GCDH Promotes Breast Cancer Glutaminolysis Reprogramming by Inducing GLS1 Expression Through Histone Crotonylation at Its Promoter Region.

Objective: Glutaryl-CoA dehydrogenase (GCDH) is a mitochondrial enzyme involved in lysine and tryptophan catabolism, yet its role in cancer metabolism remains poorly understood. This study aimed to investigate the function of GCDH in regulating glutamine metabolism and proliferation in breast cancer cells, and to elucidate its molecular mechanism via epigenetic modulation of glutaminase 1 (GLS1). Methods: GCDH expression was silenced using siRNAs in human breast cancer cell lines MCF-7 and MDA-MB-231. Cell proliferation was assessed using CCK-8 and EdU assays. Glutamine metabolism was analyzed by quantifying intracellular levels of glutamine, glutamate, α-ketoglutarate (α-KG), and ATP. In vivo effects were evaluated using a xenograft model in BALB/c nude mice. Chromatin immunoprecipitation (ChIP), luciferase reporter assays, and Western blotting were performed to explore the epigenetic regulation of GLS1. Functional interaction between GCDH and GLS1 was further validated through overexpression and knockdown studies, and the requirement for GCDH's enzymatic activity was tested using a catalytically inactive mutant. Results: GCDH knockdown significantly suppressed proliferation in MCF-7 and MDA-MB-231 cells (p<0.001), decreased EdU incorporation (p<0.01), and impaired glutamine metabolism, as indicated by elevated intracellular glutamine and reduced levels of glutamate, α-KG, and ATP (all p<0.05). In vivo, GCDH depletion led to reduced tumor growth and weight (p<0.001), with altered metabolic profiles consistent with impaired glutaminolysis (decreased α-KG, p<0.05). Mechanistically, GCDH silencing reduced global and GLS1 promoter-specific H3K27 crotonylation (p<0.01), suppressing GLS1 transcriptional activity (p<0.001). Overexpression of GLS1 reversed the metabolic and proliferative deficits induced by GCDH knockdown. Furthermore, wild-type GCDH overexpression, but not a catalytically inactive mutant, partially restored glutamate production and ATP levels in GLS1-deficient cells (p<0.05), indicating a functional interplay that depends on GCDH's enzymatic activity. Conclusion: GCDH promotes breast cancer cell proliferation and metabolic activity by enhancing glutaminolysis through epigenetic upregulation of GLS1 via histone crotonylation. Critically, this novel metabolic-epigenetic axis requires the catalytic function of GCDH. These findings not only reveal a novel metabolic-epigenetic axis driven by a specific mitochondrial enzyme but also suggest GCDH as a potential therapeutic target in breast cancer.