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Wnt signaling and hepatocarcinogenesis: Molecular targets for the development of innovative anticancer drugs

  • Author Footnotes
    † These authors contributed equally to this work.
    Floriane Pez
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69008 Lyon, France

    Université Lyon-1, F-69622 Villeurbanne, France

    Centre Léon Bérard, F-69008 Lyon, France
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  • Author Footnotes
    † These authors contributed equally to this work.
    Anaïs Lopez
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69008 Lyon, France

    Université Lyon-1, F-69622 Villeurbanne, France

    Centre Léon Bérard, F-69008 Lyon, France
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  • Miran Kim
    Affiliations
    The Liver Research Center, Brown University, Providence, RI, USA
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  • Jack R. Wands
    Affiliations
    The Liver Research Center, Brown University, Providence, RI, USA
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  • Claude Caron de Fromentel
    Affiliations
    INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69008 Lyon, France

    Université Lyon-1, F-69622 Villeurbanne, France

    Centre Léon Bérard, F-69008 Lyon, France
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  • Philippe Merle
    Correspondence
    Corresponding author. Address: UMR Inserm U1052 CNRS5286, Centre de Recherche en Cancérologie de Lyon, 151 Cours Albert Thomas, 69424 Lyon Cedex 03, France. Tel.: +33 472681954; fax: +33 472681971.
    Affiliations
    INSERM U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, F-69008 Lyon, France

    Université Lyon-1, F-69622 Villeurbanne, France

    Centre Léon Bérard, F-69008 Lyon, France

    Hospices Civils de Lyon, Service d’Hépatologie et de Gastroentérologie, Groupement Hospitalier Lyon Nord, F-69000 Lyon, France
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  • Author Footnotes
    † These authors contributed equally to this work.
Open AccessPublished:July 08, 2013DOI:https://doi.org/10.1016/j.jhep.2013.07.001

      Summary

      Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide. HCC can be cured by radical therapies if early diagnosis is done while the tumor has remained of small size. Unfortunately, diagnosis is commonly late when the tumor has grown and spread. Thus, palliative approaches are usually applied such as transarterial intrahepatic chemoembolization and sorafenib, an anti-angiogenic agent and MAP kinase inhibitor. This latter is the only targeted therapy that has shown significant, although moderate, efficiency in some individuals with advanced HCC. This highlights the need to develop other targeted therapies, and to this goal, to identify more and more pathways as potential targets. The Wnt pathway is a key component of a physiological process involved in embryonic development and tissue homeostasis. Activation of this pathway occurs when a Wnt ligand binds to a Frizzled (FZD) receptor at the cell membrane. Two different Wnt signaling cascades have been identified, called non-canonical and canonical pathways, the latter involving the β-catenin protein. Deregulation of the Wnt pathway is an early event in hepatocarcinogenesis and has been associated with an aggressive HCC phenotype, since it is implicated both in cell survival, proliferation, migration and invasion. Thus, component proteins identified in this pathway are potential candidates of pharmacological intervention. This review focuses on the characteristics and functions of the molecular targets of the Wnt signaling cascade and how they may be manipulated to achieve anti-tumor effects.

      Abbreviations:

      HCC (hepatocellular carcinoma), FZD (frizzled), CSC (cancer stem cells), PCP (planar cell polarity), TCF/LEF (T-cell factor/lymphoid enhancer factor), TLE-1 (transducin like enhancer-1), APC (adenomatous polyposis coli protein), GSK3β (glycogen synthase kinase 3β), CK1 (casein kinase 1), β-TRCP (beta-transducin repeat containing protein), SCF (Skp1/cullin F-box complex), Dvl/Dsh (disheveled), Pygo (pygopus), CBP (CREB-binding protein), PKC (protein kinase C), NFAT (nuclear factor of activated T cell), NLK (Nemo-like kinase), Wif1 (Wnt inhibitory protein-1), sFRP (secreted FZD-related proteins), Dkk (Dickkopf), Krm (Kremen), Rspo (R-spondin), Gpc3 (glypican-3), DIFs (differentiation-inducing factors)

      Keywords

      Introduction

      HCC represents a major public health problem with a high impact on society. HCC is the sixth most common tumor worldwide in terms of incidence (about one million per year). Projections are that this incidence will substantially increase during the next decades due to persistent infection with the hepatitis C virus as well as the emergence of non-alcoholic steatohepatitis as a major health problem. HCC portends a poor prognosis since ranking third in terms of “cause of death” by cancer, and often presents as a major complication of cirrhosis related to chronic hepatitis B and C infections, or non-virus related [
      • Bosch F.X.
      • Ribes J.
      • Cleries R.
      • Diaz M.
      Epidemiology of hepatocellular carcinoma.
      ,
      • Kawada N.
      • Imanaka K.
      • Kawaguchi T.
      • Tamai C.
      • Ishihara R.
      • Matsunaga T.
      • et al.
      Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis.
      ,
      • Parkin D.M.
      • Bray F.
      • Ferlay J.
      • Pisani P.
      Estimating the world cancer burden: Globocan 2000.
      ]. The dismal prognosis is generally related to a late diagnosis after HCC cells have infiltrated the liver parenchyma, have spread through the portal venous system and/or have formed distant metastases. However, if HCC is diagnosed early (<20% of patients), these smaller tumors may be cured by surgical resection, liver transplantation or radiofrequency ablation. In more advanced tumors (>80% of patients at diagnosis), only palliative approaches can be applied. In this regard, transarterial intrahepatic chemoembolization has been shown to be somewhat effective in increasing overall survival of individuals with tumors that have spread only into the liver parenchyma without extrahepatic metastasis (median overall survival is increased from 15 to 20 months compared to the best supportive care). In HCC with extrahepatic spread, only sorafenib, an anti-angiogenic and MAP kinase inhibitor, has been shown to increase overall survival of patients (from 8 to 11 months) [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ]. All other systemic approaches such as cytotoxic chemotherapy have not been shown to be effective; thus, to date, no targeted therapy except sorafenib has been proven to prolong life in patients with HCC. However, there are ongoing or ended clinical trials with agents that target FGF, VEGF, PDGF, EGF, IGF, mTOR, and TGFβ signaling pathways but none has been shown yet to have a significant impact on patient survival [
      • Villanueva A.
      • Hernandez-Gea V.
      • Llovet J.M.
      Medical therapies for hepatocellular carcinoma: a critical view of the evidence.
      ].
      Recently, cancer stem cells (CSC) have been hypothesized to play a key role in tumor maintenance as well as relapse after surgical resection. There is accumulating information that supports a role for CSC in hepatocarcinogenesis to maintain the tumor size and to initiate tumor recurrence following therapy [
      • Yamashita T.
      • Honda M.
      • Nakamoto Y.
      • Baba M.
      • Nio K.
      • Hara Y.
      • et al.
      Discrete nature of EpCAM(+) and CD90(+) cancer stem cells in human hepatocellular carcinoma.
      ]. The pool of CSC is maintained by self-renewal capabilities that are largely driven by reactivation of embryonic signaling programs mediated by Wnt, Notch, Bmi, and Hedgehog pathways, similar to what has been previously demonstrated during breast carcinogenesis [
      • Liu S.
      • Dontu G.
      • Wicha M.S.
      Mammary stem cells, self-renewal pathways, and carcinogenesis.
      ]. Preclinical studies further underline the potential value of inhibiting activation of these signaling programs in some tumor types [
      • Chen F.
      • Li Y.
      • Wang L.
      • Hu L.
      Knockdown of BMI-1 causes cell-cycle arrest and derepresses p16INK4a, HOXA9 and HOXC13 mRNA expression in HeLa cells.
      ,
      • Lu F.L.
      • Yu C.C.
      • Chiu H.H.
      • Liu H.E.
      • Chen S.Y.
      • Lin S.
      • et al.
      Sonic hedgehog antagonists induce cell death in acute myeloid leukemia cells with the presence of lipopolysaccharides, tumor necrosis factor-alpha, or interferons.
      ,
      • McAuliffe S.M.
      • Morgan S.L.
      • Wyant G.A.
      • Tran L.T.
      • Muto K.W.
      • Chen Y.S.
      • et al.
      Targeting Notch, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy.
      ,
      • Nambotin S.B.
      • Lefrancois L.
      • Sainsily X.
      • Berthillon P.
      • Kim M.
      • Wands J.R.
      • et al.
      Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.
      ].
      In this review, we describe the features of a therapeutic target, i.e., the Wnt pathway, for potential therapy of HCC. We will discuss experimental and preclinical studies regarding the use of Wnt inhibitors as a therapeutic approach for HCC.

      The Wnt-mediated signaling

      The first member of the Wnt family of ligands was identified from the int-1 gene found in a mammary adenocarcinoma, located at the integration site of the mouse mammary tumor virus (MMTV); subsequently, it was demonstrated to have oncogenic properties [
      • Nusse R.
      • Varmus H.E.
      Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.
      ]. More important, int-1 homolog genes have been found in human tumors as well [
      • van Ooyen A.
      • Kwee V.
      • Nusse R.
      The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences.
      ]. In addition, a highly conserved int-1 homolog was also discovered in Drosophila and designated Wingless “Wg” [
      • Sharma R.P.
      • Chopra V.L.
      Effect of the Wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster.
      ]. The combination of int-1 and Wingless led to the common Wnt1 terminology and recently has been used to designate the Wnt family of ligands [
      • Nusse R.
      • Brown A.
      • Papkoff J.
      • Scambler P.
      • Shackleford G.
      • McMahon A.
      • et al.
      A new nomenclature for int-1 and related genes: the Wnt gene family.
      ].
      Wnt proteins are secreted extracellular auto-paracrine glycoproteins that interact with Frizzled receptors (FZD), a seven transmembrane domain protein, resembling the G-protein-coupled receptor (GPCR) family. Vinson and colleagues revealed that FZD contains an extracellular cysteine-rich domain (CRD) which is the putative binding site for the Wnt ligands. These investigators demonstrated the functional role of the frizzled locus to coordinate development of the cytoskeleton in Drosophila epidermal cells [
      • Vinson C.R.
      • Conover S.
      • Adler P.N.
      A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains.
      ]. Subsequently, Wnt/FZD-mediated signaling has been extensively studied, and although it has been widely implicated in cellular homeostasis, these ligand/receptor interactions have now been appreciated as key factors during the oncogenesis process and therefore, could serve as new therapeutic targets.
      Thus, Wnt proteins represent members of a highly conserved family that is involved in several processes including embryonic development, cell fate determination, proliferation, polarity, migration, and stem cell maintenance. In addition, Wnt/beta-catenin signaling has been found to play key roles in metabolic zonation of adult liver, regeneration [
      • Gebhardt R.
      • Hovhannisyan A.
      Organ patterning in the adult stage: the role of Wnt/beta-catenin signaling in liver zonation and beyond.
      ]. In adult organisms, deregulation of Wnt signaling may lead to tumor development [
      • Cadigan K.M.
      • Nusse R.
      Wnt signaling: a common theme in animal development.
      ,
      • Clevers H.
      Wnt/beta-catenin signaling in development and disease.
      ]. The Wnt-mediated pathway is activated through the binding of one Wnt ligand to a FZD receptor. Ten different FZD receptors and 19 Wnt ligands have been identified in humans. The binding of Wnt to an FZD receptor can trigger activation of at least three different pathways. The first is the Wnt/β-catenin cascade, also called the Wnt-canonical pathway; the remaining two are the planar cell polarity (PCP) and the Wnt/calcium pathways, respectively. The two latter are β-catenin independent and represent examples of the non-canonical cascades. In this regard, a multitude of combinations between the 19 Wnt ligands and the 10 FZD receptors, such as co-receptors and other molecules, are theoretically possible. Classically, Wnt1/2/3/3a/8a/8b/10a/10b and FZD1/5/7/9 are classified as the canonical elements, whereas Wnt4/5a/5b/6/7a/7b/11 and FZD2/3/4/6 are designated as non-canonical components. The remaining Wnt2b/9a/9b/16 and FZD8/10 proteins remain unclassified [
      • Clevers H.
      Wnt/beta-catenin signaling in development and disease.
      ,
      • Huelsken J.
      • Behrens J.
      The Wnt signalling pathway.
      ]. However, it remains elusive how selectivity between Wnt/FZD as well as specificity of downstream signaling is achieved. Some Wnt/FZD elements can share dual canonical and non-canonical functions. For instance, it has been shown that in absence of Ror2 co-receptors, Wnt5a can activate β-catenin signaling with FZD4 and Lrp5 [
      • Armstrong A.
      • Ryu Y.K.
      • Chieco D.
      • Kuruvilla R.
      Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development.
      ]. FZD3 has been described to act likely through canonical pathways in mice neurogenesis [
      • Armstrong A.
      • Ryu Y.K.
      • Chieco D.
      • Kuruvilla R.
      Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development.
      ]. Zhang et al. demonstrated that in Xenopus foregut, FZD7 can activate low level of β-catenin and non-canonical JNK signaling in which both pathways contributed to foregut fate and proliferation while JNK pathway regulated cell morphology [
      • Zhang Z.
      • Rankin S.A.
      • Zorn A.M.
      Different thresholds of Wnt-Frizzled 7 signaling coordinate proliferation, morphogenesis and fate of endoderm progenitor cells.
      ]. It is of interest that canonical and non-canonical pathways can not only be driven by specific Wnt/FZD combinations, but also by cell type, differentiation status, localization and composition of the microenvironment [
      • Mikels A.J.
      • Nusse R.
      Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context.
      ].

