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1.
J Hepatol ; 2024 Aug 20.
Article in English | MEDLINE | ID: mdl-39173955

ABSTRACT

BACKGROUND & AIMS: Liver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC-oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis - a rising global unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

2.
Am J Physiol Gastrointest Liver Physiol ; 327(2): G123-G139, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38771154

ABSTRACT

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.


Subject(s)
Interleukin-22 , Interleukins , Intestinal Mucosa , Interleukins/metabolism , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Animals , Cell Proliferation , Signal Transduction , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/pathology , Mice , Up-Regulation , Proto-Oncogene Proteins c-akt/metabolism , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase IX/genetics , Antigens, Neoplasm
3.
iScience ; 27(3): 109301, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38469563

ABSTRACT

Persistent liver injury triggers a fibrogenic program that causes pathologic remodeling of the hepatic microenvironment (i.e., liver fibrosis) and portal hypertension. The dynamics of gene regulation during liver disease progression and early regression remain understudied. Here, we generated hepatic transcriptome profiles in two well-established liver disease models at peak fibrosis and during spontaneous regression after the removal of the inducing agents. We linked the dynamics of key disease readouts, such as portal pressure, collagen area, and transaminase levels, to differentially expressed genes, enabling the identification of transcriptomic signatures of progressive vs. regressive liver fibrosis and portal hypertension. These candidate biomarkers (e.g., Tcf4, Mmp7, Trem2, Spp1, Scube1, Islr) were validated in RNA sequencing datasets of patients with cirrhosis and portal hypertension, and those cured from hepatitis C infection. Finally, deconvolution identified major cell types and suggested an association of macrophage and portal hepatocyte signatures with portal hypertension and fibrosis area.

4.
J Hepatol ; 78(2): 343-355, 2023 02.
Article in English | MEDLINE | ID: mdl-36309131

ABSTRACT

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Animals , Mice , Carcinoma, Hepatocellular/genetics , Claudin-1/genetics , Liver Neoplasms/genetics , Carcinogens , Tumor Microenvironment , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Cell Line, Tumor
5.
Sci Transl Med ; 14(676): eabj4221, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36542691

ABSTRACT

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.


Subject(s)
Antibodies, Monoclonal , Cell Plasticity , Animals , Mice , Humans , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Claudin-1 , Liver Cirrhosis/drug therapy
6.
Viruses ; 14(5)2022 05 17.
Article in English | MEDLINE | ID: mdl-35632812

ABSTRACT

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence.


Subject(s)
DNA, Circular , Hepatitis B, Chronic , DNA, Circular/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , Gene Expression , Hepatitis B e Antigens/genetics , Hepatitis B virus , Humans
7.
Gut ; 2022 Dec 09.
Article in English | MEDLINE | ID: mdl-36591611

ABSTRACT

OBJECTIVES: Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation. One major challenge to address this urgent question is the absence of robust models for the study of cccDNA biology. DESIGN: We established a cell-based HBV cccDNA reporter assay and performed a loss-of-function screen targeting 239 genes encoding the human DNA damage response machinery. RESULTS: Overcoming the limitations of current models, the reporter assay enables to quantity cccDNA levels using a robust ELISA as a readout. A loss-of-function screen identified 27 candidate cccDNA host factors, including Y box binding protein 1 (YBX1), a DNA binding protein regulating transcription and translation. Validation studies in authentic infection models revealed a robust decrease in HBV cccDNA levels following silencing, providing proof-of-concept for the importance of YBX1 in the early steps of the HBV life cycle. In patients, YBX1 expression robustly correlates with both HBV load and liver disease progression. CONCLUSION: Our cell-based reporter assay enables the discovery of HBV cccDNA host factors including YBX1 and is suitable for the characterisation of cccDNA-related host factors, antiviral targets and compounds.

8.
Nat Commun ; 12(1): 5525, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34535664

ABSTRACT

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.


