Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation

Abstract

Although cancer cells are frequently faced with nutrient-and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anti-cancer strategy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) was downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low glucose condition. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo UTP synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-Lnorleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation. Recent findings emphasize the role of the hexosamine-biosynthesis pathway (HBP), a sub-branch of glucose metabolism, in carcinogenesis (9, 10). The HBP and glycolysis share the first two steps and diverge at fructose-6-phosphate (F6P). Glutamine-fructose-6-phosphate aminotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP, converts F6P and glutamine to glucosamine-6-phosphate and glutamate. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end products of HBP, is a donor substrate for O-linked β-N-acetylglucosamine (O-GlcNAc) modification (also known as O-GlcNAcylation) (11). O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation is highly dependent on the intracellular concentration of the donor substrate UDP-GlcNAc, which is proposed to be a nutrient sensor that couples metabolic and signaling pathways (12, 13). Increased glucose flux through the HBP and elevated UDP-GlcNAc contribute to hyper-O-GlcNAcylation in cancer cells (14). Previous data suggested that elevated O-GlcNAcylation may serve as a hallmark of cancer (15). Similar to phosphorylation, O-GlcNAcylation is a dynamic post-translational modification that regulates protein subcellular localization, stability, protein-protein interactions, or enzymatic activity according to the nutrient demands of cells (16). OGT and O-GlcNAcase (OGA) are the only enzymes known to be responsible for adding and removing N-acetylglucosamine (GlcNAc) on ...

Phosphoenolpyruvate carboxykinase in cell metabolism: Roles and mechanisms beyond gluconeogenesis

“…Huh7 human HCC cells carrying a PCK1 S90A mutation, which abolishes the activity of phosphorylating INSIGs, fails to grow after in vivo xenografting [ 33 ]. Except for compartment-specific activity, this may also depend on glucose availability, since neither PCK1 overexpression nor depletion promotes HCC growth under high-glucose conditions [ 106 ]. Although much remains to be explored, these studies, in concert, demonstrate the metabolic vulnerability of cancer cells and indicate that both PCK1 and PCK2 may be promising therapeutic targets in clinic practice.…”

Targeting Pyrimidine Metabolism in the Era of Precision Cancer Medicine

“…Published studies have suggested that cellular O-glycosylation level is highly associated with the activities of OGT or OGA and the level of substrate UDP-GlcNAc (85)(86)(87)(88). However, the direct and detailed role of pyrimidine synthesis in the regulation of Oglycosylation is not well established.…”

Moonlighting functions of metabolic enzymes and metabolites in cancer

“…Nevertheless, whether the PCK1 protein kinase activity plays an essential role in this context remains unclear. In human hepatocellular carcinoma where PCK1 is markedly depleted, accumulation of the PCK1 substrate oxaloacetate activates checkpoint kinase 2 to promote cancer cell proliferation, reinforcing the functional importance of PCK1 metabolic activity (Xiang et al, 2021).…”