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Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB., Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies., Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth., Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB., Lay Summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer., Competing Interests: Conflict of interest Prof. Josep M. Llovet is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and consulting fees from Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Eli Lilly, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Ltd, Fortress Biotech, Sprink Pharmaceuticals and Nucleix and CANFITE. CA has a research contract with CHIOME Biosciences Inc. The other authors report no conflicts of interest in this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus infection is a major cause of liver diseases including hepatocellular carcinoma (HCC). The viral regulatory protein HBx is essential for viral replication and has been involved in the development of HCC. Recently, we characterized a subset of HCCs that replicate HBV. Our aim was to characterize HBx encoded by the full-length HBV DNA (cccDNA) in HCC and non-HCC liver. HBx genes were amplified and sequenced from eight paired HCC and non-HCC tissues in which HBV cccDNA and pgRNA were both present. Sequence analyses identified twelve amino acid positions mutated between HCC and non-HCC liver, and detected in at least three cases. We next assessed the impact of these mutations on HBx function by testing their transcriptional activity. We examined their ability to rescue the transcription of HBV virus deficient for HBx in differentiated HepaRG cells and to induce Smc5/6 degradation, which is mandatory for viral replication. We assessed their capacity to activate a CREB-dependent reporter. Finally we analyzed their growth suppressive activity using colony formation assays. Our results showed that most HBx variants isolated from HCC retain their ability to support HBV cccDNA transcription and to degrade Smc5/6. Strikingly, HCC specific HBx variants are impaired in their antiproliferative activity, which may be detrimental for tumor growth. In conclusion, in contrast to previous observations that tumor HBx variants lack transcriptional activity, we showed here that HBx variants have retained their ability to counteract Smc5/6 and thus to activate cccDNA transcription although they tend to lose antiproliferative activity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription., Methods: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differentiated HepaRG cells with wild-type virus or virus deficient (HBVX-) for the expression of hepatitis B virus X protein (HBx), that is required for HBV expression., Results: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA., Conclusion: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2)., Methods: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice., Results: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/β-catenin signaling in Apc(lox/lox) animals., Conclusions: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) is a widespread human pathogen responsible for acute and chronic liver diseases. The hepatitis B burden is particularly heavy in endemic countries, where liver cirrhosis and hepatocellular carcinoma are leading causes of death. However, the oncogenic role of HBV remains enigmatic. As the virus has no cytopathic effect, liver damage is attributed to immune responses that induce inflammation, apoptosis and regeneration, fostering the accumulation of genetic and epigenetic alterations. In a more direct action, frequent integration of HBV DNA into host chromosomes may lead to insertional mutagenesis of cancer-related genes and chromosomal instability. HBV proteins, notably the HBx transactivator, participate as co-factors in oncogenesis. Better understanding of hepatitis B pathogenesis is mandatory for improving disease management., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The hepatitis B virus (HBV) is a small enveloped DNA virus, which primarily infects hepatocytes and causes acute and persistent liver disease. Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma, but the molecular mechanisms underlying virally-induced tumourigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular cancer-related genes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long-term effects of viral proteins in enhancing immune-mediated liver disease. Recent genetic studies indicate that HBV-related tumours display a distinctive profile with a high rate of chromosomal alterations and low frequency of beta-catenin mutations. This review will discuss the evidence implicating chronic HBV infection as a causal risk factor of primary liver cancer. It will also discuss the molecular mechanisms that are critical for the tumourigenic process due to long lasting infection with HBV.

Despite the existence of effective vaccines, HBV infection remains a major health problem with 2 billion people infected worldwide. Among them, 350 million are chronically infected, a major risk factor for the development of hepatocellular carcinoma (HCC). There is a strong need to develop new and efficient treatments against chronic infection and HCC. It is therefore important to understand HBV replication and persistence as well as the role of HBV in liver carcinogenesis. This chapter focuses on the regulatory protein HBx which is thought to play a central role in HBV regulation and pathogenesis. HBx has been shown to modulate a myriad of viral and cellular functions, yet its role in virus replication and pathogenesis in infected individuals remains far from being completely understood.