The Wnt/ β-Catenin Pathway
Satdarshan P.S. Monga, George K. Michalopoulos
15
15.1
Background
Genetic studies in species such as Xenopus, Dro-
sophila and Caenorhabditis have lent themselvesquite well to further our understanding of the mo- lecular basis of several diseases. A classical exam- ple is the identification and characterization of the Wnt/ β-catenin pathway, which is crucial in normal development including embryogenesis, organo- genesis and epithelial–mesenchymal interactions;
at the same time, its deregulation is implicated in disorders such as cancers (reviewed in [5, 129, 150]) (Fig. 15.1). This pathway also remains one of the most conserved pathways through the evolutionary process. In Drosophila, the role of Wnt or Wingless (Wg) was initially identified in normal wing devel- opment; however, it was later recognized for multi- ple functions such as inducing segment polarity and anterior–posterior patterning, which are imperative for a viable embryo [9, 154, 183]. As the importance of Wnt emerged, several key components of this pathway were identified. The discovery of armadil- lo (or β-catenin) added a significant player to this orchestra. Although there had been circumstantial evidence suggesting such a relationship existed, β-catenin was not positively identified as a central component of the Wg pathway until a few years later [146, 151, 154, 166]. These studies led to the emer- gence of a model system for cell adhesion and signal transduction [149]. This was also just the begin- ning of understanding of the Wnt/ β-catenin path- way and its role in complex cellular processes such as cell–cell adhesion, mitogenesis, motogenesis and morphogenesis in the vertebrates.
The next several years focused on the discovery of various novel pathway components that improved our understanding of the regulation of this pathway in normal physiology and disease. Several impor- tant players and their interactions were identified, such as the Wnt receptor frizzled (Fz), zeste-white 3 kinase or glycogen synthase kinase 3 β (GSK3β), adenomatous polyposis gene product (APC), axin
and disheveled (Dsh); these were directly influ- enced by the Wnt signaling [19, 59, 89, 139, 152, 172, 189]. Other newer components and interactions, as well as an expanding list of target genes, have since been identified. Research is also focused on their role in regulation of this pathway in health and dis- ease. In addition, several crosstalks have now been established between the Wnt pathway and other prominent pathways such as the Jagged/Notch (see Chapter 16), hepatocyte growth factor (HGF)/Met, epidermal growth factor (EGF) and transforming growth factor (TGF) pathways (see Chapter 12) [45, 64, 65, 125, 138, 142, 240].
Presently, the role of the Wnt/ β-catenin pathway is well established in vertebrates in embryogenesis and carcinogenesis [150, 153]. β-Catenin knockout yields an embryonic lethal phenotype in mice due to a defect in gastrulation [58]. Other studies in verte- brates have also shown its role in anterior–posterior axis specification and mesoderm formation [75, 76].
Availability of conditional knockouts to overcome embryonic lethality has been key to understanding a more ubiquitous role of β-catenin and other Wnt components in the development of many organs such as the kidneys, lungs, brain, limbs, muscles and skin [27, 61, 77, 88, 106, 130]. Its role in liver development is beginning to be uncovered and is discussed in Sect. 15.3.1. This pathway is crucial in stem cell biology, where it is known to regulate stem cell renewal in multiple tissues including hemat- opoietic, epidermal and intestinal compartments [37, 50, 93, 105, 164]. It has also been identified as a prominent player in angiogenesis and vasculogen- esis and maintenance of endothelial cell adhesion [30, 53, 132, 215].
15.2
The Wnt/ β-Catenin
Signal Transduction Pathway
The binding of an extracellular secreted glyco-
protein Wnt to its cell surface receptor Fz induces
specific downstream events consisting of many intricate protein–protein interactions involving meaningful changes in their binding, phosphoryla- tion and localization [14]. Although the most un- derstood and predictable events are the result of the activation of the canonical Wnt pathway (Fig. 15.2), the signaling can be transduced to at least two other branches, which, though independent, still influ- ence and regulate each other. These two pathways are the planar cell polarity pathway and the Wnt/
Ca
2+pathway (Fig. 15.3). How the diversification of these signals is modulated remains under investiga- tion. Recent studies have shown that interactions of the Wnt–Fz complex with the Wnt co-receptor LDL- related protein (LRP) instruct the signaling to follow the canonical route [180]. Also, the serine/threonine kinase Par-1 induces the canonical Wnt/ β-catenin signaling, at the same time inhibiting the planar cell polarity pathway [197]. On the other hand, signals involving cuticle and strabismus (Stbm) or diversin alone detour the pathway to the planar cell polarity path, at the same time inhibiting the canonical Wnt pathway [143, 178, 235]. Finally, other independent pathways are able to significantly impact the state of the Wnt pathway as well.
15.2.1
The Canonical Wnt Pathway
In a normal steady state where excess of β-catenin, a key component of this pathway and a powerful
"oncoprotein", is not needed or in the absence of a Wnt signal, the free monomeric form of β-catenin
Fig. 15.1. Two-state canonical pathway signaling. On the left, in the absence of Wnt or the presence of its inhibitors, β-catenin is phosphorylated at ser/thr residues to be degraded. The right panel shows activation of the pathway in the presence of Wnt, which allows β-catenin to be released from its cytoplasmic complex to translocate to the nucleus and bind to TCF family members and induce target genes. Black boxes oncogenes, gray boxes tumor suppressor genes
Fig. 15.2. All-component signaling in the canonical Wnt path- way. This comprehensive schematic includes most of the com- ponents of this pathway, demonstrating various protein–protein interactions occurring in the extracellular region, subcellular and cytoplasmic region as well as in the nucleus. (This figure has been borrowed with Dr. Roel Nusse’s permission from his web- site at www. stanford.edu/~rnusse/wntwindow.html.)
in the cytoplasm is actively targeted for degrada- tion by ubiquitination. This is comparable to the pathway being in "off" mode (reviewed in [150]). In this situation, β-catenin is being phosphorylated at serine and threonine residues in its amino-termi- nal region, specifically at serine-45 (Ser45), Ser33, Ser33 and threonine-41 (Thr41) by casein kinase I α (CK Ια) and GSK3β [4, 17, 108, 229]. CK and GSK3β are part of a larger multiprotein degradation com- plex that includes axin, which acts as a scaffold to form homodimeric or heterodimeric complexes with axin2/conductin, APC and diversin and each of these plays a role in phosphorylating β-catenin [59, 81, 133]. Once phosphorylated, this larger complex enables recognition and ubiquitination of β-catenin by β-transducin repeat-containing protein (βTrCP) and its ensuing proteosomal degradation [1]. Thus free levels of β-catenin are kept low and it is pre- vented from translocating to the nucleus to induce target gene transcription. These events are also ob- served if Wnts are sequestered or prevented from binding to their receptors. Several such modulators have now been identified. Fz-related proteins (FRPs) are smaller proteins (30 kDa) with Fz-like cysteine- rich domains that bind and sequester Wnts [163].
Similarly, Wnt inhibitory factors (WIFs) bind Wnts to inactivate the pathway [69]. Cerebrus is a more non-specific inhibitor that represses Wnt, nodal and bone morphogenic protein (BMP) signaling [156].
Any of the Wnts (19 members in humans) in the absence of their negative regulators, bind to their seven-transmembrane receptor Fz, which further induces a ternary complex formation with LRP5/6 (or arrow) [19, 158, 204, 217]. This complex is cru- cial in dictating the downstream canonical Wnt/ β- catenin signaling. One of the inhibitors, Dickkopf (Dkk), prevents Wnt-induced Fz–LRP complex for-
mation and hence Wnt signaling [180]. Upon forma- tion of the ternary complex, signal is transduced through multiple intermediate proteins finally to induce hypophosphorylation of β-catenin at the APC-axin-GSK3 β-CK complex. One such interac- tion is the activation of Dsh, which blocks β-catenin degradation by recruiting GSK3 β-binding protein (GBP)/Frat-1, which displaces GSK3 β from axin, re- sulting in its inactivation [104, 174]. Also, Dsh can bind to phosphatase PP2C, which enables it to de- phosphorylate axin [195]. Dsh can also potentiate β- catenin stabilization following its activation by the serine/threonine kinase Par-1 [197]. CKs are yet an- other group of proteins that consist of two unrelated kinases (CK1 ε and CK2), which associate to, phos- phorylate and thus activate Dsh [155, 173, 192, 221].
