Primary Hepatoma Research Articles

Activated hepatic stellate cells promote angiogenesis in hepatocellular carcinoma by secreting angiopoietin-1.

Angiogenesis is the central pathological process in hepatocellular carcinoma (HCC), and its progression is affected by tumor cells and the microenvironment. Activated hepatic stellate cells (aHSCs) are the most significant stromal cells involved in HCC. This study was aimed to explore the effects and mechanisms of aHSCs on angiogenesis in HCC. We isolated primary hepatoma cells, aHSCs, and hepatic vascular endothelial cells from human HCC samples. Then, we performed a novel in vitro assay and in vivo experiment in a nude mouse HCC model to investigate the functions of aHSCs on angiogenesis in HCC. Our results demonstrated that aHSCs are the primary sources of angiopoietin-1 (Ang-1) in human HCC in vitro, and aHSCs could promote hepatic vascular endothelial cell (HVEC) growth by secreting Ang-1. Furthermore, aHSCs could induce HVEC microtubule formation, and this ability was reduced when Ang-1 expression was silenced in aHSCs. In addition, CD34 expression in a nude mouse HCC model was downregulated when Ang-1 messenger RNA was silenced in aHSCs. Our data also indicated that HSC Ang-1 expression could be inhibited by overexpressing Raf kinase inhibitor protein. Therefore, we suggest that aHSCs promote angiogenesis through secreting Ang-1, potentially providing an interesting target for antiangiogenic therapies for HCC.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas

Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

Initial Clinical Experience of Stereotactic Body Radiation Therapy (SBRT) For Liver Metastases, Primary Liver Malignancy, and Pancreatic Abdominal Nodal Recurrence with 4D-MRI based Online Adaptation and Real-Time MRI Monitoring using a 1.5 Tesla MR-Linac

The commercial introduction of a 1.5 MR equipped linear accelerator presents an opportunity to acquire MR images while simultaneously treating with external beam radiation therapy. We present the first in human experience treating patients for both liver and abdominal tumors using a 1.5 MR Linac. A commercially available 1.5 MR Linac was used to treat a total of 4 patients with abdominal tumors in our institution. Each patient consented to enroll in an advanced imaging protocol. A treatment checklist was followed during each fraction, with tasks time-stamped upon completion. Adaptive plans were based on pre-beam motion-averaged or mid-position images derived from respiratory-correlated 4D-MRI. Methodology applied included either adapt-to-position, or adapt-to-shape workflows. A parallel contouring strategy was employed using independent delineation software. Quantitative diffusion-weighted images, T2 mapping, and B0 maps were acquired during plan adaptation. Real-time motion monitoring was performed during beam-on. A comparison between the doses to normal organs at risk on the reference plan and the daily delivered plan was conducted. The median patient age was 70.5, two patients were male and two were female. Primary tumors treated (doses prescribed/fractions) included 1 liver melanoma metastatic lesion (45 Gy/3), 2 primary hepatocellular carcinomas (40-45 Gy/5), 1 upper abdominal nodal (pancreatic) recurrence (30 Gy/6). Two patients were treated with the adapt-to-position work flow, two patients were treated with the adapt-to-shape workflow. The median treatment time duration was approximately 50 minutes. For the liver lesions treated, the liver mean radiation dose was found to be lower on the adaptive plan as compared to the reference plan. In addition, for the abdominal nodal recurrence,accumulated dose to at risk GI structures was sufficiently below the reference plan dose, which permitted administration of an additional treatment fraction. We present a successful first in human experience of treating both an upper abdominal (pancreatic) nodal recurrence along with primary and secondary liver malignancies with a 1.5 MR Linear accelerator using adapt-to-position and adapt-to-shape strategies. Treatments were well tolerated by all patients. Fully quantitative MR imaging was acquired during the treatment course. Unique work flow elements and custom MR sequence utilization will be presented.

