1. Fig. 6. A FOXO3a-BIM cascade mediates sensitivity to PARP and MEK inhibition.
A FOXO3a-BIM cascade mediates sensitivity to PARP and MEK inhibition. (A) OVCAR8 cells were treated with 10 nM control siRNA, BIM siRNA, or FOXO3a siRNA. The next day, cells were treated with increasing doses of BMN673 (left) or combination with increasing doses of BMN673 and 5 μM MEKi (AZD6244) (right). Cell viability was assessed with PrestoBlue after 96 hours. (B) OVCAR8 cells were transfected with BIM siRNA or control siRNA. Western blotting for BIM demonstrated effective BIM knockdown by siRNA (top). OVCAR8 cells were transfected with FOXO3a siRNA or control siRNA. Western blotting for FOXO3a demonstrated effective FOXO3a knockdown by siRNA. BIM is down-regulated by FOXO3a knockdown (bottom). 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 hours. In the combination, cells were treated with increasing doses of BMN673 and a constant 5 μM MEKi (AZD6244). (D) OVCAR8 cells were transfected with BIM or control plasmid. Western blotting for BIM demonstrated effective BIM overexpression (OE; top). OVCAR8 cells were transfected with FOXO3a or control plasmid. Western blotting for FOXO3a demonstrated effective FOXO3a overexpression. BIM is up-regulated by FOXO3a overexpression (bottom). ERK2 was used as loading control. (E) OVCAR8 cells 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–fluorescein isothiocyanate/propidium iodide 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 carried out after 96 hours of combination therapy with BMN673 dosed as indicated and a constant dose of 5 μM MEKi (AZD6244). (G) Left: BIM/FOXO3a proteins 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 hours. Western blot detection of cleaved PARP1 (Cl-PARP1) and cleaved caspase-3 (Cl-Caspase3) was used to measure apoptotic cell death (left). Right: BIM/FOXO3a cells 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 hours. Western blot detection of cleaved PARP and cleaved caspase-3 was used to measure apoptotic cell death (right). 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 hours. Western blot detection of cleaved PARP and cleaved caspase-3 was used to measure apoptotic cell death. ERK2 was used as loading control. (I) OVCAR8 cells were transfected with FOXO reporter for 24 hours and then treated as indicated. FOXO activity was assayed 48 hours after treatments (n = 3). In all reporter assays, the luciferase-based reporter signal was normalized to the expression of a cotransfected Renilla luciferase control plasmid. (J) OVCAR8 cells were treated with BMN673 for 48 hours. ChIP-PCR was used to determine percentage input of the BIM promoter precipitated with endogenous FOXO3a. Results are means ± SEM of three independent experiments. Student’s t test: ***P <
Chaoyang Sun et al., Sci Transl Med 2017;9:eaal5148
Published by AAAS