      The canonical Wnt/FZD pathway

      The β-catenin protein, encoded by the CTNNB1 gene, is a key component of Wnt-canonical pathway signaling. β-catenin has a central region which presents armadillo domain repeats important for the binding of partners, such as Axin1 and adenomatous polyposis coli protein (APC) as well as transcription factors [
      • Graham T.A.
      • Weaver C.
      • Mao F.
      • Kimelman D.
      • Xu W.
      Crystal structure of a beta-catenin/Tcf complex.
      ]. The C- and N-terminal regions are important. C-terminus of β-catenin serves as a binding factor for a multitude of complexes promoting β-catenin-mediated transcription, whereas phosphorylation of the N-terminus promotes degradation of β-catenin. Indeed, β-catenin may be present in several cellular compartments, such as the inner plasma membrane having a role in cell-cell junctions, the cytoplasm and the nucleus where it forms an active complex containing TCF/LEF transcription factors (T-cell factor/lymphoid enhancer factor) [
      • Ozawa M.
      • Baribault H.
      • Kemler R.
      The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.
      ]. In the absence of nuclear β-catenin, TCF/LEF interact with the transcriptional co-repressor transducin like enhancer-1 (TLE-1) (Drosophila homolog Groucho), thus preventing β-catenin target gene expression [
      • Brantjes H.
      • Roose J.
      • van De Wetering M.
      • Clevers H.
      All Tcf HMG box transcription factors interact with Groucho-related co-repressors.
      ]. Following translocation into the nucleus, β-catenin binds to TCF/LEF and replaces the TLE-1 repressor to form a transcriptional complex that activates the expression of its target genes (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Canonical Wnt/FZD signaling pathway. (A) In the absence of Wnt signaling, soluble β-catenin is phosphorylated by a degradation complex consisting of the kinases GSK3β and CK1α and the scaffolding proteins APC and Axin1. Phosphorylated β-catenin is targeted for proteasomal degradation after ubiquitination by the SCF protein complex. In the nucleus and in the absence of β-catenin, TCF/LEF transcription factor activity is repressed by TLE-1; (B) activation of the canonical Wnt/FZD signaling leads to phosphorylation of Dvl/Dsh, which in turn recruits Axin1 and GSK3β adjacent to the plasma membrane, thus preventing the formation of the degradation complex. As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it promotes the expression of target genes via interaction with TCF/LEF transcription factors and other proteins such as CBP, Bcl9, and Pygo.
      In absence of the canonical Wnt signaling, cytosolic β-catenin is targeted for degradation by a complex composed of a scaffold of proteins named axin1, APC, and two serine/threonine kinases: the glycogen synthase kinase 3β (GSK3β) and the casein kinase 1 (CK1) [
      • MacDonald B.T.
      • Tamai K.
      • He X.
      Wnt/beta-catenin signaling: components, mechanisms, and diseases.
      ] (Fig. 1A). Axin1 and APC act together as scaffolding proteins through binding of β-catenin, and enhance its N-terminal phosphorylation by GSK3β and CK1. The first phosphorylation event is generated by CK1 at Ser45 which allows the GSK3β-mediated sequential phosphorylation of Thr41, Ser37, and Ser33 [
      • Liu C.
      • Kato Y.
      • Zhang Z.
      • Do V.M.
      • Yankner B.A.
      • He X.
      Beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation.
      ,
      • Liu C.
      • Li Y.
      • Semenov M.
      • Han C.
      • Baeg G.H.
      • Tan Y.
      • et al.
      Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.
      ]. Ser37 and Ser33 phosphorylations provide a binding site for the E3 ubiquitin ligase β-TRCP (β-transducin repeat containing protein), leading to β-catenin ubiquitination in a β-TRCP/Skp1/cullin F-box complex (SCF) dependent manner followed by proteasomal degradation [
      • Hart M.
      • Concordet J.P.
      • Lassot I.
      • Albert I.
      • Del los Santos R.
      • Durand H.
      The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell.
      ,
      • Kimelman D.
      • Xu W.
      Beta-catenin destruction complex: insights and questions from a structural perspective.
      ].
      Activation of the canonical Wnt signaling cascade leads to disruption of the β-catenin degradation complex, resulting in β-catenin accumulation in the cytoplasm followed by translocation into the nucleus where it serves as a transcription factor to activate downstream target genes (Fig. 1B). In brief, this process is as follows: Wnt ligand binds to the extracellular domain of an FZD receptor and Lrp5/6 co-receptors. This ternary complex (Wnt/FZD/Lrp) recruits the scaffolding phosphoprotein dishevelled (Dvl/Dsh) at the plasma membrane which in turn traps the axin-bound-GSK3β complex, thus preventing proteasomal degradation of cytosolic β-catenin. When stabilized, β-catenin is able to translocate into the nucleus, where it binds to TCF/LEF transcription factors and then forms a transcriptionally active complex with pygopus (Pygo), CBP (CREB-Binding Protein) and Bcl9 proteins [
      • Daniels D.L.
      • Weis W.I.
      Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation.
      ]. In mammals, four TCF genes have been described, which adds further complexity to the mechanism(s) of activation of the Wnt canonical cascade [
      • Arce L.
      • Yokoyama N.N.
      • Waterman M.L.
      Diversity of LEF/TCF action in development and disease.
      ]. Of notice is the β-catenin pool localized at the plasma membrane that plays a key role in cell-cell junctions. To this aim, a complex including either p120 catenin/γ-catenin(plakoglobin)/α-catenin or p120 catenin/β-catenin/α-catenin [
      • Ozawa M.
      • Baribault H.
      • Kemler R.
      The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.
      ] binds to the cytoplasmic carboxyl terminus domain of E-cadherin adhesion molecule, in order to join cadherins to the actin cytoskeleton. More precisely, p120 catenin binds to the juxtamembrane and then β-catenin or γ-catenin binds to the cytoplasmic domain of E-cadherin. The remaining α-catenin serves as a link between actin and β/γ-catenin which leads to the stabilization of cell adhesion [
      • Hirano S.
      • Kimoto N.
      • Shimoyama Y.
      • Hirohashi S.
      • Takeichi M.
      Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization.
      ]. The possible consequences of inhibiting β-catenin at adherent junctions have to be discuss in respect of their role in epithelio-mesenchymal transition (EMT). Disruption of E-cadherin-mediated adherent junctions is a major event in EMT [
      • Yang J.
      • Weinberg R.A.
      Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis.
      ] and because of the interplay between cadherin-mediated cell adhesion and canonical/β-catenin signaling [
      • Heuberger J.
      • Birchmeier W.
      Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling.
      ], targeting β-catenin could also promote the disruption of these junctions leading to enhance EMT. However, Wickline et al. have shown that in hepatocyte-specific β-catenin-conditional null mice, γ-catenin is upregulated and associated with E-cadherin and actin to maintain adherent junctions. In addition, no nuclear γ-catenin was detected in liver of KO mice, leading to the conclusion that despite armadillo domains on γ-catenin, there is no compensation at nuclear level. Nevertheless, authors warn us about preventing concurrent γ-catenin suppression that may increase tumor cell invasion [
      • Wickline E.D.
      • Awuah P.K.
      • Behari J.
      • Ross M.
      • Stolz D.B.
      • Monga S.P.
      Hepatocyte gamma-catenin compensates for conditionally deleted beta-catenin at adherens junctions.
      ]. More recent study confirmed these results in in vitro experiments with HCC cell lines and identified the mechanism of γ-catenin stabilization as serine/threonine phosphorylation induced by protein kinase A [
      • Wickline E.D.
      • Du Y.
      • Stolz D.B.
      • Kahn M.
      • Monga S.P.
      Gamma-Catenin at adherens junctions: mechanism and biologic implications in hepatocellular cancer after beta-catenin knockdown.
      ]. With regard to this recent data, targeting β-catenin in HCC therapies may not disturb cell junctions since the design of Wnt inhibitors for therapeutic intervention, specifically designates soluble active β-catenin as preferential target.

      The non-canonical Wnt/FZD pathways

      In contrast to the canonical Wnt pathway, non-canonical signaling does not depend on β-catenin and requires Ror2/Ryk co-receptors instead of Lrp5/6 (Fig. 2). In the Wnt/PCP pathway, Wnt/FZD interaction promotes the recruitment of Dvl/Dsh, which in turn binds to the small GTPase protein called Rac, leading to both the induction of ROCK (Rho-associated protein kinase) pathway and the activation of the MAP kinase cascade and subsequently to the activation of AP1-mediated target gene expression [
      • Jones C.
      • Chen P.
      Planar cell polarity signaling in vertebrates.
      ,
      • Oishi I.
      • Suzuki H.
      • Onishi N.
      • Takada R.
      • Kani S.
      • Ohkawara B.
      • et al.
      The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway.
      ]. In the Wnt/calcium pathway, the complex formation between FZD, Dvl/Dsh and G proteins results in PLC (Phospho Lipase C) activation which cleaves PIP2 (Phosphatidyl Inositol 4,5 biphosphate) into DAG (DiAcylGlycerol) and IP3 (Inositol 1,4,5-triphosphate). This process results in the activation of PKC (Protein Kinase C) through DAG while IP3 promotes calcium release from the endoplasmic reticulum. Increased intracellular concentration of calcium enhances phosphorylation and activation of PKCs. This also triggers the activation of Ca2+-calmodulin-dependent calcineurin and CAMKII (Ca2+-calmodulin dependent kinase II), leading to NFAT (Nuclear Factor of Activated T-cell) and NLK (Nemo Like Kinase) translocation, respectively. NLK acts as a β-catenin pathway inhibitor through phosphorylation and degradation of TCF/LEF transcription factors [
      • Ishitani T.
      • Kishida S.
      • Hyodo-Miura J.
      • Ueno N.
      • Yasuda J.
      • Waterman M.
      • et al.
      The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling.
      ].
      Figure thumbnail gr2
      Fig. 2Non-canonical Wnt/FZD signaling pathways. Interaction of Wnt, FZD, and ROR2/RYK co-receptors leads to either (1) JNK activation, (2) PKCs activation, (3) NFAT transactivation, or (4) inhibition of β-catenin activity through binding of NLK to TCF/LEF.