Stereotactic body radio therapy for inoperable large hepatocellular cancers: results from a clinical audit.

To evaluate the outcomes of stereotactic radiotherapy (SBRT) in the treatment of inoperable hepatocellular carcinomas (HCC) that are unsuitable for, or refractory to other liver-directed therapies. Between March 2015 and June 2018, patients with primary HCCs refractory to or unsuitable for treatment with other liver-directed therapies were treated with SBRT. Patients of Child status A5-B7 and with normal liver reserve ≥ 700 cc were preferred. Local control (LC), overall survival (OS), progression free survival (PFS) and effect of prognostic factors were analysed. 21 patients with inoperable HCCs were treated. The median tumour diameter was 9.6 cm (5-21) and median tumour volume was 350 cc (32.9 - 2541). The median SBRT dose prescription was 42 Gy/6 fractions (25 - 54 Gy/6#). The 1- and 2-year LC rate was 88 and 43 % respectively. Overall rate of > grade III toxicity was 14 %. Patients with Child A5 liver function had a better median OS than A6 and B7 patients [21 vs 11 vs 8 months]. Also, tumours with GTV < 350 cc volumes had a better OS compared to GTV of greater than 350 cc [24 months vs 8 months, p value = 0.004]. This study showed that SBRT can be used safely and effectively to treat inoperable HCCs with or without prior loco-regional therapies, resulting in good local control and survival with acceptable toxicity. Use of SBRT in inoperable HCC is safe and effective.

The Optimal Management for Sub-Centimeter Hepatocellular Carcinoma: Curative Treatments or Follow-Up?

BackgroundThe optimal strategy for dealing with sub-centimeter hepatic nodules has not yet been established. This study aimed to assess whether there was a need to provide curative treatments for sub-centimeter hepatocellular carcinomas (HCCs) to patients at risk for high false positives.Material/MethodsWe identified patients with primary pathologically diagnosed HCC ≤2 cm from 2004 to 2015 in the Surveillance, Epidemiology and End Results (SEER) database. They were divided according to the interventions they received: local ablation, surgical resection, or liver transplantation. In each group, overall survival and cancer-specific survival were used as endpoints to compare the prognoses between patients with sub-centimeter HCC and patients with HCC measuring 1 to 2 cm by Kaplan-Meier. Propensity score matching was performed to reduce bias. We also compared the survival of patients with a primary solitary HCC based on interventions, in the different tumor size groups. Bootstrapping was performed to validate the findings.ResultsOverall, 10.4% of patients (197 out of 1894) had HCCs <1 cm, and 89.6% of patients (1697 out of 1894) had HCCs in the 1 to 2 cm range. There was no significant difference in overall and cancer-specific survival between patients with HCCs <1 cm and those with HCCs in the 1 to 2 cm range, in all treatment groups. After adjusting confounding factors, no significant correlation was found between tumor size and survival time. In patients with HCCs measuring ≤2 cm, overall survival and cancer-specific survival were superior in liver transplantation compared with surgical resection and local ablation. Surgical resection provided better survival than local ablation.ConclusionsCompared to patients with HCCs measuring 1 to 2 cm, the survival rates of patients with sub-centimeter HCCs was not improved through curative treatments, risking high false positives.

TNF-derived peptides inhibit tumour growth and metastasis through cytolytic effects on tumour lymphatics.

