Maria Isabel Fiel

Pivotal Role of mTOR Signaling in Hepatocellular Carcinoma

BACKGROUND & AIMS The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway. METHODS The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model. RESULTS Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade. CONCLUSIONS MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

Autophagy Releases Lipid That Promotes Fibrogenesis by Activated Hepatic Stellate Cells in Mice and in Human Tissues

BACKGROUND & AIMS The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells. METHODS We analyzed hepatic stellate cells from C57BL/6 wild-type, Atg7(F/F), and Atg7(F/F)-GFAP-Cre mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl(4) or thioacetamide (TAA); liver tissues and stellate cells were analyzed. Autophagy was blocked in fibrogenic cells from liver and other tissues using small interfering RNAs against Atg5 or Atg7 and chemical antagonists. Human pulmonary fibroblasts were isolated from samples of lung tissue from patients with idiopathic pulmonary fibrosis or from healthy donors. RESULTS In mice, induction of liver injury with CCl(4) or TAA increased levels of autophagy. We also observed features of autophagy in activated stellate cells within injured human liver tissue. Loss of autophagic function in cultured mouse stellate cells and in mice following injury reduced fibrogenesis and matrix accumulation; this effect was partially overcome by providing oleic acid as an energy substrate. Autophagy also regulated expression of fibrogenic genes in embryonic, lung, and renal fibroblasts. CONCLUSIONS Autophagy of activated stellate cells is required for hepatic fibrogenesis in mice. Selective reduction of autophagic activity in fibrogenic cells in liver and other tissues might be used to treat patients with fibrotic diseases.

A system of classifying microvascular invasion to predict outcome after resection in patients with hepatocellular carcinoma.

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) recurs in approximately 70% of cases after resection. Vascular invasion by tumor cells can be classified as gross or microscopic (microvascular invasion [mVI]) and is a risk factor for recurrence. We examined a large cohort of patients with HCC who were treated by resection to identify features of mVI that correlated with recurrence and survival. METHODS We reviewed the records of all HCC resections performed at the Mount Sinai School of Medicine between January 1990 and March 2006 to identify those with mVI, established by histologic analysis. The numbers and sizes of vessels invaded, invasion of a vessel with a muscular wall, distance from the tumor, and satellite nodules were recorded. RESULTS Of the 384 patients who underwent resection for HCC, 131 (34.1%) met the entry criteria. The median follow-up period was 28.9 months. There were 68 recurrences and 54 deaths. In multivariate analysis, invasion of a vessel with a muscular wall predicted recurrence (hazard ratio, 1.8; P = .02), and invasion of a vessel with a muscular wall (hazard ratio, 2.2; P = .018) and invasion of a vessel that was more than 1 cm from the tumor (hazard ratio, 2.1; P = .015) predicted survival. A risk score that assigned points for the presence of each variable correlated with recurrence (P = .028) and survival (P < .0001). CONCLUSIONS A novel classification system that includes invasion of a vessel with a muscular wall and invasion of a vessel that is more than 1 cm from the tumor can accurately predict risk of recurrence and survival of patients with mVI after resection of HCC.

OSTEOPONTIN, AN OXIDANT STRESS-SENSITIVE CYTOKINE, UP-REGULATES COLLAGEN-I VIA INTEGRIN αVβ3 ENGAGEMENT AND PI3K-pAkt-NFκB SIGNALING

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen‐I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro‐fibrogenic phenotype and Collagen‐I expression. Recombinant OPN (rOPN) up‐regulated Collagen‐I protein in primary HSCs in a transforming growth factor beta (TGFβ)–independent fashion, whereas it down‐regulated matrix metalloprotease‐13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α‐smooth muscle actin (αSMA) expression and enhanced their invasive and wound‐healing potential. HSCs isolated from wild‐type (WT) mice were more profibrogenic than those from OPN knockout (Opn−/−) mice and infection of primary HSCs with an Ad‐OPN increased Collagen‐I, indicating correlation between both proteins. OPN induction of Collagen‐I occurred via integrin αvβ3 engagement and activation of the phosphoinositide 3‐kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)–signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70‐kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin αvβ3 prevented the OPN‐mediated activation of the PI3K/pAkt/NFκB–signaling cascade and Collagen‐I up‐regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN‐mediated Collagen‐I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen‐I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl4 injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up‐regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (OpnHEP Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl4 injection and TAA treatment caused more liver fibrosis to WT than to Opn−/− mice and the reverse occurred in OpnHEP Tg mice. Conclusion: OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen‐I protein contributing to scarring and liver fibrosis. (HEPATOLOGY 2012)

