Mcl-1 deficiency in murine livers leads to nuclear polyploidisation and mitotic errors: Implications for hepatocellular carcinoma.

Background & Aims: Mcl-1, an antiapoptotic protein overexpressed in many tumours, including hepatocellular carcinoma (HCC), represents a promising target for cancer treatment. Although Mcl-1 non-apoptotic roles might critically influence the therapeutic potential of Mcl-1 inhibitors, these functions remain poorly understood. We aimed to investigate the effects of hepatic Mcl-1 deficiency (Mcl-1Δhep) on hepatocyte ploidy and cell cycle in murine liver in vivo and the possible implications on HCC. Methods: Livers of young Mcl-1Δhep and wild-type (WT) mice were analysed for ploidy profile, mitotic figures, in situ chromosome segregation, gene set enrichment analysis and were subjected to two-thirds partial hepatectomy to assess Mcl-1 deficiency effect on cell cycle progression in vivo. Mcl-1Δhep tumours in older mice were analysed for ploidy profile, chromosomal instability, and mutational signatures via whole exome sequencing. Results: In young mice, Mcl-1 deficiency leads to nuclear polyploidy and to high rates of mitotic errors with abnormal spindle figures and chromosome mis-segregation along with a prolonged spindle assembly checkpoint activation signature. Chromosomal instability and altered ploidy profile are observed in Mcl-1Δhep tumours of old mice as well as a characteristic mutational signature of currently unknown aetiology. Conclusions: Our study suggests novel non-apoptotic effects of Mcl-1 deficiency on nuclear ploidy, mitotic regulation, and chromosomal segregation in hepatocytes in vivo. In addition, the Mcl-1 deficiency characteristic mutational signature might reflect mitotic issues. These results are of importance to consider when developing anti-Mcl-1 therapies to treat cancer. Impact and implications: Although Mcl-1 inhibitors represent promising hepatocellular carcinoma treatment, the still poorly understood non-apoptotic roles of Mcl-1 might compromise their successful clinical application. Our study shows that Mcl-1 deficiency leads to nuclear polyploidy, mitotic errors, and aberrant chromosomal segregation in hepatocytes in vivo, whereas hepatocellular tumours spontaneously induced by Mcl-1 deficiency exhibit chromosomal instability and a mutational signature potentially reflecting mitotic issues. These results have potential implications for the development of anti-Mcl-1 therapies to treat hepatocellular carcinoma, especially as hyperproliferative liver is a clinically relevant situation.

JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

IκB kinaseα/ß control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell-death mediator receptor-interacting protein kinase 1.

UNLABELLED: The IκB-Kinase (IKK) complex-consisting of the catalytic subunits, IKKα and IKKß, as well as the regulatory subunit, NEMO-mediates activation of the nuclear factor κB (NF-κB) pathway, but previous studies suggested the existence of NF-κB-independent functions of IKK subunits with potential impact on liver physiology and disease. Programmed cell death is a crucial factor in the progression of liver diseases, and receptor-interacting kinases (RIPKs) exerts strategic control over multiple pathways involved in regulating novel programmed cell-death pathways and inflammation. We hypothesized that RIPKs might be unrecognized targets of the catalytic IKK-complex subunits, thereby regulating hepatocarcinogenesis and cholestasis. In this present study, mice with specific genetic inhibition of catalytic IKK activity in liver parenchymal cells (LPCs; IKKα/ß(LPC-KO) ) were intercrossed with RIPK1(LPC-KO) or RIPK3(-/-) mice to examine whether RIPK1 or RIPK3 might be downstream targets of IKKs. Moreover, we performed in vivo phospho-proteome analyses and in vitro kinase assays, mass spectrometry, and mutagenesis experiments. These analyses revealed that IKKα and IKKß-in addition to their known function in NF-κB activation-directly phosphorylate RIPK1 at distinct regions of the protein, thereby regulating cell viability. Loss of this IKKα/ß-dependent RIPK1 phosphorylation in LPCs inhibits compensatory proliferation of hepatocytes and intrahepatic biliary cells, thus impeding HCC development, but promoting biliary cell paucity and lethal cholestasis. CONCLUSIONS: IKK-complex subunits transmit a previously unrecognized signal through RIPK1, which is fundamental for the long-term consequences of chronic hepatic inflammation and might have potential implications for future pharmacological strategies against cholestatic liver disease and cancer. (Hepatology 2016;64:1217-1231).

Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.

Hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8(+) T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8(+) T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8(+) and NKT cells cooperatively induce liver damage. Hepatocellular LTßR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development.

Chronic liver inflammation and hepatocellular carcinoma: persistence matters.

Inflammatory responses in the liver--a central constituent of hepatic wound healing--can be self-limited or persistent depending on the aetiology, liver health state, concentration of toxins or pathogens, and the time frame of exposure to toxins or infection. In case the immune system eradicates a pathogen or in case toxin-exposure is transient, acute hepatitis resolves and the affected liver tissue regenerates ad integrum. However, in many cases liver damage remains chronic. Irrespective of the aetiology, chronic liver damage drives chronic hepatitis and hepatocyte death as well as compensatory proliferation, reflecting liver regeneration. Over time this potentially promotes further hepatic damage, fibrosis, cirrhosis and liver cancer. Here, we review the current knowledge on how chronic liver injury and inflammation is triggered and maintained, and how inflammation is linked to liver cancer. We also discuss the most frequently used animal models for damage or inflammation induced liver cancer and their suitability for conducting clinically relevant research.

TAK1 suppresses a NEMO-dependent but NF-kappaB-independent pathway to liver cancer.

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.