Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: Novel insights from interspecies comparison

Biochimica et Biophysica Acta 1826 (2012) 215–237

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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbacan

Review

Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: Novel insights from interspecies comparison Diego F. Calvisi, Maddalena Frau, Maria L. Tomasi, Francesco Feo ⁎, Rosa M. Pascale Department of Clinical and Experimental Medicine, Division of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy

a r t i c l e

i n f o

Article history: Received 21 January 2012 Received in revised form 12 April 2012 Accepted 14 April 2012 Available online 26 April 2012 Keywords: Hepatocarcinogenesis Signal transduction MicroRNA Prognostic marker Therapeutic target Interspecies comparison

a b s t r a c t Hepatocellular carcinoma is a frequent and fatal disease. Recent researches on rodent models and human hepatocarcinogenesis contributed to unravel the molecular mechanisms of hepatocellular carcinoma dedifferentiation and progression, and allowed the discovery of several alterations underlying the deregulation of cell cycle and signalling pathways. This review provides an interpretive analysis of the results of these studies. Mounting evidence emphasises the role of up-regulation of RAS/ERK, PI3K/AKT, IKK/NF-kB, WNT, TGF-β, NOTCH, Hedgehog, and Hippo signalling pathways as well as of aberrant proteasomal activity in hepatocarcinogenesis. Signalling deregulation often occurs in preneoplastic stages of rodent and human hepatocarcinogenesis and progressively increases in carcinomas, being most pronounced in more aggressive tumours. Numerous changes in signalling cascades are involved in the deregulation of carbohydrate, lipid, and methionine metabolism, which play a role in the maintenance of the transformed phenotype. Recent studies on the role of microRNAs in signalling deregulation, and on the interplay between signalling pathways led to crucial achievements in the knowledge of the network of signalling cascades, essential for the development of adjuvant therapies of liver cancer. Furthermore, the analysis of the mechanisms involved in signalling deregulation allowed the identification of numerous putative prognostic markers and novel therapeutic targets of specific hepatocellular carcinoma subtypes associated with different biologic and clinical features. This is of prime importance for the selection of patient subgroups that are most likely to obtain clinical benefit and, hence, for successful development of targeted therapies for liver cancer. © 2012 Elsevier B.V. All rights reserved.

Abbreviations: ACAC, acetyl-CoA carboxylase; ACLY, ATP citrate lyase; AEG-1, astrocyte elevated gene-1; AKT, v-AKT murine thymoma viral oncogene homolog; AMPK, AMP kinase; APC/C(CDH1), Anaphase Promoting Complex/Cyclosome, and its activator CDH1); AUF1, AUrich RNA binding factor 1; AURKA, Aurora A; BHMT, betaine-homocysteine methyltransferase; CD25, interleukin 2 receptor, alpha); CDC2, cell cycle controller-2; CDC37, cell division cycle 37; CDC25B, cell division cycle 25B; CDC14B, cell division cycle 14, Saccharomyces cerevisiae homolog B; CDK, cyclin-dependent kinase; CDKN1α, cyclin-dependent kinase inhibitor 1α,p21WAF1; chREBP, (carbohydrate responsive element binding protein); CK1δ/ε, casein kinase 1δ/ε; CKS1, Cdc28 protein kinase 1; COL1A2, type I collagen a2; COXII, cytochrome oxidase subunit II; CRM1, required for Chromosome region maintenance 1; CTGF, connective tissue growth factor; DELTEX, DELTEX Drosophila homolog; DMBT1, deleted in malignant brain tumours 1; DN, dysplastic nodule; DUSP1, dualspecificity phosphatase 1; EGFR, epidermal growth factor receptor; EMT, epithelial to mesenchymal transition; EpCAM, epithelial cell adhesion molecule; ERK1/2, extracellular signal-regulated kinase 1/2; EZH2, enhancer of ZESTE, drosophila, homolog 2; FASN, fatty acid synthase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FOXM1, Forkhead box M1; FOXO1, forkhead box O1; G6PD, glucose-6-phosphate dehydrogenase; GADD45g, growth arrest and DNA-damage-inducible-γ; GI, genomic instability; GLI, glioma-associated oncogene homolog 1; GNMT, glycine N-methyltransferase; GSK3β, glycogen synthase kinase-3β; GLUT, glucose transporter; HDAC10, histone deacetylase 10; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCCB, HCC with better prognosis; HCCP, HCC with poorer prognosis; HCV, hepatitis C virus; HES-1, hairy/enhancer of split, Drosophila homolog 1; HINT1, histidine triad nucleotide-binding protein 1; HIF-1a, hypoxia-inducible factor 1α; HKII, hexokinase II; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; HSP90, heat shock protein 90; HuR, AUrich RNA binding factor 1; IGFR, insulin-like growth factor receptor; IKK, inhibitor of kappa light chain gene enhancer in b cells, kinase of, beta; IL1A, interleukin 1A; LATS1/2, Wts homologues; LDH, lactic dehydrogenase; LINC, MYBL2–LIN9 complex; LKB1, liver kinase b1; LXR-β, liver X receptor β; MAPK, Mitogen Activated Protein Kinase; MAT, methionine adenosyltransferase; MDK, Midkine; ME, malic enzyme; MET, hepatocyte growth factor receptor; MICA, major histocompatibility complex class I chain-related gene A; 5-MTHF-HMT, N5-methyltetrahydrofolate homocysteine methyltransferase; mTOR, mammalian target of Rapamycin; mTORC1, mTOR complex 1; MST1/2, homologues of Hpo; MVK, mevalonate kinase; NADPH, nicotinamide adenine dinucleotide phosphate; NASH, non-alcoholic steatohepatitis; NCOA1, nuclear receptor coactivator 1; NEK2, never in mitosis gene A-related kinase 2; NF-kB, nuclear factor kB; NO, Nitric oxide; NOS, nitric oxide synthase; NRDG3, N-myc downstream-regulated gene 3; NRIP1, nuclear receptor-interacting protein 1; PAI-1, plasminogen activator inhibitor-1; PAPSS1, 3′-phosphoadenosine 50-phosphosulfate synthase-1; PFK2, phosphofructokinase 2; PDGFRα, PDGFRβ, platelet derived growth factor α,β; PDK-1, pyruvate dehydrogenase kinase-1; PK, pyruvate kinase; PI3K, phosphatydilinositol 3-kinase; PP1CA, protein phosphatase 1 catalytic subunit alpha; PTCH, patched; PTEN, Phosphatase and tensin homologue deleted on chromosome 10; RASSF1A, Ras association domain family 1A; RhoGDIA, RhoGDP dissociation inhibitor; ROCK2, RHO-associated coiled-coil-containing protein kinase 2; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; Sav1, Salvador; SCD1, stearoyl-CoA desaturase 1; SCO-2, synthesis of cytochrome c oxidase 2; SKP2, S-phase kinase-associated protein 2; SMAD, mothers against decapentaplegic, drosophila, homolog of; SMO, smoothened; SNAIL, snail Drosophila homolog; SQS, squalene synthetase; SREBP2, sterol regulatory element binding protein 2; STMN1, Stathmin; SUFU, Suppressor of fused; TGF-β, tumour growth factor-β; TIGAR, TP53-induced glycolysis and apoptosis regulator; TIMP3, tissue inhibitor of metalloproteinase 3; TKL-1, transketolase 1; TNFAIP3, tumour necrosis factor alpha-induced protein 3; VEGF-α, vascular endothelial growth factor-α; ZEB1/2, zinc finger e box-binding homeobox 1/2 ⁎ Corresponding author. Tel.: + 39 079 228307; fax: + 39 079 228485. E-mail address: [email protected] (F. Feo). 0304-419X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2012.04.003

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Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk factors and cancer heterogeneity . . . . . . . . . . . . . . . Cell cycle deregulation and the proliferation signature . . . . . . . Deregulation of signalling pathways . . . . . . . . . . . . . . . . 4.1. Mitogen Activated Protein Kinase (MAPK) pathway . . . . . 4.2. PI3K/AKT pathway . . . . . . . . . . . . . . . . . . . . 4.3. MYBL2 and LINC in liver tumours with mutated p53 . . . . . 4.4. Inducible nitric oxide synthase . . . . . . . . . . . . . . . 4.5. TGF-β expression and HCC dedifferentiation . . . . . . . . 4.6. Deregulation of NOTCH receptors . . . . . . . . . . . . . 4.7. Hedgehog signalling . . . . . . . . . . . . . . . . . . . . 4.8. WNT/β-catenin signalling pathway . . . . . . . . . . . . . 4.9. Hippo signalling . . . . . . . . . . . . . . . . . . . . . . 5. Signalling deregulation as master producer of altered metabolism . 5.1. Carbohydrate metabolism . . . . . . . . . . . . . . . . . 5.2. AKT/mTOR signalling and lipid metabolism . . . . . . . . . 5.3. Methionine metabolism and hepatocarcinogenesis progression 6. Hepatocellular carcinoma gene signatures . . . . . . . . . . . . . 7. A new frontier of research on HCC: the role of microRNAs . . . . . 7.1. MicroRNAs as signalling regulators . . . . . . . . . . . . . 7.2. MicroRNAs as prognostic markers and therapeutic targets . . 8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Hepatocellular carcinoma (HCC) is one of the most frequent human cancers, with 0.25–1 million of newly diagnosed cases each year [1–3]. Highest frequencies of HCC occur in sub-Saharan Africa and far eastern Asia, where hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are endemic, and in regions where food contaminated with Aflatoxin B1 is consumed [1–4]. HCC incidence exhibits striking differences related to age, gender, ethnic group, and geographic region [4,5], and is rising even in countries with relatively low incidence [1,6–8]. HCC is a fatal disease, with a life expectancy of about 6 months from the time of diagnosis. Partial liver resection or liver transplantation is potentially curative. Ultrasonography is sufficiently sensitive to detect small HCC lesions, which may be efficiently treated by resection or radiofrequency ablation [9]. However, only a minority of cases is amenable to these treatments [2,9,10]. Furthermore, therapies with pharmacological agents (i.e. Sorafenib alone or in combination with other signalling inhibitors) or alternative strategies, including trans-arterial chemo-embolisation or yttrium-90 microspheres, and percutaneous ethanol injection, do not improve substantially the prognosis of patients with locally advanced disease [2,9,10]. It is widely accepted [3,11–14] that interaction of DNA with carcinogens and reactive oxygen and nitrogen species, generated during carcinogen metabolism and/or inflammation accompanying early stages of hepatocarcinogenesis, results in genomic instability leading to somatic point mutations, copy number alterations of individual genes, and gain/loss of chromosomal arms. Numerous excellent reviews have summarised the progresses made in the identification of chromosome aberrations as well as oncogene and oncosuppressor mutations in HCC [4,13,15,16]. Several lines of evidence indicate that the progressive accumulation of genomic alterations, leading to the progressive deregulation of assorted signalling pathways, allows initiated cells to evolve to dysplastic nodules and, finally, to malignant lesions [11–14]. In recent years, a better knowledge of the signalling pathways regulating tumour progression and the definition of distinct genetic variants of HCC to predict the disease outcome led to the identification of new potential prognostic markers and targets for molecular-based

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therapies [10–13]. Studies performed in transgenic mice differently prone to HCC progression, rodent strains with different inherited susceptibilities to hepatocarcinogenesis, and different human HCC subtypes, selected according to their clinicopathologic features, strongly contributed to the knowledge of changes leading to the deregulation of signalling pathways and their role in HCC development [11,14,17]. This review provides an interpretive analysis of recent advances in the implication of the deregulation of major signalling pathways for the determination of distinct prognostic subtypes of HCC. We explore the contribution of these studies both for the identification of new putative prognostic markers and possible opportunities for targeted therapies. 2. Risk factors and cancer heterogeneity Major risk factors associated with the development of HCC are chronic HBV and HCV infections, alcoholic hepatitis, Aflatoxin B1 [3–5], and some diseases inherited by monogenic and polygenic mechanisms [18–30] (Table 1). However, HCC frequency shows great differences within a human population in response to risk factors [31], suggesting a pathogenetic role of additional environmental and/or genetic factors. The association of increased risk of HCC [32–34] with genetic polymorphisms of Cytochromes P450 2E1 and 2D6, and Aldehyde dehydrogenase, involved in ethanol metabolism, CYP1A2, CYP3A4, and CYP3A5, involved in Aflatoxin activation, and the detoxification enzyme Glutathione S-transferase is controversial [35]. Recent evidence indicates that miRNA polymorphisms may contribute to cirrhosis-related HCC susceptibility in Chinese patients (see Section 6). Studies of families at risk suggest the implication of a polygenic control of HCC incidence [36–39]. Familial aggregations have been explained in a pedigree study from China [40] by the interaction of HBV infection and a major gene. Present evidence points to an analogous genetic model of predisposition to chemically induced rodent hepatocarcinogenesis [14,41]. A locus involved in genetic susceptibility to HCC has been mapped on chromosome 4q25 in Chinese families [42]. It was suggested that PAPSS1 (3′-phosphoadenosine 50phosphosulfate synthase-1) [43] is a cancer-associated gene. A recent genome-wide association study led to the discovery of a previously

D.F. Calvisi et al. / Biochimica et Biophysica Acta 1826 (2012) 215–237 Table 1 Genetic syndromes associated with the development of hepatocellular carcinoma. Syndrome Monogenic Porphyria acuta intermittens Porpyria cutanea tarda α1-Antitrypsin deficiency Glycogen storage disease type 1a Glycogen storage disease type 1b Glycogen storage disease type 3 Hemochromatosis Hereditary Tyrosinemia acuta Polygenic Autoimmune hepatitis Diabetes type 2 Hypothyroidism Metabolic syndromeg

