CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis

Abstract

Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.

Keywords: cancer; cell death; ferroptosis; lipids; nanoparticles.

Fig. 1.

HDL NP reduces GPx4 and induces ferroptosis in OCCC cells. (A) ES2 and (B) TOV21G ovarian clear cell carcinoma (OCCC) cells treated with increasing concentrations of HDL NP show a time- and dose-dependent reduction in GPx4 expression at 48 h and 72 h. (C) Cell viability curves of wild-type ES2 and (D) TOV21G cells treated with indicated concentrations of HDL NP. Data shown are the mean ± SD of n = 6. (E) Cell membrane lipid peroxidation levels measured by C11-BODIPY assay show a time- and dose-dependent increase upon HDL NP treatment in ES2 and (F) TOV21G cells. Data shown are the mean ± SD of n = 3. (G) Cell death induced by HDL NP treatment is rescued by treatment with 1 μM ferrostatin-1 or 1 μM deferoxamine (DFO) in ES2 and (H) TOV21G cells. Data shown are the mean ± SD of n = 5. For (E–H), *P < 0.05 and ****P < 0.0001.

Fig. 2.

A CRISPR-based positive selection screen in ES2 OCCC cells identifies metabolic genes whose loss confers resistance to HDL NP. (A) Schematic of the CRISPR-based positive selection screen, PD = Population Doublings. (B) A dot plot depicting the gene scores in untreated and HDL NP-treated (10 nM) ES2 cells. The gene score is the postscreen median log2 fold change in the abundance of all sgRNAs targeting a particular gene. (C) Western blots showing ACSL4 expression in ES2 wild-type (WT), clonal nontargeting (NT), and clonal ACSL4 knockout (KO) cell lines derived using three independent sgRNAs. (D) Cell viability of ES2 clonal nontargeting and clonal ACSL4 KO cell lines treated with increasing concentrations of HDL NP following 48 h treatment. Data shown are the mean ± SD of n = 3. (E) Representative western blots showing TXNRD1 and GPx4 expression in ES2 pool sgNontargeting, sgTXNRD1_4, and sgTXNRD1_7 cell lines. (F) Cell viability of ES2 sgTXNRD1 pool KO cells treated with increasing concentrations of HDL NP following 48 h treatment. Data shown are the mean ± SD of n = 6.

Fig. 3.

ACSL4 is necessary for HDL NP to induce cell membrane lipid peroxidation and cell death. (A) Western blots of ES2 clonal nontargeting and ACSL4 KO cells treated with PBS (vehicle) or 5 nM HDL NP for 48 h. (B) Lipid peroxidation levels measured by C11-BODIPY in ES2 clonal nontargeting and ACSL4_KO1 cells treated with increasing concentrations of HDL NP at 48 h, ****P < 0.0001. Data shown are the mean ± SD of n = 4. (C) Cell viability of ES2 clonal nontargeting and ACSL4_KO1 overexpressing ACSL4 or GFP control treated with increasing concentrations of HDL NP at 48 h posttreatment, and western blots showing ACSL4 protein expression in corresponding cell lines. Data shown are the mean ± SD of n = 6. (D) Western blots of shControl and shACSL4 knockdown TOV21G cells treated with vehicle (PBS) or 10 nM HDL NP for 48 h. (E) Lipid peroxidation levels measured by C11-BODIPY in TOV21G shControl and shACSL4 knockdown cells treated with increasing concentrations of HDL NP for 72 h, ****P < 0.0001. Data shown are the mean ± SD of n = 4. (F) Cell viability of shControl and shACSL4 knockdown TOV21G cells treated with increasing concentrations of HDL NP for 72 h. Data shown are the mean ± SD of n = 6.

Fig. 4.

TXNRD1 KO in ES2 OCCC cells prevents HDL NP-induced cell death by increasing GPx4 expression. (A) GPx4 protein levels are increased in ES2 ovarian cancer cells upon TXNRD1 KO and elevated GPx4 expression is maintained upon 24 h HDL NP treatment. (B) Lipid peroxidation levels measured by C11-BODIPY in ES2 pool sgNontargeting and sgTXNRD1_4 KO cells treated with increasing concentrations of HDL NP at 48 h, ***P < 0.001 and ****P < 0.0001. Data shown are the mean ± SD of n = 4. (C) Cell viability of ES2 sgTXNRD1_4 and sgTXNRD1_7 pool KO cell lines treated with small-molecule GPx4 inhibitors, RSL3 and ML210, at 24 h posttreatment, and the system x_c– inhibitor, erastin, at 48 h posttreatment. Data shown are the mean ± SD of n = 6. (D) Western blot showing pharmacologic inhibition by the TXNRD1 small-molecule inhibitor, auranofin, does not increase GPx4 expression in ES2 cells. (E) Cell viability of ES2 ovarian cancer cells with cotreatment of HDL NP and 50 nM or 100 nM auranofin at 48 h posttreatment. Data shown are the mean ± SD of n = 6.

Fig. 5.

HDL NP reduces cellular selenium and downregulates expression of various selenoproteins. (A) Western blots showing expression of various selenoproteins treated with and without 10 nM HDL NP and indicated concentrations of sodium selenite (Na₂SeO₃) in ES2 and (B) TOV21G ovarian cancer cells. (C) Lipid peroxidation levels measured by C11-BODIPY at indicated HDL NP concentrations, with and without 50 nM Na₂SeO₃ in ES2 at 48 h and (D) in TOV21G cells at 72 h posttreatment. Data shown are the mean ± SD of n = 4. (E) Cell viability of ES2 cells treated with increasing concentrations of HDL NP, with and without 50 nM Na₂SeO₃ in ES2 (F) and TOV21G cells. Data shown are the mean ± SD of n = 6. (G) Selenium levels measured by ICP-MS upon treatment with 10 nM HDL NP, with and without 50 nM Na₂SeO₃ in ES2 at 24 h posttreatment and (H) in TOV21G cells at 48 h posttreatment. Data shown are the mean ± SD of n = 3. For all panels, *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 6.

HDL NP enhances steady-state mitochondrial ROS. (A) Representative images and image quantification of oxidized (405 nm)/reduced (488 nm) fluorescent signal from ES2 cells stably expressing the mitochondrial matrix-targeted HyPer7 hydrogen peroxide (H₂O₂) probe imaged immediately after HDL NP treatment, (B) 24 h posttreatment, and (C) 48 h posttreatment. (Scale bar, 50 µm.) Data shown are mean ± SD of n = 20. For all panels, **P < 0.01, and ****P < 0.0001.