Abstract New advancements have been made in recent years in the understanding of the molecular mechanisms that govern human liver tumorigenesis. Experimental ani-mal models have been widely used, especially mouse models. In this review we highlight some of the genetically engineered mouse models that have proved to be excellent tools to study the intracellular signalling pathways altered in hepatocarcinogenesis and establish potential correlations with data from humans, with special focus on hepatocel-lular carcinoma (HCC), the most common type of primary liver cancer. Information obtained from these animal mod-els will help to design future therapeutic approaches to HCC, particularly those that explore drugs that specifically target the altered molecular pathways.

Keywords Liver cancer · HCC · Growth factors · TGF-beta · p53 · Mdr-2

Introduction

Liver cancer comprises diverse, histologically distinct primary hepatic neoplasms. Among these hepatocellular carcinoma (HCC) is the most common, representing 83% of all cases and one of the world’s deadliest cancers [1]. In recent years impressive progress has been made in under-standing its molecular pathogenesis and several experimen-tal animal models have contributed to the definition of the signal transduction pathways involved [2]. In spite of the interspecies differences and the obvious fact that there is no mouse model that can possibly mirror all human HCC variants, there is no doubt that animal models have allowed us to learn much about the molecular mechanisms underly-ing the pathogenesis of HCC. In fact, common alterations in specific growth factors and signal transduction pathways playing a critical role in the development and progression of liver cancer in mice and humans have been identified, thus providing promising targets for HCC therapies. Here, we highlight some of the genetically engineered mouse models that have proved to be excellent tools to study the intracellular molecular mechanisms altered in liver tumori-genesis and summarise the most important and clinically relevant findings achieved.

Experimental models recapitulating overactivation of growth factor pathways

Both in vitro and in vivo studies provided early evidence of an association of transforming growth factor-alpha (TGF-α) overexpression and TGF-α-driven signalling activation with hepatocarcinogenesis. Thus, deregulation of the TGF-α signalling pathway is correlated with the

*Supported by an unrestricted educational grantfrom Merck Serono

A. SánchezDep. Bioquímica y Biología Molecular IIFacultad de FarmaciaUniversidad ComplutenseES-28040 Madrid, Spain

I. Fabregat (Y) Laboratori d’Oncologia MolecularInstitut d’Investigació Biomèdica de Bellvitge (IDIBELL)and Universitat de Barcelona (IDIBELL-UB)Gran Via de l’Hospitalet, 199ES-08907 L’Hospitalet, Barcelona, Spaine-mail: ifabregat@idibell.org

Clin Transl Oncol (2009) 11:208-214DOI 10.1007/s12094-009-0342-x

E D U C AT I O N A L S E R I E S R e d S e r i e s *

Genetically modified animal models recapitulating molecular events altered in human hepatocarcinogenesis

Aránzazu Sánchez · Isabel Fabregat

Received: 8 January 2009 / Accepted: 10 February 2009

CURRENT TECHNOLOGY IN CANCER RESEARCH AND TREATMENT

Clin Transl Oncol (2009) 11:208-214 209

transformation of liver epithelial cell lines by carcinogens and oncogenes [3, 4]. Mice overexpressing TGF-α showed increased hepatocyte proliferation and hyperplasia, which ultimately resulted in development of hepatocellular tu-mours [5–8]. Molecular characterisation of these tumours evidenced an enhanced expression of the endogenous c-myc and insulin-like growth factor II genes, suggesting that these, and possibly other factors, may collaborate in TGF-α-induced hepatocarcinogenesis [7].

Liver tumours occur in transgenic mice selectively expressing c-myc in the liver, but the tumour incidence in these c-myc transgenic mice is relatively low with a long latency period [9]. However, coexpression of TGF-α and c-myc transgenes leads to a tremendous acceleration of tumour growth [10], proving the cooperation or synergy between growth factors and oncogenes in tumorigenesis. Double transgenic c-myc/TGF-α mice have been particu-larly useful for delineating the molecular events occurring during HCC formation and progression. A quite thorough analysis of the preneoplastic and neoplastic lesions has been performed and a few important molecules and signal-ling pathways have been found to be altered and contribute to the neoplasia development and malignant conversion. The highly malignant phenotype of the HCC arising in these mice is the result of the increased proliferation via a disruption of the cyclin D/pRb/E2F pathway and the TGF-α-dependent reduced apoptosis [11, 12].