      Antagonists and agonists of Wnt/FZD-mediated signaling

      Several secreted proteins are known to negatively or positively regulate the Wnt/FZD complex. Four classes of antagonistic molecules have been described. Wnt inhibitory protein-1 (Wif1) and secreted FZD-related proteins (sFRP1, 2, 3, 4, 5) bind to and sequester the soluble Wnt ligands, thus inhibiting their interaction and binding to FZD receptors [
      • Bafico A.
      • Gazit A.
      • Pramila T.
      • Finch P.W.
      • Yaniv A.
      • Aaronson S.A.
      Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling.
      ,
      • Jones S.E.
      • Jomary C.
      Secreted Frizzled-related proteins: searching for relationships and patterns.
      ,
      • Patthy L.
      The WIF module.
      ,
      • Uren A.
      • Reichsman F.
      • Anest V.
      • Taylor W.G.
      • Muraiso K.
      • Bottaro D.P.
      • et al.
      Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling.
      ]. The Dickkopf family is composed of four members (Dkk1, 2, 3, 4) that can interact with both Lrp5/6 and Krm1,2 (Kremen1,2) co-receptors [
      • Mao B.
      • Wu W.
      • Davidson G.
      • Marhold J.
      • Li M.
      • Mechler B.M.
      • et al.
      Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.
      ]. The ternary complex Lrp-Dkk-Krm prevents β-catenin stabilization by promoting Lrp5/6 endocytosis [
      • Wang K.
      • Zhang Y.
      • Li X.
      • Chen L.
      • Wang H.
      • Wu J.
      • et al.
      Characterization of the Kremen-binding site on Dkk1 and elucidation of the role of Kremen in Dkk-mediated Wnt antagonism.
      ]. Wise and Sost proteins form the other class of secreted antagonists. They bind to Lrp5/6 and thus disrupt the Wnt-induced FZD-Lrp5/6 interaction [
      • Itasaki N.
      • Jones C.M.
      • Mercurio S.
      • Rowe A.
      • Domingos P.M.
      • Smith J.C.
      • et al.
      Wise, a context-dependent activator and inhibitor of Wnt signalling.
      ,
      • Semenov M.
      • Tamai K.
      • He X.
      SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor.
      ].
      Three agonistic molecules have recently been identified; the R-spondins (Rspo1, 2, 3, 4), norrin and glypican-3 (Gpc3). The Gpc3 is a heparan sulfate proteoglycan bound to the cell membrane through a glycosyl-phosphatidylinositol anchor. Gpc3 increases autocrine/paracrine canonical Wnt signaling by binding to Wnt ligands, thus facilitating the interaction between Wnt ligands and FZD receptors [
      • Capurro M.I.
      • Xiang Y.Y.
      • Lobe C.
      • Filmus J.
      Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling.
      ]. Mechanisms by which Rspo and Norrin activate the canonical Wnt pathway have not been clarified. Rspo1 is able to bind to both Lrps and FZDs but it has also been proposed that Rspo prevents Lrp6 internalization through binding to Krm instead of Dkk [
      • Binnerts M.E.
      • Kim K.A.
      • Bright J.M.
      • Patel S.M.
      • Tran K.
      • Zhou M.
      • et al.
      R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6.
      ,
      • Nam J.S.
      • Turcotte T.J.
      • Smith P.F.
      • Choi S.
      • Yoon J.K.
      Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression.
      ,
      • Xu Q.
      • Wang Y.
      • Dabdoub A.
      • Smallwood P.M.
      • Williams J.
      • Woods C.
      • et al.
      Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.
      ].

