Nonalcoholic fatty liver disease and hepatocellular carcinoma

Nonalcoholic fatty liver disease and hepatocellular carcinoma

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX Available online at www.sciencedirect.com Metabolism www.metabolismjou...

907KB Sizes 0 Downloads 151 Views

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX

Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

Nonalcoholic fatty liver disease and hepatocellular carcinoma Heinz Zoller a , Herbert Tilg b,⁎ a b

Department of Medicine II, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Department of Medicine I, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria

A R T I C LE I N FO

AB S T R A C T

Article history:

The fastest growing cause of cancer-related death is hepatocellular carcinoma (HCC), which is at

Received 21 August 2015

least partly attributable to the rising prevalence of non-alcoholic fatty liver disease. Non-alcoholic

Accepted 20 January 2016

fatty liver disease (NAFLD) encompasses a broad spectrum of conditions, ranging from nonprogressive bland steatosis to malignant transformation into hepatocellular cancer. The estimated annual HCC incidence in the progressive form of NAFLD – non-alcoholic

Keywords:

steatohepatitis (NASH) – is about 0.3%. The risk of HCC development is higher in men and

Metabolic liver disease

increases with age, more advanced fibrosis, progressive obesity, insulin resistance and diabetes

Hepatoma

mellitus. Studies on the molecular mechanism of HCC development in NAFLD have shown that

Fatty liver

hepatocarcinogenesis is associated with complex changes at the immunometabolic interface. In

Carcinogenesis

line with these clinical risk factors, administration of a choline-deficient high-fat diet to mice over

Primary liver tumor

a prolonged period results in spontaneous HCC development in a high percentage of animals. The role of altered insulin signaling in tumorigenesis is further supported by the observation that components of the insulin-signaling cascade are frequently mutated in hepatocellular cancer cells. These changes further enhance insulin-mediated growth and cell division of hepatocytes. Furthermore, studies investigating nuclear factor kappa B (NF-κB) signaling and HCC development allowed dissection of the complex links between inflammation and carcinogenesis. To conclude, NAFLD reflects an important risk factor for HCC, develops also in non-cirrhotic livers and is a prototypic cancer involving inflammatory and metabolic pathways. Strengths/weaknesses and summary of the translational potential of the messages in the paper. The systematic review summarizes findings from unbiased clinical and translational studies on hepatocellular cancer in non-alcoholic fatty liver disease. This provides a concise overview on the epidemiology, risk factors and molecular pathogenesis of the NAFL-NASH-HCC sequence. One limitation in the field is that few HCC studies stratify patients by underlying etiology, although the etiology of the underlying liver disease is an important co-determinant of clinical disease course and molecular pathogenesis. Molecular profiling of NAFL and associated HCC holds great translational potential for individualized surveillance, prevention and therapy. © 2016 Elsevier Inc. All rights reserved.

1.

Background and Introduction

Fatty liver is histologically characterized by increased hepatocellular storage of triglycerides, where – according to a recent

consensus paper – the histo-pathological diagnosis “steatosis” is defined by the finding of lipid deposition in >5% of hepatocytes whereas the involvement of more than 50% of hepatocytes is referred to as “fatty liver” [1]. Hepatic steatosis

⁎ Corresponding author. Tel.: + 43 512 504 23539; fax: + 43 512 504 23538. E-mail address: [email protected] (H. Tilg). http://dx.doi.org/10.1016/j.metabol.2016.01.010 0026-0495/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

2

M ET ABOL I S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–XX X

and fatty liver are typically associated with high alcohol intake, but can also occur in patients without significant alcohol consumption. Based on this observation, the term “non-alcoholic steatohepatitis” NASH had been coined by Jürgen Ludwig in 1980 [2]. Follow up studies with serial liver biopsies have shown that the association of hepatic steatosis with inflammation (“steatohepatitis”), apoptosis, fibrosis, Mallory–Denk bodies and hepatocellular ballooning was not universally associated with progressive liver disease [3,4]. Further long-term follow up studies led to the recognition that in the absence of significant inflammation “non-alcoholic fatty liver” indicates no increased risk of liver-related complications [5]. The implications from these observations for clinical practice are that non-alcoholic fatty liver encompasses a broad spectrum of disorders ranging from a benign and reversible condition with increased triglyceride storage to a progressive and potentially lethal liver disease. To appropriately reflect the variable course of the disease, non-alcoholic fatty liver disease (NAFLD) is categorized into the non progressive form nonalcoholic fatty liver (NAFL) and a potentially progressive form termed non-alcoholic steatohepatitis (NASH) [6]. This classification follows the concept that inflammation in patients with NASH can progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The concept of linear progression of NAFLD where fibrosis and cirrhosis result from repair mechanisms induced by inflammation (NASH) and hepatocellular carcinoma is a complication of cirrhosis has been recently challenged mainly for two reasons. First, more recent studies have shown that even NAFL can progress to NASH in 44% even in patients without histological inflammation at baseline [7]. Second, HCC has been increasingly recognized in patients without cirrhosis [8,9]. Although fibrosis, cirrhosis and hepatocellular carcinoma appear to be the generic responses to any kind of liver injury, the time-course and sequence of events appear to be even less predictable in NAFLD than in chronic hepatitis C or alcoholic liver disease. The aim of this article is to review the distinct pathogenesis of hepatocellular carcinoma in patients with non-alcoholic fatty liver disease for research and patient care.

2. Epidemiology of Hepatocellular Carcinoma in Non-Alcoholic Fatty Liver and Risk Factors for Hepato-Carcinogenesis 2.1.

Prevalence and Incidence

The incidence and prevalence of HCC in NAFLD depend on the stage of underlying fatty liver disease, patient characteristics and comorbidities (Table 1 and Table 2). According to a recent meta-analysis in cohorts of patients with non-cirrhotic stages of NAFLD, the cumulative HCC mortality was 0%–3% for study periods up to 20 years. In cohorts with NASH cirrhosis the cumulative incidence ranges from 2.4% over 7 years to 12.8% over 3 years [10]. Hence, disease stage is the most important risk factor and the HCC risk is highest in patients with NASH-cirrhosis. However, studies comparing NASH cohorts with HCV-cirrhosis have shown that the relative risk for the development of HCC is actually lower in NASH cirrhosis than in HCV cirrhosis [11–14].

