Publications by authors named "Jesus M Banales"

150 Publications

Analysis of SARS-CoV-2 RT-qPCR Ct values vis-à-vis anti-SARS-CoV-2 antibodies from a high incidence region.

Int J Infect Dis 2021 Jul 14. Epub 2021 Jul 14.

Institute of Clinical Chemistry, University Hospital Schleswig-Holstein, Kiel/Lübeck, Germany.

Objectives: To examine the relationship between antibody status and Ct values and the prognostic value of the latter for COVID-19 patients, and the inter-assay comparability of SARS-CoV-2 Ct values.

Methods: In 347 COVID-19 inpatients, SARS-CoV-2 Ct values (via RT-qPCR) on admission were compared in between two assays and correlated with the antibody response (in the course of the disease), the clinical course and the time since onset of symptoms.

Results: Ct values for two of three target genes showed significant differences between the two assays used (p=0.012 and p<0.0001). Ct values were significantly higher for antibody positive patients (p<0.0001). Ct values were positively correlated with the amount of time since onset of symptoms (R: 0.332-0.363; p<0.001). Patients with fatal outcomes showed higher viral loads than survivors (p<0.0001).

Conclusions: Ct values depend strongly on the assay used and the target gene examined and should not be used as quantitative values to guide therapeutic or diagnostic decisions. The inverse association between the antibody status and the viral load suggests that antibodies contribute to the elimination of the virus, independent of the outcome, which is influenced by the viral load on admission and might depend more strongly on other parts of the immune response.
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http://dx.doi.org/10.1016/j.ijid.2021.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278831PMC
July 2021

Adiponectin, Leptin, and IGF-1 Are Useful Diagnostic and Stratification Biomarkers of NAFLD.

Front Med (Lausanne) 2021 23;8:683250. Epub 2021 Jun 23.

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, < 0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; < 0.01). Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.
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http://dx.doi.org/10.3389/fmed.2021.683250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260936PMC
June 2021

Next-Generation Biomarkers for Cholangiocarcinoma.

Cancers (Basel) 2021 Jun 28;13(13). Epub 2021 Jun 28.

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), 20014 San Sebastian, Spain.

The increasing mortality rates of cholangiocarcinoma (CCA) registered during the last decades are, at least in part, a result of the lack of accurate non-invasive biomarkers for early disease diagnosis, making the identification of patients who might benefit from potentially curative approaches (i.e., surgery) extremely challenging. The obscure CCA pathogenesis and associated etiological factors, as well as the lack of symptoms in patients with early tumor stages, highly compromises CCA identification and to predict tumor development in at-risk populations. Currently, CCA diagnosis is accomplished by the combination of clinical/biochemical features, radiological imaging and non-specific serum tumor biomarkers, although a tumor biopsy is still needed to confirm disease diagnosis. Furthermore, prognostic and predictive biomarkers are still lacking and urgently needed. During the recent years, high-throughput omics-based approaches have identified novel circulating biomarkers (diagnostic and prognostic) that might be included in large, international validation studies in the near future. In this review, we summarize and discuss the most recent advances in the field of biomarker discovery in CCA, providing new insights and future research directions.
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http://dx.doi.org/10.3390/cancers13133222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269024PMC
June 2021

Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis.

Mol Metab 2021 Jun 18;53:101275. Epub 2021 Jun 18.

Liver Disease Lab, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), 48160, Derio, Bizkaia, Spain. Electronic address:

Objective: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD.

Methods: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8.

Results: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models CONCLUSIONS: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.
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http://dx.doi.org/10.1016/j.molmet.2021.101275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8280515PMC
June 2021

YAP Accelerates Notch Driven Cholangiocarcinogenesis via mTORC1 in Mice.

