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Transcriptional regulators in hepatocarcinogenesis – Key integrators of malignant transformation

Open AccessPublished:March 22, 2012DOI:https://doi.org/10.1016/j.jhep.2011.11.029

      Summary

      Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies with poor prognosis and increasing incidence in the Western world. Only for a minority of HCC patients, surgical treatment options offer potential cure and therapeutic success of pharmacological approaches is limited. Highly specific approaches (e.g., kinase inhibitors) did not significantly improve the situation so far, possibly due to functional compensation, genetic heterogeneity of HCC, and development of resistance under selective pressure. In contrast, transcriptional regulators (especially transcription factors and co-factors) may integrate and process input signals of different (oncogenic) pathways and therefore represent cellular bottlenecks that regulate tumor cell biology. In this review, we want to summarize the current knowledge about central transcriptional regulators in human hepatocarcinogenesis and their potential as therapeutic target structures. Genomic and transcriptomic data of primary human HCC revealed that many of these factors showed up in subgroups of HCCs with a more aggressive phenotype, suggesting that aberrant activity of transcriptional regulators collect input information to promote tumor initiation and progression. Therefore, expression and dysfunction of transcription factors and co-factors may gain relevance for diagnostics and therapy of HCC.

      Keywords

      Introduction

      With more than 500,000 new cases each year, hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death [
      • Breuhahn K.
      • Gores G.
      • Schirmacher P.
      Strategies for hepatocellular carcinoma therapy and diagnostics: lessons learned from high throughput and profiling approaches.
      ]. Its risk factors are well defined (e.g., chronic viral hepatitis, alcoholic and non-alcoholic steatohepatitis, aflatoxin incorporation, and several hereditary diseases) and are detectable in up to 90% of all cases. Unfortunately, genetic heterogeneity of HCC severely complicates the development of effective and specific drugs, which is reflected by the current lack of therapeutic options.
      For few patients, partial hepatic resection or liver transplantation offers potential cure and only the multi-kinase inhibitor sorafenib has been established as the first effective and approved systemic treatment for progressed HCC [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ]. Interestingly, functional genomics revealed that dysregulation of some signalling pathways (e.g., WNT-, IGF/IGF-1R, HGF/c-MET) characterizes subgroups of HCCs with specific clinical and biological features [
      • Breuhahn K.
      • Gores G.
      • Schirmacher P.
      Strategies for hepatocellular carcinoma therapy and diagnostics: lessons learned from high throughput and profiling approaches.
      ,
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ,
      • Kaposi-Novak P.
      • Lee J.S.
      • Gomez-Quiroz L.
      • Coulouarn C.
      • Factor V.M.
      • Thorgeirsson S.S.
      Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype.
      ], suggesting that receptor-mediated signalling cascades may represent promising therapeutic target structures. However, the first clinical trials indicated that administration of monoclonal antibodies and kinase inhibitors targeting different tumor-relevant receptors did not further improve the situation [
      • Villanueva A.
      • Minguez B.
      • Forner A.
      • Reig M.
      • Llovet J.M.
      Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy.
      ]. For this reason, the identification of targets that modulate/regulate different oncogenic pathways in HCC and accordingly direct drug development are of high relevance for improvement of the current dismal and unsatisfactory situation.
      Transcription factors and transcriptional co-factors convert and integrate extra- and intracellular signals and therefore represent central regulators of cellular processes in normal and malignantly transformed cells. About 10% of all human genes code for proteins containing DNA-binding domains; most of these proteins may function as transcription factors and co-factors, which are pivotal for fine-tuning regulation of the transcriptional machinery. Basically, eukaryotic mRNA transcription is divided into three steps: (1) binding of transcriptional activators to cis-acting DNA sequences, (2) recruitment of the pre-initiation complex to the core promoter, and (3) elongation of the mRNA molecule [
      • Morse R.H.
      Transcription factor access to promoter elements.
      ]. In this system, general transcription factors (e.g., TFIIA and TFIIH) are sufficient for basal transcription and form (together with RNA polymerase II) the pre-initiation complex at the core promoter region. Specific transcription factors bind additional DNA motifs (e.g., enhancers and silencers), which are in most cases located in the vicinity of the core promoter [
      • Baumann M.
      • Pontiller J.
      • Ernst W.
      Structure and basal transcription complex of RNA polymerase II core promoters in the mammalian genome: an overview.
      ]. Initiation, progression, and termination of this highly complex process as well as classification of the different specific transcription factors and co-factors have been studied in detail (see [
      • Brivanlou A.H.
      • Darnell Jr., J.E.
      Signal transduction and the control of gene expression.
      ,
      • Juven-Gershon T.
      • Kadonaga J.T.
      Regulation of gene expression via the core promoter and the basal transcriptional machinery.
      ,
      • MacQuarrie K.L.
      • Fong A.P.
      • Morse R.H.
      • Tapscott S.J.
      Genome-wide transcription factor binding: beyond direct target regulation.
      ]). One key feature of transcriptional regulation is that most of these proteins remain as inactive forms in the cytoplasm until they translocate in the nucleus upon stimulation. In tumor cells, activation or aberrant expression of these factors frequently represents the last step in a number of signalling pathways that affect proliferation, apoptosis, differentiation, migration, or senescence in an oncogenic manner.
      Figure thumbnail fx2

      Transcriptional regulators in human hepatocarcinogenesis

      Identification of key integrators using functional genomics

      Dysregulation of several transcription factors and co-factors has been described in the context of human HCC; however, functional genomics approaches on primary human HCCs revealed that aberrant expression of some factors was characteristic of subgroups of HCCs with specific biological and clinical features (Table 1). Transcriptional analyses as well as integrative approaches (further including genomic, mutational, and protein data) demonstrated that c-Myc, b-Myb, β-catenin, AP-1, p53, HIF-1α, E2F, and HOX may play key roles in hepatocarcinogenesis [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ,
      • Lee J.S.
      • Chu I.S.
      • Heo J.
      • Calvisi D.F.
      • Sun Z.
      • Roskams T.
      • et al.
      Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.
      ,
      • Lee J.S.
      • Heo J.
      • Libbrecht L.
      • Chu I.S.
      • Kaposi-Novak P.
      • Calvisi D.F.
      • et al.
      A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
      ]. Many of these factors are known regulators of central protumorigenic cellular processes in HCC cells such as proliferation (c-Myc, b-Myb, β-catenin, AP-1, p53, HIF-1α, E2F), (anti-) apoptosis (c-Myc, b-Myb, AP-1, p53, E2F, HOX), and migration/invasion (β-catenin, HOX).
      Table 1Transcriptional regulators in human hepatocarcinogenesis
      • Moghaddam S.J.
      • Haghighi E.N.
      • Samiee S.
      • Shahid N.
      • Keramati A.R.
      • Dadgar S.
      • et al.
      Immunohistochemical analysis of p53, cyclinD1, RB1, c-fos and N-ras gene expression in hepatocellular carcinoma in Iran.
      ,
      • de La Coste A.
      • Romagnolo B.
      • Billuart P.
      • Renard C.A.
      • Buendia M.A.
      • Soubrane O.
      • et al.
      Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.
      ,
      • Merle P.
      • de la Monte S.
      • Kim M.
      • Herrmann M.
      • Tanaka S.
      • Von Dem Bussche A.
      • et al.
      Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma.
      ,
      • Shih Y.L.
      • Shyu R.Y.
      • Hsieh C.B.
      • Lai H.C.
      • Liu K.Y.
      • Chu T.Y.
      • et al.
      Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma.
      ,
      • Wirths O.
      • Waha A.
      • Weggen S.
      • Schirmacher P.
      • Kuhne T.
      • Goodyer C.G.
      • et al.
      Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms’ tumors.
      ,
      • Yu B.
      • Yang X.
      • Xu Y.
      • Yao G.
      • Shu H.
      • Lin B.
      • et al.
      Elevated expression of DKK1 is associated with cytoplasmic/nuclear beta-catenin accumulation and poor prognosis in hepatocellular carcinomas.
      ,
      • Pang R.
      • Yuen J.
      • Yuen M.F.
      • Lai C.L.
      • Lee T.K.
      • Man K.
      • et al.
      PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma.
      ,
      • Yau T.O.
      • Chan C.Y.
      • Chan K.L.
      • Lee M.F.
      • Wong C.M.
      • Fan S.T.
      • et al.
      HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing.
      ,
      • Ladu S.
      • Calvisi D.F.
      • Conner E.A.
      • Farina M.
      • Factor V.M.
      • Thorgeirsson S.S.
      E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR and COX-2 in a mouse model of human liver cancer.
      ,
      • Li W.
      • Ni G.X.
      • Zhang P.
      • Zhang Z.X.
      • Wu Q.
      Characterization of E2F3a function in HepG2 liver cancer cells.
      up, upregulated; down, downregulated; n.d., not determined.
      Based on meta-analysis of existing gene expression profiles, preferential accumulation of E2F1, c-Myc, and increased nuclear expression of p53 were detected in groups of HCCs characterized by high tumor cell proliferation, dedifferentiation, and dismal prognosis [
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ]. The central role for c-Myc has been further substantiated by the identification of c-Myc-dependent target gene signatures (based on copy number gains of 8q24), which were significantly enriched in early human HCCs but not in premalignant lesions, suggesting that dysregulation of c-Myc and its target genes is also critically involved in malignant transformation [
      • Kaposi-Novak P.
      • Libbrecht L.
      • Woo H.G.
      • Lee Y.H.
      • Sears N.C.
      • Coulouarn C.
      • et al.
      Central role of c-Myc during malignant conversion in human hepatocarcinogenesis.
      ]. In addition, c-Myc-dependent signatures of four microRNAs are characteristic of HCCs with a more aggressive phenotype [
      • Cairo S.
      • Wang Y.
      • de Reynies A.
      • Duroure K.
      • Dahan J.
      • Redon M.J.
      • et al.
      Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.
      ]. Comprehensive expression profiling revealed overexpression of b-Myb in HCCs where it belongs to a cluster of genes associated with cell cycle progression and proliferation [
      • Chen X.
      • Cheung S.T.
      • So S.
      • Fan S.T.
      • Barry C.
      • Higgins J.
      • et al.
      Gene expression patterns in human liver cancers.
      ]. The role of p53 is supported by previous integrative studies, which revealed that mutations in TP53 define a subgroup of HCCs characterized by chromosome instability and poor differentiation [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Laurent-Puig P.
      • Legoix P.
      • Bluteau O.
      • Belghiti J.
      • Franco D.
      • Binot F.
      • et al.
      Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
      ]. In addition, elevated HIF-1α and HOXA13 levels in a subgroup of patients correlated with poor prognosis [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Lee J.S.
      • Chu I.S.
      • Heo J.
      • Calvisi D.F.
      • Sun Z.
      • Roskams T.
      • et al.
      Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.
      ,
      • Cillo C.
      • Schiavo G.
      • Cantile M.
      • Bihl M.P.
      • Sorrentino P.
      • Carafa V.
      • et al.
      The HOX gene network in hepatocellular carcinoma.
      ]. HCCs characterized by a fetal hepatoblast-like expression signature and poor patient prognosis showed overexpression of AP-1 subunits (e.g., c-Fos and Fra-2) and upregulation of c-Jun/c-Fos-dependent target genes [
      • Lee J.S.
      • Heo J.
      • Libbrecht L.
      • Chu I.S.
      • Kaposi-Novak P.
      • Calvisi D.F.
      • et al.
      A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
      ]. However, these analyses did not yield a uniform picture for an activation of β-catenin. On one hand, it has been shown that HCCs with β-catenin mutations are characterized by large tumor size and low genomic instability [
      • Laurent-Puig P.
      • Legoix P.
      • Bluteau O.
      • Belghiti J.
      • Franco D.
      • Binot F.
      • et al.
      Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
      ] and that elevated β-catenin expression (associated with a WNT signature) defined a group of dedifferentiated HCCs with a more aggressive phenotype [
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ]. In contrast, in another study, subgroups with WNT activation exhibited better prognosis than groups without activation [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ].

