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

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Figure 2

PCK1 knockout promotes UDP-GlcNAc synthesis partially through oxaloacetate accumulation.

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PCK1 knockout promotes UDP-GlcNAc synthesis partially through oxaloaceta...
Heatmap of metabolites (A) and fold-changes in intermediate metabolites of the HBP (B). Relative UDP-GlcNAc levels were measured by LC-MS in PCK1-OE SK-Hep1 cells (C) and PKO cells (D). (E) Schematic representation of the HBP. Glucose intake feed into the HBP that produces UDP-GlcNAc. N-Acetylglucosamine-1-phosphate (GlcNAc1P) and UTP, terminal metabolites of the HBP and pyrimidine synthesis, represent the final rate-limiting steps of UDP-GlcNAc synthesis. (F) Fold-changes in the intermediate metabolites of uridine synthesis. (G) Relative OAA levels, as measured by LC-MS in PCK1 -OE SK-Hep1 cells. (H) Relative levels of UDP-GlcNAc, as measured by LC-MS in PKO cells treated with 1 mM OAA. m+3 labeled UDP-GlcNAc levels in PKO cells (I) and PCK1 overexpressing SK-Hep1 cells (J) cultured with 13C5-glutamine. (K) Protein O-GlcNAcylation levels in SK-Hep1 cells cultured for 12 hours in medium containing 5 mM glucose and PEP (left), OAA (middle), or Asp (right). Protein O-GlcNAcylation levels in PKO-cells treated with 20 μM AOA for 12 hours (L) or transfected with a GOT2 shRNA1/2 plasmid for 48 hours (M). (N) Immunoblots of SK-Hep1 lysates treated for 12 hours with OAA (1 mM), Asp (1 mM), or AOA (20 μM), as indicated. (O) Proliferation ability of SK-Hep1 cells treated as indicated. Data are mean ± SD (n ≥ 3 experiments). *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student’s t test (2 groups) or 1-way ANOVA followed by Tukey’s test (more than 2 groups). Data are representative of at least 3 independent experiments.