Katrin Hoffmann

Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors

To identify new tumor‐suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome‐wide methylation profiling and vertical integration with array‐based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well‐characterized human hepatocellular carcinomas (HCCs). Bisulfite‐converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy‐number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor‐suppressor gene (period homolog 3 [PER3], insulin‐like growth‐factor–binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down‐regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5‐aza‐dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor‐suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high‐resolution genomic and transcriptomic data facilitates the identification of new tumor‐suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817–1827)

Melatonin promotes sorafenib-induced apoptosis through synergistic activation of JNK/c-jun pathway in human hepatocellular carcinoma

Melatonin has been shown to exert anticancer activity on hepatocellular carcinoma (HCC) through its antiproliferative and pro‐apoptotic effect in both experimental and clinical studies, and sorafenib is the only approved drug for the systemic treatment of HCC. Thus, this study was designed to investigate the combined effect of melatonin and sorafenib on proliferation, apoptosis, and its possible mechanism in human HCC. Here, we found that both melatonin and sorafenib resulted in a dose‐dependent growth inhibition of HuH‐7 cells after 48 hours treatment, and the combination of them enhanced the growth inhibition in a synergistic manner. Colony formation assay indicated that co‐treatment of HuH‐7 cells with melatonin and sorafenib significantly decreased the clonogenicity compared to the treatment with single agent. Furthermore, FACS and TUNEL assay confirmed that melatonin synergistically augmented the sorafenib‐induced apoptosis after 48 hours incubation, which was in accordance with the activation of caspase‐3 and the JNK/c‐jun pathway. Inhibition of JNK/c‐jun pathway with its inhibitor SP600125 reversed the phosphorylation of c‐jun and the activation of caspase‐3 induced by co‐treatment of HuH‐7 cells with melatonin and sorafenib in a dose‐dependent manner. Furthermore, SP600125 exhibited protective effect against apoptosis induced by the combination of melatonin and sorafenib. This study demonstrates that melatonin in combination with sorafenib synergistically inhibits proliferation and induces apoptosis in human HCC cells; therefore, supplementation of sorafenib with melatonin may serve as a potential therapeutic choice for advanced HCC.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. METHODS PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. CONCLUSIONS In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.

Long term follow up after resection emphasizes the role of surgery in Primary Hepatic Epithelioid Hemangioendothelioma

Background Primary Hepatic Epithelioid Hemangioendothelioma is an extremely rare tumor of vascular origin. Patients In this work we present clinical data and long term results of eight patients who were surgically treated in our institution. Liver transplantation was performed in five patients (four cadaveric and one living related) and major liver resection in three patients. Three patients are alive with recurrent tumor. After a median follow-up of 100 months (48–266) all patients are alive. Conclusions Only surgery can provide cure in HEH. If the extent of the disease prohibits primary resection liver transplantation might offer the most valuable option.

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll Like Receptor 3 in Hepatocytes

BACKGROUND & AIMS Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. METHODS We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. RESULTS HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. CONCLUSIONS Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence.

A rare case of recurrent malignant triton tumor in a male with NF1: Case report and mini-review

Highlights • We report on a rare case of metastasizing malignant triton tumor.• We report on the first case of a malignant triton tumor with concurrent atypical neurofibroma and malignant peripheral nerve sheath tumor.• The coexistence of atypical neurofibroma, malignant peripheral nerve sheath tumor and malignant triton tumor in the context of NF1 provides valuable information on the pathogenesis of malignant triton tumors.