HACD1-driven long-chain fatty acid metabolic reprogramming enhances esophageal cancer tumorigenicity.
Cancer Stem Cells (CSCs) are the core drivers of esophageal cancer recurrence, metastasis, and treatment resistance, and their metabolic reprogramming characteristics provide an important entry point for targeted therapy. This study aimed to explore the role and regulatory mechanism of HACD1 (3-Hydroxyacyl-CoA Dehydrogenase 1), a key enzyme in lipid metabolism, in maintaining the stemness of esophageal cancer. Using the Patient-Derived Xenograft (PDX) cell line HD-12 and laboratory cell lines TE-1 and KYSE-150, a Tumoursphere (TS) stemness enrichment model was constructed, and systematic analysis was conducted by combining quantitative Polymerase Chain Reaction (qPCR), transcriptome sequencing, lipidomics, functional experiments, and animal models. The results showed that the upregulation of stemness markers (Oct4(Octamer-binding transcription factor 4), Sox2 (SRY-box transcription factor 2), etc.) in PDX-derived HD-12 tumourspheres was significantly higher than that in laboratory cell lines, with unique transcriptomic characteristics. Lipidomics confirmed that lipid metabolites such as Triglycerides (TG) and Long-Chain Fatty Acids (LCFA) were significantly enriched in tumourspheres. The HACD (3-Hydroxyacyl-CoA Dehydrogenase) family was specifically highly expressed in stem cells, and the enzymatic activity of HACD1 (3-hydroxyacyl-CoA dehydratase 1) was positively correlated with the stemness phenotype. HACD1 knockdown significantly inhibited esophageal cancer tumoursphere formation, cell migration, and in vivo tumourigenic ability, and downregulated the expression of stemness markers; exogenous supplementation of Docosahexaenoic Acid (DA) could reverse this inhibitory effect in a concentration-dependent manner. Metabolic analysis indicated that tumoursphere cells rely on both glycolysis and lipid oxidation for energy supply and possess metabolic flexibility, and blocking a single pathway cannot effectively inhibit stemness. This study is the first to confirm that HACD1 maintains the stemness phenotype of esophageal cancer by regulating lipid metabolism. The PDX model exhibits a unique stemness regulatory pattern closer to clinical practice, providing new molecular targets and experimental basis for the metabolic targeted therapy of esophageal cancer.