Targeting NRF2 and FSP1 to Overcome Ferroptosis Resistance in TSC2-Deficient and Cancer Cells

Background/Objectives: Ferroptosis, an iron-dependent form of cell death, shows promise as a target for therapy-resistant cancers that exhibit increased iron metabolism. Ferroptosis is primarily characterised by lipid peroxidation within cell membranes. However, many cancers evade ferroptosis by upregulation of specialised ferroptosis defence mechanisms. This study investigates ferroptosis susceptibility in Tuberous Sclerosis Complex (TSC) cell models, and ovarian and breast cancer cell lines to identify ferroptosis resistance mechanisms and therapeutic targets. Methods: We assessed ferroptosis susceptibility using the ferroptosis inducers, RSL3 and erastin. We explored ferroptosis resistance genes using inhibitors of NRF2 (ML385) and FSP1 (iFSP1). RNA-sequencing was conducted to identify dysregulated ferroptosis resistance genes and to better characterise NRF2 target genes. RNA sequencing was also performed to identify gene expression after treatment with pro-ferroptotic iron-oxide nanoparticles. Results: TSC2-deficient cells exhibited resistance to RSL3 and erastin-induced ferroptosis, which correlated with increased ferroptosis defence gene expression, including NRF2 and downstream targets. NRF2 inhibition re-sensitised TSC2-deficient cells to ferroptosis, confirming its protective role. However, FSP1 inhibition did not re-sensitise TSC2-deficient angiomyolipoma kidney tumor cells to RSL3. In contrast, FSP1 knockdown significantly enhanced ferroptosis sensitivity in ovarian (PEO1, PEO4, OVCAR3) and breast (MDA-MB-436) cancer cell lines. Notably, in MDA-MB-436 cells, FSP1 knockdown was more effective than NRF2 inhibition in reversing ferroptosis resistance. Conclusions: Our findings highlight NRF2 and FSP1 as key regulators of ferroptosis resistance in TSC2-deficient and cancer cells. However, the differential efficacy of targeting these pathways suggests that patient stratification may be necessary for optimal therapeutic strategies. Targeting NRF2 and FSP1 could enhance ferroptosis susceptibility, offering a potential therapeutic approach for ferroptosis resistance cancers. Overall design: MDA-MB-436 cells. Cells were cultured in DMEM with 15% (v/v) FBS, 2mM L-Glutamine, 0.01mg/mL Insulin, and 100 U/mL penicillin/streptomycin. Cells were subject to reverse transfection with siRNA SMARTpools (DharmaconTM ON-TARGETplus siRNA) targeting NFE2L2, AIFM2, or non-targeting siRNA (all at 50 nM), using DharmaFECT-1 transfection reagent (DharmaconTM, Horizon Discovery Ltd.). Alternatively, cells were treated with 500 µM (Fe2+) of iron-oxide nanoparticles coated in DLin-MC3-DMA for 48 h, with reverse transfection of non-targeting siRNA. After 72 h target knockdown, samples were collected in RNAprotect reagent (Qiagen, West Sussex, UK) prior to total RNA extraction with an RNEasy kit (Qiagen). Total RNA was quantified using a QubitTM RNA HS Assay.