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Background and Aims: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors., Approach and Results: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl 4 treatment (Jnk Δhepa + DEN + CCl 4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in Jnk Δhepa + DEN + CCl 4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status., Conclusions: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies., (© 2020 by the American Association for the Study of Liver Diseases.)

Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib., (© 2019 by the American Association for the Study of Liver Diseases.)

Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low-molecular-weight metabolites by new high-throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19-9 (CA 19-9) and alpha-fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy-proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14-15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases., (© 2018 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

In cirrhosis, increased intrahepatic vascular resistance (IHVR) is the primary factor for portal hypertension (PH) development. Hepatic stellate cells (HSCs) play a major role increasing IHVR because, when activated, they are contractile and promote fibrogenesis. Protease-activated receptors (PARs) can activate HSCs through thrombin and factor Xa, which are known PAR agonists, and cause microthrombosis in liver microcirculation. This study investigates the effects of the oral anticoagulant, rivaroxaban (RVXB), a direct antifactor Xa, on HSC phenotype, liver fibrosis (LF), liver microthrombosis, and PH in cirrhotic rats. Hepatic and systemic hemodynamic, nitric oxide (NO) bioavailability, LF, HSC activation, and microthrombosis were evaluated in CCl 4 and thioacetamide-cirrhotic rats treated with RVXB (20 mg/kg/day) or its vehicle for 2 weeks. RVXB significantly decreased portal pressure (PP) in both models of cirrhosis without changes in portal blood flow, suggesting a reduction in IHVR. RVXB reduced oxidative stress, improved NO bioavailability, and ameliorated endothelial dysfunction. Rivaroxaban deactivated HSC, with decreased alpha-smooth muscle actin and mRNA expression of other HSC activation markers. Despite this marked improvement in HSC phenotype, no significant changes in LF were identified. RVXB markedly reduced fibrin deposition, suggesting reduced intrahepatic microthrombosis., Conclusion: RVXB decreases PP in two rat models of cirrhosis. This effect is mostly associated with decreased IHVR, enhanced NO bioavailability, HSC deactivation, and reduced intrahepatic microthrombosis. Our findings suggest that RVXB deserves further evaluation as a potential treatment for cirrhotic PH. (Hepatology 2017;65:2031-2044)., (© 2017 by the American Association for the Study of Liver Diseases.)

Unlabelled: Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10-deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C-X-C chemokine receptor-4 (CXCR4), was reduced in DEN-induced MMP10-deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10-stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal-derived factor-1 (SDF1), through the extracellular signal-regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter., Conclusion: MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: The identification of molecular mechanisms involved in the maintenance of the transformed phenotype of hepatocellular carcinoma (HCC) cells is essential for the elucidation of therapeutic strategies. Here, we show that human HCC cells display an autocrine loop mediated by connective tissue growth factor (CTGF) that promotes DNA synthesis and cell survival. Expression of CTGF was stimulated by epidermal growth factor receptor (EGFR) ligands and was dependent on the expression of the transcriptional coactivator, Yes-associated protein (YAP). We identified elements in the CTGF gene proximal promoter that bound YAP-enclosing complexes and were responsible for basal and EGFR-stimulated CTGF expression. We also demonstrate that YAP expression can be up-regulated through EGFR activation not only in HCC cells, but also in primary human hepatocytes. CTGF contributed to HCC cell dedifferentiation, expression of inflammation-related genes involved in carcinogenesis, resistance toward doxorubicin, and in vivo HCC cell growth. Importantly, CTGF down-regulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 expression and was involved in the reduced sensitivity of these cells toward TRAIL-mediated apoptosis., Conclusion: We have identified autocrine CTGF as a novel determinant of HCC cells' neoplastic behavior. Expression of CTGF can be stimulated through the EGFR-signaling system in HCC cells in a novel cross-talk with the oncoprotein YAP. Moreover, to our knowledge, this is the first study that identifies a signaling mechanism triggering YAP gene expression in healthy and transformed liver parenchymal cells., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a condition characterized by excessive deposition of extracellular matrix. Fibrogenic cells, including hepatic stellate cells and myofibroblasts, are activated in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin (AR) is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. Here, we have addressed the in vivo role of AR in experimental liver fibrosis. To this end, liver fibrosis was induced in AR+/+ and AR-/- mice by chronic CCl(4) administration. Histological and molecular markers of hepatic fibrogenesis were measured. Additionally, the response of cultured human and mouse liver fibrogenic cells to AR was evaluated. We observed that AR was expressed in isolated Kupffer cells and liver fibrogenic cells in response to inflamatory stimuli and platelet-derived growth factor, respectively. We demonstrate that the expression of alpha-smooth muscle actin and collagen deposition were markedly reduced in AR-/- mice compared to AR+/+ animals. AR-/- mice also showed reduced expression of tissue inhibitor of metalloproteinases-1 and connective tissue growth factor, two genes that responded to AR treatment in cultured fibrogenic cells. AR also stimulated cell proliferation and exerted a potent antiapoptotic effect on isolated fibrogenic cells., Conclusion: These results indicate that among the different EGFR ligands, AR plays a specific role in liver fibrosis. AR may contribute to the expression of fibrogenic mediators, as well as to the growth and survival of fibrogenic cells. Additionally, our data lend further support to the role of the EGFR system in hepatic fibrogenesis.

