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.

Background: Hepatocellular Carcinoma (HCC) is a matter of great global public health importance; however, its current therapeutic effectiveness is deemed inadequate, and the range of therapeutic targets is limited. The aim of this study was to identify early growth response 1 (EGR1) as a transcription factor target in HCC and to explore its role and assess the potential of gene therapy utilizing EGR1 for the management of HCC. Methods: In this study, both in vitro and in vivo assays were employed to examine the impact of EGR1 on the growth of HCC. The mouse HCC model and human organoid assay were utilized to assess the potential of EGR1 as a gene therapy for HCC. Additionally, the molecular mechanism underlying the regulation of gene expression and the suppression of HCC growth by EGR1 was investigated. Results: The results of our investigation revealed a notable decrease in the expression of EGR1 in HCC. The decrease in EGR1 expression promoted the multiplication of HCC cells and the growth of xenografted tumors. On the other hand, the excessive expression of EGR1 hindered the proliferation of HCC cells and repressed the development of xenografted tumors. Furthermore, the efficacy of EGR1 gene therapy was validated using in vivo mouse HCC models and in vitro human hepatoma organoid models, thereby providing additional substantiation for the anti-cancer role of EGR1 in HCC. The mechanistic analysis demonstrated that EGR1 interacted with the promoter region of phosphofructokinase-1, liver type (PFKL), leading to the repression of PFKL gene expression and consequent inhibition of PFKL-mediated aerobic glycolysis. Moreover, the sensitivity of HCC cells and xenografted tumors to sorafenib was found to be increased by EGR1. Conclusion: Our findings suggest that EGR1 possesses therapeutic potential as a tumor suppressor gene in HCC, and that EGR1 gene therapy may offer benefits for HCC patients.

Histone deacetylases 1 and 2 (HDAC1 and HDAC2) are ubiquitously expressed in tissues, including the liver, and play critical roles in numerous physiopathological processes. Little is known regarding the role of HDAC1 and HDAC2 in liver regeneration. In this study we generated mice in which Hdac1, Hdac2 or both genes were selectively knocked out in hepatocytes to investigate the role of these genes in liver regeneration following hepatic injury induced by partial hepatectomy or carbon tetrachloride administration. The loss of HDAC1 and/or HDAC2 (HDAC1/2) protein resulted in impaired liver regeneration. HDAC1/2 inactivation did not decrease hepatocytic 5-bromo-2-deoxyuridine uptake or the expression of proliferating cell nuclear antigen, cyclins, or cyclin-dependent kinases. However, the levels of Ki67, a mitotic marker that is expressed from the mid-G₁ phase to the end of mitosis and is closely involved in the regulation of mitotic progression, were greatly decreased, and abnormal mitosis lacking Ki67 expression was frequently observed in HDAC1/2-deficient livers. The down-regulation of either HDAC1/2 or Ki67 in the mouse liver cancer cell line Hepa1-6 resulted in similar mitotic defects. Finally, both HDAC1 and HDAC2 proteins were associated with the Ki67 gene mediated by CCAAT/enhancer-binding protein β. Conclusion: Both HDAC1 and HDAC2 play crucial roles in the regulation of liver regeneration. The loss of HDAC1/2 inhibits Ki67 expression and results in defective hepatocyte mitosis and impaired liver regeneration. (Hepatology 2013; 58:2089–2098)

Although the dysregulation of bile acid (BA) composition has been associated with fibrosis progression, its precise roles in liver fibrosis is poorly understood. This study demonstrates that solute carrier family 27 member 5 (SLC27A5), an enzyme involved in BAs metabolism, is substantially downregulated in the liver tissues of patients with cirrhosis and fibrosis mouse models. The downregulation of SLC27A5 depends on RUNX family transcription factor 2 (RUNX2), which serves as a transcriptional repressor. The findings reveal that experimental SLC27A5 knockout (Slc27a5^−/−) mice display spontaneous liver fibrosis after 24 months. The loss of SLC27A5 aggravates liver fibrosis induced by carbon tetrachloride (CCI₄) and thioacetamide (TAA). Mechanistically, SLC27A5 deficiency results in the accumulation of unconjugated BA, particularly cholic acid (CA), in the liver. This accumulation leads to the activation of hepatic stellate cells (HSCs) by upregulated expression of early growth response protein 3 (EGR3). The re‐expression of hepatic SLC27A5 by an adeno‐associated virus or the reduction of CA levels in the liver using A4250, an apical sodium‐dependent bile acid transporter (ASBT) inhibitor, ameliorates liver fibrosis in Slc27a5^−/− mice. In conclusion, SLC27A5 deficiency in mice drives hepatic fibrosis through CA‐induced activation of HSCs, highlighting its significant implications for liver fibrosis treatment.