Fig 2: Combination of PARP and GPX4 inhibitors synergistically inhibits BRCA1-deficient cancers. (A) UWB1.289 or HCC1937 cells were combined treated with serial dosages of Olaparib and ML210 for three days, followed by Bliss synergy analysis. (B) Colony formation analysis of UWB1.289 or HCC1937 cells treated with 0.5 μM Olaparib, 0.5 μm ML210 or their combination. (C–D) UWB.1289 cells were treated with 100 μM Olaparib, 2 μM ML210, or their combination for 48 h, and lipid peroxidation (C) and PI staining (D) assays were performed. (E–F) BRCA1-mutant ovarian cancer organoids (PDO) were treated with serial concentrations of olaparib, ML210, or their combination for 5 days, then the organoid viability was determined. The representative organoid after treatment (E) The ZIP synergy score (F) were shown. (G) BRCA1-mutant ovarian cancer organoids were treated with Olaparib (50 μM), ML210 (5 μM), ferrostatin-1(Fer-1) (10 μM) or their combination as indicated for 5 days, and organoid viability was measured. (H–I) BRCA1-KO A2780 xenograft bearing-mice were administrated with PBS (ctrl), olaparib (100 mg/kg), ML210 (30 mg/kg) or their combination, tumor volumes were measured and tumor growth curve (H) and tumor wights (I) were shown. (J) IHC analysis of 4-HNE in indicating drug-treated xenograft tumor tissues. The representative IHC staining (left) and quantification of IHC staining (right) were shown. Data were presented as means ± SD, n = 6. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA test.

Fig 3: BRCA1 negatively regulates GPX4 protein stability. (A) Western blot analysis of indicated protein levels in UWB1.289, UWB1.289 re-expressing BRCA1(UWB-BRCA1), control (ctrl) or BRCA1 siRNA transfected-A2780 cells. (B–C) Western blot analysis of GPX4 protein levels in Ctrl/BRCA1-KO A2780 cells (B), SKOV3 and OVCAR-3 cells transfected with ctrl or BRCA1 siRNA(C). (D–E) Relative mRNA levels of BRCA1 and GPX4 were quantified in UWB1.289/UWB-BRCA1 cells (D), or BRCA1-silenced A2780, SKOV3 and OVCAR-3 cells (E). (F) UWB1.289 and UWB-BRCA1 cells were treated with 100 μg/ml cycloheximide (CHX) for 0–8 h, followed by Western blot analysis of indicated proteins. (G) A2780 cells were transfected with ctrl or BRCA1 siRNA, 48 h after transfection, cells were treated with 100 μg/ml cycloheximide (CHX) for 0–8 h, then indicated proteins were assessed by Western blot. (H–I) IHC(H) and Western blot (I) analysis of GPX4 expression in WT or BRCA1-mutant ovarian cancer tissues. (J–K) UWB1.289, UWB-BRCA1 or UWB-BRCA1 cells stably expressing flag-GPX4 were treated with or without 0.5 μM RSL3 for 24 h, followed by analysis of lipid peroxidation using BODIY 581/591 C11 labeling (J) and cell death using PI staining (K). The GPX4 expressions in above cells were confirmed by Western blot. Data were presented as mean ± standard deviation (SD). *p < 0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA test. n.s. stands for no significance.

Fig 4: GPX4 promotes growth in BRCA1-deficient ovarian cancer. (A) IHC analysis of BRCA1 and GPX4 protein expression in ovarian cancer tissue. The representative IHC staining (left) and correlation analysis of GPX4 immunoscore and BRCA1 immunoscore (right) were shown. N = 96. (B) BRCA1-KO A2780 cells were transfected with or without cholesterol-modified in vivo GPX4 siRNA, followed by Western blot analysis of indicated proteins. (C–D) Tumor growth curve(C) and tumor weights (D) of indicated A2780 xenografts treated with control (ctrl) or GPX4 siRNA. (E) IHC analysis of GPX4 and 4-HNE levels in indicated xenograft tumors. The representative IHC staining (left) and their quantification (right) were shown. Data were presented as mean ± standard deviation (SD), n = 6. **p < 0.01by two-way ANOVA test.

Fig 5: Phospholipase PLA2G7 acts complementary to GPX4 to protect PCa cells from punicic-acid-triggered ferroptosis(A) Relative viability of 22RV1 cells treated with either vehicle (DMSO 0.1% v/v, control), punicic acid (PunA) 3 μM, Ras-selective lethal (RSL3) 0.1 μM, Darapladib (Dara) 0.1 μM, or combinations thereof for 24 h, normalized to the control.(B) Relative viability of 22RV1 cells transfected with either a negative control siRNA (siNeg) or a siRNA pool targeting GPX4 (siGPX4), treated for 24 h with vehicle (DMSO 0.1% v/v, control), PunA 10 μM, Dara 0.1 μM, or a combination thereof, normalized to the control.(C) Lipid peroxidation levels in siNeg and siGPX4 22RV1 cells after 4 h of treatment with vehicle (DMSO 0.1% v/v, control), PunA 10 μM, Dara 0.1 μM, or a combination thereof. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control.(D) Relative viability of 22RV1 cells transfected with either a negative control siRNA (siNeg) or a siRNA pool targeting PLA2G7 (siPLA2G7) treated for 24 h with vehicle (DMSO 0.1% v/v, control), PunA 50 μM, RSL3 0.3 μM, or a combination thereof, normalized to the control.(E) Lipid peroxidation levels in siNeg and siPLA2G7 22RV1 cells after 4 h of treatment with vehicle (DMSO 0.1% v/v, control), PunA 50 μM, RSL3 0.3 μM, or a combination thereof. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control.(F) Relative viability of PC3 cells overexpressing either a negative control vector (OE-CTL) or PLA2G7 (OE-PLA2G7) and transfected with either a negative control vector (-CTL) or a GPX4-expressing vector (-GPX4), treated for 24 h with the indicated doses of PunA, normalized to the control (untreated cells).(G) Lipid peroxidation levels in OE-CTL-CTL, OE-PLA2G7-CTL, OE-CTL-GPX4, and OE-PLA2G7-GPX4 PC3 cells treated for 4 h with the indicated doses of PunA. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control (untreated cells).(H) Correlation between the cell resistance score and the PunA IC50 of PC3, DU145, LNCaP, C4-2B, VCaP, and 22RV1 cell lines. Cell resistance score was defined as the product of the abundances of GPX4 and PLA2G7 proteins over the one of ACSL4 protein, determined by western blot and normalized to β-ACTIN (for GPX4 and ACSL4) or valosin-containing protein (VCP) (for PLA2G7) protein abundance. Data are represented as mean ± SEM of N ≥ 3 independent cultures (A–H). Significance was established by one-way ANOVA with Sidak’s multiple comparisons (A) or two-way ANOVA with Sidak’s multiple comparisons (B–G). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. For two-way ANOVA comparing both treatments and cell clones, statistical significance is indicated by letters, with capital letters (A) for comparing different treatments for the same clone (e.g., OE-CTL-CTL) and small letters (a) for comparing clones for the same treatment (e.g., OE-CTL-CTL vs. OE-CTL-GPX4). See also Figure S7.