      v-myc avian myelocytomatosis viral oncogene homolog (c-Myc)

      The proto-oncogene c-Myc potentially interacts with up to 15% of mammalian gene promoters; thus it is involved in a plethora of biological processes such as cell growth and cell cycle, metabolism, programmed cell death, cell adhesion, DNA repair, as well as microRNA regulation. Heterodimerization with its binding partner Max and interaction with several co-regulators (e.g., SWI/SNF) are essential for efficient induction of transcription and probably target gene specificity. While Max is ubiquitously expressed in many cell types, c-Myc abundance is tightly controlled and therefore defines the activity of the c-Myc/Max complex [
      • Eilers M.
      • Eisenman R.N.
      Myc’s broad reach.
      ].
      In HCC, c-Myc is reported to be overexpressed in 47–65% of all cases [
      • Prange W.
      • Breuhahn K.
      • Fischer F.
      • Zilkens C.
      • Pietsch T.
      • Petmecky K.
      • et al.
      Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes.
      ,
      • Saegusa M.
      • Takano Y.
      • Kishimoto H.
      • Wakabayashi G.
      • Nohga K.
      • Okudaira M.
      Comparative analysis of p53 and c-myc expression and cell proliferation in human hepatocellular carcinomas – an enhanced immunohistochemical approach.
      ]. Elevated c-Myc levels are predominantly based on chromosomal gains of the Myc gene locus (8q24.21), which were detected in up to 60% of all the cases [
      • Wang Y.
      • Wu M.C.
      • Sham J.S.
      • Zhang W.
      • Wu W.Q.
      • Guan X.Y.
      Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad survey using high-throughput tissue microarray.
      ,
      • Abou-Elella A.
      • Gramlich T.
      • Fritsch C.
      • Gansler T.
      C-myc amplification in hepatocellular carcinoma predicts unfavorable prognosis.
      ] and which correlated with dedifferentiation and poor patient prognosis [
      • Abou-Elella A.
      • Gramlich T.
      • Fritsch C.
      • Gansler T.
      C-myc amplification in hepatocellular carcinoma predicts unfavorable prognosis.
      ]. Interestingly, array-CGH data revealed significantly elevated copy number gains and c-Myc overexpression in alcohol-related HCCs, while no chromosomal changes were detectable in cryptogenic HCCs that are thought to be caused by non-alcoholic steatohepatitis [
      • Schlaeger C.
      • Longerich T.
      • Schiller C.
      • Bewerunge P.
      • Mehrabi A.
      • Toedt G.
      • et al.
      Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.
      ]. Besides genomic gains, HBV- and HCV-derived proteins (HBx and core protein, respectively) may induce c-Myc expression [
      • Balsano C.
      • Avantaggiati M.L.
      • Natoli G.
      • De Marzio E.
      • Will H.
      • Perricaudet M.
      • et al.
      Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cmyc protooncogene at the transcriptional level.
      ,
      • Ma H.C.
      • Lin T.W.
      • Li H.
      • Iguchi-Ariga S.M.
      • Ariga H.
      • Chuang Y.L.
      • et al.
      Hepatitis C virus ARFP/F protein interacts with cellular MM-1 protein and enhances the gene trans-activation activity of c-Myc.
      ], indicating that hepatocarcinogenic viruses may have developed independent “epigenomic” mechanisms to further support c-Myc expression.

      v-myb myeloblastosis viral oncogene homolog (avian)-like 2 (b-Myb)

      b-Myb (synonym: MYBL2) belongs to the Myb proto-oncogene family. It directly binds to the consensus MYB-binding site of gene promoters or interacts with other transcription factors (e.g., SP1) to regulate the expression of target genes [
      • Sala A.
      • Saitta B.
      • De Luca P.
      • Cervellera M.N.
      • Casella I.
      • Lewis R.E.
      • et al.
      B-MYB transactivates its own promoter through SP1-binding sites.
      ]. Moreover, in conjunction with E2F, b-Myb regulates gene transcription control during cell cycle progression [
      • Zhu W.
      • Giangrande P.H.
      • Nevins J.R.
      E2Fs link the control of G1/S and G2/M transcription.
      ].
      In HCC, b-Myb overexpression was detectable in more than 90% of all the analyzed cases predominantly based on genomic gains of the MYBL2 gene locus (chr. 20q13.1) and transcriptional activation by E2F1 [
      • Nakajima T.
      • Yasui K.
      • Zen K.
      • Inagaki Y.
      • Fujii H.
      • Minami M.
      • et al.
      Activation of B-Myb by E2F1 in hepatocellular carcinoma.
      ]. Its elevated expression in HCC tissues correlated with p53 staining (indicating mutations) and vascular invasion [
      • Chen X.
      • Cheung S.T.
      • So S.
      • Fan S.T.
      • Barry C.
      • Higgins J.
      • et al.
      Gene expression patterns in human liver cancers.
      ]. In addition, overexpression of b-Myb increased cell viability of HCC cell lines [
      • Frau M.
      • Ladu S.
      • Calvisi D.F.
      • Simile M.M.
      • Bonelli P.
      • Daino L.
      • et al.
      Mybl2 expression is under genetic control and contributes to determine a hepatocellular carcinoma susceptible phenotype.
      ]. Recently, it was shown that in HCC tissues the expression and especially the phosphorylation/activation of b-Myb significantly and positively correlated with proliferation, genomic instability, and microvessel density. In addition, b-Myb levels were higher in HCC patients with poor prognosis [
      • Calvisi D.F.
      • Simile M.M.
      • Ladu S.
      • Frau M.
      • Evert M.
      • Tomasi M.L.
      • et al.
      Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53.
      ]. Interestingly, b-Myb seems to be involved in the induction of DNA repair mechanisms and integrity of a b-Myb/LIN9 complex contributes to the survival of p53-deficient HCC cells [
      • Calvisi D.F.
      • Simile M.M.
      • Ladu S.
      • Frau M.
      • Evert M.
      • Tomasi M.L.
      • et al.
      Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53.
      ].

      β-catenin

      Activation of the Wingless (Wnt) pathway leads to the nuclear translocation of the transcriptional co-regulator β-catenin, where it interacts with the T-cell factor (TCF)-/lymphoid-enhancer factor (LEF) transcription factor that regulates cell cycle progression, differentiation, and cell motility. In the absence of pathway activation, aminoterminal phosphorylation of β-catenin by the APC/Axin/GSK-3β complex leads to its polyubiquitination and subsequent degradation [
      • Gavert N.
      • Ben-Ze’ev A.
      Beta-catenin signaling in biological control and cancer.
      ].
      More than 40% of all HCCs exhibit nuclear accumulation of β-catenin, which is in most cases explained by stabilizing point mutations and deletions in its N-terminal domain [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Prange W.
      • Breuhahn K.
      • Fischer F.
      • Zilkens C.
      • Pietsch T.
      • Petmecky K.
      • et al.
      Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes.
      ,
      • Breuhahn K.
      • Longerich T.
      • Schirmacher P.
      Dysregulation of growth factor signaling in human hepatocellular carcinoma.
      ]. In addition, inactivating mutations in components regulating β-catenin degradation such as Axin-1, Axin-2 [
      • Taniguchi K.
      • Roberts L.R.
      • Aderca I.N.
      • Dong X.
      • Qian C.
      • Murphy L.M.
      • et al.
      Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas.
      ] and GSK-3β have been detected as well as overexpression of the APC-complex regulating factors (e.g., Dvl3 [
      • Chan D.W.
      • Chan C.Y.
      • Yam J.W.
      • Ching Y.P.
      • Ng I.O.
      Prickle-1 negatively regulates Wnt/beta-catenin pathway by promoting dishevelled ubiquitination/degradation in liver cancer.
      ]), and different WNT ligands and receptors (e.g., FRZ7; [
      • Bengochea A.
      • de Souza M.M.
      • Lefrancois L.
      • Le Roux E.
      • Galy O.
      • Chemin I.
      • et al.
      Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma.
      ]). Interestingly, APC mutations – which are frequent events in gastric and colorectal cancer [
      • Lea I.A.
      • Jackson M.A.
      • Li X.
      • Bailey S.
      • Peddada S.D.
      • Dunnick J.K.
      Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns.
      ] – are rare in human HCCs [
      • Csepregi A.
      • Rocken C.
      • Hoffmann J.
      • Gu P.
      • Saliger S.
      • Muller O.
      • et al.
      APC promoter methylation and protein expression in hepatocellular carcinoma.
      ,
      • Legoix P.
      • Bluteau O.
      • Bayer J.
      • Perret C.
      • Balabaud C.
      • Belghiti J.
      • et al.
      Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.
      ]. Next to the aberrant activation of β-catenin, accumulation of its transcriptional binding partners has been shown in many HCCs (e.g., nuclear accumulation of LEF-1/TCF in 52% [
      • Schmitt-Graeff A.
      • Ertelt-Heitzmann V.
      • Allgaier H.P.
      • Olschewski M.
      • Nitschke R.
      • Haxelmans S.
      • et al.
      Coordinated expression of cyclin D1 and LEF-1/TCF transcription factor is restricted to a subset of hepatocellular carcinoma.
      ]). Several additional factors which are known modifiers of Wnt/β-catenin activity such as DKK, HDPR1/dapper, and PIN1 are also dysregulated in HCC, clearly demonstrating that in most cases synchronous activation of different pathway components and regulators is necessary to achieve complete pro-tumorigenic capacity of the pathway.
      Both, HBV and HCV infections may stimulate the Wnt/β-catenin pathway by independent and virus-specific mechanisms. While HCV-positive HCC patients showed high frequency mutations in CTNNB1 [
      • Huang H.
      • Fujii H.
      • Sankila A.
      • Mahler-Araujo B.M.
      • Matsuda M.
      • Cathomas G.
      • et al.
      Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection.
      ,
      • Wong C.M.
      • Fan S.T.
      • Ng I.O.
      Beta-catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance.
      ], other data suggested that HBV may activate this pathway in a mutation-independent manner due to expression of HBx-induced host proteins that stabilize β-catenin [
      • Wong C.M.
      • Fan S.T.
      • Ng I.O.
      Beta-catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance.
      ,
      • Lian Z.
      • Liu J.
      • Li L.
      • Li X.
      • Clayton M.
      • Wu M.C.
      • et al.
      Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with upregulation of beta-catenin.
      ].

