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Supplementary Figure 4 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure 2 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure Legends from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib. Mol Cancer Ther; 10(6); 1007–17. ©2011 AACR.

Supplementary Figure 3 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Supplementary Figure 1 from Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Background: While osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is the standard treatment in patients with advanced non-small-cell lung cancer (NSCLC) with sensitising EGFR and acquired T790M mutations, progression inevitably occurs. The angiogenic pathway is implicated in EGFR TKI resistance. Patients and methods: BOOSTER is an open-label randomised phase II trial investigating the efficacy and safety of combined osimertinib 80 mg daily and bevacizumab 15 mg/kg every 3 weeks, versus osimertinib alone, in patients with EGFR-mutant advanced NSCLC and acquired T790M mutations after failure on previous EGFR TKI therapy. Primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints were overall survival (OS), objective response rate (ORR) and adverse events (AEs). Results: Between May 2017 and February 2019, 155 patients were randomised (combination: 78; osimertinib: 77). At data cut-off of 22 February 2021, median follow-up was 33.8 months [interquartile range (IQR): 26.5-37.6 months] and 129 (83.2%) PFS events were reported in the intention-to-treat population. There was no difference in median PFS between the combination [15.4 months; 95% confidence interval (CI) 9.2-18.0 months] and osimertinib arm (12.3 months; 95% CI 6.2-17.2 months; stratified log-rank P = 0.83), [hazard ratio (HR) = 0.96; 95% CI 0.68-1.37]. Median OS was 24.0 months (95% CI 17.8- 32.1 months) in the combination arm and 24.3 months (95% CI 16.9-37.0 months) in the osimertinib arm (stratified log-rank P = 0.91), (HR = 1.03; 95% CI 0.67-1.56). Exploratory analysis revealed a significant interaction of smoking history with treatment for PFS (adjusted P = 0.0052) with a HR of 0.52 (95% CI 0.30-0.90) for smokers, and 1.47 (95% CI 0.92-2.33) for never smokers. ORR was 55% in both arms and the median time to treatment failure was significantly shorter in the combination than in the osimertinib arm, 8.2 months versus 10.8 months, respectively (P = 0.0074). Safety of osimertinib and bevacizumab was consistent with previous reports with grade >= 3 treatment-related AEs (TRAEs) reported in 47% and 18% of patients on combination and osimertinib alone, respectively. Conclusions: No difference in PFS was observed between osimertinib plus bevacizumab and osimertinib alone. Grade >= 3 TRAEs were more common in patients on combination.

AIM Non-alcoholic fatty liver disease (NAFLD) patients benefit from physical exercise. This study aimed to investigate the effect of acute exercise on hepatic gene expression in different mouse models of NAFLD. METHODS C57BL/6J mice were fed with a control (CD) or a high fat (HFD) diet. AlbCrePtenflox/flox (Pten-KO) and Fxr-/- mice, two genetic models of NAFLD with insulin hypersensitivity and resistance, respectively, were fed with CD. After 4 weeks, mice were randomly assigned to exercise or sedentariness. Mice were killed 15 min or 3 h after the running/sedentary period. Genome-wide hepatic gene expression was evaluated with the Illumina Micro-array platform. Quantitative polymerase chain reaction confirmed changes in gene expression. RESULTS Acute exercise transiently affected the expression of genes involved in the immune response in C57BL/6 mice fed with CD and this effect normalized in the recovery phase. Acute exercise affected genes involved in gluconeogenesis in the insulin resistant Fxr-/- model. Genes involved in lipid metabolism were affected in C57BL/6 mice fed with CD, but not in mouse models of NAFLD. Genes involved in DNA damage response pathways were deregulated only in C57BL/6 mice fed with CD and not in mouse models of NAFLD. CONCLUSION The simultaneous analysis of different NAFLD models revealed that an acute exercise bout affects hepatic gene expression differentially according to animal models and that most of the differentially expressed genes are involved in glucose and fatty acid metabolism, immune regulation, and DNA damage response.

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth.