Am J Pathol 2021 Jun 12. Epub 2021 Jun 12.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California; School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China. Electronic address:

Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignant neoplasm with limited therapeutic options. Previous studies have found that Notch1 overexpression alone suffices to induce iCCA in the mouse, albeit after long latency. This study found that activation of the Yes-associated protein (Yap) proto-oncogene occurs during Notch1-driven iCCA progression. After co-expressing activated Notch1 intracellular domain (Nicd) and Yap (YapS127A) in the mouse liver, rapid iCCA formation and progression occurred in Nicd/Yap mice. Mechanistically, an increased expression of amino acid transporters and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway was detected in Nicd/Yap mouse liver tumors. Significantly, the genetic deletion of Raptor, the major mTORC1 component, completely suppressed iCCA development in Nicd/Yap mice. Elevated expression of Notch1, YAP, amino acid transporters, and members of the mTORC1 pathway was also detected ubiquitously in a collection of human iCCA specimens. Their levels were associated with a poor patient outcome. This study demonstrates that Notch and YAP concomitant activation is frequent in human cholangiocarcinogenesis. Notch and YAP synergize to promote iCCA formation by activating the mTORC1 pathway.
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http://dx.doi.org/10.1016/j.ajpath.2021.05.017DOI Listing
June 2021

Optimizing the use of twitter for research dissemination: The "Three Facts and a Story" randomized-controlled trial.

J Hepatol 2021 Aug 29;75(2):271-274. Epub 2021 May 29.

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute - Donostia University Hospital -, University of the Basque Country (UPV/EHU), CIBERehd, Ikerbasque, San Sebastian, Spain.

Background & Aims: Published research promoted on twitter reaches more readers. Tweets with graphics are more engaging than those without. However, data are limited regarding how to optimize multimedia tweets for engagement.

Methods: The "Three facts and a Story" trial is a randomized-controlled trial comparing a tweet featuring a graphical abstract to paired tweets featuring the personal motivations behind the research and a summary of the findings. Fifty-four studies published by the Journal of Hepatology were randomized at the time of online publication. The primary endpoint was assessed at 28-days from online publication with a primary outcome of full-text downloads from the website. Secondary outcomes included page views and twitter engagement including impressions, likes, and retweets.

Results: Overall, 31 studies received standard tweets and 23 received story tweets. Five studies were randomized to story tweets but crossed over to standard tweets for lack of author participation. Most papers tweeted were original articles (94% standard, 91% story) and clinical topics (55% standard, 61% story). Story tweets were associated with a significant increase in the number of full text downloads, 51 (34-71) vs. 25 (13-41), p = 0.002. There was also a non-significant increase in the number of page views. Story tweets generated an average of >1,000 more impressions than standard tweets (5,388 vs. 4,280, p = 0.002). Story tweets were associated with a similar number of retweets, and a non-significant increase in the number of likes.

Conclusion: Tweets featuring the authors and their motivations may increase engagement with published research.
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http://dx.doi.org/10.1016/j.jhep.2021.05.020DOI Listing
August 2021

Anti-miR-518d-5p overcomes liver tumor cell death resistance through mitochondrial activity.

Cell Death Dis 2021 May 28;12(6):555. Epub 2021 May 28.

Liver Disease Laboratory, Precision Medicine and Metabolism Laboratory, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain.

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient's overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.
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http://dx.doi.org/10.1038/s41419-021-03827-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163806PMC
May 2021

Reply to (20-1119.R1) Surgery for advanced intrahepatic cholangiocarcinoma warrants further investigation.

Hepatology 2021 May 17. Epub 2021 May 17.

Medical Oncology / Institute of Cancer Sciences, The Christie NHS Foundation Trust / University of poManchester, Manchester, United Kingdom.

We thank Jansson & Sparrelid for their Letter to the Editor(1)regarding our publication(2). We stated that "based on our results, patients with liver metastases should not be offered therapeutic strategies suitable for early stages, in view of poor survival". Jansson(1) argues that "the role of surgery in multiple intrahepatic cholangiocarcinoma (iCCA) should not be dismissed without further analysis"; similar to Zhang's views(3).
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http://dx.doi.org/10.1002/hep.31904DOI Listing
May 2021

Unscrambling a novel pathogenic role for interleukin-20 in acute hepatitis and bacterial infection: A double-edged sword?