The discovery of abnormally elevated E2F activity in the HCC lesions in the double transgenic c-myc/TGF-α mice prompted Conner et al. [13] to generate liver specific-E2F transgenic mice. These animals displayed a 100% in-cidence of adenomas, but only some of these (about 30%) showed malignant transformation to HCC at 12 months. The authors argue that this is a reflection of the dual role of E2F-1, as oncogene, during the early stages of hepatocar-cinogenesis, mediated by a persistent increase in cell pro-liferation, and tumour suppressor, during the tumour pro-gression phase, mediated by a steady-state increase in the apoptotic rate, and potentially responsible for the delayed or diminished malignant conversion [13]. Similarly to the TGF-α transgenic mice, a strong induction of endogenous c-myc was observed in the E2F-transgenic livers, sug-gesting cooperation between these two oncogenes in liver oncogenesis. Supporting this hypothesis, double transgenic c-myc/E2F mice showed accelerated HCC development with 100% incidence of tumours by 6–8 months.

Interestingly, molecular characterisation of the tumours developed in the various animal models has revealed im-

portant differences. Thus, the c-myc/TGF-α tumours, char-acterised as fast growing and very aggressive, displayed extensive genomic instability and a low rate of beta-catenin activation while the c-myc or c-myc/E2F-1 tumours were characterised by a high frequency of beta-catenin activa-tion in a relatively stable genome [14–16]. These data were consistent with observations in human HCC showing that ß-catenin activation occurs in a HCC subset with a rela-tively stable genome [17, 18] and a more favourable prog-nosis [19]. Direct comparison of the global gene expression patterns of HCCs from these different mouse models and human HCCs showed that gene expression signatures re-flecting similar phenotypes are indeed conserved in mice and humans during tumour development. Thus, HCCs from c-myc, E2F1 and c-myc/E2F1 mice grouped together in the better survival subset of human HCC, while c-myc/TGF-α mouse HCCs fit into the poorer survival set of human HCC [20] (Table 1). These studies were exceedingly important for several reasons. On the one hand, they served to identify two classes of mouse models that appear to closely reca-pitulate the molecular patterns of the two subclasses of hu-man HCC: the c-myc/TGF-α HCCs typically having a poor prognosis, including earlier and higher incidence of HCC development, higher mortality and genomic instability; and the c-myc, E2F1 and c-myc/E2F1, which have a relatively higher frequency of ß-catenin mutations and nuclear accu-mulation, which are indicative of lower genomic instability and better prognosis. On the other hand, they demonstrated the usefulness of the genetically engineered mice for the study of human cancer and provided the basis for future studies aimed at better understanding the molecular patho-genesis of human neoplastic development. In addition to this, they hold promise for the discovery of relevant thera-peutic targets and the testing of potential therapeutic agents.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that regulates multiple cellular functions, including proliferation, differentiation, migration, morphogenesis, survival and angiogenesis, through binding to its tyrosine kinase receptor, Met [21–23]. The protooncogene c-Met is implicated in many types of malignant tumours [24, 25]. Activating mutations, amplification and increases in c-met and HGF mRNA and protein expression have been described in rodent and human HCC [26–29] but the rele-vance of these alterations for tumour formation or progres-sion remains somehow elusive. Transforming activity of HGF has been demonstrated in vitro utilising immortalised mouse hepatocytes [30]. However, both direct and inverse correlations between the expression of c-Met and HGF and

Table 1 TGF-α/c-Myc/E2F transgenic mouse models

TGF-α/c-Myc mice c-Myc/E2F mice

Highly malignant phenotype as a result of disruption Accelerated HCC development. 100% incidence of tumoursin the cyclinD/pRb/E2F pathway. Extensive genomic instability, by 6–8 months. High frequency of beta catenin activationlow rate of beta-catenin activation. in a relatively stable genome.Fit into the poorer survival set of human HCC Fit into the better survival subset of human HCC

210 Clin Transl Oncol (2009) 11:208-214

the growth, malignancy and metastatic potential of HCC cell lines have been reported [31, 32]. Moreover, HGF en-hances motility, but inhibits growth and induces apoptosis in spontaneously and aflatoxin-transformed rat liver epithe-lial (RLE) cells [33].