Subject(s)
Drug Discovery , Liver/pathology , Models, Biological , Animals , Carcinogenesis/pathology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Chemoprevention , Cohort Studies , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Disease Models, Animal , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Hepacivirus/physiology , Hepatitis C/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Immunologic Surveillance/drug effects , Inflammation/pathology , Liver/drug effects , Liver/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice, Knockout , Nizatidine/pharmacology , Prognosis , Signal Transduction/drug effects , Transcriptome/genetics
9.
Life Sci Alliance ; 4(9)2021 09.
Article in English | MEDLINE | ID: mdl-34290079

ABSTRACT

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) world-wide. The molecular mechanisms of viral hepatocarcinogenesis are still partially understood. Here, we applied two complementary single-cell RNA-sequencing protocols to investigate HBV-HCC host cell interactions at the single cell level of patient-derived HCC. Computational analyses revealed a marked HCC heterogeneity with a robust and significant correlation between HBV reads and cancer cell differentiation. Viral reads significantly correlated with the expression of HBV-dependency factors such as HLF in different tumor compartments. Analyses of virus-induced host responses identified previously undiscovered pathways mediating viral carcinogenesis, such as E2F- and MYC targets as well as adipogenesis. Mapping of fused HBV-host cell transcripts allowed the characterization of integration sites in individual cancer cells. Collectively, single-cell RNA-Seq unravels heterogeneity and compartmentalization of both, virus and cancer identifying new candidate pathways for viral hepatocarcinogenesis. The perturbation of pro-carcinogenic gene expression even at low HBV levels highlights the need of HBV cure to eliminate HCC risk.


Subject(s)
Carcinoma, Hepatocellular/etiology , Cell Transformation, Viral , Hepatitis B virus/physiology , Hepatitis B/complications , Hepatitis B/virology , Liver Neoplasms/etiology , Adult , Aged , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Cell Differentiation , Cell Line, Tumor , Disease Susceptibility , Female , Gene Expression Profiling , Gene Expression Regulation, Viral , Hepatitis B, Chronic/complications , Hepatitis B, Chronic/virology , Humans , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Grading , RNA, Viral , Single-Cell Analysis/methods , Transcriptome , Viral Load
10.
Gut ; 70(1): 157-169, 2021 01.
Article in English | MEDLINE | ID: mdl-32217639

ABSTRACT

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.


Subject(s)
Carcinoma, Hepatocellular/prevention & control , Epigenesis, Genetic , Hepatitis C, Chronic/complications , Liver Neoplasms/prevention & control , Non-alcoholic Fatty Liver Disease/complications , Animals , Carcinoma, Hepatocellular/etiology , Disease Models, Animal , Hepatitis C, Chronic/genetics , Hepatitis C, Chronic/pathology , Humans , Liver Neoplasms/etiology , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/pathology
11.
Nat Commun ; 11(1): 2707, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32483149

ABSTRACT

Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p18/genetics , Gain of Function Mutation , Genetic Testing/methods , Genome-Wide Association Study/methods , Hepatitis B/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p18/metabolism , Gene Expression Profiling/methods , HEK293 Cells , Hep G2 Cells , Hepatitis B/metabolism , Hepatitis B/virology , Hepatitis B virus/physiology , Host Microbial Interactions , Humans , Kaplan-Meier Estimate , Liver/metabolism , Liver/pathology , Liver/virology , RNA Interference , Virus Replication/physiology
12.
Article in English | MEDLINE | ID: mdl-31710888

ABSTRACT

Polyaromatic hydrocarbons (PAH) are common pollutants of water ecosystems originating from incineration processes and contamination with mineral oil. Water solubility of PAHs is generally low; for toxicity tests with aquatic organisms, they are therefore usually dissolved in organic solvents. Here we examined the effects of a typical model PAH, phenanthrene, and a solvent, acetone, on amphipods as relevant aquatic invertebrate models. Two of these species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, are common endemics of the oligotrophic and pristine Lake Baikal, while one, Gammarus lacustris, is widespread throughout the Holarctic and inhabits smaller and more eutrophic water bodies in the Baikal area. Neither solvent nor phenanthrene caused mortality at the applied concentrations, but both substances affected gene expression in all species. Differential gene expression was more profound in the species from Lake Baikal than in the Holarctic species. Moreover, in one of the Baikal species, E. cyaneus, we found that many known components of the cellular xenobiotic detoxification system reacted to the treatments. Finally, we detected a negative relationship between changes in transcript abundances in response to the solvent and phenanthrene. This mixture effect, weaker than the impact by a single mixture component, needs further exploration.


