HOXA9 drives lymphatic metastasis by activating the c-MYC-glycolysis-lactate axis in gastric cancer.

BACKGROUND: The molecular mechanisms underlying lymph node metastasis (LNM) in gastric cancer (GC) remain poorly understood. This study investigated HOXA9’s role in driving LNM via metabolic reprogramming. METHODS: Integrated analysis of gastric cancer RNA sequencing data and clinical specimens was performed. Functional validation involved HOXA9 overexpression and knockdown in AGS and HGC-27 cell lines, c-MYC silencing by siRNA, and glycolytic inhibition using 2-deoxyglucose (2-DG, 2.5 mM). In vitro assays evaluated proliferation (CCK-8), apoptosis (Annexin V/PI), migration/invasion (Transwell), lymphangiogenesis (HLEC tubulogenesis), and metabolism (Seahorse analyser). In vivo, effects were evaluated using a popliteal LNM mouse model (n = 6/group) and administered exogenous lactate (20 mM) to restore levels. RESULTS: HOXA9 was significantly upregulated in LNM-positive GC tissues (1.3-fold, p = 0.0006) and predicted poor survival (HR = 1.57, p = 1.7 × 10⁻⁵). HOXA9 overexpression enhanced GC cell proliferation (2.5-fold, p < 0.0001), invasion (1.6-fold, p = 0.0002), and migration (2.0-fold, p < 0.0001), while suppressing apoptosis. Mechanistically, HOXA9 directly bound the c-MYC promoter, thereby upregulating glycolytic enzymes (HIF-1α, HK2, GLUT1, PDK1, LDHA) and increasing lactate secretion (1.7-fold, p = 0.005). The resultant lactate-rich microenvironment stimulated lymphangiogenesis (1.4-fold, p < 0.01) and endothelial cell migration (1.8-fold, p < 0.001). These effects were significantly reversed by c-MYC knockdown or 2-DG treatment, with 2-DG reducing lymphangiogenesis by 37.56% (p < 0.0001). In vivo, HOXA9 knockdown reduced LNM burden (66% reduction in node volume, 83% lower metastasis rate), and this effect was markedly rescued by lactate supplementation. CONCLUSIONS: HOXA9 promotes GC LNM by activating the c-MYC-glycolysis-lactate axis, which remodels the lymphatic niche. This axis represents a targetable pathway for GC therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-025-07326-0.