Figure 6.

High-OXPHOS Metabolism Enhances Chemosensitivity by Modulating ROS Levels

For a Figure360 author presentation of Figure 6, see https://dx.doi:10.1016/j.cmet.2018.09.002#mmc5.

(A) Representative dose-response curve showing variation in cell viability of low- (IGROV1, SKOV3, OVCAR8) and high- (CAOV3, OC314, OVCAR4) OXPHOS OCCLs after 48 hr of treatment. Cells were exposed to carboplatin + paclitaxel at concentrations of 0.01 to 1,000 μM. Data relative to vehicle-treated controls are means ± SEM (n = 3 independent experiments). Note that IC₅₀^high-OXPHOS = 3.5 μM, IC₅₀^low-OXPHOS = 13 μM. p value from Student’s t test.

(B and C) Relative cell viability of high-OXPHOS OCCLs (CAOV3, OC314, and OVCAR4) transfected with non-targeting siRNA (siCTL), siRNA targeting PML (siPML#1 and #2) (B), or siRNA targeting PGC-1α (siPGC-1α#1 and #2) (C). Cells were exposed to carboplatin [5.10⁻⁵ M] and paclitaxel [10⁻⁶ M] during 96 hr. Data relative to vehicle-treated controls are means ± SEM (n = 3 independent experiments). p values from paired t test.

(D) Waterfall plot showing change to baseline per mouse at the end of carboplatin + paclitaxel treatment in mice engrafted with high-OXPHOS (OC314) stable cell lines expressing either non-targeting (shCTRL) or PML-targeting shRNA (shPML). Baseline is the mean of untreated control group of mice. Change to baseline is calculated as (RTV from carboplatin + paclitaxel treated mice/RTV from control mice) − 1 × 100.

(E) Change to baseline comparing shCTRL and shPML mouse models. Medians are indicated. p value from Mann-Whitney test.

(F) Specific MFI of CellROX probe in low- (IGROV1, SKOV3, OVCAR8, blue) and high- (CAOV3, OC314, OVCAR4, red) OXPHOS OCCLs following carboplatin + paclitaxel treatment ([5.10⁻⁵ M] carboplatin+[10⁻⁶ M] paclitaxel, 24 hr). Data are means ± SEM (n = 3 independent experiments). p values from paired t test.

(G) Percent (%) of healthy (white) or altered (black) mitochondria morphology following carboplatin + paclitaxel treatment in low- (OV21) and high- (OV26) PDX models. (n ≥ 9 e.m. pictures).

(H) Specific MFI using CellROX probe in high-OXPHOS cells (CAOV3, OC314, and OVCAR4) transfected with shCRTL, shPML#1, or shPML#2. Each dot is the mean value for each cell line (n = 3 independent experiments). Bar plots show means ± SEM of the three cell lines per condition (shCTRL, shPML#1, and shPML#2). p values from paired t test.

(I) Same as in (H) using Bodipy C11 probe.

(J) Same as in (H) using RhoM probe normalized to the lysosomal content, assessed by lystrocker probe.

(K) Left: representative dose-response curve showing variation in cell viability of low- (IGROV1, SKOV3, OVCAR8) and high- (CAOV3, OC314, OVCAR4) OXPHOS OCCLs after 72 hr of ironomycin from 0.0001 to 1,000 μM. Data relative to vehicle-treated controls are means ± SEM (n = 3 independent experiments). Right: bar plot showing the corresponding ironomycin IC50 values (n = 3 independent experiments). p values from Student’s t test.

(L–N) As in (H)–(J) in low- (IGROV1, SKOV3, OVCAR8) and high- (CAOV3, OC314, OVCAR4) OXPHOS cells upon ironomycin treatment (6 μM, 24 hr). Data are means ± SEM (n = 3 independent experiments). p values from paired t test.

(O) Ironomycin IC50 values in high-OXPHOS cells, CAOV3 (left), OC314 (middle), and OVCAR4 (right) transfected either with non-targeting shRNA (shCTRL) or with shRNA targeting PML (shPML#1 and #2). Data are shown as mean ± SEM (n = 3 independent experiments). p values from paired t test.

(P) Around half of HGSOCs are characterized by elevated levels of carbonylated proteins and lysophospholipids, with decreased abundance of glutathione intermediates, all hallmarks of redox imbalance. Oxidative stress promotes PML and PML-NB accumulation, leading to PGC-1α activation through its deacetylation. PGC-1α activation in turn increases transcription of ETC components, further enhancing mitochondrial respiration. High-OXPHOS HGSOCs rely on OXPHOS, as well as glutamine- and fatty acid-fueled TCA cycle. Mitochondrial respiration might participate in ROS production, thereby leading to a potential positive feedback loop in high-OXPHOS HGSOCs. High-OXPHOS HGSOCs exhibit an enhanced sensitivity to conventional therapies, an effect mediated at least in part through the ROS-PML axis described here.

ROS, reactive oxygen species; NB, nuclear bodies; Ac, acetylated lysine; ETC, electron transport chain; TF, transcription factor; CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; CV, complex V; TCA, tricarboxylic acid; NADH, H⁺, nicotinamide adenine dinucleotide reduced form; FADH2, flavin adenine dinucleotide reduced form; OXPHOS, oxidative phosphorylation.