There are also conflictive reports in HGF and c-Met transgenic and knock-out mice. Overexpression of mouse HGF in transgenic mice under the metallothionein (MT-HGF) gene promoter resulted in spontaneous formation of benign and malignant liver tumours in most transgenic mice after 17 months of age, which was associated with a chronic activation of c-met [34]. MT-HGF overexpression also promoted DEN-induced hepatocarcinogenesis, which was often accompanied by abnormal blood vessel forma-tion and up-regulation of the expression of vascular en-dothelial growth factor (VEGF), suggesting that HGF can directly promote tumour progression through enhancement of angiogenesis and/or indirectly through VEGF induc-tion [35]. Evidence that Met can contribute to genesis and maintenance of HCC was provided by targeting expression of human Met in mouse hepatocytes. Tumours initiated by Met transgene regressed when the transgene was inactivat-ed, suggesting that continuous expression of Met is needed for the maintenance of the lesions [36].

In contrast, c-myc-driven hepatocarcinogenesis is strik-ingly inhibited by coexpression of HGF, which delayed the appearance of preneoplastic lesions and prevented malig-nant conversion. Furthermore, c-myc/HGF double trans-genic mice were completely resistant to tumour promotion by phenobarbital. These results suggested that HGF might function as a tumour suppressor during the early stages of liver carcinogenesis [37]. A similar inhibitory effect of HGF on HCC development was found in double transgenic mice HGF/TGF-α [38]. The puzzle is further amplified by recent data from Takami et al. showing that hepatocyte-specific c-Met conditional knockout mice display greatly increased susceptibility to N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis [39]. The tumour-promoting effect of c-Met signalling deficiency was associated with dysregula-tion of genes associated with stress responses and increased oxidative stress, and was reversed by administration of the antioxidant N-acetyl-L-cysteine, suggesting that intact HGF/c-Met signalling is critical for maintaining normal re-dox homeostasis and has tumour suppressor properties dur-ing the early stages of DEN-induced hepatocarcinogenesis.

The most illustrative demonstration of the importance of the HGF/c-Met signalling pathway in HCC has been provided by Kaposi-Novak et al. These authors defined a Met-dependent gene expression signature by comparing wt- and Met-deficient primary hepatocytes through mi-croarray analysis, and used it for comparative functional genomic analysis applied to human HCC samples [40]. This approach has revealed a clinically significant sub-group of human HCCs with a prominent Met-dependent gene expression signature, associated with aggressive phenotype and poor prognosis as evidenced by increased vascular invasion rate and microvessel density as well as

decreased mean survival time. These results demonstrated the predictive value of the Met expression signature on the disease outcome in HCC patients. Although further under-standing of the significance of HGF/c-Met signalling in carcinogenesis is needed, these data have important clinical implications and help validate Met as a potential target in human HCC therapy.

Experimental models recapitulating loss in tumour suppressor pathways

TGF-ß1 is a multifunctional cytokine that regulates many biological processes, including proliferation, differentiation and death, being considered a tumour suppressor. However, it also mediates disruption of cell adhesion, increase in mi-gration and invasion, immune suppression and angiogen-esis, being considered a tumour promoter [41]. The final effects might depend on cell context and microenviron-ment. In hepatocytes, TGF-ß1 is well known as an impor-tant regulatory suppressor factor that inhibits proliferation and induces cell death [42–44]. However, an overexpres-sion of TGF-ß1 has been found in human HCC [27, 45, 46] and hepatoblastomas [47], and during the early stages of hepatocarcinogenesis in mice co-expressing c-myc and TGF-α transgenes [11]. Furthermore, HCC cell lines show autocrine expression of TGF-ß [48].