Dendritic cell regulation of carbon tetrachloride–induced murine liver fibrosis regression

Although hepatic fibrosis typically follows chronic inflammation, fibrosis will often regress after cessation of liver injury. In this study, we examined whether liver dendritic cells (DCs) play a role in liver fibrosis regression using carbon tetrachloride to induce liver injury. We examined DC dynamics during fibrosis regression and their capacity to modulate liver fibrosis regression upon cessation of injury. We show that conditional DC depletion soon after discontinuation of the liver insult leads to delayed fibrosis regression and reduced clearance of activated hepatic stellate cells, the key fibrogenic cell in the liver. Conversely, DC expansion induced either by Flt3L (fms‐like tyrosine kinase‐3 ligand) or adoptive transfer of purified DCs accelerates liver fibrosis regression. DC modulation of fibrosis was partially dependent on matrix metalloproteinase (MMP)‐9, because MMP‐9 inhibition abolished the Flt3L‐mediated effect and the ability of transferred DCs to accelerate fibrosis regression. In contrast, transfer of DCs from MMP‐9–deficient mice failed to improve fibrosis regression. Conclusion: Taken together, these results suggest that DCs increase fibrosis regression and that the effect is correlated with their production of MMP‐9. The results also suggest that Flt3L treatment during fibrosis resolution merits evaluation to accelerate regression of advanced liver fibrosis. (HEPATOLOGY 2012;55:243–255)

Risk for immune-mediated graft dysfunction in liver transplant recipients with recurrent HCV infection treated with pegylated interferon.

BACKGROUND & AIMS Patients with recurrent hepatitis C virus infection treated with pegylated interferon (PEG) after liver transplantation can develop severe immune-mediated graft dysfunction (IGD) characterized by plasma cell hepatitis or rejection. METHODS We conducted a multicenter case-control study of 52 liver transplant recipients with hepatitis C to assess the incidence of, risk factors for, and outcomes of PEG-IGD. Data from each patient were compared with those from 2 matched patients who did not develop PEG-IGD (n = 104). We performed a multivariate analysis of risk factors and analyzed treatment and outcomes of graft dysfunction subtypes. RESULTS Overall incidence of PEG-IGD during a 10-year study period was 7.2%. Risk factors included no prior PEG therapy (odds ratio = 5.3; P < .0001), therapy with PEGα-2a (odds ratio = 4.7; P = .03), and immune features (mainly plasma cell hepatitis) on pre-PEG therapy liver biopsies (odds ratio = 3.9; P = .005). The PEG-IGD group had lower long-term patient (61.5% vs 91.3% of controls) and graft (38.5% vs 85.6% of controls) survival and higher rates of retransplantation (34.6% vs 6.7% of controls) (all, P < .0001), without increases in sustained virologic response. Variables associated with increased mortality included acute rejection as the PEG-IGD sub-type (hazard ratio [HR] = 2.4; P = .002), a high level of alkaline phosphatase at PEG initiation (HR = 1.003; P = .005), and lack of a sustained virologic response (HR = 3.3; P = .04). Variables associated with graft failure included a high level of alkaline phosphatase at PEG initiation (HR = 1.002; P = .04) and lack of a sustained virologic response (HR = 2.1; P = .04). CONCLUSIONS PEG-IGD has high morbidity and mortality and is not associated with increased rates of virologic response. It is important to avoid PEG therapy in liver transplant recipients with specific clinical, biochemical, and histologic risk factors for PEG-IGD.

Impact of donor age on survival and fibrosis progression in patients with hepatitis C undergoing liver transplantation using HCV+ allografts