2.2.

Obesity

Observational studies have further shown that obesity [15], diabetes [16], high iron [17] and alcohol consumption [18] are further risk factors for HCC development in NAFLD [19,20]. Obesity is generally associated with an increased risk of developing malignancies. In particular the risk for the development of gastrointestinal tumors, urinary tract malignancies, non-Hodgkin's lymphoma and myeloma increases with body mass index. For liver cancer, a clear relationship between the degree of obesity and cancer risk has been shown. The relative risk for HCC development increases from 1.13 in patients with BMI 25–29.9 kg/m2 to 4.52 in individuals with BMI 35–39.9 kg/m2 [21]. A more recent and detailed study shows, that especially childhood obesity predisposes to the development of hepatocellular cancer [22]. Epidemiological evidence also supports the positive correlation between obesity and HCC. A potential limitation of such studies is that controlling for confounders associated with obesity such as diabetes is notoriously difficult [23]. For a comprehensive and recent review on the molecular

Table 1 – HCC incidence in NAFLD. Author

Year n

Population

Diagnostic tool

Median follow up Cumulative Estimated annual (*mean follow up) incidence incidence

Ascha et al. [18]

2010

195

NASH cirrhosis

3.2 years

12.8%

4.0%

Adams et al. [72] Hui et al. [12]

2005 2003

420 23

NAFLD NASH cirrhosis

7.6 years 5 years

0.5% 0%

0.1% 0.0%

Teli et al. [5] Powell et al. [4] Ratziu et al. [13] Sanyal et al. [73]

1995 1990 2002 2006

40 42 27 152

NAFL NASH Cryptogenic cirrhosis NASH cirrhosis

11 years 4.5 years 0.78 years 10 years

0% 2.4% 8.0% 2.0%

0.0% 0.5% 10% 0.2%

Yatsuji et al. [14] Bhala et al. [11]

2009 2011

69 257

NASH cirrhosis NASH cirrhosis and advanced fibrosis

Abdominal CT and AFP every 6 months Review of medical records Clinical monitoring every 6 months Review of medical records Review of medical records Review of medical records Ultrasound and AFP yearly to 6 monthly Not reported Not reported

5 years 7.1 years*

11.3% 2.4%

2.3% 0.3%

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

3

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX

Table 2 – HCC prevalence in NAFLD cohorts. Author

Year

n

Study population

Prevalence

Bugianesi et al. [74] Hashimoto et al. [75] Takuma and Nouso [19] Hai et al. [76] Maeda et al. [77] Kawada et al. [78] Malik et al. [79] Chagas et al. [80]

2002 2009 2010 2006 2008 2009 2009 2009

641 382 445 481 242 1168 98 394

Patients with HCC in cirrhotic livers NASH population Patients curatively treated for HCC Patients with resected HCC Patients with resected HCC Patients with resected HCC Patients transplanted with NASH cirrhosis HCC detected by ultrasound

23/641 34/382 11/445 2/418 3/242 8/1168 17/98 7/394

mechanisms of HCC development the interested reader is referred to Karagozian et al. [24].

2.3.

Diabetes

The association of HCC with insulin resistance and diabetes is well established. Evidence for this association comes from case series and large epidemiological studies [19]. Pooled case control studies from the Veterans Affairs database in the United stated including over 173,000 patients with diabetes and over 650,000 controls showed that the HCC incidence rate is significantly higher among diabetic patients (2.39 vs. 0.87 per 10,000 person-years), which translates into a relative risk of 1.86 for diabetic individuals. Similar results were obtained from large epidemiological studies across Europe [25–27]. In a Greek study, a total of 333 patients with HCC were investigated and their characteristics were compared with 360 patients admitted for ear, nose and throat problems [28]. The risk of having diabetes was 1.83 (95% confidence interval 1.18–2.84) in the HCC cohort. More recently, Asian investigators could further demonstrate that in a cohort of patients with chronic HCV infection the presence of diabetes was associated with a two-fold increased risk for HCC development [29]. By comparison the risk for chronic viral hepatitis (HBV or HCV) was 98.6 with a 95% CI ranging from 51.9 to 187.3 [26].

2.4.

Iron

In search for factors associated with HCC in NAFLD, iron was identified as risk factor for HCC progression. This observation is in accord with the finding that increased hepatocellular iron is also associated with more advanced fibrosis in NAFLD [30]. In an Italian study histochemically stainable iron was semiquantitatively assessed in a group of 51 patients with HCC in NASH cirrhosis and 102 controls without HCC. Significant differences between groups were noted in the following variables: grade of inflammation, serum ferritin and prevalence of diabetes. Each of these variables was higher in the HCC cohort. Further, the amount of stainable iron in hepatocytes, endothelial cells and biliary epithelial cells was also significantly higher in the HCC cohort than control subjects [17]. Although the evidence for increased hepatic iron in HCC associated with NAFLD is strong and plausible, it remains unclear if this finding is a cause or consequence of more advanced disease stage that predisposes to the development of HCC.

3.6% 8.9% 2.5% 0.5% 0.8% 0.7% 17% 1.8%

3. Molecular Mechanism of NAFLD-Induced Hepatocarcinogenesis Clinical, observational and epidemiological studies support a concept where multiple mechanisms drive tumorigenesis and HCC development in NAFLD. Among these, inflammation and endocrine alterations as well as abnormalities in carbohydrate, lipid, and iron metabolism will be reviewed here. Molecular studies in human HCC tissue and animal models of HCC associated with NAFLD show that carcinoma development is not only associated with significant changes in hepatocyte biology but also with profound changes in local and systemic immunologic, endocrine and metabolic pathways.

