Tryptophan metabolites drive ferroptosis resistance in triple-negative breast cancer cells

Introduction Tryptophan (Trp) metabolites are increased in various cancer types due to elevated expression of Trp dioxygenases, TDO2, IDO1, and IDO2. While recent studies have highlighted the immunosuppressive functions of these metabolites, their intrinsic significance to cancer cell survival remains poorly understood. This study explores the role of formylkynurenine and kynurenine, the first two metabolites in Trp catabolism on triple-negative breast cancer (TNBC) resistance to ferroptosis. Methods We performed comprehensive gene expression analysis using 46 ER+, 103 TNBC tumours, and 21 adjacent normal tissue samples from the SRA database. BRCA-TCGA RNA-seq and DNA-seq data were analysed to identify alterations in three Trp dioxygenases in TNBC patients. RNA-seq was conducted on BT549 cell line cultured with 50 μM formylkynurenine and kynurenine. Gene expression changes were validated using western blotting and real-time qPCR. Intracellular glutathione (GSH) and oxidized glutathione (GSSG) levels were quantified by LC-MS and MS/MS approach. Cell sensitivity to ferroptosis was evaluated by treating cells with 300 nM RSL3. Results Our analysis revealed increased expression of all three Trp dioxygenases, TDO2, IDO1, and IDO2, in TNBC tumours compared to luminal and adjacent normal tissues. DNA amplification was detected in IDO1 and IDO2, and reduced IDO1 promoter methylation in TNBC samples compared to luminal subtypes. Formylkynurenine and kynurenine both at concentrations of 50-100 μM significantly enhanced BT549 cell line proliferation. Transcriptomic analysis indicated that these metabolites regulate genes involved in ROS metabolism and iron transport. Metabolomics analysis further demonstrated alterations in the intracellular GSH/GSSG ratio. Notably, we report for the first time that kynurenine upregulates GPX4 protein expression, while formylkynurenine does not. However, both metabolites increased cellular resistance to RSL3-induced ferroptosis. Conclusion Formylkynurenine and kynurenine both increase GSH/GSSG ratio. While kynurenine upregulates GPX4 expression, formylkynurenine does not alter its levels. These findings establish a novel metabolic axis linking Trp catabolism to ferroptosis resistance in TNBC, suggesting that targeting this pathway could represent a promising therapeutic strategy for this aggressive subtype of breast cancer.