      Wnt signaling deregulation in human hepatocarcinogenesis

      Similar to other tumor tissue types, the canonical Wnt/FZD signaling is a critical contributor to HCC pathogenesis. Indeed, 40–70% of HCCs harbor nuclear accumulation of the β-catenin protein, one of the hallmarks of the Wnt/β-catenin pathway activation [
      • Lachenmayer A.
      • Alsinet C.
      • Savic R.
      • Cabellos L.
      • Toffanin S.
      • Hoshida Y.
      • et al.
      Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.
      ,
      • Wong C.M.
      • Fan S.T.
      • Ng I.O.
      Beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance.
      ,
      • 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.
      ]. Activating mutations of the β-catenin gene (CTNNB1) occur in 8–30% of tumors, while loss-of-function/mutations in APC and Axin genes occur in 1–3% and 8–15%, respectively and are mutually exclusive to CTNNB1 mutations [
      • Lachenmayer A.
      • Alsinet C.
      • Savic R.
      • Cabellos L.
      • Toffanin S.
      • Hoshida Y.
      • et al.
      Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.
      ,
      • 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.
      ,
      • 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.
      ,
      • Guichard C.
      • Amaddeo G.
      • Imbeaud S.
      • Ladeiro Y.
      • Pelletier L.
      • Maad I.B.
      • et al.
      Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
      ,
      • Park J.Y.
      • Park W.S.
      • Nam S.W.
      • Kim S.Y.
      • Lee S.H.
      • Yoo N.J.
      • et al.
      Mutations of beta-catenin and AXIN I genes are a late event in human hepatocellular carcinogenesis.
      ,
      • 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.
      ,
      • Amaddeo G.
      • Guichard C.
      • Imbeaud S.
      • Zucman-Rossi J.
      Next-generation sequencing identified new oncogenes and tumor suppressor genes in human hepatic tumors.
      ]. Some observations suggest that the CTNNB1 mutation could be a late event during hepatocarcinogenesis. However, accumulation of β-catenin was detected in the early stage of HCC development, suggesting that other mechanisms could contribute to β-catenin stabilization (Table 1) [
      • Park J.Y.
      • Park W.S.
      • Nam S.W.
      • Kim S.Y.
      • Lee S.H.
      • Yoo N.J.
      • et al.
      Mutations of beta-catenin and AXIN I genes are a late event in human hepatocellular carcinogenesis.
      ,
      • Suzuki T.
      • Yano H.
      • Nakashima Y.
      • Nakashima O.
      • Kojiro M.
      Beta-catenin expression in hepatocellular carcinoma: a possible participation of beta-catenin in the dedifferentiation process.
      ]. Strikingly, extrinsic activation of Wnt/β-catenin pathway and CTNNB1 mutation do not lead to the same molecular expression pattern, supporting different roles for wild type and mutated β-catenin. The Wnt/β-catenin activated HCC subclass with a CTNNB1 mutation is characterized by upregulation of liver-specific Wnt-targets, low grade and well-differentiated tumors, with chromosome stability and a favorable prognosis. The Wnt/β-catenin activated HCC subclass without CTNNB1 mutation is characterized by dysregulation of classical Wnt targets, high chromosomal instability, aggressive phenotype, and is preferentially associated with chronic HBV infection [
      • Lachenmayer A.
      • Alsinet C.
      • Savic R.
      • Cabellos L.
      • Toffanin S.
      • Hoshida Y.
      • et al.
      Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.
      ,
      • Suzuki T.
      • Yano H.
      • Nakashima Y.
      • Nakashima O.
      • Kojiro M.
      Beta-catenin expression in hepatocellular carcinoma: a possible participation of beta-catenin in the dedifferentiation process.
      ,
      • 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.
      ].
      Table 1Most prevalent potential mechanisms involved in activation of beta-catenin found so far in HCCs.
      Modulation of Wnt ligands or FZD receptor expression could account for Wnt/β-catenin pathway activation without any other mutations in CTNNB1, APC, or Axin genes. Indeed, upregulation of activators, such as ligands (Wnt1/3/4/5a/10b) or receptors/co-receptors (FZD3/6/7, Lrp6), and downregulation of inhibitors (sFRP1/4/5, Wif1, Dkk3, Dkk4) have been reported both in HCC tumors and surrounding precancerous liver tissues, which emphasizes that their over and/or underexpression may be early molecular events during hepatocarcinogenesis [
      • 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.
      ,
      • Kim M.
      • Lee H.C.
      • Tsedensodnom O.
      • Hartley R.
      • Lim Y.S.
      • Yu E.
      • et al.
      Functional interaction between Wnt3 and Frizzled-7 leads to activation of the Wnt/beta-catenin signaling pathway in hepatocellular carcinoma cells.
      ,
      • Wei W.
      • Chua M.S.
      • Grepper S.
      • So S.K.
      Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells.
      ,
      • Lee H.H.
      • Uen Y.H.
      • Tian Y.F.
      • Sun C.S.
      • Sheu M.J.
      • Kuo H.T.
      • et al.
      Wnt-1 protein as a prognostic biomarker for hepatitis B-related and hepatitis C-related hepatocellular carcinoma after surgery.
      ,
      • Tung E.K.
      • Wong B.Y.
      • Yau T.O.
      • Ng I.O.
      Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion.
      ,
      • Yoshikawa H.
      • Matsubara K.
      • Zhou X.
      • Okamura S.
      • Kubo T.
      • Murase Y.
      • et al.
      WNT10B functional dualism: beta-catenin/Tcf-dependent growth promotion or independent suppression with deregulated expression in cancer.
      ,
      • Deng Y.
      • Yu B.
      • Cheng Q.
      • Jin J.
      • You H.
      • Ke R.
      • et al.
      Epigenetic silencing of WIF-1 in hepatocellular carcinomas.
      ,
      • Ding Z.
      • Qian Y.B.
      • Zhu L.X.
      • Xiong Q.R.
      Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma.
      ,
      • Fatima S.
      • Lee N.P.
      • Tsang F.H.
      • Kolligs F.T.
      • Ng I.O.
      • Poon R.T.
      • et al.
      Dickkopf 4 (DKK4) acts on Wnt/beta-catenin pathway by influencing beta-catenin in hepatocellular carcinoma.
      ].
      Although β-catenin activation is crucial for liver development and regeneration, it is not sufficient per se for initiation of hepatocarcinogenesis. Indeed, animal models overexpressing an active β-catenin protein do not spontaneously form HCC [
      • Cadoret A.
      • Ovejero C.
      • Saadi-Kheddouci S.
      • Souil E.
      • Fabre M.
      • Romagnolo B.
      • et al.
      Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin.
      ,
      • Harada N.
      • Miyoshi H.
      • Murai N.
      • Oshima H.
      • Tamai Y.
      • Oshima M.
      • et al.
      Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.
      ,
      • Tan X.
      • Apte U.
      • Micsenyi A.
      • Kotsagrelos E.
      • Luo J.H.
      • Ranganathan S.
      • et al.
      Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver.
      ]. However, β-catenin activation may cooperate with other oncogenic pathways such as insulin/IGF-1/IRS-1/MAPK, H-RAS, MET, AKT and chemicals to induce HCC formation in mice [
      • Harada N.
      • Miyoshi H.
      • Murai N.
      • Oshima H.
      • Tamai Y.
      • Oshima M.
      • et al.
      Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.
      ,
      • Stauffer J.K.
      • Scarzello A.J.
      • Andersen J.B.
      • De Kluyver R.L.
      • Back T.C.
      • Weiss J.M.
      • et al.
      Coactivation of AKT and beta-catenin in mice rapidly induces formation of lipogenic liver tumors.
      ,
      • Nejak-Bowen K.N.
      • Thompson M.D.
      • Singh S.
      • Bowen Jr., W.C.
      • Dar M.J.
      • Khillan J.
      • et al.
      Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin.
      ,
      • Longato L.
      • de la Monte S.
      • Kuzushita N.
      • Horimoto M.
      • Rogers A.B.
      • Slagle B.L.
      • et al.
      Overexpression of insulin receptor substrate-1 and hepatitis Bx genes causes premalignant alterations in the liver.
      ]. It is described that beta-catenin mutation is a late event in hepatocarcinogenesis since present in some HCC tumors whereas absent in preneoplastic lesions, thus prompting us to speculate that only non-mutated beta-catenin could play a role in very early steps of hepatocarcinogenesis such as initiation and promotion. However, mutated forms of beta-catenin are used in experimental models to assess the role of activated beta-catenin in hepatocarcinogenesis. In these experimental mouse models, it is well shown that mutated beta-catenin is insufficient alone and per se for initiation of HCC but only enhance tumor promotion either in a context of chromosomal instability and increase of susceptibility to DEN-induced HCC formation [
      • Nejak-Bowen K.N.
      • Thompson M.D.
      • Singh S.
      • Bowen Jr., W.C.
      • Dar M.J.
      • Khillan J.
      • et al.
      Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin.
      ,
      • Aleksic K.
      • Lackner C.
      • Geigl J.B.
      • Schwarz M.
      • Auer M.
      • Ulz P.
      • et al.
      Evolution of genomic instability in diethylnitrosamine-induced hepatocarcinogenesis in mice.
      ], or in a context of Lkb1+/− mice that spontaneously develop multiple hepatic nodular foci (NdFc) followed by HCC [
      • Miyoshi H.
      • Deguchi A.
      • Nakau M.
      • Kojima Y.
      • Mori A.
      • Oshima M.
      • et al.
      Hepatocellular carcinoma development induced by conditional beta-catenin activation in Lkb1+/- mice.
      ], or in a context of H-Ras transgenic mice where mutated beta-catenin appears as a strong carcinogenic co-factor collaborating with the mutated Ras oncogene [
      • Harada N.
      • Oshima H.
      • Katoh M.
      • Tamai Y.
      • Oshima M.
      • Taketo M.M.
      Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations.
      ]. In contrast and apparently paradoxically, invalidation of beta-catenin in hepatic beta-catenin conditional knockout mice has been found as enhancing DEN-induced tumorigenesis [
      • Zhang X.F.
      • Tan X.
      • Zeng G.
      • Misse A.
      • Singh S.
      • Kim Y.
      • et al.
      Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling.
      ]. Of interest is another model of HCC developing in mice under exposure to phenobarbital (PB, potent tumor promoter in mouse liver) and DEN as tumor initiator. A tumor initiation–promotion study was conducted in mice with conditional hepatocyte-specific knockout (KO) of Ctnnb1 and in Ctnnb1 wild type controls. As expected, DEN + PB strongly enhanced liver tumor formation in Ctnnb1 wild type mice. Amazingly, the prevalence of tumors in Ctnnb1 KO mice was 7-fold higher than in wild type mice, suggesting an enhancing effect of the gene KO on liver tumor development [
      • Rignall B.
      • Braeuning A.
      • Buchmann A.
      • Schwarz M.
      Tumor formation in liver of conditional beta-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen.
      ]. Thus there is a paradox where the absence of wild type beta-catenin or presence of the mutated form, both lead to enhanced DEN-induced hepatocarcinogenesis. The issue is that the discussion is speculative since the mechanism of increased HCC in conditional beta-catenin KO is unknown. In the design of Wnt inhibitors for therapeutic intervention, these agents do target the Wnt pathway through the soluble beta-catenin cascade, but do not impact on invalidation of the beta-catenin pool involved in the membrane catenin/cadherin complexes involved in cell homeostasis. The beta-catenin therapeutic targeting may need to be personalized, based on the unexpected findings of enhanced tumorigenesis after chemical exposure in hepatocyte-specific beta-catenin conditional knockout mice.
      Although the role of Wnt/β-catenin pathway is debated with respect to the initiation of hepatocarcinogenesis, it is definitively implicated in determining HCC aggressiveness, due to its promotion of increased cell proliferation, migration and invasion. This finding has been further substantiated by ectopic expression of Wnt3 and FZD7, Lrp6 or downregulation of sFRP1, Dkk1 and Dkk4 in HCC cell lines [
      • Kim M.
      • Lee H.C.
      • Tsedensodnom O.
      • Hartley R.
      • Lim Y.S.
      • Yu E.
      • et al.
      Functional interaction between Wnt3 and Frizzled-7 leads to activation of the Wnt/beta-catenin signaling pathway in hepatocellular carcinoma cells.
      ,
      • Tung E.K.
      • Wong B.Y.
      • Yau T.O.
      • Ng I.O.
      Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion.
      ,
      • Fatima S.
      • Lee N.P.
      • Tsang F.H.
      • Kolligs F.T.
      • Ng I.O.
      • Poon R.T.
      • et al.
      Dickkopf 4 (DKK4) acts on Wnt/beta-catenin pathway by influencing beta-catenin in hepatocellular carcinoma.
      ,
      • Qin X.
      • Zhang H.
      • Zhou X.
      • Wang C.
      • Zhang X.
      • Ye L.
      Proliferation and migration mediated by Dkk-1/Wnt/beta-catenin cascade in a model of hepatocellular carcinoma cells.
      ,
      • 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.
      ]. Moreover, recent studies have revealed that the Wnt/β-catenin pathway is also involved in the self-renewal and expansion of HCC initiating cells (i.e., the so-called liver CSC) which also influences tumor aggressiveness and resistance to chemo- radio-therapeutic agents [
      • Yamashita T.
      • Ji J.
      • Budhu A.
      • Forgues M.
      • Yang W.
      • Wang H.Y.
      • et al.
      EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.
      ,
      • Yuzugullu H.
      • Benhaj K.
      • Ozturk N.
      • Senturk S.
      • Celik E.
      • Toylu A.
      • et al.
      Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells.
      ]. Furthermore, Wnt/FZD-mediated signaling could influence tumor microenvironment that supports tumor survival, growth, and size. Recent investigations emphasize the role of sFRP1 in the induction of senescence of tumor-associated fibroblasts after chemotherapeutic treatment [
      • Elzi D.J.
      • Song M.
      • Hakala K.
      • Weintraub S.T.
      • Shiio Y.
      Wnt antagonist SFRP1 functions as a secreted mediator of senescence.
      ,
      • Krtolica A.
      • Campisi J.
      Cancer and aging: a model for the cancer promoting effects of the aging stroma.
      ,
      • Schmitt C.A.
      Senescence, apoptosis and therapy–cutting the lifelines of cancer.
      ].
      It is noteworthy that the canonical and non-canonical Wnt/FZD pathways may have complementary roles in the pathogenesis of HCC. Indeed, β-catenin activation appears to be involved in the tumor initiation phase of hepatic oncogenesis, whereas subsequent activation of non-canonical pathways associated with inactivation of β-catenin may enhance tumor promotion and progression [
      • Yuzugullu H.
      • Benhaj K.
      • Ozturk N.
      • Senturk S.
      • Celik E.
      • Toylu A.
      • et al.
      Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells.
      ]. However, non-canonical pathways can also exhibit opposite effects on tumor behavior, since specific Wnt/FZD combinations are able to function as tumor suppressors [
      • Toyama T.
      • Lee H.C.
      • Koga H.
      • Wands J.R.
      • Kim M.
      Noncanonical Wnt11 inhibits hepatocellular carcinoma cell proliferation and migration.
      ]. Although little is known about the role of Wnt/PKC pathway in HCC, it has been demonstrated that inhibition of PKCβ activity reduces motility and invasion properties of HCC cells [
      • Guo K.
      • Li Y.
      • Kang X.
      • Sun L.
      • Cui J.
      • Gao D.
      • et al.
      Role of PKCbeta in hepatocellular carcinoma cells migration and invasion in vitro: a potential therapeutic target.
      ]. Finally, activation of the Wnt/JNK pathway during HCC progression would presumably support tumor growth, since enhanced JNK activity appears to be involved in HCC cell proliferation both in vitro and in vivo [
      • 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.
      ].

      Identification of molecular targets for therapeutic interventions

      There is some evidence to link the Wnt pathway activation to tumor cell properties characteristic of the malignant phenotype, such as enhanced cell proliferation, migration and invasion, which raises the possibility to target members of this signaling cascade as an attractive therapeutic approach for treatment of HCC [
      • Anastas J.N.
      • Moon R.T.
      WNT signalling pathways as therapeutic targets in cancer.
      ,
      • King T.D.
      • Zhang W.
      • Suto M.J.
      • Li Y.
      Frizzled7 as an emerging target for cancer therapy.
      ] (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Potential Wnt-component targets for therapeutic intervention on tumor development and growth. Inactivation of Wnt signaling pathway could be achieved by: (1) targeting extracellular signaling molecules with monoclonal antibodies, soluble factors or small molecules; (2) preventing the FZD/Dvl interaction; (3) stabilizing the destruction complex or (4) increasing β-catenin proteasomal degradation and (5) preventing the interaction between β-catenin and its co-factors for transactivity in the nucleus. The relationship between therapeutic molecules and their protein targets is indicated by a color code. Molecules in bold have been tested in HCC model, those in italics in other models of tumor growth.

