Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation
Jin Xiang, … , Kai Wang, Ni Tang
Jin Xiang, … , Kai Wang, Ni Tang
Published March 9, 2021
Citation Information: J Clin Invest. 2021;131(8):e144703. https://doi.org/10.1172/JCI144703.
View: Text | PDF
Research Article Metabolism Oncology

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

Abstract

Although cancer cells are frequently faced with a 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 anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is 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 conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate 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-L-norleucine 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.

Authors

Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang

×

Figure 1

PCK1 deficiency enhances protein O-GlcNAcylation.

Options: View larger image (or click on image) Download as PowerPoint
PCK1 deficiency enhances protein O-GlcNAcylation. Immunoblotting analysi...
Immunoblotting analysis of global O-GlcNAcylation levels in PCK1-knockout PLC/PRF/5 cells (PKO cells) treated with medium containing different levels of glucose for 12 hours (A). Densitometric analysis was performed with Image-Pro Plus software (B). (C) Representative Western blot analysis of the indicated proteins in SK-Hep1 cells overexpressing green fluorescence protein (GFP; control cells), WT PCK1, or an enzymatically deficient mutant (PCK1 G309R) after incubation in medium containing 5 mM glucose for 12 hours. Mock-treated cells served as a blank control. To determine OGT and protein O-GlcNAcylation levels in PKO cells, cells were transfected with shRNA targeting OGT mRNA or a scrambled control shRNA (shcon) for 48 hours (D), or treated with 50 μM ST (ST045849, OGT inhibitor) for 12 hours (E). For immunoblotting analysis of SK-Hep1 cells, PCK1-expressing cells were transfected with an OGA shRNA1/2 plasmid for 48 hours (F), or treated with 25 μM TG (Thiamet G, OGA inhibitor) for 12 hours (G). Immunoblotting (H) and densitometric analysis (I) of liver tumors from DEN/CCl4-induced WT and LKO mice after fasting for 12 hours. Data are mean ± SD (n = 6 experiments). **P < 0.01, 2-tailed unpaired Student’s t test.