      Activator protein-1 (AP-1)

      The activating protein-1 (AP-1) transcription factor collectively describes a family of functionally and structurally related heterodimeric complexes composed of varying combinations of, for example, Jun (c-Jun, Jun-B, Jun-D) and Fos (c-Fos, Fos-B, Fra-1, Fra-2) proteins [
      • Hess J.
      • Angel P.
      • Schorpp-Kistner M.
      AP-1 subunits: quarrel and harmony among siblings.
      ]. They are typically activated by growth factor/receptor tyrosine kinase pathways and, based on their specific composition, regulate proliferation, apoptosis, and differentiation under physiological conditions and in tumorigenesis [
      • Eferl R.
      • Wagner E.F.
      AP-1: a double-edged sword in tumorigenesis.
      ].
      Although overexpression of c-Fos and c-Jun has been described in human HCC [
      • Arbuthnot P.
      • Kew M.
      • Fitschen W.
      c-fos and c-myc oncoprotein expression in human hepatocellular carcinomas.
      ,
      • Chang Y.S.
      • Yeh K.T.
      • Yang M.Y.
      • Liu T.C.
      • Lin S.F.
      • Chan W.L.
      • et al.
      Abnormal expression of JUNB gene in hepatocellular carcinoma.
      ,
      • Yuen M.F.
      • Wu P.C.
      • Lai V.C.
      • Lau J.Y.
      • Lai C.L.
      Expression of c-Myc, c-Fos, and c-jun in hepatocellular carcinoma.
      ], increasing evidence indicates that AP-1 constituents may also provide tumor-suppressive activity under specific conditions [
      • Eferl R.
      • Wagner E.F.
      AP-1: a double-edged sword in tumorigenesis.
      ]. For HCC, this was exemplified by JunB, which is frequently reduced in HCCs compared to surrounding non-tumorous tissues [
      • Guo C.
      • Liu Q.G.
      • Zhang L.
      • Song T.
      • Yang X.
      Expression and clinical significance of p53, JunB and KAI1/CD82 in human hepatocellular carcinoma.
      ]. Nevertheless, a gradual increase in the DNA binding capacity of AP-1 has been detected in peritumoral and HCC tissues compared to histologically normal liver samples, indicating that aberrant activation of AP-1 during human hepatocarcinogenesis may be involved in hepatocyte transformation and tumor progression [
      • Liu P.
      • Kimmoun E.
      • Legrand A.
      • Sauvanet A.
      • Degott C.
      • Lardeux B.
      • et al.
      Activation of NF-kappa B, AP-1 and STAT transcription factors is a frequent and early event in human hepatocellular carcinomas.
      ].
      In addition to the aberrant expression of AP-1 subunits, phosphorylation/activity of AP-1 constituents is regulated by a broad spectrum of stimuli that control the MAPK pathway. Especially Jun N-terminal kinase (JNK)-1 has been described to contribute to hepatocarcinogenesis. Studies demonstrated increased activation of JNK-1 in more than 50% of HCCs [
      • Hui L.
      • Zatloukal K.
      • Scheuch H.
      • Stepniak E.
      • Wagner E.F.
      Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation.
      ] and correlation of high JNK-1 level with poor patient survival [
      • Chang Q.
      • Chen J.
      • Beezhold K.J.
      • Castranova V.
      • Shi X.
      • Chen F.
      JNK1 activation predicts the prognostic outcome of the human hepatocellular carcinoma.
      ].

      p53

      Mutations in the TP53 gene (which codes for the tumor suppressor p53) are likely to be the most frequent tumor-relevant mutations defined in human malignancies. In human hepatocarcinogenesis, multiple mechanisms can be associated with functional inactivation of wild type p53 (p53wt). First, more than 30% of all HCCs show chromosomal losses of the TP53 gene locus (17p13.1) [
      • Moinzadeh P.
      • Breuhahn K.
      • Stutzer H.
      • Schirmacher P.
      Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade – results of an explorative CGH meta-analysis.
      ]. Second, viral proteins with transforming capacity (e.g., HBV-derived HBx-Ag) bind and inactivate p53wt [
      • Feitelson M.A.
      • Duan L.X.
      Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma.
      ]. Third, inactivation of p53 by point mutations or small deletions is detectable in 10–28% of Western world HCCs. However, in areas with high aflatoxin-B1 exposure, the incidence of codon 249 (p53mut249) transversions may be present in as much as 50% of all HCCs and represents one of the earliest and most clear-cut examples of molecular cancer epidemiology [
      • Lasky T.
      • Magder L.
      Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure?.
      ,
      • Ozturk M.
      P53 mutation in hepatocellular carcinoma after aflatoxin exposure.
      ]. Since p53 binds DNA as a tetramer, mutated p53mut249 exhibits a dominant negative effects on p53wt with regard to cell survival and cell cycle control [
      • Lee M.K.
      • Sabapathy K.
      The R246S hot-spot p53 mutant exerts dominant-negative effects in embryonic stem cells in vitro and in vivo.
      ]. In addition, various gain of function mutations have been identified in human (hepato-)carcinogenesis that facilitate tumor-promoting activity of p53mut at different stages of tumor progression as well as resistance to cancer treatment [
      • Oren M.
      • Rotter V.
      Mutant p53 gain-of-function in cancer.
      ]. Lastly, aberrant expression and activity of factors that regulate p53 stability (e.g., the E3 ubiquitin ligase MDM2, which targets p53 for proteasomal degradation) may affect its availability [
      • Schlaeger C.
      • Longerich T.
      • Schiller C.
      • Bewerunge P.
      • Mehrabi A.
      • Toedt G.
      • et al.
      Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.
      ,
      • Higashitsuji H.
      • Itoh K.
      • Sakurai T.
      • Nagao T.
      • Sumitomo Y.
      • Masuda T.
      • et al.
      The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53.
      ]. Because concentrations and activity of p53 are affected by many molecular mechanisms at different levels in HCC, it is likely that in most (if not all) HCCs the biological integrity of the p53 pathway is somehow impaired. However, respective comprehensive studies comparing the frequency of the different modes of p53 dysregulation in human HCC are missing so far.
      Increasing data indicate that full length isoforms of p63 and p73 (TAp63 and TAp73), which are structurally related to p53wt, can partly mimic its function. In HCC cells, p53mut directly binds to TAp63/TAp73 and subsequently decreases their pro-apoptotic features [
      • Gaiddon C.
      • Lokshin M.
      • Ahn J.
      • Zhang T.
      • Prives C.
      A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain.
      ,
      • Schilling T.
      • Kairat A.
      • Melino G.
      • Krammer P.H.
      • Stremmel W.
      • Oren M.
      • et al.
      Interference with the p53 family network contributes to the gain of oncogenic function of mutant p53 in hepatocellular carcinoma.
      ]. It is therefore likely that next to the mutational status of p53, an altered ratio between p53 and p73 (TAp73 and the shorter ΔNp73) and/or p63 (TAp63 and the shorter ΔNp63) influences p53-dependent effects and chemosensitivity in HCC patients.

      Hypoxia-inducible factor-1 (HIF-1)

      Hypoxia induces the activity of the heterodimeric transcription factor HIF-1. In contrast to the constitutively expressed subunit HIF-1β, HIF-1α is degraded in a proteasome-dependent manner under normoxic conditions by post-transcriptional prolyl hydroxylation. Hypoxia prevents this protein from being degraded, resulting in increased HIF-1α levels and induction of HIF-1-regulated genes that may promote angiogenesis, metabolic processes, and cell survival [
      • Fandrey J.
      • Gassmann M.
      Oxygen sensing and the activation of the hypoxia inducible factor 1 (HIF-1) – invited article.
      ].
      Hypoxic tumor microenvironment, as a result of uncontrolled tumor cell growth, and subsequent hypervascularisation are frequently observed in moderately and poorly differentiated tumors [
      • Keith B.
      • Simon M.C.
      Hypoxia-inducible factors, stem cells, and cancer.
      ]. In these cases, high-level expression of HIF-1α was observed in more than 60% of HCC tissues, which correlated with poor patient prognosis [
      • Huang G.W.
      • Yang L.Y.
      • Lu W.Q.
      Expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in hepatocellular carcinoma: impact on neovascularization and survival.
      ]. Interestingly, elevated HIF-1α transcript levels in adjacent non-tumorous liver tissues correlated with poor disease-free and overall survival of HCC patients, indicating a paracrine effect of hypoxia even on non-tumorous cells [
      • Simon F.
      • Bockhorn M.
      • Praha C.
      • Baba H.A.
      • Broelsch C.E.
      • Frilling A.
      • et al.
      Deregulation of HIF1-alpha and hypoxia-regulated pathways in hepatocellular carcinoma and corresponding non-malignant liver tissue – influence of a modulated host stroma on the prognosis of HCC.
      ].
      In HCC, stabilization of HIF-1α in hypoxic-dependent and independent manners is achieved by different mechanisms. First, activation of typical growth factor-pathways promotes HIF-1α stabilization (e.g., HGF/c-MET signalling) [
      • Tacchini L.
      • Dansi P.
      • Matteucci E.
      • Desiderio M.A.
      Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells.
      ,
      • Lee T.K.
      • Poon R.T.
      • Yuen A.P.
      • Man K.
      • Yang Z.F.
      • Guan X.Y.
      • et al.
      Rac activation is associated with hepatocellular carcinoma metastasis by up-regulation of vascular endothelial growth factor expression.
      ]. Second, HIF-1α is stabilized after infection with HCV through impairment of mitochondrial phosphorylation and subsequent metabolite-mediated inhibition of HIF-1α-prolyl hydroxylation [
      • Ripoli M.
      • D’Aprile A.
      • Quarato G.
      • Sarasin-Filipowicz M.
      • Gouttenoire J.
      • Scrima R.
      • et al.
      Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation.
      ]. Third, overexpression of the oncoprotein gankyrin (p28GANK) in HCCs induces HIF-1α [
      • Fu J.
      • Chen Y.
      • Cao J.
      • Luo T.
      • Qian Y.W.
      • Yang W.
      • et al.
      P28GANK overexpression accelerates hepatocellular carcinoma invasiveness and metastasis via phosphoinositol 3-kinase/AKT/hypoxia-inducible factor-1alpha pathways.
      ].

