In vitro transformation of primary human hepatocytes: Epigenetic changes and stemness properties.

Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

Toll-like receptor 3 downregulation is an escape mechanism from apoptosis during hepatocarcinogenesis.

BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; p = 0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; p = 0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.

Down-regulation of the Wnt/ß-catenin signaling pathway by Cacnb4.

The ß4 isoform of the ß-subunits of voltage-gated calcium channel regulates cell proliferation and cell cycle progression. Herein we show that coexpression of the ß4-subunit with actors of the canonical Wnt/ß-catenin signaling pathway in a hepatoma cell line inhibits Wnt-responsive gene transcription and decreases cell division, in agreement with the role of the Wnt pathway in cell proliferation. ß4-subunit-mediated inhibition of Wnt signaling is observed in the presence of LiCl, an inhibitor of glycogen synthase kinase (GSK3) that promotes ß-catenin translocation to the nucleus. Expression of ß4-subunit mutants that lost the ability to translocate to the nucleus has no effect on Wnt signaling, suggesting that ß4-subunit inhibition of Wnt signaling occurs downstream from GSK3 and requires targeting of ß4-subunit to the nucleus. ß4-subunit coimmunoprecipitates with the TCF4 transcription factor and overexpression of TCF4 reverses the effect of ß4-subunit on the Wnt pathway. We thus propose that the interaction of nuclear ß4-subunit with TCF4 prevents ß-catenin binding to TCF4 and leads to the inhibition of the Wnt-responsive gene transcription. Thereby, our results show that ß4-subunit is a TCF4 repressor and therefore appears as an interesting candidate for the regulation of this pathway in neurons where ß4-subunit is specifically expressed.

EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes.

BACKGROUND & AIMS: Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. METHODS: Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. RESULTS: EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. CONCLUSIONS: The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. LAY SUMMARY: In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.

Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major risk factor for developing liver cancer, and the HBV X protein (pX) has been implicated as a cofactor in hepatocyte transformation. We have shown that HBV replication as well as in vitro transformation by pX are associated with induction of the mitotic polo-like kinase 1 (Plk1) and down-regulation of the chromatin remodeling components Suz12 and Znf198. Herein, we demonstrate the same inverse relationship between Plk1 and Suz12/Znf198 in liver tumors from X/c-myc bitransgenic mice and woodchuck hepatitis virus (WHV)-infected woodchucks. Employing these animal models and the HBV replicating HepAD38 cells we examined the effect of Suz12/Znf198 down-regulation on gene expression. Genes analyzed include hepatic cancer stem cell markers BAMBI, DKK1,2, DLK1, EpCAM, MYC, and proliferation genes CCNA1, CCND2, IGFII, MCM4-6, PLK1, RPA2, and TYMS. Suz12 occupancy at the promoters of BAMBI, CCND2, DKK2, DLK1, EpCAM, and IGFII was demonstrated by chromatin immunoprecipitation in untransformed hepatocytes, but was markedly reduced in pX-transformed and Suz12 knockdown cells. Accordingly, we refer to these genes as "Suz12 repressed" genes in untransformed hepatocytes. The Suz12 repressed genes and proliferation genes were induced in HBV-replicating HepAD38 cells and, interestingly, they exhibited distinct expression profiles during hepatocellular carcinoma (HCC) progression in X/c-myc bitransgenics. Specifically, CCND2, EpCAM, and IGFII expression was elevated at the proliferative and preneoplastic stages in X/c-myc bitransgenic livers, whereas BAMBI and PLK1 were overexpressed in hepatic tumors from X/c-myc bitransgenics and WHV-infected woodchucks. Importantly, most of these genes were selectively up-regulated in HBV-induced HCCs. CONCLUSION: The distinct expression profile of the identified Suz12 repressed genes in combination with the proliferation genes hold promise as biomarkers for progression of chronic HBV infection to HCC.

Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.

BACKGROUND & AIMS: We previously reported the frequent overexpression of the FZD7 membrane receptor in hepatocellular carcinoma (HCC) and its role for controlling cancer phenotype. Herein, this study aimed at assessing the anticancer properties of compounds inhibiting FZD7 activity by disrupting its binding with the cytosolic Dishevelled (DVL) adaptator. METHODS: We have designed small interfering peptides (RHPDs) that are able to enter within cells and to competitively antagonize the binding of FZD7 to the PDZ domain of DVL. Their anti-neoplastic properties were assessed in vitro on a panel of human HCC cell lines and in vivo on the SV40-TAg transgenic mouse model of HCC. RESULTS: We have shown that RHPDs decrease cell viability via apoptosis depending on their affinity for PDZ, with a therapeutic index between cancerous and non-cancerous cells. RHPD properties were linked to ß-catenin degradation and PKCδ activation. In transgenic mice, intra-tumor injection of RHPDs inhibited HCC progression. CONCLUSIONS: We have completed a proof-of-concept showing that in vitro and in vivo the pharmacological inhibition of FZD7 displays anti-cancerous properties against HCC. The mechanisms can involve ß-catenin and PKCδ modulations. Further studies are warranted to design protocols showing the compatibility with systemic in vivo applications.