Gene

Inheritance

Reference

HMBSa URODb SERPINA 1 G6PCc G6PTd AGLe HFE FAHf

AD AD AR AR AR AR AR AR

[18] [19] [20] [21,22] [23,24] [23] [25] [26] [27] [28] [29] [30,31]

a

Hydroxymethylbilane synthase. Uroporphyrinogen decarboxylase. c Glucose-6-phosphatase, catalytic. d Glucose-6-phosphatase translocase. e Amylo-1,6-glucosidase. f Fumarylacetoacetate hydrolase. g Includes non-alcoholic fatty liver disease and non-alcoholic steatohepatitis associated with a cluster of interrelated metabolic risk factors such as raised fasting glucose, central obesity, dyslipoproteinemia, and hypertension [31]. b

unidentified locus in the 5′ flanking region of MICA (Major histocompatibility complex class I chain-related gene A), on 6p21.33, strongly associated with HCV-induced HCC, and with progression from chronic hepatitis C to HCC [44]. Thus, according to these researches, a complex combination and interplay of susceptibility/resistance alleles determine the individual risk. Some individuals may inherit a predominance of susceptibility alleles and/or a major allele and be highly cancer prone. However, since human individuals are generally casually assorted, a situation of high or low genetic risk should be rare. A corollary of this situation is that the effect of polygenic inheritance can be masked by a predominant presence of environmental high risk factors. The complex relationships of genetic and etiologic factors, and interaction with various environmental risk factors, involved in different steps of hepatocarcinogenesis, create a wide genotypic and phenotypic heterogeneity within human HCC [45]. Consequently, the evaluation of the role of deregulation of single genes and signalling pathways in HCC pathogenesis and identification of HCC prognostic subtypes may be difficult. A valuable contribution to explore HCC pathogenesis is provided by rodent models [46,47]. Preclinical experimentation in these models has the advantage of obtaining premalignant and malignant lesions with low grade of heterogeneity and differences in phenotypic propensity to progression, in the absence of disturbing environmental influences. These models allow the simultaneous implementation of various technologies and biomarkers to monitor tumour progression and response to treatment, which cannot be determined in HCC patients. However, substantial challenges in the use of animal models include the difficulties to reproduce the inflammatory and cirrhotic lesions, which precede HCC development in most patients. Only 10– 20% of human HCC, including malignant transformation of hepatocellular adenomas, develop in healthy livers [48]. Nevertheless, comparative evaluation of the results of preclinical studies in human lesions strongly contributed to dissect the molecular pathogenesis of HCC and identify new prognostic HCC subtypes [11,15,17]. 3. Cell cycle deregulation and the proliferation signature Numerous observations link the pervasive deregulation of c-MYC, CYCLIN D1 and CYCLIN E to hepatocarcinogenesis progression. c-MYC down-regulation by antisense strategy leads to a decrease in E2F1 level and blocks in vitro growth of human and rat HCCs [49].

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Dysplastic nodules (DNs) and HCCs, chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis, undergo fast progression and exhibit highest c-Myc amplification and/or overexpression, compared to slowly progressing lesions of BN and Wistar resistant rats [50]. A central role of c-MYC amplification/overexpression in the progression of human DN to HCC has also been documented [51,52]. Interestingly, HCCs developing in c-Myc transgenic mice undergo regression, associated with tumour cells redifferentiation, following inactivation of the c-Myc transgene [53]. Over-expression of CYCLIN D1, CYCLIN A, and CYCLIN E, associated with Cyclin-dependent kinases (CDKs) activation leading to pRb hyperphosphorylation occurs in human HCC [54,55] and in DN and HCC of genetically susceptible rats [56]. The consequent restraint in the formation of inactive pRb–E2F1 complex allows the formation of E2F1–Dp1 complexes activating DNA synthesising genes [57]. In DN and/or HCC of resistant rats, low or no increases in Cyclins D1, E, and A, and E2f1 expression, and Cyclin–Cdks complex levels are associated with p16 INK4A over-expression and pRb hypophosphorylation [56]. These findings envisage a block of G1–S transition in liver lesions of resistant rat liver, and indicate a role of genes involved in G1 and S phases of cell cycle in the progression of preneoplastic and neoplastic liver lesions. Accordingly, CYCLIN D1, CYCLIN A, CYCLIN E, and CDC2 (Cell cycle controller-2) are known to be prognostic markers for human HCC [58]. Active proliferation in fast progressing DN and HCC requires inactivity of cell cycle inhibitors. p16 INK4 is a specific inhibitor of CDK4 and CDK6, which prevents pRb phosphorylation by CDK4/6 and blocks G1 phase progression. p16 INK4 is inactivated in 60–85% of human HCC via GpG methylation of its promoter [59]. Other mechanisms of p16 INK4 inactivation include the formation of complexes of Cyclin D1 kinases with Heat shock protein 90 (HSP90) and Cell division cycle 37 (CDC37) protein [60], and the nuclear export of E2F4, a p16 INK4 effector, by CRM1 (required for Chromosome region maintenance 1; Exportin 1) [61,62] (Fig. 1). These mechanisms are sharply

Fig. 1. Protection by CDC37–HSP90 and CRM1 of cell cycle G1–S progression from inhibition by p16INK4A. In the nucleus, p16INK4A forms inhibitory complexes with the kinases CDK4 and CDK6, hindering their activation by Cyclins D and pRb phosphorylation. The chaperons CDC37 and HSP90 form a complex with CDKs, that competes with p16INK4A thus impeding the formation of the inhibitory complex. The protein CRM1 complexes the p16INK4A effector E2F4, and relocates it to the cytoplasm, thus inactivating p16INK4A. The blunt arrow indicates inhibition.

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with CDK2-dependent serine phosphorylation (which inhibits the interaction between CDH1 and SKP2), and HINT1 inactivation, impede SKP2 degradation [65]. Recent studies on the transcription factor FOXM1 (Forkhead box M1; Fig. 3), a major downstream effector of ERK1/2 (extracellular signal-regulated kinase 1/2) proto-oncogenes, suggest the existence of differences in G2/M regulation in HCC subtypes. FOXM1 induces the transcription of AURKA (Aurora A) and NEK2 (never in mitosis gene A-related kinase 2) genes that generate genomic instability, CYCLIN B1, CDC2, and CDC25B (cell division cycle 25B) that regulate G2–M transition, ERYTROPOIETIN involved in micro-vascularisation, and the anti-apoptotic SURVIVIN [67]. Up-regulation of FoxM1 and its targets occurs earlier and is more pronounced in DN and HCC of susceptible F344 than resistant BN rats. This is associated with highest Cdc25B expression, Cdc2–Cyclin B1 complexes, and G2–M transition in F344 than BN rat liver lesions [68]. Studies on human HCC showed that FOXM1 levels progressively increase from surrounding non-tumourous livers to HCC, reaching highest values in HCCP,

Fig. 2. Post-translational regulation of CDK2 inhibitors. CDK2, activated by Cyclin E, is involved in the transition of G1 to S phases of cell cycle. G1–S transition is curtailed by CDK2 inhibitors P21WAF1, P27KIP1, P27KIP2, P130, RASSF1A, and FOXO1. CDK2mediated phosphorylation of Ser64, and to a lesser extent Ser72, stabilises SKP2 by interfering with its association with the ubiquitin ligase CDH1-APC-C. Consequently, SKP2 in complex with CKS1 may ubiquitinate CDK2 inhibitors thus allowing their proteasomal degradation. This mechanism is limited by the SKP2–CKS1 ubiquitin ligase inhibitor HINT-1, as well as by SKP2 dephosphorylation operated by the phosphatase CDC-154B. The blunt arrows indicate inhibition.

enhanced in DN and HCC of genetically susceptible rats [63]. Notably, higher up-regulation of HSP90/CDC37 and formation of protective complexes occur in human HCC subtypes with poorer prognosis (based on survival length after partial liver resection; HCCP) with respect to HCC with better prognosis (HCCB) [63]. These findings suggest that HSP90 and CDC37 genes could be targets for HCC chemoprevention and therapy. Cell cycle inhibitors of Cyclin E and Cyclin A dependent Cdk2, p21 WAF1, p27 KIP1, p57 KIP2, p130, p107, RassF1A (Ras association domain family 1A), active during late G1 and S phases, undergo a consistent ubiquitination by the ubiquitin ligase complex Skp2–Cks1 (S-phase kinase-associated protein 2–Cdc28 protein kinase 1), followed by proteasomal degradation during the G1–S transition in DN and HCC of genetically susceptible rats [64]. Low Skp2–Csk1 activity and proteasomal degradation of cell cycle inhibitors occur in resistant rat lesions [64]. Down-regulation by promoter methylation of CDK2 inhibitors encoding genes P21 WAF1, P27 KIP1, P57KIP2, P130, RASSF1A, and FOXO1 (Forkhead box O1), occurs in variable percentages of human HCC [65]. Recent research showed that, in unmethylated cases, ubquitination of inhibiting proteins by SKP2–CSK1 ligase, followed by their proteasomal degradation, allows active G1–S transition and fast growth [65]. Promoter hypermethylation or proteasomal degradation of CDK2 inhibitors occurs more frequently in HCCP than HCCB, and the level of SKP2 expression is directly correlated with the rate of HCC cell proliferation and level of micro-vascularisation of samples, and inversely correlated with apoptosis [65]. Analysis of the mechanisms responsible for interstrain difference in degradation of CDK2 inhibitors showed upregulation of the SKP2 suppressor, HINT1 (Histidine triad nucleotidebinding protein 1), and SKP2 dephosphorylation by CDC14B (cell division cycle 14, Saccharomyces cerevisiae homolog B) phosphatase, followed by ubiquitin ligase (APC/C)CDH1- (Anaphase Promoting Complex/Cyclosome and its activator CDH1)-mediated degradation [66], in HCCB [65] (Fig. 2). In HCCP, down-regulation of CDC14B associated

Fig. 3. Crosstalk between RAS–ERK pathway and FOXM1. The active RAS (RAS–GTP), RAF, MEK1/2, and ERK1/2 cascade may be limited by the ERK1/2 inhibitor DUSP1. This mechanism is controlled by DUSP1 phosphorylation of Ser296 residue, followed by its ubiquitination by the SKP2–CKS1 ubiquitin ligase and proteasomal degradation, as well as by SKP2–CKS1 activation operated by FOXM1, a major target of ERK1/2. FOXM1 strongly influences cell proliferation and survival, by targeting AURKA and NEK2 genes, involved in genomic instability (GI), CDC2, CYCLIN B1, and CDC25B genes, involved in G2–M progression, antiapoptotic SURVIVIN (SURV), and angiogenesis genes such as ERYTROPOIETIN (EPO) and VEGF. Therefore, FOXM1 is involved in a positive feedback loop, reinforcing the ERK cascade by its ability to inhibit DUSP1.

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compared to HCCB, thus substantiating a role of FOXM1 in HCC development and progression [68]. FOXM1 expression correlates positively with HCC proliferation and micro-vascularisation, and negatively with apoptosis. Interestingly, FOXM1 may trigger the activation of the SKP2–CSK1 ubiquitin ligase, whereas its down-regulation suppresses the ligase expression [69], which suggests that its activity could also contribute to the deregulation of G1–S transition in HCC [68]. Overall, the studies on cell cycle deregulation during hepatocarcinogenesis indicate the early alteration of genes regulating G1–S and G2–M transitions, partly dependent on alterations of the transcriptional and post-translational control of cell cycle inhibitors. Moreover, they strongly suggest a prognostic value of the deregulation of cell cycle inhibitors. 4. Deregulation of signalling pathways Several studies aimed at identifying the molecular mechanisms underlying cell cycle deregulation point towards the deregulation of growth factors and their receptors in preneoplastic and neoplastic liver cells. Growth factor binding to these receptors results in receptor phosphorylation, followed by activation of adapter proteins and activation of different signal transduction pathways. Numerous signalling pathways, including MAPK, PI3K/AKT, iNOS/IKK/NF-kB, TGF-β, NOTCH, Hedgehog, WNT/β-Catenin, and Hippo pathways are involved in the deregulation of HCC growth and have a pivotal role in the determination of HCC progression and aggressivity. 4.1. Mitogen Activated Protein Kinase (MAPK) pathway One of the major branches of the MAPK signalling, the RAS–ERK cascade, involved in cell growth stimulation, transduces signals from tyrosine kinase receptors, such as EGFR (epidermal growth factor receptor) [70,71], IGFR (insulin-like growth factor receptor) [72,73], PDGFRα and PDGFRβ (platelet derived growth factors α and β) [74], and MET (hepatocyte growth factor receptor, HGFR) [75,76]. These receptors are up-regulated in variable percentages of human HCC, due to over-activity of the corresponding genes or, as for EGFR, to overproduction of ligands such as TGF-α, EGF and Amphiregulin [77,78]. Up-regulation of the RAS–ERK cascade (Fig. 3) in human HCC leads to over-expression of active ERK1/2 (phosphorylated ERK1/2), which transactivates numerous genes involved in cell proliferation (c-JUN, C-FOS, C-MYC, FOXO, and ETS), angiogenesis [hypoxia-inducible factor 1α (HIF-1a), vascular endothelial growth factor-α (VEGF-a)], and glycolysis [hexokinaseii (HkII)] [79–81]. MAPK cascade genes and their targets are generally overexpressed in foci of altered hepatocytes, DN, and HCC of rodents and humans [3]. Ras mutations infrequently occur in rodent HCC, induced by various chemicals [82], and in human HCC, but they are common in HCC of workers exposed to vinyl chloride [83]. Recent studies have shown down-regulation of Dusp1 (dual-specificity phosphatase 1), a specific Erk inhibitor, in DN and HCC of susceptible F344 rats, which consequently exhibit elevated active Erk levels [84]. Conversely, elevated Erk activation phosphorylates the ser296 residue of Dusp1, thus contributing to its ubiquitination by SKP2–CKS1 ubiquitin ligase, followed by proteasomal degradation [84]. On the other hand, ERK1/2 sustains SKP2–CKS1 activity through its target FOXM1 [68] (Fig. 3). In DN and HCC of resistant BN rats, high Dusp1 expression is associated with its relatively low ubiquitination [84], with consequent inhibition of pErk1/2, and its target genes, suggesting that Dusp1 is a gene involved in the acquisition of a resistant phenotype. Activated ERK proteins and their targets HIF1-α, VEGF-α, and HKII, exhibit the highest up-regulation in HCCP when compared to HCCB [85]. In agreement with this finding, ERK1/2 activation has been associated with HCC aggressivity in a series of 208 patients [86]. Elevated RAF/MEK/ERK signalling also occurs in HBV and HCV

