Macrophage autophagy protects against hepatocellular carcinogenesis in mice

Autophagy is a lysosomal degradation pathway of cellular components that regulates macrophage properties. Macrophages are critically involved in tumor growth, metastasis, angiogenesis and immune suppression. Here, we investigated whether macrophage autophagy may protect against hepatocellular carcinoma (HCC). Experiments were performed in mice with deletion of the autophagy gene Atg5 in the myeloid lineage (ATG5Mye−/− mice) and their wild-type (WT) littermates. As compared to WT, ATG5Mye−/− mice were more susceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis, as shown by enhanced tumor number and volume. Moreover, DEN-treated ATG5Mye−/− mice exhibited compromised immune cell recruitment and activation in the liver, suggesting that macrophage autophagy invalidation altered the antitumoral immune response. RNA sequencing showed that autophagy-deficient macrophages sorted from DEN mice are characterized by an enhanced expression of immunosuppressive markers. In vitro studies demonstrated that hepatoma cells impair the autophagy flux of macrophages and stimulate their expression of programmed cell death-ligand 1 (PD-L1), a major regulator of the immune checkpoint. Moreover, pharmacological activation of autophagy reduces hepatoma cell-induced PD-L1 expression in cultured macrophages while inhibition of autophagy further increases PD-L1 expression suggesting that autophagy invalidation in macrophages induces an immunosuppressive phenotype. These results uncover macrophage autophagy as a novel protective pathway regulating liver carcinogenesis.

Phosphorylation of NF-κBp65 drives inflammation-mediated hepatocellular carcinogenesis and is a novel therapeutic target

Background Nuclear factor­κB (NF-κB) plays a vital role in hepatocellular carcinoma (HCC). β-arrestin1 (ARRB1) has been proved to enhance the activity of NF-κBp65, and our previous study indicated that ARRB1 promotes hepatocellular carcinogenesis and development of HCC. However, it remains unknown whether p65 is involved in hepatocellular carcinogenesis through the ARRB1-mediated pathway. Methods The levels of NF-κBp65 and NF-κBp65 phosphorylation (p-p65) were assessed in including normal liver, primary HCC and paired paracancerous tissues. Liver-specific p65 knockout mice were used to examine the role of p65 and p-p65 in hepatocarcinogenesis. The mechanism of NF-κBp65 and p-p65 in hepatocarcinogenesis via ARRB1 was also studied both in vitro and in vivo. Results Phosphorylation of NF-κBp65 was markedly upregulated in inflammation-related HCC patients and was significantly increased in mouse hepatic inflammation models, which were induced by tetrachloromethane (CCl4), diethylnitrosamine (DEN), TNF-α, as well as DEN-induced HCC. Hepatocyte-specific p65-deficient mice markedly decreased in the HCC incidence and size of tumours by the repressing of the proliferation of malignant cells in a DEN-induced HCC model. Furthermore, ARRB1 directly bounds p65 to promote the phosphorylation of NF-κBp65 at ser536, resulted in cell malignant proliferation through GSK3β/mTOR signalling. Conclusion The data demonstrated that phosphorylation of NF-κBp65 drives hepatocellular carcinogenesis in response to inflammation-mediated ARRB1, and that inhibition of the phosphorylation of NF-κBp65 restrains the hepatocellular carcinogenesis. The results indicate that phosphorylation of NF-κBp65 is a novel therapeutic target for HCC.

Inhibition of androgen/AR signaling inhibits diethylnitrosamine (DEN) induced tumour initiation and remodels liver immune cell networks

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.