Subject(s)
Acetone/adverse effects , Amphipoda/drug effects , Phenanthrenes/adverse effects , Transcriptome/drug effects , Water Pollutants, Chemical/adverse effects , Amphipoda/genetics , Amphipoda/physiology , Animals , Solvents/adverse effects , Species Specificity
13.
Gut ; 69(2): 380-392, 2020 02.
Article in English | MEDLINE | ID: mdl-31076402

ABSTRACT

OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.


Subject(s)
Hepacivirus/pathogenicity , Hepatitis C, Chronic/genetics , N-Acetylglucosaminyltransferases/physiology , Gene Expression Regulation/physiology , Gene Knockdown Techniques/methods , Genome-Wide Association Study/methods , Hepacivirus/physiology , Hepatitis C, Chronic/virology , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Life Cycle Stages/genetics , MicroRNAs/genetics , Morphogenesis/physiology , N-Acetylglucosaminyltransferases/genetics , Up-Regulation , Virulence/genetics
14.
Nucleic Acids Res ; 47(20): 10543-10552, 2019 11 18.
Article in English | MEDLINE | ID: mdl-31584075

ABSTRACT

With the rapid increase of sequenced metazoan mitochondrial genomes, a detailed manual annotation is becoming more and more infeasible. While it is easy to identify the approximate location of protein-coding genes within mitogenomes, the peculiar processing of mitochondrial transcripts, however, makes the determination of precise gene boundaries a surprisingly difficult problem. We have analyzed the properties of annotated start and stop codon positions in detail, and use the inferred patterns to devise a new method for predicting gene boundaries in de novo annotations. Our method benefits from empirically observed prevalances of start/stop codons and gene lengths, and considers the dependence of these features on variations of genetic codes. Albeit not being perfect, our new approach yields a drastic improvement in the accuracy of gene boundaries and upgrades the mitochondrial genome annotation server MITOS to an even more sophisticated tool for fully automatic annotation of metazoan mitochondrial genomes.


Subject(s)
Mitochondrial Proteins/genetics , Molecular Sequence Annotation/methods , Animals , Genetic Code , Genome, Mitochondrial , Mitochondrial Proteins/metabolism , Molecular Sequence Annotation/standards , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
BMC Genomics ; 20(1): 712, 2019 Sep 13.
Article in English | MEDLINE | ID: mdl-31519144

ABSTRACT

BACKGROUND: Lake Baikal is one of the oldest freshwater lakes and has constituted a stable environment for millions of years, in stark contrast to small, transient bodies of water in its immediate vicinity. A highly diverse endemic endemic amphipod fauna is found in one, but not the other habitat. We ask here whether differences in stress response can explain the immiscibility barrier between Lake Baikal and non-Baikal faunas. To this end, we conducted exposure experiments to increased temperature and the toxic heavy metal cadmium as stressors. RESULTS: Here we obtained high-quality de novo transcriptome assemblies, covering mutiple conditions, of three amphipod species, and compared their transcriptomic stress responses. Two of these species, Eulimnogammarus verrucosus and E. cyaneus, are endemic to Lake Baikal, while the Holarctic Gammarus lacustris is a potential invader. CONCLUSIONS: Both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles. G. lacustris reacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions.


Subject(s)
Amphipoda/genetics , Amphipoda/physiology , Lakes , Stress, Physiological/genetics , Transcriptome , Amphipoda/drug effects , Animals , Cadmium/toxicity , Geography , Heat-Shock Response/genetics , Species Specificity , Stress, Physiological/drug effects , Transcriptome/drug effects
16.
Gastroenterology ; 157(2): 537-551.e9, 2019 08.
Article in English | MEDLINE | ID: mdl-30978357

ABSTRACT

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.