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induced hepatitis as well as in alcoholic hepatitis, which may evolve to HCC, suggesting an early deregulation of MAPK in human hepatocarcinogenesis [87–89]. However, the role of DUSP1 in early stages of human HCC has not been evaluated. Of note, DUSP1 expression is higher in HCCB than in normal and non-tumourous surrounding liver, whereas DUSP1 expression is low in HCCP due to promoter hypermethylation, loss of heterozygosity at the DUSP1 locus, and phosphorylation followed by ubiquitination and proteasomal degradation [85]. In HCC, DUSP1 expression is inversely correlated with the expression of active ERK1/2, proliferation rate, and micro-vessel density, and directly correlated with apoptosis [85]. These observations point to a putative prognostic role of pERK1/2 and DUSP1 and indicate that the cooperation between pERK1/2 and SKP2–CKS1 ligase [68], through DUSP1 phosphorylation and FOXM1 activation, provides a positive feedback regulation of HCC proliferation [90] (Fig. 3). 4.2. PI3K/AKT pathway Stimulation of tyrosine kinase receptors on cell surface, leads to PI3K phosphorylation followed by AKT/PKB activation. Active AKT is channelled into a plethora of downstream biological responses from angiogenesis, cell survival, proliferation, translation, to metabolism (Supplementary Fig. S1). In normal tissues, the PI3K/AKT/mTOR (phosphatydilinositol 3-kinase/AKT/mammalian target of Rapamycin) pathway is negatively regulated by the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), which dephosphorylates the lipid products of PI3K [91]. Recent microarray analysis showed that 23% of HCC patients have elevated levels of AKT phosphorylation on Ser473 [92]. The mechanisms activating PI3K/AKT/mTOR pathway in HCC include: over-expression of IGF and EGF and their receptors and related growth factors [93,94], PI3K mutation [95], PTEN mutation, deletion, or downregulation by HBX protein in HBV-infected patients [96]. PTEN down-regulation and AKT activation have been implicated in poor prognosis [86,97,98]. A crucial role of PI3K/AKT/mTOR signalling in HCC is supported by the observation that its inhibition strongly reduces in vitro growth and orthotopic implantation of HCC cells [99–101]. Partial remission and stable disease at 3 months were found in a phase II study on patients with HCC treated with the mTOR inhibitor Sirolimus [102]. Akt up-regulation, associated with inactive Gsk3β (glycogen synthase kinase-3β), occurs in chemically-induced preneoplastic and neoplastic rat liver lesions [103] and in HCC of c-Myc/TGF-α double transgenic mice [104]. Up-regulation of the PI3K/AKT/mTOR pathway, in diethylnitrosamine-induced mouse HCC, is associated with the down-regulation of Metallothionein expression, suggesting a role of this pathway in Metallothionein regulation and reactive oxygen species production [105]. A role of PI3K/AKT/mTOR signalling deregulation in liver disease predisposing to human HCC is suggested by the link between PTEN down-regulation and development of several hepatic diseases, including non-alcoholic steatohepatitis (NASH) and HCV hepatitis [106]. The stimulation of inflammation, cell cycle progression, and cell proliferation following PTEN down-regulation and over-activity of PI3K/AKT/mTOR signalling [106] indicates that impaired PTEN expression may represent an important step in the progression of NASH and viral hepatitis towards HCC. Recent observations suggest a connection between the expression of the transcription factor MYBL2 and AKT signalling. Highest MYBL2 expression occurs in fast growing DN and HCC of F344 rats, HCC of E2F1 transgenic mice, and human HCCP than in slow progressing lesions of BN rats, c-Myc transgenic mice, and HCCB [107,108]. Accordingly, phosphorylated MYBL2 expression is positively correlated with human HCC growth and micro-vascularisation, and negatively correlated with apoptosis, suggesting a prognostic role of active MYBL2 in HCC [109]. HCC cells transfected with MYBL2 exhibit increased

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proliferation and G1–S and G2–M cell cycle phases, whereas the opposite occurs when MYBL2 is silenced by specific siRNA [108]. MYBL2 transfection in Huh7 cells transcriptionally activates a variety of genes involved in signal transduction and cell proliferation, including MDK (Midkine) [108], which, in turn, may activate cell cycle and AKT and ERK1/2 pathways [109]. Accordingly, correlative studies showed a positive correlation of MYBL2 with AKT expression in human HCC (Frau net al., unpublished results). Among the genes targeted by MYBL2, the transcription regulator HDAC10 (histone deacetylase 10) is strongly up-regulated, and the oncosuppressor PP1CA (protein phosphatase 1 catalytic subunit alpha) is down-regulated in Huh7 cells [108]. Moreover, Hdac10 protein expression progressively increases, whereas Pp1CA expression progressively decreases from normal liver, to dysplastic liver, and HCC of c-Myc and E2f1 transgenic mice, with highest changes in the more aggressive HCC of E2f1 mice [108]. Interestingly, protein phosphatase-1 inhibits AKT phosphorylation at Thr-450 [110]. This interferes with the capacity of PI3K/AKT cascade to promote cell survival and cell proliferation through stimulation of WNT/β-catenin and IKK/NF-kB pathways [110] (Fig. S1B). 4.3. MYBL2 and LINC in liver tumours with mutated p53 Numerous studies indicate that the p53 tumour suppressor gene plays a major role in hepatocarcinogenesis. In geographical areas where food contaminated by Aflatoxin B1 is consumed, p53 mutation at the third position of codon 249 is an early and frequent event [3,13,16]. In contrast, p53 mutation in HCC may occur as a late event without a typical mutational pattern in areas with low Aflatoxin B1 intake [3,13,16]. A recent work [108] showed a role of the active MYBL2–LIN9 complex (LINC) in the survival of DNA damaged HCC cell harbouring mutant p53. Higher MYBL2 and LINC levels were found in HCC with a mutant p53 gene, than in HCC with wild-type p53. Functional experiments on HCC cell lines harbouring wild-type p53 (Huh6 and HepG2) or mutant p53 (Huh7 and Hep3B) showed that although MYBL2 suppression reduced proliferation, induced apoptosis, and increased DNA damage at similar levels in the four cell lines, strongest growth restraint and apoptosis, and massive DNA damage occurred only in p53 mutant cell lines when MYBL2 or LIN9 silencing was associated with doxorubicin-induced DNA damage. At the molecular level, doxorubicin treatment did not affect MYBL2 and LIN9 levels in any cell line, but induced the inactive complex LIN9–p130 and gradual dissociation of MYBL2 from LIN9 in p53 wild-type cells, whereas MYBL2–LIN9 binding was not reduced by doxorubicin in p53 mutant cells. Silencing of p53 or p21 WAF1 abolished the DNA damage response in p53 wild-type cell lines and enhanced apoptosis and growth restraint. These results assign a crucial role to the integrity of the MYBL2–LIN9 complex for survival of HCC cells with mutant p53 in the presence of DNA damage. 4.4. Inducible nitric oxide synthase Nitric oxide (NO) is a product of the conversion by nitric oxide synthase (NOS) of L-arginine to L-citrulline. The inducible iNOS isoform is present in hepatocytes, Kupffer and stellate cells, and cholangiocytes. In general, iNOS is not expressed at a significant level in normal cells, but is highly induced in many cancer cell types [111]. NO causes a variety of severe damages, including DNA strand breaks and oxidation, and inhibition of DNA repair. In particular, NO may favour HCC development by inducing DNA mutations, in hepatocytes surviving to oxidative stress, and vasodilatation that can provide premalignant and malignant cells with sufficient metabolites and oxygen. Overproduction of inflammatory cytokines and growth factors during early stages of hepatocarcinogenesis deregulates iNOS [112,113]. Reactive

nitrogen species produced via iNOS during chronic hepatitis may play a key role in carcinogenesis by causing DNA damage. Suppression of iNOS by aminoguanidine results in decreased growth of HCC cell lines and in NF-kB (nuclear factor kB) and RAS/ERK down-regulation as well as in increased apoptosis in vivo and in vitro [114]. Conversely, blocking of NF-kB signalling by sulfalazine or specific siRNA causes iNOS down-regulation in HCC cell lines. Analogous results have been obtained by blocking ERK signalling by UO126 [114]. These findings document the existence of an active iNOS cross-talk with IKK/NF-kB (inhibitor of kappa light chain gene enhancer in b cells, kinase of, beta/NF-kB) and ERK signalling in HCC. Recent research indicates that iNOS cross-talk with IKK/NFkB cascade is involved in HCC progression. The progressive induction of iNOS, IKK, and NF-kB occurred in mouse and liver preneoplastic and neoplastic lesions, with highest levels in most aggressive HCC of F344 rats and c-Myc-Tgf-α transgenic mice, compared to the lesions of BN rats and c-Myc transgenics, as well as in HCCP than HCCB [114]. Interestingly, in human HCC, iNOS expression was found to correlate positively with genomic instability, cell proliferation, and micro-vascularisation, and negatively with apoptosis [114]. These observations suggest that the components of iNOS signalling could represent putative prognostic markers and therapeutic targets for human HCC. 4.5. TGF-β expression and HCC dedifferentiation Binding of mammalian TGF-β (tumour growth factor-β) isoforms, TGF-β1, TGF-β2 and TGF-β3, to a receptor system constituted by RI, RII, and RIII, is followed by the activation of SMAD (mothers against decapentaplegic, drosophila, homolog of) protein signalling. Heteromeric complexes of SMAD2 and SMAD3 with SMAD4 translocate to the nucleus and repress or activate specific DNA sequences [115,116] (Fig. S2). TGF-β1 is a tumour suppressor with true haploinsufficiency [117]. Disruption of Tgf-β1 signalling is associated with the acceleration of hepatocarcinogenesis in c-Myc/TGF-α transgenic mice [118]. Haploinsufficiency of tumour suppression by Tgf-β1 in mice heterozygous for TgβR-II deletion enhances the susceptibility to hepatocarcinogenesis by N-diethylnitrosamine [119]. A reduction of TGF-β receptors occurs in up to 70% of HCC [120]. Recent studies [121] aimed at illustrating the mechanisms of TGF-β oncosuppression, showed that the SMAD3/4 adaptor β2-Spectrin (Fig. S2) interacts with CDK4 and SMAD3 in a competitive and TGF-β-dependent manner, and markedly reduces CDK4 expression to a greater extent than other CDKs and cyclins, and inhibits pRb phosphorylation, thus interfering with cell cycle activity, cellular proliferation, and HCC formation. In apparent contrast with these findings, TGF-β1 is overexpressed in a consistent number of HCCs and its expression correlates with HCC dedifferentiation [122]. TGF-β1 may be considered a prognostic marker for HCC because of the correlation of its serum levels with poorer prognosis and increased tumour angiogenesis [123,124]. The mechanisms of HCC induction by TGF-β are poorly known. A mechanism linked to TGF-β effect on fibrogenesis has been proposed. The phosphorylation of SMAD2/3 leads to the secretion of multiple fibrogenic mediators, such as CTGF (connective tissue growth factor), cytokeratin, vimentin, fibronectin, α-smooth muscle actin, as well as to loss of E-cadherin and PAI-1 (plasminogen activator inhibitor-1) [125]. This may induce the production of extracellular matrix proteins, fibrosis and precancerous lesions. Continuous TGF-β secretion could support neo-angiogenesis, loss of E-cadherin-dependent adhesion, and β1-integrin activation that may favour the formation of tumour metastases [124,125]. Of note, TGF-β signalling induces epithelial to mesenchymal transition (EMT), which leads to enhanced cell migration and invasiveness

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[126]. Thus, tumour cells that have lost the responsiveness to TGF-β but retain an active TGF-β signalling may exhibit enhanced migration and invasiveness. Interestingly, according to a recent report [127], transfection of iNOS to mouse hepatocytes or treatment of these cells with the NO donor S-nitroso-N-acetylpenicillamine inhibits TGF-β-induced EMT and apoptosis. Depletion of intracellular ATP by the mitochondrial uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) inhibits TGF-β induced EMT, apoptosis, and Stat3, suggesting that ATP depletion and Stat3 activity are involved in EMT and apoptosis induction by TGF-β. These observations together with analogous findings relative to other tumour types [128] point to a dual role of TGF-β. Different mechanisms could protect HCC cells from inhibition by TGF-β. In liver tissues of patients with HCC, TGF-βR-II level may be lower than in patients with chronic hepatitis or cirrhosis, and transfection of TGF-βR-II cDNA into the HCC cell line Huh7 induces cell growth inhibition and apoptosis [129]. In carcinogen-treated rats, progressive rise in Smad7 expression from early lesions to HCC is associated with a decrease in expression of TGF-β-R1, Smad2, and Smad4 [130]. The presence of elevated SMAD7 expression in human HCC suggests that an analogous mechanism may occur in human hepatocarcinogenesis [131]. Another possible mechanism of resistance to TGF-β, found in a small cohort of HCC patients, consists in mutations of either SMAD2 or SMAD4 [132]. Notably, TGF-β signalling dysfunction occurs in STAT3/Oct4-positive HCC cells [133], which are considered liver cancer progenitors, indicating a possible role of TGF-β/SMAD signalling in cancer stem cell-driven HCC progression. Emerging evidence indicates that TGFβ plays a crucial role in the transition of a stem cell to a progenitor cell phenotype, and then to a differentiated phenotype. Mice heterozygous for the adaptor protein Elf (Elf +/− mice; Fig. S2) develop HCC [134], and inactivation of at least one of TGF-β signalling components occurs in gastrointestinal tumours [135]. These important observations suggest that tumours may develop in tissues where the transition of progenitor cells to more differentiated stages takes place in the absence of differentiating factors from the TGF-β cascade. Therefore, abnormalities in TGF-β-driven epithelial differentiation should favour carcinogenesis. 4.6. Deregulation of NOTCH receptors NOTCH genes encode highly conserved transmembrane glycoproteins that are activated by interaction with transmembrane ligands of the Delta/Jagged family expressed on the surface of neighbouring cells [136–138] (Fig. S3). Upon interaction, NOTCH receptors are cleaved by metalloproteases and the intracellular NOTCH domain translocates to the nucleus and activates the transcription of target genes including HES-1 (hairy/enhancer of split, Drosophila homolog 1), DELTEX (DELTEX Drosophila homolog), CD25 (interleukin 2 receptor, alpha), CDKN1α (cyclin-dependent kinase inhibitor 1α; p21 WAF1), and SNAIL (snail Drosophila homolog) [137,139,140]. Accumulating evidence established that the NOTCH1 plays vital roles in cell differentiation, embryonic development, EMT, and tissue self-renewal, as well as in the pathogenesis of some human cancers and genetic disorders [139–142]. Of note, NOTCH1 signalling has been found to posses both oncogene and oncosuppressor gene activities [141]. A link between RAS/ERK signalling and transcription of the NOTCH target gene, HES-1, has been discovered in the neuroblastoma cell line SK-N-BE[2]c [143]. Studies aimed at identifying signal transduction pathways playing additional roles in malignant transformation in concert with activated NOTCH4 in tumour cell lines showed that transformation by NOTCH requires active signals from the ERK/MAPK and PI3K pathways downstream of RAS [144]. Higher NOTCH1, JAGGED, and HES-1 mRNA and NOTCH1–4 protein levels were found in HCC than in surrounding non-tumourous liver [145,146]. However, according to other observations, NOTCH3 protein accumulates in HCC, but not in

