and a constant dose of 5 µM MEKi (AZD6244).

(G) On the left, BIM/FOXO3a were knocked down by siRNA in OVCAR8. Forty hours later, cells were treated with BMN673 (1 µM), AZD6244 (5 µM), or combination therapy with BMN673 (1 µM) and AZD6244 (5 µM) for 96 h. Western blot detection of cleaved-PARP and cleaved-Caspase-3 was used to measure apoptotic cell death (left panel). On the right, BIM/FOXO3a were overexpressed in OVCAR8 for 48 hours. Cells were then treated with BMN673 (1 µM), AZD6244 (5 µM), or combination therapy with BMN673 (1 µM) and AZD6244 (5 µM) for 96 h. Western blot detection of cleaved-PARP and cleaved-Caspase-3 was used to measure apoptotic cell death (right panel). ERK2 was used as loading control.

(H) Two RAS-mutant cell lines (HOC1 and HOC7) were treated with BMN673 (1 µM), AZD6244 (1 µM), or combination therapy with BMN673 (1 µM) and AZD6244 (1 µM) for 96 h. WB detection of cleaved-PARP and cleaved-Caspase-3 was used to measure apoptotic cell death. ERK2 was used as loading control.

(I) OVCAR8 were transfected with FOXO reporter for 24 h, then treated as indicated. FOXO activity was assayed 48 h after treatments (n = 3). In all reporter assays, the luciferase-based reporter signal was normalized to the expression of a co-transfected renilla luciferase control plasmid.

(J) OVCAR8 were treated with BMN673 for 48 h. CHIP-PCR was used to determine percentage input of the BIM promoter precipitated with endogenous FOXO3a.

Results are shown as means±SEM of three independent experiments. Student’s t test: *P<0.05, **P<0.01, and ***P<0.001.