Potential utility of l-carnitine for preventing liver tumors derived from metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. METHODS: l-carnitine (2 g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. RESULTS: Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. CONCLUSIONS: Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.

Mint3-depletion-induced energy stress sensitizes triple-negative breast cancer to chemotherapy via HSF1 inactivation.

Given the lack of therapeutic targets, the conventional approach for managing triple-negative breast cancer (TNBC) involves the utilization of cytotoxic chemotherapeutic agents. However, most TNBCs acquire resistance to chemotherapy, thereby lowering the therapeutic outcome. In addition to oncogenic mutations in TNBC, microenvironment-induced mechanisms render chemoresistance more complex and robust in vivo. Here, we aimed to analyze whether depletion of Munc18-1 interacting protein 3 (Mint3), which activates hypoxia-inducible factor 1 (HIF-1) during normoxia, sensitizes TNBC to chemotherapy. We found that Mint3 promotes the chemoresistance of TNBC in vivo. Mint3 depletion did not affect the sensitivity of human TNBC cell lines to doxorubicin and paclitaxel in vitro but sensitized tumors of these cells to chemotherapy in vivo. Transcriptome analyses revealed that the Mint3-HIF-1 axis enhanced heat shock protein 70 (HSP70) expression in tumors of TNBC cells. Administering an HSP70 inhibitor enhanced the antitumor activity of doxorubicin in TNBC tumors, similar to Mint3 depletion. Mint3 expression was also correlated with HSP70 expression in human TNBC specimens. Mechanistically, Mint3 depletion induces glycolytic maladaptation to the tumor microenvironment in TNBC tumors, resulting in energy stress. This energy stress by Mint3 depletion inactivated heat shock factor 1 (HSF-1), the master regulator of HSP expression, via the AMP-activated protein kinase/mechanistic target of the rapamycin pathway following attenuated HSP70 expression. In conclusion, Mint3 is a unique regulator of TNBC chemoresistance in vivo via metabolic adaptation to the tumor microenvironment, and a combination of Mint3 inhibition and chemotherapy may be a good strategy for TNBC treatment.

Beta-Hydroxyisovaleryl-Shikonin Eradicates Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells by Suppressing dUTP Pyrophosphatase Expression.

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (ß-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, ß-HIVS treatment decreased dUTPase expression. ß-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. ß-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.

Functional involvement of endothelial lipase in hepatitis B virus infection.

BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.

Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus.

BACKGROUND & AIMS: The risk of hepatocellular carcinoma (HCC) remains after achieving a sustained virological response (SVR) in patients with chronic hepatitis C (CHC). Epigenetic abnormalities might be key regulators in the development of HCC. This study aimed to identify the genes involved in hepatocarcinogenesis after an SVR. METHODS: DNA methylation in liver tissue was compared between 21 CHC patients without HCC and 28 CHC patients with HCC, all of whom had achieved an SVR. Additional comparisons with 23 CHC patients before treatment and 10 normal livers were performed. The characteristics of a newly identified gene were explored in vitro and in vivo. RESULTS: We found that the transmembrane protein no. 164 (TMEM164) gene was demethylated by hepatitis C virus infection and HCC development after achieving an SVR. TMEM164 was expressed mainly in endothelial cells, alpha smooth muscle actin-positive cells, and some capillarized liver sinusoidal endothelial cells. TMEM164 expression was significantly correlated with liver fibrosis and relapse-free survival in HCC patients. TMEM164 was induced by shear stress, interacted with GRP78/BiP, accelerated ATF6 (activating transcription factor 6)-mediated endoplasmic reticulum (ER) stress signaling, and activated interleukin-6/STAT3 (signal transducer and activator of transcription 3) signaling in the TMNK1 liver endothelial cell line. Therefore, we termed TMEM164 "shear stress-induced transmembrane protein associated with ER stress signaling" (SHERMER). SHERMER knockout mice were protected against CCL4-induced liver fibrosis. SHERMER overexpression in TMNK1 cells accelerated HCC growth in a xenograft model. CONCLUSIONS: We identified a new transmembrane protein, SHERMER, in CHC patients with HCC after achieving an SVR. SHERMER was induced by shear stress and accelerated ATF6-mediated ER stress signaling in endothelial cells. Thus, SHERMER is a novel endothelial marker associated with liver fibrosis, hepatocarcinogenesis, and progression of HCC.

