44 Ferroptosis Suppressor Protein 1 (FSP1) Drives Ferroptosis Resistance in Chromophobe Renal Cell Carcinoma

Abstract

Abstract Background Chromophobe renal cell carcinoma (ChRCC) is characterized by a marked sensitivity to ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation. Ferroptosis can be induced by inhibition of SLC7A11, leading to reduced cystine uptake and depletion of reduced glutathione (GSH). The mechanisms by which ChRCC cells adapt to evade ferroptotic death remain poorly understood. Methods Ferroptosis-resistant ChRCC cell lines (UOK276 and RCJ-T2) were generated through continuous treatment with Imidazole Ketone Erastin (IKE), an inhibitor of SLC7A11, for 45 days (1 μM for UOK276, 5 μM for RCJ-T2). Results ChRCC cells exposed to continuous ferroptotic stress through long-term culture with IKE developed stable resistance, forming colonies by day 25 and achieving proliferation rates comparable to untreated controls by day 45. Quantification of resistance demonstrated a marked increase in IKE IC₅₀, from 0.85 μM to 6.2 μM in UOK276 cells and from 0.08 μM to 4.45 μM in RCJ-T2 cells (p < 0.0001), confirming strong resistance to ferroptosis induction. Resistant cells showed significant upregulation of SLC7A11 (up to 10-fold) at both the mRNA and protein levels. Under ferroptotic stress, resistant cells maintained stable intracellular GSH levels and preserved GSH/GSSG ratios, in contrast to sensitive cells where GSH levels dropped significantly when treated with ferroptosis inducers (up to 7-fold in UOK276 and 3-fold in RCJ-T2). These findings indicate the maintenance of a stable intracellular redox environment under ferroptotic stress in the IKE-resistant cells. Interestingly, the IKE-resistant cells showed reduced GPX4 at the protein level, but demonstrated cross-resistance to GPX4 inhibition by RSL3, with IC₅₀ values increasing from 3.4 nM to 37 nM in UOK276 cells and from 8.2 nM to 19.7 nM in RCJ-T2 cells (p < 0.0001), suggesting evasion of ferroptosis through GPX4-independent antioxidant mechanisms. Forty-eight ferroptosis-associated genes were differentially expressed between resistant and sensitive cells. Among these, FSP1 mRNA was increased by 5-fold in UOK276 and 17-fold in RCJ-T2 cells, which was confirmed at the protein level. Functionally, pharmacological inhibition of FSP1 using icFSP1 or FSEN-1 restored sensitivity to ferroptosis. Treatment of IKE-resistant cells with IKE or RSL3 in combination with icFSP1 significantly reduced viability of resistant cells (p < 0.01–0.001), demonstrating a functional dependence on FSP1-mediated ferroptosis defense. To assess the clinical relevance of ferroptosis resistance, an 18-gene ferroptosis resistance score (FerrResistScore) derived from genes upregulated in resistant ChRCC cells was developed. Application of this signature showed significantly higher FerrResistScores in metastatic tumors compared to matched primary tumors (p = 0.0031), indicating enrichment of ferroptosis resistance-associated transcriptional programs in metastatic ChRCC. These findings suggests that adaptation to ferroptotic stress may facilitate tumor progression. In the TCGA-KICH cohort, patients with higher FerrResistScores exhibited reduced overall survival compared to those with lower scores (log-rank p = 0.068; Cox p = 0.0533), supporting an association between ferroptosis resistance and adverse clinical outcomes. Together, these findings highlight the clinical relevance of ferroptosis resistance programs in ChRCC and their potential utility as prognostic markers. Conclusions ChRCC-derived cells that acquire ferroptosis resistance upregulate SLC7A11 and activate a GPX4-independent antioxidant program mediated by FSP1, enabling maintenance of redox homeostasis under sustained ferroptotic stress. Pharmacologic inhibition of FSP1 restores ferroptosis sensitivity, identifying FSP1 as a key mediator of resistance in ChRCC. In addition, a ferroptosis resistance signature is enriched in metastatic ChRCC and associated with poorer clinical outcomes, suggesting a role in tumor progression and potential utility as prognostic markers.