Mitochondrial HSP90 Paralog TRAP1 Deletion Drives Glutamine Addiction in Tumor Cells via Destablization of the Cys/Glu Antiporter SLC7A11/xCT.

TRAP1, the mitochondrial isoform of HSP90, has emerged as a key regulator of cancer cell metabolism, yet the mechanisms by which it rewires nutrient utilization remain poorly understood. We previously reported that TRAP1 loss increases glutamine (Gln) dependency of mitochondrial respiration following glucose (Glc) withdrawal. In this study, we investigate how TRAP1 deletion impacts Glc metabolism and the mechanisms enabling Gln retention to support mitochondrial respiration via reductive carboxylation and the oxidative TCA cycle. TRAP1 knockout (KO) in bladder and prostate cancer cells recapitulates the carbon source-specific metabolic rewiring previously observed. Stable isotope tracing reveals that although Glc oxidation remains functional, TRAP1 KO reduces overall Glc uptake and its contribution to glycolysis and the pentose phosphate pathway. This effect is consistent across multiple cell lines. Concurrently, TRAP1-deficient cells exhibit increased Gln retention and reliance, potentially due to downregulation of the cystine/glutamate antiporter SLC7A11/xCT. Supporting this, xCT overexpression reduces Gln-dependent respiration in TRAP1 KO cells. qPCR and proteasome inhibition assays suggest that xCT is regulated posttranslationally via protein stability. Notably, xCT suppression does not trigger ferroptosis, indicating a selective adaptation rather than induction of cell death. Together, our findings suggest that TRAP1 loss decreases Glc uptake while preserving its metabolic fate, promoting Gln conservation through xCT downregulation to maintain mitochondrial respiration without inducing ferroptosis. IMPLICATIONS: These results reveal a TRAP1-dependent mechanism of metabolic rewiring in cancer cells and identify xCT-mediated Gln conservation as a key adaptive response, underscoring TRAP1 as a potential metabolic vulnerability and therapeutic target in tumors with altered nutrient utilization.