Cyclin E1 in Murine and Human Liver Cancer: A Promising Target for Therapeutic Intervention during Tumour Progression.

Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation, migration and malignant signalling events in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of ß1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.

Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti-Fibrotic Effects in Hepatic Stellate Cells In Vitro.

Liver fibrosis is a wound healing process in response to chronic liver injury, which is characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase in complex with E-and A-type cyclins. We have demonstrated that cyclin E1 is essential for liver fibrogenesis in mice, but it is not known if this is dependent on Cdk2 or related Cdks. Here, we aimed to evaluate the benefit of the pan-Cdk inhibitor CR8 for treatment of liver fibrosis in vitro. CR8-treatment reduced proliferation and survival in immortalized HSC lines and in addition attenuated pro-fibrotic properties in primary murine HSCs. Importantly, primary murine hepatocytes were much more tolerant against the cytotoxic and anti-proliferative effects of CR8. We identified CR8 dosages mediating anti-fibrotic effects in primary HSCs without affecting cell cycle activity and survival in primary hepatocytes. In conclusion, the pharmacological pan-Cdk inhibitor CR8 restricts the pro-fibrotic properties of HSCs, while preserving proliferation and viability of hepatocytes at least in vitro. Therefore, CR8 and related drugs might be beneficial for the treatment of liver fibrosis.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Inhibition of Caspase-8 does not protect from alcohol-induced liver apoptosis but alleviates alcoholic hepatic steatosis in mice.

Hepatic apoptosis is involved in the progression of alcoholic liver disease (ALD). Caspase-8, the apical initiator in death receptor-mediated apoptosis, has been implicated in acute liver injury and in non-alcoholic steatohepatitis. However, the relevance of Caspase-8 in the pathogenesis of ALD remains unclear. In the present study, we investigated the impact of Caspase-8 in human and murine alcohol-induced apoptosis and in ALD. We investigated human samples from ALD patients, primary mouse hepatocytes, and hepatocyte-specific Caspase-8 knockout (Casp8Δhepa) mice in acute and chronic models of ethanol (EtOH) administration. Caspase-8 activation was detected in liver biopsies from ALD patients, as well as in livers of wild-type (WT) mice after chronic ethanol feeding for 8 weeks using the Lieber-DeCarli model. Lack of Caspase-8 expression in Casp8Δhepa animals failed to prevent alcohol-induced liver damage and apoptosis. Instead, inhibition of Caspase-8 shifted the ethanol-induced death signals towards pronounced activation of the intrinsic, mitochondria-dependent apoptosis pathway in Casp8Δhepa livers involving enhanced release of cytochrome c, stronger Caspase-9 activation and specific morphological changes of mitochondria. In vitro and in vivo intervention using a pan-caspase inhibitor markedly attenuated alcohol-induced hepatocyte damage in a Caspase-8-independent manner. Surprisingly, EtOH-fed Casp8Δhepa mice displayed significantly attenuated steatosis and reduced hepatic triglyceride and free fatty acids content. Caspase-8 is dispensable for alcohol-induced apoptosis, but plays an unexpected role for alcohol-dependent fat metabolism. We provide evidence that simultaneous inhibition of extrinsic and intrinsic apoptosis signaling using pan-caspase inhibitors in vivo might be an optimal approach to treat alcohol-induced liver injury.

Targeting CCl4 -induced liver fibrosis by RNA interference-mediated inhibition of cyclin E1 in mice.

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4 -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. CONCLUSION: Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257).

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation.

UNLABELLED: The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8(Δhepa) ) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8(Δhepa) mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8(Δhepa) mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8(Δhepa) mice but resulted in incomplete termination of the regeneration process and hepatomegaly. CONCLUSION: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly.

Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury.

BACKGROUND & AIMS: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.

Role of spinal microglia in myositis-induced central sensitisation: an immunohistochemical and behavioural study in rats.

