Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence.

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration.

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.

CXCR2 inhibition enables NASH-HCC immunotherapy.

OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

Beneficial Noncancerous Mutations in Liver Disease.

Chronic liver disease results in fibrosis and cancer. While injury is associated with mutational burden, a recent study (Zhu et al. Cell 2019;177:608-621) highlights that not all positively selected mutations in the liver are precancerous. Indeed, some may be beneficial to the ability of the liver to not only withstand injury , but also to regenerate.

The landscape of gene mutations in cirrhosis and hepatocellular carcinoma.

Chronic liver disease and primary liver cancer are a massive global problem, with a future increase in incidences predicted. The most prevalent form of primary liver cancer, hepatocellular carcinoma, occurs after years of chronic liver disease. Mutations in the genome are a causative and defining feature of all cancers. Chronic liver disease, mostly at the cirrhotic stage, causes the accumulation of progressive mutations which can drive cancer development. Within the liver, a Darwinian process selects out dominant clones with selected driver mutations but also leaves a trail of passenger mutations which can be used to track the evolution of a tumour. Understanding what causes specific mutations and how they combine with one another to form cancer is a question at the heart of understanding, preventing and tackling liver cancer. Herein, we review the landscape of gene mutations in cirrhosis, especially those paving the way toward hepatocellular carcinoma development, that have been characterised by recent studies capitalising on technological advances in genomic sequencing. With these insights, we are beginning to understand how cancers form in the liver, particularly on the background of chronic liver disease. This knowledge may soon lead to breakthroughs in the way we detect, diagnose and treat this devastating disease.

Outcomes and prognostic factors of multimodality treatment for locally recurrent rectal cancer with curative intent.

PURPOSE: Radical management of locally recurrent rectal cancer (LRRC) can lead to prolonged survival. This study aims to assess outcomes and identify prognostic factors for patients with LRRC treated using a multimodality treatment protocol. METHODS: An analysis of a prospectively maintained institutional database of consecutive patients who underwent radical surgical resection for LRRC was performed. Potential prognostic factors were investigated using a Cox proportional hazards model. RESULTS: Ninety-eight patients were included in this study. A multimodality approach was taken in the majority, including preoperative chemoradiation (78%), intraoperative radiation therapy (47%) and adjuvant chemotherapy (41%). Extended resection was performed where required: bone resection (34%) and lateral pelvic sidewall dissection (31%). The rate of R0 resection was 66%. Estimated rates of 5-year overall survival (OS) and progression-free survival (PFS) were 41.8% (95% CI 32.5-53.7) and 22.5% (95% CI 15.3-33.1). On multivariate analysis, stage III disease at initial primary surgery, a positive margin at initial primary surgery, synchronous or previously resected oligometastases, a lateral or sacral invasive-type pelvic recurrence and the requirement for IORT all predicted for inferior PFS (p < 0.05). Eleven percent of patients subsequently underwent further pelvic surgery for pelvic re-recurrence and had an estimated 5-year OS rate of 54.5% (95% CI 29.0-100.0) from repeat surgery. CONCLUSIONS: Radical multimodality management of LRRC leads to prolonged survival in approximately 40% of patients. Those with sacral or lateral invasive-type recurrence or oligometastatic disease have inferior outcomes and further research is needed to optimise treatment for these groups.

Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling.

Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine.

How the liver keeps itself in shape.

After fasting, hepatocytes proliferate to help the liver grow back to its original size.

Neutrophils as potential therapeutic targets in hepatocellular carcinoma.

The success of atezolizumab plus bevacizumab treatment contributed to a shift in systemic therapies for hepatocellular carcinoma (HCC) towards combinations that include cancer immunotherapeutic agents. Thus far, the principal focus of cancer immunotherapy has been on interrupting immune checkpoints that suppress antitumour lymphocytes. As well as lymphocytes, the HCC environment includes numerous other immune cell types, among which neutrophils are emerging as an important contributor to the pathogenesis of HCC. A growing body of evidence supports neutrophils as key mediators of the immunosuppressive environment in which some cancers develop, as well as drivers of tumour progression. If neutrophils have a similar role in HCC, approaches that target or manipulate neutrophils might have therapeutic benefits, potentially including sensitization of tumours to conventional immunotherapy. Several neutrophil-directed therapies for patients with HCC (and other cancers) are now entering clinical trials. This Review outlines the evidence in support of neutrophils as drivers of HCC and details their mechanistic roles in development, progression and metastasis, highlighting the reasons that neutrophils are well worth investigating despite the challenges associated with studying them. Neutrophil-modulating anticancer therapies entering clinical trials are also summarized.

p53-mediated redox control promotes liver regeneration and maintains liver function in response to CCl4.