Tumour necrosis factor (TNF) is a multi-functional cytokine with profound and diverse effects on physiology and pathology. Identifying the molecular determinants underlying the functions and pathogenic effects of TNF is key to understanding its mechanisms of action and identifying new therapeutic opportunities based on this important molecule. Previously, we showed that some evolutionarily conserved peptides derived from TNF could induce cell death (e.g. apoptosis and/or necrosis), a feature of immune defence mechanisms shared by many vertebrates. In this study, we demonstrated that necrosis-inducing peptide P16 kills human glioblastoma cancer cells and primary human hepatoma or renal cancer cells isolated from patients who had not responded to standard treatments. Importantly, we show that the necrosis-inducing peptide P1516 significantly improves survival by inhibiting tumour metastasis in a 4T1 breast cancer syngeneic graft mouse model. Because the lymphatic system is an important metastatic route in many cancers, we also tested the effect of TNF-derived peptides on monolayers of primary human lymphatic endothelial cells (hDLEC) and found that they increased junctional permeability by inducing cytoskeletal reorganization, gap junction formation and cell death. Transmission electron microscopy imaging evidence, structural analysis and in-vitro liposome leakage experiments strongly suggest that this killing is due to the cytolytic nature of these peptides. P1516 provides another example of a pro-cytotoxic TNF peptide that probably functions as a cryptic necrotic factor released by TNF degradation. Its ability to inhibit tumour metastasis and improve survival may form the basis of a novel approach to cancer therapy.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response

Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

ANXA2Tyr23 and FLNASer2152 phosphorylation associate with poor prognosis in hepatic carcinoma revealed by quantitative phosphoproteomics analysis

Hepatoma is one of the most common malignant tumors, and most patients have very poor prognosis. Early prediction and intervention of the hepatoma recurrence/metastasis are the most effective way to improve the patients' clinical outcomes. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative phospho-proteomics approach to identify biomarkers associated with hepatoma recurrence/metastasis in hepatoma cell lines with increasing metastasis ability. In total, 75 phosphorylated peptides corresponding to 60 phosphoproteins were significantly dysregulated and the participated biological processes of these phosphoproteins were tightly associated with tumor metastasis. Further signaling pathway analysis revealed that key signaling pathways which play crucial roles in cancer metastasis have been significantly over activated in the highly metastatic cells. Furthermore, the phosphorylation of FLNASer2152 and ANXA2Tyr23 were validated to be significantly up regulated in the high-metastatic cells comparing with the low-metastatic cells. By further investigation the clinical significance of the phosphorylation of FLNASer2152 and ANXA2Tyr23 in large-scale clinical samples, revealed that the over phosphorylation of FLNASer2152 and ANXA2Tyr23 were associated with poor prognosis and might be potential prognostic biomarkers for the primary hepatoma. When FLNASer2152 combined with ANXA2Tyr23, it had a better prognostic value for both OS and TTR.

Cylindrospermopsin induces cellular stress and activation of ERK1/2 and p38 MAPK pathways in adult human liver stem cells

Cyanobacterial toxin cylindrospermopsin (CYN) is an emerging freshwater contaminant, whose expanding environmental occurrence might result into increased human health risks. CYN is potent hepatotoxin, with cytotoxicity and genotoxicity documented in primary hepatocytes or hepatoma cell lines. However, there is only limited information about CYN effects on adult human liver stem cells (LSCs), which play an important role in liver tissue development, regeneration and repair. In our study with human liver cell line HL1-hT1 which expresses characteristics of LSCs, CYN was found to be cytotoxic and increasing cell death after 24–48 h exposure to concentrations >1 μM. Subcytotoxic 1 μM concentration did not induce cell death or membrane damage, but inhibited cellular processes related to energy production, leading to a growth stagnation after >72 h. Interestingly, these effects were not associated with increased DNA damage, reactive oxygen species production, or endoplasmic reticulum stress. However, CYN induced a sustained (24–48 h) activation of mitogen-activated protein kinases ERK1/2 and p38, and increased expression of stress-related transcription factor ATF3. Thus, LSCs were not primarily affected by CYN-induced genotoxicity and oxidative stress, but via activation of signaling and transcriptional pathways critical for regulation of cell proliferation, stress responses, cell survival and inflammation. Alterations of LSCs during CYN-induced liver injury, including the role of nongenotoxic mechanisms, should be therefore considered in mechanistic assessments of chronic CYN hepatotoxicity and hepatocarcinogenicity.