Experiments in mouse models have revealed that over-expression of TGF-ß1 in the liver predisposes to both spon-taneous and chemically induced hepatocarcinogenesis [49]. Spontaneous development of hepatocellular tumours with a 59% incidence occurred at 16–18 months of age, which was enhanced and accelerated in the c-myc/TGF-ß1 mice. DEN treatment dramatically accelerated hepatocarcinogen-esis in the double transgenic mice compared to both single parental lines, leading to a higher tumour frequency and size, and a high frequency of malignant conversion. Inter-estingly, progression from benign to malignant phenotype correlated with an early and maintained loss of TGF-ßRII expression (78% of HCCs). Inactivation of TGF-ßRII seemed necessary but not sufficient for tumour progression because no tumours were detected in the single transgenic TGF-ß1 mice. These results suggested that TGF-ß1-in-duced transformation requires cooperation with additional mutations or genetic alterations, i.e., c-myc up-regulation, to overcome the inhibitory actions of TGF-ß1 and facilitate tumour development [49].

The importance of disruption of TGF-ß1 suppressor signalling, particularly at the receptor level, for hepatocel-lular carcinogenesis has also been demonstrated by using TßRII heterozygous knock-out mice. These mice did not develop tumours spontaneously but exhibited increased susceptibility to DEN-induced liver tumorigenesis [50], highlighting a role for TβRII as a suppressor of hepato-carcinogenesis. In agreement with this, reintroduction of the TβRII receptor in hepatoma cells from TβRII-negative

Clin Transl Oncol (2009) 11:208-214 211

cancers restores sensitivity to TGF-β1 and reduces their tumorigenicity [51]. Additionally, Santoni-Rugiu et al. demonstrated that enhancement of c-myc-induced hepato-carcinogenesis by co-expression of TGF-α is also associ-ated with early and frequent occurrence of TßRII-negative preneoplastic and neoplastic lesions and with reduced levels of p27 in HCC cells, indicating that disruption of TGF-ß1 growth-suppressive pathways may play a cru-cial role in the promotion and progression of liver cancer [52]. These observations supported a model in which the autocrine production of active TGF-ß1 by the hepatocytes creates a selective environment that facilitates transformed cells with down-regulated TßRII expression to escape from the growth-inhibitory effects of this cytokine and progress towards a more malignant phenotype.

It is now very well established that TGF-ß1 can induce both tumour suppressive and oncogenic properties [41]. Consistent with this, experiments with cultured hepatocytes and hepatoma cells have demonstrated that apart from inducing cell death, TGF-ß1 can activate antiapoptotic sig-nals [53–56]. Furthermore, TGF-ß1 induces an epithelial–mesenchymal transition [56–58], an important late event during development of epithelial tumours [59]. An associa-tion between the TGF-ß-induced growth arrest/apoptosis and EMT is suggested, since cells that have acquired a mesenchymal phenotype become refractory to TGF-ß sup-pressor effects [60–62]. These results support a crucial role for TGF-ß in tumour development and progression. The mechanisms that allow cells to escape from the inhibitory effects of TGF-ß are not completely understood. Interest-ingly, preneoplastic and neoplastic lesions arising from TGF-ß1, TGF-α/c-myc transgenic mice and human HCC cell lines showed constitutive activation of NF-kB signal-ling, which might contribute to accelerate epithelial neo-plastic progression, by protecting from TGF-ß1-induced apoptosis [63, 64]. In agreement with this, ROS-dependent

NFkB activation induced by TGF-ß1 in foetal hepatocytes mediates the up-regulation of EGFR ligands and the anti-apoptotic signals in TGF-ß-treated cells [65].