In regenerating liver, hepatocytes accumulate lipids before the major wave of parenchymal growth. This transient, regeneration-associated steatosis (TRAS) is required for liver recovery, but its purpose is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is a key inhibitor of the protein kinase B/mammalian target of rapamycin axis that regulates growth and metabolic adaptations after hepatectomy. In quiescent liver, PTEN causes pathological steatosis when lost, whereas its role in regenerating liver remains unknown. Here, we show that PTEN down-regulation promotes liver growth in a TRAS-dependent way. In wild-type mice, PTEN reduction occurred after TRAS formation, persisted during its disappearance, and correlated with up-regulated β-oxidation at the expense of lipogenesis. Pharmacological modulation revealed an association of PTEN with TRAS turnover and hypertrophic liver growth. In liver-specific Pten(-/-) mice shortly after induction of knockout, hypertrophic regeneration was accelerated and led to hepatomegaly. The resulting surplus liver mass was functional, as demonstrated by raised survival in a lethal model of resection-induced liver failure. Indirect calorimetry revealed lipid oxidation as the primary energy source early after hepatectomy. The shift from glucose to lipid usage was pronounced in Pten(-/-) mice and correlated with the disappearance of TRAS. Partial inhibition of β-oxidation led to persisting TRAS in Pten(-/-) mice and abrogated hypertrophic liver growth. PTEN down-regulation may promote β-oxidation through β-catenin, whereas hypertrophy was dependent on mammalian target of rapamycin complex 1. CONCLUSION PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

Background & Aims Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD. Methods PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD. Results Genotype frequencies were significantly different between NAFLD-HCC cases (CC=28, CG=43, GG=29) and NAFLD-controls (CC=125, CG=117, GG=33) ( p =0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23–4.14], p =0.0082), with GG homozygotes exhibiting a 5-fold [1.47–17.29], p =0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n=1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55–4.10], p =0.0002; GG vs. CC: OR 12.19 [6.89–21.58], p Conclusions Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

Background & Aims The combination of erlotinib with sorafenib is currently being investigated in a phase III RCT. We studied the effect of erlotinib and sorafenib on HCC in a preclinical model. Methods The Morris Hepatoma (MH) and HepG2 cells were treated in vitro with sorafenib (1–10μM) and erlotinib (1–5μM) and evaluated for tumor cell viability, apoptosis, and target regulation. Antiangiogenic effects were studied by measuring VEGF levels, endothelial cell viability, apoptosis, migration, and the aortic ring assay. In vivo , MH cells were implanted into the liver of syngeneic rats and treated with vehicle, sorafenib 5–10mg/kg, erlotinib 10mg/kg, and respective combinations. Results In vitro , erlotinib downregulated p-ERK but showed no significant effect on tumor cell viability in MH and HEPG2 cells. Despite a similar target regulation, sorafenib significantly reduced cell viability of HCC cells by induction of apoptosis, in a dose-dependent manner (11±5%; 20±10%; 51±5% for sorafenib 1, 5, 10μM). No additional effect was observed upon combination with erlotinib. Of note, erlotinib treatment resulted in endothelial cell migration and vascular sprouting of aortic rings through induction of VEGF mRNA and protein levels in endothelial and tumor cells, which was blocked by sorafenib. In vivo , erlotinib had no single agent antitumor activity, raised serum-VEGF levels, and lacked a synergistic effect in combination with sorafenib. Conclusions Erlotinib had no antitumor effect on HCC in vitro nor in vivo, but induced VEGF, which may reflect a resistance mechanism to erlotinib monotherapy. No improvement of sorafenib efficacy was observed upon combination with erlotinib.

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O2 (hypoxic) or 21% O2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial β-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60±3% compared with 50±2% in controls; P

The process of blood vessel proliferation, known as angiogenesis, is essential during embryonic development and organogenesis. In adult life, it participates in normal tissue repair, wound healing, and cyclical growth of the corpus luteum and the endometrium. Crucial as it is, angiogenesis can become pathological, and abnormal angiogenesis contributes to the pathogenesis of inflammatory and neoplasic diseases. The present review highlights the evidence for the role of angiogenesis in HCC (hepatocellular carcinoma) and discusses the increasing importance of inhibitors of angiogenesis in HCC therapy.

Background/Aims: Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model. Methods: Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays. Results: Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed. Conclusions: mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment. � 2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective bile acid frequently prescribed to patients with cholestatic diseases. Several mechanisms of action have been investigated, but the possibility that cyclic adenosine monophosphate responsive element binding protein (CREB), a transcription factor promoting cell survival, mediates TUDCA's protective effects has not been considered. We examined whether TUDCA activates CREB and whether this activation can protect biliary epithelial cells. Cholangiocytes were stressed by exposure to CCI-779, which inhibits signaling though the kinase mTOR (mammalian target of rapamycin), resulting in cell cycle arrest and apoptosis. Incubation of normal rat cholangiocytes (NRC) cells, with TUDCA resulted in phosphorylation of CREB (Western blotting analysis) and activation of CREB transcription activity (luciferase reporter assay). Inhibition of calcium signals and inhibition of protein kinase C prevented the TUDCA-induced activation of CREB. CCI-779 decreased the viability of rat cholangiocytes in a dose-dependent manner (MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay). TUDCA protected against CCI-779 cytotoxicity. A dominant negative form of CREB was stably transduced in NRC cells (NRC-M1). TUDCA protection was decreased in NRC-M1. While CCI-779 induced apoptosis in NRC cells as determined by caspase 3 activity, TUDCA attenuated CCI-779-induced apoptosis, an effect absent in NRC-M1. Finally, CCI-779 blocked proliferation of both NRC and NRC-M1 (thymidine incorporation) and this was unaffected by TUDCA. In conclusion, TUDCA activates CREB in cholangiocytes, reducing the apoptotic effect of CCI-779. These findings suggest a novel cytoprotective mechanism for this bile acid.