      E2F transcription factors

      The activity of many E2F transcription factor family members (E2F1-E2F8) is tightly regulated by the tumor suppressor retinoblastoma (Rb). While hypophosphorylated Rb binds and inactivates E2Fs, hyperphosphorylated Rb releases E2Fs which heterodimerize with DP (E2F dimerization partner) transcription factor family members. E2F1-E2F3a predominantly activate gene expression, while E2F3b-E2F8 are referred to as transcriptional repressors. Since E2Fs regulate key tumor-relevant processes in a differential manner (e.g., cell proliferation and apoptosis), these factors may exert oncogenic and tumor suppressive functions [
      • van den Heuvel S.
      • Dyson N.J.
      Conserved functions of the pRB and E2F families.
      ].
      So far, E2F1, E2F3, and E2F8 have been described as overexpressed in human HCC compared to non-tumorous liver tissues [
      • Nakajima T.
      • Yasui K.
      • Zen K.
      • Inagaki Y.
      • Fujii H.
      • Minami M.
      • et al.
      Activation of B-Myb by E2F1 in hepatocellular carcinoma.
      ,
      • Deng Q.
      • Wang Q.
      • Zong W.Y.
      • Zheng D.L.
      • Wen Y.X.
      • Wang K.S.
      • et al.
      E2F8 contributes to human hepatocellular carcinoma via regulating cell proliferation.
      ,
      • Xu T.
      • Zhu Y.
      • Xiong Y.
      • Ge Y.Y.
      • Yun J.P.
      • Zhuang S.M.
      MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells.
      ]; however, different modes of dysregulation have been detected so far. First, reduction of Rb levels (e.g., due to expression of viral proteins and chromosomal losses of the Rb gene) may lead to enhanced E2F activity [
      • Moinzadeh P.
      • Breuhahn K.
      • Stutzer H.
      • Schirmacher P.
      Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade – results of an explorative CGH meta-analysis.
      ,
      • Choi B.H.
      • Choi M.
      • Jeon H.Y.
      • Rho H.M.
      Hepatitis B viral X protein overcomes inhibition of E2F1 activity by pRb on the human Rb gene promoter.
      ]. Second, reduction of regulatory microRNAs (e.g., miR-195), which may lead to increased E2F3 expression [
      • Xu T.
      • Zhu Y.
      • Xiong Y.
      • Ge Y.Y.
      • Yun J.P.
      • Zhuang S.M.
      MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells.
      ]. Third, genomic gains, microsatellite instability, and deletion mutations in the E2F genes (e.g., for E2F1 and E2F4) are commonly detected in HCC [
      • Park Y.M.
      • Choi J.Y.
      • Bae S.H.
      • Byun B.H.
      • Ahn B.M.
      • Kim B.S.
      • et al.
      Microsatellite instability and mutations of E2F-4 in hepatocellular carcinoma from Korea.
      ,
      • Midorikawa Y.
      • Tsutsumi S.
      • Nishimura K.
      • Kamimura N.
      • Kano M.
      • Sakamoto H.
      • et al.
      Distinct chromosomal bias of gene expression signatures in the progression of hepatocellular carcinoma.
      ].

      Homeobox (HOX) proteins

      The homeobox gene family is involved in specifying positional and spatio-temporal information during embryogenesis in mammalian cells. The 39 HOX genes are organized in four clusters on separated chromosomes and are expressed in an organ- and tissue-specific manner in embryonic and adult cells [
      • Zacchetti G.
      • Duboule D.
      • Zakany J.
      Hox gene function in vertebrate gut morphogenesis: the case of the caecum.
      ].
      Accumulating data suggest the relevance of HOX gene dysregulation in human carcinogenesis as was shown for different solid tumors and leukemia [
      • Shah N.
      • Sukumar S.
      The Hox genes and their roles in oncogenesis.
      ]. Recent publications also linked aberrant HOX protein expression with human hepatocarcinogenesis [
      • Cillo C.
      • Schiavo G.
      • Cantile M.
      • Bihl M.P.
      • Sorrentino P.
      • Carafa V.
      • et al.
      The HOX gene network in hepatocellular carcinoma.
      ,
      • Kanai M.
      • Hamada J.
      • Takada M.
      • Asano T.
      • Murakawa K.
      • Takahashi Y.
      • et al.
      Aberrant expressions of HOX genes in colorectal and hepatocellular carcinomas.
      ]. Most HOX genes – especially HOXA5 and HOXA13 – were upregulated at the mRNA level in HCC compared to non-cancerous livers, possibly via a common mechanism [
      • Kanai M.
      • Hamada J.
      • Takada M.
      • Asano T.
      • Murakawa K.
      • Takahashi Y.
      • et al.
      Aberrant expressions of HOX genes in colorectal and hepatocellular carcinomas.
      ]. Functional data of HOX proteins in HCC cells are missing so far, but studies in other tumor entities suggested that these factors regulate apoptosis, receptor signalling, and invasion [
      • Shah N.
      • Sukumar S.
      The Hox genes and their roles in oncogenesis.
      ]. For HCC, a physical interaction of HOXA13 with eIF4E and its possible role in the selective nuclear export of tumor-relevant transcripts have been described (e.g., Cyclin D1, c-Myc, and VEGF) [
      • Cillo C.
      • Schiavo G.
      • Cantile M.
      • Bihl M.P.
      • Sorrentino P.
      • Carafa V.
      • et al.
      The HOX gene network in hepatocellular carcinoma.
      ].
      In summary, high throughput screening approaches revealed groups of HCCs that are characterized by activation of transcriptional regulators; for some proteins their tumor-supporting properties have been analyzed in detail (e.g., β-catenin, p53, c-Myc), while for others comprehensive expression data, mode of dysregulation, and functional effects in HCC cells are largely missing (e.g., HOX family members).
      More importantly, these data suggest that the coordinated activation of transcriptional regulators is a frequent characteristic of more aggressive HCCs (high proliferation, dedifferentiation, large tumor size, poor survival), while it is not a key feature of less aggressive HCCs. These signatures are more than surrogate markers of specific signalling pathways dysregulated in HCC, since their activation may be regulated by very different mechanisms and partly by signalling pathway-independent stimuli (e.g., E2F overexpression due to genomic gains, aberrant micro-RNA expression, and loss of Rb function).
      One limitation is that current expression profiling analysis may consider genomic, transcriptomic, and (to a lesser extent) proteomic information; however, the activity of transcriptional regulators not only is defined by their expression but also by their subcellular distribution. Although some studies linked the dysregulation of specific factors with their cellular distribution (e.g., β-catenin [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ]), a comprehensive analysis integrating mRNA and protein levels together with subcellular localization is missing so far. Some potentially relevant factors such as NF-kB, FBPs, STATs, YAP, and SMAD2/4 have not shown up in any of the profiling studies so far, since they are only moderately regulated or their oncogenic activation is not dysregulated at the expression level. In these cases, mutations or post-transcriptional/translational regulation and modification may account for aberrant accumulation of transcriptional regulators. It would be interesting to see whether activation or nuclear localization of these factors is more or less frequent in subgroups of HCCs with specific gene signatures.

      Implications for targeted therapy

      The differential diagnostic and clinical relevance of transcription factors and co-factors in hepatocarcinogenesis was supported by different profiling approaches partly integrating genomic and transcriptomic data. These studies not only defined subgroups of HCCs with characteristic clinical and biological features but also allocated aberrant activation/expression of signature transcriptional regulators (β-catenin, AP-1, HIF1, c-Myc, b-Myb, E2F, and p53) to HCCs with a more aggressive phenotype [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ,
      • Lee J.S.
      • Chu I.S.
      • Heo J.
      • Calvisi D.F.
      • Sun Z.
      • Roskams T.
      • et al.
      Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.
      ,
      • Lee J.S.
      • Heo J.
      • Libbrecht L.
      • Chu I.S.
      • Kaposi-Novak P.
      • Calvisi D.F.
      • et al.
      A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
      ,
      • Laurent-Puig P.
      • Legoix P.
      • Bluteau O.
      • Belghiti J.
      • Franco D.
      • Binot F.
      • et al.
      Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
      ]. These observations have further substantiated the integrative character of transcriptional regulators and their ability to incorporate independent stimuli into a final oncogenic stimulus. It also implicates that dysregulation of these factors may put their stamps on the expression profiles and the biology of specific HCC subgroups – this qualifies these factors as potential molecular markers.
      In highly heterogeneous carcinomas, broader inhibition of oncogenic mechanisms may turn out to be more efficient compared to highly specific blocking of single pathways components such as receptor tyrosine kinases [
      • Villanueva A.
      • Minguez B.
      • Forner A.
      • Reig M.
      • Llovet J.M.
      Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy.
      ]. In principle, transcriptional regulators may represent attractive target structures in cancer therapy, since their activity integrates the activation of many pathways representing the bottleneck and final instance before transcriptional activation results in an oncogenic phenotype. However, targeting transcription factors in cancer raises important issues:
      • (1)
        Although global inhibition of transcription factors may cause acceptable unwanted effects [
        • Frank D.A.
        Targeting transcription factors for cancer therapy.
        ], tumor cell-specific targeting is preferable. Systemic approaches might cause severe side effects under specific physiological conditions (e.g., regeneration).
      • (2)
        At least in some cases, targeting approaches have to discriminate between structurally related proteins, since different family members facilitate distinct biological features (e.g., AP-1 subunits). Blocking entire protein families may lead to uncontrolled and even opposite effects.
      • (3)
        Transcriptional regulators lack enzymatic activity; thus, protein-directed inhibitors may have to disturb large surface protein/protein or protein/DNA interaction structures (e.g., c-Myc/Max heterodimers) posing additional challenge, e.g., on lead substance optimization.
      Despite these obstacles, successful methods targeting oncogenic transcription factors’ activity have been developed leading to promising strategies (Fig. 1), which do not rely on the pharmacological inhibition of upstream regulators (e.g., receptor tyrosine kinase inhibitors [
      • Villanueva A.
      • Minguez B.
      • Forner A.
      • Reig M.
      • Llovet J.M.
      Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy.
      ]) or virus-mediated re-expression of pathway antagonists that are silenced in HCC (e.g., the WNT pathway antagonist Wst-1 [
      • Hu J.
      • Dong A.
      • Fernandez-Ruiz V.
      • Shan J.
      • Kawa M.
      • Martinez-Anso E.
      • et al.
      Blockade of Wnt signaling inhibits angiogenesis and tumor growth in hepatocellular carcinoma.
      ,
      • Huang L.
      • Li M.X.
      • Wang L.
      • Li B.K.
      • Chen G.H.
      • He L.R.
      • et al.
      Prognostic value of Wnt inhibitory factor-1 expression in hepatocellular carcinoma that is independent of gene methylation.
      ]).
      Figure thumbnail gr1
      Fig. 1Modes of dysregulation of transcriptional regulators (TR) in human HCC cells (red) including mutations of upstream signalling pathway components (e.g., receptors and pathway constituents), mutations, and genomic alterations. Possibilities of pathway interference (blue) include the inhibition of receptor tyrosine kinase (RTK) inhibitors, expression of pathway antagonists, and inhibitors of TR-dimerization and TR/DNA interaction. Virotherapy links TRs activity with viral replication and oncolysis.

