Abstract

Glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1), the first rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), is a pivotal regulator of HBP flux. Despite its established significance, the molecular underpinnings of GFAT1's role in hepatocellular carcinoma (HCC) remain to be elucidated. Here, we found that GFAT1 was upregulated in HCC, and high GFAT1 level was correlated with poor patient prognosis. Our in vitro and in vivo studies demonstrated that GFAT1 facilitated hepatoma cell proliferation and invasion by enhancing HBP and O-GlcNAcylation through its enzymatic activity. Global profiling of O-GlcNAcylation identified vascular endothelial zinc finger protein 1 (VEZF1) as a key substrate heavily O-GlcNAcylated in GFAT1-overexpressing hepatoma cells. Notably, O-GlcNAcylation at specific serine residues (Ser123 and Ser124) within VEZF1 attenuated its proteasomal degradation, thereby enhancing its protein stability and promoting tensin 1 (TNS1) transcription in HCC. In addition, we designed a bioactive VEZF1-derived peptide to competitively inhibit GFAT1-mediated O-GlcNAcylation of VEZF1. This intervention effectively reduced TNS1 expression and suppressed the progression of HCC in a mouse model. Collectively, our findings underscore the therapeutic potential of targeting the GFAT1-VEZF1-TNS1 signaling axis in HCC.