3.1. Hypothesis Free Approaches in Understanding NAFLD-Associated HCC Development The complex molecular biology of hepatocarcinogenesis has been comprehensively studied in unbiased approaches by “omic” methods, which aim at large-scale characterization of all changes in the genome, transcriptome, metabolome and proteome of tumor cells as compared to normal hepatocytes (reviewed in [31]). The currently most comprehensive hypothesis-free approach to study the genetic changes associated with HCC has probably been achieved by full exome sequencing, where quantitative and qualitative information on somatic genetic alterations has been obtained. In a recent study, the mutational signatures of 243 liver tumors studied included 18% patients in whom NASH was an indefinable risk factor. The molecular signatures of NAFLD associated HCC were heterogeneous with no discernible mutation profile. In this respect NAFLD HCC is similar to hemochromatosis- and HCV-associated HCC and differs from HBV- and alcoholic liver disease-associated HCCs where distinct mutational signatures could be identified. Full exome sequencing provides quantitative information on amplifications/deletions of specific regions and qualitative information on the sequence identity of somatic mutations. Quantitative analysis revealed DNA amplifications, where most abundantly amplified regions covered the genes encoding telomerase reverse transcriptase (TERT), vascular endothelial growth factor A (VGFA), and the oncogenes MET and MYC. Qualitative analysis showed that the highest prevalence of mutation was present in genes of the βcatenin/WNT signaling pathway (CTNNB1, AXIN1), albumin (ALB), the cell cycle regulator TP53 and CDKN2A [32].

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

4

M ET ABOL I S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–XX X

Table 3 – Genes with epigenetic changes in HCC. Gene

Gene name

HBV/HCV-HCC

NASH-HCC

Reference

CDKN2A CHD1 APC SOCS1 RASSF1 RARB PRDM2

Cyclin-dependent kinase inhibitor 2 A – p16 Cadherin-1 Adenomatous polyposis coli Suppressor of cytokine signaling 1 Ras association domain-containing protein 1 Retinoic acid receptor beta PR domain zinc finger protein 2

Hypermethylation Hypermethylation Hypermethylation Hypermethylation Hypermethylation Hypermethylation Demethylation

Not affected Differential expression Studied in animal model – no effect Not reported, but included in meta-analysis Not reported Not reported Not reported

[81] [82] [62] [39] [83] [84] [85]

Before full exome sequencing became available, amplifications or deletions of genomic DNA were analyzed by comparative genomic hybridization (CGH). These studies showed that the genetic instability in patients with NASH is about 10- to 20-fold higher than in NAFL. When NASH specific variants were searched for alterations known to be associated with HCC, 2 CNVs located at 13q12.11 and 12q13.2 were identified by CGH. These regions harbor the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes [33]. The exportin 4 gene, whose copy number was amplified in NASH, encodes a nuclear transporter that can bind to a broad range of substrates and acts as a cargo protein between the cytoplasm and the nucleus [34]. XPO4 inactivation causes HCC in mice and XPO4 overexpression was shown to be associated with a better prognosis and increased survival in humans with HCC [35,36]. The other gene whose copy number was significantly different in NASH is the calmodulin dependent phosphodiesterase 1B (PDE1B). This gene was significantly reduced in NASH and also shown to be significantly down-regulated in cirrhosis [33]. Another unbiased approach that allows dissection of HCC pathogenesis is genome-wide mRNA expression studies. The high number of reports on this subject allows for a metaanalytical approach, where genes reproducibly up- or downregulated can be identified [37]. None of the studies included in this meta-analysis was stratified by underlying etiology or included only NAFLD-associated HCC. It is therefore impossible to draw direct conclusions on the pathogenesis of NAFLD associated HCC. To assess the implications of metabolic changes in HCC development, the list of reproducibly and

differentially expressed genes was searched for genes known to be implicated in fat or glucose metabolism. Of the genes with most abundant changes in expression, genes directly link to carbohydrate or lipid metabolism are listed in Table 4. Differential gene expression can be caused by gene amplification/deletion, mutations in regulatory elements or epigenetic changes. The most important tumor specific epigenetic change that can result in differential gene expression is DNA methylation. Epigenetic studies have shown, that the methylation status of the CpG island near the PDE1B is associated with survival in patients with HCC [38]. Additional epigenetic changes that have been identified in HCCs are listed in Table 3, where only few studies report on specific epigenetic changes for NAFLDassociated HCC. The majority of studies have reported on the HCCs associated with viral hepatitis or on pooled analysis of HCCs from mixed etiological background. According to recent meta-analyses, HCC is reproducibly associated with epigenetic changes in the genes adenomatous polyposis coli gene (APC), glutathione-S-transferase P1 gene (GSTP1) and the suppressor of the cytokine signaling 1 gene (SOCS1) [31,39], but as shown in Table 3, the only epigenetic change that has clearly been associated with NAFLD HCC is the gene encoding the chromodomain helicase DNA binding protein 1 (CDH1). Finally, gene expression is also controlled by micro RNAs. Similar to other large-scale molecular profiling studies, which were mostly done in cohorts of HCCs with mixed etiological background, miRNA expression profiling was also not exclusively investigated in NASH-HCC. The results from genome-wide studies on miRNA expression in HCC have been summarized

Table 4 – List of genes differentially expressed in HCC directly implicated in carbohydrate and fat metabolism [37]. Upregulated TPI1

Triosephosphate isomerase 1

GBE1 FDFT1

Glucan (1,4-alpha-), branching enzyme 1 Farnesyl-diphosphate farnesyltransferase 1

Catalyzes the isomerization of glyceraldehydes 3-phosphate (G3P) and dihydroxy-acetone phosphate (DHAP) in glycolysis and gluconeogenesis Glycogen branching enzyme Enzyme at the branch point in the mevalonate pathway (first specific enzyme in cholesterol biosynthesis)

Downregulated PON3

Paraoxonase 3

ACAT1

Acetyl-CoA acetyltransferase 1

MTTP SCP2

Microsomal triglyceride transfer protein Sterol carrier protein 2

Encoded protein is secreted into the bloodstream and associates with high-density lipoprotein (HDL). The protein also rapidly hydrolyzes lactones and can inhibit the oxidation of low-density lipoprotein (LDL) Catalyzes the reversible formation of acetoacetyl-coenzyme A from two molecules of acetyl-coenzyme A Central role in lipoprotein assembly. Peroxisome-associated thiolase that is involved in the oxidation of branched chain fatty acids

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX

and it has been reported that PTEN is a target gene of differentially regulated miRNAs. These miRNAs include miR-21 [40] and miR-29a [41]. In addition, studies in a mouse model of NASH-HCC show that miRNA122, which accounts for approximately 70% of all miRNA in hepatocytes is suppressed during early HCC development [42]. Hence it is reasonable to assume that deregulation in miRNA expression plays a significant role in HCC development, but the exact role of miRNAs in the progression from NAFLD to HCC remains to be determined. In conclusion, unbiased studies have shown that hepatocarcinogenesis in general is associated with changes in a broad range of metabolic functions. Although no study has yet focused exclusively on human NASH associated hepatocarcinogenesis, studies on HCCs of various etiologic background have found that the central pathway regulating energy homeostasis – the phosphoinositide 3-kinase (PI3K)– AKT–mTOR pathway – is mutated in over 50% of HCCs studied [32]. This finding places energy homeostasis at the center of hepatocarcinogenesis and ties in well with the results from hypothesis-driven studies to construct a comprehensive model of progression from NAFLD to HCC.