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cirrhotic liver, whereas NOTCH4 accumulation occurs in both cirrhotic and tumourous liver [147]. In addition, higher NOTCH3 and HES-6, but not HES-1, mRNA levels were found in HCC than cirrhotic liver, whereas NOTCH4 mRNA levels were lower in HCC than cirrhotic liver [148]. Activated NOTCH signalling is required for hepatitis B virus X protein to promote proliferation and survival of human hepatic cells [148]. Active RAS/ERK and PI3K/AKT cascades are up-regulated in HCC (see Sections 4.1 and 4.2), but the mechanistic evaluation of the effects of the up-regulation of these pathways on NOTCH signalling is lacking. Moreover, in contrast with above results, it was reported that NOTCH1 down-regulates cell cycle genes, including Cyclins A, D1 and E, up-regulates p21 WAF1, inhibits the proliferation and induces apoptosis in human HCC cells [149,150]. Consistent with the latter observation, the oncosuppressor PTEN is up-regulated and the PI3K/AKT signalling is inhibited in HEK293 fibroblasts overexpressing NOTCH1 [151]. These contradictory findings are partly unexplained. However, recent results [152] suggest that NOTCH1 may differently regulate oncogenesis according to the p53 status. An oncogenic link that increases with HCC grade was found between p53, NOTCH1, and SNAIL in HCC. A correlation between NOTCH1 and p53 expression occurred in HCC cells expressing p53WT, but not in p53Mut-expressing cells. In the presence of p53WT, SNAIL/NOTCH1 activation increased the invasiveness of HCC cells, whereas in the absence of p53WT, NOTCH1 decreased the HCC invasiveness. These observations imply that the combinatorial expression pattern of NOTCH1, SNAIL, and p53WT proteins and the mutational status of p53 could predict HCC behaviour, and encourage efforts to target the NOTCH1/SNAIL axis therapeutically. Interestingly, higher expression of genes involved in NOTCH pathway occurs in CD133[+] than in CD133[−] HCC cells [153], and recent results showed that DELTEX and p300 are highly expressed in HepG2 stem-like cells [154]. Notably, CD133 positivity correlates with higher aggressivity of HCC and shorter patients' survival [155–157]. Moreover, in human HCC cell lines, the presence of cells positive for the combination of CD133 and CD44 markers is associated with higher clonogenicity and in vivo carcinogenesis with respect to cells expressing CD133 alone [158]. Taken together, these results support a role of NOTCH signalling in the maintenance, differentiation and propagation of liver cancer stem cells and HCC aggressivity. 4.7. Hedgehog signalling Two transmembrane proteins, Patched (PTCH) and Smoothened (SMO), transduce the signal of the evolutionarily conserved Hedgehog pathway (Fig. S4). In the absence of an Hh ligand, PTCH represses SMO [159–163]. When hedgehog ligand binds to PTCH and releases the repression of SMO by PTCH, SMO transduces signals intracellularly, and this results in the nuclear localization of the transcription factor GLI (glioma-associated oncogene homolog 1). Three GLI proteins, GLI1, GLI2, and GLI3 are present in vertebrates. GLI1 and 2 are the major positive intermediaries of Hh signalling, which induce transcription of cell cycle- and growth-related genes, including Cyclins, IGF-2, β-Catenin, whereas GLI3 has a repressor effect [164]. Recent evidence demonstrated that Hh signalling plays an important role in multiple tumour types, including basal cell carcinoma, medulloblastoma, small cell lung, breast, prostate, gastric, oesophageal, pancreatic and liver cancers [165,166]. Over-expression of various Hh genes has been found in up to 60% of human HCC and specific inhibition of Hh pathway leads to downregulation of GLI targeted genes [167–170]. Blocking of Hh pathway inhibits proliferation, induces apoptosis and represses c-MYC and CYCLIN D1 expression in HCC cell lines [166–170], suggesting that this pathway may be a candidate for therapeutic targeting in HCC. Notably, recent studies [171] in which Hh signalling was manipulated in cultured liver cells, showed increased hepatic expression of Hh

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ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC, including progenitors expressing markers of tumour stem/ initiating cells, are Hh-responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC, in accordance with the hypothesised oncogenic role of the Hh, and growth. Recent observations [68] indicate the existence of a functional interplay between ERK and GLI1 activity leading to FOXM1 upregulation, resulting in increased proliferation and angiogenesis and reduced apoptosis in Hh-responsive HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of these cell lines. A reciprocal activation of ERK2 and GLI1 has been found: GLI1 has a MEK1 (mitogen-activated protein kinase kinase 1)-responsive N-terminal domain, and the activation of the ERK pathway by basic FGF (fibroblast growth factor) stimulates GLI1 activity through this domain [172]. Indirect ERK activation through GLI-mediated induction of PDGF receptor has been postulated [173]. Furthermore, FOXM1 activation is mediated by an additive activity of ERK and GLI1 in HCC cell lines [68]. Conversely, FOXM1 sustains ERK activity via inhibition of the degradation of the ERK inhibitor, DUSP1 [68]. A significant association between the expressions of GLI2 and FOXM1 proteins in HCC has been described [174]. Interestingly, combined over-expression of HSP90 and CDC37 sustains elevated SUFU (suppressor of fused) expression [175]. Accordingly, our preliminary data indicate the upregulation of the SUFU gene in human HCC (unpublished results), and a previous report showed a strong induction of HSP90 and CDC37 in F344 rat liver lesions and human HCCP [63]. Thus, a role for combined activity of HSP90 and CDC37 in the highest activation of GLI1 observed in aggressive neoplastic lesions of F344 rats and human HCCP might be hypothesised. Increased nuclear immunolabelling of GLI2 protein is significantly correlated with poorer HCC differentiation, and with portal vein tumour thrombosis [169,174]. In addition, over-expression of FOXM1 protein is significantly associated with increased HCC progression [68,174]. Altogether, these findings strongly suggest that Hh signalling is involved in the differentiation and proliferation of tumour cells, at least in part through inducing FOXM1 protein up-regulation. 4.8. WNT/β-catenin signalling pathway The canonical WNT pathway has emerged as one of the main signalling cascades involved in human hepatocarcinogenesis [176]. The central player of this pathway is the β-catenin gene [176,177]. βCatenin is a component of the sub-membranous plaque of adherens junctions and desmosomes in mammalian cells (Fig. S5). It is physiologically involved in two major functions: cell–cell adhesion by association with E-cadherin, and transmission of the proliferative signal of the Wingless/WNT pathway. Protein levels of β-catenin are regulated by phosphorylation at the NH2-terminal region by GSK-3β complex consisting of APC protein, axin/conductin and GSK-3β. Once phosphorylated, β-catenin is rapidly degraded through the ubiquitin– proteasome pathway [177–179]. Mutations of APC or β-catenin lead to elevated free pools of βcatenin, which translocates into the nucleus where it interacts with transcription factors of the Tcf/lymphoid enhancer factor family. βCatenin/TCF complex activates target genes that are involved in the control of cell growth and apoptosis [176–185] (Fig. S5). WNT signalling is up-regulated in a subset of HCC via either mutations in the genes encoding components of the WNT pathway, including βcatenin (the most frequently observed), AXIN1 and AXIN2 (rare), or in the absence of WNT/β-catenin mutations [176,178]. In transgenic mouse models, activation of β-catenin was most frequent in liver tumours from c-Myc transgenic mice, whereas it was very rare in faster growing and histologically more aggressive HCCs developed in c-

Myc/TGF-α mice [180]. c-Myc and c-Myc/TGF-α HCCs with nuclear accumulation of β-catenin displayed a higher proliferation rate and tumour size when compared with HCCs without β-catenin activation, suggesting that activation of β-catenin provides proliferative rather than survival advantages in transgenic hepatocarcinogenesis [180]. The reason for the low incidence of β-catenin activation in c-Myc/ TGF-α driven hepatocarcinogenesis is unclear. One possible explanation is that c-Myc/TGF-α neoplastic clones without activation of βcatenin possess selective growth advantages over β-catenin positive clones. In support of this hypothesis, treatment with phenobarbital significantly increases the rate of β-catenin activation in c-Myc/TGFα HCCs by favouring the growth of clones with an intact β-catenin locus [180]. Of note, loss of heterozygosity at the β-catenin locus is frequent in untreated c-Myc/TGF-α HCCs and might explain the low rate of β-catenin activation in this mouse model. Since c-Myc/TGF-α pre-neoplastic and neoplastic lesions are characterised by a high degree of genomic instability [181], the low rate of β-catenin activation in this model is consistent with the observations that β-catenin activation occurs in a subset of human HCCs with a relatively stable genome [181,182] and a more favourable prognosis [183]. However, more recent findings indicate that β-catenin activation may also be negatively associated to the patient's survival length, due to its ability to promote EMT and metastasis following hypoxic stimuli [184]. Also, WNT/β-catenin activation promotes the up-regulation of the hepatic stem cell marker, epithelial cell adhesion molecule (EpCAM), whose presence is associated with poor prognosis in HCC [185]. Based on this contrasting results, it is tempting to speculate that activation of the WNT/β-catenin pathway might influence the prognosis of HCC patients depending on the interaction with other pathways or the tumour [micro]environment. 4.9. Hippo signalling The Hippo pathway is a conserved signalling pathway involved in the regulation of organ growth in Drosophila and vertebrates. The upstream inputs of the pathway are still poorly known. The core components of the Drosophila pathway are conserved in mammals (Fig. S6): Mst1/2 (homologues of Hpo), Sav1 (Salvador), Lats1/2 (Wts homologues), MOBKL1A and MOBKL1B (collectively referred to as Mob1; homologues of Mob-as-a-tumour-suppressor, Mats), and YAP and its paralog TAZ (homologues of Yorki, Yki). Various inputs feeding into the core of the Hippo pathway can also act at different levels within the cascade. A complex network of mechanisms regulates Hippo signalling. Some inputs are associated with the plasma membrane and might transmit information from the extracellular milieu or from cell–cell contacts (Fig. S6) [186–188]. Binding to RASSF proteins also activates MST1/2. MST1/2 and LATS1/2 form a kinase cascade, are regulated by SAV1 and MOB1A/B, respectively, and phosphorylate YAP and TAZ (Fig. S6) [186–188]. Activated LATS1/2 in turn phosphorylates and inhibits YAP/TAZ transcription coactivators [186–188]. A mechanism of YAP/TAZ inhibition involves the phosphorylation of Ser127 in YAP or the corresponding sites in TAZ, which promotes 14-3-3 binding and subsequent cytoplasmic sequestration and inactivation [189,190]. Moreover, YAP phosphorylation at Ser381 by LATS1/2 primes YAP for subsequent phosphorylation by another kinase, presumably casein kinase 1 (CK1δ/ε), activating a phosphorylation-dependent degradation motif. Subsequently, the E3 ubiquitin ligase SCFβ-TRCP is recruited to YAP, leading to its ubiquitination and proteolysis [190,191]. YAP has been regarded to as an oncogene, whose sustained overexpression leads to tumour formation. Elevated YAP protein levels and nuclear localization have been observed in multiple human cancers, including HCC [192–195]. Clinically, high YAP expression in HCC was found to be significantly associated with poor differentiation and high serum AFP in the HCC patients, and YAP was an independent predictor for HCC-specific disease-free survival [196]. Recently, a