J Hepatol 2021 Jul 10;75(1):22-24. Epub 2021 May 10.

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), ISCIII, Madrid, Spain; Ikerbasque, Basque Foundation for Science, Bilbao, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2021.03.011DOI Listing
July 2021

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells.

Hepatology 2021 May 6. Epub 2021 May 6.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Background And Aims: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA.

Approach And Results: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA.

Conclusions: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment.
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http://dx.doi.org/10.1002/hep.31888DOI Listing
May 2021

Applications of organoids in regenerative medicine: a proof-of-concept for biliary injury.

Nat Rev Gastroenterol Hepatol 2021 Jun;18(6):371-372

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute - Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain.

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http://dx.doi.org/10.1038/s41575-021-00459-9DOI Listing
June 2021

Definite and indeterminate nonalcoholic steatohepatitis share similar clinical features and prognosis: A longitudinal study of 1893 biopsy-proven nonalcoholic fatty liver disease subjects.

Liver Int 2021 Apr 25. Epub 2021 Apr 25.

Digestive Department, Hospital Universitario San Cecilio, Granada, Spain.

Background And Aim: Histological score systems may not fully capture the essential nonalcoholic steatohepatitis (NASH) features, which is one of the leading causes of screening failure in clinical trials. We assessed the NASH distribution and its components across the fibrosis stages and their impact on the prognosis and their relationship with the concept of metabolic-associated fatty liver disease (MAFLD).

Methods: Spanish multicenter study including 1893 biopsy-proven nonalcoholic fatty liver disease (NAFLD) patients from HEPAmet registry. NASH was diagnosed by NAS score ≥4 (including steatosis, ballooning and lobular inflammation) and fibrosis by Kleiner score. The presence of MAFLD was determined. Progression to cirrhosis, first episode of decompensated cirrhosis and death were collected during the follow-up (4.7 ± 3.8 years).

Results: Fibrosis was F0 34.3% (649/1893), F1 27% (511/1893), F2 16.5% (312/1893), F3 15% (284/1893) and F4 7.2% (137/1893). NASH diagnosis 51.9% (982/1893), and its individual components (severe steatosis, ballooning and lobular inflammation), increased from F0 (33.6%) to F2 (68.6%), and decreased significantly in F4 patients (51.8%) (P = .0001). More than 70% of non-NASH patients showed some inflammatory activity (ballooning or lobular inflammation), showing a similar MAFLD rate than NASH (96.2% [945/982] vs. 95.2% [535/562]) and significantly higher than nonalcoholic fatty liver (NAFL) subjects (89.1% [311/349]) (P < .0001). Progression to cirrhosis was similar between NASH (9.5% [51/539]) and indeterminate NASH (7.9% [25/316]), and higher than steatosis (5% [14/263]) (logRank 8.417; P = .015). Death and decompensated cirrhosis were similar between these.

Conclusions: The prevalence of steatohepatitis decreased in advanced liver disease. However, most of these patients showed some inflammatory activity histologically and had metabolic disturbances. These findings should be considered in clinical trials whose main aim is to prevent cirrhosis progression and complications, liver transplant and death.
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http://dx.doi.org/10.1111/liv.14898DOI Listing
April 2021

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted.

J Hepatol 2021 Aug 20;75(2):363-376. Epub 2021 Apr 20.

University of Navarra, Centre for Applied Medical Research, Program in Solid Tumours, Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, Pamplona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC, Instituto de Salud Carlos III), Madrid, Spain; University of Navarra, Department of Pathology, Anatomy and Physiology, Pamplona, Spain. Electronic address:

Background & Aims: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA.

