THE HEXOSAMINE BIOSYNTHETIC PATHWAY ON METABOLIC DYSREGULATION LINKED TO Β1,6-GLCNAC-BRANCHED N-GLYCANS IN COLORECTAL CANCER

INTRODUCTION AND OBJECTIVES: Malignant tumors often exhibit metabolic alterations, including increased glucose (Glc) and glutamine (Gln) consumption. A common feature is the heightened activity of the hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine (UDP-GlcNAc). Glycans dependent on UDP-GlcNAc, such as branched N-glycans, O-GlcNAc in proteins (OGP), and hyaluronic acid (HA), are associated with tumorigenesis and metastasis. This study investigates the influence of the HBP on metabolic dysregulation related to glycosylation, with a focus on the role of MGAT5-mediated synthesis of branched N-glycans in colorectal cancer (CRC). MATERIAL AND METHODS: We used metabolomics analyses to assess the impact on cell metabolism, specifically intermediates of the HBP and UDP-GlcNAc production, following the inhibition of β1,6-GlcNAc-branched N-glycan synthesis through Mgat5 knockout (KO Mgat5 cells). To determine which processes — N-glycans with β1,6-GlcNAc, O-GlcNAc, or HA branches —are most affected by UDP-GlcNAc restriction, we inhibited GFAT, a key HBP enzyme. To assess the effects of impaired branched N-glycan synthesis and GFAT inhibition on Glc or Gln influx, we quantified the uptake of these molecules. Additionally, TCGA data were used to correlations analyses regarding MGAT5 and/or GFAT (GFPT1 and GFPT2) expression and key genes in the HBP, Glc/Gln transport, and glycan synthesis pathways, and their relation to CRC progression. RESULTS AND CONCLUSION: Metabolomics analyses revealed that inhibiting β1,6-GlcNAc-branched N-glycan synthesis negatively impacts intermediates of the HBP, including UDP-GlcNAc, as well as several other activated monosaccharides. These alterations led to the formation of two distinct groups of metabolically different cells, with α-ketoglutarate, an intermediate of glutamine metabolism, being one of the most important discriminant metabolites responsible for this metabolic separation. Additionally, we observed a decrease in glutamine (Gln) consumption by cells in the absence of β1,6-GlcNAc-branched N-glycans. Potential reductions in OGP and HA synthesis were also observed. Moreover, UDP-GlcNAc restriction caused by GFAT inhibition resulted in a significant impairment of OGP levels, with no changes in HA levels. Interestingly, we also observed increased levels of branched N-glycans, along with an increased Gln consumption. Finally, we found that MGAT5, GFPT1, and GFPT2 expression levels are distinctly related to CRC survival, demonstrating MGAT5 as a potential prognostic marker, given the association of its increased expression with poorer disease-free survival. Together, our results suggest an interrelationship between different demands for UDP-GlcNAc in tumor cells, contributing to a better understanding of the interconnection between biochemical processes that require UDP-GlcNAc in the context of colorectal cancer.