Another recent addition is LKB1 (XEEK1), which associates to and regulates GSK3 β activity by phos- phorylation, activating the pathway. The end result is the hypophosphorylation of β-catenin at specific serine and threonine residues, its release from the multi-protein complex, cytoplasmic stabilization of its monomeric form and ensuing nuclear transloca- tion where it bind to an HMG box containing DNA- binding protein T cell factor/lymphoid-enhancing factor (TCF/LEF) family member [26, 165].
Once the TCF– β-catenin complex is formed in the nucleus, there is transcriptional activation of several target genes that have now been identified (Table 15.1). At the same time, tissue- and stage- specificity in the target gene transactivation is be- coming evident. Another important aspect is to understand how the transcriptional activation is being regulated. Apart from the targets listed in Ta- ble 15.1, several Wnt components such as dFz7, Fz2, FRP2, WISP, βTrCP and TCF are themselves targets, suggesting the existence of several regulatory loops
Fig. 15.3. Left panel Wnt/Ca2+ path- way. Wnt signaling incites intracyto- plasmic Ca2+ accumulation through the Ins(1,4,5)P3 receptor. This in turn activates Ca2+-calmodulin-dependent protein kinase II, protein kinase C or calcineurin. Calcineurin induces nu- clear translocation of NF-AT to acti- vate target genes. Right panel Planar polarity pathway where frizzled and disheveled (or axin) via Daam1 acti- vate the JNK or Rho-associated kinase (ROCK) to induce cytoskeletal changes to achieve planar polarity
within this pathway. In the absence of Wnt signal- ing, TCF inside the nucleus acts as a repressor of the target genes and it does so at least in conjunc- tion with a co-repressor Groucho and interactions with histone deacetylase Rpd3 [20, 31, 32]. In the presence of a Wnt signal, β-catenin can induce the transcriptional activation capability of TCF and the two important players identified at this level are the legless or Bcl9 and pygopos. Legless promotes
recruitment of pygopos to β-catenin in the nucleus and permits it to become transcriptionally active [95, 145]. Another positive regulator to be identi- fied is Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodeling complexes. It has been shown that β-catenin recruits Brg-1 to the TCF target gene promoters to assist in chromatin remod- eling, which is necessary for transcriptional activa- tion [13]. CREB-binding protein (CBP), which is a
Table 15.1. List of prominent target genes of the Wnt/β-catenin pathway
Target genes Model Regulation Reference
Axin-2 Human colon cancer Upregulated [228]
BMP4 Human colon cancer Upregulated [86]
Xenopus Downregulated [10]
C-Jun Human colon cancer Upregulated [110]
C-Myc Human colon cancer Upregulated [62]
Cdx1 Mouse Wnt3A Upregulated [105]
Cdx4 Zebrafish Upregulated [42]
Connexin-43 Xenopus, mouse Upregulated [213]
Cyclo-oxygenase-2 Mouse (Wnt1) Upregulated [68]
3T3L1 preadipocytes Upregulated [109]
Cyclin-D1 Human colon cancer Upregulated [188, 208]
E-Cadherin Mouse hair follicles Downregulated [82]
FGF4 Mouse tooth bud Upregulated [96]
Fibronectin Xenopus Upregulated [55]
G-Protein-coupled receptor 49 (Gpr49) Hepatocellular cancer Upregulated [227]
Glutamate transporter-1 (GLT-1) Mouse liver Upregulated [29]
Glutamine synthetase (GS) Mouse liver Upregulated [29]
IGF-I/IGF-II 3T3L1 Preadipocytes Upregulated [109]
Keratin Mouse hair follicle Upregulated [41]
MMP-7 Human colon cancer Upregulated [23, 38]
Ornithine aminotransferase Mouse liver Upregulated [29]
PPAR-δ Human colon cancer Upregulated [10]
Survivin Human colon cancer Upregulated [236]
TCF-1 Human colon cancer Upregulated [168]
uPAR Human colon cancer Upregulated [110]
VEGF Human colon cancer Upregulated [237]
known co-activator for several transcription fac- tors, was shown to repress TCF in Drosophila [216].
However, in vertebrates, CBP and another related acetyltransferase p300 acted as a transcriptional co- activator in β-catenin-TCF transcription machinery [63, 203]. Two other homologous proteins, pontin52 and reptin52, bind to β-catenin, and function as its antagonistic regulators [15, 16]. Finally, another protein that deserves a mention is Chibby, which functions as a competitive inhibitor of β-catenin- mediated transcriptional activation by competing with LEF-1 [202].
15.2.2
The Wnt/Ca2+ Pathway
The first evidence of the existence of such a path- way came from a comparable phenotype that was observed in Xenopus following overexpression of Wnt5A and overexpression of the 5HT1c serotonin receptor [7, 191]. At that time this serotonin receptor was known to stimulate Ca
2+release in a G-protein- dependent fashion, suggesting the possibility that a similar pathway might be occurring in response to Wnt5A [7]. Further analysis identified a rat Fz- 2 (rFz2) that induced intracellular Ca
2+release in response to Wnt5A activation via interaction with the phosphatidylinositol pathway in a G-protein- dependent manner [190, 231]. This induced an in- crease in intracellular Ca
2+that in turn stimulated two major Ca
2+-sensitive enzymes, Ca
2+/calmodu- lin-dependent protein kinase II (CamKII) and pro- tein kinase C (PKC) [97, 184]. CamKII activation was shown by in vitro kinase activity and increased autophosphorylation and the activation of PKC was demonstrated by in vitro kinase activity and mem- brane translocation. These events occurred in a β-catenin-independent manner as shown by the in- ability of Wnt-8 and Rfz-1 (activate canonical sign- aling) to activate either CamKII or PKC activation (reviewed in [98]). More functional evidence came from the examination of this pathway as a “ventral- izing” inducer in Xenopus. It was shown that the elevated levels of intracellular Ca
2+in response to Wnt also activated phosphatase calcineurin, which initiated dephosphorylation of the transcription factor NF-AT, allowing its nuclear translocation and activation of target genes [175]. How this pathway is regulated in relation to the canonical Wnt path- way is still unclear but it is suggested that NF-AT or its targets might influence the canonical pathway downstream of Dsh and upstream of β-catenin to balance the dorsal–ventral axis formation [175].
15.2.3
The Planar Cell Polarity Pathway
Additional studies uncovered yet another pathway that involves Wnt signaling and is distinct from the two pathways described so far (reviewed in [214]).
Clues for the existence of this branch of the path- way emerged initially from the studies in Drosphila wing. As with all epithelial cells, these specialized cells are polarized along their apical–basal axis. In addition, they exhibit planar polarity that arranges the cells within the epithelial plane of the wing in a proximal–distal axis. This involves rearrangement of the cellular cytoskeleton along the proximal–dis- tal axis such that actin is polymerized at its distal tip, which forms wing hairs that uniformly point distally [reviewed in [148]. The quest for such genes led to the discovery of Fz and Dsh as central play- ers in this rearrangement [211]. However, no role of β-catenin could be identified in planar polarity [8].
This triggered intense research to elucidate bifurca- tion of the pathway at the level of Fz and Dsh [3].
The summary that emerges shows activation of Jun- N-terminal kinase (JNK) in response to Fz. In the wing, Rho and Rho kinase are important intermedi- ates that are downstream of Fz, whereas in the eye, another tissue exhibiting planar polarity, a small guanosine triphosphatase Rho and JNK-mitogen- activated protein kinase (MAPK) are prominent players [22, 56, 112, 207, 224]. Also, Dsh and axin have now been shown directly to activate the JNK pathway, suggesting that they might function in co- operation [238]. Interestingly, loss of function stud- ies of JNK and JNKK show no compromise in planar polarity and it has been suggested that other MAPK components might have a redundant role in this process [116]. This also led to identification of two additional kinases, TAK1 (MAPKKK homolog) and MAPK family member Nemo, which are proposed to function in parallel to the canonical Wnt pathway to confer planar polarity [122, 123]. As it stands now, this pathway branches off from the canonical path- way at Dsh; and involves cadherin-related trans- membrane protein flamingo (Fmi) or Starry Night;
the proteoglycan knypek (Kny); and the PDZ mol- ecule Stbm [143, 186, 209]. Dsh is connected to Rho and Rho-associated kinase (ROCK) via Daam [57].
