Teriflunomide modulates proteomic and phospho-kinase landscapes in breast cancer: evidence for non-canonical effects beyond DHODH inhibition

Teriflunomide, the active metabolite of leflunomide, has attracted interest as a candidate for cancer repurposing because, in addition to inhibiting dihydroorotate dehydrogenase (DHODH), it may also modulate non-canonical signalling pathways implicated in tumour growth and survival. This study investigated the anticancer effects of teriflunomide in luminal and triple-negative breast cancer models and examined whether its downstream molecular effects differ according to subtype. Luminal MCF-7 and triple-negative MDA-MB-231 breast cancer cells were treated with teriflunomide and analysed using cell viability, clonogenicity, migration assays, label-free proteomics, KEGG pathway analysis, and phospho-kinase profiling. Treated cancer cells were also compared with untreated counterparts and with non-tumorigenic MCF-10 A cells to evaluate treatment-associated proteomic remodelling. Teriflunomide reduced tumour-cell fitness in both models, but downstream molecular responses were subtype dependent. In MCF-7 cells, treatment was associated with a clearer shift in protein abundance and suppression of growth-related phospho-signalling, particularly within the PI3K/AKT/mTOR/p70S6K/RSK axis. In contrast, MDA-MB-231 cells showed a more limited abundance-level proteomic shift but broader activation of phospho-signalling involving EGFR/Src/STAT3, MAPK, and AKT-related pathways, consistent with adaptive rewiring under drug-induced stress. Integration of proteomic and phospho-kinase data showed that changes in kinase abundance were often uncoupled from changes in activation state. Proteomic analysis identified MFF, TOMM20, CSNK1A1, DKC1, ANPEP, and DDAH1 as notable regulated markers. TCGA-based validation showed that most top-regulated proteins differed between breast cancer and normal tissue, while only a subset varied across pathological stages, supporting their relevance as potential combination-strategy candidates. Teriflunomide exerts subtype-dependent anticancer effects in breast cancer cells and reshapes signalling networks in a context-specific manner. These findings support further evaluation of teriflunomide in subtype-informed combination strategies targeting adaptive kinase and metabolic pathways.