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

Background/Objectives: Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and holds promise as a therapeutic strategy against cancers with elevated iron metabolism. However, many tumors evade ferroptosis through the upregulation of specialized antioxidant defense mechanisms. Here, we investigated ferroptosis susceptibility and resistance mechanisms in TSC models and in ovarian and breast cancer cell lines, aiming to identify potential therapeutic targets. Methods: Ferroptosis sensitivity was assessed using RSL3 and erastin. We explored the contribution of ferroptosis defense pathways using inhibitors of NRF2 (ML385) and FSP1 (iFSP1). RNA sequencing was performed to evaluate the expression of ferroptosis resistance genes and to explore NRF2-regulated transcriptional programs. Results: TSC2-deficient cells were resistant to RSL3- and erastin-induced ferroptosis. This resistance correlated with upregulation of ferroptosis defense genes, including NRF2 and its downstream targets. Pharmacological inhibition of NRF2 resensitized TSC2-deficient cells to ferroptosis, confirming a protective role for NRF2. However, FSP1 inhibition did not restore ferroptosis sensitivity in TSC2-deficient angiomyolipoma cells. In contrast, FSP1 knockdown significantly enhanced ferroptosis sensitivity in ovarian (PEO1, PEO4, OVCAR3) and breast (MDA-MB-436) cancer cells. Notably, in MDA-MB-436 cells, FSP1 knockdown was more effective than NRF2 inhibition to enhance ferroptosis sensitivity. FSP1 expression was not regulated by NRF2, suggesting that NRF2-targeted therapies alone may be insufficient to overcome ferroptosis resistance in certain cancer contexts. Conclusions: TSC2-deficient cells resist ferroptosis via an adaptive antioxidant response that protects against elevated iron-mediated lipid peroxidation. Our findings identify NRF2 and FSP1 as key, but mechanistically distinct, regulators of ferroptosis resistance. The differential efficacy of targeting these pathways across cancer types highlights the potential need for patient stratification. Dual targeting of NRF2 and FSP1 may offer an effective therapeutic strategy for iron-dependent, ferroptosis-resistant cancers.