Methods: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Follow-up RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition.

Results: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss.

Conclusions: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies.

Lay Summary: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.
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http://dx.doi.org/10.1016/j.jhep.2021.03.028DOI Listing
August 2021

E2F1 and E2F2-Mediated Repression of CPT2 Establishes a Lipid-Rich Tumor-Promoting Environment.

Cancer Res 2021 Jun 26;81(11):2874-2887. Epub 2021 Mar 26.

Department of Gastroenterology and Hepatology, Marqués de Valdecilla University Hospital, Santander, Spain.

Lipid metabolism rearrangements in nonalcoholic fatty liver disease (NAFLD) contribute to disease progression. NAFLD has emerged as a major risk for hepatocellular carcinoma (HCC), where metabolic reprogramming is a hallmark. Identification of metabolic drivers might reveal therapeutic targets to improve HCC treatment. Here, we investigated the contribution of transcription factors E2F1 and E2F2 to NAFLD-related HCC and their involvement in metabolic rewiring during disease progression. In mice receiving a high-fat diet (HFD) and diethylnitrosamine (DEN) administration, and expressions were increased in NAFLD-related HCC. In human NAFLD, E2F1 and E2F2 levels were also increased and positively correlated. and mice were resistant to DEN-HFD-induced hepatocarcinogenesis and associated lipid accumulation. Administration of DEN-HFD in and mice enhanced fatty acid oxidation (FAO) and increased expression of , an enzyme essential for FAO, whose downregulation is linked to NAFLD-related hepatocarcinogenesis. These results were recapitulated following knockdown in liver, and overexpression of elicited opposing effects. E2F2 binding to the promoter was enhanced in DEN-HFD-administered mouse livers compared with controls, implying a direct role for E2F2 in transcriptional repression. In human HCC, and expressions inversely correlated with expression. Collectively, these results indicate that activation of the E2F1-E2F2-CPT2 axis provides a lipid-rich environment required for hepatocarcinogenesis. SIGNIFICANCE: These findings identify E2F1 and E2F2 transcription factors as metabolic drivers of hepatocellular carcinoma, where deletion of just one is sufficient to prevent disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2874/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2052DOI Listing
June 2021

Clinical correlates of anti-SARS-CoV-2 antibody profiles in Spanish COVID-19 patients from a high incidence region.

Sci Rep 2021 02 23;11(1):4363. Epub 2021 Feb 23.

Institute of Clinical Chemistry, University Hospital Schleswig-Holstein, Kiel/Lübeck, Germany.

Laboratory testing for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of two pillars: the detection of viral RNA via rt-PCR as the diagnostic gold standard in acute cases, and the detection of antibodies against SARS-CoV-2. However, concerning the latter, questions remain about their diagnostic and prognostic value and it is not clear whether all patients develop detectable antibodies. We examined sera from 347 Spanish COVID-19 patients, collected during the peak of the epidemic outbreak in Spain, for the presence of IgA and IgG antibodies against SARS-CoV-2 and evaluated possible associations with age, sex and disease severity (as measured by duration of hospitalization, kind of respiratory support, treatment in ICU and death). The presence and to some degree the levels of anti-SARS-CoV-2 antibodies depended mainly on the amount of time between onset of symptoms and the collection of serum. A subgroup of patients did not develop antibodies at the time of sample collection. Compared to the patients that did, no differences were found. The presence and level of antibodies was not associated with age, sex, duration of hospitalization, treatment in the ICU or death. The case-fatality rate increased exponentially with older age. Neither the presence, nor the levels of anti-SARS-CoV-2 antibodies served as prognostic markers in our cohort. This is discussed as a possible consequence of the timing of the sample collection. Age is the most important risk factor for an adverse outcome in our cohort. Some patients appear not to develop antibodies within a reasonable time frame. It is unclear, however, why that is, as these patients differ in no respect examined by us from those who developed antibodies.
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http://dx.doi.org/10.1038/s41598-021-83969-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902674PMC
February 2021

Targeted therapies for extrahepatic cholangiocarcinoma: preclinical and clinical development and prospects for the clinic.