BACKGROUND AIMS: Unhealthy lifestyles predispose people to non alcoholic steatohepatitis (NASH) which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity it is unknown whether regular exercise reduces the risk of developing HCC. Therefore we studied the effect of regular exercise on the development of HCC in male hepatocyte specific PTEN deficient mice (AlbCrePten(flox/flox)) which develop steatohepatitis and HCC spontaneously. METHODS: Mice were fed a standardized 10 fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day 5 days/week during 32 weeks. RESULTS: After 32 weeks of regular exercise 71 of exercised mice developed nodules larger than 15 mm(3)vs. 100 of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically exercise stimulated the phosphorylation of AMPK and its substrate raptor which decreased the kinase activity of mTOR. CONCLUSIONS: These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo. 2012 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved Although many of us hope for a long life, this wish is undoubtedly tied to the concept of healthy life. Thus, an anticipated thinking is that health is a prerequisite for longevity. This corollary has been recently challenged in a study published in Science, where Lamming and colleagues report that rapamycin extends lifespan of mice by inhibition of mTORC1, despite impairing glucose homeostasis through the disruption of mTORC2 signaling [1]. The mammalian target of rapamycin (mTOR) is a protein kinase linked to two distinct multi-protein complexes: mTORC1 and mTORC2. Within these complexes, the proteins raptor and rictor are bound Journal of Hepatology 20 Received 6 June 2012; received in revised form 26 June 2012; accepted 28 June 2012 * Corresponding author. Tel.: +34 93 260 7280. E-mail address: sara.kozma@uc.edu (S.C. Kozma). to mTORC1 or mTORC2, respectively, while mLST8 is associated with both mTORC1 and mTORC2. mTORC1 regulates pathways involved in cell growth, metabolism and aging, while mTORC2 is involved in cytoskeleton and insulin signaling regulation [2]. Downstream of mTORC1, the ribosomal S6 protein kinase (S6K1) has been identified as a key player for the role of mTORC1 [2]. Genetic attenuation of mTORC1 signaling was shown to extend lifespan in yeast, nematodes, fruit flies, as well as mice [4]. Interestingly, knocking out S6K1 led to improved insulin sensitivity [3] and induced gene expression patterns similar to those seen in caloric restriction [4]. For several years, caloric restriction has been known to increase lifespan in mammals [5]. Caloric restriction reduced mTORC1 signaling but also improved glucose tolerance as well as insulin sensitivity and these effects have been shown to positively influence longevity [5]. Along the line that implies the role of mTORC1 in longevity, the activity of the mTORC1/S6K1 signaling pathway increases with aging, while S6K1 deletion leads to increased lifespan and resistance to agerelated pathologies [4]. On the other hand, mTORC2 is largely insensitive to rapamycin in an acute setting; however, it can be inhibited by chronic exposure to rapamycin [6]. It was demonstrated that chronic treatment with rapamycin caused glucose intolerance in humans and rodents [7,8]. One therefore would predict that the deleterious effects of chronic rapamycin treatment on glucose homeostasis would hamper the life-extending effects of mTORC1 inhibition. However, mice fed rapamycin late in life had their median and maximal lifespan extended [9]. Having this paradox in mind, Lamming and colleagues [10] verified whether chronic rapamycin treatment, at a dose used to increase lifespan, would induce glucose intolerance and increased gluconeogenesis. They demonstrated in vivo that rapamycin not only disrupted the mTORC1 complex in muscle, adipose and liver tissues, but also affected mTORC2 assembling in the same tissues. This was further confirmed by finding reduced phosphorylation of the mTORC2 substrates PKCa-S657, AKT-S473, and NDRG1-T346 in all tissues studied upon refeeding of mice or insulin stimulation [1]. Using hyperinsulinemic-euglycemic clamps, the authors showed that rapamycin specifically affected the efficiency of insulin to suppress hepatic glucose production [1]. By conditionally deleting either raptor or rictor in the mouse liver, or by using livers from rictor knockout animals, the authors established that disruption of mTORC2 signaling, but not mTORC1, impaired glucose tolerance and induced hepatic gluconeogenesis by increased hepatic glucose production [1]. This 12 vol. 57 j 1368–1369 Rapamycin mT ORC1 Longevity mT ORC2 Glucose intolerance Fig. 1. The two sides of a dagger represent both effects of rapamycin treatment: on one side, inhibition of mTORC1 signaling increases lifespan and, on the other side, inhibition of mTORC2 affects glucose homeostasis. JOURNAL OF HEPATOLOGY effect was not further affected by rapamycin treatment. However, livers from raptor knockout mice had normal glucose homeostasis and remained rapamycin sensitive. Together these observations suggested that chronic rapamycin treatment at a dose used to extend lifespan, impaired hepatic glucose homeostasis by induction of gluconeogenesis, an effect mediated by mTORC2 disruption. However, can the paradoxical effects of mTORC1 and mTORC2 inhibition be dissociated? To test that, Lamming and colleagues generated several mice carrying only one allele of different components of the mTORC1 (Mtor , Raptor , Mlst8 ) or double-mutants (Mtor Raptor and Mtor Mlst8 mice). Similar to what described for S6K1 deletion [4], the female Mtor Mlst8 mice showed an increased lifespan and this was accompanied by normal glucose tolerance. Interestingly, although both mTOR and mlst8 are present in both complexes, hepatic mTORC1 signalling was reduced in Mtor Mlst8 female mice, whereas mTORC2 signalling was not affected. This corresponded to a lower raptor–mTOR ratio compared to rictor–mTOR ratio, bringing the authors to hypothesize a recruitment of mTOR and mlst8 to mTORC2 when protein amounts were limited. The data from Lamming and colleagues strongly suggest that selective inhibition of mTORC1 or mTORC2 signaling results in lifespan extension and glucose homeostasis impairment, respectively (Fig. 1). However, it is unclear whether rapamycin treatment extends lifespan by postponing death from age-related pathologies and/or by retarding other cellular mechanisms of aging. For instance, while Harrison et al. [9] showed that mice fed rapamycin live longer, they did not report on the mouse glucose-homeostasis status, their body weight or food intake. However, more recent studies have shown that feeding or intraperitonealy injections of rapamycin reduce body weight and food intake of rodents [6]. Conversely, while Lamming et al. reported a clear effect of rapamycin treatment on glucose homeostasis, the changes in body weight and lifespan under the rapamycin-induced insulin resistance were not determined. Indeed, female Mtor Mlst8 mice, which lived longer, had apparently normal glucose tolerance. All these data together suggest that the ability of mTORC1 inhibition to extend lifespan may involve maintenance of glucose homeostasis. Accordingly, it has been shown that fat-specific insulin receptor knockout mice have reduced fat mass without caloric restriction, which is associated Journal of Hepatology 2012 with increased longevity; possibly through improvement of insulin signaling in the remaining body tissues [10]. The determination of whether the effects of rapamycin treatment on longevity require changes in body weight and composition, food intake, and glucose homeostasis would help gain insight into the mechanism by which mTOR complexes influence lifespan. Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib. Mol Cancer Ther; 10(6); 1007–17. ©2011 AACR.