On the basis of the dual role of TGF-ß1 in tumorigene-sis, e.g., tumour-suppressive and tumour-promoting proper-ties, Coulouarn et al. have recently applied gene expression microarray technology to identify human HCC subgroups based on a TGF-ß1 signalling pathway-specific signa-ture, by direct comparison between TGF-ß1-treated wt vs. TßRII knock-out primary hepatocytes [66]. Interestingly, two subsets of TGF-ß-responsive genes, reflecting both the suppressive and oncogenic properties of TGF-ß, were iden-tified. Comparative functional genomic analysis identified subgroups of human HCC either positive or negative for the TGF-ß gene expression signature. Furthermore, within the TGF-ß-positive HCC subgroup, two distinct subsets of tumours that preferentially expressed early or late TGF-ß-responsive genes were discriminated. Importantly, these two distinct HCC subgroups differed greatly in survival and recurrence. Patients with the late TGF-ß signature had a considerably shortened mean survival time and increased tumour recurrence compared to the patients with the early TGF-ß signature (Fig. 1). These results constituted more proof for the usefulness of the experimental models and comparative functional genomic approaches for the mo-lecular classification of human HCC, and the clinical relevance and predictive and prognostic value of these ap-proaches for the development of personalised therapies.

Among the most common alterations observed in hu-man HCC are mutations in the p53 tumour suppressor gene (TP53) [67]. The presence of specific mutational hotspots in TP53 in different types of human cancer implicates en-vironmental carcinogens and endogenous processes. In this sense, somatic mutations at the third base in codon 249 of TP53 in HCC have been related to exposure to aflatoxin B1 (AFB1), in cooperation with HBV infection [67]. Chronic

Early stagesTGF-beta

Suppressor arm

Protumorigenic pathways

Late stagesTGF-beta

Suppressor arm

Protumorigenic pathways

Early TGF-β signature in HCC patients. Better prognosis

Late TGF-β signature in HCCpatients. Shortened mean survival

time and increased tumor recurrence

Fig. 1 Dual role of TGF-ß in liver tumorigenesis. In early stages preneoplastic cells respond to TGF-ß inhibiting growth and inducing apoptosis, being considered a tumour suppressor factor. Later stages correlate with molecular alterations that allow cells to escape from its suppressor effects, favouring responses that mediate cell migration and invasion

212 Clin Transl Oncol (2009) 11:208-214

infection with HBV and HCV viruses and exposure to oxi-dative stress, including haemochromatosis or inflammation, induce damage in the DNA and mutations in cancer-related genes, including TP53. Thus, it would seem plausible that p53 mutation might operate in either HCC initiation or pro-gression, depending on the context. Indeed, Trp53 knockout mice develop more metastatic tumours than the wild-type mice when HCC is induced by using somatic delivery of oncogene-bearing avian retroviral vectors to the liver [68]. Concomitant loss of the Ink4a/Arf tumour suppressor locus accelerated tumour formation and metastasis in the same animal model, suggesting potential roles for the p16 and p19 tumour suppressors in this process [69]. However, in spite of these clear results, adenoviral delivery of p53 re-combinant DNA into mouse models bearing HCCs did not apparently suppress tumour growth [70]. Farazi et al., in a recent work [71], have helped to clarify this point. They have demonstrated that the effect of p53 loss in chronic liver disease-associated HCC is dependent on cellular context, in particular, intact or dysfunctional telomeres. In mice, reduction in telomere length is not clearly observed, which is related to the existence of long telomeres and high expression of telomerase (mTert). However, in Tert–/– mice p53 mutation enabled advanced HCC disease [71]. These authors propose that in the face of chronic liver damage, at-tenuated p53 function and telomere-induced chromosomal instability might play critical and cooperative roles in the progression of HCC. In the context of intact telomeres, p53 mutation might not have such a relevant effect on liver tum-origenesis.

Experimental models recapitulating inflammation- associated hepatocellular carcinoma

Knock-out animals of the multidrug-resistant gene Mdr2 represent a model of inflammation-associated HCC. The liver-specific phospho-glycoprotein responsible for the transport of phosphatidylcholine through the bile canali-cular membrane is absent in these animals, which results in bile regurgitation into the portal tracts, with the sub-sequent portal inflammation and fibrosis, reminiscent of human intrahepatic cholestasis [72, 73]. Due to the liver inflammation the Mdr2–/– mice develop hepatocyte dys-plasia and show liver tumorigenic foci at 16 months of age (almost 100%). Gene expression profiling has shown that Mdr2–/– animals differ from other published murine HCC models and are of great interest because they share several important deregulated pathways and many coordi-nately differentially expressed genes with human data sets [74]. The Mdr2–/– mouse may serve as a model for the ß-catenin-negative subgroup of human HCCs characterised by low nuclear cyclin D1 levels and down-regulation of multiple tumour suppressor genes. Most Mdr2–/– liver samples were more similar to the better-survival human HCC samples and better-survival-like mouse models.

