Celastrol induces ferroptosis in ccRCC through TEF-Driven ACOX2 upregulation and metabolic reprogramming

BACKGROUND: Celastrol, a bioactive triterpenoid from Tripterygium wilfordii, exhibits antitumor properties across multiple malignancies, while the molecular mechanisms linking it to metabolic regulation and ferroptosis in clear cell renal cell carcinoma (ccRCC) remain insufficiently defined. PURPOSE: This study aimed to identify the key molecular mediator through which celastrol induces ferroptosis in ccRCC and to evaluate its clinical and prognostic relevance. METHODS: Transcriptomics, bioinformatics, and functional assays were combined with clinical validation in ccRCC tissues (n=278) and TCGA datasets. Mechanistic analyses included chromatin immunoprecipitation, luciferase reporter assays, cellular thermal shift, molecular docking, and ubiquitination assays, with ferroptosis evaluated in vitro and in xenograft models. RESULTS: ACOX2 was identified as a celastrol-responsive lipid metabolic enzyme with reduced expression in ccRCC. ACOX2 showed prognostic value for overall survival prediction (AUC = 0.8019), with low expression corresponding to unfavorable outcomes in both univariable (HR = 0.169, CI 0.064-0.440) and multivariable analyses (HR = 0.186, CI 0.067-0.517). Functionally, ACOX2 enhanced lipid peroxidation and ferroptotic sensitivity while restraining malignant phenotypes. Celastrol stabilized the transcription factor TEF by inhibiting its ubiquitination and proteasomal degradation, thereby promoting transcriptional activation of ACOX2. ChIP-qPCR demonstrated increased TEF occupancy at the ACOX2 promoter, with TEF/Input rising from 4.72 ± 1.23 to 8.08 ± 0.94 and TEF/IgG increasing from 4.71 ± 1.24 to 7.94 ± 2.02, indicating a ∼1.70-fold enhancement. In vivo, celastrol suppressed tumor growth and induced ferroptosis in an ACOX2-dependent manner without systemic toxicity. CONCLUSION: A TEF-ACOX2 regulatory axis is defined through which celastrol induces ferroptosis in ccRCC, linking peroxisomal lipid metabolism to tumor suppression. These findings identify ACOX2 as both a mechanistic mediator and a prognostic biomarker and support celastrol as a phytochemical candidate for ccRCC therapy.