How the signal is detoured towards the planar po-
larity pathway remains obscure. Recently, a product
of the Wnt target gene naked (Nkd) has been shown
to bind to Dsh and stimulate the JNK pathway, at
the same time blocking β-catenin [171]. It is not yet
known which upstream proteins or which Wnts or
Fz specifically, if at all, favor one pathway versus the
other, or whether it is more of a tissue- or stage-spe-
cific decision. However, it is now evident that planar polarity might be a function of establishing a gra- dient of Wnt and Fz signaling along a specific axis within the sheet of epithelial cells. This has led to the discovery of a role of the cadherin superfamily of adhesion molecules: Fat (Ft), Dachsous (Ds) and Four-jointed (Fj) in Drosophila eye [230]. Wnt regu- lates expression of Ds and Fj, which further generate a gradient of Ft activity; in turn, this establishes an Fz activity gradient along the desired axis. Although further analysis is vital, similar conserved pathways might operate in mammals, conferring cell polarity to specialized cells such as hepatocytes.
15.2.4
β-Catenin–E-Cadherin Interactions
Apart from playing a central role in the canoni- cal Wnt pathway as a transcriptional co-activator, β-catenin performs yet another crucial function by acting as a bridge between the cytoplasmic do- main of the cadherins and the actin-containing cy- toskeleton [90, 136, 220]. It is interesting that each of these two roles is played by a distinct β-catenin in C. elegans [94]. We have reported a smaller spe- cies of β-catenin during liver development, which appears to be located at the membrane and associ- ating to E-cadherin; a functional characterization is pending [121]. Cadherins consist of an extracel- lular domain, a transmembrane domain, and a cy- toplasmic tail, which is the most conserved region among various subtypes. Type I cadherins are the most characterized and consist of E-cadherin and N-cadherin. Structurally, the cytoplasmic tails of cadherins show dimerization and connect to the ac-
tin cytoskeleton via p120, β-catenin and α-catenin (Fig. 15.4). Specific β-catenin-binding sites on the cytoplasmic domain of cadherins have been charac- terized [74, 84]. The significance of regulation of β- catenin–cadherin interactions is not only important in modulating cell–cell adhesion but has been ex- tended to the transcriptional activation function of β-catenin as well. These interactions are regulated by tyrosine phosphorylation and not phosphoryla- tion at serine/threonine residues (reviewed in [107]).
A large body of literature has shown the significance of such interaction by multiple means. Phosphoryla- tion of β-catenin destabilizes the β-catenin–cadher- in bond, and the α-catenin–β-catenin complex, un- coupling cadherin from the actin cytoskeleton and promoting loss of intracellular adhesion [141, 170].
Dephosphorylating β-catenin at tyrosine residues enhanced E-cadherin, β-catenin and α-catenin re- assembly [71]. Following tyrosine phosphorylation of β-catenin, its cytosolic pool is greatly increased, as is its ability to bind to TATA-box-binding protein (TBP) and increased transcriptional activity of the β-catenin/TCF complex [157]. This has also been narrowed down to tyrosine residue 654. Another important ramification of tyrosine phosphorylation of β-catenin and dissociation of the β-catenin–E- cadherin complex is that it leaves the cytoplasmic domain of E-cadherin unstructured and vulnerable to degradation [74].
Fig 4
How is the β-catenin–cadherin complex be- ing regulated? The answer to this question is quite complex, so only the most pertinent regulators are mentioned here. One level of regulation of the caten- in–cadherin complex is via the GTP-bound form of the G α subunit of heterotrimeric G proteins; it has been shown that overexpression of G α12/13 results in dissociation of this complex [114, 115]. Another key regulator of this complex with important intrac- ellular adhesion implications is the protein tyrosine phosphatase 1B (PTP1B) that directly associates to the intracytoplasmic tail of cadherins [11, 225]. It is interesting to note that there is a partial overlap in the binding domains of PTP1B, β-catenin and Gα12, thus adding complexity to the regulation of cadher- in function (reviewed in [107]). Other specific inter- actions that regulate phosphotyrosine- β-catenin in- clude: (a) non-receptor kinases src and Fer [18, 169];
(b) transmembrane kinases EGF receptor (EGFR) and Met (HGF receptor) [21, 65, 85, 185, 200]; (c) protein tyrosine phosphatases including LAR-PTP, the chondroitin sulfate proteoglycan PTP β/ζ, and members of the meprin/A5/Mu (MAM) domain- containing family [24, 25, 51, 117, 131].
Fig. 15.4. Schematic showing the cadherin–catenin complex at the cell membrane. The key regulation sites are marked by as- terisks
15.2.5
Miscellaneous Interactions/Crosstalks
A few other interactions are worth mentioning. We reported a novel Met– β-catenin complex at the hepa- tocyte membrane that appears to be independent of the β-catenin–E-cadherin complex and is liver-spe- cific [125]. HGF induced tyrosine phosphorylation- dependent nuclear translocation of β-catenin with an increase in c-myc by interactions involving the Met– β-catenin complex. Other reports have also identified a similar effect of HGF to induce nuclear translocation and β-catenin/TCF transactivation via the canonical pathway [40, 64, 142]. These ob- servations are relevant as high levels of HGF have been observed in patients with liver pathologies that might be influencing β-catenin redistribution and altering the disease course [187, 226].
Another key crosstalk that has been reported is with the transforming growth factor β (TGFβ). A physical interaction exists between the β-catenin–
TCF complex and smad4. Smad4 is a mediator of the TGF β signaling that interacts with smad2-smad3 heterodimers following TGF β signaling. This cross- talk is likely to be very relevant in the liver, consid- ering the role of TGF β signaling in liver growth and regeneration and also the phenotypes observed in the "loss of function" studies involving this path- way's components, such as embryonic lethality due to compromised liver development and alterations in β-catenin, E-cadherin and β1-integrin in mice lacking a copy of smad2 and smad3 [118, 119, 194, 219].
15.3
Wnt/ β-Catenin Signaling in Liver:
Physiological Relevance
The importance of the Wnt/ β-catenin pathway in liver began to be recognized only in the late 1990s.
Earlier studies had focused on the altered immuno- histochemical expression of β-catenin and E-cad- herin in hepatocellular cancer [HCC]. Other groups initiated studies to examine the mechanism of such an increase, which led to recognition of mutations in the Ctnnb1 ( β-catenin gene) as well as in other com- ponents of the multiprotein-degradation machin- ery. Concurrent studies in cancers of other tissues also contributed significantly to our understand- ing of molecular mechanisms involving this path- way. This also led to the identification of aberrant Wnt/ β-catenin signaling in pediatric liver tumors and hepatic adenomas. Studies were also focused on
liver growth and development, to understand bet- ter the regulation of this pathway in liver physiology and pathology.
15.3.1
Role in Liver Development
Lack of β-catenin yielded an embryonic lethal phe- notype as a result of defects in gastrulation [58]. Due to the availability of "floxed" β-catenin mice, it can be conditionally knocked out utilizing tissue-spe- cific cre recombinase mice such as the albumin-cre or the α-fetoprotein-albumin-cre mice. This work is ongoing and although a definite role of the Wnt/
β-catenin pathway in liver development cannot be inferred at this time, we have performed studies uti- lizing in vitro organ cultures and a comprehensive ontogenic analysis that demonstrate a crucial role of this pathway in early liver development. Liver is first specified from foregut endoderm at around somite stage 5–6 and involves instructive signals from mes- oderm including cardiac mesenchyme and septum transversum specifically in the form of fibroblast growth factors (FGFs) and bone morphogenic pro- tein-4 (BMP4) [233, 234]. We are unaware of the role of the Wnt/ β-catenin pathway in liver initiation at this stage, although strong circumstantial evidence does exist, as both FGF4 and BMP4 are transcrip- tional targets of the canonical Wnt/ β-catenin path- way [86, 96]. This was further strengthened by an- other observation that, although Wnt-1 was present in the liver throughout development, β-catenin was highly temporally regulated. We found high levels of Ctnnb1 and normal β-catenin protein (97 kDa) at embryonic day 10 (E10)–E11 livers, followed by a gradual decrease and disappearance after E16 stage until the perinatal period in ICR/CD-1 mice [121].