Subject(s)
Hepacivirus/pathogenicity , Hepatitis C, Chronic/pathology , Hepatocytes/pathology , Liver/pathology , Animals , Cell Line, Tumor , Datasets as Topic , Disease Models, Animal , Gene Expression Profiling , Glucose/metabolism , Hepatitis C, Chronic/metabolism , Hepatitis C, Chronic/virology , Hepatocytes/transplantation , Hepatocytes/virology , Humans , Liver/cytology , Liver/virology , Metabolomics , Mice , Peroxisomes/metabolism , Peroxisomes/pathology , Proteomics , STAT3 Transcription Factor/metabolism , Transplantation Chimera
17.
Cell Rep ; 26(12): 3416-3428.e5, 2019 03 19.
Article in English | MEDLINE | ID: mdl-30893612

ABSTRACT

In eukaryotes, tRNAs are transcribed in the nucleus and exported to the cytosol, where they deliver amino acids to ribosomes for protein translation. This nuclear-cytoplasmic movement was believed to be unidirectional. However, active shuttling of tRNAs, named tRNA retrograde transport, between the cytosol and nucleus has been discovered. This pathway is conserved in eukaryotes, suggesting a fundamental function; however, little is known about its role in human cells. Here we report that, in human cells, oxidative stress triggers tRNA retrograde transport, which is rapid, reversible, and selective for certain tRNA species, mostly with shorter 3' ends. Retrograde transport of tRNASeC, which promotes translation of selenoproteins required to maintain homeostatic redox levels in cells, is highly efficient. tRNA retrograde transport is regulated by the integrated stress response pathway via the PERK-REDD1-mTOR axis. Thus, we propose that tRNA retrograde transport is part of the cellular response to oxidative stress.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Oxidative Stress , RNA Transport , RNA, Transfer/metabolism , Signal Transduction , HeLa Cells , Humans , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , eIF-2 Kinase/metabolism
18.
Gastroenterology ; 156(8): 2313-2329.e7, 2019 06.
Article in English | MEDLINE | ID: mdl-30836093

ABSTRACT

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.


Subject(s)
Antiviral Agents/therapeutic use , Carcinoma, Hepatocellular/virology , Hepatitis C, Chronic/pathology , Liver Neoplasms/genetics , Liver Neoplasms/virology , Adult , Animals , Carcinoma, Hepatocellular/genetics , Case-Control Studies , Cohort Studies , Disease Models, Animal , Epigenesis, Genetic , Europe , Female , Gene Expression Regulation, Neoplastic , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Humans , Japan , Liver Neoplasms/pathology , Male , Mice , Mice, SCID , Random Allocation , Sustained Virologic Response
19.
RNA Biol ; 15(1): 144-155, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29099323

ABSTRACT

Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.


Subject(s)
DNA-Directed RNA Polymerases/chemistry , RNA Nucleotidyltransferases/chemistry , RNA, Transfer/genetics , RNA/chemistry , Adaptation, Physiological/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence/genetics , Bacillales/chemistry , Bacillales/genetics , Catalytic Domain/genetics , Cold Temperature , DNA-Directed RNA Polymerases/genetics , Enzyme Stability , Nucleic Acid Conformation , Nucleotides/genetics , RNA/biosynthesis , RNA/genetics , RNA Nucleotidyltransferases/genetics , RNA, Transfer/chemistry , Stress, Physiological/genetics
20.
J Pathol ; 243(2): 242-254, 2017 10.
Article in English | MEDLINE | ID: mdl-28727142

ABSTRACT

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Subject(s)
Colorectal Neoplasms/genetics , Mutation/genetics , Antigens, Neoplasm/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/immunology , Gene Expression/genetics , Genes, Neoplasm/genetics , Genome, Human/genetics , Humans , Immunity, Cellular , Phenotype , Recurrence , Transcriptome/genetics , Tumor Escape/genetics , Tumor Escape/immunology
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