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Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator beta-catenin. Recent studies have demonstrated that axin, which coordinates beta-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3beta, APC, axin, beta-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein-protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on beta-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of beta-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity. Show less

The components of the Wnt-signaling pathway are reported to be mutated in human cancer cells, but the relationship between the components and oral squamous carcinoma (SCC) is still unknown. In this study, we analyzed the epigenetic changes and expression patterns of four member proteins of the Wnt-signaling pathway and analyzed the mutations of beta-catenin and AXIN 1 genes, in order to explore the roles of the pathway in the development of oral cancer. The results showed that there are no beta-catenin and AXIN 1 gene mutations and no methylation of the CpG island of beta-catenin, AXIN I and GSK3beta genes in oral cancer cells; methylation of the CpG island of APC occurs in the precancerous stage and it is a dynamic change; the aberrant expressions or abnormal localization of the Wnt-signaling pathway proteins have no relationship with methylation status or mutation. From our results, we suggest that the Wnt pathway related genes play a very limited role in the development of oral SCC. Show less

The components of the Wnt-signaling pathway are mutated in tumors, but the relationship between these components and cervical cancer has not been elucidated. In this study, we used immunohistochemistry, single strand confirmation polymorphism (SSCP) and direct sequencing methods to analyze the mutation and protein expressions of both CTNNB1 and AXIN1 in cervical cancer. Among the 30 tested cervical cancers, no mutation of CTNNB1 but 3 polymorphisms were found. Mutation analysis of AXIN1 revealed that one specimen had a heterozygous mutation at codon 740 (GCC right curved arrow ACC) and six polymorphisms were also found. Immunohistochemistry showed no relationship between the protein expression patterns and mutation of AXIN1 and CTNNB1. Mutations of CTNNB1 may not be a factor, whereas mutations of AXIN1 may play a limited role in tumorigenesis of cervical cancer. In addition, aberrant expression patterns are not mutation related, so that other factors may be responsible for these changes. Show less

Axin is a negative regulator of the Wnt signalling pathway, and genetic alterations of AXIN1 have been suggested to be an important factor of carcinogenesis in some tumours. The objective of this study was to clarify the clinicopathologic and prognostic significance of Axin in oesophageal squamous cell carcinoma (SCC). Immunohistochemical staining for Axin was performed on surgical specimens obtained from 81 patients with oesophageal SCC. Western and Northern blottings were performed on proteins and RNA from oesophageal SCC cell lines. Then polymerase chain reaction-single-strand conformational analysis (PCR-SSCP) was performed on DNA from oesophageal SCC patients and cell lines. Axin expression was found to be correlated inversely with depth of invasion, lymph node metastasis, and lymphatic invasion. Although univariate analysis showed Axin to be a negative predictor, multivariate analysis showed that it was not an independent prognostic marker. In all but one of the seven cell lines examined, the levels of protein expression were equivalent to RNA expression. PCR-SSCP showed that five patients and three cell lines had polymorphisms in exon 4 or 5 of the AXIN1 gene, but none of the 81 patients with oesophageal SCC had mutations. Our findings suggest that reduced expression of Axin is correlated with tumour progression of oesophageal SCC. However, additional studies will be necessary to elucidate the mechanism responsible for loss of Axin expression in tumour cells. Show less

Despite high rates of loss of heterozygosity affecting various chromosomes, the number of tumor suppressor genes (TSGs) found to be consistently involved in primary liver cancer is low. In the past decade, characterization of homozygous deletions (HDs) in tumors has become instrumental to identify new TSGs or to reveal the influence of a particular TSG on the development of a specific tumor type. We performed a detailed HD profiling at 238 critical loci on a collection of 57 hepatobiliary tumor cell lines (hepatocellular, cholangiocellular, and bile duct carcinomas, hepatoblastomas, and immortalized hepatocytes). We identified HDs at 9 independent loci, the analysis of which was extended to 17 additional hepatobiliary tumor cell lines. In total, 34 homozygous losses involving 9 distinct genes were detected in the 74 cell lines analyzed. Besides expected deletions at the p16-INK4A/p14-ARF, FHIT, AXIN1, and p53 genes, we detected HDs at the PTEN, NF2, STK11, BAX, and LRPDIT genes that were formerly not known to be implicated in human liver tumorigenesis. In conclusion, our data suggest that these genes may represent novel liver tumor suppressive targets. Additional tumorigenic pathways should be carefully considered in hepatocarcinogenesis. Show less

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1. Show less

Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, beta-catenin, leading to the nuclear accumulation of beta-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. Here we identified a novel zebrafish gene encoding Ccd1, which possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 formed homomeric and heteromeric complexes with Dvl and Axin and activated TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos led to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) caused the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 was inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype was rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning. Show less

