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Hepatocellular carcinoma (HCC) is the second leading cause of cancer‐related deaths worldwide. Treatment options for patients with advanced‐stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient‐derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma‐c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor‐associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

Background & Aims HBeAg seroconversion during the natural history of chronic hepatitis B (CHB) is associated with a strong drop in serum HBV DNA levels and a reduction of intrahepatic covalently closed circular DNA (cccDNA) content. Of particular interest is the transition to HBeAg-negative chronic infection (ENCI). ENCI, previously known as inactive carrier state, is characterized by very low or negative viremia and the absence of liver disease. The molecular mechanisms responsible for the transition to ENCI and for the control of viral replication in ENCI are still poorly understood. Methods To identify which step(s) in the viral life cycle are controlled during the transition to ENCI, we quantified cccDNA, pre-genomic RNA (pgRNA), total HBV RNA and DNA replicative intermediates in 68 biopsies from patients in different phases of CHB. Results HBeAg seroconversion is associated with a reduction of cccDNA amounts as well as transcriptional activity. Silencing of cccDNA is particularly pronounced in ENCI, where there was ~46 times less pgRNA per cccDNA compared to HBeAg-negative CHB. Furthermore, a subgroup of patients with HBeAg-negative CHB can be characterized by reduced replication efficiency downstream of pgRNA. Conclusions The reduction in serum viral load during the transition to ENCI seems to primarily result from strong inhibition of the transcriptional activity of cccDNA which can be maintained in the absence of liver disease. Lay summary During the natural course of chronic hepatitis B virus infections, the immune response can gain control of viral replication. Quantification of viral DNA and RNA in liver biopsies of patients in different stages of chronic hepatitis B allowed us to identify the steps in the viral life cycle that are affected during the transition from active to inactive disease. Therapeutic targeting of these steps might induce sustained inhibition of viral transcription.

Background & Aims Most viruses are detected at early stages of cell infection and induce an innate immune response mediated by production of interferons (IFNs). IFNs induce expression of hundreds of IFN-stimulated genes (ISGs). Infection of chimpanzees with hepatitis C virus, but not hepatitis B virus (HBV), induces ISG expression in the liver. HBV might not induce an innate immune response because it is not detected by pattern recognition receptors (the stealth properties of HBV) or because HBV suppresses IFN production or signaling despite detection by pattern recognition receptors. We studied innate immune signaling in liver biopsies from patients with different stages of chronic HBV infection and uninfected individuals (controls). Methods We obtained liver within 10 minutes after collection from 30 patients with chronic HBV infection (hepatitis B e antigen-positive or -negative, with or without hepatitis) and 42 controls (most with fatty liver disease). The liver tissues were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, in situ hybridization, HBV RNA quantification, and HBV genotyping; some specimens were incubated with toll-like receptor (TLR) ligands (polyinosinic-polycytidylic acid) or infected with Sendai virus and then analyzed. Results Liver specimens from patients with HBV infection were not expressing more IFN or ISGs than those from control patients, indicating that chronic HBV infection did not activate an innate immune response. However, liver specimens from patients with HBV infection did produce IFN and induce expression of ISGs following activation of TLR3 with poly(I:C) or Sendai virus infections, so the innate immune response is not suppressed in these tissues. Conclusion Liver tissues from patients with chronic HBV infection do not have induction of an innate immune response, but this response can be activated by other factors (TLR3 binding, Sendai virus infection) in HBV-infected liver tissue. These findings support the hypothesis that HBV is invisible to pattern recognition receptors.

In order to search for alternative agents to overcome chemoresistance during the treatment of ovarian cancer, this study aimed to examine the anticancer effects and action mechanism of salinomycin, a selective inhibitor of cancer stem cells, on cisplatin-resistant human ovarian cancer cell lines in vitro and in vivo. The concentration- (0.01-200 µM) and time‑dependent (24-72 h) growth inhibitory effects of salinomycin were observed in the ovarian cancer cell lines OV2008, C13, A2780, A2780-cp, SKOV3 and OVCAR3, by measuring cell viability using the resazurin reduction assay. The IC50 (24 h) range of salinomycin on the six cell lines was found to be 1.7-7.4 µM. After cisplatin-resistant C13 cells were treated with salinomycin, the percentage of apoptotic cells determined by flow cytometry was significantly increased, in a concentration- and time‑dependent manner. However, no cell cycle arrest was detected in the G1/G0, S and G2/M phases in the salinomycin‑treated and control cells. The Bio-Plex phosphoprotein 5-plex assay (Akt, IκB-α, ERK1/2, JNK and p38 MAPK) demonstrated a marked time- and concentration‑dependent increase in the phosphorylation of p38 MAPK, subsequent to salinomycin treatment. Moreover, salinomycin significantly suppressed tumor growth in a tumor xenograft model. These findings suggested that salinomycin efficiently inhibits the cisplatin-resistant human ovarian cancer cell line growth through the induction of apoptosis, potentially associated with the p38 MAPK activation.

