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Background & Aims The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types due to its ability to promote the degradation of numerous oncogenic target proteins. Herein, we aimed to elucidate its role in intrahepatic cholangiocarcinoma (iCCA). Methods Herein, we first confirmed that FBXW7 gene expression was reduced in human iCCA specimens. To identify the molecular mechanisms by which FBXW7 dysfunction promotes cholangiocarcinogenesis, we generated a mouse model by hydrodynamic tail vein injection of Fbxw7ΔF, a dominant negative form of Fbxw7, either alone or in association with an activated/myristylated form of AKT (myr-AKT). We then confirmed the role of c-MYC in human iCCA cell lines and its relationship to FBXW7 expression in human iCCA specimens. Results FBXW7 mRNA expression is almost ubiquitously downregulated in human iCCA specimens. While forced overexpression of Fbxw7ΔF alone did not induce any appreciable abnormality in the mouse liver, co-expression with AKT triggered cholangiocarcinogenesis and mice had to be euthanized by 15 weeks post-injection. At the molecular level, a strong induction of Fbxw7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-MYC was consistently confirmed as a FBXW7 target in human CCA cell lines. Most importantly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 only delayed tumorigenesis in the same model. In human iCCA specimens, an inverse correlation between the expression levels of FBXW7 and c-MYC transcriptional activity was observed. Conclusions Downregulation of FBXW7 is ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice via c-Myc-dependent mechanisms. Targeting c-MYC might represent an innovative therapy against iCCA exhibiting low FBXW7 expression. Lay summary There is mounting evidence that FBXW7 functions as a tumor suppressor in many cancer types, including intrahepatic cholangiocarcinoma, through its ability to promote the degradation of numerous oncoproteins. Herein, we have shown that the low expression of FBXW7 is ubiquitous in human cholangiocarcinoma specimens. This low expression is correlated with increased c-MYC activity, leading to tumorigenesis. Our findings suggest that targeting c-MYC might be an effective treatment for intrahepatic cholangiocarcinoma.

Background & Aims Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. Methods We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo . Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. Results Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro , inducing strong ICC cell apoptosis and only marginally affecting proliferation. Conclusions This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Background & Aims Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. Results Overexpression of FASN , either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT -overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with a strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway.

Background & Aims Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo . Methods Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. Results The transgenic mice revealed SHH expression in 2–5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53 R172H and KRAS G12D , we found that the SHH expression promoted the transition from HCA to HCC. Conclusions SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.

Background/Aims: An increase in proliferative activity and other distinct hepatocellular alterations — resembling preneoplastic foci and progressing to hepatocellular tumors — have been shown to develop in liver acini draining the blood from islets of Langerhans, transplanted through the portal vein into the liver of streptozotocin-diabetic rats. Methods: Altered and unaltered liver acini were investigated for possible changes in hepatic stellate cells 4–76 days after islet transplantation. Results: Corresponding to a significant increase in the hepatocellular volume, the volume density of total non-parenchymal cells was significantly reduced in altered compared to unaltered liver acini. With regard to the total nonparenchymal cell volume, the hepatic stellate cell fraction was not different, whereas the fraction of Kupffer cells was significantly reduced and the fraction of sinusoidal endothelial cells was significantly increased in altered compared to unaltered liver acini, respectively. The volume density as well as the single volume of the hepatic stellate cell mitochondria increased significantly in altered compared to unaltered liver acini. Hepatic stellate cell lipid droplets did not show significant differences between altered and unaltered liver acini. In situ hybridization for hepatocyte growth factor mRNA showed no differences in intensity of the specific signals in hepatic stellate cells of altered versus unaltered liver acini. The transplanted islets were negative for hepatic growth factor mRNA. Conclusions: The results suggest that hepatic growth factor production by hepatic stellate cells or by islet cells is not relevant to hepatocellular proliferative activity in altered liver acini.

