Blocking necroptosis reduces inflammation and tumor incidence in a mouse model of diet-induced hepatocellular carcinoma

Background & Aims Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. As necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis- induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet- induced HCC. Methods Male and female wild-type (WT) mice or mouse models where necroptosis is blocked ( Ripk3 -/- or Mlkl -/- mice) were fed a control diet or choline-deficient low fat diet (CD-LFD) or CD-high fat diet (CD-HFD) for 6 months. Changes in inflammation, immune cell infiltration, activation of oncogenic pathways, and tumor incidence were assessed by gene expression analysis, western blotting, and flow cytometry. RNA sequencing (RNA-seq) was performed to assess the changes in liver transcriptome. Results Blocking necroptosis by deleting either Ripk3 or Mlkl reduced markers of inflammation [proinflammatory cytokines (TNFα, IL-6, and IL-1β), F4/80 +ve macrophages, CCR2 +ve infiltrating monocytes], inflammation associated oncogenic pathways (JNK, PD-L1/PD-1, β-catenin), and HCC in male mice. In female mice, blocking necroptosis reduced HCC independent of inflammation. Blocking necroptosis reduced cell senescence markers in males and females, suggesting a novel cross-talk between necroptosis and cell senescence. Conclusions Our data show that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. In female mice necroptosis contributes to HCC independent of inflammation. Thus, our study suggests that necroptosis is a valid target for NAFLD-mediated HCC. Synopsis Necroptosis is a cell death pathway that mediate inflammation. Blocking necroptosis attenuated chronic inflammation by reducing recruitment and activation of liver macrophages, which in turn reduced activation of oncogenic pathways and progression of NAFLD to HCC in mice. Graphical Abstract