3.2.

Candidate Gene Studies on NAFLD Associated HCC

Clinical observation and the results from unbiased large scale profiling support the concept that insulin resistance is the key to unlock the pathogenesis of HCC development in NAFLD. The molecular link between energy balance and cell cycle

5

control in hepatocytes is probably provided through insulin receptor signaling via PI3K and PTEN (Fig. 1). As discussed above, diabetes mellitus and insulin resistance are commonly associated with NAFLD. In animal models of insulin resistance such as the choline-deficient L-amino-aciddefined-diet (CDAA) mouse low doses of carbon tetrachloride accelerate fibrosis progression and are associated with HCC development [43]. Such an experimental model lends support to a multiple hit hypothesis whereby liver disease and especially HCC development can be viewed as a multistep process that requires several hits; i.e. CDAA and carbon tetra chloride. This concept is not only helpful in understanding such animal models but also has clinical utility in that patients with a bright liver on ultrasound should be investigated for other coincident liver diseases (2nd hits), contributing to progressive fibrosis and HCC development. Experimental evidence supports the role of altered insulin receptor signaling for HCC development. Studies investigating insulin signaling showed that tissue adjacent to HCCs exhibits strong overexpression of the insulinreceptor substrate-1 (IRS-1) [44]. Tyrosine phosphorylated IRS-1 displays binding sites for numerous down-stream partners. Among these are phosphoinositide 3-kinases (PI3K), which are specific lipid kinases that catalyze phosphorylation at the 3′-OH group of the inositol ring of specific membrane phosphoinsitides. In particular phosphatidylinositol-3.4-bisphosphate (PIP2) is as substrate for PI3K and the reaction results in the production of phosphatidylinositol-3.4.5-triphosphate (PIP3) opening a binding

Fig. 1 – Insulin- and PDGF C signaling. Binding of insulin or platelet derived factor C (PDGF C) to their respective receptors triggers confirmational changes and protein phosphorylation and activation of a signaling cascade that results in activation of a family of proteins known as phosphoinositide 3-kinases (PI3K). Activation of PI3K results in phosphorylation at the 3′-OH group of the inositol ring of the membrane phosphoinsitides phosphatidylinositol-3.4-bisphosphate (PIP2). This reaction results in the production of phosphatidylinositol-3.4.5-triphosphate (PIP3), which opens a binding site for protein kinase B (AKT). Binding of AKT to PIP3 localizes AKT to the plasma membrane. AKT at the membrane phosphorylates a different substrates that control diverse cellular functions including energy metabolism and cellular proliferation. The main inhibitor of the PI3K/AKT pathway is the tumor suppressor phosphatase and tensin homologue on chromosome 10 gene (PTEN). Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

6

M ET ABOL I S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–XX X

site for protein kinase B (AKT). Binding of AKT to PIP3 localizes AKT to the plasma membrane [45] (Fig. 2). Once correctly localized at the membrane, the serine/threonine kinase AKT phosphorylates a range of different substrates that control diverse cellular functions including energy metabolism and cellular proliferation [46]. The main inhibitor of the PI3K/AKT pathway is the tumor suppressor phosphatase and tensin homologue on chromosome 10 gene (PTEN) named after its genetic localization on chromosome 10q23.3. Somatic mutations in PI3-kinases were found in 11% of human HCCs and PTEN mutations were present in 3% [32]. Accordingly, PI3K transgenic and PTEN deficient mice have been found to develop steatosis, hepatomegaly and HCCs [47]. In 80 months old PTEN knock-out animals 100% of all animals had adenomas and 66% developed spontaneous HCCs [48]. These effects of PI3K/AKT signaling on hepatocarcinogenesis are mainly mediated through its effect on cell cycle (cyclin D1), apoptosis Mdm2/p53 and cell growth (mTOR) [49]. Similar effector pathways that include increased expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and MDM2 in association with steatosis and spontaneous HCC development

are found in association with NAFLD and HCC development in another animal model – the embryonic liver fodrin (ELF) knock out mouse [50]. ELF is a 2nd messenger in transforming-growth factor beta (TGF-β) signaling, which – after activation in hepatic stellate cells – results in fibrogenesis. Similarly, stable or transient overexpression of plateletderived growth factor C also results in hepatic steatosis and spontaneous hepatocellular carcinomas probably via receptor mediated activation of PI3K and further downstream signaling [51] (Fig. 1). A further genetic animal model of steatosis and hepatocarcinogenesis is the fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout mouse, where the lack of this specific peroxisomal enzyme that is required for degradation of very long chain fatty acid results in hepatic lipid accumulation, steatohepatitis and hepatocellular carcinoma [52]. The molecular mechanism of carcinoma development in this model was found to be mediated via endoplasmic reticulum stress related effectors [53]. An animal model that recapitulates human NASH to HCC progression without transgenic overexpression or targeted gene disruption was recently described, where a choline-deficient

gene amplification:

epigenetic changes: Altered methylation of promoter regions of PDE1B, APC, glutathione-S-transferase