Expert Opin Investig Drugs 2021 Apr 23;30(4):377-388. Epub 2021 Feb 23.

Department of Molecular Medicine (DMM), University of Padua, Padua. Italy.

Until recently, cholangiocarcinoma (CCA) was a largely overlooked disease, and among CCAs, extrahepatic CCA (eCCA) was even more neglected. Despite the growing impact of molecularly targeted therapies and immunotherapy, prognosis of eCCA is dismal. Therefore, unraveling the complex molecular landscape of eCCA has become an urgent need. Deep phenotyping studies have revealed that eCCA is a heterogeneous tumor, harboring specific alterations categorizable into four classes, 'Mesenchymal', 'Proliferation', 'Immune', 'Metabolic'. Molecular alterations convey the activation of several pro-oncogenic pathways, where either actionable drivers or outcome predictors can be identified. We offer insights on perturbed pathways, molecular profiling, and actionable targets in eCCA and present a perspective on the potential stepping-stones to future progress. A systematic literature search in PubMed/ClinicalTrials.gov websites was performed by authors from different disciplines according to their specific topic knowledge to identify the newest and most relevant advances in precision medicine of eCCA. eCCA is a distinct entity with unique features in terms of molecular classes, oncogenic drivers, and tumor microenvironment. Since more prevalent mutations are currently undruggable, and immunotherapy can be offered only to a minority of patients, international collaborations are instrumental to improve the understanding of the molecular underpins of this disease.
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http://dx.doi.org/10.1080/13543784.2021.1880564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194059PMC
April 2021

Reply to "Does multiple intrahepatic cholangiocarcinoma worsen prognosis as "M1" stage?"; multiple primaries vs liver metastases.

Hepatology 2021 Feb 7. Epub 2021 Feb 7.

Medical Oncology / Institute of Cancer Sciences, The Christie NHS Foundation Trust / University of Manchester, Manchester, United Kingdom.

We thank Zhang and colleagues for their Letter to the Editor (1) regarding our work (2). Our manuscript ("Liver Metastases of Intrahepatic Cholangiocarcinoma: Implications for an Updated Staging System"(2)), suggested changes to the American Joint Committee on Cancer (AJCC) staging classification for intrahepatic cholangiocarcinoma (iCCA), by classifying "liver metastases" as stage IV rather than stage II/III in the absence/presence of lymph node metastases, respectively, as per AJCC v.8 (3).
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http://dx.doi.org/10.1002/hep.31740DOI Listing
February 2021

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease.

Gut 2020 Dec 24. Epub 2020 Dec 24.

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

Objective: Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD.

Design: RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or -deficient ( ) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks.

Results: RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in mice. Furthermore, 3 deficiency hampered tumourigenesis. Intriguingly, mice displayed increased body weight gain, while lipidomics showed that deletion of shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis.

Conclusion: Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.
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http://dx.doi.org/10.1136/gutjnl-2020-321767DOI Listing
December 2020

Melatonin Protects Cholangiocytes from Oxidative Stress-Induced Proapoptotic and Proinflammatory Stimuli via miR-132 and miR-34.

Int J Mol Sci 2020 Dec 18;21(24). Epub 2020 Dec 18.

Department of Medical Biology, Pomeranian Medical University, 71-111 Szczecin, Poland.

