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 6

O-GlcNAcylation promotes DEN/CCl4-induced hepatocellular carcinogenesis in PCK1-knockout mice.

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O-GlcNAcylation promotes DEN/CCl4-induced hepatocellular carcinogenesis...
(A) Reproductive strategy for generating AlbCre(–/–), Pck1(fl/fl) (WT), and AlbCre(+/–), Pck1(fl/fl) (liver-specific knockout, LKO) mice. (B) PCK1 protein expression in WT and LKO mouse organs involving the heart, liver, spleen, and kidney were confirmed by immunoblotting. (C) Schematic representation of the experimental procedures used with WT and LKO mice. Mice were injected intraperitoneally with 75 mg/kg DEN or 4% CCl4 (every 3 days) as indicated, followed by combined administration of 5 mg/kg AOA or 1 mg/kg DON (twice per week) for 16 weeks. Control mice were provided a normal diet (ND). Gross appearances (D) and hematoxylin and eosin staining (E) (scale bar: 100 μm) of liver samples with tumors, and the numbers of tumor nodules (F), n = 6/group. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Tukey’s test. The yellow dotted-line circles represent tumors. AST (G) and ALT (H) levels in mouse serum samples (n = 6/group). (I) Glycolysis-metabolite profiles, derived from liver tumors of WT or LKO mice, were determined by performing LC-MS/MS metabolomics assays. (J) Relative UDP-GlcNAc levels (n = 6/group). The indicated proteins in liver tumors were assessed by immunohistochemical labeling (K) (scale bar: 100 μm) and Western blotting (L).