Telomerase, MET, MYC vascular endothelial growth factor A

Growth & Regeneration sustained growth and regenerativ signals

gene mutations: β-catenin, Axin1, albumin, TP53, cyclin dependen Kinases 2A (CDKN2A)

normal liver

replicative immortality

non-alcoholic fatty liver disease

altered receptor signalling: Insulin & proinflmmatory Signalling

sustaining proliferative signaling Diabetes

Hormones

hepatocellular carcinoma

Microbial Metabolites e.g. LPS, deoxycholic acid

e.g. Insulin Gut Microbiome

Proinflammatory Cytokines e.g. Interleukin-1β

Obesity

Inflammation

Fig. 2 – Drivers of HCC Development in NAFLD. Inflammation, diabetes, obesity (and iron) are systemic risk factors for NAFLD and HCC and can be considered ‘fertile soil’ for HCC development – i.e. such changes provide an appropriate microenvironment for the progression of malignant lesions. The progression from the normal liver to NAFLD and further progression to NASH and HCC is not always linear and some patients can progress from NAFL to advanced fibrosis in the absence of significant inflammation [86–88]. On a cellular level, epigenetic and genetic events provide a procarcinogenic environment by maintaining sustained growth and regenerative signals. The next step in HCC development is replicative immortality as a consequence of several gene mutations. Inflammation and hyperinsulinism in NAFLD and especially in NASH further provide sustained proliferative signaling which causes more rapid HCC growth [89]. Soluble immune and endocrine mediators derived from the gut microbiome provide a molecular mechanism that is implicated in HCC progression and hence provide a link between ‘seed’ and ‘soil’. Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX

high-fat diet (CD-HFD) or a high-fat diet alone was fed to C57B/6 mice for 12 months. The HCC incidence in HFD fed animals was 2.5% compared with 25% in CD-HFD fed animals. The link between steatosis and HCC development was inflammation, mediated by signaling via the lymphotoxin-β receptor (LTB). Interestingly, LTB signaling is not only crucial for the transition from NAFLD to NASH but also for hepatic fat accumulation in this animal model [54]. Taken together, animal models of steatosis-associated HCC development show that tumorigenesis is invariably associated with inflammation. This sequence is reminiscent of the “human NAFL-NASH-HCC sequence” and is in accord with the fact that altered nuclear factor kappa B (NF-κB) signaling is associated with notable changes in HCC development. NF-κB is a transcription factor that is activated by binding of T-cell derived cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β). However, the association between NF-κB and HCC development is not straight forward, because inhibition of this proinflammatory signaling pathway has apparently contradictory effects on HCC development in different mouse models [55]. Constitutive inhibition of NF-κB activation by genetic deletion of essential components of its upstream activators such as p50 or IKK strongly enhances dietinduced liver injury and HCC development in a chemical carcinogenesis model, where mice were injected the mutagen diethylnitrosamine (DEN) [56,57]. In contrast, inhibition of NF-κB was associated with reduced hepatocarcinogenesis in the Mdr2knockout mouse [58]. To resolve this contradictory role of NF-κB signaling, conditional knockouts have been employed. These studies showed that global deletion of the NF-κB activator IKKβ results in protection from HCC development, whereas hepatocyte specific deletion of NF-κB enhances susceptibility of liver damage and HCC development (reviewed in [59]). Taken together, these results clearly show that the cellular location of the proinflammatory response is essential, whereas NF-κB activation in hepatocytes appears to be protective. Animal models of HCC development also include genetically engineered mice. These models and mechanisms are beyond the scope of this review as such animals develop HCCs without steatosis [60]. Finally, β-catenin/WNT signaling, which is affected by somatic mutation in >37% of patients with HCC [32], is also not covered in detail here because animal models interrogating this pathway by transgenic overexpression of mutant β-catenin have thus far failed to produce spontaneous HCCs [61–65]. Finally, another approach to understand the association of specific genes with HCC development is genetic association studies. The only genetic association study clearly demonstrating that a specific single nucleotide polymorphism confers an increased HCC risk was recently published [66]. In this case control study including 100 HCC cases and 275 controls with histologically characterized NAFLD carriage of one G allele in position rs738409 of the PNPLA3 gene was associated with a 2.26-fold increased risk, whereas GG homozygotes had a 5-fold increased HCC risk when compared with patients homozygous for CC. These risk effects were even higher when compared with a control population. Whether polymorphisms in other genes associated with progressive fibrosis in NAFLD such as TM6SF2 are also associated with an increased HCC risk is currently unknown [67].

7

4. Gut Microbiota in NAFLD-Induced Hepatocarcinogenesis Recent results from studies in animal models of hepatocellular cancer suggest a strong link between tumor progression and gut microbiota. Clinical studies validating findings from animal models in human hepatocellular carcinoma are emerging. In one study fecal microbial composition of 105 patients with early HCC and 45 with advanced HCC was compared with that of 131 healthy controls. In accordance with the results from animal studies, moderate dysbiosis was associated with more advanced HCC. In particular, phylum Bacteroidetes was decreased, while Fusobacteria and Proteobacteria were increased in the HCC group [68]. Although these data suggest that significant differences in the gut microbiome of patients with HCC and healthy patients exist, it is difficult to ascertain if these differences reflect changes induced by the underlying liver disease or true HCC associated alterations of the intestinal microbiome. Results from comprehensive animal studies suggest that specific pathogen associated microbial patterns (PAMPs) such as lipopolysaccharide (LPS), which are recognized by Toll like receptor 4 (TLR4) promote HCC cell growth but are dispensable for HCC initiation [69]. In this study the growth promoting effect of LPS was associated by increased expression of hepatocyte growth factor (HGF) and the hepatic mitogen Epiregulin. Another animal study further suggests that the increased tumor risk associated with obesity could – at least in part – also be conferred by the intestinal microbiome. In accordance with the concept that the intestinal microbiome is a codeterminant of tumor progression rather than tumor induction, obesity induced by high-fat diet in wild-type mice was not associated with increased cancer development. However, after chemical induction of HCC with the chemical dimethylbenz(a)anthracene DMBA, obesity induced by high fat diet or by genetic defects in Lepob/ob mice was associated with HCC development in 100% of the population as compared to 5% in lean control mice. Studies on the underlying mechanism showed that genetic deletion of IL1β or antibiotic treatment could reduce the tumor burden. In obese mice the bacterial metabolite deoxycholic acid (DCA), which is known to mediate DNA damage was increased in obese mice and could be reduced by antibiotic treatment or IL1β [70]. Taken together, animal studies show that the gut microbiome is an important determinant of HCC progression. While the biological signals mediating HCC tumor progression are being unraveled in animal studies, translation and validation of these findings on crosstalk between the intestinal microbiome and HCC development in human studies will be the next level (Fig. 2).

5.

