006 | Iron-mediated mitochondrial dynamics shape ferroptosis and drug resistance in ovarian cancer cells

Anna Martina Battaglia¹, Emanuele Giorgio¹, Lavinia Petriaggi¹, Giuseppe Natali¹, Cristiana Galeano¹, Sebastiano Vaccarella², Giorgia Federico³, Sandra Mota⁴, Paulo Jorge Oliveira⁴, Flavia Biamonte¹ | ¹Department of Experimental and Clinical Medicine, “Magna Graecia” University of Catanzaro, Italy; ²Translational Oncology Research Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy; ³Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", Naples, Italy; ⁴CNC-UC, Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.

Non-genetic signaling plasticity is a central driver of therapy resistance in cancer. Here, to dissect iron-dependent signaling circuits governing ferroptosis susceptibility in ovarian cancer (OVCA), we treated KURAMOCHI, PEO1, COV362, and PEA1 cell lines with two iron-based class IV ferroptosis inducers FINs (Ferlixit or ferric ammonium citrate, 100 μM for 24h, following dose-time optimization). KURAMOCHI and PEO1 cells were resistant to FINs and activated an adaptive iron-buffering program characterized by coordinated downregulation of the transferrin receptor CD71, upregulation of ferritin heavy chain (FtH1), and suppression of the mitochondrial iron importer mitoferrin-1 (MFRN1) protein levels, thereby limiting mitochondrial iron flux. In contrast, COV362 and PEA1 cells underwent ferroptosis (≈30% and >60% PI⁺ cells, respectively), accompanied by marked lipid peroxidation (≈18% and ≈40%). Notably, ferroptosis occurred despite FtH1 induction, due to sustained CD71 expression and MFRN1 upregulation, resulting in mitochondrial iron overload. Mechanistically, ferroptosis-sensitive cells displayed a mitochondria-centric signaling collapse, marked by excessive mitochondrial ROS accumulation, membrane hyperpolarization (assessed by flow cytometry), loss of TOM20 and VDAC2 (assessed by immunofluorescence), and DRP1-dependent mitochondrial fission. These alterations were associated with the suppression of both oxidative phosphorylation and glycolytic ATP production, as assessed by Seahorse metabolic flux analysis. Conversely, resistant cells preserved mitochondrial structure and bioenergetic function. In vivo validation using subcutaneous PEA1 xenografts demonstrated that iron treatment (100 mg/kg, intraperitoneally, twice weekly), initiated at comparable tumor sizes, consistently delayed tumor growth, resulting in a ~32% reduction in tumor volume at day 36. Finally, WB analysis revealed that resistance in PEO1 and KURAMOCHI correlated with enrichment of cancer stem cell (CSC) markers (NANOG, OCT4, CD44, ALDH1A1). Accordingly, CSC-enriched populations generated under 3D non-adherent conditions exhibited enhanced iron buffering capacity and broad resistance to iron-based FINs. Collectively, these findings define iron-regulated mitochondrial signaling as a non-genetic determinant of ferroptosis sensitivity and therapy tolerance in OVCA.