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.
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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 4

PCK1 deficiency promotes CHK2 O-GlcNAcylation at T378.

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PCK1 deficiency promotes CHK2 O-GlcNAcylation at T378. IP-LC-MS/MS analy...
IP-LC-MS/MS analysis of O-GlcNAc-modified proteins, represented by (A) flowchart describing the processes used for IP-LC-MS/MS analysis, and (B) Kyoto Encyclopedia of Genes and Genomes–based analysis of significantly enriched pathways represented by proteins that bound to Flag-tagged OGT. Co-IP of OGT-HA and CHK2-Flag was examined using an anti-HA antibody (C) or an anti-Flag antibody (D) in PKO cells. (E) Co-IP of endogenous OGT and CHK2 in PKO cells. (F) Subcellular colocalization of OGT and CHK2 in SK-Hep1 cell was determined with immunofluorescence staining. Scale bar: 50 μm. (G) Schematic representation of the CHK2 constructs. WT CHK2 contains 3 domains, including a SQ/TQ cluster domain, a Forkhead-associated (FHA) domain, and a kinase domain. Truncation mutants of CHK2, comprising amino acids (aa) 69–543 or 1–221, were designated as ΔN and ΔC, respectively. (H) Interactions between OGT and full-length WT, the ΔN, or the ΔC in HEK293 cells were determined by co-IP. (I) CHK2 IP with anti-Flag M2 agarose beads in HEK293 cells transfected with CHK2-Flag or a vector control. (J) PKO cells were treated with 50 μM PUGNAc or 50 μM ST for 24 hours, incubated in 5 mM glucose, and followed by a sWGA pull-down assay. Western blot was determined by anti-CHK2. Cell lysates of PCK1-OE cells (K), PKO cells (L), or SK-Hep1 cells treated with 1 mM PEP or OAA (M) were immunoprecipitated with anti-Flag agarose beads and immunoblotted, as indicated. (N) LC-MS analysis of CHK2-Flag identified residue T378 as the CHK2 O-GlcNAcylation site, which corresponded to the O-GlcNAcylated CHK2 peptide ILGETSLMR. (O) IP with anti-Flag M2 agarose beads in PKO cells. Cells were transfected with vectors encoding Flag-tagged versions of WT CHK2, T378A CHK2, or T383A CHK2. (P) Cross-species sequence alignment of CHK2.