Super-Resolution Microscopy Analysis of Hepatitis B Viral cccDNA and Host Factors.

Infection with hepatitis B virus (HBV) cannot be cured completely because of the persistence of covalently closed circular DNA (cccDNA). We previously found that the host gene dedicator of cytokinesis 11 (DOCK11) was required for HBV persistence. In this study, we further investigated the mechanism that links DOCK11 to other host genes in the regulation of cccDNA transcription. cccDNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) in stable HBV-producing cell lines and HBV-infected PXB-cells®. Interactions between DOCK11 and other host genes were identified by super-resolution microscopy, immunoblotting, and chromatin immunoprecipitation. FISH facilitated the subcellular localization of key HBV nucleic acids. Interestingly, although DOCK11 partially colocalized with histone proteins, such as H3K4me3 and H3K27me3, and nonhistone proteins, such as RNA Pol II, it played limited roles in histone modification and RNA transcription. DOCK11 was functionally involved in regulating the subnuclear distribution of host factors and/or cccDNA, resulting in an increase in cccDNA closely located to H3K4me3 and RNA Pol II for activating cccDNA transcription. Thus, it was suggested that the association of cccDNA-bound Pol II and H3K4me3 required the assistance of DOCK11. DOCK11 facilitated the association of cccDNA with H3K4me3 and RNA Pol II.

Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.

FOXM1 Is a Novel Molecular Target of AFP-Positive Hepatocellular Carcinoma Abrogated by Proteasome Inhibition.

Alpha-fetoprotein (AFP) is an oncofetal protein that is elevated in a subset of hepatocellular carcinoma (HCC) with poor prognosis, but the molecular target activated in AFP-positive HCC remains elusive. Here, we demonstrated that the transcription factor forkhead box M1 (FOXM1) is upregulated in AFP-positive HCC. We found that FOXM1 expression was highly elevated in approximately 40% of HCC cases, and FOXM1-high HCC was associated with high serum AFP levels, a high frequency of microscopic portal vein invasion, and poor prognosis. A transcriptome and pathway analysis revealed the activation of the mitotic cell cycle and the inactivation of mature hepatocyte metabolism function in FOXM1-high HCC. The knockdown of FOXM1 reduced AFP expression and induced G2/M cell cycle arrest. We further identified that the proteasome inhibitor carfilzomib attenuated FOXM1 protein expression and suppressed cell proliferation in AFP-positive HCC cells. Carfilzomib in combination with vascular endothelial growth factor receptor 2 (VEGFR2) blockade significantly prolonged survival by suppressing AFP-positive HCC growth in a subcutaneous tumor xenotransplantation model. These data indicated that FOXM1 plays a pivotal role in the proliferation of AFP-positive liver cancer cells. Carfilzomib can effectively inhibit FOXM1 expression to inhibit tumor growth and could be a novel therapeutic option in patients with AFP-positive HCC who receive anti-VEGFR2 antibodies.

Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: The lipid oxidation is a key factor for damaging hepatocytes and causing cell death. However, the mechanisms underlying hepatocyte death and the role of the most popular lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in nonalcoholic steatohepatitis (NASH) remains unclear. METHODS: We demonstrated using hepatoma cell lines, a NASH mouse model, HNE-treated monkeys, and biopsy specimens from patients with NASH that HNE induced hepatocyte death by disintegrating the lysosomal limiting membrane. RESULTS: The degree of HNE deposition in human NASH hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores, and was associated with enlargement of the staining of lysosomes in hepatocytes. In in vitro experiments, HNE activated µ-calpain via G-protein coupled receptor (GPR) 120. The resultant rupture/permeabilization of the lysosomal limiting membrane induced the leakage of cathepsins from lysosomes and hepatocyte death. The blockade of G-protein coupled receptor 120 (GPR120) or µ-calpain expression suppressed lysosomal membrane damage and hepatocyte death by HNE. Alda-1, which activates aldehyde dehydrogenase 2 to degrade HNE, prevented HNE-induced hepatocyte death. Intravenous administration of HNE to monkeys for 6 months resulted in hepatocyte death by a mechanism similar to that of cultured cells. In addition, intraperitoneal administration of Alda-1 to choline-deficient, amino-acid defined treated mice for 8 weeks inhibited HNE deposition, decreased liver inflammation, and disrupted lysosomal membranes in hepatocytes, resulting in improvement of liver fibrosis. CONCLUSIONS: These results provide novel insights into the mechanism of hepatocyte death in NASH and will contribute to the development of new therapeutic strategies for NASH.