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liver-specific Yap transgenic mice model showed a reversible overgrowth of liver and the development of liver tumours, indicating that YAP might play a role in hepatocarcinogenesis [197]. HCC also develop in mice given diethylnitrosamine and then subjected to repeated treatments with the mitogen 1,4-bis[2-[3,5-dichloropyridyloxy]]benzene. YAP and its targets, α-fetoprotein and CTGF, were up-regulated in these tumours, whereas microRNA-375 was downregulated [198]. miR-375 is a negative regulator of YAP expression that reduces the proliferation and invasion of HCC cells, and could have a potential therapeutic role in HCC treatment [199]. These findings indicate that YAP activation plays an important role in hepatocarcinogenesis. This is confirmed by various observations of liver carcinogenesis induced by impaired Hippo pathway associated with YAP activation. Liver-specific ablation of mouse WW45 (SAV1), an adaptor for the MST kinase (Fig. S6), leads to increased liver size and expansion of hepatic oval cells and to eventual development of liver cancer [200]. WW45 ablation increases YAP level and induces its localization to the nucleus in oval cells, likely accounting for their increased proliferative activity, but not in hepatocytes [200]. Liver tumours that develop in mice heterozygous for WW45 deletion or with liver-specific WW45 ablation show a mixed pathology combining characteristics of HCC and cholangiocarcinoma and seem to originate from oval cells [200]. Similar observations were found in mice containing a combined Mst1 and Mst2 deletion [201]. Furthermore, transcriptional profiling of liver tissues from both Mst1/2 and Sav1 conditional mutants shows a specific enrichment of Hippo signalling regulated genes involved in immune and inflammatory responses [201]. Histological and immunological characterization of Mst1/2 double mutant liver tissues revealed abundant periductal accumulation of adult facultative oval cells [201]. Importantly, 70% of human HCC samples show a conspicuous decrease in MST1/2 activity [197]. Several observations have documented a crosstalk between Hippo signalling and different pathways controlling cell proliferation and survival, thus integrating morphogenesis with growth control signalling. RASSF1A disrupts the inhibitory complex RAF1–MST2, thus activating LATS1 with subsequent release of the transcriptional coactivator YAP. Nuclear translocation and binding of YAP to p73 result in transcription of the pro-apoptotic target gene PUMA [202]. Pi3K/AKT signalling interacts with MST1/2 [203]. AKT phosphorylates MST2 in response to mitogens, oncogenic RAS, or depletion of PTEN. MST2 phosphorylation by AKT limits the activity of MST2 by preventing MST2 homodimerization necessary for its activation, as well as by blocking its binding to RASSF1A and promoting its association with the RAF-1 inhibitory complex [204]. MST1 exerts pro-apoptotic function through cleavage by caspases, autophosphorylation of Thr183 and subsequent translocation to the nucleus where it phosphorylates LATS1/2, FOXO3, JNK, and histone H2B [205,206]. The cleavage of MST1 is inhibited by its interaction with AKT followed by phosphorylation of Thr387 and Thr120, which leads to inhibition of MST1 kinase activity, nuclear translocation, and autophosphorylation of Thr183 [205,206]. According to recent observations [207] CTGF expression is stimulated by EGFR ligands and is dependent on the expression of YAP. The formation of YAP complexes with CTGF proximal promoter is responsible for basal and EGFR-stimulated CTGF expression. YAP up-regulation through EGFR activation occurs both in HCC cells and primary human hepatocytes [207]. The Hippo pathway promotes Notch signalling in regulation of cell differentiation, proliferation, and oocyte polarity in Drosophila [208]. Due to the role of Notch signalling in HCC differentiation status and aggressivity, the study of crosstalk between Hippo and Notch pathways could give new insights on HCC progression. Cytoplasmic localization of TAZ, secondary to the sequestration of inactive phospho-TAZ/YAP in the cytoplasm, inhibits canonical WNT signalling [209,210]. Finally, TAZ binds heteromeric SMAD2/3/4 complexes in the presence of TGF-β, and YAP and TAZ determine the nuclear retention of SMAD2/3 [210]. Thus, in the

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presence of impaired Hippo pathway, YAP/TAZ could contribute to the effects of TGF-β/SMAD signalling activation, including EMT and enhanced cell migration and invasiveness (see Section 4.5). These few examples of crosstalk of Hippo cascade with other pathways suggest a critical role of this cascade in carcinogenesis as well as a potentially important contribution of the suppression of YAP/TAZ activity to a networked molecular therapeutic approach to hepatocarcinogenesis. 5. Signalling deregulation as master producer of altered metabolism The attempts to explain the uncontrolled tumour cell growth before the oncogene revolution have intensively focused on the study of metabolism. The observation of an enhanced rate of glucose uptake by tumour cells has been a starting point for understanding the differential metabolism of cancer. Several studies explored the needs of fast growing cancer cells including rapid ATP generation to maintain energy status, biosynthesis of carbohydrates, proteins, lipids and nucleic acids, and maintenance of appropriate cellular redox status [211]. Accumulating evidence indicates that many oncogenic signalling pathways regulate downstream targets connected to metabolic regulation, and some of these metabolic alterations are apparently required for malignant transformation [212]. The adaptation of cancer cell metabolism to support fast growth and survival gives the opportunity to exploit tumour-specific metabolism for therapeutic purposes. Recent preclinical studies have shown some promising results of attempts of pharmacological targeting of enzymes that regulate restructured metabolism of cancer cells [212]. 5.1. Carbohydrate metabolism In 1924, Otto Warburg [213] observed that cancer cells produce most of their ATP through glycolysis, even under aerobic conditions. This behaviour of cancer cells, subsequently denominated “Warburg phenomenon” (or less appropriately “Warburg effect”) has been the object of several studies showing that even if mitochondria isolated from HCC may efficiently consume oxygen and produce ATP [214], intact HCC cells use prevalently glycolytic ATP for protein synthesis [215], and there is a correlation between glycolytic ATP production and aggressiveness of the tumour cells [216]. Thus, the Warburg phenomenon could be considered a positive modifier of cancer, such that it may not be causative but rather facilitates tumour progression [216]. Indeed, glycolytic metabolism although theoretically ≈18 fold less efficient than mitochondrial respiratory chain for ATP production, allows the cells to produce ATP and survive in hypoxic conditions, during the early avascular phase of HCC development, as well as in late stages, when tumour mitochondria are often relatively few and small, lack cristae, and are deficient in the b-F1 subunit of the ATP synthase [217]. Furthermore, lactate generation in cancer cells, as principal end product of aerobic glycolysis, creates acid conditions that favour tumour invasion [218], and could suppress anticancer immune effectors [219]. Glucose can also be metabolised through the pentose phosphate pathway that generates nicotinamide adenine dinucleotide phosphate (NADPH), thus ensuring the cell's antioxidant defences against a hostile microenvironment and chemotherapeutic agents [220]. NADPH contributes to fatty acid synthesis, whereas the non-oxidative portion of the pentose phosphate pathway leads to ribose 5-phosphate synthesis, which may also be fuelled into glycolysis, and is necessary for nucleotide synthesis. The oxidative and non-oxidative parts of the pentose phosphate pathway are controlled by glucose-6-phosphate dehydrogenase (G6PD) and transketolase-1 (TKL-1) activities, respectively, which are both elevated in HCC [221,222]. Several oncogenes and signalling pathways are implicated in the Warburg phenomenon. The AKT/mTOR pathway (Fig. S1A,B)

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contributes to create a tumour glycolytic phenotype by enhancing glucose uptake though the increase in expression of glucose transporters GLUT1, GLUT2, and GLUT4, and the activation by phosphorylation of key glycolytic enzymes, such as HKII and phosphofructokinase 2 (PFK2; Fig. S7) [223–225]. AKT signalling includes mTOR activation through the inhibition by phosphorylation of its negative regulator complex TSC2/TSC1 (Fig. S1B) [226]. mTOR constitutes, with RAPTOR and GβL, the mTORC1 that stimulates protein and lipid biosynthesis, as well as glycolysis, through the HIF-1α. HIF-1α activation may be also triggered by H-RAS [227,228]. HIF-1α regulates the expression of GLUT1 [228] and induces the expression of PFK and lactate dehydrogenase (LDH), as well as of pyruvate dehydrogenase kinase-1 (PDK-1), which inhibits pyruvate dehydrogenase complex by phosphorylating its E1 subunit (Fig. S7) [226]. Furthermore, HIF-1α stimulates, together with other transcription factors, such as SP1 and SP2, and RAS, the expression of pyruvate kinase (PK) M2 isozyme, characteristic of lung tissues and all cells with high rates of nucleic acid synthesis, which substitutes the liver isozyme L in HCC [229]. Finally, HIF-1α sustains the pentose phosphate pathway, by inducing the expression of G6PD and TKL-1 [230]. Inhibition of FOXOs by AKT may also contribute to glycolysis activation: mTORC1dependent glycolysis is increased by FOXO3a knockdown due to decreased expression of the TSC1 tumour suppressor [231]. The MYC oncogene, which is amplified and/or over-expressed in DN and HCC [56], encodes a transcription factor that directly binds numerous glycolytic genes, including those encoding HKII, enolase, and LDH-A, and contributes to inhibit pyruvate dehydrogenase by activating PDK-1 [232]. On the other hand, glutamine flux into the Krebs cycle was found to be in part regulated in cancer cell lines by expression of MYC and p53 [233,234]. Finally, MYC is involved in mitochondrial biogenesis which, when sustained by high MYC levels, can result in high mitochondrial ROS and NO production, which may induce mtDNA mutations that in turn contribute to dysfunctional mitochondria [235]. NO has been found to be an endogenous AMP kinase (AMPK) activator [236]. AMPK regulates energy dynamics by limiting anabolic pathways (to prevent further ATP consumption) and by facilitating catabolic pathways (to increase ATP generation) thus inhibiting gluconeogenesis and cell proliferation, together with AKT [237,238]. On the other hand AMPK may phosphorylate and strongly inhibit mTOR, thus opposing the effects of AKT [239]. It has been proposed that cancer cells, including HCC, exhibit a loss of AMPK activator liver kinase b1 (LKB1), which has been identified as a tumour suppressor gene [239]. Indeed, LKB1 gene exhibits different genetic alterations, including nonsense and missense mutations and allelic loss, in 11–22% of HCC [240]. However, recent observations [241] showed that LKB1 is required for cell survival in an S-adenosylmethioninedeficient cell line, derived from MAT1A (methionine adenosyltransferase 1A)-KO mice that spontaneously develop HCC (see Section 5.3) [242]. Furthermore, LKB1 regulation of AKT-mediated survival, independently of PI3K, AMPK and mTORC2, was demonstrated in these cells, and cytoplasmic staining of p-LKB1(Ser428) was found in a nonalcoholic steatohepatitis (NASH)-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC with both ASH (alcoholic steatohepatitis) and NASH aetiology [241]. These observations indicate that the effects of AKT/mTOR and LKB1/AMPK signalling are often contradictory and not completely clear. However, molecular and biochemical approaches indicate prevalent activation of genes encoding glycolytic rate-limiting enzymes such as HKII, PFK, and PK, as well as enolase and LDH [223–232]. Moreover, induction of PDK-1 by MYC and HIF-1α, with consequent inhibition of pyruvate dehydrogenase complex, blocks pyruvate decarboxylation and inhibits the Krebs cycle and mitochondrial electron-transport chain [226,227]. The latter may be also inhibited, in HCC cells, by p53 deletion/mutation, through a decrease in the synthesis of cytochrome c oxidase 2 (SCO-2). SCO-2 is required for the

assembly of the COXII subunit (cytochrome oxidase subunit II) into the cytochrome c oxidase complex, which is integral to the respiratory chain [243]. In contrast, in early stages of HCC development, when no p53 mutation may be found (except in the Aflatoxin B1-induced tumours), p53 may inhibit the glycolytic pathway by up-regulating the expression of TIGAR (TP53-induced glycolysis and apoptosis regulator), an enzyme that decreases the activity of the glycolytic enzyme fructose-2,6-bisphosphate, a well as by supporting PTEN expression and activating the expression of SCO-2 [239]. Overall, available evidence indicates that RAS/ERK, AKT/mTOR, and MYC signalling, and the limited capacity of cancer cells to maintain enough mitochondrial mass to support sufficient flux through the electron-transport chain, favour the glycolytic metabolism of HCC cells. Noticeably, the activation of these pathways may even be observed, although at a lower level than in HCC, in early preneoplastic and/or dysplastic nodules (see Section 4). This agrees with previous observations indicating a positive molecular correlation of the activity of key glycolytic enzymes (HKII, PFK, and PK), and a negative correlation of the activity of gluconeogenesis enzymes (pyruvate decarboxylase, phosphoenolpyruvate carboxykinase, fructose-biphosphatase, and glucose-6-phosphatase) with the growth rate and progression of HCC [244]. 5.2. AKT/mTOR signalling and lipid metabolism A major downstream effector of the AKT proto-oncogene is the lipogenic cascade. It is now widely accepted that highly proliferating cancer cells require de novo fatty acid synthesis to continually provide lipids for membrane production [245,246]. Also, the newly synthesised fatty acids are necessary for energy production through lipid β-oxidation, as anchors to target proteins to membranes, and as precursors in the synthesis of lipid second messengers [245,246]. At the molecular level, the exacerbated fatty acid biosynthesis is reflected by the increased activity and expression of several lipogenic enzymes in neoplastic cells, including ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACAC), fatty acid synthase (FASN), malic enzyme (ME), and stearoyl-CoA desaturase (SCD)1. In human HCC, a concomitant up-regulation of the lipogenic pathway enzymes, including the ones involved in fatty acid (FASN, ACAC, ACLY, ME, SCD1) and cholesterol synthesis, including SREBP2 (sterol regulatory element binding protein 2), HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), and SQS (squalene synthetase) as well as their upstream inducers chREBP (carbohydrate responsive element binding protein), SREBP1, and LXR-β (liver X receptor β) was detected in non-neoplastic surrounding livers and HCC when compared with normal livers [247,248]. The same lipogenic proteins were further up-regulated in the HCCP subgroup, thus indicating that increased lipogenesis is associated with HCC development and might predict patient's prognosis [248,249]. Subsequent experiments in human HCC cell lines showed that over-expression of an activated form of AKT led to increased cell growth and reduced apoptosis, and induced a rise in lipid synthesis and up-regulation of the major lipogenic proteins [248]. Conversely, a remarkable growth inhibition, associated with a fall in lipogenesis and in the levels of lipogenic proteins followed suppression of AKT in HCC cells [248]. At the molecular level, activation of de novo lipogenesis was shown to be dependent on the mTORC1. The latter data were further substantiated by using an in vivo approach. The activated/myristylated Akt form was stably over-expressed into the hepatocytes of wild-type FVB/N mice by hydrodynamic transfection [248]. AKT-transfected hepatocytes exhibited a massively enlarged clear cytoplasm, owing to increased glycogen and fat storage, which displayed proliferative activity. Groups of AKT-positive cells progressively expanded with the time, forming preneoplastic lesions that ultimately underwent malignant conversion and occurrence of HCC [248]. Noticeably, mice injected through the tail vein with AKT expressing plasmids