5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases.

The Wilms' tumor suppressor WT1 is a transcriptional regulator present in the fetal but not in the mature liver. Its expression and functional role in liver diseases remains unexplored. In this study, we analyzed WT1 expression by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal and diseased livers. In addition, we performed in vitro studies in isolated rat hepatocytes to investigate WT1 regulation and function. We detected WT1 messenger RNA (mRNA) in 18% of normal livers, 17% of chronic hepatitis with minimal fibrosis, 49% of chronic hepatitis with bridging fibrosis, and 71% of cirrhotic livers. In cirrhosis, WT1 immunoreactivity was localized to the nucleus of hepatocytes. WT1 mRNA abundance correlated inversely with prothrombin time (P =.04) and directly with serum bilirubin (P =.002) and with the MELD score (P =.001) of disease severity. In rats, WT1 expression was present in fetal hepatocytes and in the cirrhotic liver but not in normal hepatic tissue. In vitro studies showed that isolated primary hepatocytes express WT1 when stimulated with transforming growth factor beta (TGF-beta) or when the cells undergo dedifferentiation in culture. Moreover, we found that WT1 down-regulates hepatocyte nuclear factor 4 (HNF-4), a factor that is essential to maintain liver function and metabolic regulation in the mature organ. Hepatic expression of HNF-4 was impaired in advanced human cirrhosis and negatively correlated with WT1 mRNA levels (P =.001). In conclusion, we show that WT1 is induced by TGF-beta and down-regulates HNF-4 in liver cells. WT1 is reexpressed in the cirrhotic liver in relation to disease progression and may play a role in the development of hepatic insufficiency in cirrhosis.

S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5'-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and well-tolerated drug.

We investigated the modulation of rat liver S-adenosylmethionine (SAM) synthetase in a model of acute sepsis. Our results show that animals treated with bacterial lipopolysaccharide experience a marked decrease in liver SAM synthetase activity. No changes were detected in the hepatic levels of SAM synthetase protein, suggesting that inactivation of the existing enzyme was the cause of the observed activity loss. Lipopolysaccharide treatment resulted in the expression of calcium-independent/cytokine-inducible nitric oxide (NO) synthase in liver and the accumulation in plasma of the NO-derived species nitrite and nitrate. NO implication in the in vivo regulation of SAM synthetase was evaluated in animals treated with the NO donor molecule 3-morpholinosydnonimine. The analysis of liver enzymatic activity, along with protein and messenger RNA levels yielded results similar to those obtained with lipopolysaccharide treatment. To assess directly the sensitivity of SAM synthetase to NO, the rat liver-purified high- and low-molecular weight forms of the enzyme were exposed to various doses of 3-morpholinosydnonimine and other NO donors such as S-nitroso-N-acetylpenicillamine, resulting in a dose-dependent inhibition of enzymatic activity. This effect was reversed by addition of the reducing agents beta-mercaptoethanol and glutathione. Finally, cysteine 121 was identified as the site of molecular interaction between NO and rat liver SAM synthetase that is responsible for the inhibition of the enzyme. To reach this conclusion, the 10 cysteine residues of the enzyme were changed to serine by site-directed mutagenesis, and the effect of NO on the various recombinant enzymes was measured.