Leukocyte cell-derived chemotaxin 2 is an antiviral regulator acting through the proto-oncogene MET.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

A single hepatitis B virus genome with a reporter allows the entire viral life cycle to be monitored in primary human hepatocytes.

For the development of antiviral agents to eliminate hepatitis B virus (HBV), it is essential to establish an HBV cell culture system that can easily monitor HBV infection. Here, we created a novel HBV infection monitoring system using a luminescent 11-amino acid reporter, the high-affinity subunit of nano-luciferase binary technology (HiBiT). The HiBiT-coding sequence was inserted at the N-terminus of preS1 in a 1.2-fold plasmid encoding a genotype C HBV genome. After transfection of HepG2 cells with this HiBiT-containing plasmid, the supernatant was used to prepare a recombinant cell culture-derived virus (HiBiT-HBVcc). Primary human hepatocytes (PXB) were inoculated with HiBiT-HBVcc. Following inoculation, intracellular and extracellular HiBiT activity and the levels of various HBV markers were determined. Reinfection of naive PXB cells with HiBiT-HBVcc prepared from HiBiT-HBVcc-infected PXB cells was analyzed. When PXB cells were infected with HiBiT-HBVcc at several titers, extracellular HiBiT activity was detected in a viral titer-dependent manner and was correlated with intracellular HiBiT activity. Inhibitors of HBV entry or replication suppressed extracellular HiBiT activity. Viral DNA, RNA, and proteins were detectable, including covalently closed circular DNA, by Southern blot analysis. The synthesis of relaxed-circular DNA from single-stranded DNA in HiBiT-HBV decreased to one third of that of wild-type HBV, and the infectivity of HiBiT-HBVcc decreased to one tenth of that of wild-type HBVcc. HiBiT-HBVcc prepared from PXB cells harboring HiBiT-HBV was able to infect naive PXB cells. Conclusions: Recombinant HiBiT-HBV can undergo the entire viral life cycle, thus facilitating high-throughput screening for HBV infection in vitro using supernatants. This system will be a powerful tool for developing antiviral agents.

Dickkopf-1 Promotes Angiogenesis and is a Biomarker for Hepatic Stem Cell-like Hepatocellular Carcinoma.

Cancer stemness evinces interest owing to the resulting malignancy and poor prognosis. We previously demonstrated that hepatic stem cell-like hepatocellular carcinoma (HpSC-HCC) is associated with high vascular invasion and poor prognosis. Dickkopf-1 (DKK-1), a Wnt signaling regulator, is highly expressed in HpSC-HCC. Here, we assessed the diagnostic and prognostic potential of serum DKK-1. Its levels were significantly higher in 391 patients with HCC compared with 205 patients with chronic liver disease. Receiver operating characteristic curve analysis revealed the optimal cutoff value of DKK-1 to diagnose HCC and predict the 3-year survival as 262.2 and 365.9 pg/mL, respectively. HCC patients with high-serum DKK-1 levels showed poor prognosis. We evaluated the effects of anti-DKK-1 antibody treatment on tumor growth in vivo and of recombinant DKK-1 on cell proliferation, invasion, and angiogenesis in vitro. DKK-1 knockdown decreased cancer cell proliferation, migration, and invasion. DKK-1 supplementation promoted angiogenesis in vitro; this effect was abolished by an anti-DKK-1 antibody. Co-injection of the anti-DKK-1 antibody with Huh7 cells inhibited their growth in NOD/SCID mice. Thus, DKK-1 promotes proliferation, migration, and invasion of HCC cells and activates angiogenesis in vascular endothelial cells. DKK-1 is a prognostic biomarker for HCC and a functional molecule for targeted therapy.

BMP9-ID1 Signaling Activates HIF-1α and VEGFA Expression to Promote Tumor Angiogenesis in Hepatocellular Carcinoma.

Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

Establishment of liver tumor cell lines from atherogenic and high fat diet fed hepatitis C virus transgenic mice.