The p53 transcription factor coordinates wide-ranging responses to stress that contribute to its function as a tumour suppressor. The responses to p53 induction are complex and range from mediating the elimination of stressed or damaged cells to promoting survival and repair. These activities of p53 can modulate tumour development but may also play a role in pathological responses to stress such as tissue damage and repair. Using a p53 reporter mouse, we have previously detected strong induction of p53 activity in the liver of mice treated with the hepatotoxin carbon tetrachloride (CCl4). Here, we show that p53 functions to support repair and recovery from CCl4-mediated liver damage, control reactive oxygen species (ROS) and limit the development of hepatocellular carcinoma (HCC), in part through the activation of a detoxification cytochrome P450, CYP2A5 (CYP2A6 in humans). Our work demonstrates an important role for p53-mediated redox control in facilitating the hepatic regenerative response after damage and identifies CYP2A5/CYP2A6 as a mediator of this pathway with potential prognostic utility in human HCC.

A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo.

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are suboptimal in vivo because of poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled noninvasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol- or CCl4-induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver.

BACKGROUND: Stem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling. OBJECTIVE: To examine the composition and formation of stem progenitor cell niches. METHODS: The composition of the hepatic progenitor cell niche in independent models of liver injury and hepatic progenitor cell activation in rodents and humans was studied. To identify the origin of the progenitor and niche cells, sex-mismatched bone marrow transplants in mice, who had received the choline-ethionine-deficient-diet to induce liver injury and progenitor cell activation, were used. The matrix surrounding the progenitor cells was described by immunohistochemical staining and its functional role controlling progenitor cell behaviour was studied in cell culture experiments using different matrix layers. RESULTS: The progenitor cell response in liver injury is intimately surrounded by myofibroblasts and macrophages, and to a lesser extent by endothelial cells. Hepatic progenitor cells are not of bone marrow origin; however, bone marrow-derived cells associate intimately with these cells and are macrophages. Laminin always surrounds the progenitor cells. In vitro studies showed that laminin aids maintenance of progenitor and biliary cell phenotype and promotes their gene expression (Dlk1, Aquaporin 1, gammaGT) while inhibiting hepatocyte differentiation and gene expression (CEPB/alpha). CONCLUSIONS: During liver damage in rodents and humans a stereotypical cellular and laminin niche forms around hepatic progenitor cells. Laminin helps maintenance of undifferentiated progenitor cells. The niche links the intrahepatic progenitor cells with bone marrow-derived cells and links tissue damage with progenitor cell-mediated tissue repair.

Specificity and off-target effects of AAV8-TBG viral vectors for the manipulation of hepatocellular gene expression in mice.

Mice are a widely used pre-clinical model system in large part due to their potential for genetic manipulation. The ability to manipulate gene expression in specific cells under temporal control is a powerful experimental tool. The liver is central to metabolic homeostasis and a site of many diseases, making the targeting of hepatocytes attractive. Adeno-associated virus 8 (AAV8) vectors are valuable instruments for the manipulation of hepatocellular gene expression. However, their off-target effects in mice have not been thoroughly explored. Here, we sought to identify the short-term off-target effects of AAV8 administration in mice. To do this, we injected C57BL/6J wild-type mice with either recombinant AAV8 vectors expressing Cre recombinase or control AAV8 vectors and characterised the changes in general health and in liver physiology, histology and transcriptomics compared to uninjected controls. We observed an acute and transient trend for reduction in homeostatic liver proliferation together with induction of the DNA damage marker γH2AX following AAV8 administration. The latter was enhanced upon Cre recombinase expression by the vector. Furthermore, we observed transcriptional changes in genes involved in circadian rhythm and response to infection. Notably, there were no additional transcriptomic changes upon expression of Cre recombinase by the AAV8 vector. Overall, there was no evidence of liver injury, and only mild T-cell infiltration was observed 14 days following AAV8 infection. These data advance the technique of hepatocellular genome editing through Cre-Lox recombination using Cre expressing AAV vectors, demonstrating their minimal effects on murine physiology and highlight the more subtle off target effects of these systems.

Primary liver cancer in the UK: Incidence, incidence-based mortality, and survival by subtype, sex, and nation.