      Chemical compounds targeting transcription factor activity and expression

      Some methodological approaches utilize small organic molecules that specifically inhibit the interaction between transcription factors and their respective DNA-binding domains. In addition, several transcription factors form hetero- or homodimers (e.g., AP-1, Myc/Max, HIF-1α/HIF-1β) as well as multimers (e.g., p53) in order to recognize and bind their respective DNA binding sites. Disturbing these protein/protein interactions by specific substances may destabilise transcriptional complex assembly and thereby inhibit dysfunctional transcriptional activity.
      For both approaches, low-molecular-weight compounds should overcome high-affinity interaction between transcription factors and their DNA recognition motif or protein/protein interfaces. For many transcriptional regulators, various pharmacological substances have been developed and tested in different in vitro and/or in vivo cancer models. These include compounds targeting c-Myc/Max (e.g., substance 10058-F4 [
      • Lin C.P.
      • Liu C.R.
      • Lee C.N.
      • Chan T.S.
      • Liu H.E.
      Targeting c-Myc as a novel approach for hepatocellular carcinoma.
      ]), FBP (compound 1 [
      • Huth J.R.
      • Yu L.
      • Collins I.
      • Mack J.
      • Mendoza R.
      • Isaac B.
      • et al.
      NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.
      ]), STAT3 (e.g., ISS 610 [
      • Turkson J.
      • Kim J.S.
      • Zhang S.
      • Yuan J.
      • Huang M.
      • Glenn M.
      • et al.
      Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity.
      ,
      • Coleman D.R.t.
      • Ren Z.
      • Mandal P.K.
      • Cameron A.G.
      • Dyer G.A.
      • Muranjan S.
      • et al.
      Investigation of the binding determinants of phosphopeptides targeted to the SRC homology 2 domain of the signal transducer and activator of transcription 3. Development of a high-affinity peptide inhibitor.
      ]), and β-catenin/TCF (e.g., PKF115–584 [
      • Lepourcelet M.
      • Chen Y.N.
      • France D.S.
      • Wang H.
      • Crews P.
      • Petersen F.
      • et al.
      Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex.
      ]). Some of these substances have already been successfully tested using HCC cells (e.g., 10058-F4 [
      • Lin C.P.
      • Liu J.D.
      • Chow J.M.
      • Liu C.R.
      • Liu H.E.
      Small-molecule c-Myc inhibitor, 10058-F4, inhibits proliferation, downregulates human telomerase reverse transcriptase and enhances chemosensitivity in human hepatocellular carcinoma cells.
      ]).
      For some factors, such as HIF-1, for which the design of specific drugs inhibiting protein/DNA or protein/protein interaction has been less successful (reviewed in [
      • Onnis B.
      • Rapisarda A.
      • Melillo G.
      Development of HIF-1 inhibitors for cancer therapy.
      ]), the lack of specificity of the employed substances favoured approaches that indirectly regulate HIF-1 concentrations, e,g. compounds that affect HIF-1 expression (transcription and translation) and stability (e.g., YC-1 [
      • Yeo E.J.
      • Chun Y.S.
      • Cho Y.S.
      • Kim J.
      • Lee J.C.
      • Kim M.S.
      • et al.
      YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.
      ]).
      Because most p53 mutations represent missense mutations affecting its DNA binding domain, pharmacological reactivation of p53mut isoforms may induce apoptosis and senescence of tumor cells. Screening of chemical libraries and rational design of substances that stabilize the wild type conformation of mutated p53 have led to the identification of small molecules that bind p53mut and restore its tumor suppressive function (e.g., PhiKan083 and PRIMA-1; reviewed in [
      • Essmann F.
      • Schulze-Osthoff K.
      Translational approaches targeting the p53 pathway for anticancer therapy.
      ]). In addition, reconstitution of wild type p53 activity in tumors without p53 mutations (but e.g., with MDM2 overexpression) was achieved by substances that block MDM2-dependent degradation of p53 (e.g., Nutlin and MI-219 [
      • Shangary S.
      • Qin D.
      • McEachern D.
      • Liu M.
      • Miller R.S.
      • Qiu S.
      • et al.
      Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition.
      ,
      • Vassilev L.T.
      • Vu B.T.
      • Graves B.
      • Carvajal D.
      • Podlaski F.
      • Filipovic Z.
      • et al.
      In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.
      ]). Many of these substances have already been tested in HCC models (e.g., PRIMA-1 [
      • Shi H.
      • Lambert J.M.
      • Hautefeuille A.
      • Bykov V.J.
      • Wiman K.G.
      • Hainaut P.
      • et al.
      In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249.
      ], Nutlin [
      • Lee Y.M.
      • Lim J.H.
      • Chun Y.S.
      • Moon H.E.
      • Lee M.K.
      • Huang L.E.
      • et al.
      Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1alpha.
      ], 10058-F4 [
      • Lin C.P.
      • Liu J.D.
      • Chow J.M.
      • Liu C.R.
      • Liu H.E.
      Small-molecule c-Myc inhibitor, 10058-F4, inhibits proliferation, downregulates human telomerase reverse transcriptase and enhances chemosensitivity in human hepatocellular carcinoma cells.
      ], and YC-1 [
      • Yeo E.J.
      • Chun Y.S.
      • Cho Y.S.
      • Kim J.
      • Lee J.C.
      • Kim M.S.
      • et al.
      YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.
      ]) showing promising preclinical anti-tumorigenic effects. In case of Nutlin, a first clinical trial for the treatment of retinoblastoma has been initiated.

      Gene therapeutic approaches

      Both retroviral and adenoviral approaches have been established for the delivery of proteins and peptides in tumor cells. However, because genomic retrovirus integration is believed to increase the risk of tumor development in patients, most approaches focussed on adenovirus-based strategies. One example of the successful application of this method is the restoration of p53wt function in tumor cells. Replication-impaired, non-integrating adenoviral vectors carrying p53 under the control of the CMV promoter has been approved in China (trademarked as Gendicine) and used for the treatment of head and neck cancer [
      • Guo J.
      • Xin H.
      Chinese gene therapy. Splicing out the West?.
      ]. Although not approved by the FDA, a recent report from China demonstrated that combination of Gendicine with radiotherapy showed high response rate and increased survival in HCC patients [
      • Yang Z.X.
      • Wang D.
      • Wang G.
      • Zhang Q.H.
      • Liu J.M.
      • Peng P.
      • et al.
      Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma.
      ].
      Oncolytic virotherapeutics link the loss of p53 activity with anti-neoplastic viral replication [
      • Scheffner M.
      • Werness B.A.
      • Huibregtse J.M.
      • Levine A.J.
      • Howley P.M.
      The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.
      ]. Since adenoviral E1B inhibits p53wt activity and prevents infected cells from undergoing apoptosis, the infection with E1B-deficient viruses should selectively kill p53mut or p53del tumor cells as employed in the genetically engineered adenovirus Onyx-015 (with E1B-55K deletion), which replicates specifically in p53 dysfunctional cells [
      • Bischoff J.R.
      • Kirn D.H.
      • Williams A.
      • Heise C.
      • Horn S.
      • Muna M.
      • et al.
      An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.
      ]. Although first trials demonstrated that Onyx-015 was tolerated well, and in combination with chemotherapy resulted in some anti-tumoral activity [
      • Reid T.
      • Galanis E.
      • Abbruzzese J.
      • Sze D.
      • Wein L.M.
      • Andrews J.
      • et al.
      Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral, immunologic, and clinical endpoints.
      ], other studies casted doubts about virus-specificity and therefore challenged the applicability of E1B-55K/p53-dependent virotherapy [
      • Hall A.R.
      • Dix B.R.
      • O’Carroll S.J.
      • Braithwaite A.W.
      P53-dependent cell death/apoptosis is required for a productive adenovirus infection.
      ]. In 2005, H101 was approved in China (trademarked as Oncorine) which showed a high degree of similarity with Onyx-015 [
      • Guo J.
      • Xin H.
      Chinese gene therapy. Splicing out the West?.
      ]. A recent HCC case report demonstrated beneficial effects in a patient with recurrent HCC after administration of TACE and H101 [
      • He Q.
      • Liu Y.
      • Zou Q.
      • Guan Y.S.
      Transarterial injection of H101 in combination with chemoembolization overcomes recurrent hepatocellular carcinoma.
      ].
      Recently, p53-dependent oncolytic viruses (Adp53sensor) without any alterations in the E1B gene have been designed; here p53mut or p53del selectivity was achieved by chemotherapy-induced and p53-dependent expression of the transcriptional repressor Gal4-KRAB, which itself inhibits viral E1A gene expression [
      • Kuhnel F.
      • Gurlevik E.
      • Wirth T.C.
      • Struver N.
      • Malek N.P.
      • Muller-Schilling M.
      • et al.
      Targeting of p53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by p53-homologues.
      ]. This system allowed efficient viral replication in p53 dysfunctional tumor cells but not in cells with p53wt in vitro as well as in vivo and showed a favourable liver toxicity profile in animals. Importantly, p63 and p73 isoforms were not sufficient to compromise Adp53sensor replication, demonstrating that this approach was not restricted by other p53 family members in HCC cells [
      • Kuhnel F.
      • Gurlevik E.
      • Wirth T.C.
      • Struver N.
      • Malek N.P.
      • Muller-Schilling M.
      • et al.
      Targeting of p53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by p53-homologues.
      ]. Thus, novel virotherapeutic strategies based on p53 integrity may overcome limitations of previous oncolytic approaches and some studies suggested that HCC cells are promising targets [
      • Chen W.
      • Wu Y.
      • Liu W.
      • Wang G.
      • Wang X.
      • Yang Y.
      • et al.
      Enhanced antitumor efficacy of a novel fiber chimeric oncolytic adenovirus expressing p53 on hepatocellular carcinoma.
      ,
      • Xue X.B.
      • Xiao C.W.
      • Zhang H.
      • Lu A.G.
      • Gao W.
      • Zhou Z.Q.
      • et al.
      Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines.
      ].
      Because this methodological approach links anti-tumorigenic viral activity with the activity of oncogenic transcriptional regulators and does not rely on the perturbation of protein/protein and protein/DNA interaction, it represents a promising tool for targeting non-druggable factors. Indeed, different studies demonstrated the applicability of this method in cells with aberrant STAT3 [
      • Han Z.
      • Hong Z.
      • Chen C.
      • Gao Q.
      • Luo D.
      • Fang Y.
      • et al.
      A novel oncolytic adenovirus selectively silences the expression of tumor-associated STAT3 and exhibits potent antitumoral activity.
      ], HIF-1α [
      • Cherry T.
      • Longo S.L.
      • Tovar-Spinoza Z.
      • Post D.E.
      Second-generation HIF-activated oncolytic adenoviruses with improved replication, oncolytic, and antitumor efficacy.
      ,
      • Post D.E.
      • Van Meir E.G.
      A novel hypoxia-inducible factor (HIF) activated oncolytic adenovirus for cancer therapy.
      ], E2F [
      • Jakubczak J.L.
      • Ryan P.
      • Gorziglia M.
      • Clarke L.
      • Hawkins L.K.
      • Hay C.
      • et al.
      An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy.
      ], and β-catenin [
      • Dvory-Sobol H.
      • Sagiv E.
      • Kazanov D.
      • Ben-Ze’ev A.
      • Arber N.
      Targeting the active beta-catenin pathway to treat cancer cells.
      ] activity. In case of E2F, oncolytic adenoviruses using the tumor-specific E2F1 promoter have successfully been tested in HCC cells [
      • Jakubczak J.L.
      • Ryan P.
      • Gorziglia M.
      • Clarke L.
      • Hawkins L.K.
      • Hay C.
      • et al.
      An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy.
      ].