Biosynthesis of melatonin by cholangiocytes is essential for maintaining the function of biliary epithelium. However, this cytoprotective mechanism appears to be impaired in primary biliary cholangitis (PBC). MiR-132 has emerged as a mediator of inflammation in chronic liver diseases. The effect of melatonin on oxidative stress and bile acid-induced apoptosis was also examined in cholangiocyes overexpressing miR506, as a PBC-like cellular model. In PBC patients the serum levels of melatonin were found increased in comparison to healthy controls. Whereas, in cholangiocytes within cirrhotic PBC livers the melatonin biosynthetic pathway was substantially suppressed even though the expressions of melatonin rate-limiting enzyme aralkylamine N-acetyltransferase (AANAT), and CK-19 (marker of cholangiocytes) were enhanced. In cholangiocytes exposed to mitochondrial oxidative stress melatonin decreased the expression of proapoptotic stimuli (PTEN, Bax, miR-34), which was accompanied by the inhibition of a pivotal mediator of inflammatory response Nf-κB-p65 and the activation of antiapoptotic signaling (miR-132, Bcl2). Similarly, melatonin reduced bile acid-induced proapoptotic caspase 3 and Bim levels. In summary, the insufficient hepatic expression of melatonin in PBC patients may predispose cholangiocytes to oxidative stress-related damage. Melatonin, via epigenetic modulation, was able to suppress NF-κB signaling activation and protect against biliary cells apoptotic signaling.
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http://dx.doi.org/10.3390/ijms21249667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766218PMC
December 2020

Primary biliary cholangitis: pathogenic mechanisms.

Curr Opin Gastroenterol 2021 Mar;37(2):91-98

Unit of Medical Training, School of Medicine, University of Navarra, Pamplona, Spain.

Purpose Of Review: Primary biliary cholangitis (PBC) is characterized by autoimmune damage of intrahepatic bile ducts associated with a loss of tolerance to mitochondrial antigens. PBC etiopathogenesis is intriguing because of different perplexing features, namely: a) although mitochondria are present in all cell types and tissues, the damage is mainly restricted to biliary epithelial cells (BECs); b) despite being an autoimmune disorder, it does not respond to immunosuppressive drugs but rather to ursodeoxycholic acid, a bile salt that induces HCO3- rich choleresis; c) the overwhelming female preponderance of the disease remains unexplained. Here we present an etiopathogenic view of PBC which sheds light on these puzzling facts of the disease.

Recent Findings: PBC develops in patients with genetic predisposition to autoimmunity in whom epigenetic mechanisms silence the Cl-/HCO3- exchanger AE2 in both cholangiocytes and lymphoid cells. Defective AE2 function can produce BECs damage as a result of decreased biliary HCO3- secretion with disruption of the protective alkaline umbrella that normally prevents the penetration of toxic apolar bile salts into cholangiocytes. AE2 dysfunction also causes increased intracellular pH (pHi) in cholangiocytes, leading to the activation of soluble adenylyl cyclase, which sensitizes BECs to bile salt-induced apoptosis. Recently, mitophagy was found to be inhibited by cytosolic alkalization and stimulated by acidification. Accordingly, we propose that AE2 deficiency may disturb mitophagy in BECs, thus, promoting the accumulation of defective mitochondria, oxidative stress and presentation of mitochondrial antigens to the immune cells. As women possess a more acidic endolysosomal milieu than men, mitophagy might be more affected in women in an AE2-defective background. Apart from affecting BECs function, AE2 downregulation in lymphocytes may also contribute to alter immunoregulation facilitating autoreactive T-cell responses.

Summary: PBC can be considered as a disorder of Cl-/HCO3- exchange in individuals with genetic predisposition to autoimmunity.
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http://dx.doi.org/10.1097/MOG.0000000000000703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879833PMC
March 2021

Dual Pharmacological Targeting of HDACs and PDE5 Inhibits Liver Disease Progression in a Mouse Model of Biliary Inflammation and Fibrosis.

Cancers (Basel) 2020 Dec 13;12(12). Epub 2020 Dec 13.

Program of Hepatology, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated -KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor β (TGFβ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.
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http://dx.doi.org/10.3390/cancers12123748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763137PMC
December 2020

Pathogenesis of Cholangiocarcinoma.