Unequivocal evidence for endogenous geranylgeranoic acid biosynthesized from mevalonate in mammalian cells

Geranylgeranoic acid (GGA) has been reported to induce autophagic cell death via upregulation of lipid-induced unfolded protein response in several human hepatoma-derived cell lines, and its 4,5-didehydro derivative has been developed as a preventive agent against second primary hepatoma in clinical trials. We have previously reported that GGA is a natural diterpenoid synthesized in several medicinal herbs. Here, we provide unequivocal evidence for de novo GGA biosynthesis in mammals. First, with normal male Wistar rats, the levels of GGA in liver were found to be far greater than those in other organs analyzed. Second, we demonstrated the metabolic GGA labeling from the 13C-labeled mevalonolactone in the human hepatoma-derived cell line, HuH-7. Isotopomer spectral analysis revealed that approximately 80% of the cellular GGA was newly synthesized from mevalonate (MVA) in 12 h and the acid picked up preexisting farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), suggesting that GGA is derived from FPP and GGPP through the MVA pathway. Third, zaragozic acid A, a squalene synthase inhibitor, induced dose-dependent upregulation of endogenous GGA content in HuH-7 cells and their concomitant cell death. These results strongly suggest that a cancer-preventive GGA is biosynthesized via the MVA pathway in mammals.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Genomics of lipid-laden human hepatocyte cultures enables drug target screening for the treatment of non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures.MethodsPHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways.ResultsWhole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance.ConclusionThe genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates.

Exogenous exosomes from mice with acetaminophen-induced liver injury promote toxicity in the recipient hepatocytes and mice

Exosomes are small extracellular membrane vesicles released from endosomes of various cells and could be found in most body fluids. The main functions of exosomes have been recognized as important mediators of intercellular communication and as potential biomarkers of various disease states. This study investigated whether exogenous exosomes from mice with acetaminophen (APAP)-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in mice. We observed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. In addition, confocal microscopy of ex vivo liver section showed that exogenously added exosomes were accumulated in recipient hepatocytes. Furthermore, plasma reactive oxygen species and hepatic TNF-α/IL-1β production were elevated in APAP-exosomes recipient mice compared to control-exosomes recipient mice. The levels of apoptosis-related proteins such as phospho-JNK/JNK, Bax, and cleaved caspase-3 were increased in mouse liver received APAP-exosomes. These results demonstrate that exogenous exosomes from APAP-exposed mice with acute liver injury are functional and stimulate cell death or toxicity of the recipient hepatocytes and mice.

Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3β/β-Catenin Signaling Pathway.

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3β (GSK-3β) and increased β-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3β/β-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased β-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased β-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates β-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3β/β-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3β, and increases β-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases β-catenin levels. Although HBx activates AKT to upregulate β-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Solute Carrier Organic Anion Transporter Family Member 3A1 Is a Bile Acid Efflux Transporter in Cholestasis

Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of sometransporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed moreliver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.

Cytotoxicity of anticancer candidate salinomycin and identification of its metabolites in rat cell cultures

Salinomycin (SAL) is a polyether antibiotic, which is commonly used as a coccidiostat and has recently shown to exhibit anticancer activity. The toxic action of the drug may be connected with the extent and routes of its biotransformation. The cytotoxic potential of SAL and its combination with tiamulin and prednisolone was investigated using three cell models from rat: primary hepatocytes, hepatoma cells (FaO) and myoblasts (L6). The four biochemical endpoints were assessed: mitochondrial and lysosomal activity, total cell protein content and membrane integrity. The metabolites of SAL in the medium from cell cultures were determined using LC-MS/MS. The cytotoxicity of SAL was time-, concentration- and cells dependent. The most sensitive endpoint was the inhibition of lysosomal activity. Tiamulin increased SAL cytotoxicity, whereas the opposite results were observed for prednisolone. Primary hepatocytes were the most efficient in SAL biotransformation both in terms of its intensity and number of produced metabolites. The range of the cytotoxicity and mode of salinomycin interaction with tiamulin and prednisolone cannot be explained by the biotransformation alone.

Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB.

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells.

The first study on therapeutic efficacies of a vascular disrupting agent CA4P among primary hepatocellular carcinomas with a full spectrum of differentiation and vascularity: Correlation of MRI-microangiography-histopathology in rats.

To better inform the next clinical trials of vascular disrupting agent combretastatin-A4-phosphate (CA4P) in patients with hepatic malignancies, this preclinical study aimed at evaluating CA4P therapeutic efficacy in rats with primary hepatocellular carcinomas (HCCs) of a full spectrum of differentiation and vascularity by magnetic resonance imaging (MRI), microangiography and histopathology. Ninety-six HCCs were raised in 25 rats by diethylnitrosamine gavage. Tumor growth was monitored by T2-/T1-weighted-MRI (T2WI, T1WI) using a 3.0 T scanner. Early vascular response and later intratumoral necrosis were detected by dynamic-contrast-enhanced (DCE) MRI and diffusion-weighted-imaging (DWI) before, 1 and 12 hr after CA4P iv-administration. In vivo MRI-findings were validated by postmortem-techniques. Multi-parametric MRI revealed rapid CA4P-induced tumor vascular shutdown within 1 hr, followed by variable intratumoral necrosis at 12 hr. Tumor volumes decreased by 10% at 1 hr (p < 0.05), but resumed at 12 hr. Correlations of semi-quantitative DCE parameter initial-area-under-the-gadolinium-curve (IAUGC30) with histopathology proved partial vascular closure and compensational reopening (p < 0.05). The higher grades of vascularity prevented those residual tumor tissues from CA4P-caused ischemic necrosis. By histopathology using a 4-scale cellular-differentiation criteria and a 4-grade tumor-vascularity classification, percentage of CA4P-induced necrosis negatively correlated with HCC differentiation (r = -0.404, p < 0.001) and tumor vascularity (r = -0.370, p < 0.001). Ordinal-logistic-regression helped to predict early tumor responses to CA4P in terms of tumoral differentiation and vascularity. Our study demonstrated that CA4P could induce vascular shutdown in primary HCCs within 1 hr, resulting in various degrees of tumor necrosis at 12 hr. MRI as a real-time imaging biomarker may help to define tumor vascularity and differentiation and further to predict CA4P therapeutic outcomes.

ADAR1 affects HCV infection by modulating innate immune response

The hepatitis C virus (HCV) is a globally prevalent infectious pathogen. As many as 80% of people infected with HCV do not control the virus and develop a chronic infection. Response to interferon (IFN) therapy is widely variable in chronic HCV infected patients, suggesting that HCV has evolved mechanisms to suppress and evade innate immunity responsible for its control and elimination. Adenosine deaminase acting on RNA 1 (ADAR1) is a relevant factor in the regulation of the innate immune response. The loss of ADAR1 RNA-editing activity and the resulting loss of inosine bases in RNA are critical in producing aberrant RLR-mediated innate immune response, mediated by RNA sensors MDA5 and RIG-I. Here, we describe ADAR1 role as a regulator of innate and antiviral immune function in HCV infection, both in vitro and in patients. Polymorphisms within ADAR1 gene were found significantly associated to poor clinical outcome to HCV therapy and advanced liver fibrosis in a cohort of HCV and HIV-1 coinfected patients. Moreover, ADAR1 knockdown in primary macrophages and Huh7 hepatoma cells enhanced IFN and IFN stimulated gene expression and increased HCV replication in vitro. Overall, our results demonstrate that ADAR1 regulates innate immune signaling and is an important contributor to the outcome of the HCV virus–host interaction. ADAR1 is a potential target to boost antiviral immune response in HCV infection.