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displayed AKT and mTORC1 up-regulation, which was paralleled by induction of lipogenic enzymes involved in fatty acid and cholesterol biosynthesis [248]. Using the hydrodynamic transfection method, another mouse model co-expressing AKT and an oncogenic form of β-Catenin has been established [250]. Of note, these mice developed HCC that were characterised by a lipogenic phenotype. Also, interspecies comparison of microarray data from AKT/β-Catenin liver tumours and human HCC showed that the mouse tumour expression profiles closely resemble those of human HCC characterised by a poor clinical prognosis [250]. Thus, these data indicate that activation of the AKT cascade leads to lipogenesis activation and hepatocarcinogenesis in the mouse as well as to a dismal prognosis of human HCC. 5.3. Methionine metabolism and hepatocarcinogenesis progression The development of HCC in rats fed a choline-deficient diet was first observed by Copeland and Salmon [251]. Although these observations were difficult to interpret, because of the possible presence of Aflatoxin B1 and other carcinogens contaminating the diet, they focused the attention on the role of labile methyl group deficiency in carcinogenesis. This role has been subsequently confirmed by the observation that rat HCC develop even when methyl-deficient diets and rats environment are accurately checked for contamination of carcinogens [252]. The treatment of rats with a methyl-deficient diet results in pronounced fall in S-adenosylmethionine (SAM), without significant changes in S-adenosylhomocysteine (SAH) content, and with consequent decrease in SAM:SAH ratio [253]. However, significant decrease in SAM level and SAM:SAH ratio also occurs in DN and HCC developing in rats fed adequate diet, several weeks after arresting carcinogen administration [254,255], as well as in human cirrhotic liver and HCC [256]. The liver is the main source of SAM biosynthesis from methionine and ATP, catalysed by methionine adenosyltransferase (MAT) isozymes (Fig. S8) [257]. SAM may be channelled after decarboxylation to polyamine synthesis, or converted to SAH during transmethylation reactions. SAH hydrolase catalyses the reversible hydrolysis of SAM to yield homocysteine and adenosine. In the liver, homocysteine may be channelled to transsulfuration pathway by a reaction catalysed by cystathionine β-synthase leading to cystathionine that is a precursor of cysteine, further utilised for glutathione biosynthesis. Alternatively, homocysteine may be utilised for methionine re-synthesis by a reaction catalysed by BHMT, or by a reaction catalysed by 5-methyltetrahydrofolate homocysteine methyltransferase (MTHF-HMT). Interestingly, low SAM levels favour homocysteine re-methylation, whereas high SAM levels activate cystathionine βsynthase, whose Km for SAM is 1.2–2 mM, much higher than that of MTHF-HMT (60 μM) [257], thus favouring glutathione synthesis. Of note, SAH is a potent competitive inhibitor of transmethylation reactions that are also inhibited by methylthioadenosine, a reaction product of polyamine synthesis. In mammals, the liver-specific MAT1A gene encodes MATI/III isoforms, whereas the widely expressed MAT2A encodes MATII isozyme [257]. MATI and MATIII isozymes have, respectively, intermediate (23 μM–1 mM) and highest (215 μM–7 mM) Km for methionine, whereas the MATII isoform has the lowest Km (≈4–10 μM) [258]. Fall in MAT1A expression with concomitant MAT2A up-regulation occurs in liver cirrhosis and HCC of rodents and humans, leading to a decrease in MAT1A:MAT2A ratio (called MAT1A/MAT2A switch) [256,259]. The up-regulation of MATII isoform cannot compensate for the decrease in MATI/III isozyme, being inhibited by its reaction product [260]. As a consequence, the MATI/III:MATII activity ratio strongly contributes, together with the increase in SAM decarboxylation and polyamine synthesis to the sharp decrease in SAM level [254]. The investigation of the role of SAM decrease in preneoplastic and neoplastic liver, showed a strong chemopreventive effect of the

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reconstitution of normal SAM levels in liver cells, by administration of SAM to rats after the cessation of treatments with carcinogens, independently of the protocol utilised for HCC induction [255,259,261,262]. Moreover, human HCC cell lines transfected with MAT1A or cultured in the presence of SAM undergo a strong restraint in proliferation rate [263,264]. These observations were recently confirmed by Lu et al. [265] that injected in rat liver parenchyma the human HCC cell line, H4IIE. After two weeks, the animals developed tumours in the liver. Continuous SAM intravenous infusion after tumour cell injection inhibited HCC formation in these rats. However SAM infusion for 24 days did not affect the size of already established tumours. It should be noted, in this respect, that in these rats chronic SAM administration led to the compensatory induction of hepatic GNMT expression that prevented SAM accumulation (Fig. S8), in contrast to 10-fold higher hepatic SAM levels induced by a 24–38 hour infusion. However, the efficacy of SAM treatment in patients with HCC remains to be examined because GNMT is silenced in most HCC [264]. These important findings suggest that the MAT1A/MAT2A switch and the consequent fall in SAM level are strongly involved in hepatocarcinogenesis. In agreement with this conclusion, MAT1A knockout mouse model, characterised by chronic SAM deficiency even in the presence of MAT2A induction, exhibits hepatomegaly without histologic abnormalities at 3 months of age, and macrovesicular steatosis involving 25–50% of hepatocytes and mononuclear cell infiltration in periportal areas, at 8 months [242]. HCC develop in many of these mice at 18 months of age [242]. Researches aimed at elucidating the molecular mechanisms underlying MAT1A/MAT2A switch in hepatocarcinogenesis, showed the association of MAT1A down-regulation in cirrhotic liver of CCl4-treated rats and in human HepG2 cell line with CCGG sequence methylation in MAT1A promoter [265]. In Huh7 cells, MAT1A down-regulation was attributed to CCGG methylation at +10 and +80 of coding region [266]. MAT2A up-regulation in human HCC was associated with CCGG hypomethylation of gene promoter [267]. Recent work [268] confirmed these results and showed Mat1A/Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation of Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast growing HCC of F344 rats. In slowly growing HCC induced in BN rats, very low changes in Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred [268]. Furthermore, the highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis. Notably, levels of AUF1 (AUrich RNA binding factor 1), enhancing mRNA decay [269,270] and destabilising MAT1A mRNA [271], sharply increase in F344 and human HCC [268]. This is associated with a rise in HuR (AUrich RNA binding factor 1), selectively binding to AUrich elements promoting mRNA stabilisation [269,270], and stabilising MAT2A mRNA [271]. Accordingly, a consistent increase in MAT1AAUF1 and MAT2A-HuR ribonucleoproteins occurs in F344 and human HCC. These changes are very low or absent in BN HCC. These observations indicate that both transcriptional and post-transcriptional mechanisms contribute to MAT1A/MAT2A switch and SAM decrease during hepatocarcinogenesis. Moreover, they suggest that MAT1A/MAT2A switch and SAM reduction may have a prognostic value for hepatocarcinogenesis. This is further supported by the observation of highest decrease in MAT1A:MAT2A expression and MATI/III:MAT/II activity ratios, associated with highest SAM fall, in F344 HCC and HCCP, with respect to BN HCC and HCCB [259]. DNA hypomethylation promotes genomic instability (GI) [272], and GI correlates with tumour stage [273]. In human HCC, MAT1A:MAT2A expression and MATI/III:MATII activity ratios correlate negatively with cell proliferation and GI, and positively with apoptosis and DNA methylation, and MATI/III:MATII ratio strongly predicts patients' survival length [268]. The mechanisms underlying the effects of MAT1A/MAT2A switch and SAM decrease on tumour cell growth have been the object of

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several studies. MAT1A KO mice exhibit increased oxidative stress [274] and GI [275], and down-regulation of Apurinic/Apyrimidinic Endonuclease 1 (APEX1) [275], which is a protein involved in DNA base excision repair [276]. These changes, which may favour tumourigenesis, have been attributed to SAM deficiency. Prevention of SAM depletion associated with de-differentiation of hepatocytes placed in culture, by addition of SAM to the medium, stabilises APEX1 protein [275]. Another mechanism rendering MAT1A KO mice prone to hepatocarcinogenesis involves ERK activation though a decreased mRNA and protein levels of its inhibitor DUSP1 (see Section 4.1) [277]. The decrease in DUSP1 protein was attributed to its proteasomal degradation, due to the up-regulation of the E3 ligase, SKP2. Noticeably, exogenous SAM was found to correct the effects of SAM deficiency in MAT1A KO mice by interfering with the degradation of DUSP1 protein, which resulted in normalisation of DUSP1 mRNA levels and reduction of ERK activity [277]. Other mechanisms may be involved in SAM antitumour effect. SAM has an antioxidant effect [274] and inhibits the activity of different growth-related genes and proteins [278] including iNOS [279]. Furthermore, SAM level modulates IKK-β activity of in vitrogrowing cells [280] and counteracts NF-kB activation in rat preneoplastic foci [280]. Accordingly, HepG2 and Huh7 HCC cell lines, transiently transfected with MAT1A exhibit sharp increases in SAM level associated with significant reduction in cell proliferation, increase in apoptosis, significant down-regulation of Cyclin D1, E2F1, IKK, NFkB, and the antiapoptotic BCL2 and XIAP genes, and up-regulation of the proapoptotic BAK and BAX genes [269]. Thus, low MatI/III activity and SAM content in precancerous liver and HCC of rats susceptible to hepatocarcinogenesis should result in increased oxidative and nitrative stress, GI, overexpression of NF-kB and its targets, including c-Myc, Cyclin D1, iNos and VegfA. All of these changes have been indeed observed in F344 HCC and HCCP, whereas they are low/absent in BN HCC and HCCB. Finally, MAT1A has been found to target osteopontin [281], a growth factor that activates ERK and PI3K/AKT pathways [281]. In complex, the studies on the deregulation of methionine metabolism in hepatocarcinogenesis, revealed a multifaceted ensemble of relationships between the changes in MAT1A:MAT2A ratio and SAM content and the activity of multiple signalling pathways. This emphasises the role of the MAT1A:MAT2A ratio in HCC progression. Moreover, all these findings underscore the importance of molecular therapies of HCC aimed at maintaining MAT1A:MAT2A ratio and cellular SAM content to normal levels. 6. Hepatocellular carcinoma gene signatures More than 300 microarray analyses, published so far, have made available a wealth of information on gene deregulation in HCC [282–288]. These studies provided a snapshot of the transcriptional state of healthy or diseased tissue, identified common aberrant molecular pathways involved in human hepatocarcinogenesis, intrahepatic metastases, HCC recurrence, and different stages, as well as molecular signatures of molecular subclasses of HCC, predicting HCC outcome and survival after surgical hepatectomy [282–288]. Here we focus on researches specifically dealing with the identification of novel HCC prognostic subgroups. Previous research established the usefulness of the comparative functional genomics to evaluate rodent models for human prostate [289], breast [290], and liver [291,292] cancers. This strategy is based on the neutral theory of molecular evolution [293,294] according to which the frequency of a gene variant follows a stochastic process of genetic drift through a population, whereas deleterious mutations are rapidly removed by selection. According to this theory, the functionally important genomic elements evolve at a slower rate than less important elements. It has been hypothesised [292,295] that if the regulatory elements of evolutionarily related species are conserved, it is reasonable to

believe that the gene expression signatures representing similar phenotypes in different species are also conserved. Therefore, attempts have been made by the Thorgeisson's group to identify aberrant phenotypes reflecting molecular pathways conserved during the development of liver cancer in rodents and humans [292]. Specifically, the gene expression profiles of human HCCs were compared to those of mouse HCC induced in different models. Integrated gene expression data showed high similarity of HCC of c-Myc, E2f1 and cMyc/E2f1 transgenic mice to human HCCB (B subgroup), whereas the expression patterns of c-Myc/Tgfα and diethynitrosamineinduced mouse HCC were similar to HCCP (A subgroup). Tumour phenotypic behaviour of mouse models, evaluated by proliferation rate, apoptosis, and prognosis, corresponded to those of paired human subtypes. In a subsequent research, the same group [295] compared gene expression data from rat foetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Notably, the gene expression profile of rat hepatoblasts was well separated from that of mouse hepatocytes (obtained from the c-Myc/E2F1 and c-Myc/TGFα models, in which transgenes were expressed from the albumin promoter transcriptionally active in differentiated hepatocytes). Furthermore, gene expression profiling identified two subtypes of human HCCs. One subtype (HB) exhibited features of hepatoblasts, coclustered with rat hepatoblasts, suggesting a similar cell developmental stage, and expressed markers of hepatic progenitors, oval cells. The other subtype (HC) showed features of differentiated hepatocytes. Both HB and HC subtypes expressed α-fetoprotein to similar levels. The HB subtype co-clustered with the proliferation group of genes which characterised the poor survival Cluster A of human HCC [295]. On the basis of the expression of proliferation-related genes, the HC subtype was further subdivided into Cluster A (proliferation signature-positive) and Cluster B (proliferation signaturenegative). The HB subtype in Cluster A exhibited the poorest survival with respect to subtype HC. These results were confirmed in a microarray study by the Zucman-Rossi's group [296] that identified two major HCC clusters, sub-divided into six distinct groups [G1–G6]. Subgroups G1–G3, similar to the Cluster A sub-type of HCCs described by Lee et al. [295], exhibited high chromosomal instability and poor prognosis, and over-expressed proliferation, cell cycle, and DNA metabolism genes. Interestingly, subgroups G1 and G2 originated from patients with chronic HBV infection, low HBV copy, and exhibited over-expression of foetal genes, including α-fetoprotein and parentally imprinted genes, thus resembling the poor prognosis HB Cluster A subtype [295]. In a recent study [297], unsupervised hierarchical clustering of the expression profiles identified a cluster of 1308 differentially expressed genes versus normal liver in persistent preneoplastic lesions of F344 rats, induced by the “resistant hepatocyte” model [298]. These lesions express CK19, a marker of hepatoblasts. In remodelling, CK-negative lesions, only 156 differently expressed genes were found [297]. Comparative functional genomics showed co-clustering of CK19-positive early lesions and advanced HCCs of rat with human HCCs characterised by poor prognosis. Furthermore, the CK19-associated gene expression signature predicted patient survival and tumour recurrence [297]. These findings identify CK19 as a prognostic marker of early neoplastic lesions and suggest a progenitor derivation of HCC in the rat “resistant hepatocyte” model. Furthermore, they establish an experimental system to examine the potential contribution of hepatocyte progenitor cells to HCC and confirm the usefulness of the comparative evaluation of gene expression data sets from rodent and human liver lesions. In this context, a recent study in our laboratory [299] on gene expression patterns of DN and HCC induced by “resistant hepatocyte” model in genetically susceptible F344 rats and resistant BN rats, showed a signature predicting DN and HCC progression and disclosed, for the first time, a major role of oncosuppressor genes as effectors of genetic resistance to hepatocarcinogenesis. Integrated gene expression data