A syngeneic mouse model bearing a transplanted tumor is indispensable for the evaluation of the efficacy of immune checkpoint inhibitors (ICIs). However, few syngeneic mouse models of liver cancer are available. We established liver tumor cell lines (MHCF1 and MHCF5) from hepatitis C virus transgenic mice fed an atherogenic high-fat diet. MHCF1 and MHCF5 were successfully transplanted into the subcutaneous space of syngeneic C57BL/6 mice, in addition, they efficiently developed orthotopic tumors in the liver of syngeneic C57BL/6 mice. MHCF5 grew rapidly and showed a more malignant phenotype compared with MHCF1. Histologically, MHCF1-derived tumors were a combined type of hepatocellular carcinoma and MHCF5-derived tumors showed a sarcomatous morphology. Interestingly, MHCF1 and MHCF5 showed different sensitivity against an anti-PD1 antibody and MHCF5-derived tumors were resistant to this antibody. CD8 T cells infiltrated the MHCF1-derived tumors, but no CD8 T cells were found within the MHCF5-derived tumors. Gene expression profiling and whole-exon sequencing revealed that MHCF5 displayed the features of an activated cancer stem cell-like signature of sonic hedgehog and Wnt signaling. Therefore, these cell lines could be useful for the identification of new biomarkers and molecular mechanisms of ICI resistance and the development of new drugs against liver cancer.

BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma.

The malignant nature of hepatocellular carcinoma (HCC) is closely related to the presence of cancer stem cells (CSCs). Bone morphologic protein 9 (BMP9), a member of the transforming growth factor-beta (TGF-ß) superfamily, was recently reported to be involved in liver diseases including cancer. We aimed to elucidate the role of BMP9 signaling in HCC-CSC properties and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have identified that high BMP9 expression in tumor tissues or serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in epithelial cell adhesion molecule (EpCAM)-positive HCC subtype via enhancing inhibitor of DNA-binding protein 1 (ID1) expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9-promoted HCC-CSC properties by suppressing Wnt/ß-catenin signaling. Interestingly, cells treated with BMP receptor inhibitors K02288 and LDN-212854 blocked HCC-CSC activation by inhibiting BMP9-ID1 signaling, in contrast to cells treated with the TGF-ß receptor inhibitor galunisertib. Treatment with LDN-212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9-ID1 signaling in promoting HCC-CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM-positive HCC. Therefore, targeting BMP9-ID1 signaling could offer novel therapeutic options for patients with malignant HCC.

Chronic liver disease enables gut Enterococcus faecalis colonization to promote liver carcinogenesis.

Gut dysbiosis is observed in chronic hepatobiliary diseases and is frequently associated with liver carcinogenesis; however, the extent and specific mechanisms triggered by alterations in the microbiota mediating tumorigenesis in these patients remain unclear. Here we show that Enterococcus faecalis is abundant in the microbiota of patients with hepatitis C virus-related chronic liver disease. Xenotransplantation of gut microbiota from these patients increased the number of spontaneous liver tumors in mice and enhanced susceptibility to liver carcinogens. Hepatic colonization by gelE-positive E. faecalis increased liver expression of proliferative genes in a TLR4-Myd88-dependent manner, leading to liver tumorigenesis. Moreover, decreased fecal deoxycholic acid levels were associated with colonization by E. faecalis. Overall, these data identify E. faecalis as a key promoter of liver carcinogenesis.

Fatty acid-driven modifications in T-cell profiles in non-alcoholic fatty liver disease patients.

BACKGROUND: The interaction between T-cells/fatty acids involved in non-alcoholic fatty liver disease (NAFLD) and liver fibrosis progression is poorly understood. In this study, we conducted a comprehensive analysis of T-cell profiles of NAFLD patients to better understand their relationship with fatty acids and relevance to liver fibrosis. METHODS: We analyzed the differences in T-cell profiles of peripheral blood mononuclear cells (PBMCs) between 40 NAFLD patients and 5 healthy volunteers (HVs), and their relationship with liver fibrosis stage or progression. Moreover, we analyzed the relationship between T-cell profiles and fatty acid compositions in vivo, and changes in T-cell profiles after treatment with fatty acids in vitro. RESULTS: T-cell profiles of NAFLD patients were different from those of HVs. The CD25+CD45+CD4+ T-cell frequency was increased in NAFLD patients with high liver fibrosis stage and progression, and this indicated immune activation. Despite such a state of immune activation, the PD1+CD4+ T-cell frequency was decreased in the same patients group. The PD1+CD4+ T-cell frequency had a significantly negative correlation with the serum fatty acid composition ratio C16:1n7/C16:0. Moreover, the PD1+CD4+ T-cell frequency was significantly decreased by in vitro treatment with fatty acids. In addition, its rate of frequency change was significantly different between C16:0 and C16:1n7 and decreased by artificially increasing the C16:1n7/C16:0 ratio. CONCLUSIONS: The analysis of PBMCs in NAFLD patients showed that T-cell profiles were different from those of HVs. And, it suggested that fatty acids modified T-cell profiles and were involved in liver fibrosis in NAFLD patients.

