Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO^LPC-KO mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. To address potential functional redundancies between death receptors we generated and analysed NEMO^LPC-KO mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO^LPC-KO mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO^LPC-KO mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO^LPC-KO mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.Liver cancer is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide.^{1, 2} Liver cancer predominantly arises in the context of chronic inflammatory conditions, most notably in virus hepatitis (HBV and HCV).^{1, 2} Although infectious agents are the primary cause of liver cancer worldwide, the incidence in western countries is rising due to the increase in obesity and non-alcoholic steatohepatitis.³ The pathogenesis of hepatocellular carcinoma (HCC) is incompletely understood and it is plausible that the different underlying aetiologies determine a distinct context for liver carcinogenesis. However, the prevailing universal concept is that continuous liver parenchymal damage and hepatocyte cell death drive compensatory proliferation and within the context of a chronically inflamed liver tissue mutations and epigenetic changes accumulate eventually transforming hepatocytes into malignant cells. Therefore, understanding the tissue-intrinsic processes that determine cell death and chronic inflammation resulting in hepatocarcinogenesis is a critical need in order to design more effective therapeutic strategies.The nuclear factor κB (NF-κB) pathway is implicated in cancer development in particular in the context of chronic inflammation.^{4, 5} In relation to liver cancer, NF-κB signalling has been implicated in the pathogenesis of hepatitis, liver fibrosis, cirrhosis and HCC.^{6, 7} The IKK complex, composed of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ, and a regulatory subunit termed NEMO/IKKγ, activates NF-κB by phosphorylating inhibitor of NF-κB (IκB) proteins targeting them for degradation by the proteasome and thus allowing the nuclear accumulation of NF-κB dimers.⁵ IKK2 is primarily responsible for targeting and degrading IκBα thus inducing canonical NF-κB activation, although the two kinases show some degree of functional redundancy in controlling canonical NF-κB signalling.^{5, 8} NEMO/IKKγ is indispensable for activation of canonical NF-κB signalling.^{9, 10, 11}NF-κB signalling was proposed to exhibit tumour promoter or tumour suppressor properties in different models of liver cancer. In the Mdr2^−/− mouse model of inflammation-driven liver carcinogenesis, NF-κB inhibition caused by transgenic IκBα super–repressor expression in hepatocytes inhibited HCC progression.¹² Moreover, hepatocyte-restricted ablation of IKK2 prevented hepatitis and liver tumorigenesis induced by overexpression of lymphotoxins α and β in hepatocytes.¹³ However, mice with hepatocyte-specific IKK2 ablation developed more tumours induced by a single injection of the chemical carcinogen diethylnitrosamine,¹⁴ revealing a tumour suppressor role of NF-κB in this context.Studies in mice lacking NEMO specifically in liver parenchymal cells (LPCs) further supported a tumour suppressor function of IKK/NF-κB signalling in liver cancer. NEMO^LPC-KO mice showed spontaneous hepatocyte apoptosis resulting in chronic steatohepatitis and the development of HCC by the age of 1 year.¹⁵ LPC-specific ablation of Fas-Associated with Death Domain (FADD or MORT1), an adapter protein essential for the recruitment of caspase-8 to the Death Inducing Signalling Complex and the induction of death receptor-mediated apoptosis,¹⁶ prevented both spontaneous and LPS-induced apoptosis of NEMO-deficient hepatocytes and the development of steatohepatitis.¹⁵ In addition, LPC-specific knockout of caspase-8 inhibited spontaneous hepatocyte apoptosis and HCC development in NEMO^LPC-KO mice, although it caused non-apoptotic hepatocyte death and cholestasis.¹⁷ Given the essential role of FADD and caspase-8 in mediating apoptosis downstream of death receptors,¹⁶ we hypothesized that death receptor-mediated apoptosis of NEMO-deficient hepatocytes drives the development of hepatitis and HCC in NEMO^LPC-KO mice. The three main death receptors of the TNF receptor superfamily that are capable of inducing caspase-8-mediated apoptosis are TNFR1, Fas/CD95 and TRAIL-R/DR5.¹⁶ To address the role of death receptor-induced apoptosis in triggering the spontaneous death of NEMO-deficient hepatocytes and the development of steatohepatitis and HCC, we generated and analysed NEMO^LPC-KO mice lacking TNFR1, Fas or TRAIL-R specifically in LPCs. Surprisingly, we found that LPC-specific knockout of each of the death receptors alone but also combined deficiency of TNFR1, Fas and TRAIL-R in LPCs did not prevent spontaneous hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. In addition, knockout of TNF in all cells also did not protect NEMO^LPC-KO mice from hepatocyte death, hepatitis and HCC. Collectively, these results demonstrate that TNFR1, Fas and TRAIL-R are not required for the development of chronic liver damage and HCC in NEMO^LPC-KO mice.