NMR-based metabolomics reveals that TG2 inhibition triggers cell line-specific metabolic reprogramming in triple-negative breast cancer

Background: Transglutaminase 2 (TG2) is a multifunctional enzyme involved in cellular and metabolic processes. In cancer, TG2 promotes epithelial-mesenchymal transition, invasiveness, adhesion, and metastatic competence. In breast cancer (BC), TG2 is frequently overexpressed and associated with metastasis, therapy resistance, and poor prognosis, making it a promising therapeutic target. Aim: This study investigates the metabolic effects of TG2 inhibition by AA9, a novel inhibitor, in two aggressive triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and MDA-MB-436. By integrating NMR-based metabolomic profiling of both intracellular and extracellular compartments, we provide a comprehensive characterization of the metabolic alterations induced by TG2 inhibition. Results: AA9 induced cell-specific metabolic responses in both TNBC cell models. In MDA-MB-231, TG2 inhibition reduced glucose consumption, lactate production, and TCA intermediates, suggesting compensatory anaplerotic adaptations. Intracellular branched-chain amino acids (BCAAs) were accumulated alongside reduced glutamate and increased secretion of BCAA catabolites. In MDA-MB-436, glucose consumption was unchanged, while lactate increased and TCA intermediates decreased, indicating enhanced aerobic glycolysis. Myo-inositol and taurine increased, suggesting osmotic adaptation. In both cell lines, GSSG decreased, consistent with impaired GPX4 activity, whereas NAD+ increased. Changes in glycine and glutamine further suggest redox stress and amino acid rewiring. AA9 also altered lipid metabolism: MDAMB-231 showed changes consistent with phospholipid turnover and membrane stress, whereas MDA-MB-436 displayed a distinct lipid adaptation pattern. Conclusions: TG2 inhibition induces cell-specific metabolic rewiring in aggressive TNBC cells, revealing distinct metabolic vulnerabilities and supporting TG2 as a promising therapeutic target. Funding: FIL - Bando di Ateneo per la Ricerca 2025 - Azione B dell'Università di Parma.