High-Glucose Microenvironment Promotes Canine Osteosarcoma Cell Stemness via the HBP/O-GlcNAc Signaling Axis.

Osteosarcoma (OS) is characterized by high metastatic potential and marked chemoresistance, with cancer stem cells (CSCs) serving as major drivers of malignant progression. Canine osteosarcoma (cOS) is considered an ideal comparative medicine model for human osteosarcoma (hOS). Accumulating evidence indicates that aberrant glucose metabolism and hexosamine biosynthetic pathway (HBP, hexosamine biosynthetic pathway)/O-linked N-acetylglucosamine (O-GlcNAc)ylation are involved in tumor progression; however, the precise mechanisms by which they regulate stemness in canine osteosarcoma cells remain unclear. In this study, we comprehensively employed glucose gradient culture, untargeted metabolomics, O-GlcNAc-modified proteomics, in vitro gene silencing, and a subcutaneous xenograft model in nude mice. Cellular functional assays revealed that high glucose significantly enhanced malignant phenotypes and stemness properties of canine osteosarcoma cells. Metabolomic analyses confirmed aberrant activation of the HBP in osteosarcoma cells. Further experiments demonstrated that high glucose enhances HBP flux and O-GlcNAcylation in a dose-dependent manner; silencing of glutamine-fructose-6-phosphate transaminase 1 (GFPT1), O-GlcNAc transferase (OGT), and O-GlcNAcase (OGA) verified that both the HBP pathway and O-GlcNAcylation positively regulate malignant biological behaviors and stemness maintenance. In vivo tumorigenesis assays demonstrated that OGT knockdown markedly suppressed osteosarcoma growth. O-GlcNAc-modified proteomics identified transducin-like enhancer of split 3 (TLE3), nuclear receptor corepressor 1 (NCOR1), and neurogenic locus notch homolog protein 2 (NOTCH2) as key differentially modified proteins, predominantly enriched in the Wingless/Integrated (Wnt) and Notch signaling pathways. Collectively, our findings demonstrate that high glucose activates the HBP pathway, elevates global O-GlcNAcylation levels, and modifies TLE3/NCOR1/NOTCH2, thereby promoting stemness maintenance in canine osteosarcoma stem cells. This study provides novel metabolic targets for precision therapy of osteosarcoma.