Studies have suggested that the use of hepatitis C virus (HCV)‐positive (HCV+) donor allografts has no impact on survival. However, no studies have examined the effect that HCV+ donor histology has upon recipient and graft survival. We evaluated the clinical outcome and impact of histological features in HCV patients transplanted using HCV+ livers. We reviewed all patients transplanted for HCV at our institution from 1988 to 2004; 39 received HCV+ allografts and 580 received HCV‐negative (HCV−) allografts. Survival curves compared graft and patient survival. Each HCV+ allograft was stringently matched to a control of HCV− graft recipients. No significant difference in survival was noted between recipients of HCV+ livers and controls. Patients receiving HCV+ allografts from older donors (age ≥50 yr) had higher rates of graft failure (hazard ratio, 2.74) and death rates (hazard ratio, 2.63) compared to HCV− allograft recipients receiving similarly‐aged older donor livers. Matched case‐control analysis revealed that recipients of HCV+ allografts had more severe fibrosis post‐liver transplantation than recipients of HCV− livers (P = 0.008). More advanced fibrosis was observed in HCV+ grafts from older donors compared to HCV+ grafts from younger donors (P = 0.012). In conclusion, recipients of HCV+ grafts from older donors have higher rates of death and graft failure, and develop more extensive fibrosis than HCV− graft recipients from older donors. Recipients of HCV+ grafts, regardless of donor age, develop more advanced liver fibrosis than recipients of HCV− grafts. Liver Transpl 12:1496‐1503, 2006.

The presence of JAK2V617F mutation in the liver endothelial cells of patients with Budd-Chiari syndrome.

Patients with myeloproliferative disorders are at a high risk of developing thrombotic events. Several investigators have hypothesized that endothelial cell (EC) abnormalities might contribute to this prothrombotic state. Budd-Chiari syndrome (BCS) and portal vein thrombosis have been reported to be associated with JAK2V617F-positive hematopoiesis. We explored whether JAK2V617F was present in ECs in the vessels of polycythemia vera (PV) patients with BCS using laser capture microdissection followed by nested polymerase chain reaction or reverse-transcribed polymerase chain reaction. The ECs of the 2 BCS patients with PV were homozygous for the JAK2V617F and were shown to express transcripts characteristic of ECs but not hematopoietic cells. ECs of the other BCS patient with PV and 2 patients with hepatoportal sclerosis without PV contained exclusively wild-type JAK2. The presence of JAK2V617F in both ECs and hematopoietic cells belonging to BCS patients with PV indicate that ECs in PV are involved by the malignant process and that in a subpopulation of the patients the disease might originate from a common cell of origin for hematopoietic and ECs.

High Mobility Group Box-1 (HMGB1) Participates in the Pathogenesis of Alcoholic Liver Disease (ALD)

Background: HMGB1 is a proinflammatory cytokine produced in response to tissue injury, but its role in ALD is unknown. Results: HMGB1 increases; translocates; and undergoes acetylation, phosphorylation, and oxidation in ALD. HMGB1 ablation in hepatocytes protects against steatosis and injury in ALD. Conclusion: HMGB1 plays a key role in ALD. Significance: Dissecting how the increase in HMGB1 causes hepatotoxicity is key for understanding the pathogenesis of ALD. Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5′AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.

A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice

We have developed a novel model for depleting mouse hepatic stellate cells (HSCs) that has allowed us to clarify their contributions to hepatic injury and fibrosis. Transgenic (Tg) mice expressing the herpes simplex virus thymidine kinase gene (HSV‐Tk) driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir (GCV). Effects of GCV were explored in primary HSCs and in vivo. Panlobular damage was provoked to maximize HSC depletion by combining CCl4 (centrilobular injury) with allyl alcohol (AA) (periportal injury), as well as in a bile duct ligation (BDL) model. Cell depletion in situ was quantified using dual immunofluorescence (IF) for desmin and GFAP. In primary HSCs isolated from both untreated wild‐type (WT) and Tg mice, GCV induced cell death in ∼50% of HSCs from Tg, but not WT, mice. In TG mice treated with CCl4+AA+GCV, there was a significant decrease in GFAP and desmin‐positive cells, compared to WT mice (∼65% reduction; P < 0.01), which was accompanied by a decrease in the expression of HSC‐activation markers (alpha smooth muscle actin, beta platelet‐derived growth factor receptor, and collagen I). Similar results were observed after BDL. Associated with HSC depletion in both fibrosis models, there was marked attenuation of fibrosis and liver injury, as indicated by Sirius Red/Fast Green, hematoxylin and eosin quantification, and serum alanine/aspartate aminotransferase. Hepatic expression of interleukin‐10 and interferon‐gamma was increased after HSC depletion. No toxicity of GCV in either WT or Tg mice accounted for the differences in injury. Conclusion: Activated HSCs significantly amplify the response to liver injury, further expanding this cell types repertoire in orchestrating hepatic injury and repair. (HEPATOLOGY 2013)