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revealed lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressive rat and human HCC. Importantly, BHMT, DUSP1, and GADD45g expression was found to predict patients' survival. At present, few studies analysed the role of gene expression signatures on overall survival after surgical HCC resection. Two prognostic subgroups with different survival were identified by a 406-gene expression signature [300], and a patient's subgroup with short survival was characterised by a 111-Met regulated gene cluster [300]. A gene expression profile resembling that of foetal hepatoblasts allowed identifying a patient's subgroup with particularly short survival [76]. In a recent study, a 186-gene expression profile failed to yield a significant association with survival of HCC patients, whereas profiles of the non-tumourous SL were highly correlated with survival [301]. Patient populations in this study had early HCC, whereas other studies discovering outcome-predicting tumour-derived signatures [76,291,292] analysed patients that tended to have more advanced disease. It was hypothesised [301] that late recurrence, typical of early small HCC [2], could result from new primary tumours arising in a damaged SL rather than from the proliferation of residual cells derived from original tumour [301]. These observations emphasise the importance of outcome-predicting signatures derived from early stages of hepatocarcinogenesis. In the rat model, a signature characterised by higher expression of cell proliferation genes and lower expression of oncosuppressors discriminate fast-proliferating and fast-progressing DN from slowly-proliferating DN, unable to progress to HCC [299]. Few studies focused on the molecular signature of human DN. A 240-gene signature was reported to discriminate low-grade DN, high grade DN and grade 1 HCC in HBV patients [302]. A study on liver lesions from HCV cirrhotic patients, led to the identification of 12 genes differently expressed between early HCCs and DNs [303]. A 3-gene set including GPC3, LYVE1, and SURVIVIN had elevated discriminative accuracy [303]. Functional analysis identified the MYC oncogene as a plausible driver gene for malignant conversion of human DN [304], in accordance with observations on rat DN [49,299], indicating that the deregulation of MYC driven signalling underlies the progression of DN and HCC in humans and rodents. 7. A new frontier of research on HCC: the role of microRNAs MiRNA constitute a family of non-coding minute RNAs involved in post-transcriptional regulation of gene expression. In the nucleus, RNA Polymerase II transcribes primary miRNAs (pri-miRNA) consisting of a 5′-7-methyl guanylate cap, a characteristic imperfect stemloop secondary structure, and a 32-poly(A) tail [305,306]. The endoribonuclease III, Drosha, with its co-factor Pasha, cleaves pri-miRNAs to precursor miRNAs (pre-miRNA) of 50–150 nucleotides. Exportin5 exports pre-miRNAs to cytoplasm, where the RNAse III Dicer cleaves them into double-stranded duplexes of 20–23 nucleotides. Each miRNA duplex separates into single-stranded mature miRNA and forms with Argonaute proteins the RNA-induced silencing complex (RISC). RISC binds to the 3′-UTR of its target(s) inhibiting translation of single or multiple proteins [307]: complete miRNA complementarity induces mRNA degradation, whereas partial complementarity represses translation. Numerous studies suggest interference with the aetiology, and early and late stages of hepatocarcinogenesis [305,306,308,309]. Many miRNAs are down-regulated in HCC [310]. Accordingly, DICER down-regulation occurs in many tumours, including HCC [311]. MiR-126* down-regulation has been suggested to be directly linked to alcohol-induced hepatocarcinogenesis [312]. Microarray profiling studies showed reduction in miRNAs expression specific of HCVand HBV-associated cases: down-regulation of miR-190, miR-134, miR-151 occurs in HCV cases, and of miR-23a, miR-142-5p, miR34c, in HBV cases [313]. MiR-96 was reported to be distinctively upregulated in HBV-associated HCC [314], whereas miR-193b upregulation has been found upon transfection of HCV genome [315].

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MiR-122, a liver-specific miRNA abundantly expressed in liver tissues, accelerates ribosome binding to HCV RNA, which in turn stimulates viral translation [316]. MiR-122 inactivation leads to 80% reduction in HCV RNA replication in HCC cell lines [317]. Animal studies have confirmed the role of miRNAs in early stages of hepatocarcinogenesis. Up-regulation of miRNAs, including miR-1792 cluster, miR-106a, and miR-34, occurs during tamoxifen-induced hepatocarcinogenesis in female rats [318]. In mice administered a choline-deficient and amino acid-defined diet, in which steatohepatitis precedes HCC development, microarray analysis identified 30 differential expressed miRNAs [319]. Among these, miR-155 was consistently up-regulated during the course of deficient diet intake [319]. Research on c-Myc-transgenic mice showed down-regulation of a number of miRNAs, including miR-15/16, miR-26a, miR-34a, miR-150 and miR-195 [320]. MiR-26a expression was most significantly perturbed, and its ectopic expression induced G1 arrest in HepG2 HCC cell line [321]. Interestingly, consistent reduction of miR-26a has been found in human liver cancer biopsies, compared to paired samples of normal liver [321].

7.1. MicroRNAs as signalling regulators Numerous recent studies focused on cell cycle activation by miRNAs in HCC (Table 2). MiR-423 [322] and miR-221 [323] target, respectively, cell cycle inhibitors of WAF and KIP families. Their overexpression in HCC [322,323], associated with the down-regulation of miR-26a [320,321,324], miR-195 [325], and miR-122a [326] which target, respectively, Cyclins D2 and E, Cyclin D1, CDK6 and E2F3, and Cyclin G1, results in enhanced cell cycle progression. Furthermore, down-regulation of miR-223 leads to STMN1 (Stathmin) over-expression [327] and microtubules stabilisation, which should favour G2–M transition. Paradoxically, miR-93 and miR-106B, upregulated in HCC, target E2F1 [328], a key gene of G1–S transition. It has been hypothesised [307] that miR-93 and miR-106b upregulation may prevent excessive accumulation of high levels of the apoptogenic E2F1 [329]. However, the effects of miR-93 and miR106B knockdown on E2F1 cellular levels and apoptosis have not been evaluated. A body of evidence documents the impact of miRNAs' deregulation with signal transduction pathways in HCC (Table 2). Upregulation of miR-221, miR-222, and miR-21, leads to PPA2 and/or PTEN inhibition with consequent activation of AKT signalling [330–333]. Moreover, miR-221, miR-222, and miR-181b/d overexpression leads, through TIMP3 (tissue inhibitor of metalloproteinase 3) inhibition, to over-expression of MMP2 and MMP9 and enhanced cancer cell migration [331,334]. HCC cell migration can also be stimulated by the down-regulation of miR-200b and miR-124 that facilitates EMT transition, through activation of respectively, ZEB1/2 (zinc finger e box-binding homeobox 1/2) [335], ROCK2 (RHO-associated coiled-coil-containing protein kinase 2) and EZH2 (enhancer of ZESTE, drosophila, homolog 2) [336]. MiR-221 also contributes to AKT activation via inhibition of DDIT4 (DNA damageinducible transcript 4), an essential regulator of the mTOR kinase through stimulation of the tuberous sclerosis tumour TSC1/2 complex (Fig. S1B), which is a putative tumour suppressor [337]. Down-regulation of miR-122, leading to over-expression of NRDG3 (N-Myc downstream-regulated gene 3) [338], plays an important role in HBV-related hepatocarcinogenesis. An inverse correlation between the expression of miR-122 and the NDRG3 protein was noted in HBV-related HCC specimens. The transfection of the miR-122 expression vector into the HepG2.2.15 cell line repressed the transcription and expression of NDRG3, which subsequently reversed the malignant phenotype of cells. Restoration of miR-122 inhibited replication of HBV, expression of viral antigens, and proliferation of HCC cells [338].

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Table 2 Targets and functional effects of selected microRNAs in hepatocarcinogenesis. microRNA

Targetsa

Functional effectsb

Ref

Upregulation miR-423

P21/WAF1

[322]

PP2A PTEN

Enhanced CDK2 activity (G1–S transition) Enhanced CDK2 activity (G1–S transition) Prevention of excessive E2F1 accumulation (?) Enhanced AKT pathway Enhanced AKT pathway

TIMP3

Enhanced MMP2 and MMP9

[331,334]

miR-192

ERCC3, ERCC4

Apoptosis inhibition Apoptosis inhibition Inhibition of caspases 3, 6, 7, and 8 TSC1/2 complex inhibition and AKT activation Inhibition of DNA excision repair

[345] [328] [331]

miR-221

BMF BIM TRAIL-induced apoptosis DDIT4

Enhanced G1–S transition Enhanced G1–S transition

[320,324] [325]

Enhanced cell cycle progression Enhanced HCC proliferation

[326]

Enhanced cell migration Microtubules stabilisation (G1–M transition) NCOA1, NRIP1 Enhanced NF-kB signalling STAT3 Active STAT3 NDRG3 Stimulation of HCC proliferation AEG-1 Stimulation of HCC proliferation YAP Stimulation of HCC proliferation SMO Stimulation of HCC proliferation c-MET Stimulation of HCC proliferation ZEB1, ZEB2 Stimulation of EMT ROCK2, EZH2 Stimulation of EMT MCL-I Apoptosis inhibition BCL-2, MCL-I Apoptosis inhibition BCL-W Apoptosis inhibition TNFAIP3 Apoptosis inhibition AFP, HSP90, NF- Inhibition of TNF-α-related kB apoptosis

[358] [327]

miR-221 miR-93, miR-106B miR-222 miR-221, miR-222, miR-21 miR-221, miR-222 miR-181b/d miR-221 miR-25 miR-221, miR-222

Downregulation miR26a miR195 miR-122 Let-7g Let-7g miR-223 miR-22 miR-637 miR-122 miR-375 miR-375 miR-338-3p miR34a miR-200b miR-124 miR-101 miR-29 miR-122 miR-29c miR-491-5p

P27/KIP1, p57/ KIP2 E2F1

Cycl D2, E2, IL6 Cycl D1, CDK6, E2F3 Cycl G1 c-MYC, P16/ INK4a Collagen I-α2 STMN1

[323] [328] [330] [331–333]

[337] [379]

[356]

[351] [352] [338] [339] [199] [341] [342] [355,356] [357] [346] [347] [348] [349] [350]

a TIMP3, tissue inhibitor of metalloproteinase 3; DDIT4, DNA damage-inducible transcript 4; ERCC3/4, excision-repair, complementing defective, in Chinese hamster, 3/4; NCOA1, nuclear receptor coactivator 1; NRIP1, nuclear receptor-interacting protein 1; STNM1, Stathmin 1; NRDG3, N-myc downstream-regulated gene 3; AEG-1, astrocyte elevated gene-1; SMO, smoothened; ZEB1/2, zinc finger e box-binding homeobox 1/ 2; ROCK2, RHO-associated coiled-coil-containing protein kinase 2; EZH2, enhancer of ZESTE, drosophila, homolog 2; TNFAIP3, tumour necrosis factor alpha-induced protein 3. Ref, references. b Functional effects of miRNA up- or down-regulation.