Also, the E10–E12 livers showed various percent- ages of resident non-hematopoietic cells displaying membranous, cytoplasmic and nuclear localization of β-catenin that transformed to predominant mem- branous localization at all later stages. Interestingly, membranous localization as well as interaction with E-cadherin persisted even after E16 stage, when normal 97-kDa protein was virtually undetectable and the only form detected was a lower species of around 65 kDa. Another interesting outcome was a significant stage-specific correlation of nuclear and cytoplasmic β-catenin to cell proliferation in devel- oping liver. What would be the impact of decreasing β-catenin levels on liver development? Although a definitive answer of the in vivo significance is being pursued, we answered this question in an ex vivo organ culture system using two different strategies.
We used a modified embryonic liver culture system
from the 1960s and 1970s to investigate the effect of growth factors and to perform in vitro “loss-of- function” studies of developmentally relevant genes using antisense oligonucleotides [66, 67, 101, 124, 128]. Utilizing a similar approach, E10 livers were cultured for 72 h in the presence of phospho-mor- pholino-oligomers against Ctnnb1 [126]. There was a significant ablation of β-catenin protein and a resulting compromise in cell proliferation and cell survival in these cultures. In addition, there was an absence of CK-19 (biliary marker)-positive cells, in- dicating a role of β-catenin in biliary specification of the hepatic bipotential progenitors that constitute the E10 liver [126]. Also, β-catenin inhibition affect- ed hepatocyte maturation. A second strategy uti- lized the same E10 embryonic liver culture system, albeit the culture media was serum-free Wnt3A- conditioned media (containing active Wnt3A), negative control media or the Wnt3A-conditioned media in the presence of sFRP1 (Wnt inhibitor). The results were analogous to the antisense studies and showed survival and proliferation of predominantly CK-19-positive cells in the Wnt-conditioned me- dia as compared to both other conditions that dis- played extensive cell death [79]. Thus there appears to be a definite role of the Wnt/ β-catenin pathway in biliary epithelial cell growth and propagation in the ex vivo liver development model. Utilizing the same model, we have also demonstrated the proc- ess of hepatic progenitor enrichment by exogenous FGF treatment. Further analysis yielded important mechanistic insights into this process by means of β-catenin stabilization and redistribution within the progenitors [179].
15.3.2
Liver Regeneration
Due to the importance of aberrant Wnt/ β-catenin signaling in liver cancer, it is imperative to under- stand the regulation of this pathway in a "regulated"
growth environment. One such widely accepted system in the liver is the two-thirds partial hepatec- tomy model, which involves removal of three of the five lobes (rats), following which the remnant liver is able to restore the lost hepatic mass within 7 days [120]. The Wnt/ β-catenin pathway was comprehen- sively examined in this model [127]. There was a significant increase in the total β-catenin protein within the first few minutes of hepatectomy, which was mediated by an epigenetic or post-translational mechanism and was transcription independent.
There was a concurrent increase in its nuclear trans- location. Interestingly this increase in total protein was transient and there was activation of the β-cat-
enin degradation complex including axin and APC, which led to a significant decrease in total β-catenin protein by 15 minutes of liver regeneration. How- ever, β-catenin persisted in the nuclei of the hepa- tocytes until around 48 h. Normal β-catenin levels were restored at around the same time, probably secondary to increased β-catenin gene expression at 6 h onwards, following hepatectomy. This leads us to believe that there are crucial modulators of the pathway that are able to monitor β-catenin levels in a regulated growth milieu. This is not surprising be- cause of the abundance of this potent "oncoprotein"
mitogen at the membrane of normal hepatocytes. It would be devastating not to have a stringent moni- toring and efficient degradation system to limit unnecessary or sustained β-catenin activation. An- other inference drawn from this study was that the Wnt/ β-catenin pathway might be one of the earliest pathways to become activated following hepatecto- my that might initiate a cascade of events including but not limited to inducing gene expression of c-myc,
cyclin-D1 and uPAR or yet-undiscovered targets.The Met– β-catenin complex in hepatocytes might also be one of the contributing sources of nuclear β-catenin, as elevated tyrosine phosphorylation of Met and activation of HGF are also observed during early liver regeneration [125, 147, 193].
15.3.3 Liver Growth
Other studies have addressed the effect of β-catenin on liver growth. One such study examined the in vivo effect of overexpressing a stable, mutant β-cat- enin that is truncated at the amino-terminal, mak- ing it resistant to degradation due to the absence of serine/threonine phosphorylation sites. Such a construct was originally described in an intestinal tumor model [167]. Mutant β-catenin transgenic mice generated under transcriptional control of cal- bindin-D9K (CaBP9K) promoter and liver-specific enhancer of the aldolase B gene displayed three to four times larger livers due to increased cell prolif- eration [28]. Interestingly the authors did not detect changes in any of the conventional target genes of the pathway such as c-myc and cyclin-D1. Subse- quent analysis of transgenic livers and subtractive hybridization led to identification of three genes that are involved in glutamine metabolism as tar- gets of the Wnt/ β-catenin pathway (Table 15.1).
However, no signs of liver transformation, such as hepatocyte dysplasia or nodule formation were ob- served in these animals. This might suggest the re- quirement of or cooperation with other pathways.
However, the increase in cell proliferation in these
mice is an attractive preneoplastic event that might foster secondary genetic events and eventually lead to β-catenin dysregulation and malignant transfor- mation. Similar studies are ongoing in our labora- tory where we have generated normal β-catenin transgenic (non-truncated form) mice under albu- min promoter/enhancer. These mice show a milder hepatomegaly with a 15%–25% increase in liver weight to body weight ratio due to enhanced cell pro- liferation. More importantly, subsets of animals are able to increase serine/threonine phosphorylation of β-catenin and maintain normal liver size. This model has also revealed EGFR as a potential target of β-catenin in liver. Transgenic livers display around 2.2-fold higher EGFR expression as well as a concur- rent increase in EGFR protein. Ongoing analysis has detected activated EGFR (tyrosine-phosphorylated) in transgenic livers showing β-catenin stabilization that might be therapeutically relevant.
15.4
Wnt/ β-Catenin Signaling in Liver:
Pathological Relevance
The role of the Wnt/ β-catenin pathway in carcino- genesis is unquestionable. Apart from cancer, this pathway is also being implicated in inflammatory and autoimmune pathologies. Any such role in pathogenesis of other liver disease remains under investigation. In the forthcoming sections, we will discuss the aberrations in this pathway in liver tu- mors, with an emphasis on its role in hepatoblas- toma, HCC, and cholangiocarcinoma; other miscel- laneous hepatic disorders are also discussed.
15.4.1
Hepatoblastomas
Hepatoblastomas are the most common malig- nant hepatic tumor found during early childhood.
These embryonal tumors are frequently sporadic;
however, the incidence is highest in patients suffer- ing from familial adenomatous polyposis coli [78].
This led to the identification of APC mutations as the molecular etiology for hepatoblastomas in fa- milial cases [99]. An increased frequency of diverse APC mutations (57%) was then reported in the spo- radic form of the disease as well [140]. Since APC regulates β-catenin levels, the next set of analyses focused on and revealed abnormal β-catenin ac- cumulation and associated amino-terminal muta- tions (exon 3) in around 50% of all sporadic hepa- toblastomas [91]. A number of reports that followed
illustrated nuclear and cytoplasmic localization of β-catenin in 90%–100% of all hepatoblastomas [83, 212, 218]. Predominantly in-frame mutations in the β-catenin gene in the form of deletions or missense were observed in 70%–90% of such cases [83, 218].