      Outlook

      Based on genomic and transcriptomic information on human HCC, a defined number of transcriptional regulators, which are predominantly activated in a group of phenotypically aggressive HCCs, have been identified. Despite methodological limitations, for some of these factors, first perturbation strategies have been successfully established and tested in HCC; however, in order to improve their applicability and to avoid the development of resistance in tumor cells, more transcriptional regulators have to be made druggable. In this regard, it will be important to overcome the increased challenges of lead substance optimization imposed by the lack of active enzymatic centres in transcription factors and transcriptional co-factors. Optimized tumor cell targeting will probably further improve the activity and specificity of anti-tumoral viruses.
      It is worth mentioning, that previous profiling data suggested the existence of HCC derived from hepatic progenitor cells/stem cells of the liver [
      • Lee J.S.
      • Heo J.
      • Libbrecht L.
      • Chu I.S.
      • Kaposi-Novak P.
      • Calvisi D.F.
      • et al.
      A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
      ], and it is now believed that about 40% of all HCCs are of clonal origin and therefore potentially arise from progenitor/stem cells [
      • Mishra L.
      • Banker T.
      • Murray J.
      • Byers S.
      • Thenappan A.
      • He A.R.
      • et al.
      Liver stem cells and hepatocellular carcinoma.
      ]. These cells perpetuate themselves through self-renewal and are responsible for tumor formation and progression through activation of distinct pathways including, e.g., TGFβ-/IL6-, Notch-, and Hedgehog signalling [
      • Oishi N.
      • Wang X.W.
      Novel therapeutic strategies for targeting liver cancer stem cells.
      ,
      • Yao Z.
      • Mishra L.
      Cancer stem cells and hepatocellular carcinoma.
      ]. In addition, transcriptional regulators and pathways identified by functional genomics such as Wnt/β-catenin and c-Myc are central regulators of stemness. Therefore, targeting additional transcriptional regulators (e.g., SMADs, NICD, or Gli) might cover the whole field of potential targets in terms of pathways and specific hepatocytic cell types.

      Financial support

      Research on HCC by P.S. and K.B. has been sponsored by the German Research Foundation (SFB/TRR77), the Helmholtz Alliance on Immunotherapy of Cancer, and the BMBF (Virtual Liver).