Over-expression of AEG-1 (astrocyte elevated gene-1) [339] and Hippo signalling [199,340] may be related to miR-375 downregulation. MiR-375 expression is inversely correlated to AEG-1 expression in HCC tissues, and AEG-1 knockdown in HCC cells, similar to miR-375 over-expression, suppresses tumour properties, indicating a role of AEG-1 in hepatocarcinogenesis. Down-regulation of miR-338-3p is associated with activation of Hedgehog signalling [341]. Interestingly, SMO and MMP9 are over-expressed and

associated with invasion and metastasis in HCC tissues, and forced over-expression or down-regulation of miR-338-3p, leads to their inhibition or up-regulation in HCC cell lines [341]. These data indicate that targeting the smoothened gene by miR-338-3p might be a novel potential therapeutic strategy for liver. An inverse correlation between miR-34a and c-MET-protein has been found in 76% of HCC patients [342]. Ectopic expression of miR-34a in HepG2 cells inhibits tumour cell migration and invasion in a c-MET-dependent manner [342]. MiR-34a targeting of c-MET results in reduction of both cMET mRNA and protein levels, as well as in decrease of c-METinduced phosphorylation of ERK1/2 [342]. The interaction of TGF-β signalling with miRNAs is worthy of note. TGF-β induces the expression of the miR-23a-27a-24 cluster in HCC cells [343]. As a consequence the anti-proliferative and proapoptotic effects of TGF-β are significantly attenuated [343]. Furthermore, in mice fed a choline-deficient and amino acid-defined diet, TGF-β stimulates the expression of miR-181b and d, which repress their target TIMP3 thus promoting the proliferation, migration, invasion and tumourigenicity of HCC cells through stimulation of MMP2 and MMP9 activities [334]. Notably, over-expression of hepatic TGFβ as well as of SMAD2, 3 and 4 is correlated with elevated miR181b/d [334]. Functional experiments revealed that the exposure of liver cells to TGF-β induces increase in miR-181b level, which is abrogated by SMAD4 silencing [334]. These results and the observation that miR-106b-25/miR-17-92 clusters abrogate cell cycle inhibition and apoptosis induced by TGF-β [344], strongly suggest that miRNAs may render HCC cells resistant to oncosuppression by TGF-β [344]. Modulation of apoptosis by miRNAs may also contribute to HCC development (Table 2). MiR-221 and miR-25 target the proapoptotic genes BMF and BIM [328,345], and down-regulation of miR-122, miR-101, and miR-29 favours the expression of the antiapoptotic genes BCL-2, MCL-I, and BCL-w [346–348]. This might lead to the release of cytochrome from mitochondria to cytoplasm, with consequent activation of caspase 9 and of effector caspases 3, 6, and 7. Interestingly, according to recent observations, miR-29c down-regulation in HBV-related HCC cell lines and clinical tissues, leads to up-regulation of its target TNFAIP3 (tumour necrosis factor alpha-induced protein 3), a key regulator in inflammation and immunity [349]. Forced over-expression of miR-29c in HepG2.2.15 cells suppresses TNFAIP3 expression and HBV DNA replication, inhibits cell proliferation, and induces apoptosis. Up-regulation of miR-221, miR-222, and miR-181b/d in HCC results in down-regulation of inhibitors of TIMP3 and in increased resistance to TRAIL-induced apoptosis [331,334]. Furthermore, acute liver injury up-regulates microRNA491-5p in mice [349]. miR-491-5p sensitises HCC cells to TNF-α induced apoptosis, probably through the down-regulation of αfetoprotein, HSP-90, and NF-kB [350]. Anti-apoptotic signalling may be elicited by down-regulation, in HCC, of miR-22 [351], which targets NCOA1 (nuclear receptor coactivator 1) and NRIP1 (nuclear receptor-interacting protein 1), as well as miR-637, which targets STAT3 [352]. NCOA1 and NRIP1 over-expression leads to activation of NF-kB and its anti-apoptotic targets XIAP, c-IAP, and SURVIVIN (Fig. S1B). The induction of STAT3 by cytokines favours HCC expansion by increasing cell survival, cell proliferation and migration, and angiogenesis, via activation of CYCLIN D1, MMP2/9, and HIF-1α and VEGF [353]. Finally, according to recent research [354] miR-22 and miR-29b are over-expressed in liver of 2-Acetylaminofluorene-treated male rats, containing large preneoplastic foci evenly distributed throughout the entire section of the liver and exhibiting up-regulation of Mthfr and Mat1A (Fig. S8). Significant down-regulation of these genes occurred in hepatic untransformed TRL1215 cells after transfection with pre-miR-29b and pre-miR-22. These findings suggest an additional mechanism of post-transcriptional regulation of MAT1A, and implicates miR-29b and miR-22 in the epigenetic abnormalities, induced in preneoplastic liver and probably throughout

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the hepatocarcinogenesis process, by MAT1A/MAT2A switch (see Section 5.3). Overall, the studies on the role of miRNAs on hepatocarcinogenesis have shown that, presumably starting in early stages of this process, several miRNAs strongly contribute to the deregulation of cell cycle and different (maybe all) signalling pathways, acting synergistically as oncogenes or oncosuppressors. This opens new interesting perspectives on the pathogenic mechanisms of hepatocarcinogenesis, underlining the role of post-transcriptional regulation of signalling pathways. 7.2. MicroRNAs as prognostic markers and therapeutic targets Growing evidence documents the emerging role of miRNAs in the identification of putative prognostic markers and therapeutic targets of specific HCC subtypes associated with different biologic and clinical features. MiRNAs deregulation leading to increment in AKT and c-MET signalling (Table 2) favours migration and invasion of tumour cells and formation of metastases. The down-regulation of miR-200b [335,355,356] and miR-124 [357] favours cell migration by activating, respectively, ZEB1 and ZEB2, as well as ROCK2 and EZH2. The latter induces epigenetic silencing of multiple tumor suppressor miRNAs, including miR-200b [355]. Let-7 microRNA family plays pivotal roles in carcinogenesis as potential growth suppressor. Let-7g is under-expressed in metastatic HCC, compared with metastasis-free HCC [358]. Transfection of Let7g significantly inhibits HCC cell migration but not invasion. Let-7g targets COL1A2 (type I collagen a2), and its inhibitory effect on cell migration can be counteracted by type I collagen a2, suggesting that Let-7g suppresses HCC metastasis at least in part through targeting COL1A2 [287]. hsa-Let-7g expression is markedly lowered in HepG2, Hep3B and Huh7 cells, yet higher in the Bel-7404 HCC cell line [359]. Proliferation of HCC cell line is significantly inhibited after the transfection of hsa-Let-7g mimics, whereas the transfection of hsaLet-7g inhibitor exerts an opposite effect. hsa-Let-7g inhibits the proliferation of HCC cell line via negative and positive regulations of, respectively, c-MYC and p16 INK4A [358]. These observations envisage the possibility of up-regulation by hsa-Let-7g of the oncosuppressor gene p16INK4A. This agrees with previous observations of positive modulation of RNA replication by miR-122 binding to the 5′ noncoding region of HCV RNA [360–363]. MiR-122 highest downregulation occurs in HCC with intrahepatic metastases [362]. This suggests a role of miR-122 in metastases, which has been validated in orthotopic murine models of liver cancer [362]. Evidence has been presented [363] indicating that ADAM17 (disintegrin and metalloprotease 17) mediates miR-122 effect on metastases. HCC cell migration and invasion are also promoted by miR-151, frequently over-expressed in HCC, through inhibition of RhoGDP dissociation inhibitor (RhoGDIA) [364]. The RhoGTPases, including RhoA, Rac1, and Cdc42 act in multiple ways to regulate cell migration, affecting actin and microtubule dynamics, cell–cell and cell–extracellular matrix adhesion [365]. RhoGDIA binds to the GDP-bound form of RhoGTPase thus preventing the activation of the metastasis-promoting pathway. Above observations clearly indicate a contribution of miRNAs to HCC progression. This is confirmed by several studies showing a correlation between the modulation of gene expression by miRNAs and HCC aggressivity. Thus, targeting of p57 KIP2 by miR-221 correlates with advanced stage, high proliferation of HCC, and lower diseasefree survival [323]. MiR-155 expression levels are highest in patients with poorer recurrence-free survival after liver transplantation and miR-155 is an independent predictor of poor prognosis [366]. Several other microRNAs correlate with HCC prognosis. These include miR-29 [347], miR-99a [367], miR-203 [368], miR-124 [357], miR-122 [369], Let-7g [357], and miR-26 [324]. Interestingly, recent work has shown a positive correlation of the over-expression of DLK1–DIO3 miRNA cluster, on chromosome 14q32, with HCC stem cell markers (CD133, CD90, EpCAM, Nestin), and association with poor survival

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rate in HCC patients [370]. This suggests that coordinate upregulation of the DLK1–DIO3 miRNA cluster may define a novel stem cell-like subtype of HCC associated with poor prognosis [370]. Three clusters of oncogenic miRNAs' molecular subclasses have been identified by microarray analysis [371]: Cluster A was enriched in HCC with β-catenin gene mutations, Cluster B included interferonresponse-related genes, and Cluster C was enriched by samples with abnormal IGF-1R and Akt signalling activation. Three subsets of Class C tumours were identified, among which C2 HCCs showed over-expression of several miRNAs from chr19q13.42, including miR-517a and miR-520c [371]. Expression of miR-517a and miR520c increased HCC cell proliferation, and invasion in vitro, and MiR-517a promoted carcinogenesis in vivo [371]. The mechanisms of miRNA deregulation in tumours are poorly known. MiR-151 is encoded in the 8q24.3 chromosomal region, frequently amplified in HCC [364], and miR-122 is encoded in the 18q21.31 region, frequently lost, in HCC [372]. Polymorphisms of pri-miR-146a [373], miRNA-196a2 [374,375], pri-miR-218 [376], and miR-34b/c [377] have been associated with increased susceptibility to HCC. Insertion of “TTCA” in the 3′UTR of interleukin-1 alpha (IL1A) disrupts miR-122 and miR-378 binding, resulting in an IL1A over-expression, likely increasing susceptibility to HCC [378]. An important aspect of the mechanisms of carcinogenesis is the possibility that miRNA deregulation contributes to the accumulation of DNA damage that characterises tumour progression. MiR-192 has been shown to inhibit nucleotide excision repair by targeting ERCC3 and ERCC4 in HepG2.2.15 cells [379]. Analogous observations in human colorectal cancer showed that miR-155 down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and microsatellite instability [380]. Some observations suggest the possibility that epigenetic changes may also be involved in miRNAs deregulation in liver tumours. In primary human HCC, miR-1 down-regulation is associated with promoter methylation [381]. Ectopic expression of miR-1 in HCC cells inhibits cell growth and reduces the replication potential and clonogenic survival. Several miR-1 targets including FoxP1, MET, and HDAC4 are up-regulated in HCCs, and their down-regulation, induced by the hypomethylating agent 5-azacytidine, inhibits cell cycle progression and induces apoptosis [381]. These interesting findings, and the observation of epigenetic regulation of miRNA involved in other tumour types [308] envisage new possibilities of the epigenetic approach to HCC treatment. The contribution of several miRNAs to HCC progression, suggests the possibility of their role in HCC therapy. Over-expression of miR122 modulates the sensitivity of HCC cells to chemotherapeutic drugs, including sorafenib, adriamycin or vincristine, through downregulating MDR related genes MDR-1, GST-π, and MRP, antiapoptotic gene Bcl-w and cell cycle related gene cyclin B1 [382,383]. Depletion of miR-181b sensitises SK-Hep1 cells to doxorubicin [334]. Furthermore, patients with low miR-26 expression show better responses to interferon therapy [384]. In a mouse model, miR-26 administration to mice, using a self-complementary adeno-associated virus vector, strongly induces apoptosis and suppresses HCC progression [321]. Similarly, therapeutic administration of cholesterol-conjugated 2′-Omethyl-modified miR-375 mimics was found to suppress the growth of hepatoma xenografts in nude mice [339]. The analysis of the implication of miRNAs in HCC therapy is at its beginning, but the results of relatively few studies on this topic support to the idea that this could be a promising approach to molecular therapy of HCC. 8. Concluding remarks HCC is a frequent and fatal disease worldwide, with limited therapeutic options when diagnosed at late stage. Studies aimed at detecting the alterations of signal transduction during hepatocarcinogenesis are of prime importance both for the implementation of new diagnostic tools helping to predict the prognosis of the HCC patients, and for

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the development of novel, more specific therapeutic strategies against this deadly disease. A number of molecular biomarkers have been identified, in recent years, allowing more accurate prediction of prognosis for HCC patients and also providing targets for novel therapeutic agents. However, the approaches to molecular HCC treatment have given contradictory results, limited therapeutic effects, or major adverse effects [10,385]. Encouraging but still limited results in the treatment of human HCC have been obtained with the use of the multi-kinase inhibitor Sorafenib, as well as in phase II studies particularly with Sutinib, Brivanib and mTOR inhibitors [11,386]. However, mounting evidence indicates that not only the deregulation of single signalling pathways, but also the interplay between different altered signalling cascades affects tumour cell function. Therefore, the complexity of the molecular changes present in HCC, and the crosstalks between different signalling pathways predict the impossibility to cure HCC development by interfering with a single signalling pathway. Better results could be obtained by addressing adjuvant molecular therapies towards crucial targets of the different signalling pathways involved in hepatocarcinogenesis. Networked biologic therapies have been suggested [387] in which a combination of non-cytotoxic interventions must be performed to interrupt the damage. Recently, combined molecular therapies, aimed at the blockade of several survival pathways, have been proposed. The combination of Sorafenib and Rapamycin, to inhibit the RAS/ERK and PI3K/AKT pathways, leads to growth suppression in mouse models of HCC, and enhances tumour necrosis and ulceration of human HCC xenografts, with respect to Sorafenib alone [388,389]. Moreover, the association of Bevacizumab, a monoclonal antibody to VEGF, and the EGFR inhibitor Erlotinib [390], and that of Mapatumumab [391], a fullyhuman monoclonal antibody to TRAIL-R1, in combination with Sorafenib were found to have a therapeutic effect in subjects with advanced HCC. In this regard, a better understanding of the interactive regulatory network involving distinct signalling cascades will presumably provide the indications necessary to identify the best targets for HCC treatment and develop more effective therapeutic strategies against human liver cancer. Accumulating evidence of cross-talk between metabolic regulation and signalling pathways suggests that the combination of direct metabolic inhibitors and inhibitors of signalling pathways might be a powerful approach to treat cancer. Numerous inhibitors are known that target glycolytic enzymes (i.e. HK, PK, PDH, PDK1) and fatty acid synthesis (i.e. ACAC, ACLY, FASN) [392–394]. However, they are generally not very potent, and high toxic doses should be required. This could be avoided by targeting crucial metabolic steps through inhibition of both genes and enzymes involved in a given step, by the contemporary use of different inhibitors or of single inhibitor acting on different targets. The latter is the case of statins that inhibit HMGCR, thereby interfering with the membrane formation, as well as with MYC activation by phosphorylation, cell adhesion molecules, and cell cycle key genes, such as Cyclins D1 and E, CDK1, CDK2, and CDK4 [395–397]. Interestingly, Pitavastatin was found to be effective in inhibiting diethylnitrosamine-induced liver preneoplastic lesions in C57BL/KsJ-db/db (db/db) obese mice and, therefore, could be useful in the chemoprevention of liver cancer in obese individuals [398]. Furthermore, the clinical information gathered in generated some clinical questions regarding the patients eligible for new treatments. Mounting studies on the molecular mechanism responsible for liver tumour development and progression are allowing the identification of specific HCC subtypes associated with different biologic and clinical features. This is of prime importance for the selection of patient subgroups that are most likely to obtain clinical benefit and, hence, for the successful development of future targeted therapies of HCC. The present review examines recent achievements in the knowledge of the deregulation and interplay of signalling cascades in prognostic subtypes of HCC in order to provide a collection

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