Mutations in AXIN1 were also identified in less than 10% of these tumors [205]. Hepatoblastomas as a component of syndromes such as Beckwith-Wiede- mann syndrome have also revealed abnormal Wnt/
β-catenin activation [52, 218]. Thus there is compel- ling data that shows Wnt/ β-catenin aberrations as an obligatory event in the etiopathogenesis of hepa- toblastomas. Use of β-catenin nuclear reactivity as a prognostic indicator for the disease was suggested but is not a widespread practice [144]. Interesting analysis of new members of the Wnt pathway such as the pathway inhibitor Dkk1 has shown overex- pression in hepatoblastomas and is believed to be due to negative feedback related to uncontrolled Wnt signaling [222].
15.4.2
Benign Liver Neoplasms
These consist of hepatocellular adenoma and fo-
cal nodular hyperplasia (FNH). There are only a
few reports that have examined the Wnt/ β-catenin
pathway in these rare tumors. Initially studies in-
volving chemical carcinogenesis in mice revealed β-
catenin stabilization in adenomas due to mutations
in various degradation components [47]. Patient
studies followed and demonstrated abnormal cyto-
plasmic and/or nuclear localization of β-catenin in
30%–46% of all hepatic adenomas [34, 210]. None
of these studies detected any mutations in the β-
catenin gene, especially in the exon 3 that contains
the area interacting with GSK3 β and containing the
phosphorylation sites. Other degradation compo-
nents including axin, APC and GSK3 β did not show
any mutations either. However, while one study re-
ported interstitial deletions from exon 3 to exon 4,
the other showed only two APC polymorphisms of
unknown significance. Thus, while activation of
the Wnt/ β-catenin pathway was observed in a sig-
nificant subset of hepatic adenomas, the mechanism
remains unknown. Also, no significant changes in
the pathway were observed in FNH. Finally, occa-
sional case reports have implicated this pathway in
isolated pediatric liver cell adenoma or as a part of
Prader-Willi syndrome by means of redistribution
of β-catenin or identification of mutation of the β-
catenin gene [201].
15.4.3
Hepatocellular Cancer
A disease of extremely poor prognosis, HCC remains one of the leading causes of mortality and morbidity around the globe. The disease bears a strong etiolog- ical association with viral hepatitis, hemochromato- sis, chemical carcinogens, and toxins (mycotoxins) (reviewed in [206]). Pre-existing cirrhosis due to any number of factors such as concurrent metabolic disease or infection also predisposes to HCC. We are now beginning to understand the molecular mecha- nisms of this devastating disease. Inappropriate Wnt/ β-catenin activation has been implicated in many cancers and is one of the important aberrant pathways identified in HCC in animals and man [100, 150, 159]. Abnormal localization of cadherins and catenins in liver cancer was first shown by im- munohistochemistry [80]. A more comprehensive study identified anomalous β-catenin expression as well as mutations in the Ctnnb1 gene in around 25%
of all HCC cases and up to 50% of all hepatic tumors in transgenic lines such as c-myc or H-ras [44]. Sev- eral subsequent studies corroborated these observa- tions, although the mutations in the β-catenin gene ranged from 12% to 34%, whereas abnormal β-cat- enin redistribution always exceeded that number by as much as 15%–20%, suggesting additional mecha- nisms [92, 102, 135, 223]. Table 15.2 shows the sites of mutations in Ctnnb1 in some of the reported stud- ies on human HCC.
Discrepancy between frequencies of Ctnnb1 mutations and their aberrant immunohistochemi- cal localization led to identification of mutations in other important degradation components of the Wnt pathway. AXIN1 and AXIN2 mutations were also detected in around 5%–10% and 3% of HCCs respectively [176, 205]. Reports analyzing GSK3 β studies are conflicting although elevated levels of inactive GSK3 β are observed in HCCs harboring β- catenin accumulation [12, 39, 54].
Aberrant immunohistochemical findings for β- catenin in HCC include nuclear and/or cytoplasmic with or without membranous localization and rep- resent heterogeneity in mechanisms inducing this redistribution. Similarly, variations in frequency of mutations appear to be reflective of differences in geographical, dietary and other factors influ- encing the molecular pathogenesis of this disease.
One study detected an inverse correlation between β-catenin mutations and loss of heterozygosity in the genome, suggesting chromosomal instability (involving tumor suppressor genes) and mutations in Ctnnb1 representing alternative modes of tumor progression [102]. Interestingly a much higher fre- quency of Ctnnb1 mutations are observed in HCC associated with hepatitis C virus (HCV) infection.
More than 40% of HCV-associated HCCs demon- strate stabilizing mutations in the β-catenin gene (mostly at Ser45) as well as nuclear accumulation of its protein [72]. HBV-related HCC has an overall lower frequency of β-catenin mutations [70]. Also, although mutations in its gene were infrequent, afla-
Table 15.2. List of studies showing spectra of mutations in the Ctnnb1 gene.
Study Cases with
mutations Mutation sites Additional information
S33 S37 S45 T41 Others
(including deletions)a
[39] 15/45 5 10 No GSK3β mutations
[227] 16/38 2 2 3 1 10 Multiple mutations in two patients
[205] 14/73 1 7 1 5 One insertion between S33 and G34
[48] 5/62 1 2 2 Aflatoxin study
[72] 9/22 1 3 2 1 3 Multiple mutations in one patient
[135] 12/35 1 2 2 7 Multiple mutations in two patients
[102] 21/119 3 2 8 4 5 Multiple mutations in one patient
[44] 6/26 2 1 1 2 One patient had deletion
aDeletions usually involved one of the key sites: S33, S37, S45 or T41.
toxin-associated HCC showed increased accumu- lation of β-catenin in around 45% of tumors [48].
Analysis has also extended to identify distinct mo- lecular signatures of HCC arising in cirrhotic ver- sus non-cirrhotic livers and, although preliminary, this analysis suggests unique pathogenetic events in the two subsets. While HCC in non-cirrhotic livers demonstrates more frequent Wnt/ β-catenin involvement along with other pathways, HCC aris- ing in cirrhosis shows mainly p53 alterations [206].
Along similar lines, another study reported more frequent Wnt/ β-catenin aberrations in HCV-asso- ciated HCCs as compared to alcoholism-associated HCC, which more frequently involves RB1 and p53 pathways [49].
Prognostic implications of aberrant β-catenin localization have also been addressed in patients;
however, the reports are once again conflicting.
Earlier studies indicated a poorer prognosis asso- ciated with nuclear accumulation of β-catenin in HCC [72, 135]. Other studies have correlated nucle- ar β-catenin with a non-invasive form of the tumor and better prognosis; they implied that mutant and wild-type nuclear β-catenin protein do not function alike [70, 111]. Another report shows a non-nuclear type of β-catenin overexpression related to poor cell differentiation, larger tumor size and significantly shorter disease-free survival time [223]. A recent study found a significant relationship between nu- clear cellular retinol-binding protein-1, nuclear β- catenin, low Ki-67 positivity, favorable prognosis and 2-year survival [177].
This all said, the role of the Wnt/ β-catenin path- way in HCC in animal models is yet to be charac- terized optimally. The studies involving β-catenin transgenic mice that overexpress truncated β-cat- enin in liver do not show any evidence of sponta- neous carcinogenesis [28]. Our unpublished work utilizing normal (non-truncated) human β-catenin gene under transcriptional control of albumin pro- moter/enhancer also yielded no tumors in the liver.
Similar lack of tumorigenesis was also observed following adenoviral-mediated overexpression of dominant stable β-catenin mutant in liver [60].