BACKGROUND & AIMS: The incidence of primary liver cancer (PLC) is increasing in Western Europe. To understand trends over time and the current burden in the UK, a detailed analysis of the epidemiology of PLC and its subtypes was conducted. METHODS: Data on PLCs diagnosed during 1997-2017 were obtained from population-based, nationwide registries in the UK. European age-standardised incidence (ASR) and incidence-based mortality rates (ASMR) per 100,000 person-years were calculated overall and by sex and UK-nation. Annual percentage change in rates was estimated using Joinpoint regression. One-, 2-, and 5-year age-standardised net survival was estimated. RESULTS: A total of 82,024 PLCs were diagnosed. Both hepatocellular carcinoma (HCC) incidence and mortality rates trebled (ASR 1.8-5.5 per 100,000, ASMR 1.3-4.0). The rate of increase appeared to plateau around 2014/2015. Scottish men consistently had the highest HCC incidence rates. PLC survival increased, driven by a substantial increase in the proportion that are HCC (as prognosis is better than other PLCs) and in HCC survival (change in 1-year survival 24-47%). Intrahepatic cholangiocarcinoma was the most common PLC in women and 1-year survival improved from 22.6% to 30.5%. CONCLUSIONS: PLC incidence has been increasing rapidly but, as most risk factors are modifiable, it is largely a preventable cancer. This rate of increase has slowed in recent years, possibly attributable to effective treatment for hepatitis C. As other risk factors such as obesity and diabetes remain prevalent in the UK, it is unlikely the considerable burden of this disease will abate. While improvements in survival have been made, over half of patients are not alive after 1 year, therefore further progress in prevention, early detection, and treatment innovation are needed. LAY SUMMARY: Many more people are getting liver cancer, particularly the subtype hepatocellular carcinoma, than 20 years ago. Men in Scotland are most likely to get liver cancer and to die from it. Survival after liver cancer diagnosis is getting longer but still less than half are alive after 1 year.

Autophagy suppresses the formation of hepatocyte-derived cancer-initiating ductular progenitor cells in the liver.

Hepatocellular carcinoma (HCC) is driven by repeated rounds of inflammation, leading to fibrosis, cirrhosis, and, ultimately, cancer. A critical step in HCC formation is the transition from fibrosis to cirrhosis, which is associated with a change in the liver parenchyma called ductular reaction. Here, we report a genetically engineered mouse model of HCC driven by loss of macroautophagy and hemizygosity of phosphatase and tensin homolog, which develops HCC involving ductular reaction. We show through lineage tracing that, following loss of autophagy, mature hepatocytes dedifferentiate into biliary-like liver progenitor cells (ductular reaction), giving rise to HCC. Furthermore, this change is associated with deregulation of yes-associated protein and transcriptional coactivator with PDZ-binding motif transcription factors, and the combined, but not individual, deletion of these factors completely reverses the dedifferentiation capacity and tumorigenesis. These findings therefore increase our understanding of the cell of origin of HCC development and highlight new potential points for therapeutic intervention.

Effect of Hepatocellular Carcinoma Surveillance Programmes on Overall Survival in a Mixed Cirrhotic UK Population: A Prospective, Longitudinal Cohort Study.

INTRODUCTION: Surveillance for hepatocellular carcinoma (HCC) is recommended by national and international guidelines. However, there are no trial data on whether surveillance improves clinical outcomes in a UK cirrhosis population of mixed aetiology. Our aim was to determine the impact of, and adherence to, surveillance on overall survival. METHODS: We prospectively collected data on consecutive patients diagnosed with HCC between January 2009 and December 2015 at two large UK centres. We assessed outcomes depending on whether they had been entered into an HCC surveillance programme, and if they had adhered to that. RESULTS: Out of 985 patients diagnosed with HCC in this study, 40.0% had been enrolled in a surveillance programme. Of these, 76.6% were adherent with surveillance and 24.4% were not. Adherence to surveillance was significantly associated with improved overall survival, even when accounting for lead-time bias using different approaches (HR for 270 days lead-time adjustment 0.64, 0.53 to 0.76, p < 0.001). CONCLUSIONS: When adjusted for lead-time bias, HCC surveillance is associated with improved overall survival; however, the beneficial effect of surveillance on survival was lower than reported in studies that did not account fully for lead-time bias.

Cellular senescence as a potential mediator of COVID-19 severity in the elderly.

SARS-CoV-2 is a novel betacoronavirus which infects the lower respiratory tract and can cause coronavirus disease 2019 (COVID-19), a complex respiratory distress syndrome. Epidemiological data show that COVID-19 has a rising mortality particularly in individuals with advanced age. Identifying a functional association between SARS-CoV-2 infection and the process of biological aging may provide a tractable avenue for therapy to prevent acute and long-term disease. Here, we discuss how cellular senescence-a state of stable growth arrest characterized by pro-inflammatory and pro-disease functions-can hypothetically be a contributor to COVID-19 pathogenesis, and a potential pharmaceutical target to alleviate disease severity. First, we define why older COVID-19 patients are more likely to accumulate high levels of cellular senescence. Second, we describe how senescent cells can contribute to an uncontrolled SARS-CoV-2-mediated cytokine storm and an excessive inflammatory reaction during the early phase of the disease. Third, we discuss the various mechanisms by which senescent cells promote tissue damage leading to lung failure and multi-tissue dysfunctions. Fourth, we argue that a high senescence burst might negatively impact on vaccine efficacy. Measuring the burst of cellular senescence could hypothetically serve as a predictor of COVID-19 severity, and targeting senescence-associated mechanisms prior and after SARS-CoV-2 infection might have the potential to limit a number of severe damages and to improve the efficacy of vaccinations.