Summary and Conclusions

HCC is an increasingly recognized complication of NAFLD, where clinical and histopathological studies have led to the identification of male sex, age, degree of inflammation, hepatic iron accumulation and fibrosis as risk factors. In addition, comorbidities that increase the risk of progression

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

8

M ET ABOL I S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–XX X

from NAFL to HCC include obesity, diabetes and insulin resistance. Insulin signaling is also at the heart of the molecular changes associated with hepatocarcinogenesis in NAFL. Qualitative genetic changes in signaling pathways that link inflammation cell cycle and metabolic control affect PI3K genes as second messengers of the insulin receptor in just over 10% of HCCs. Accordingly, not only diabetes but also insulin therapy has been identified as a risk factor for the development of HCC. Metformin can inhibit the progression of NAFL to HCC [71]. Intracellular signal transduction from insulin binding to its receptor converges with NF-κB signaling building a link with inflammation and highlighting T- and NKT-cell infiltration as essential for HCC development. Specifically, lymphotoxin-β appears to be the key signal from NKT-cells to hepatocytes required for fat accumulation and hepatocarcinogenesis in an insulin-resistant animal model. Although genetically engineered mice have helped to dissect the molecular details of hepatocarcinogenesis in NAFLD, translation of these findings into clinical practice is just beginning. The wide range of molecular changes that can induce steatosis and spontaneous HCC development in mice is reflected by diverse risk factors and different molecular and genetic alterations in human HCCs. A major challenge will be the translation of these profiles into individualized follow up and therapy and ultimately improved outcomes.

Disclosures The authors declare no conflict of interest in relation to this work.

REFERENCES

[1] Tannapfel A, Denk H, Dienes HP, Langner C, Schirmacher P, Trauner M, et al. Histopathological diagnosis of non-alcoholic and alcoholic fatty liver disease. Virchows Arch 2011;458: 511–23. [2] Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980;55:434–8. [3] Powell EE, Cooksley WG, Hanson R, Searle J, Halliday JW, Powell LW. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years. Hepatology 1990;11:74–80. [4] Abdelmalek M, Ludwig J, Lindor KD. Two cases from the spectrum of nonalcoholic steatohepatitis. J Clin Gastroenterol 1995;20:127–30. [5] Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural history of nonalcoholic fatty liver: a follow-up study. Hepatology 1995;22:1714–9. [6] Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology 2012;142:1592–609. [7] McPherson S, Hardy T, Henderson E, Burt AD, Day CP, Anstee QM. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: implications for prognosis and clinical management. J Hepatol 2015;62: 1148–55.

[8] Guzman G, Brunt EM, Petrovic LM, Chejfec G, Layden TJ, Cotler SJ. Does nonalcoholic fatty liver disease predispose patients to hepatocellular carcinoma in the absence of cirrhosis? Arch Pathol Lab Med 2008;132:1761–6. [9] Leung C, Yeoh SW, Patrick D, Ket S, Marion K, Gow P, et al. Characteristics of hepatocellular carcinoma in cirrhotic and non-cirrhotic non-alcoholic fatty liver disease. World J Gastroenterol 2015;21:1189–96. [10] White DL, Kanwal F, El-Serag HB. Association between nonalcoholic fatty liver disease and risk for hepatocellular cancer, based on systematic review. Clin Gastroenterol Hepatol 2012;10:1342–1359.e2. [11] Ratziu V, Bonyhay L, Di Martino V, Charlotte F, Cavallaro L, Sayegh-Tainturier MH, et al. Survival, liver failure, and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis. Hepatology 2002;35:1485–93. [12] Hui JM, Kench JG, Chitturi S, Sud A, Farrell GC, Byth K, et al. Long-term outcomes of cirrhosis in nonalcoholic steatohepatitis compared with hepatitis C. Hepatology 2003; 38:420–7. [13] Bhala N, Angulo P, van der Poorten D, Lee E, Hui JM, Saracco G, et al. The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology 2011;54: 1208–16. [14] Yatsuji S, Hashimoto E, Tobari M, Taniai M, Tokushige K, Shiratori K. Clinical features and outcomes of cirrhosis due to non-alcoholic steatohepatitis compared with cirrhosis caused by chronic hepatitis C. J Gastroenterol Hepatol 2009; 24:248–54. [15] Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology 2010;51:1820–32. [16] Loria P, Lonardo A, Anania F. Liver and diabetes. A vicious circle. Hepatol Res 2013;43:51–64. [17] Sorrentino P, D'Angelo S, Ferbo U, Micheli P, Bracigliano A, Vecchione R. Liver iron excess in patients with hepatocellular carcinoma developed on non-alcoholic steato-hepatitis. J Hepatol 2009;50:351–7. [18] Ascha MS, Hanouneh IA, Lopez R, Tamimi TA, Feldstein AF, Zein NN. The incidence and risk factors of hepatocellular carcinoma in patients with nonalcoholic steatohepatitis. Hepatology 2010;51:1972–8. [19] Yasui K, Hashimoto E, Komorizono Y, Koike K, Arii S, Imai Y, et al. Characteristics of patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma. Clin Gastroenterol Hepatol 2011;9:428–33 [quiz e50]. [20] Takuma Y, Nouso K. Nonalcoholic steatohepatitis-associated hepatocellular carcinoma: our case series and literature review. World J Gastroenterol 2010;16:1436–41. [21] Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med 2003; 348:1625–38. [22] Hassan MM, Abdel-Wahab R, Kaseb A, Shalaby A, Phan AT, El-Serag HB, et al. Obesity Early in Adulthood Increases Risk but Does Not Affect Outcomes of Hepatocellular Carcinoma. Gastroenterology 2015;149:119–29. [23] Saunders D, Seidel D, Allison M, Lyratzopoulos G. Systematic review: the association between obesity and hepatocellular carcinoma - epidemiological evidence. Aliment Pharmacol Ther 2010;31:1051–63. [24] Karagozian R, Derdak Z, Baffy G. Obesity-associated mechanisms of hepatocarcinogenesis. Metabolism 2014;63: 607–17. [25] Adami HO, Chow WH, Nyren O, Berne C, Linet MS, Ekbom A, et al. Excess risk of primary liver cancer in patients with diabetes mellitus. J Natl Cancer Inst 1996;88:1472–7.

