Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA–DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.

Summary Hepatocellular carcinoma (HCC) is a deadly tumour whose causative agents are generally well known, but whose pathogenesis remains poorly understood. Nevertheless, key genetic alterations are emerging from a heterogeneous molecular landscape, providing information on the tumorigenic process from initiation to progression. Among these molecular alterations, those that affect epigenetic processes are increasingly recognised as contributing to carcinogenesis from preneoplastic stages. The epigenetic machinery regulates gene expression through intertwined and partially characterised circuits involving chromatin remodelers, covalent DNA and histone modifications, and dedicated proteins reading these modifications. In this review, we summarise recent findings on HCC epigenetics, focusing mainly on changes in DNA and histone modifications and their carcinogenic implications. We also discuss the potential drugs that target epigenetic mechanisms for HCC treatment, either alone or in combination with current therapies, including immunotherapies.

ObjectiveHepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis.DesignG9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology.ResultsG9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity.ConclusionsDual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

Liver fibrosis is an essential component of chronic liver disease (CLD) and hepatocarcinogenesis. The fibrotic stroma is a consequence of sustained liver damage combined with exacerbated extracellular matrix (ECM) accumulation. In this context, activation of hepatic stellate cells (HSCs) plays a key role in both initiation and perpetuation of fibrogenesis. These cells suffer profound remodeling of gene expression in this process. This review is focused on the epigenetic alterations participating in the transdifferentiation of HSCs from the quiescent to activated state. Recent advances in the field of DNA methylation and post-translational modifications (PTM) of histones (acetylation and methylation) patterns are discussed here, together with altered expression and activity of epigenetic remodelers. We also consider recent advances in translational approaches, including the use of epigenetic marks as biomarkers and the promising antifibrotic properties of epigenetic drugs that are currently being used in patients.

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Ayudas para apoyar a grupos de investigación del sistema Universitario Vasco (IT971-16);

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy.

Chronic liver diseases (CLD) represent a worldwide health problem. While CLDs may have diverse etiologies, a common pathogenic denominator is the presence of liver fibrosis. Cirrhosis, the end-stage of CLD, is characterized by extensive fibrosis and is markedly associated with the development of hepatocellular carcinoma. The most important event in hepatic fibrogenesis is the activation of hepatic stellate cells (HSC) following liver injury. Activated HSCs acquire a myofibroblast-like phenotype becoming proliferative, fibrogenic, and contractile cells. While transient activation of HSCs is part of the physiological mechanisms of tissue repair, protracted activation of a wound healing reaction leads to organ fibrosis. The phenotypic changes of activated HSCs involve epigenetic mechanisms mediated by non-coding RNAs (ncRNA) as well as by changes in DNA methylation and histone modifications. During CLD these epigenetic mechanisms become deregulated, with alterations in the expression and activity of epigenetic modulators. Here we provide an overview of the epigenetic alterations involved in fibrogenic HSCs transdifferentiation with particular focus on histones acetylation changes. We also discuss recent studies supporting the promising therapeutic potential of histone deacetylase inhibitors in liver fibrosis.