Oncogenic role of the frizzled-7/beta-catenin pathway in hepatocellular carcinoma.

BACKGROUND/AIMS: The molecular mechanisms of hepatocarcinogenesis remain largely unknown. Previous studies suggest that activation of the Wnt/beta-catenin pathway is important during hepatocyte transformation but the role of Frizzled receptor (FZD) in this process has not been defined. Here we investigate activation of this pathway by FZD using transgenic hepatocellular carcinoma (HCC) murine models. METHODS: We employed single (c-myc, SV40-Tag) and established double [insulin receptor substrate-1 (IRS-1/c-myc) and hepatitis Bx protein (X/c-myc)] transgenic lines and all developed HCC. Expression of 9 FZD was measured by real time RT-PCR and Western blot analysis. Phosphorylation and cellular accumulation of beta-catenin were assessed in both dysplastic tissue and tumors. We investigated the effect of a dominant negative (DN) FZD7 on TCF transcriptional activity in a SV40 derived HCC cell line. RESULTS: FZD7 was highly overexpressed at the mRNA and protein level(s) in HCC and occurred in dysplasia. Upregulation of FZD7 was associated with reduced phosphorylation of beta-catenin and led to nuclear accumulation in HCC tumors. Ectopic expression of a DN FZD7 construct decreased TCF transcriptional activity in tumor cells. CONCLUSIONS: These observations suggest that upregulation of FZD7 receptors in association with activation of the canonical Wnt/beta-catenin pathway is a common molecular event in HCC.

Increase of doxorubicin sensitivity by doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is known to be chemoresistant to anticancer drugs due to the multidrug resistant (MDR) transporters expression. Here, we compared in vitro and in vivo the anti-tumor efficacy of doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles (PIHCA-Dox) versus free doxorubicin (Dox). These nanoparticles are known to overcome the MDR phenotype. METHODS: We first determined in vitro the 50% inhibition concentration (IC(50)) of these drugs on different human hepatoma cell lines. Secondly, the efficacy of the drugs in vivo was determined on the X/myc transgenic murine model of HCC by histological counting of apoptotic tumorous hepatocytes and by TUNEL labeling. We characterized by semi-quantitative RT-PCR the MDR-related gene (mdr1, mdr3, mrp1) expression pattern in this model. RESULTS: In vitro, IC(50) was reduced with PIHCA-Dox versus Dox for Huh7 (1.7-fold reduction; P<0.001), HepaRG (4.5-fold reduction; P<0.01), HepG2 (1.5-fold reduction; P<0.001), and HepG2.2.15 (1.5-fold reduction; P=0.059). In vivo, HCC in transgenic mice overexpressed the mdr1 and mdr3 genes and the antitumor drugs efficacy was greatly enhanced after injection of PIHCA-Dox (9.0+/-5.0%; n=15) versus Dox (4.6+/-3.3%; n=13; P=0.01) for apoptotic bodies count. CONCLUSIONS: These promising data showing a higher anti-tumor efficacy on HCC of PIHCA-Dox versus Dox, warrant further studies in both animals and humans.

Long-term high-dose interferon-alpha therapy delays Hepadnavirus-related hepatocarcinogenesis in X/myc transgenic mice.

The role of interferon-alpha (IFN-alpha) remains unclear in prevention of virus-induced hepatocellular carcinoma in humans. We have investigated it herewith in the X/myc transgenic mouse model of Hepadnavirus-related hepatocarcinogenesis because of upregulation of c-myc oncogene in the liver. We have demonstrated that IFN-alpha can downregulate dose-dependently hepatocyte proliferation and c-myc overexpression at early premalignant stages, while it does not affect either hepatocyte apoptosis or telomerase activity at these steps. However, continuous and long-term administration of IFN-alpha dose-dependently delays tumor onset in dysplastic livers and increases overall survival of animals, more efficiently whether started before the onset of dysplasia. The present study therefore highlights that early preventive administration of IFN-alpha can slow down evolution towards hepatocellular carcinoma via repression of c-myc and hepatocyte proliferation at premalignant steps in experimental c-myc-induced hepatocarcinogenesis. However, the transient effect observed in this study emphasizes a need to clarify the possible mechanisms of acquired resistance and subsequent therapeutic escape. Our experimental model may be a pertinent tool to explore antioncogenic properties of IFN-alpha in human cirrhotic livers showing c-myc upregulation.