These suggest that while Wnt/ β-catenin aberrations are significant in HCC, they might be insufficient on their own and require cooperation of other pathways or additional mutations or epigenetic changes to in- duce hepatocarcinogenesis. An analogous observa- tion in patients is increased nuclear localization of β-catenin in HCC and not in dysplastic nodules, in- dicating a role in tumor progression rather than in- duction [160]. In the same report nuclear β-catenin levels did not correlate with conventional nuclear targets such as cyclin D1, c-myc and MMP7, but they did correspond to loss of E-cadherin and nuclear
p53 [160]. However, a rational explanation for the discrepancy in nuclear target expression could be a multitude of signaling aberrations. Alternatively, this is yet another reiteration of the need to identify novel liver-specific targets of this pathway, such as a recent identification of orphan G-protein-coupled receptor-Gpr49 [227]. Devising newer liver models, including transgenic lines, as well as newer chemical carcinogenesis strategies, such as tumors induced by 2-amino-3,4-dimethylimidazo(4,5-f)quinoline, which selectively induces anomalies in the Wnt/ β- catenin pathway, would allow a better understand- ing of the role and regulation of this pathway, as well as having significant therapeutic implications [73].
15.4.4
Bile Duct Tumors
The most common tumor that arises in the biliary tree is the cholangiocarcinoma, which can originate from either the intrahepatic portion – intrahepatic cholangiocarcinoma (ICC) – or the hilum (hilar cholangiocarcinoma) (reviewed in [134]). The mo- lecular pathogenesis has not yet been characterized and, along with several other oncogenic pathways analyzed, there have been reports implicating aber- rant Wnt/ β-catenin signaling in a subset of these tu- mors. It is worth mentioning that this pathway has a definite role in biliary development and survival [79, 126]. There is also significant crosstalk of this pathway with the Notch/jagged pathway, which is associated with developmental defects in the biliary tree [33, 36, 43, 113, 137]. In cholangiocarcinoma, reduced expression of β-catenin and E-cadherin at the membrane is observed as compared to the sur- rounding non-cancerous ducts [6]. More impor- tantly, nuclear localization of β-catenin is seen in a subset of tumors based on histology and location of the tumor (reviewed in [161]). For most ICCs, aber- rant nuclear localization is observed in around 15%
and a decrease in membranous localization is relat- ed to poorer histological differentiation [196]. This study failed to identify any mutations in exon 3 of the β-catenin gene, although it did not analyze mu- tations in any other components of the Wnt pathway.
A larger study detected exon 3 mutations in 7.5% of
biliary tract cancer and in 57% of gall bladder ad-
enomas [162]. A higher frequency of mutations is
seen in ampullary and gall bladder carcinomas than
the bile duct cancers. A higher correlation of Ctnnb1
mutation and papillary adenocarcinoma is also ob-
served. Intraductal papillary neoplasms also show
anomalous nuclear localization of β-catenin in
around 25% of patients without any β-catenin gene
mutation in the GSK3 β-phosphorylation region [2].
Again, other components of the pathway were not analyzed for mutations in this study. Thus, while we can incriminate the Wnt/ β-catenin pathway in a subset of biliary tract neoplasms, more studies are needed to comprehend the mechanism of its ob- served deregulation.
15.4.5
Miscellaneous Pathologies
The Wnt/ β-catenin pathway is also gaining impor- tance in the molecular pathogenesis of several non- malignant conditions. Autoimmune and inflamma- tory conditions such as osteoarthritis, rheumatoid arthritis, idiopathic pulmonary fibrosis and renal fibrosis are a few examples that show activation of this pathway [35, 87, 198, 199]. In liver, increased expression of β-catenin protein as well as a Wnt pathway gene (apoptosis-related protein 3) has been identified in HCV-associated cirrhosis and not au- toimmune hepatitis cirrhosis [182]. Although we have previously discussed activation of this pathway in HCC and cirrhosis, we should point out that the frequency of activation of Wnt/ β-catenin in cirrho- sis-associated HCCs and non-HCV hepatitis-associ- ated HCC is generally lower [49, 206]. We must reit- erate that these are recent studies and would require more corroboration. cDNA array analysis was also utilized to examine the alterations in gene expres- sion in primary biliary cirrhosis (PBC) as compared to disease-free livers and primary sclerosis cholan- gitis (PSC)-associated cirrhosis [181]. This analysis revealed overexpression of numerous genes of the Wnt pathway, prominently Wnt5A, Wnt13, FRITZ and β-catenin in the PBC samples, again implicat- ing the Wnt pathway in the pathogenesis of PBC by probably contributing to the accompanying inflam- mation, fibrosis and regeneration.
15.5
Therapeutic Implications
Because the Wnt/ β-catenin pathway is involved in multiple pathologies in other organs as well as the liver, it is essential to address its therapeutic impli- cations. The most crucial component of this path- way, especially in the liver, is perhaps β-catenin, and most of the relevant pathologies are an effect of either β-catenin loss from the membrane or its cytoplasmic stabilization and nuclear translocation resulting in an increase in target gene expression.
The overall impact of launching this pathway re- sults in unnecessary changes in cell proliferation,
apoptosis and adhesion contributing to the dis- ease pathogenesis. Thus, a few therapeutic efforts made so far have been aimed at achieving β-catenin downregulation. Successful inhibition of β-catenin has been achieved by antisense and RNAi applica- tions [46, 126]. However, issues such as cost-ef- fectiveness and better in vitro and in vivo efficacy need to be resolved. Selective cox-2 inhibitors have also shown an anti- β-catenin role. A recent study in implanted colorectal cancer cells has demonstrated the ability of rofecoxib to decrease β-catenin levels and shrink tumors [232]. Gleevec has been shown to decrease tyrosine-phosphorylated β-catenin lev- els only [239]. Another group of agents including Exisulind and analogues, which are inhibitors of cyclic GMP phosphodiesterases (PDE), have been shown to activate protein kinase G (PKG), which in turn decreases β-catenin levels via a novel GSK3β- independent processing mechanism [103]. Another important strategy will be to identify novel tissue- specific targets of the pathway that are contribut- ing to the disease to develop therapies against such molecules. Thus, a successful therapy directed at the Wnt/ β-catenin pathway would be able to normalize β-catenin in terms of both its quantity and localiza- tion within a cell.
Selected Reading
Gutkind JS, ed. Signaling networks and cell cycle control: the molecular basis of cancer and other diseases. In: Cancer drug discovery and development, ch. 5. Humana Press, 2000. ISBN: 089603710X. (This book provides information on the Wnt/β-catenin pathway and interactions among various components of the pathway, with emphasis on their role in cancer.)
Thomson AW, Lotze MT, eds. The cytokine handbook (4th ed).
Elsevier Science, Ltd., 2003. ISBN: 0126896631. (This com- pilation of useful and unbiased information on various cy- tokines and their regulation provides comprehensive details on many important proteins and cytokines, along with their roles in physiology and disease.)
Website “The Wnt Homepage” at http://www.stanford.edu/
~rnusse/wntwindow.html (This site gives in-depth and updated information about the Wnt pathway. An absolute must for anybody working in the Wnt field.)
References
1. Aberle H, Bauer A, Stappert J et al. Beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J 1997;16:3797–
3804.
2. Abraham SC, Lee JH, Hruban RH et al. Molecular and immu- nohistochemical analysis of intraductal papillary neoplasms of the biliary tract. Hum Pathol 2003;34:902–910.
3. Adler PN, Lee H. Frizzled signaling and cell-cell interactions in planar polarity. Curr Opin Cell Biol 2001;13:635–640.
4. Amit S, Hatzubai A, Birman Y et al. Axin-mediated CKI phos- phorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 2002;16:1066–1076.
5. Arias AM. Epithelial mesenchymal interactions in cancer and development. Cell 2001;105:425–431.
6. Ashida K, Terada T, Kitamura Y, Kaibara N. Expression of E- cadherin, alpha-catenin, beta-catenin, and CD44 (standard and variant isoforms) in human cholangiocarcinoma: an im- munohistochemical study. Hepatology 1998;27:974–982.
7. Ault KT, Durmowicz G, Galione A et al. Modulation of Xeno- pus embryo mesoderm-specific gene expression and dor- soanterior patterning by receptors that activate the phos- phatidylinositol cycle signal transduction pathway. Devel- opment 1996;122:2033–2041.