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      References

        • Breuhahn K.
        • Gores G.
        • Schirmacher P.
        Strategies for hepatocellular carcinoma therapy and diagnostics: lessons learned from high throughput and profiling approaches.
        Hepatology. 2011; 53: 2112-2121
        • Llovet J.M.
        • Ricci S.
        • Mazzaferro V.
        • Hilgard P.
        • Gane E.
        • Blanc J.F.
        • et al.
        Sorafenib in advanced hepatocellular carcinoma.
        N Engl J Med. 2008; 359: 378-390
        • Boyault S.
        • Rickman D.S.
        • de Reynies A.
        • Balabaud C.
        • Rebouissou S.
        • Jeannot E.
        • et al.
        Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
        Hepatology. 2007; 45: 42-52
        • Hoshida Y.
        • Nijman S.M.
        • Kobayashi M.
        • Chan J.A.
        • Brunet J.P.
        • Chiang D.Y.
        • et al.
        Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
        Cancer Res. 2009; 69: 7385-7392
        • Kaposi-Novak P.
        • Lee J.S.
        • Gomez-Quiroz L.
        • Coulouarn C.
        • Factor V.M.
        • Thorgeirsson S.S.
        Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype.
        J Clin Invest. 2006; 116: 1582-1595
        • Villanueva A.
        • Minguez B.
        • Forner A.
        • Reig M.
        • Llovet J.M.
        Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy.
        Annu Rev Med. 2010; 61: 317-328
        • Morse R.H.
        Transcription factor access to promoter elements.
        J Cell Biochem. 2007; 102: 560-570
        • Baumann M.
        • Pontiller J.
        • Ernst W.
        Structure and basal transcription complex of RNA polymerase II core promoters in the mammalian genome: an overview.
        Mol Biotechnol. 2010; 45: 241-247
        • Brivanlou A.H.
        • Darnell Jr., J.E.
        Signal transduction and the control of gene expression.
        Science. 2002; 295: 813-818
        • Juven-Gershon T.
        • Kadonaga J.T.
        Regulation of gene expression via the core promoter and the basal transcriptional machinery.
        Dev Biol. 2010; 339: 225-229
        • MacQuarrie K.L.
        • Fong A.P.
        • Morse R.H.
        • Tapscott S.J.
        Genome-wide transcription factor binding: beyond direct target regulation.
        Trends Genet. 2011; 27: 141-148
        • Malz M.
        • Weber A.
        • Singer S.
        • Riehmer V.
        • Bissinger M.
        • Riener M.O.
        • et al.
        Overexpression of far upstream element binding proteins: a mechanism regulating proliferation and migration in liver cancer cells.
        Hepatology. 2009; 50: 1130-1139
        • Dong J.
        • Feldmann G.
        • Huang J.
        • Wu S.
        • Zhang N.
        • Comerford S.A.
        • et al.
        Elucidation of a universal size-control mechanism in Drosophila and mammals.
        Cell. 2007; 130: 1120-1133
        • Calvisi D.F.
        • Ladu S.
        • Gorden A.
        • Farina M.
        • Conner E.A.
        • Lee J.S.
        • et al.
        Ubiquitous activation of Ras and Jak/Stat pathways in human HCC.
        Gastroenterology. 2006; 130: 1117-1128
        • Longerich T.
        • Breuhahn K.
        • Odenthal M.
        • Petmecky K.
        • Schirmacher P.
        Factors of transforming growth factor beta signalling are co-regulated in human hepatocellular carcinoma.
        Virchows Arch. 2004; 445: 589-596
        • O’Neil B.H.
        • Buzkova P.
        • Farrah H.
        • Kashatus D.
        • Sanoff H.
        • Goldberg R.M.
        • et al.
        Expression of nuclear factor-kappaB family proteins in hepatocellular carcinomas.
        Oncology. 2007; 72: 97-104
        • Calvisi D.F.
        • Pinna F.
        • Ladu S.
        • Pellegrino R.
        • Simile M.M.
        • Frau M.
        • et al.
        Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC.
        Gut. 2009; 58: 679-687
        • Gho J.W.
        • Ip W.K.
        • Chan K.Y.
        • Law P.T.
        • Lai P.B.
        • Wong N.
        Re-expression of transcription factor ATF5 in hepatocellular carcinoma induces G2-M arrest.
        Cancer Res. 2008; 68: 6743-6751
        • Conner E.A.
        • Lemmer E.R.
        • Omori M.
        • Wirth P.J.
        • Factor V.M.
        • Thorgeirsson S.S.
        Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis.
        Oncogene. 2000; 19: 5054-5062
        • Lee J.S.
        • Chu I.S.
        • Mikaelyan A.
        • Calvisi D.F.
        • Heo J.
        • Reddy J.K.
        • et al.
        Application of comparative functional genomics to identify best-fit mouse models to study human cancer.
        Nat Genet. 2004; 36: 1306-1311
        • Lee J.S.
        • Chu I.S.
        • Heo J.
        • Calvisi D.F.
        • Sun Z.
        • Roskams T.
        • et al.
        Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.
        Hepatology. 2004; 40: 667-676
        • Lee J.S.
        • Heo J.
        • Libbrecht L.
        • Chu I.S.
        • Kaposi-Novak P.
        • Calvisi D.F.
        • et al.
        A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
        Nat Med. 2006; 12: 410-416
        • Kaposi-Novak P.
        • Libbrecht L.
        • Woo H.G.
        • Lee Y.H.
        • Sears N.C.
        • Coulouarn C.
        • et al.
        Central role of c-Myc during malignant conversion in human hepatocarcinogenesis.
        Cancer Res. 2009; 69: 2775-2782
        • Cairo S.
        • Wang Y.
        • de Reynies A.
        • Duroure K.
        • Dahan J.
        • Redon M.J.
        • et al.
        Stem cell-like micro-RNA signature driven by Myc in aggressive liver cancer.
        Proc Natl Acad Sci USA. 2010; 107: 20471-20476
        • Chen X.
        • Cheung S.T.
        • So S.
        • Fan S.T.
        • Barry C.
        • Higgins J.
        • et al.
        Gene expression patterns in human liver cancers.
        Mol Biol Cell. 2002; 13: 1929-1939
        • Laurent-Puig P.
        • Legoix P.
        • Bluteau O.
        • Belghiti J.
        • Franco D.
        • Binot F.
        • et al.
        Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
        Gastroenterology. 2001; 120: 1763-1773
        • Cillo C.
        • Schiavo G.
        • Cantile M.
        • Bihl M.P.
        • Sorrentino P.
        • Carafa V.
        • et al.
        The HOX gene network in hepatocellular carcinoma.
        Int J Cancer. 2011; 129: 2577-2587
        • Eilers M.
        • Eisenman R.N.
        Myc’s broad reach.
        Genes Dev. 2008; 22: 2755-2766
        • Prange W.
        • Breuhahn K.
        • Fischer F.
        • Zilkens C.
        • Pietsch T.
        • Petmecky K.
        • et al.
        Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes.
        J Pathol. 2003; 201: 250-259
        • Saegusa M.
        • Takano Y.
        • Kishimoto H.
        • Wakabayashi G.
        • Nohga K.
        • Okudaira M.
        Comparative analysis of p53 and c-myc expression and cell proliferation in human hepatocellular carcinomas – an enhanced immunohistochemical approach.
        J Cancer Res Clin Oncol. 1993; 119: 737-744
        • Wang Y.
        • Wu M.C.
        • Sham J.S.
        • Zhang W.
        • Wu W.Q.
        • Guan X.Y.
        Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad survey using high-throughput tissue microarray.
        Cancer. 2002; 95: 2346-2352
        • Abou-Elella A.
        • Gramlich T.
        • Fritsch C.
        • Gansler T.
        C-myc amplification in hepatocellular carcinoma predicts unfavorable prognosis.
        Mod Pathol. 1996; 9: 95-98
        • Schlaeger C.
        • Longerich T.
        • Schiller C.
        • Bewerunge P.
        • Mehrabi A.
        • Toedt G.
        • et al.
        Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.
        Hepatology. 2008; 47: 511-520
        • Balsano C.
        • Avantaggiati M.L.
        • Natoli G.
        • De Marzio E.
        • Will H.
        • Perricaudet M.
        • et al.
        Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cmyc protooncogene at the transcriptional level.
        Biochem Biophys Res Commun. 1991; 176: 985-992
        • Ma H.C.
        • Lin T.W.
        • Li H.
        • Iguchi-Ariga S.M.
        • Ariga H.
        • Chuang Y.L.
        • et al.
        Hepatitis C virus ARFP/F protein interacts with cellular MM-1 protein and enhances the gene trans-activation activity of c-Myc.
        J Biomed Sci. 2008; 15: 417-425
        • Sala A.
        • Saitta B.
        • De Luca P.
        • Cervellera M.N.
        • Casella I.
        • Lewis R.E.
        • et al.
        B-MYB transactivates its own promoter through SP1-binding sites.
        Oncogene. 1999; 18: 1333-1339
        • Zhu W.
        • Giangrande P.H.
        • Nevins J.R.
        E2Fs link the control of G1/S and G2/M transcription.
        EMBO J. 2004; 23: 4615-4626
        • Nakajima T.
        • Yasui K.
        • Zen K.
        • Inagaki Y.
        • Fujii H.
        • Minami M.
        • et al.
        Activation of B-Myb by E2F1 in hepatocellular carcinoma.
        Hepatol Res. 2008; 38: 886-895
        • Frau M.
        • Ladu S.
        • Calvisi D.F.
        • Simile M.M.
        • Bonelli P.
        • Daino L.
        • et al.
        Mybl2 expression is under genetic control and contributes to determine a hepatocellular carcinoma susceptible phenotype.
        J Hepatol. 2011; 55: 111-119
        • Calvisi D.F.
        • Simile M.M.
        • Ladu S.
        • Frau M.
        • Evert M.
        • Tomasi M.L.
        • et al.
        Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53.
        Hepatology. 2011; 53: 1226-1236
        • Gavert N.
        • Ben-Ze’ev A.
        Beta-catenin signaling in biological control and cancer.
        J Cell Biochem. 2007; 102: 820-828
        • Breuhahn K.
        • Longerich T.
        • Schirmacher P.
        Dysregulation of growth factor signaling in human hepatocellular carcinoma.
        Oncogene. 2006; 25: 3787-3800
        • Taniguchi K.
        • Roberts L.R.
        • Aderca I.N.
        • Dong X.
        • Qian C.
        • Murphy L.M.
        • et al.
        Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas.
        Oncogene. 2002; 21: 4863-4871
        • Chan D.W.
        • Chan C.Y.
        • Yam J.W.
        • Ching Y.P.
        • Ng I.O.
        Prickle-1 negatively regulates Wnt/beta-catenin pathway by promoting dishevelled ubiquitination/degradation in liver cancer.
        Gastroenterology. 2006; 131: 1218-1227
        • Bengochea A.
        • de Souza M.M.
        • Lefrancois L.
        • Le Roux E.
        • Galy O.
        • Chemin I.
        • et al.
        Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma.
        Br J Cancer. 2008; 99: 143-150
        • Lea I.A.
        • Jackson M.A.
        • Li X.
        • Bailey S.
        • Peddada S.D.
        • Dunnick J.K.
        Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns.
        Carcinogenesis. 2007; 28: 1851-1858
        • Csepregi A.
        • Rocken C.
        • Hoffmann J.
        • Gu P.
        • Saliger S.
        • Muller O.
        • et al.
        APC promoter methylation and protein expression in hepatocellular carcinoma.
        J Cancer Res Clin Oncol. 2008; 134: 579-589
        • Legoix P.
        • Bluteau O.
        • Bayer J.
        • Perret C.
        • Balabaud C.
        • Belghiti J.
        • et al.
        Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.
        Oncogene. 1999; 18: 4044-4046
        • Schmitt-Graeff A.
        • Ertelt-Heitzmann V.
        • Allgaier H.P.
        • Olschewski M.
        • Nitschke R.
        • Haxelmans S.
        • et al.
        Coordinated expression of cyclin D1 and LEF-1/TCF transcription factor is restricted to a subset of hepatocellular carcinoma.
        Liver Int. 2005; 25: 839-847
        • Huang H.
        • Fujii H.
        • Sankila A.
        • Mahler-Araujo B.M.
        • Matsuda M.
        • Cathomas G.
        • et al.
        Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection.
        Am J Pathol. 1999; 155: 1795-1801
        • Wong C.M.
        • Fan S.T.
        • Ng I.O.
        Beta-catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance.
        Cancer. 2001; 92: 136-145
        • Lian Z.
        • Liu J.
        • Li L.
        • Li X.
        • Clayton M.
        • Wu M.C.
        • et al.
        Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with upregulation of beta-catenin.
        Hepatology. 2006; 43: 415-424
        • Hess J.
        • Angel P.
        • Schorpp-Kistner M.
        AP-1 subunits: quarrel and harmony among siblings.
        J Cell Sci. 2004; 117: 5965-5973
        • Eferl R.
        • Wagner E.F.
        AP-1: a double-edged sword in tumorigenesis.
        Nat Rev Cancer. 2003; 3: 859-868
        • Arbuthnot P.
        • Kew M.
        • Fitschen W.
        c-fos and c-myc oncoprotein expression in human hepatocellular carcinomas.
        Anticancer Res. 1991; 11: 921-924
        • Chang Y.S.
        • Yeh K.T.
        • Yang M.Y.
        • Liu T.C.
        • Lin S.F.
        • Chan W.L.
        • et al.
        Abnormal expression of JUNB gene in hepatocellular carcinoma.
        Oncol Rep. 2005; 13: 433-438
        • Yuen M.F.
        • Wu P.C.
        • Lai V.C.
        • Lau J.Y.
        • Lai C.L.
        Expression of c-Myc, c-Fos, and c-jun in hepatocellular carcinoma.
        Cancer. 2001; 91: 106-112
        • Guo C.
        • Liu Q.G.
        • Zhang L.
        • Song T.
        • Yang X.
        Expression and clinical significance of p53, JunB and KAI1/CD82 in human hepatocellular carcinoma.
        Hepatobiliary Pancreat Dis Int. 2009; 8: 389-396
        • Liu P.
        • Kimmoun E.
        • Legrand A.
        • Sauvanet A.
        • Degott C.
        • Lardeux B.
        • et al.
        Activation of NF-kappa B, AP-1 and STAT transcription factors is a frequent and early event in human hepatocellular carcinomas.
        J Hepatol. 2002; 37: 63-71
        • Hui L.
        • Zatloukal K.
        • Scheuch H.
        • Stepniak E.
        • Wagner E.F.
        Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation.
        J Clin Invest. 2008; 118: 3943-3953
        • Chang Q.
        • Chen J.
        • Beezhold K.J.
        • Castranova V.
        • Shi X.
        • Chen F.
        JNK1 activation predicts the prognostic outcome of the human hepatocellular carcinoma.
        Mol Cancer. 2009; 8: 64
        • Moinzadeh P.
        • Breuhahn K.
        • Stutzer H.
        • Schirmacher P.
        Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade – results of an explorative CGH meta-analysis.
        Br J Cancer. 2005; 92: 935-941
        • Feitelson M.A.
        • Duan L.X.
        Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma.
        Am J Pathol. 1997; 150: 1141-1157
        • Lasky T.
        • Magder L.
        Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure?.
        Environ Health Perspect. 1997; 105: 392-397
        • Ozturk M.
        P53 mutation in hepatocellular carcinoma after aflatoxin exposure.
        Lancet. 1991; 338: 1356-1359
        • Lee M.K.
        • Sabapathy K.
        The R246S hot-spot p53 mutant exerts dominant-negative effects in embryonic stem cells in vitro and in vivo.
        J Cell Sci. 2008; 121: 1899-1906
        • Oren M.
        • Rotter V.
        Mutant p53 gain-of-function in cancer.