Annu Rev Pathol 2021 01 2;16:433-463. Epub 2020 Dec 2.

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), 20014 San Sebastian, Spain; email:

Cholangiocarcinoma (CCA) encompasses a group of malignancies that can arise at any point in the biliary tree. Although considered a rare cancer, the incidence of CCA is increasing globally. The silent and asymptomatic nature of these tumors, particularly in their early stages, in combination with their high aggressiveness, intra- and intertumor heterogeneity, and chemoresistance, significantly compromises the efficacy of current therapeutic options, contributing to a dismal prognosis. During the last few years, increasing efforts have been made to unveil the etiologies and pathogenesis of these tumors and to develop more effective therapies. In this review, we summarize current findings in the field of CCA, mainly focusing on the mechanisms of pathogenesis, cells of origin, genomic and epigenetic abnormalities, molecular alterations, chemoresistance, and therapies.
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http://dx.doi.org/10.1146/annurev-pathol-030220-020455DOI Listing
January 2021

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl-Induced Liver Fibrosis.

Int J Mol Sci 2020 Nov 27;21(23). Epub 2020 Nov 27.

Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), 48160 Derio, Bizkaia, Spain.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process "protein polyubiquitination" is enriched after CCl-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (Ub mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.
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http://dx.doi.org/10.3390/ijms21239043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729774PMC
November 2020

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

Hepatology 2021 Jun;73(6):2380-2396

Instituto de Investigaciones Sanitarias de Navarra IdiSNA, Pamplona, Spain.

Background And Aims: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors.

Approach And Results: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl treatment (Jnk + DEN + CCl mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in Jnk + DEN + CCl mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status.

Conclusions: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.
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http://dx.doi.org/10.1002/hep.31642DOI Listing
June 2021

Neutrophils interact with cholangiocytes to cause cholestatic changes in alcoholic hepatitis.

Gut 2021 Feb 19;70(2):342-356. Epub 2020 Nov 19.

Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut, USA

Background & Objectives: Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease.

Design: Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings.

Results: Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin β1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes.

Conclusions: Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.
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http://dx.doi.org/10.1136/gutjnl-2020-322540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906004PMC
February 2021

Novel GANAB variants associated with polycystic liver disease.

Orphanet J Rare Dis 2020 10 23;15(1):302. Epub 2020 Oct 23.

Department of Gastroenterology and Hepatology, Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Background: Polycystic liver disease (PLD) is an inherited disorder characterized by numerous cysts in the liver. Autosomal dominant polycystic kidney and liver disease (ADPKD and ADPLD, respectively) have been linked to pathogenic GANAB variants. GANAB encodes the α-subunit of glucosidase II (GIIα). Here, we report the identification of novel GANAB variants in an international cohort of patients with the primary phenotype of PLD using molecular inversion probe analysis.

Results: Five novel GANAB variants were identified in a cohort of 625 patients with ADPKD or ADPLD. In silico analysis revealed that these variants are likely to affect functionally important domains of glucosidase II α-subunit. Missense variant c.1835G>C p.(Arg612Pro) was predicted to disrupt the structure of the active site of the protein, likely reducing its activity. Frameshift variant c.687delT p.(Asp229Glufs*60) introduces a premature termination codon predicted to have no activity. Two nonsense variants (c.2509C>T; p.(Arg837*), and c.2656C>T; p.(Arg886*)) and splice variant c.2002+1G>C, which causes aberrant pre-mRNA splicing and affecting RNA processing, result in truncated proteins and are predicted to cause abnormal binding of α- and β-subunits of glucosidase II, thus affecting its enzymatic activity. Analysis of glucosidase II subunits in cell lines shows expression of a truncated GIIα protein in cells with c.687delT, c.2509C>T, c.2656C>T, and c.2002+1G>C variants. Incomplete colocalization of the subunits was present in cells with c.687delT or c.2002+1G>C variants. Other variants showed normal distribution of GIIα protein.