There is increasing evidence that spinal glial cells play an important role in chronic pain states. However, so far no data on the role of microglia in muscle pain are available. The aim of the present study was to investigate the involvement of spinal microglial cells in chronic muscle pain. In a rat model of chronic muscle inflammation (injection of complete Freund s adjuvant into the gastrocnemius-soleus muscle) alterations of microglia were visualized with quantitative OX-42 immunohistochemistry in the dorsal horn of the segments L4 and L5 12 days after induction of inflammation. In behavioural experiments the influence of chronic intrathecally applied minocycline - a specific microglia inhibitor - or an antibody against tumour necrosis factor-alpha (TNF-alpha; a cytokine released from microglia) on pain-related behaviour was investigated after 1, 3, 6, and 12 days. The immunhistochemical data show that in the deep laminae of the spinal dorsal horn microglial cells reacted with morphological changes to the muscle inflammation. Following inflammation, the mean boundary length surrounding the OX-42 immunostained area was significantly shorter. This indicates that microglial cells were activated by the myositis and withdrew their processes. Chronic intrathecal administration of minocycline or anti TNF-alpha with an osmotic mini-pump largely normalised the inflammation-induced changes in spontaneous exploratory behaviour and attenuated the hypersensitivity to mechanical stimulation. Both the immunohistochemical and behavioural data show that spinal microglial cells are involved in nociceptive processes in the cause of a chronic muscle inflammation.

Molecular mechanism of Mitomycin C-dependent caspase-8 regulation: implications for apoptosis and synergism with interferon-alpha signalling.

Caspase-8 is frequently mutated or silenced in several tumors including hepatocellular carcinomas (HCC) thereby potentially contributing to chemoresistance. The aim of our present study was to evaluate if chemotherapeutic drugs may mediate their effects through up-regulation of caspase-8 gene transcription. Huh7 hepatoma cells were transfected with a caspase-8 promoter construct fused to a luciferase reporter gene followed by stimulation with a subset of different chemotherapeutic drugs. Several drugs slightly induced caspase-8 promoter activity. However, strong caspase-8 promoter induction was found after Mitomycin C (MMC) treatment and this correlated with an increase in endogenous caspase-8 mRNA expression. Further molecular analysis demonstrated that MMC controls caspase-8 transcription via a c-jun/AP1 site located in the promoter in close proximity to the transcription start site. Inactivation of this c-jun/AP1 site using a dominant-negative c-jun adenovirus or site-directed mutagenesis inhibited MMC-dependent promoter induction. MMC treatment resulted in higher caspase-8 enzymatic activity and apoptosis and could be synergistically enhanced by co-stimulation with interferon-alpha (IFNalpha) via independent transcriptional mechanisms. In summary MMC controls caspase-8 expression via a c-jun/AP1 element in its promoter region. MMC-induced up-regulation of caspase-8 triggers apoptosis in target cells which can be further enhanced by IFNalpha. Therefore these findings also provide a potential new therapeutic approach to treat cancer cells.

Effects on c-Fos expression in the PAG and thalamus by selective input via tetrodotoxin-resistant afferent fibres from muscle and skin.

Nociceptive information from skin and muscle is differently processed at many levels of the central nervous system. However, with regard to nociceptive input from muscle to the thalamus, only few data are available. Here, we investigated the c-Fos expression in the thalamus and the periaqueductal grey matter (PAG) induced by electrical stimulation of tetrodotoxin-resistant (TTX-r), presumably nociceptive, afferent fibres. In addition, a comparison between the effects of TTX-r input from muscle and skin was made. In anaesthetised rats, a skin or a muscle nerve was stimulated electrically for 1h at an intensity supramaximal for unmyelinated fibres. To block TTX-sensitive afferents, TTX was applied to the sciatic nerve. c-Fos was visualized using DAB immunohistochemistry. Here we report for the first time that in the PAG and medial thalamus, the main effect of TTX-r input from muscle was a reduction in c-Fos expression, and that in some thalamic nuclei (e.g. posterior, reuniens, and central medial nuclei), significant differences in the number of c-Fos-positive cells were found after muscle and cutaneous input, respectively. The thalamic regions with the strongest effects of muscle input were the VL bilaterally and the VPL contralaterally (increase in c-Fos expression) as well as the rhomboid nucleus (decrease in c-Fos expression).