        Cold Spring Harb Perspect Biol. 2010; 2: a001107
        • Higashitsuji H.
        • Itoh K.
        • Sakurai T.
        • Nagao T.
        • Sumitomo Y.
        • Masuda T.
        • et al.
        The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53.
        Cancer Cell. 2005; 8: 75-87
        • Gaiddon C.
        • Lokshin M.
        • Ahn J.
        • Zhang T.
        • Prives C.
        A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain.
        Mol Cell Biol. 2001; 21: 1874-1887
        • Schilling T.
        • Kairat A.
        • Melino G.
        • Krammer P.H.
        • Stremmel W.
        • Oren M.
        • et al.
        Interference with the p53 family network contributes to the gain of oncogenic function of mutant p53 in hepatocellular carcinoma.
        Biochem Biophys Res Commun. 2010; 394: 817-823
        • Fandrey J.
        • Gassmann M.
        Oxygen sensing and the activation of the hypoxia inducible factor 1 (HIF-1) – invited article.
        Adv Exp Med Biol. 2009; 648: 197-206
        • Keith B.
        • Simon M.C.
        Hypoxia-inducible factors, stem cells, and cancer.
        Cell. 2007; 129: 465-472
        • Huang G.W.
        • Yang L.Y.
        • Lu W.Q.
        Expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in hepatocellular carcinoma: impact on neovascularization and survival.
        World J Gastroenterol. 2005; 11: 1705-1708
        • Simon F.
        • Bockhorn M.
        • Praha C.
        • Baba H.A.
        • Broelsch C.E.
        • Frilling A.
        • et al.
        Deregulation of HIF1-alpha and hypoxia-regulated pathways in hepatocellular carcinoma and corresponding non-malignant liver tissue – influence of a modulated host stroma on the prognosis of HCC.
        Langenbecks Arch Surg. 2010; 395: 395-405
        • Tacchini L.
        • Dansi P.
        • Matteucci E.
        • Desiderio M.A.
        Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells.
        Carcinogenesis. 2001; 22: 1363-1371
        • Lee T.K.
        • Poon R.T.
        • Yuen A.P.
        • Man K.
        • Yang Z.F.
        • Guan X.Y.
        • et al.
        Rac activation is associated with hepatocellular carcinoma metastasis by up-regulation of vascular endothelial growth factor expression.
        Clin Cancer Res. 2006; 12: 5082-5089
        • Ripoli M.
        • D’Aprile A.
        • Quarato G.
        • Sarasin-Filipowicz M.
        • Gouttenoire J.
        • Scrima R.
        • et al.
        Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation.
        J Virol. 2010; 84: 647-660
        • Fu J.
        • Chen Y.
        • Cao J.
        • Luo T.
        • Qian Y.W.
        • Yang W.
        • et al.
        P28GANK overexpression accelerates hepatocellular carcinoma invasiveness and metastasis via phosphoinositol 3-kinase/AKT/hypoxia-inducible factor-1alpha pathways.
        Hepatology. 2011; 53: 181-192
        • van den Heuvel S.
        • Dyson N.J.
        Conserved functions of the pRB and E2F families.
        Nat Rev Mol Cell Biol. 2008; 9: 713-724
        • Deng Q.
        • Wang Q.
        • Zong W.Y.
        • Zheng D.L.
        • Wen Y.X.
        • Wang K.S.
        • et al.
        E2F8 contributes to human hepatocellular carcinoma via regulating cell proliferation.
        Cancer Res. 2010; 70: 782-791
        • Xu T.
        • Zhu Y.
        • Xiong Y.
        • Ge Y.Y.
        • Yun J.P.
        • Zhuang S.M.
        MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells.
        Hepatology. 2009; 50: 113-121
        • Choi B.H.
        • Choi M.
        • Jeon H.Y.
        • Rho H.M.
        Hepatitis B viral X protein overcomes inhibition of E2F1 activity by pRb on the human Rb gene promoter.
        DNA Cell Biol. 2001; 20: 75-80
        • Park Y.M.
        • Choi J.Y.
        • Bae S.H.
        • Byun B.H.
        • Ahn B.M.
        • Kim B.S.
        • et al.
        Microsatellite instability and mutations of E2F-4 in hepatocellular carcinoma from Korea.
        Hepatol Res. 2000; 17: 102-111
        • Midorikawa Y.
        • Tsutsumi S.
        • Nishimura K.
        • Kamimura N.
        • Kano M.
        • Sakamoto H.
        • et al.
        Distinct chromosomal bias of gene expression signatures in the progression of hepatocellular carcinoma.
        Cancer Res. 2004; 64: 7263-7270
        • Zacchetti G.
        • Duboule D.
        • Zakany J.
        Hox gene function in vertebrate gut morphogenesis: the case of the caecum.
        Development. 2007; 134: 3967-3973
        • Shah N.
        • Sukumar S.
        The Hox genes and their roles in oncogenesis.
        Nat Rev Cancer. 2010; 10: 361-371
        • Kanai M.
        • Hamada J.
        • Takada M.
        • Asano T.
        • Murakawa K.
        • Takahashi Y.
        • et al.
        Aberrant expressions of HOX genes in colorectal and hepatocellular carcinomas.
        Oncol Rep. 2010; 23: 843-851
        • Frank D.A.
        Targeting transcription factors for cancer therapy.
        IDrugs. 2009; 12: 29-33
        • Hu J.
        • Dong A.
        • Fernandez-Ruiz V.
        • Shan J.
        • Kawa M.
        • Martinez-Anso E.
        • et al.
        Blockade of Wnt signaling inhibits angiogenesis and tumor growth in hepatocellular carcinoma.
        Cancer Res. 2009; 69: 6951-6959
        • Huang L.
        • Li M.X.
        • Wang L.
        • Li B.K.
        • Chen G.H.
        • He L.R.
        • et al.
        Prognostic value of Wnt inhibitory factor-1 expression in hepatocellular carcinoma that is independent of gene methylation.
        Tumour Biol. 2011; 32: 233-240
        • Lin C.P.
        • Liu C.R.
        • Lee C.N.
        • Chan T.S.
        • Liu H.E.
        Targeting c-Myc as a novel approach for hepatocellular carcinoma.
        World J Hepatol. 2010; 2: 16-20
        • Huth J.R.
        • Yu L.
        • Collins I.
        • Mack J.
        • Mendoza R.
        • Isaac B.
        • et al.
        NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.
        J Med Chem. 2004; 47: 4851-4857
        • Turkson J.
        • Kim J.S.
        • Zhang S.
        • Yuan J.
        • Huang M.
        • Glenn M.
        • et al.
        Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity.
        Mol Cancer Ther. 2004; 3: 261-269
        • Coleman D.R.t.
        • Ren Z.
        • Mandal P.K.
        • Cameron A.G.
        • Dyer G.A.
        • Muranjan S.
        • et al.
        Investigation of the binding determinants of phosphopeptides targeted to the SRC homology 2 domain of the signal transducer and activator of transcription 3. Development of a high-affinity peptide inhibitor.
        J Med Chem. 2005; 48: 6661-6670
        • Lepourcelet M.
        • Chen Y.N.
        • France D.S.
        • Wang H.
        • Crews P.
        • Petersen F.
        • et al.
        Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex.
        Cancer Cell. 2004; 5: 91-102
        • Lin C.P.
        • Liu J.D.
        • Chow J.M.
        • Liu C.R.
        • Liu H.E.
        Small-molecule c-Myc inhibitor, 10058-F4, inhibits proliferation, downregulates human telomerase reverse transcriptase and enhances chemosensitivity in human hepatocellular carcinoma cells.
        Anticancer Drugs. 2007; 18: 161-170
        • Onnis B.
        • Rapisarda A.
        • Melillo G.
        Development of HIF-1 inhibitors for cancer therapy.
        J Cell Mol Med. 2009; 13: 2780-2786
        • Yeo E.J.
        • Chun Y.S.
        • Cho Y.S.
        • Kim J.
        • Lee J.C.
        • Kim M.S.
        • et al.
        YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.
        J Natl Cancer Inst. 2003; 95: 516-525
        • Essmann F.
        • Schulze-Osthoff K.
        Translational approaches targeting the p53 pathway for anticancer therapy.
        Br J Pharmacol. 2012; 165: 328-344
        • Shangary S.
        • Qin D.
        • McEachern D.
        • Liu M.
        • Miller R.S.
        • Qiu S.
        • et al.
        Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition.
        Proc Natl Acad Sci USA. 2008; 105: 3933-3938
        • Vassilev L.T.
        • Vu B.T.
        • Graves B.
        • Carvajal D.
        • Podlaski F.
        • Filipovic Z.
        • et al.
        In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.
        Science. 2004; 303: 844-848
        • Shi H.
        • Lambert J.M.
        • Hautefeuille A.
        • Bykov V.J.
        • Wiman K.G.
        • Hainaut P.
        • et al.
        In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249.
        Carcinogenesis. 2008; 29: 1428-1434
        • Lee Y.M.
        • Lim J.H.
        • Chun Y.S.
        • Moon H.E.
        • Lee M.K.
        • Huang L.E.
        • et al.
        Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1alpha.
        Carcinogenesis. 2009; 30: 1768-1775
        • Guo J.
        • Xin H.
        Chinese gene therapy. Splicing out the West?.
        Science. 2006; 314: 1232-1235
        • Yang Z.X.
        • Wang D.
        • Wang G.
        • Zhang Q.H.
        • Liu J.M.
        • Peng P.
        • et al.
        Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma.
        J Cancer Res Clin Oncol. 2010; 136: 625-630
        • Scheffner M.
        • Werness B.A.
        • Huibregtse J.M.
        • Levine A.J.
        • Howley P.M.
        The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.
        Cell. 1990; 63: 1129-1136
        • Bischoff J.R.
        • Kirn D.H.
        • Williams A.
        • Heise C.
        • Horn S.
        • Muna M.
        • et al.
        An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.
        Science. 1996; 274: 373-376
        • Reid T.
        • Galanis E.
        • Abbruzzese J.
        • Sze D.
        • Wein L.M.
        • Andrews J.
        • et al.
        Hepatic arterial infusion of a replication-selective oncolytic adenovirus (dl1520): phase II viral, immunologic, and clinical endpoints.
        Cancer Res. 2002; 62: 6070-6079
        • Hall A.R.
        • Dix B.R.
        • O’Carroll S.J.
        • Braithwaite A.W.
        P53-dependent cell death/apoptosis is required for a productive adenovirus infection.
        Nat Med. 1998; 4: 1068-1072
        • He Q.
        • Liu Y.
        • Zou Q.
        • Guan Y.S.
        Transarterial injection of H101 in combination with chemoembolization overcomes recurrent hepatocellular carcinoma.
        World J Gastroenterol. 2011; 17: 2353-2355
        • Kuhnel F.
        • Gurlevik E.
        • Wirth T.C.
        • Struver N.
        • Malek N.P.
        • Muller-Schilling M.
        • et al.
        Targeting of p53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by p53-homologues.
        Mol Ther. 2010; 18: 936-946
        • Chen W.
        • Wu Y.
        • Liu W.
        • Wang G.
        • Wang X.
        • Yang Y.
        • et al.
        Enhanced antitumor efficacy of a novel fiber chimeric oncolytic adenovirus expressing p53 on hepatocellular carcinoma.
        Cancer Lett. 2011; 307: 93-103
        • Xue X.B.
        • Xiao C.W.
        • Zhang H.
        • Lu A.G.
        • Gao W.
        • Zhou Z.Q.
        • et al.
        Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines.
        World J Gastroenterol. 2010; 16: 4677-4684
        • Han Z.
        • Hong Z.
        • Chen C.
        • Gao Q.
        • Luo D.
        • Fang Y.
        • et al.
        A novel oncolytic adenovirus selectively silences the expression of tumor-associated STAT3 and exhibits potent antitumoral activity.
        Carcinogenesis. 2009; 30: 2014-2022
        • Cherry T.
        • Longo S.L.
        • Tovar-Spinoza Z.
        • Post D.E.
        Second-generation HIF-activated oncolytic adenoviruses with improved replication, oncolytic, and antitumor efficacy.
        Gene Ther. 2010; 17: 1430-1441
        • Post D.E.
        • Van Meir E.G.
        A novel hypoxia-inducible factor (HIF) activated oncolytic adenovirus for cancer therapy.
        Oncogene. 2003; 22: 2065-2072
        • Jakubczak J.L.
        • Ryan P.
        • Gorziglia M.
        • Clarke L.
        • Hawkins L.K.
        • Hay C.
        • et al.
        An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy.
        Cancer Res. 2003; 63: 1490-1499
        • Dvory-Sobol H.
        • Sagiv E.
        • Kazanov D.
        • Ben-Ze’ev A.
        • Arber N.
        Targeting the active beta-catenin pathway to treat cancer cells.
        Mol Cancer Ther. 2006; 5: 2861-2871
        • Mishra L.
        • Banker T.
        • Murray J.
        • Byers S.
        • Thenappan A.
        • He A.R.
        • et al.
        Liver stem cells and hepatocellular carcinoma.
        Hepatology. 2009; 49: 318-329
        • Oishi N.
        • Wang X.W.
        Novel therapeutic strategies for targeting liver cancer stem cells.
        Int J Biol Sci. 2011; 7: 517-535
        • Yao Z.
        • Mishra L.
        Cancer stem cells and hepatocellular carcinoma.
        Cancer Biol Ther. 2009; 8: 1691-1698
        • Moghaddam S.J.
        • Haghighi E.N.
        • Samiee S.
        • Shahid N.
        • Keramati A.R.
        • Dadgar S.
        • et al.
        Immunohistochemical analysis of p53, cyclinD1, RB1, c-fos and N-ras gene expression in hepatocellular carcinoma in Iran.
        World J Gastroenterol. 2007; 13: 588-593
        • de La Coste A.
        • Romagnolo B.
        • Billuart P.
        • Renard C.A.
        • Buendia M.A.
        • Soubrane O.
        • et al.
        Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.
        Proc Natl Acad Sci USA. 1998; 95: 8847-8851
        • Merle P.
        • de la Monte S.
        • Kim M.
        • Herrmann M.
        • Tanaka S.
        • Von Dem Bussche A.
        • et al.
        Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma.
        Gastroenterology. 2004; 127: 1110-1122
        • Shih Y.L.
        • Shyu R.Y.
        • Hsieh C.B.
        • Lai H.C.
        • Liu K.Y.
        • Chu T.Y.
        • et al.
        Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma.
        Cancer. 2006; 107: 579-590
        • Wirths O.
        • Waha A.
        • Weggen S.
        • Schirmacher P.
        • Kuhne T.
        • Goodyer C.G.
        • et al.
        Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms’ tumors.
        Lab Invest. 2003; 83: 429-434
        • Yu B.
        • Yang X.
        • Xu Y.
        • Yao G.
        • Shu H.
        • Lin B.
        • et al.
        Elevated expression of DKK1 is associated with cytoplasmic/nuclear beta-catenin accumulation and poor prognosis in hepatocellular carcinomas.
        J Hepatol. 2009; 50: 948-957
        • Pang R.
        • Yuen J.
        • Yuen M.F.
        • Lai C.L.
        • Lee T.K.
        • Man K.
        • et al.
        PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma.
        Oncogene. 2004; 23: 4182-4186
        • Yau T.O.
        • Chan C.Y.
        • Chan K.L.
        • Lee M.F.
        • Wong C.M.
        • Fan S.T.
        • et al.
        HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing.
        Oncogene. 2005; 24: 1607-1614
        • Ladu S.
        • Calvisi D.F.
        • Conner E.A.
        • Farina M.
        • Factor V.M.
        • Thorgeirsson S.S.
        E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR and COX-2 in a mouse model of human liver cancer.
        Gastroenterology. 2008; 135: 1322-1332
        • Li W.
        • Ni G.X.
        • Zhang P.
        • Zhang Z.X.
        • Wu Q.
        Characterization of E2F3a function in HepG2 liver cancer cells.
        J Cell Biochem. 2010; 111: 1244-1251