A major link between inflammation and cancer is pro-vided by NF-kappaB transcription factors. Different stud-ies in genetically modified animals have indicated that IkappaB kinase beta (IKKbeta), required for NF-kappaB activation, links chronic inflammation with liver carcino-genesis. Indeed, mice lacking IKKbeta only in hepatocytes (Ikkbeta(Deltahep) mice) exhibited a marked increase in chemical hepatocarcinogenesis caused by DEN. This corre-lated with enhanced reactive oxygen species (ROS) produc-tion, increased c-Jun N-terminal kinase (JNK) activation and hepatocyte death, which give rise to augmented compensato-ry proliferation of surviving hepatocytes [75, 76]. However, decreased hepatocarcinogenesis was found in mice lacking IKKbeta in both hepatocytes and haematopoietic-derived Kupffer cells, due to the lack of inflammatory processes. These results indicated that chemical liver carcinogenesis depends on inflammation and suggested the usefulness of anti-inflammatory intervention targeting Kupffer cells in chemoprevention of HCC. Chemicals or viruses that inter-fere with NF-κB activation in hepatocytes, but not in sur-rounding cells, might promote HCC development (Fig. 2).

A relevant aspect of HCC epidemiology is the gender difference of incidence. Men are about three to five times more likely to develop HCC than women. Similar, or even more pronounced, gender differences are observed in rodent chemical-induced HCC models. Interleukin-6 knock-out (IL-6–/–) mice have helped to understand this gender disparity. IL-6 is a multifunctional cytokine largely responsible for the hepatic response to systemic inflam-mation (acute phase response). IL-6 concentrations are increased in patients with HCC [77]. IL-6 is necessary for compensatory regeneration in chemical models of hepatocarcinogenesis [78] and deletion of the suppressor of cytokine signalling-3 (SOCS3), a negative regulator of IL-6-related cytokines, enhances hepatitis- or chemical-induced liver carcinogenesis [79, 80]. In a recent work, Naugler et al. have elegantly described that ablation of IL-6 in IL-6–/– mice abolished the gender differences in a model of DEN-induced hepatocarcinogenesis. Oestrogen inhibited secretion of IL-6 from Kupffer cells exposed to necrotic hepatocytes and reduced circulating concentra-tions of IL-6 in DEN-treated male mice [81]. These results further evidence the connection between inflammation and HCC and open the way for therapeutic anti-inflammatory approaches to prevent or control this malignancy.

In conclusion, genetic modification of growth fac-tor pathways has revealed the relevance of the epidermal growth factor receptor (EGFR) ligands in hepatocarcino-genesis, which in cooperation with c-myc might mimic the poorer survival set of human HCC. Results obtained after genetic modification of the HGF/c-Met or TGF-ß pathways have revealed that they play dual roles both as suppres-sor, in early stages, or promoters, in later stages, of HCC. Genetic modification of the p53 pathway has revealed its relevance in hepatocarcinogenesis, particularly under telomere-induced chromosomal instability. Finally, differ-ent genetically modified mice have pointed out the straight

Clin Transl Oncol (2009) 11:208-214 213

link between inflammation and liver carcinogenesis, NF-kappaB and IL-6 pathways playing pivotal roles.

Acknowledgements The authors acknowledge the help of Javier Marquez in the writing of this manuscript.

NF-kappaB Kupffer cellsViral infections

Chemicals

Inflammatory cytokinesGrowth and survival factors

Angiogenic factors

PreneoplasticLiver cell

Growth, survival, invasive properties

Fig. 2 NF-kappaB activation in inflammatory cells can promote malignant conversion and liver tumour progression. Viral infections or toxic reactions activate inflammatory cells to produce cytokines and growth factors that aug-ment compensatory proliferation of surround-ing cells. Chemicals or viruses that interfere with NF-kappaB activation in hepatocytes, but not in preneoplastic cells, would promote HCC development

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