8. Axelrod JD. Strabismus comes into focus. Nat Cell Biol 2002;4:E6–E8.
9. Babu P. Early developmental subdivisions of the wing disk in Drosophila. Mol Gen Genet 1977;151:289–294.
10. Baker JC, Beddington RS, Harland RM. Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development. Genes Dev 1999;13:3149–3159.
11. Balsamo J, Leung T, Ernst H et al. Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin- mediated adhesion by dephosphorylation of beta-catenin. J Cell Biol 1996;134:801–813.
12. Ban KC, Singh H, Krishnan R, Seow HF. GSK-3beta phosphor- ylation and alteration of beta-catenin in hepatocellular car- cinoma. Cancer Lett 2003;199:201–208.
13. Barker N, Hurlstone A, Musisi H et al. The chromatin remod- elling factor Brg-1 interacts with beta-catenin to promote target gene activation. EMBO J 2001;20:4935–4943.
14. Barker N, Morin PJ, Clevers H. The Yin-Yang of TCF/beta-cat- enin signaling. Adv Cancer Res 2000;77:1–24.
15. Bauer A, Chauvet S, Huber O et al. Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signal- ling activity. EMBO J 2000;19:6121–6130.
16. Bauer A, Huber O, Kemler R. Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein. Proc Natl Acad Sci USA 1998;95:14787–14792.
17. Behrens J, Jerchow BA, Wurtele M. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 1998;280:596–599.
18. Behrens J, Vakaet L, Friis R et al. Loss of epithelial differentia- tion and gain of invasiveness correlates with tyrosine phos- phorylation of the E-cadherin/beta-catenin complex in cells
transformed with a temperature-sensitive v-SRC gene. J Cell Biol 1993;120:757–766.
19. Bhanot P, Brink M, Samos CH et al. A new member of the friz- zled family from Drosophila functions as a Wingless recep- tor. Nature 1996;382:225–230.
20. Bienz M. TCF: transcriptional activator or repressor? Curr Opin Cell Biol 1998;10:366–372.
21. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF.
Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003;4:915–925.
22. Boutros M, Paricio N, Strutt DI, Mlodzik M. Dishevelled acti- vates JNK and discriminates between JNK pathways in pla- nar polarity and wingless signaling. Cell 1998;94:109–118.
23. Brabletz T, Jung A, Dag S et al. Beta-catenin regulates the ex- pression of the matrix metalloproteinase-7 in human color- ectal cancer. Am J Pathol 1999;155:1033–1038.
24. Brady-Kalnay SM, Mourton T, Nixon JP et al. Dynamic inter- action of PTPmu with multiple cadherins in vivo. J Cell Biol 1998;141:287–296.
25. Brady-Kalnay SM, Rimm DL, Tonks NK. Receptor protein ty- rosine phosphatase PTPmu associates with cadherins and catenins in vivo. J Cell Biol 1995;130:977–986.
26. Brannon M, Gomperts M, Sumoy L et al. A beta-catenin/XTcf- 3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. Genes Dev 1997;11:2359–
2370.
27. Brault V, Moore R, Kutsch S et al. Inactivation of the beta- catenin gene by Wnt1-Cre-mediated deletion results in dra- matic brain malformation and failure of craniofacial devel- opment. Development 2001;128:1253–1264.
28. Cadoret A, Ovejero C, Saadi-Kheddouci S et al. Hepatome- galy in transgenic mice expressing an oncogenic form of beta-catenin. Cancer Res 2001;61:3245–3249.
29. Cadoret A, Ovejero C, Terris B et al. New targets of beta-cat- enin signaling in the liver are involved in the glutamine me- tabolism. Oncogene 2002;21:8293–8301.
30. Cattelino A, Liebner S, Gallini R et al. The conditional inacti- vation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol 2003;162:1111–1122.
31. Cavallo RA, Cox RT, Moline MM et al. Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Na- ture 1998;395:604–608.
32. Chen G, Fernandez J, Mische S, Courey AJ. A functional in- teraction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development. Genes Dev 1999;13:2218–2230.
33. Chen Q, Schubert D. Presenilin-interacting proteins. Expert Rev Mol Med 2002;4:1–18.
34. Chen YW, Jeng YM, Yeh SH, Chen PJ. P53 gene and Wnt sig- naling in benign neoplasms: beta-catenin mutations in he- patic adenoma but not in focal nodular hyperplasia. Hepa- tology 2002;36:927–935.
35. Chilosi M, Poletti V, Zamo A et al. Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol 2003;162:1495–1502.
36. Colombo C, Okolicsanyi L, Strazzabosco M. Advances in fa- milial and congenital cholestatic diseases. Clinical and diag- nostic implications. Dig Liver Dis 2000;32:152–159.
37. Constantinescu S. Stemness, fusion and renewal of he- matopoietic and embryonic stem cells. J Cell Mol Med 2003;7:103–112.
38. Crawford HC, Fingleton BM, Rudolph-Owen LA et al. The met- alloproteinase matrilysin is a target of beta-catenin transac- tivation in intestinal tumors. Oncogene 1999;18:2883–2891.
39. Cui J, Zhou X, Liu Y et al. Alterations of beta-catenin and Tcf-4 instead of GSK-3beta contribute to activation of Wnt pathway in hepatocellular carcinoma. Chin Med J (Engl) 2003;116:1885–1892.
40. Danilkovitch-Miagkova A, Miagkov A, Skeel A et al. Onco- genic mutants of RON and MET receptor tyrosine kinases cause activation of the beta-catenin pathway. Mol Cell Biol 2001;21:5857–5868.
41. DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 1999;126:4557–4568.
42. Davidson AJ, Ernst P, Wang Y et al. cdx4 mutants fail to spec- ify blood progenitors and can be rescued by multiple hox genes. Nature 2003;425:300–306.
43. De Ferrari GV, Inestrosa NC. Wnt signaling function in Alzhe- imer’s disease. Brain Res Rev 2000;33:1–12.
44. de La Coste A, Romagnolo B, Billuart P et al. Somatic muta- tions of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci USA 1998;95:8847–8851.
45. De Strooper B, Annaert W. Where Notch and Wnt signaling meet. The presenilin hub. J Cell Biol 2001;152:F17–F20.
46. Deveraux QL, Aza-Blanc P, Wagner KW et al. Exposing on- cogenic dependencies for cancer drug target discovery and validation using RNAi. Semin Cancer Biol 2003;13:293–300.
47. Devereux TR, Anna CH, Foley JF et al. Mutation of beta-cat- enin is an early event in chemically induced mouse hepato- cellular carcinogenesis. Oncogene 1999;18:4726–4733.
48. Devereux TR, Stern MC, Flake GP et al. CTNNB1 mutations and beta-catenin protein accumulation in human hepato- cellular carcinomas associated with high exposure to afla- toxin B1. Mol Carcinog 2001;31:68–73.
49. Edamoto Y, Hara A, Biernat W et al. Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis. Int J Cancer 2003;106:334–341.
50. Fuchs E. Beauty is skin deep: the fascinating biology of the epidermis and its appendages. Harvey Lect 1998;94:47–77.
51. Fuchs M, Muller T, Lerch MM, Ullrich A. Association of human protein-tyrosine phosphatase kappa with members of the armadillo family. J Biol Chem 1996;271:16712–16719.
52. Fukuzawa R, Hata J, Hayashi Y et al. Beckwith-Wiedemann syndrome-associated hepatoblastoma: wnt signal activa- tion occurs later in tumorigenesis in patients with 11p15.5 uniparental disomy. Pediatr Dev Pathol 2003;6:299–306.
53. Goodwin AM, D’Amore PA. Wnt signaling in the vasculature.
Angiogenesis 2002;5:1–9.
54. Gotoh J, Obata M, Yoshie M et al. Cyclin D1 over-expression correlates with beta-catenin activation, but not with H-ras mutations, and phosphorylation of Akt, GSK3 beta and ERK1/2 in mouse hepatic carcinogenesis. Carcinogenesis 2003;24:435–442.
