SIRT3 Regulates HMGCS2 Deacetylation and Influences Cholangiocarcinoma Progression via the Metabolism of Ketone Bodies

Cholangiocarcinoma (CCA) is a highly aggressive malignancy. 3‐Hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), a mitochondrial enzyme involved in ketogenesis, has been linked to tumor progression, but its role in CCA remains unclear. HMGCS2 expression in CCA tissues was analyzed using TCGA data and immunoblotting (IB). Functional assays were performed in CCA cell lines (HuCCT‐1 and RBE) and an in vivo xenograft model. Metabolomics explored HMGCS2‐mediated metabolic changes. SIRT3–HMGCS2 interactions were examined via molecular docking, IF, CO‐IP, GST pull‐down, and CHX assays, with mutational analysis identifying interaction sites. IHC assessed clinical samples. HMGCS2 was downregulated in CCA. Overexpression inhibited proliferation and invasion, while knockdown promoted these effects, consistent in vitro and in vivo. Metabolomics showed HMGCS2 enhanced ketone body synthesis, and exogenous ketone bodies mimicked its antitumor effects. SIRT3 deacetylated HMGCS2 at K310 (with plasmid mutation assay), and low HMGCS2/SIRT3 expression correlated with poor patient survival. SIRT3‐mediated deacetylation of HMGCS2 promotes ketone body synthesis, suppressing CCA progression. HMGCS2 is a potential therapeutic target for CCA.