Nanoparticle Targeting in Chemo‐Resistant Ovarian Cancer Reveals Dual Axis of Therapeutic Vulnerability Involving Cholesterol Uptake and Cell Redox Balance

Abstract Platinum (Pt)‐based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt‐R). This work shows that Pt‐R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B‐1 (SR‐B1). SR‐B1 blockade using synthetic cholesterol‐poor HDL‐like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR‐B1 and re‐sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR‐B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR‐B1 expression through SREBF2. Targeting SR‐B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt‐R OC.


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Figure S1.(A, B) Average triglyceride content in Pt-S and Pt-R OVCAR5 (A) and OVCAR4 (B) cells as measured by the Triglyceride-Glo™ Assay (n ≥ , *p<0.05,**p<0.01).(C) Hierarchical clustering heatmap for differentially expressed (FDR<0.05) GSEA Hallmarks Cholesterol Homeostasis genes in OVCAR5 Pt-R vs. Pt-S OC cells.Gene expression was measured by RNA-seq (n=3 replicates/group).(D) Kaplan-Meier plot shows overall survival of HGSOC patients with high (n = 270) and low (n = 253) SCARB1 mRNA expression levels obtained from the TCGA and several DEO databases as described in Materials and Methods (n = 523), Survival curves were ploted by using KM Plotter.

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Figure S2.(A, B) Body weights of mice injected intraperitoneally (i.p.) with OVCAR5 Pt-R OC cells (A, n=17/group), or OC Pt-R PDX cells (B, n=7/group) and treated with PBS or HDL NPs.(C) Representative images of i.p.OC Pt-R PDX tumors treated with PBS (D) H&E staining of i.p.OC Pt-R PDX tumors collected from the abdominal wall and liver.(E) Volumes of i.p.OC PDX tumors obtained from mice treated with PBS or HDL-NPs (5 days/week, for 10 weeks) (n=7 mice per group).Values are means ± SD, *p<0.05.

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Figure S3.(A, B) OVCAR4 Pt-S (A) and Pt-R (B) cells were treated with HDL NPs for 48 or 72 hrs and percentage of oxidized/reduced lipids analyzed by C11 BODIPY staining (mean ± SD, n = 3).(C, D) OVCAR4 Pt-S (C) and Pt-R (D) cells were treated with HDL NP with or without ferrostatin-1 or DFO and cell viability assessed by MTS assay as a percentage of control (mean ± SD, n = 3).(E, F) Western blot analysis of GPx4 and β-actin (loading control) protein levels in OVCAR4 Pt-S (E) and Pt-R (F) cells treated with HDL NP for 24, 48, or 72h.Band intensities were quantified by densitometric analysis, results are shown below each blot.(G, H) (Left) Western blot analysis of HMGCS1 protein expression in OVCAR5 Pt-S (G) and Pt-R cells (H) treated with PBS and HDL NP (40 nM, 48 hours, n=3 replicates).(Right) Quantification and statistics of band intensities are shown in the bar graph.(I, J) Cell viability

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Figure S4.(A, B) Quantification by densitometric analysis of western blot bands of GPx4, SR-B1, and -actin (loading control) in OVCAR5 Pt-R and OVCAR4 Pt-R cells shown in Figure 4 A-B.(C) Mean ± SD of half maximal inhibitory concentration (IC50) of cisplatin effects on viability of OVCAR5 Pt-R cells transduced with shCTRL or shGPx4 (n=6 replicates).(D) IC50 (mean ± SD) of cisplatin effects on viability of OVCAR4 Pt-R cells transduced with shctrl or shGPx4 (n=3 replicates).(E, G) SR-B1, GPx4 and GAPDH protein levels in shGPx4 and

Table S1 .
P and q values for the three selected significantly enriched gene sets

Table S4 .
Primer sequences for selected genes in qRT-PCR analysis.