Background & Aims Activation of the AKT/mTOR and Ras/MAPK pathways and the lipogenic phenotype occurs in both a rat model of insulin-induced hepatocarcinogenesis and in human hepatocellular carcinoma (HCC). In the rat model, activation of these pathways is evident within the earliest morphologic detectable alterations, i.e., clear cell foci (CCF) of altered hepatocytes. CCF have also been described in the human liver, but molecular and metabolic alterations within these foci remain to be determined. Methods A collection of human liver specimens was examined using electron microscopy, histology, enzyme- and immunohistochemistry, and molecular analysis. Human data were compared to rat preneoplastic CCF and HCC induced by N -nitrosomorpholine administration. Results CCF occurred in ∼33% of extrafocal tissues of human non-cirrhotic livers. Electron microscopy showed massive glycogen storage within CCF, largely due to the reduced activity of the glycogenolytic enzyme glucose-6-phosphatase. Hepatocytes in CCF overexpressed the insulin receptor and glucose transporter proteins. AKT/mTOR and Ras/MAPK pathways as well as enzymes of glycolysis, de novo lipogenesis, beta-oxidation, and cholesterol synthesis were upregulated, both in human CCF, and in CCF and HCC of N -nitrosomorpholine-treated rats. The Ki-67 proliferation index was 2-fold higher in human CCF than in extrafocal tissue. Conclusions The high degree of similarity between human CCF and pre-neoplastic lesions from experimental models of hepatocarcinogenesis in terms of morphologic, molecular and metabolic features suggests a low-grade dysplastic nature of these lesions in human non-cirrhotic livers.

Background/Aims: It has previously been shown in rat liver that the gap junctional proteins connexin32 and connexin26 are downregulated when murine hepatocytes are in the S-phase of the cell cycle. Therefore, it has been hypothesized that loss of functional gap junctions could affect proliferation of hepatocytes. This study aimed to check this hypothesis. Methods: We searched for differences in liver regeneration after two-thirds partial hepatectomy between connexin32-deficient and wild-type mice. Results: The ratio of liver to body weight in regenerating liver was not affected by loss of the connexin32 gene. The peak of DNA synthesis occurred at the same time, i.e. 36 to 96 h after partial hepatectomy, in connexin32-deficient and wild-type liver. During this time, however, only about half as many nuclei of hepatocytes in connexin32-deficient liver incorporated bromodeoxyuridine, compared to wild-type liver. Furthermore, 1–2 weeks after full recovery of liver mass, we detected a higher level of bromodeoxyuridine incorporation into hepatocytes of connexin32-deficient than in wild-type liver. Conclusions: Loss of connexin32 protein and/or diminished expression of connexin26 did not promote G0/1-S transition of hepatocytes in two-thirds hepatectomized mouse livers. Instead, the extent of synchronous initiation and termination of DNA synthesis in regenerating liver was altered in connexin32-deficient mice.

The incidence of steatosis in livers retrieved for organ transplantation is up to 30%. Due to the shortage of donor organs, many of these livers are accepted for clinical transplantation, although a high rate of graft dysfunction is associated with ischemic preservation of steatotic livers. The present study was intended to reduce the ischemia/reperfusion injury of steatotic grafts by the use of venous systemic oxygen persufflation during cold storage.A histologically-documented mild to moderate steatosis was induced in livers of Wistar rats by fasting for 2 days and subsequent feeding of a fat-free diet enriched in carbohydrates. Fatty livers were retrieved and flushed via the portal vein with 60 ml of HTK. In group A, livers were then stored ischemically at 4 degrees C for 24 h. Livers of group B were additionally connected to a gaseous oxygen supply and persufflated with O2 via the venous vascular system during the cold storage period. Viability of the livers was then assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer.Venous systemic oxygen sufflation resulted in a relevant and significant reduction of parenchymal (ALT: 132+/-90 vs 434+/-172 U/l; p0.01) and mitochondrial (GLDH: 116+/-57 vs 633+/-241 U/l; p0.001) enzyme release during reperfusion. Moreover, Kupffer cell activation, as evaluated from acid phosphatase activity in the perfusate, was reduced to about 1/3 (4.0+/-1.3 vs 11.9+/-5.3 U/l; p0.01). Electron microscopic analysis revealed that the liver mitochondria and sinusoidal endothelial lining were better preserved after oxygen persufflation, which was in line with the data on enzyme release and the increased portal perfusion pressure in the untreated group, while normal values were found after venous systemic oxygen sufflation.Venous oxygen persufflation may thus represent a useful tool for the safe and improved preservation of ischemia-sensitive steatotic livers.