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.