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

M ET ABO LI S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–X XX

[26] Wideroff L, Gridley G, Mellemkjaer L, Chow WH, Linet M, Keehn S, et al. Cancer incidence in a population-based cohort of patients hospitalized with diabetes mellitus in Denmark. J Natl Cancer Inst 1997;89:1360–5. [27] Lagiou P, Kuper H, Stuver SO, Tzonou A, Trichopoulos D, Adami HO. Role of diabetes mellitus in the etiology of hepatocellular carcinoma. J Natl Cancer Inst 2000;92:1096–9. [28] Lagiou P, Rossi M, Tzonou A, Georgila C, Trichopoulos D, La Vecchia C. Glycemic load in relation to hepatocellular carcinoma among patients with chronic hepatitis infection. Ann Oncol 2009;20:1741–5. [29] Zhou YM, Zhang XF, Wu LP, Sui CJ, Yang JM. Risk factors for combined hepatocellular-cholangiocarcinoma: a hospitalbased case-control study. World J Gastroenterol 2014;20: 12615–20. [30] Nelson JE, Wilson L, Brunt EM, Yeh MM, Kleiner DE, UnalpArida A, et al. Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease. Hepatology 2011;53:448–57. [31] Pei Y, Zhang T, Renault V, Zhang X. An overview of hepatocellular carcinoma study by omics-based methods. Acta Biochim Biophys Sin (Shanghai) 2009;41:1–15. [32] Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015;47:505–11. [33] Zain SM, Mohamed R, Cooper DN, Razali R, Rampal S, Mahadeva S, et al. Genome-wide analysis of copy number variation identifies candidate gene loci associated with the progression of non-alcoholic fatty liver disease. PLoS One 2014;9:e95604. [34] Lipowsky G, Bischoff FR, Schwarzmaier P, Kraft R, Kostka S, Hartmann E, et al. Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J 2000;19: 4362–71. [35] Liang XT, Pan K, Chen MS, Li JJ, Wang H, Zhao JJ, et al. Decreased expression of XPO4 is associated with poor prognosis in hepatocellular carcinoma. J Gastroenterol Hepatol 2011;26:544–9. [36] Zhang H, Wei S, Ning S, Jie Y, Ru Y, Gu Y. Evaluation of TGFbeta, XPO4, elF5A2 and ANGPTL4 as biomarkers in HCC. Exp Ther Med 2013;5:119–27. [37] Choi JK, Choi JY, Kim DG, Choi DW, Kim BY, Lee KH, et al. Integrative analysis of multiple gene expression profiles applied to liver cancer study. FEBS Lett 2004;565:93–100. [38] Hernandez-Vargas H, Lambert MP, Le Calvez-Kelm F, Gouysse G, McKay-Chopin S, Tavtigian SV, et al. Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors. PLoS One 2010;5: e9749. [39] Liu M, Cui LH, Li CC, Zhang L. Association of APC, GSTP1 and SOCS1 promoter methylation with the risk of hepatocellular carcinoma: a meta-analysis. Eur J Cancer Prev 2015. [40] Bao L, Yan Y, Xu C, Ji W, Shen S, Xu G, et al. MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ERK pathways. Cancer Lett 2013;337:226–36. [41] Tumaneng K, Schlegelmilch K, Russell RC, Yimlamai D, Basnet H, Mahadevan N, et al. YAP mediates crosstalk between the Hippo and PI(3)K-TOR pathways by suppressing PTEN via miR-29. Nat Cell Biol 2012;14:1322–9. [42] Takaki Y, Saito Y, Takasugi A, Toshimitsu K, Yamada S, Muramatsu T, et al. Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis. Cancer Sci 2014;105:1254–60. [43] De Minicis S, Agostinelli L, Rychlicki C, Sorice GP, Saccomanno S, Candelaresi C, et al. HCC development is associated to peripheral insulin resistance in a mouse model of NASH. PLoS One 2014;9:e97136.

9

[44] Nishiyama M, Wands JR. Cloning and increased expression of an insulin receptor substrate-1-like gene in human hepatocellular carcinoma. Biochem Biophys Res Commun 1992;183: 280–5. [45] Whitman M, Downes CP, Keeler M, Keller T, Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 1988; 332:644–6. [46] Fruman DA, Rommel C. PI3K and cancer: lessons, challenges and opportunities. Nat Rev Drug Discov 2014;13:140–56. [47] Kudo Y, Tanaka Y, Tateishi K, Yamamoto K, Yamamoto S, Mohri D, et al. Altered composition of fatty acids exacerbates hepatotumorigenesis during activation of the phosphatidylinositol 3-kinase pathway. J Hepatol 2011;55:1400–8. [48] Watanabe S, Horie Y, Suzuki A. Hepatocyte-specific Ptendeficient mice as a novel model for nonalcoholic steatohepatitis and hepatocellular carcinoma. Hepatol Res 2005;33:161–6. [49] Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2: 489–501. [50] Kitisin K, Ganesan N, Tang Y, Jogunoori W, Volpe EA, Kim SS, et al. Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation. Oncogene 2007;26:7103–10. [51] Campbell JS, Hughes SD, Gilbertson DG, Palmer TE, Holdren MS, Haran AC, et al. Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci U S A 2005;102:3389–94. [52] Fan CY, Pan J, Usuda N, Yeldandi AV, Rao MS, Reddy JK. Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase. Implications for peroxisome proliferator-activated receptor alpha natural ligand metabolism. J Biol Chem 1998; 273:15639–45. [53] Huang J, Viswakarma N, Yu S, Jia Y, Bai L, Vluggens A, et al. Progressive endoplasmic reticulum stress contributes to hepatocarcinogenesis in fatty acyl-CoA oxidase 1-deficient mice. Am J Pathol 2011;179:703–13. [54] Wolf MJ, Adili A, Piotrowitz K, Abdullah Z, Boege Y, Stemmer K, et al. Metabolic activation of intrahepatic CD8 + T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes. Cancer Cell 2014;26: 549–64. [55] Vucur M, Roderburg C, Bettermann K, Tacke F, Heikenwalder M, Trautwein C, et al. Mouse models of hepatocarcinogenesis: what can we learn for the prevention of human hepatocellular carcinoma? Oncotarget 2010;1: 373–8. [56] Locatelli I, Sutti S, Vacchiano M, Bozzola C, Albano E. NFkappaB1 deficiency stimulates the progression of nonalcoholic steatohepatitis (NASH) in mice by promoting NKTcell-mediated responses. Clin Sci (Lond) 2013;124:279–87. [57] Maeda S, Kamata H, Luo JL, Leffert H, Karin M. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 2005;121:977–90. [58] Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 2004;431:461–6. [59] Karin M. NF-kappaB as a critical link between inflammation and cancer. Cold Spring Harb Perspect Biol 2009;1: a000141. [60] Martinez-Chantar ML, Corrales FJ, Martinez-Cruz LA, GarciaTrevijano ER, Huang ZZ, Chen L, et al. Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A. FASEB J 2002;16:1292–4. [61] Cadoret A, Ovejero C, Saadi-Kheddouci S, Souil E, Fabre M, Romagnolo B, et al. Hepatomegaly in transgenic mice