55. Gradl D, Kuhl M, Wedlich D. The Wnt/Wg signal transducer beta-catenin controls fibronectin expression. Mol Cell Biol 1999;19:5576–5587.
56. Habas R, Dawid IB, He X. Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrula- tion. Genes Dev 2003;17:295–309.
57. Habas R, Kato Y, He X. Wnt/Frizzled activation of Rho regu- lates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell 2001;107:843–854.
58. Haegel H, Larue L, Ohsugi M et al. Lack of beta-catenin af- fects mouse development at gastrulation. Development 1995;121:3529–3537.
59. Hamada F, Tomoyasu Y, Takatsu Y et al. Negative regulation of Wingless signaling by D-axin, a Drosophila homolog of axin. Science 1999;283:1739–1742.
60. Harada N, Miyoshi H, Murai N 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.
61. Hari L, Brault V, Kleber M et al. Lineage-specific require- ments of beta-catenin in neural crest development. J Cell Biol 2002;159:867–880.
62. He TC, Sparks AB, Rago C et al. Identification of c-MYC as a target of the APC pathway (see comments). Science 1998;281:1509–1512.
63. Hecht A, Vleminckx K, Stemmler MP et al. The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J 2000;19:1839–1850.
64. Hiscox S, Jiang WG. Association of the HGF/SF receptor, c- met, with the cell-surface adhesion molecule, E-cadherin, and catenins in human tumor cells. Biochem Biophys Res Commun 1999;261:406–411.
65. Hoschuetzky H, Aberle H, Kemler R. Beta-catenin medi- ates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J Cell Biol 1994;127:1375–
1380.
66. Houssaint E. Differentiation of the mouse hepatic primor- dium. I. An analysis of tissue interactions in hepatocyte dif- ferentiation. Cell Differ 1980;9:269–279.
67. Houssaint E. (Study of the differentiation capacities of the hepatic endoderm of mice embryos). C R Acad Sci Hebd Se- ances Acad Sci D 1972;275:461–464.
68. Howe LR, Subbaramaiah K, Chung WJ et al. Transcriptional activation of cyclooxygenase-2 in Wnt-1-transformed mouse mammary epithelial cells. Cancer Res 1999;59:1572–1577.
69. Hsieh JC, Kodjabachian L, Rebbert ML et al. A new secreted protein that binds to Wnt proteins and inhibits their activi- ties. Nature 1999;398:431–436.
70. Hsu HC, Jeng YM, Mao TL et al. Beta-catenin mutations are associated with a subset of low-stage hepatocellular carci-
noma negative for hepatitis B virus and with favorable prog- nosis. Am J Pathol 2000;157:763–770.
71. Hu P, O’Keefe EJ, Rubenstein DS. Tyrosine phosphorylation of human keratinocyte beta-catenin and plakoglobin re- versibly regulates their binding to E-cadherin and alpha- catenin. J Invest Dermatol 2001;117:1059–1067.
72. Huang H, Fujii H, Sankila A et al. Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am J Pathol 1999;155:1795–
1801.
73. Huang H, Ushijima T, Nagao M et al. Beta-catenin mutations in liver tumors induced by 2-amino-3,4-dimethylimida- zo(4,5-f)quinoline in CDF1 mice. Cancer Lett 2003;198:29–
35.
74. Huber AH, Weis WI. The structure of the beta-catenin/E- cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin. Cell 2001;105:391–402.
75. Huelsken J, Birchmeier W. New aspects of Wnt signal- ing pathways in higher vertebrates. Curr Opin Genet Dev 2001;11:547–553.
76. Huelsken J, Vogel R, Brinkmann V et al. Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol 2000;148:567–578.
77. Huelsken J, Vogel R, Erdmann B et al. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001;105:533–545.
78. Hughes LJ, Michels VV. Risk of hepatoblastoma in familial ad- enomatous polyposis. Am J Med Genet 1992;43:1023–1025.
79. Hussain SZ, Sneddon T, Tan X et al. Wnt impacts growth and differentiation in ex vivo liver development. Exp Cell Res 2004;292:157–169.
80. Ihara A, Koizumi H, Hashizume R, Uchikoshi T. Expression of epithelial cadherin and alpha- and beta-catenins in nontu- moral livers and hepatocellular carcinomas. Hepatology 1996;23:1441–1447.
81. Itoh K, Krupnik VE, Sokol SY. Axis determination in Xenopus involves biochemical interactions of axin, glycogen syn- thase kinase 3 and beta-catenin. Curr Biol 1998;8:591–594.
82. Jamora C, DasGupta R, Kocieniewski P, Fuchs E. Links be- tween signal transduction, transcription and adhesion in epithelial bud development. Nature 2003;422:317–322.
83. Jeng YM, Wu MZ, Mao TL et al. Somatic mutations of beta- catenin play a crucial role in the tumorigenesis of sporadic hepatoblastoma. Cancer Lett 2000;152:45–51.
84. Jou TS, Stewart DB, Stappert J et al. Genetic and biochemical dissection of protein linkages in the cadherin-catenin com- plex. Proc Natl Acad Sci USA 1995;92:5067–5071.
85. Kanai Y, Ochiai A, Shibata T et al. c-erbB-2 gene product directly associates with beta-catenin and plakoglobin. Bio- chem Biophys Res Commun 1995;208:1067–1072.
86. Kim JS, Crooks H, Dracheva T et al. Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells. Cancer Res 2002;62:2744–2748.
87. Kim SJ, Im DS Kim SH et al. Beta-catenin regulates expres- sion of cyclooxygenase-2 in articular chondrocytes. Bio- chem Biophys Res Commun 2002;296:221–226.
88. Kispert A, Vainio S, McMahon AP. Wnt-4 is a mesenchy- mal signal for epithelial transformation of metanephric mesenchyme in the developing kidney. Development 1998;125:4225–4234.
89. Klingensmith J, Nusse R, Perrimon N. The Drosophila seg- ment polarity gene dishevelled encodes a novel protein required for response to the wingless signal. Genes Dev 1994;8:118–130.
90. Knudsen KA, Soler AP, Johnson KR, Wheelock MJ. Interac- tion of alpha-actinin with the cadherin/catenin cell-cell ad- hesion complex via alpha-catenin. J Cell Biol 1995;130:67–
77.
91. Koch A, Denkhaus D, Albrecht S et al. Childhood hepato- blastomas frequently carry a mutated degradation target- ing box of the beta-catenin gene. Cancer Res 1999;59:269–
273.
92. Kondo Y, Kanai Y, Sakamoto M et al. Beta-catenin accumu- lation and mutation of exon 3 of the beta-catenin gene in hepatocellular carcinoma. Jpn J Cancer Res 1999;90:1301–
1309.
93. Korinek V, Barker N, Moerer P et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lack- ing Tcf-4. Nat Genet 1998;19:379–383.
94. Korswagen HC, Herman MA, Clevers HC. Distinct beta-cat- enins mediate adhesion and signalling functions in C. el- egans. Nature 2000;406:527–532.
95. Kramps T, Peter O, Brunner E et al. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 2002;109:47–
60.
96. Kratochwil K, Galceran J, Tontsch S et al. FGF4, a direct tar- get of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(-/-) mice. Genes Dev 2002;16:3173–
3185.
97. Kuhl M, Sheldahl LC, Malbon CC, Moon RT. Ca(2+)/calmodu- lin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xeno- pus. J Biol Chem 2000;275:12701–12711.
98. Kuhl M, Sheldahl LC, Park M et al. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000;16:279–283.
99. Kurahashi H, Takami K, Oue T et al. Biallelic inactivation of the APC gene in hepatoblastoma. Cancer Res 1995;55:5007–
5011.
100. Laurent-Puig P, Legoix P, Bluteau O et al. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 2001;120:1763–1773.
101. Le Douarin N, Houssaint E. (Role of the mesoderm in the in- duction of the synthesis of glycogen during differentiation of the hepatic endoderm). C R Acad Sci Hebd Seances Acad Sci D 1967;264:1872–1874.
102. Legoix P, Bluteau O, Bayer J et al. Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity. Oncogene 1999;18:4044–4046.