      Targeting extracellular molecules of the Wnt pathway

      Antibody-based therapies directed against the overexpressed Wnt ligands and FZD proteins could provide a therapeutic approach. For instance, preclinical experiments have shown that an anti-Wnt1 monoclonal antibody inhibits the Wnt signaling pathway resulting in enhanced apoptosis and inhibiting cell proliferation, both in vitro and in vivo in a xenograft model of HCC [
      • Wei W.
      • Chua M.S.
      • Grepper S.
      • So S.K.
      Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells.
      ]. These findings have been experimentally validated for several other types of tumors, such as sarcomas, colon, breast, non-small-cell lung cancer, and head-neck squamous cell carcinomas [
      • He B.
      • Reguart N.
      • You L.
      • Mazieres J.
      • Xu Z.
      • Lee A.Y.
      Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations.
      ,
      • He B.
      • You L.
      • Uematsu K.
      • Xu Z.
      • Lee A.Y.
      • Matsangou M.
      • et al.
      A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells.
      ,
      • Mikami I.
      • You L.
      • He B.
      • Xu Z.
      • Batra S.
      • Lee A.Y.
      • et al.
      Efficacy of Wnt-1 monoclonal antibody in sarcoma cells.
      ,
      • Rhee C.S.
      • Sen M.
      • Lu D.
      • Wu C.
      • Leoni L.
      • Rubin J.
      • et al.
      Wnt and frizzled receptors as potential targets for immunotherapy in head and neck squamous cell carcinomas.
      ]. Interestingly, as demonstrated with a colon cancer cell line, this anti-Wnt antibody was able to induce apoptosis even in the presence of downstream mutations in APC or CTNNB1 genes and appeared to be synergistic with docetaxel chemotherapy with respect to therapeutic response [
      • He B.
      • Reguart N.
      • You L.
      • Mazieres J.
      • Xu Z.
      • Lee A.Y.
      Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations.
      ]. Although not tested in HCC tumors thus far, anti-Wnt2 antibodies may be useful to inhibit the Wnt/β-catenin cascade. Such antibodies induce apoptosis and inhibit tumor growth in vivo in several tumor types, including melanoma, mesothelioma, and non-small-cell lung cancer [
      • Mazieres J.
      • You L.
      • He B.
      • Xu Z.
      • Twogood S.
      • Lee A.Y.
      • et al.
      Wnt2 as a new therapeutic target in malignant pleural mesothelioma.
      ,
      • You L.
      • He B.
      • Xu Z.
      • Uematsu K.
      • Mazieres J.
      • Fujii N.
      • et al.
      An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth.
      ,
      • You L.
      • He B.
      • Xu Z.
      • Uematsu K.
      • Mazieres J.
      • Mikami I.
      • et al.
      Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells.
      ]. Since non-canonical pathways seem to be implied in tumor progression, the inhibition of Wnt-related ligand could be considered for therapy. For instance, WNT5A, which seems to be involved in the non-canonical pathway in HCC [
      • Yuzugullu H.
      • Benhaj K.
      • Ozturk N.
      • Senturk S.
      • Celik E.
      • Toylu A.
      • et al.
      Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells.
      ], could be antagonized by the use of anti-WNT5a antibodies. Indeed, in gastric cancer cells where WNT5A activates the non-canonical pathway, its inhibition reduces migration and invasion activities in vitro and in vivo [
      • Hanaki H.
      • Yamamoto H.
      • Sakane H.
      • Matsumoto S.
      • Ohdan H.
      • Sato A.
      • et al.
      An anti-Wnt5a antibody suppresses metastasis of gastric cancer cells in vivo by inhibiting receptor-mediated endocytosis.
      ]. Nevertheless, since the non-canonical pathway could antagonize the canonical one, it might be deleterious to inhibit the former. Anti-FZD7 antibodies that induce apoptosis and decrease cell proliferation both in vitro and in vivo of FZD7 positive Wilms’ tumor cells are also available [
      • Pode-Shakked N.
      • Harari-Steinberg O.
      • Haberman-Ziv Y.
      • Rom-Gross E.
      • Bahar S.
      • Omer D.
      • et al.
      Resistance or sensitivity of Wilms’ tumor to anti-FZD7 antibody highlights the Wnt pathway as a possible therapeutic target.
      ]. More recently, a multispecific antibody that targets both FZD 1, 2, 5, 7, and 8 and mainly affects the canonical signaling pathway has been developed. It triggers a therapeutic reduction of breast, colon, lung and pancreas tumor growth and synergizes with other chemotherapeutic agents as well [
      • Gurney A.
      • Axelrod F.
      • Bond C.J.
      • Cain J.
      • Chartier C.
      • Donigan L.
      • et al.
      Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors.
      ]. Strikingly, this antibody remains effective even in tumor cells with APC or CTNNB1 gene mutations. In addition, FZD co-receptors could also be attractive targets for monoclonal antibody therapy since, in a retinal pigment epithelial cell line, anti-Lrp6 antibody has been shown to inhibit Wnt signaling [
      • Lee K.
      • Hu Y.
      • Ding L.
      • Chen Y.
      • Takahashi Y.
      • Mott R.
      • et al.
      Therapeutic potential of a monoclonal antibody blocking the Wnt pathway in diabetic retinopathy.
      ].
      Another therapeutic strategy would be to trap the endogenous Wnt ligands with the exogenous soluble form of FZD receptors. This approach was reported for FZD7 by Tanaka and colleagues in esophagus carcinoma cells and confirmed later in HCC cells [
      • 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.
      ,
      • Tanaka S.
      • Akiyoshi T.
      • Mori M.
      • Wands J.R.
      • Sugimachi K.
      A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals.
      ]. More recently, Wei and co-workers have developed the same approach using an FZD7 extracellular domain peptide (sFZD7) that can bind to and sequester the soluble Wnt3 ligand. This peptide decreased the viability of HCC cell lines with high specificity, since normal hepatocytes were not sensitive to sFZD7. Moreover, sFZD7 cooperates with doxorubicin to reduce HCC cell proliferation in vitro and in a xenograft murine model as well. Interestingly, it has been shown to be highly efficient and independent of the β-catenin mutational status [
      • Wei W.
      • Chua M.S.
      • Grepper S.
      • So S.K.
      Soluble Frizzled-7 receptor inhibits Wnt signaling and sensitizes hepatocellular carcinoma cells towards doxorubicin.
      ]. Inhibition of Wnt secretion by the small molecules, IWP2 and Wnt-C59 may also prevent autocrine Wnt signaling activation, as observed in colon cancer cell lines. These small molecules are also able to inhibit the progression of mammary tumors in Wnt1 transgenic mice [
      • Chen B.
      • Dodge M.E.
      • Tang W.
      • Lu J.
      • Ma Z.
      • Fan C.W.
      • et al.
      Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.
      ,
      • Proffitt K.D.
      • Madan B.
      • Ke Z.
      • Pendharkar V.
      • Ding L.
      • Lee M.A.
      • et al.
      Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.
      ]. Addition of Wnt antagonist, such as sFRP1 or Wif1, has shown encouraging therapeutic results in HCC cell lines by blocking the Wnt/β-catenin signaling. These soluble molecules induce apoptosis, reduce angiogenesis and cell proliferation both in vitro and in vivo and are not influenced by the CTNNB1 mutation status [
      • 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.
      ]. Other Wnt antagonists such as sFRP2 and sFRP5 should also be considered, since they show similar treatment effects in colon cancer as sFRP1 exhibits in HCC [
      • Suzuki H.
      • Watkins D.N.
      • Jair K.W.
      • Schuebel K.E.
      • Markowitz S.D.
      • Chen W.D.
      • et al.
      Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.
      ]. Interestingly, Dkk1 and sFRP1 addition cooperates with anti-FZD7 antibodies to increase apoptosis in Wilm’s tumor demonstrating the importance of combinatorial therapies [
      • Pode-Shakked N.
      • Harari-Steinberg O.
      • Haberman-Ziv Y.
      • Rom-Gross E.
      • Bahar S.
      • Omer D.
      • et al.
      Resistance or sensitivity of Wilms’ tumor to anti-FZD7 antibody highlights the Wnt pathway as a possible therapeutic target.
      ]. Therapeutic small molecules, such as niclosamide and silibinin, display anti-tumor activity in vitro and in vivo by suppressing Lrp6 expression, leading to inhibition of Wnt/β-catenin signaling in human prostate and breast tumor cells, as well as by promoting induction of apoptosis [
      • Lu W.
      • Lin C.
      • King T.D.
      • Chen H.
      • Reynolds R.C.
      • Li Y.
      Silibinin inhibits Wnt/beta-catenin signaling by suppressing Wnt co-receptor LRP6 expression in human prostate and breast cancer cells.
      ,
      • Lu W.
      • Lin C.
      • Roberts M.J.
      • Waud W.R.
      • Piazza G.A.
      • Li Y.
      Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/beta-catenin pathway.
      ].

      Targeting the Wnt-mediated pathway in the cytosol

      The straight-in approach to inhibit Wnt/β-catenin pathway is to directly target β-catenin by small interfering-RNA or antisense based therapy, which can reduce cell proliferation and survival of HCC cell line, providing a proof of principle for this approach [
      • Sangkhathat S.
      • Kusafuka T.
      • Miao J.
      • Yoneda A.
      • Nara K.
      • Yamamoto S.
      • et al.
      In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors.
      ,
      • Wang X.H.
      • Sun X.
      • Meng X.W.
      • Lv Z.W.
      • Du Y.J.
      • Zhu Y.
      • et al.
      Beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy.
      ,
      • Zeng G.
      • Apte U.
      • Cieply B.
      • Singh S.
      • Monga S.P.
      SiRNA-mediated beta-catenin knockdown in human hepatoma cells results in decreased growth and survival.
      ]. However, its potential use as a therapeutic tool remains unlikely since β-catenin protein is essential for cell junction. Thus, targeting the soluble active pool of β-catenin seems more appropriate.
      The interaction between the cytosolic tail of FZD and its adaptor Dvl protein is of importance in mediating Wnt signaling. A proof-of-principle has clearly been established in HCC cells, by using small interfering peptides capable of entering the tumor cells and disrupting the interaction between a specific motif on the FZD7 cytosolic tail and the PDZ domain of Dvl [
      • Nambotin S.B.
      • Lefrancois L.
      • Sainsily X.
      • Berthillon P.
      • Kim M.
      • Wands J.R.
      • et al.
      Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.
      ]. Similar results have been obtained in melanoma and non-small-cell lung cancer cells with small molecules using this same strategy [
      • Fujii N.
      • You L.
      • Xu Z.
      • Uematsu K.
      • Shan J.
      • He B.
      • et al.
      An antagonist of dishevelled protein-protein interaction suppresses beta-catenin-dependent tumor cell growth.
      ].
      Targeting the β-catenin destruction complex (APC, Axin, CK1, and GSK3β) as a therapeutic target has not been assessed in HCC so far. However, using other tumor model systems, such a strategy has demonstrated some potential. Since Axin1 overexpression induces apoptosis in HCC harboring APC, Axin1 or CTNNB1 mutations, stabilization of Axin1 would be an attractive approach to trigger β-catenin degradation [
      • Satoh S.
      • Daigo Y.
      • Furukawa Y.
      • Kato T.
      • Miwa N.
      • Nishiwaki T.
      • et al.
      AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.
      ]. This may be achieved by using inhibitors of the Axin1 or/and 2 degradation, such as the smalls peptides IWR2, JW55 or XAV939 that inhibit the Wnt/β-catenin pathway, leading to a decreased proliferation of colon and breast cancer cell lines. Nevertheless, recent findings support the idea that this decrease may be restricted to low nutriment conditions, and emphasizes that stabilization of Axin needs to be combined with other therapeutic approaches [
      • Chen B.
      • Dodge M.E.
      • Tang W.
      • Lu J.
      • Ma Z.
      • Fan C.W.
      • et al.
      Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.
      ,
      • Huang S.M.
      • Mishina Y.M.
      • Liu S.
      • Cheung A.
      • Stegmeier F.
      • Michaud G.A.
      • et al.
      Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
      ,
      • Waaler J.
      • Machon O.
      • Tumova L.
      • Dinh H.
      • Korinek V.
      • Wilson S.R.
      • et al.
      A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.
      ,
      • Bao R.
      • Christova T.
      • Song S.
      • Angers S.
      • Yan X.
      • Attisano L.
      Inhibition of tankyrases induces Axin stabilization and blocks Wnt signalling in breast cancer cells.
      ]. Preventing β-catenin stabilization through GSK3β activation would also be possible due to the discovery of differentiation-inducing factors (DIFs), which are natural metabolites expressed by Dictyostelium discoideum. Although the mechanisms of action of DIFs activity remain poorly understood, it is well known that DIFs induce β-catenin degradation and subsequently reduce cyclin D1 expression and function [
      • Takahashi-Yanaga F.
      • Sasaguri T.
      Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: inhibitors of the Wnt/beta-catenin signaling pathway as novel anticancer drugs.
      ]. CK1α, another component of the destruction complex, may be stabilized by pyrvinium that inhibits both Wnt signaling and cell proliferation, even in the presence of APC or CTNNB1 mutations, as observed in colon cancer cell lines [
      • Thorne C.A.
      • Hanson A.J.
      • Schneider J.
      • Tahinci E.
      • Orton D.
      • Cselenyi C.S.
      • et al.
      Small-molecule inhibition of Wnt signaling through activation of casein kinase 1alpha.
      ]. Another therapeutic approach would be to enhance β-catenin proteasomal degradation. In HCC, colon and prostate cancer cell lines, the small molecule antagonist CGK062 has been shown to exert such an effect, via the induction of β-catenin phosphorylation in the N-terminal domain which promotes its degradation [
      • Gwak J.
      • Lee J.H.
      • Chung Y.H.
      • Song G.Y.
      • Oh S.
      Small molecule-based promotion of PKCalpha-mediated beta-catenin degradation suppresses the proliferation of CRT-positive cancer cells.
      ]. Two chemicals agents, hexachlorophene and isoreserpine, upregulate Siah-1, an ubiquitin ligase that induces β-catenin degradation, independent of its phosphorylation status, thereby inhibiting Wnt signaling and subsequently has been shown to reduce colon cancer cell proliferation [
      • Yao H.
      • Ashihara E.
      • Maekawa T.
      Targeting the Wnt/beta-catenin signaling pathway in human cancers.
      ].