Conclusions: We identified five novel GANAB variants associated with PLD in both ADPKD and ADPLD patients supporting a common pathway in cystogenesis. These variants may lead to decreased or complete loss of enzymatic activity of glucosidase II which makes GANAB a candidate gene to be screened in patients with an unknown genetic background.
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http://dx.doi.org/10.1186/s13023-020-01585-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585303PMC
October 2020

Liver Metastases of Intrahepatic Cholangiocarcinoma: Implications for an Updated Staging System.

Hepatology 2021 Jun;73(6):2311-2325

Medical Oncology/Institute of Cancer Sciences, The Christie NHS Foundation Trust/University of Manchester, Manchester, United Kingdom.

Background And Aims: Intrahepatic cholangiocarcinoma (iCCA) with liver metastases is perceived to have a poor prognosis, but the American Joint Committee on Cancer (AJCC) classifies them as early stage in the absence of lymph nodes or extrahepatic spread.

Approach And Results: Patients with iCCA from the European Network for the Study of Cholangiocarcinoma (ENS-CCA) and Surveillance, Epidemiology, and End Results (SEER) registries with survival/staging (AJCC v.7) data were eligible. Modified staging was used (mAJCC v.7): group A: stages I-III (excluding T2bN0); group B: stage IVa (excluding T2bN1M0); group C: liver metastases (T2bN0/1); and group D: stage IVb (extrahepatic metastases). Survival analysis (Kaplan-Meier and Cox regression) was performed in an ENS-CCA training cohort (TC) and findings internally (ENS-CCA iVC) and externally (SEER) validated. The aim was to assess whether liver metastases (group C) had a shorter survival compared to other early stages (group A) to propose a modified version of AJCC v.8 (mAJCC v.8). A total of 574 and 4,171 patients from the ENS-CCA and SEER registries were included. Following the new classification, 19.86% and 17.31% of patients from the ENS-CCA and SEER registries were reclassified into group C, respectively. In the ENS-CCA TC, multivariable Cox regression was adjusted for obesity (p = 0.026) and performance status (P < 0.001); patients in group C (HR, 2.53; 95% CI, 1.18-5.42; P = 0.017) had a higher risk of death (vs. group A). Findings were validated in the ENS-CCA iVC (HR, 2.93; 95% CI, 2.04-4.19; P < 0.001) and in the SEER registry (HR, 1.88; 95% CI, 1.68-2.09; P < 0.001).

Conclusions: iCCA with liver metastases has a worse outcome than other early stages of iCCA. Given that AJCC v.8 does not take this into consideration, a modification of AJCC v.8 (mAJCC v.8), including "liver metastases: multiple liver lesions, with or without vascular invasion" as an "M1a stage," is suggested.
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http://dx.doi.org/10.1002/hep.31598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252018PMC
June 2021

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-β Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

Gastroenterology 2021 02 12;160(3):889-905.e10. Epub 2020 Oct 12.

Division of Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, Minnesota; Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota; Center for Cell Signaling in Gastroenterology Mayo Clinic and Foundation, Rochester, Minnesota. Electronic address:

Background & Aims: Transforming growth factor β (TGFβ) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFβ that mediate transcription of fibrogenic signals.

Methods: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFβ stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2 and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models.

Results: TGFβ stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFβ-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFβ. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFβ-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFβ-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis.

Conclusions: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.
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http://dx.doi.org/10.1053/j.gastro.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878301PMC
February 2021

Characterizing the Heterogeneity of Liver Cell Populations Under a NASH-Related Hepatotoxicant Using Single-Nuclei RNA Sequencing.

Cell Mol Gastroenterol Hepatol 2021 5;11(1):294-296. Epub 2020 Oct 5.

Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country, San Sebastian, Spain; National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; Ikerbasque, Basque Foundation for Science, Bilbao, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2020.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768554PMC
July 2021