Inactivation of Transcriptional Repressor Capicua Confers Sorafenib Resistance in Human Hepatocellular Carcinoma.

BACKGROUND & AIMS: Sorafenib is a multireceptor tyrosine kinase inhibitor that can prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Although most HCC patients who receive sorafenib ultimately show disease progression, it still is unclear whether and how HCC cells acquire chemoresistance during sorafenib treatment in human beings. METHODS: We analyzed surgically resected HCC tissues from a patient who received sorafenib for prevention of HCC recurrence after surgery (Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial) and established patient-derived HCC cells. Whole-exome sequence analysis was performed to detect mutations in sorafenib-resistant clones. We examined 30 advanced HCC cases immunohistochemically and 140 HCC cases enrolled in the Adjuvant Sorafenib for Hepatocellular Carcinoma after Resection or Ablation trial using microarray analysis to evaluate the association of Capicua Transcriptional Repressor (CIC) status with sorafenib treatment response. RESULTS: We found a CIC mutation in recurrent HCC specimens after sorafenib. CIC encodes Capicua, a general sensor of receptor tyrosine kinase signaling. HCC cells established from the recurrent tumor specimen showed chemoresistance to sorafenib in vitro and in vivo. Established sorafenib-resistant Huh1 and Huh7 cell lines showed reduced expression of Capicua without mutations. Immunohistochemical analysis showed that HCC patients with low Capicua expression showed poor overall survival. Microarray analysis showed that the CIC gene signature could predict the preventive effect of adjuvant sorafenib treatment on HCC recurrence. Intriguingly, although CIC knockdown induced sorafenib resistance in HCC cell lines, regorafenib suppressed growth of sorafenib-resistant, Capicua-inactivated HCC cells and inhibited extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: Evaluation of Capicua status may be pivotal to predict response to sorafenib, and regorafenib treatment could be effective to treat HCC with functional Capicua impairment.

MicroRNA-10a Impairs Liver Metabolism in Hepatitis C Virus-Related Cirrhosis Through Deregulation of the Circadian Clock Gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocator-Like 1.

The circadian rhythm of the liver plays an important role in maintaining its metabolic homeostasis. We performed comprehensive expression analysis of microRNAs (miRNAs) using TaqMan polymerase chain reaction of liver biopsy tissues to identify the miRNAs that are significantly up-regulated in advanced chronic hepatitis C (CHC). We found miR-10a regulated various liver metabolism genes and was markedly up-regulated by hepatitis C virus infection and poor nutritional conditions. The expression of miR-10a was rhythmic and down-regulated the expression of the circadian rhythm gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1) by directly suppressing the expression of RA receptor-related orphan receptor alpha (RORA). Overexpression of miR-10a in hepatocytes blunted circadian rhythm of Bmal1 and inhibited the expression of lipid synthesis genes (sterol regulatory element binding protein [SREBP]1, fatty acid synthase [FASN], and SREBP2), gluconeogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha [PGC1α]), protein synthesis (mammalian target of rapamycin [mTOR] and ribosomal protein S6 kinase [S6K]) and bile acid synthesis (liver receptor homolog 1 [LRH1]). The expression of Bmal1 was significantly correlated with the expression of mitochondrial biogenesis-related genes and reduced Bmal1 was associated with increased serum alanine aminotransferase levels and progression of liver fibrosis in CHC. Thus, impaired circadian rhythm expression of Bmal1 by miR-10a disturbs metabolic adaptations, leading to liver damage, and is closely associated with the exacerbation of abnormal liver metabolism in patients with advanced CHC. In patients with hepatitis C-related liver cirrhosis, liver tissue miR-10a levels were significantly associated with hepatic reserve, fibrosis markers, esophageal varix complications, and hepatitis C-related hepatocellular carcinoma recurrence. Conclusion: MiRNA-10a is involved in abnormal liver metabolism in cirrhotic liver through down-regulation of the expression of the circadian rhythm gene Bmal1. Therefore, miR-10a is a possible useful biomarker for estimating the prognosis of liver cirrhosis.