      Targeting the Wnt pathway in the nucleus

      Finally, an alternative way to block Wnt-mediated signaling is to target the nuclear β-catenin per se and/or the co-factors responsible for transcription of downstream Wnt-responsive genes. To accomplish this aim, several small molecules have been identified. The FH535 agent prevents both Wnt- and PPAR- (Peroxisome Proliferator-Activated Receptors) mediated signaling by suppressing the recruitment of β-catenin co-activators to target gene promoters and has been shown to be active in HCC, colon, and lung tumor cell lines [
      • Handeli S.
      • Simon J.A.
      A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities.
      ]. PKF115-584, PKF118-310, and CGP049090 are inhibitors of TCF/β-catenin binding to DNA target sequences. They induce apoptosis in vitro and in vivo, as well as cell cycle arrest at the G1/S phase and suppress tumor growth in vivo independently of the mutated status of CTNNB1 [
      • Wei W.
      • Chua M.S.
      • Grepper S.
      • So S.
      Small molecule antagonists of Tcf4/beta-catenin complex inhibit the growth of HCC cells in vitro and in vivo.
      ]. Furthermore, inhibition of β-catenin/CBP interaction by ICG-001 both selectively induces apoptosis in transformed, but not in normal colonic cells and reduces growth of colon carcinoma cells in vitro as well as in vivo [
      • Emami K.H.
      • Nguyen C.
      • Ma H.
      • Kim D.H.
      • Jeong K.W.
      • Eguchi M.
      • et al.
      A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].
      ]. A second generation ICG-001 (PRI-724) is also available and in phase-I clinical trial (http://clinicaltrials.gov/show/NCT01302405). Other β-catenin binding proteins such as TBP, Bcl9, and Pygo also represent attractive approaches for inactivating Wnt signaling. Finally, interferon can inhibit β-catenin signaling through upregulation of RanBP3 that is a nuclear export factor, serving as extruding β-catenin outside the nucleus [
      • Thompson M.D.
      • Dar M.J.
      • Monga S.P.
      Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of beta-catenin.
      ].

      Conclusions and perspectives

      Developmental regulated signaling pathways, such as Notch, Hedgehog and Wnt, have become important targets for new cancer drug development. While Notch and Hedgehog inhibitors are already in clinical trials, the Wnt inhibitors are still under preclinical assessment and only a few compounds have started to reach the phase-I clinical trials, since only recently has this pathway been recognized as playing a key role in tumor development. However, many studies have established proof-of-principle that specific targeting of molecules in this pathway can partially or fully switch off canonical as well as non-canonical Wnt signaling and lead to substantial anti-tumor activity. Thus, biotechnology and pharmaceutical organizations are currently developing Wnt signaling inhibitors. These inhibitors can target upstream or downstream proteins in this pathway. Targeting the Wnt cascade upstream of APC is controversial because downstream activating mutations in APC would, in theory, still drive tumor development. To cover the broadest number of activating mutations that occur in tumors, it seems that the ideal antagonist would be one that exerts its anti-tumor effect in the nucleus. Nevertheless, several experiments show that upstream targeting can also be very effective. Of importance is the potential toxicity of Wnt inhibitors on normal cells. Indeed, the Wnt pathway is critical for tissue and liver regeneration and for the ability of stem cells to self-renewal. Wnt pathway inhibitors could therefore have substantial and long-term side effects including anemia, immune suppression, as well as damage of the gastrointestinal tract. It is unknown what may occur in an adult mammal when this pathway is shut down or reduced in normal activity. Despite these known and unknown pitfalls, drug development is moving steadily forward to generate and characterize Wnt pathway inhibitors both in vitro and in vivo. Indeed, agents that inhibit Wnt/β-catenin signaling as a means to produce anti-tumor effects are currently being assessed in clinical trials.

      Financial support

      F. Pez is supported by the French National Institute of Cancer (INCA), A. Lopez by the French Ligue Nationale Contre le Cancer (LNCC).