Recombinant alpha interferon (IFN-alpha) is used for the treatment of viral hepatitis and some forms of cancer. During these therapies IFN-alpha is injected once daily or every second day for several months. Recently, the long-acting pegylated IFN-alpha (pegIFN-alpha) has replaced standard IFN-alpha in therapies of chronic hepatitis C because it is more effective, supposedly by inducing a long-lasting activation of IFN signaling pathways. IFN signaling in cultured cells, however, becomes refractory within hours, and little is known about the pharmacodynamic effects of continuously high IFN-alpha serum concentrations. To investigate the behavior of the IFN system in vivo, we repeatedly injected mice with IFN-alpha and analyzed its effects in the liver. Within hours after the first injection, IFN-alpha signaling became refractory to further stimulation. The negative regulator SOCS1 was rapidly upregulated and likely responsible for early termination of IFN-alpha signaling. For long-lasting refractoriness, neither SOCS1 nor SOCS3 were instrumental. Instead, we identified the inhibitor USP18/UBP43 as the key mediator. Our results indicate that the current therapeutic practice using long-lasting pegIFN-alpha is not well adapted to the intrinsic properties of the IFN system. Targeting USP18 expression may allow to exploit the full therapeutic potential of recombinant IFN-alpha.

The discovery that the human Jagged1 gene (JAG1) is the Alagille syndrome disease gene indicated that Notch signaling has an important role in bile duct homeostasis. The functional study of this signaling pathway has been difficult because mice with targeted mutations in Jagged1, Notch1, or Notch2 have an embryonic lethal phenotype. We have previously generated mice with inducible Notch1 disruption using an interferon-inducible Cre-recombinase transgene in combination with the loxP flanked Notch1 gene. We used this conditional Notch1 knockout mouse model to investigate the role of Notch1 signaling in liver cell proliferation and differentiation. Deletion of Notch1 did not result in bile duct paucity, but, surprisingly, resulted in a continuous proliferation of hepatocytes. In conclusion, within weeks after Notch1 inactivation, the mice developed nodular regenerative hyperplasia without vascular changes in the liver.

Background & aims Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. The majority of patients treated with interferon alpha do not have a sustained response with clearance of the virus. The molecular mechanisms underlying interferon resistance are poorly understood. Interferon-induced activation of the Jak-STAT (signal transducer and activator of transcription) signal transduction pathway is essential for the induction of an antiviral state. Interference of viral proteins with the Jak-STAT pathway could be responsible for interferon resistance in patients with chronic HCV. Methods We have analyzed interferon-induced signal transduction through the Jak-STAT pathway in transgenic mice that express HCV proteins in their liver cells. STAT activation was investigated with Western blots, immunofluorescence, and electrophoretic mobility shift assays. Virus challenge experiments with lymphocytic choriomeningitis virus were used to demonstrate the functional importance of Jak-STAT inhibition. Results STAT signaling was found to be strongly inhibited in liver cells of HCV transgenic mice. The inhibition occurred in the nucleus and blocked binding of STAT transcription factors to the promoters of interferon-stimulated genes. Tyrosine phosphorylation of STAT proteins by Janus kinases at the interferon receptor was not inhibited. This lack in interferon response resulted in an enhanced susceptibility of the transgenic mice to infection with a hepatotropic strain of lymphocytic choriomeningitis virus. Conclusions Interferon-induced intracellular signaling is impaired in HCV transgenic mice. Interference of HCV proteins with interferon-induced intracellular signaling could be an important mechanism of viral persistence and treatment resistance.