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010

10

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

M ET ABOL I S M CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 01 6 ) X XX–XX X

expressing an oncogenic form of beta-catenin. Cancer Res 2001;61:3245–9. Harada N, Miyoshi H, Murai N, Oshima H, Tamai Y, Oshima M, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 2002;62:1971–7. Nejak-Bowen KN, Thompson MD, Singh S, Bowen Jr WC, Dar MJ, Khillan J, et al. Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin. Hepatology 2010;51:1603–13. Tan X, Apte U, Micsenyi A, Kotsagrelos E, Luo JH, Ranganathan S, et al. Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver. Gastroenterology 2005;129:285–302. Newell P, Villanueva A, Friedman SL, Koike K, Llovet JM. Experimental models of hepatocellular carcinoma. J Hepatol 2008;48:858–79. Liu YL, Patman GL, Leathart JB, Piguet AC, Burt AD, Dufour JF, et al. Carriage of the PNPLA3 rs738409 C > G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma. J Hepatol 2014;61:75–81. Liu YL, Reeves HL, Burt AD, Tiniakos D, McPherson S, Leathart JB, et al. TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease. Nat Commun 2014;5:4309. Ren Z, Xu S, Jiang J, Zheng S. A Novel Diagnosis for Early Hepatocellular Carcinoma Based On Intestinal Microbiome. 21st Annual Meeting of the International Liver Transplant Society, Chicago, IL; 2015. Dapito DH, Mencin A, Gwak GY, Pradere JP, Jang MK, Mederacke I, et al. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 2012;21: 504–16. Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 2013;499:97–101. Chen HP, Shieh JJ, Chang CC, Chen TT, Lin JT, Wu MS, et al. Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies. Gut 2013;62:606–15. Adams LA, Crawford DH, Stuart K, House MJ, St Pierre TG, Webb M, et al. The impact of phlebotomy in nonalcoholic fatty liver disease: A prospective, randomized, controlled trial. Hepatology 2015;61:1555–64. Sanyal AJ, Banas C, Sargeant C, Luketic VA, Sterling RK, Stravitz RT, et al. Similarities and differences in outcomes of cirrhosis due to nonalcoholic steatohepatitis and hepatitis C. Hepatology 2006;43:682–9. Bugianesi E, Leone N, Vanni E, Marchesini G, Brunello F, Carucci P, et al. Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 2002;123:134–40. Hashimoto K, Hirai M, Kurosawa Y. Identification of a mouse homolog for the human hereditary haemochromatosis candidate gene. Biochem Biophys Res Commun 1997;230:35–9.

[76] Hai S, Kubo S, Shuto T, Tanaka H, Takemura S, Yamamoto T, et al. Hepatocellular carcinoma arising from nonalcoholic steatohepatitis: report of two cases. Surg Today 2006;36: 390–4. [77] Maeda T, Hashimoto K, Kihara Y, Ikegami T, Ishida T, Aimitsu S, et al. Surgically resected hepatocellular carcinomas in patients with non-alcoholic steatohepatitis. Hepatogastroenterology 2008;55:1404–6. [78] Kawada N, Imanaka K, Kawaguchi T, Tamai C, Ishihara R, Matsunaga T, et al. Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis. J Gastroenterol 2009;44:1190–4. [79] Malik SM, Gupte PA, de Vera ME, Ahmad J. Liver transplantation in patients with nonalcoholic steatohepatitis-related hepatocellular carcinoma. Clin Gastroenterol Hepatol 2009;7: 800–6. [80] Chagas AL, Kikuchi LO, Oliveira CP, Vezozzo DC, Mello ES, Oliveira AC, et al. Does hepatocellular carcinoma in nonalcoholic steatohepatitis exist in cirrhotic and non-cirrhotic patients? Braz J Med Biol Res 2009;42:958–62. [81] Kaneto H, Sasaki S, Yamamoto H, Itoh F, Toyota M, Suzuki H, et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut 2001;48:372–7. [82] Liu F, Li H, Chang H, Wang J, Lu J. Identification of hepatocellular carcinoma-associated hub genes and pathways by integrated microarray analysis. Tumori 2015;101: 206–14. [83] Zhang YJ, Ahsan H, Chen Y, Lunn RM, Wang LY, Chen SY, et al. High frequency of promoter hypermethylation of RASSF1A and p16 and its relationship to aflatoxin B1-DNA adduct levels in human hepatocellular carcinoma. Mol Carcinog 2002;35:85–92. [84] Feng Q, Stern JE, Hawes SE, Lu H, Jiang M, Kiviat NB. DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection. Exp Mol Pathol 2010;88:287–92. [85] Nishida N, Kudo M, Nagasaka T, Ikai I, Goel A. Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma. Hepatology 2012;56: 994–1003. [86] Wong VW, Wong GL, Choi PC, Chan AW, Li MK, Chan HY, et al. Disease progression of non-alcoholic fatty liver disease: a prospective study with paired liver biopsies at 3 years. Gut 2010;59:969–74. [87] Pais R, Charlotte F, Fedchuk L, Bedossa P, Lebray P, Poynard T, et al. A systematic review of follow-up biopsies reveals disease progression in patients with non-alcoholic fatty liver. J Hepatol 2013;59:550–6. [88] Singh S, Allen AM, Wang Z, Prokop LJ, Murad MH, Loomba R. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin Gastroenterol Hepatol 2015;13: 643.e1–54 [quiz e39-40]. [89] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74.

Please cite this article as: Zoller H, Tilg H, Nonalcoholic fatty liver disease and hepatocellular carcinoma, Metabolism (2016), http://dx.doi.org/10.1016/j.metabol.2016.01.010