      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

        • Bosch F.X.
        • Ribes J.
        • Cleries R.
        • Diaz M.
        Epidemiology of hepatocellular carcinoma.
        Clin Liver Dis. 2005; 9: 191-211
        • Kawada N.
        • Imanaka K.
        • Kawaguchi T.
        • Tamai C.
        • Ishihara R.
        • Matsunaga T.
        • et al.
        Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis.
        J Gastroenterol. 2009; 44: 1190-1194
        • Parkin D.M.
        • Bray F.
        • Ferlay J.
        • Pisani P.
        Estimating the world cancer burden: Globocan 2000.
        Int J Cancer. 2001; 94: 153-156
        • Llovet J.M.
        • Di Bisceglie A.M.
        • Bruix J.
        • Kramer B.S.
        • Lencioni R.
        • Zhu A.X.
        • et al.
        Design and endpoints of clinical trials in hepatocellular carcinoma.
        J Natl Cancer Inst. 2008; 100: 698-711
        • Villanueva A.
        • Hernandez-Gea V.
        • Llovet J.M.
        Medical therapies for hepatocellular carcinoma: a critical view of the evidence.
        Nat Rev Gastroenterol Hepatol. 2012; 10: 34-42
        • Yamashita T.
        • Honda M.
        • Nakamoto Y.
        • Baba M.
        • Nio K.
        • Hara Y.
        • et al.
        Discrete nature of EpCAM(+) and CD90(+) cancer stem cells in human hepatocellular carcinoma.
        Hepatology. 2013; 57: 1484-1497
        • Liu S.
        • Dontu G.
        • Wicha M.S.
        Mammary stem cells, self-renewal pathways, and carcinogenesis.
        Breast Cancer Res. 2005; 7: 86-95
        • Chen F.
        • Li Y.
        • Wang L.
        • Hu L.
        Knockdown of BMI-1 causes cell-cycle arrest and derepresses p16INK4a, HOXA9 and HOXC13 mRNA expression in HeLa cells.
        Med Oncol. 2011; 28: 1201-1209
        • Lu F.L.
        • Yu C.C.
        • Chiu H.H.
        • Liu H.E.
        • Chen S.Y.
        • Lin S.
        • et al.
        Sonic hedgehog antagonists induce cell death in acute myeloid leukemia cells with the presence of lipopolysaccharides, tumor necrosis factor-alpha, or interferons.
        Invest New Drugs. 2013; 31: 823-832
        • McAuliffe S.M.
        • Morgan S.L.
        • Wyant G.A.
        • Tran L.T.
        • Muto K.W.
        • Chen Y.S.
        • et al.
        Targeting Notch, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy.
        Proc Natl Acad Sci U S A. 2012; 109: E2939-E2948
        • Nambotin S.B.
        • Lefrancois L.
        • Sainsily X.
        • Berthillon P.
        • Kim M.
        • Wands J.R.
        • et al.
        Pharmacological inhibition of Frizzled-7 displays anti-tumor properties in hepatocellular carcinoma.
        J Hepatol. 2011; 54: 288-299
        • Nusse R.
        • Varmus H.E.
        Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.
        Cell. 1982; 31: 99-109
        • van Ooyen A.
        • Kwee V.
        • Nusse R.
        The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences.
        EMBO J. 1985; 4: 2905-2909
        • Sharma R.P.
        • Chopra V.L.
        Effect of the Wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster.
        Dev Biol. 1976; 48: 461-465
        • Nusse R.
        • Brown A.
        • Papkoff J.
        • Scambler P.
        • Shackleford G.
        • McMahon A.
        • et al.
        A new nomenclature for int-1 and related genes: the Wnt gene family.
        Cell. 1991; 64: 231
        • Vinson C.R.
        • Conover S.
        • Adler P.N.
        A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains.
        Nature. 1989; 338: 263-264
        • Gebhardt R.
        • Hovhannisyan A.
        Organ patterning in the adult stage: the role of Wnt/beta-catenin signaling in liver zonation and beyond.
        Dev Dyn. 2010; 239: 45-55
        • Cadigan K.M.
        • Nusse R.
        Wnt signaling: a common theme in animal development.
        Genes Dev. 1997; 11: 3286-3305
        • Clevers H.
        Wnt/beta-catenin signaling in development and disease.
        Cell. 2006; 127: 469-480
        • Huelsken J.
        • Behrens J.
        The Wnt signalling pathway.
        J Cell Sci. 2002; 115: 3977-3978
        • Armstrong A.
        • Ryu Y.K.
        • Chieco D.
        • Kuruvilla R.
        Frizzled3 is required for neurogenesis and target innervation during sympathetic nervous system development.
        J Neurosci. 2011; 31: 2371-2381
        • Zhang Z.
        • Rankin S.A.
        • Zorn A.M.
        Different thresholds of Wnt-Frizzled 7 signaling coordinate proliferation, morphogenesis and fate of endoderm progenitor cells.
        Dev Biol. 2013; 378: 1-12
        • Mikels A.J.
        • Nusse R.
        Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context.
        PLoS Biol. 2006; 4: e115
        • Graham T.A.
        • Weaver C.
        • Mao F.
        • Kimelman D.
        • Xu W.
        Crystal structure of a beta-catenin/Tcf complex.
        Cell. 2000; 103: 885-896
        • Ozawa M.
        • Baribault H.
        • Kemler R.
        The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.
        EMBO J. 1989; 8: 1711-1717
        • Brantjes H.
        • Roose J.
        • van De Wetering M.
        • Clevers H.
        All Tcf HMG box transcription factors interact with Groucho-related co-repressors.
        Nucleic Acids Res. 2001; 29: 1410-1419
        • MacDonald B.T.
        • Tamai K.
        • He X.
        Wnt/beta-catenin signaling: components, mechanisms, and diseases.
        Dev Cell. 2009; 17: 9-26
        • Liu C.
        • Kato Y.
        • Zhang Z.
        • Do V.M.
        • Yankner B.A.
        • He X.
        Beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation.
        Proc Natl Acad Sci U S A. 1999; 96: 6273-6278
        • Liu C.
        • Li Y.
        • Semenov M.
        • Han C.
        • Baeg G.H.
        • Tan Y.
        • et al.
        Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.
        Cell. 2002; 108: 837-847
        • Hart M.
        • Concordet J.P.
        • Lassot I.
        • Albert I.
        • Del los Santos R.
        • Durand H.
        The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell.
        Curr Biol. 1999; 9: 207-210
        • Kimelman D.
        • Xu W.
        Beta-catenin destruction complex: insights and questions from a structural perspective.
        Oncogene. 2006; 25: 7482-7491
        • Daniels D.L.
        • Weis W.I.
        Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation.
        Nat Struct Mol Biol. 2005; 12: 364-371
        • Arce L.
        • Yokoyama N.N.
        • Waterman M.L.
        Diversity of LEF/TCF action in development and disease.
        Oncogene. 2006; 25: 7492-7504
        • Hirano S.
        • Kimoto N.
        • Shimoyama Y.
        • Hirohashi S.
        • Takeichi M.
        Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization.
        Cell. 1992; 70: 293-301
        • Yang J.
        • Weinberg R.A.
        Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis.
        Dev Cell. 2008; 14: 818-829
        • Heuberger J.
        • Birchmeier W.
        Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling.
        Cold Spring Harb Perspect Biol. 2010; 2: a002915
        • Wickline E.D.
        • Awuah P.K.
        • Behari J.
        • Ross M.
        • Stolz D.B.
        • Monga S.P.
        Hepatocyte gamma-catenin compensates for conditionally deleted beta-catenin at adherens junctions.
        J Hepatol. 2011; 55: 1256-1262
        • Wickline E.D.
        • Du Y.
        • Stolz D.B.
        • Kahn M.
        • Monga S.P.
        Gamma-Catenin at adherens junctions: mechanism and biologic implications in hepatocellular cancer after beta-catenin knockdown.
        Neoplasia. 2013; 15: 421-434
        • Jones C.
        • Chen P.
        Planar cell polarity signaling in vertebrates.
        Bioessays. 2007; 29: 120-132
        • Oishi I.
        • Suzuki H.
        • Onishi N.
        • Takada R.
        • Kani S.
        • Ohkawara B.
        • et al.
        The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway.
        Genes Cells. 2003; 8: 645-654
        • Ishitani T.
        • Kishida S.
        • Hyodo-Miura J.
        • Ueno N.
        • Yasuda J.
        • Waterman M.
        • et al.
        The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling.
        Mol Cell Biol. 2003; 23: 131-139
        • Bafico A.
        • Gazit A.
        • Pramila T.
        • Finch P.W.
        • Yaniv A.
        • Aaronson S.A.
        Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling.
        J Biol Chem. 1999; 274: 16180-16187
        • Jones S.E.
        • Jomary C.
        Secreted Frizzled-related proteins: searching for relationships and patterns.
        Bioessays. 2002; 24: 811-820
        • Patthy L.
        The WIF module.
        Trends Biochem Sci. 2000; 25: 12-13
        • Uren A.
        • Reichsman F.
        • Anest V.
        • Taylor W.G.
        • Muraiso K.
        • Bottaro D.P.
        • et al.
        Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling.
        J Biol Chem. 2000; 275: 4374-4382
        • Mao B.
        • Wu W.
        • Davidson G.
        • Marhold J.
        • Li M.
        • Mechler B.M.
        • et al.
        Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.
        Nature. 2002; 417: 664-667
        • Wang K.
        • Zhang Y.
        • Li X.
        • Chen L.
        • Wang H.
        • Wu J.
        • et al.
        Characterization of the Kremen-binding site on Dkk1 and elucidation of the role of Kremen in Dkk-mediated Wnt antagonism.
        J Biol Chem. 2008; 283: 23371-23375
        • Itasaki N.
        • Jones C.M.
        • Mercurio S.
        • Rowe A.
        • Domingos P.M.
        • Smith J.C.
        • et al.
        Wise, a context-dependent activator and inhibitor of Wnt signalling.
        Development. 2003; 130: 4295-4305
        • Semenov M.
        • Tamai K.
        • He X.
        SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor.
        J Biol Chem. 2005; 280: 26770-26775
        • Capurro M.I.
        • Xiang Y.Y.
        • Lobe C.
        • Filmus J.
        Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling.
        Cancer Res. 2005; 65: 6245-6254
        • Binnerts M.E.
        • Kim K.A.
        • Bright J.M.
        • Patel S.M.
        • Tran K.
        • Zhou M.
        • et al.
        R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6.
        Proc Natl Acad Sci U S A. 2007; 104: 14700-14705
        • Nam J.S.
        • Turcotte T.J.
        • Smith P.F.
        • Choi S.
        • Yoon J.K.
        Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression.
        J Biol Chem. 2006; 281: 13247-13257
        • Xu Q.
        • Wang Y.
        • Dabdoub A.
        • Smallwood P.M.
        • Williams J.
        • Woods C.
        • et al.
        Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.
        Cell. 2004; 116: 883-895
        • Lachenmayer A.
        • Alsinet C.
        • Savic R.
        • Cabellos L.
        • Toffanin S.
        • Hoshida Y.
        • et al.
        Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.
        Clin Cancer Res. 2012; 18: 4997-5007
        • 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
        • 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
        • 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
        • 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 U S A. 1998; 95: 8847-8851
        • Guichard C.
        • Amaddeo G.
        • Imbeaud S.
        • Ladeiro Y.
        • Pelletier L.
        • Maad I.B.
        • et al.
        Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
        Nat Genet. 2012; 44: 694-698
        • Park J.Y.
        • Park W.S.
        • Nam S.W.
        • Kim S.Y.
        • Lee S.H.
        • Yoo N.J.
        • et al.
        Mutations of beta-catenin and AXIN I genes are a late event in human hepatocellular carcinogenesis.
        Liver Int. 2005; 25: 70-76
        • 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
        • Amaddeo G.
        • Guichard C.
        • Imbeaud S.
        • Zucman-Rossi J.
        Next-generation sequencing identified new oncogenes and tumor suppressor genes in human hepatic tumors.
        Oncoimmunology. 2012; 1: 1612-1613
        • Suzuki T.
        • Yano H.
        • Nakashima Y.
        • Nakashima O.
        • Kojiro M.
        Beta-catenin expression in hepatocellular carcinoma: a possible participation of beta-catenin in the dedifferentiation process.
        J Gastroenterol Hepatol. 2002; 17: 994-1000
        • 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
        • 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
        • Kim M.
        • Lee H.C.
        • Tsedensodnom O.
        • Hartley R.
        • Lim Y.S.
        • Yu E.
        • et al.
        Functional interaction between Wnt3 and Frizzled-7 leads to activation of the Wnt/beta-catenin signaling pathway in hepatocellular carcinoma cells.
        J Hepatol. 2008; 48: 780-791
        • Wei W.
        • Chua M.S.
        • Grepper S.
        • So S.K.
        Blockade of Wnt-1 signaling leads to anti-tumor effects in hepatocellular carcinoma cells.
        Mol Cancer. 2009; 8: 76
        • Lee H.H.
        • Uen Y.H.
        • Tian Y.F.
        • Sun C.S.
        • Sheu M.J.
        • Kuo H.T.
        • et al.
        Wnt-1 protein as a prognostic biomarker for hepatitis B-related and hepatitis C-related hepatocellular carcinoma after surgery.
        Cancer Epidemiol Biomarkers Prev. 2009; 18: 1562-1569
        • Tung E.K.
        • Wong B.Y.
        • Yau T.O.
        • Ng I.O.
        Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion.
        PLoS One. 2012; 7: e36565
        • Yoshikawa H.
        • Matsubara K.
        • Zhou X.
        • Okamura S.
        • Kubo T.
        • Murase Y.
        • et al.
        WNT10B functional dualism: beta-catenin/Tcf-dependent growth promotion or independent suppression with deregulated expression in cancer.
        Mol Biol Cell. 2007; 18: 4292-4303
        • Deng Y.
        • Yu B.
        • Cheng Q.
        • Jin J.
