40 Ferritin-Dependent Iron Storage and Ferritinophagy Impact Ferroptosis Sensitivity in Chromophobe Renal Cell Carcinoma

Abstract

Abstract Background Chromophobe renal cell carcinoma (ChRCC) is the second most common non-clear cell RCC, with metastatic disease associated with a median overall survival of around 2 years. ChRCC is characterized by high basal glutathione levels and marked susceptibility to ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Ferritin, composed of heavy (FTH1) and light (FTL) chains, stores intracellular iron and regulates iron homeostasis. In the ChRCC Cancer Genome Atlas (TCGA) dataset, high FTH1 expression is associated with worse disease-specific, disease free and overall survival. Ferritinophagy is a selective, NCOA4-mediated autophagic process in which ferritin is delivered to lysosomes for degradation and release of iron. The contribution of these iron-regulatory pathways to ferroptosis vulnerability have not been investigated in ChRCC. Methods TCGA RNA-seq, published single-cell RNA-Seq and proteomics datasets were analyzed. Cell viability was evaluated using crystal violet as a surrogate readout. The labile iron pool and lysosomal iron were quantified using FerroOrange and LysoFerroRed, respectively. Results Data from TCGA shows that, among the 23 cancer types, ChRCC exhibits the second highest level of FTH1 expression relative to matched normal tissue in which FTH1 is 1.9-fold higher in ChRCC compared to normal kidney (median RSEM =36877 vs 19423, p-value < 0.0001). Analysis of published single-cell RNA sequencing data (Labaki et al., JCO 2025) reveals upregulation of mTOR signaling and ferroptosis-related pathways in three ChRCC tumor cells compared to their cell of origin, α-intercalated cells. At the gene level in the same dataset, ChRCC exhibits 4-fold and 4.8-fold increased expression of FTH1 and FTL, respectively (p < 0.05). Proteomic analysis (Xiao et al., Cancer Res. 2020) further confirms this finding, showing 5-fold higher FTH1 expression in ChRCC compared to normal kidney cells (p < 0.05). To test whether lysosomal function regulates ferroptosis sensitivity in ChRCC, Chloroquine (CQ, 10 µM) or Bafilomycin A1 (Baf-A1, 2 nM) were used. Both CQ and Baf-A1 blocked ferroptosis induced by the GPX4 inhibitor RSL3 or the SLC7A11 inhibitor IKE. Western blot confirmed increased NCOA4 and FTH1. FerroOrange analysis confirmed reduced labile iron pool relative to controls, suggesting impaired ferritin-derived iron mobilization. Consistent with impaired ferritinophagy, NCOA4 knockdown increased the IC50 of RSL3 in UOK276 cells by 2.7-fold (p < 0.0001). mTOR inhibition has shown benefit in some individuals with metastatic ChRCC. To determine whether mTOR inhibition enhances ferroptosis sensitivity, UOK276 cells were treated with IKE for 48 hrs, with or without Rapalink (2nM), a next generation mTORC1 inhibitor. Combined Rapalink and IKE reduced cell viability by ∼90% compared to either agent alone, indicating increased ferroptosis sensitivity (p < 0.0001). p62 and NCOA4 protein levels were decreased by Rapalink treatment, consistent with increased ferritinophagic flux. Rapalink treatment alone for 6 hrs also increased both labile and lysosomal iron pools relative to controls, as measured by FerroOrange and LysoFerroRed assays, supporting an increase in ferritinophagy-mediated iron mobilization as a determinant of ferroptosis sensitivity. Conclusions Our findings identify ferritinophagy as a key determinant of ferroptosis sensitivity in ChRCC and support mTORC1 inhibition as a strategy to promote ferritinophagy-mediated iron mobilization and sensitize tumors to ferroptosis. Given that mTOR inhibitors are already clinically used and ferroptosis-inducing therapies are under active development, this work has strong translational potential.