ADH4 deficiency activates the NRF2-CSF1 axis by increased lipid peroxidation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is frequently associated with serious metabolic disorders, resulting in a bleak prognosis. Although ADH4 is implicated in the metabolism of fatty acids, its exact function in tumourigenesis is still unclear.

Our study initially examined proliferation potential in vitro and in vivo using various approaches, such as CCK8, EdU incorporation, colony formation assays, and a tumourigenicity assay in mice, which included orthotopic injection into the left liver lobe. Next, we conducted a quantitative analysis of oxylipins to investigate the correlation between ADH4 and lipid peroxidation in hepatoma cells. Additionally, we examined the intracellular ratio of NADP+/NADPH and measured the production of reactive oxygen species (ROS) and malondialdehyde (MDA) in other hepatoma cells. Furthermore, we employed mRNA sequencing to identify the downstream targets of ADH4 in hepatoma cells.

The results indicate that there is a significant association between ADH4 levels and lipid peroxidation in hepatoma cells. It has been shown that there is a downregulation of ADH4 expression in hepatocellular carcinoma, and that ADH4 deficiency is a contributing factor in tumour progression and overall poor prognosis. Both gain- and loss-of-function studies have demonstrated that ADH4 has an anti-proliferative effect on hepatocellular carcinoma both in vitro and in vivo. Inhibition of ADH4 leads to the accumulation of oxylipin metabolites and an increase in the NADP+/NADPH ratio, ROS and MDA production, ultimately resulting in the activation of the KEAP1/NRF2 pathway and the nuclear translocation of NRF2. Furthermore, the results of mRNA sequencing indicate a close correlation between ADH4 expression and the cytokine-cytokine receptor pathway, with the strongest negative association found with CSF1 expression. This has been validated in other hepatoma cells and clinical patient samples. Flow cytometric analysis has confirmed ADH4's ability to suppress CSF1-mediated polarization of M2-type macrophages in an NRF2 signaling-dependent manner. In conclusion, ADH4 plays a significant role in the regulation of macrophage polarization.

These findings suggest that downregulation of ADH4 may exert protumourigenic effects on hepatocellular carcinoma by increasing lipid peroxidation levels, NADP+/NADPH ratio, ROS and MDA levels, leading to activation of the KEAP1/NRF2 pathway, upregulation of CSF1 and M2-type polarisation of tumour-associated macrophages (TAMs), suggesting a potential therapeutic strategy for hepatocellular carcinoma.