        • You H.
        • Ke R.
        • et al.
        Epigenetic silencing of WIF-1 in hepatocellular carcinomas.
        J Cancer Res Clin Oncol. 2010; 136: 1161-1167
        • Ding Z.
        • Qian Y.B.
        • Zhu L.X.
        • Xiong Q.R.
        Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma.
        World J Gastroenterol. 2009; 15: 2595-2601
        • Fatima S.
        • Lee N.P.
        • Tsang F.H.
        • Kolligs F.T.
        • Ng I.O.
        • Poon R.T.
        • et al.
        Dickkopf 4 (DKK4) acts on Wnt/beta-catenin pathway by influencing beta-catenin in hepatocellular carcinoma.
        Oncogene. 2012; 31: 4233-4244
        • Cadoret A.
        • Ovejero C.
        • Saadi-Kheddouci S.
        • Souil E.
        • Fabre M.
        • Romagnolo B.
        • et al.
        Hepatomegaly in transgenic mice expressing an oncogenic form of beta-catenin.
        Cancer Res. 2001; 61: 3245-3249
        • Harada N.
        • Miyoshi H.
        • Murai N.
        • Oshima H.
        • Tamai Y.
        • Oshima M.
        • et al.
        Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.
        Cancer Res. 2002; 62: 1971-1977
        • Tan X.
        • Apte U.
        • Micsenyi A.
        • Kotsagrelos E.
        • Luo J.H.
        • Ranganathan S.
        • et al.
        Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver.
        Gastroenterology. 2005; 129: 285-302
        • Stauffer J.K.
        • Scarzello A.J.
        • Andersen J.B.
        • De Kluyver R.L.
        • Back T.C.
        • Weiss J.M.
        • et al.
        Coactivation of AKT and beta-catenin in mice rapidly induces formation of lipogenic liver tumors.
        Cancer Res. 2011; 71: 2718-2727
        • Nejak-Bowen K.N.
        • Thompson M.D.
        • Singh S.
        • Bowen Jr., W.C.
        • Dar M.J.
        • Khillan J.
        • et al.
        Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin.
        Hepatology. 2010; 51: 1603-1613
        • Longato L.
        • de la Monte S.
        • Kuzushita N.
        • Horimoto M.
        • Rogers A.B.
        • Slagle B.L.
        • et al.
        Overexpression of insulin receptor substrate-1 and hepatitis Bx genes causes premalignant alterations in the liver.
        Hepatology. 2009; 49: 1935-1943
        • Aleksic K.
        • Lackner C.
        • Geigl J.B.
        • Schwarz M.
        • Auer M.
        • Ulz P.
        • et al.
        Evolution of genomic instability in diethylnitrosamine-induced hepatocarcinogenesis in mice.
        Hepatology. 2011; 53: 895-904
        • Miyoshi H.
        • Deguchi A.
        • Nakau M.
        • Kojima Y.
        • Mori A.
        • Oshima M.
        • et al.
        Hepatocellular carcinoma development induced by conditional beta-catenin activation in Lkb1+/- mice.
        Cancer Sci. 2009; 100: 2046-2053
        • Harada N.
        • Oshima H.
        • Katoh M.
        • Tamai Y.
        • Oshima M.
        • Taketo M.M.
        Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations.
        Cancer Res. 2004; 64: 48-54
        • Zhang X.F.
        • Tan X.
        • Zeng G.
        • Misse A.
        • Singh S.
        • Kim Y.
        • et al.
        Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling.
        Hepatology. 2010; 52: 954-965
        • Rignall B.
        • Braeuning A.
        • Buchmann A.
        • Schwarz M.
        Tumor formation in liver of conditional beta-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen.
        Carcinogenesis. 2010; 32: 52-57
        • Qin X.
        • Zhang H.
        • Zhou X.
        • Wang C.
        • Zhang X.
        • Ye L.
        Proliferation and migration mediated by Dkk-1/Wnt/beta-catenin cascade in a model of hepatocellular carcinoma cells.
        Transl Res. 2007; 150: 281-294
        • 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
        • Yamashita T.
        • Ji J.
        • Budhu A.
        • Forgues M.
        • Yang W.
        • Wang H.Y.
        • et al.
        EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.
        Gastroenterology. 2009; 136: 1012-1024
        • Yuzugullu H.
        • Benhaj K.
        • Ozturk N.
        • Senturk S.
        • Celik E.
        • Toylu A.
        • et al.
        Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells.
        Mol Cancer. 2009; 8: 90
        • Elzi D.J.
        • Song M.
        • Hakala K.
        • Weintraub S.T.
        • Shiio Y.
        Wnt antagonist SFRP1 functions as a secreted mediator of senescence.
        Mol Cell Biol. 2012; 32: 4388-4399
        • Krtolica A.
        • Campisi J.
        Cancer and aging: a model for the cancer promoting effects of the aging stroma.
        Int J Biochem Cell Biol. 2002; 34: 1401-1414
        • Schmitt C.A.
        Senescence, apoptosis and therapy–cutting the lifelines of cancer.
        Nat Rev Cancer. 2003; 3: 286-295
        • Toyama T.
        • Lee H.C.
        • Koga H.
        • Wands J.R.
        • Kim M.
        Noncanonical Wnt11 inhibits hepatocellular carcinoma cell proliferation and migration.
        Mol Cancer Res. 2010; 8: 254-265
        • Guo K.
        • Li Y.
        • Kang X.
        • Sun L.
        • Cui J.
        • Gao D.
        • et al.
        Role of PKCbeta in hepatocellular carcinoma cells migration and invasion in vitro: a potential therapeutic target.
        Clin Exp Metastasis. 2009; 26: 189-195
        • 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
        • Anastas J.N.
        • Moon R.T.
        WNT signalling pathways as therapeutic targets in cancer.
        Nat Rev Cancer. 2012; 13: 11-26
        • King T.D.
        • Zhang W.
        • Suto M.J.
        • Li Y.
        Frizzled7 as an emerging target for cancer therapy.
        Cell Signal. 2012; 24: 846-851
        • He B.
        • Reguart N.
        • You L.
        • Mazieres J.
        • Xu Z.
        • Lee A.Y.
        Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations.
        Oncogene. 2005; 24: 3054-3058
        • He B.
        • You L.
        • Uematsu K.
        • Xu Z.
        • Lee A.Y.
        • Matsangou M.
        • et al.
        A monoclonal antibody against Wnt-1 induces apoptosis in human cancer cells.
        Neoplasia. 2004; 6: 7-14
        • Mikami I.
        • You L.
        • He B.
        • Xu Z.
        • Batra S.
        • Lee A.Y.
        • et al.
        Efficacy of Wnt-1 monoclonal antibody in sarcoma cells.
        BMC Cancer. 2005; 5: 53
        • Rhee C.S.
        • Sen M.
        • Lu D.
        • Wu C.
        • Leoni L.
        • Rubin J.
        • et al.
        Wnt and frizzled receptors as potential targets for immunotherapy in head and neck squamous cell carcinomas.
        Oncogene. 2002; 21: 6598-6605
        • Mazieres J.
        • You L.
        • He B.
        • Xu Z.
        • Twogood S.
        • Lee A.Y.
        • et al.
        Wnt2 as a new therapeutic target in malignant pleural mesothelioma.
        Int J Cancer. 2005; 117: 326-332
        • You L.
        • He B.
        • Xu Z.
        • Uematsu K.
        • Mazieres J.
        • Fujii N.
        • et al.
        An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth.
        Cancer Res. 2004; 64: 5385-5389
        • You L.
        • He B.
        • Xu Z.
        • Uematsu K.
        • Mazieres J.
        • Mikami I.
        • et al.
        Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells.
        Oncogene. 2004; 23: 6170-6174
        • Hanaki H.
        • Yamamoto H.
        • Sakane H.
        • Matsumoto S.
        • Ohdan H.
        • Sato A.
        • et al.
        An anti-Wnt5a antibody suppresses metastasis of gastric cancer cells in vivo by inhibiting receptor-mediated endocytosis.
        Mol Cancer Ther. 2012; 11: 298-307
        • Pode-Shakked N.
        • Harari-Steinberg O.
        • Haberman-Ziv Y.
        • Rom-Gross E.
        • Bahar S.
        • Omer D.
        • et al.
        Resistance or sensitivity of Wilms’ tumor to anti-FZD7 antibody highlights the Wnt pathway as a possible therapeutic target.
        Oncogene. 2011; 30: 1664-1680
        • Gurney A.
        • Axelrod F.
        • Bond C.J.
        • Cain J.
        • Chartier C.
        • Donigan L.
        • et al.
        Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors.
        Proc Natl Acad Sci U S A. 2012; 109: 11717-11722
        • Lee K.
        • Hu Y.
        • Ding L.
        • Chen Y.
        • Takahashi Y.
        • Mott R.
        • et al.
        Therapeutic potential of a monoclonal antibody blocking the Wnt pathway in diabetic retinopathy.
        Diabetes. 2012; 61: 2948-2957
        • Tanaka S.
        • Akiyoshi T.
        • Mori M.
        • Wands J.R.
        • Sugimachi K.
        A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals.
        Proc Natl Acad Sci U S A. 1998; 95: 10164-10169
        • Wei W.
        • Chua M.S.
        • Grepper S.
        • So S.K.
        Soluble Frizzled-7 receptor inhibits Wnt signaling and sensitizes hepatocellular carcinoma cells towards doxorubicin.
        Mol Cancer. 2011; 10: 16
        • Chen B.
        • Dodge M.E.
        • Tang W.
        • Lu J.
        • Ma Z.
        • Fan C.W.
        • et al.
        Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.
        Nat Chem Biol. 2009; 5: 100-107
        • Proffitt K.D.
        • Madan B.
        • Ke Z.
        • Pendharkar V.
        • Ding L.
        • Lee M.A.
        • et al.
        Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.
        Cancer Res. 2013; 73: 502-507
        • 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
        • Suzuki H.
        • Watkins D.N.
        • Jair K.W.
        • Schuebel K.E.
        • Markowitz S.D.
        • Chen W.D.
        • et al.
        Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer.
        Nat Genet. 2004; 36: 417-422
        • Lu W.
        • Lin C.
        • King T.D.
        • Chen H.
        • Reynolds R.C.
        • Li Y.
        Silibinin inhibits Wnt/beta-catenin signaling by suppressing Wnt co-receptor LRP6 expression in human prostate and breast cancer cells.
        Cell Signal. 2012; 24: 2291-2296
        • Lu W.
        • Lin C.
        • Roberts M.J.
        • Waud W.R.
        • Piazza G.A.
        • Li Y.
        Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/beta-catenin pathway.
        PLoS One. 2011; 6: e29290
        • Sangkhathat S.
        • Kusafuka T.
        • Miao J.
        • Yoneda A.
        • Nara K.
        • Yamamoto S.
        • et al.
        In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors.
        Int J Oncol. 2006; 28: 715-722
        • Wang X.H.
        • Sun X.
        • Meng X.W.
        • Lv Z.W.
        • Du Y.J.
        • Zhu Y.
        • et al.
        Beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy.
        Oncol Rep. 2010; 24: 1093-1099
        • Zeng G.
        • Apte U.
        • Cieply B.
        • Singh S.
        • Monga S.P.
        SiRNA-mediated beta-catenin knockdown in human hepatoma cells results in decreased growth and survival.
        Neoplasia. 2007; 9: 951-959
        • Fujii N.
        • You L.
        • Xu Z.
        • Uematsu K.
        • Shan J.
        • He B.
        • et al.
        An antagonist of dishevelled protein-protein interaction suppresses beta-catenin-dependent tumor cell growth.
        Cancer Res. 2007; 67: 573-579
        • Satoh S.
        • Daigo Y.
        • Furukawa Y.
        • Kato T.
        • Miwa N.
        • Nishiwaki T.
        • et al.
        AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.
        Nat Genet. 2000; 24: 245-250
        • Huang S.M.
        • Mishina Y.M.
        • Liu S.
        • Cheung A.
        • Stegmeier F.
        • Michaud G.A.
        • et al.
        Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.
        Nature. 2009; 461: 614-620
        • Waaler J.
        • Machon O.
        • Tumova L.
        • Dinh H.
        • Korinek V.
        • Wilson S.R.
        • et al.
        A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.
        Cancer Res. 2012; 72: 2822-2832
        • Bao R.
        • Christova T.
        • Song S.
        • Angers S.
        • Yan X.
        • Attisano L.
        Inhibition of tankyrases induces Axin stabilization and blocks Wnt signalling in breast cancer cells.
        PLoS One. 2012; 7: e48670
        • Takahashi-Yanaga F.
        • Sasaguri T.
        Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: inhibitors of the Wnt/beta-catenin signaling pathway as novel anticancer drugs.
        J Pharmacol Sci. 2009; 109: 179-183
        • Thorne C.A.
        • Hanson A.J.
        • Schneider J.
        • Tahinci E.
        • Orton D.
        • Cselenyi C.S.
        • et al.
        Small-molecule inhibition of Wnt signaling through activation of casein kinase 1alpha.
        Nat Chem Biol. 2010; 6: 829-836
        • Gwak J.
        • Lee J.H.
        • Chung Y.H.
        • Song G.Y.
        • Oh S.
        Small molecule-based promotion of PKCalpha-mediated beta-catenin degradation suppresses the proliferation of CRT-positive cancer cells.
        PLoS One. 2012; 7: e46697
        • Yao H.
        • Ashihara E.
        • Maekawa T.
        Targeting the Wnt/beta-catenin signaling pathway in human cancers.
        Expert Opin Ther Targets. 2011; 15: 873-887
        • Handeli S.
        • Simon J.A.
        A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities.
        Mol Cancer Ther. 2008; 7: 521-529
        • Wei W.
        • Chua M.S.
        • Grepper S.
        • So S.
        Small molecule antagonists of Tcf4/beta-catenin complex inhibit the growth of HCC cells in vitro and in vivo.
        Int J Cancer. 2010; 126: 2426-2436
        • Emami K.H.
        • Nguyen C.
        • Ma H.
        • Kim D.H.
        • Jeong K.W.
        • Eguchi M.
        • et al.
        A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].
        Proc Natl Acad Sci U S A. 2004; 101: 12682-12687
        • Thompson M.D.
        • Dar M.J.
        • Monga S.P.
        Pegylated interferon alpha targets Wnt signaling by inducing nuclear export of beta-catenin.
        J Hepatol. 2011; 54: 506-512