Background: Metabolic dysfunction-associated steatohepatitis-related hepatocellular carcinoma (MASH-HCC) has been reported to be less responsive to immune checkpoint inhibitors, which may be associated with metabolic reprogramming of tumour cells and abnormal tumour microenvironment. Objective: Here, we aim to investigate the role of gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) in MASH-HCC and its interplay with the tumour microenvironment. Design: Hepatocyte-specific phosphatase and tensin homologue ( Pten ) and Pck1 biallelic knockout mice were established to induce MASH-HCC. Single-cell RNA sequencing and multiparametrical flow cytometry were performed to analyse the immune landscape alterations. Untargeted metabolomics was conducted to elucidate the hepatic metabolism dysregulation. Results: PCK1 is downregulated in tumour tissues compared with adjacent non-cancerous tissues from patients with MASH-HCC. Hepatocyte-specific Pck1 knockout mice exhibited markedly increased tumorigenesis in dietary models and genetic models of spontaneous MASH-HCC, together with inhibited effector function of tumour-infiltrating CD8 ⁺ T cells. Mechanistically, PCK1 deficiency induces the accumulation of endogenous metabolite 12-hydroxyeicosatetraenoic acid (12-HETE), which can be taken up by CD8 ⁺ T cells and activate the p38 mitogen-activated protein kinase pathway by directly interacting with the BTB and CNC homology 1 transcription factor, ultimately leading to CD8 ⁺ T cells dysfunction. Notably, PCK1 restoration or 12-HETE inhibition combined with anti-PD-1 treatment increases the antitumour capability of CD8 ⁺ T cells and suppresses MASH-HCC development. Conclusion: This study reveals the pivotal role of the hepatic cell-intrinsic enzyme PCK1 in mediating CD8 ⁺ T cell dysfunction via 12-HETE-p38 signalling in MASH-HCC. PCK1 could be a metabolic checkpoint to enhance the efficacy of anti-PD-1 immunotherapy in MASH-HCC.

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.

Metabolic associated fatty liver disease (MAFLD) encompasses a broad spectrum of hepatic disorders, including steatosis, nonalcoholic steatohepatitis (NASH) and fibrosis. We demonstrated that phosphoenolpyruvate carboxykinase 1 (PCK1) plays a central role in MAFLD progression. Male mice with liver Pck1 deficiency fed a normal diet displayed hepatic lipid disorder and liver injury, whereas fibrosis and inflammation were aggravated in mice fed a high-fat diet with drinking water containing fructose and glucose (HFCD-HF/G). Forced expression of hepatic PCK1 by adeno-associated virus ameliorated MAFLD in male mice. PCK1 deficiency stimulated lipogenic gene expression and lipid synthesis. Moreover, loss of hepatic PCK1 activated the RhoA/PI3K/AKT pathway by increasing intracellular GTP levels, increasing secretion of platelet-derived growth factor-AA (PDGF-AA), and promoting hepatic stellate cell activation. Treatment with RhoA and AKT inhibitors or gene silencing of RhoA or AKT1 alleviated MAFLD progression in vivo. Hepatic PCK1 deficiency may be important in hepatic steatosis and fibrosis development through paracrine secretion of PDGF-AA in male mice, highlighting a potential therapeutic strategy for MAFLD.

Background/Aims: Acetyl coenzyme A (acetyl-CoA) is one of the most essential metabolites in cell metabolism but its function and concentration in hepatocellular carcinoma (HCC) remain elusive and controversial.Methods: A comprehensive analysis of acetyl-CoA levels and acetyl-CoA synthetase 2 (ACSS2) expression across a range of samples, including patient specimens from both hepatitis B virus (HBV) positive and HBV negative HCC individuals, HBV-transgenic mouse HCC models, and multiple cell lines. Furthermore, to evaluate the functional significance of ACSS2 in HBV-related HCC, we implemented both genetic and pharmacological inhibition strategies targeting ACSS2. Molecular mechanism and mitophagy assessment were revealed by cleavage under target and tagmentation sequencing, RNA sequencing, bioinformatic analyses, transmission electron microscopy and JC-1 staining.Results: Our study revealed a distinct metabolic signature of HBV-related HCC, marked by elevated acetyl-CoA, which was driven by ACSS2. ACSS2 was upregulated by the carbohydrate response element-binding protein in HBV-related HCC. Furthermore, ACSS2 improved tumor cell proliferation, an effect that was dependent on its enzymatic activity. Mechanistically, ACSS2-induced acetyl-CoA accumulation activated voltage-dependent anion channels 1 transcription through increased H3K27ac occupancy, which subsequently promoted mitophagy and HBV-related HCC tumorigenesis. Notably, targeting ACSS2 by depletion or inhibition with a catalytic inhibitor significantly suppressed tumor growth.Conclusions: These findings not only illustrate the interplay between metabolic reprogramming, epigenetic modification, and tumorigenesis in the context of HBV infection, but also highlight ACSS2 as a novel metabolic vulnerability in HBV-related HCC. Therefore, targeting ACSS2 could be a novel strategy against HBV-related HCC.