Background/Aims Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC. Methods Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting. Results Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro , inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation. Conclusions Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Background & Aims: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate–lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. Methods: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. Results: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; kcat:0.0223 s−1; Km:128 μmol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 ± 0.6% vs 57.7 ± 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 ± 2.35 vs 49.7 ± 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 ± 0.13 g vs 0.85 ± 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (−0.42 ± 0.58 log2 vs −0.11 ± 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. Conclusion: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

210 (+)-EPISESAMIN BLOCKS TUMOR CELL INVASIVENESS VIA SUPPRESSED ACTIVATION OF NUCLEAR FACTOR-uB AND ACTIVATOR PROTEIN-1 ALONG WITH DECREASED IL-6 RELEASE AND ENZYMATIC INHIBITION OF MMP-9 C. Freise, U. Erben, U.P. Neumann, D. Seehofer, K.Y. Kim, W. Trowitzsch-Kienast, M. Zeitz, M. Ruehl, R. Somasundaram. Department of Gastroenterology, Infectiology and Rheumatology, Charite Universitatsmedizin Berlin – Campus Benjamin Franklin, Berlin, Department of Surgery, Universitatsklinikum Aachen, Aachen, Department of Surgery, Charite Universitatsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany; Department of Biochemistry, Faculty of Beauty Design, Human Environmental Science College, Wonkwang University, Iksan City, Republic of Korea; Department of Chemical and Pharmaceutical Engineering, Beuth Hochschule fur Technik, Berlin, Germany E-mail: christian.freise@charite.de