DHODH links mitochondrial bioenergetics, one-carbon metabolism, and DNA repair to sustain aggressive prostate adenocarcinoma.

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that connects de novo pyrimidine biosynthesis with the electron transport chain (ETC) function. However, its broader role in prostate cancer (PCa) signaling and response to therapy remains unclear. METHODS: We combined transcriptomic, metabolomic, epigenomic, and functional analyses with clinical outcome data to explore the effects of DHODH depletion in ancestry-annotated European American (EA) and African American (AA) prostate cancer cell line models. RESULTS: DHODH was markedly overexpressed in malignant prostate epithelial cells and was abundant in tumors. Its knockdown hindered cell proliferation by causing pyrimidine depletion, while unexpectedly increasing oxidative phosphorylation, ETC complex activity through metabolic stress–driven mitochondrial biogenesis. This also led to oxidative stress due to an imbalance in NADP⁺/NADPH. Metabolomics indicated enhanced one-carbon metabolism and increased overall DNA methylation. Whole-genome bisulfite sequencing of DHODH KD cells identified regions with differential methylation in genes related to epithelial–mesenchymal transition (EMT) and inflammatory signaling. Functionally, the loss of DHODH decreased cell migration and metastasis and downregulated genes involved in nucleotide excision repair, mismatch repair, and base excision repair. Additionally, it upregulated a subset of Y-family DNA polymerases, which facilitate error-prone lesion bypass. This was associated with more unrepaired DNA damage and, paradoxically, higher clonogenic survival under genotoxic stress. Clinically, low DHODH expression was linked to shorter biochemical recurrence–free survival after radiotherapy combined with androgen deprivation therapy. CONCLUSIONS: DHODH functions as a crucial regulator of metabolism, epigenetics, and genomics in prostate cancer, coordinating mitochondrial respiration, one-carbon metabolism, and DNA repair signaling to promote tumor growth and improve treatment outcomes. Its absence introduces vulnerabilities in genome stability, supporting the idea of combining DHODH inhibitors with DNA repair–targeted therapies for the treatment of prostate cancer. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02691-9.