I-mfa has been identified as an inhibitor of myogenic basic helix-loop-helix transcription factors, and a related human I-mfa domain-containing protein (HIC) also has been identified as a protein that regulates Tat- and Tax-mediated expression of viral promoters. HIC and I-mfa represent a family of proteins that share a highly conserved cysteine-rich domain, termed the I-mfa domain. We show here that both I-mfa domain proteins, HIC and I-mfa, interacted in vivo with the Axin complex through their C-terminal I-mfa domains. This interaction inhibited Axin-mediated downregulation of free levels of cytosolic beta-catenin. I-mfa and HIC also both directly interacted with lymphocyte enhancer factor (LEF); however, I-mfa but not HIC significantly inhibited reporter constructs regulated by beta-catenin. The overexpression of HIC but not I-mfa decreased the inhibitory effects of Axin on beta-catenin-regulated reporter constructs, while both HIC and I-mfa decreased Axin-mediated c-Jun N-terminal kinase (JNK) activation. These data reveal for the first time that I-mfa domain proteins interact with the Axin complex and affect Axin regulation of both the Wnt and the JNK activation pathways. Interestingly, HIC differs from I-mfa in that I-mfa affects both Axin function and T-cell factor- or LEF-regulated transcription in the Wnt signaling pathway while HIC affects primarily Axin function. Show less

The activation of the APC/beta-catenin signalling pathway due to beta-catenin mutations has been implicated in the development of a subset of endometrial carcinomas (ECs). However, up to 25% of ECs have beta-catenin nuclear accumulation without evidence of beta-catenin mutations, suggesting alterations of other molecules that can modulate the Wnt pathway, such as APC, gamma-catenin, AXIN1 and AXIN2. We investigated the expression pattern of beta- and gamma-catenin in a group of 128 endometrial carcinomas, including 95 endometrioid endometrial carcinomas (EECs) and 33 non-endometrioid endometrial carcinomas (NEECs). In addition, we evaluated the presence of loss of heterozygosity and promoter hypermethylation of the APC gene and mutations in the APC, beta- and gamma-catenin, AXIN1, AXIN2, and RAS genes, and phospho-Akt expression. No APC mutations were detected but LOH at the APC locus was found in 24.3% of informative cases. APC promoter 1A hypermethylation was observed in 46.6% of ECs, and was associated with the endometrioid phenotype (P=0.034) and microsatellite instability (P=0.008). Neither LOH nor promoter hypermethylation of APC was associated with nuclear catenin expression. Nuclear beta-catenin expression was found in 31.2% of EECs and 3% of NEECs (P=0.002), and was significantly associated with beta-catenin gene exon 3 mutations (P<0.0001). beta-catenin gene exon 3 mutations were associated with the endometrioid phenotype, and were detected in 14 (14.9%) EECs, but in none of the NEECs (P=0.02). gamma-catenin nuclear expression was found in 10 ECs; it was not associated with the histological type but was associated with more advanced stages (P=0.042). No mutations in gamma-catenin, AXIN1 and 2 genes were detected in this series. Neither RAS mutations nor phospho-Akt expression, which were found in 16 and 27.6% of the cases, respectively, were associated with beta-catenin nuclear expression. Our results demonstrated a high prevalence of alterations in molecules of the APC/beta-catenin pathway, but only mutations in beta-catenin gene are associated with aberrant nuclear localization of beta-catenin. Show less

The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize beta-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3beta, and Axin1 or its homolog Axin2/Axil/conductin promotes beta-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant beta-catenin or gamma-catenin or after ligand-induced activation of a beta-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective beta-catenin regulation had elevated AXIN2 gene and protein expression. Expression of AXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of beta-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by beta-catenin-TCF. Because Axin2 can inhibit beta-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins. Show less

beta-catenin is involved in both cell-cell interactions and wnt pathway-dependent cell fate determination through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Cytoplasmic/nuclear levels of beta-catenin are important in regulated transcriptional activation of TCF/LEF target genes. Normally, these levels are kept low by proteosomal degradation of beta-catenin through Axin1- and APC-dependent phosphorylation by CKI and GSK-3beta. Deregulation of beta-catenin degradation results in its aberrant accumulation, often leading to cancer. Accordingly, aberrant accumulation of beta-catenin is observed at high frequency in many cancers. This accumulation correlates with either mutational activation of CTNNB1 (beta-catenin) or mutational inactivation of APC and Axin1 genes in some tumors. However, there are many tumors that display beta-catenin accumulation in the absence of a mutation in these genes. Thus, there must be additional sources for aberrant beta-catenin accumulation in cancer cells. Here, we provide experimental evidence that wild-type beta-catenin accumulates in hepatocellular carcinoma (HCC) cells in association with mutational inactivation of p53 gene. We also show that worldwide p53 and beta-catenin mutation rates are inversely correlated in HCC. These data suggest that inactivation of p53 is an important cause of aberrant accumulation of beta-catenin in cancer cells. Show less

To clarify the molecular mechanisms of human carcinogenesis associated with abnormal beta-catenin/T-cell factor (Tcf) signaling, we have been using cDNA microarrays to search for genes whose expression is significantly altered after introduction of wild-type APC into SW480 colon cancer cells. These experiments identified a novel human gene, termed APCDD1, that was down-regulated in the cancer cells by exogenous wild-type APC; its expression was also reduced in response to transduction of AXIN1. Moreover, we documented elevated expression of APCDD1 in 18 of 27 primary colon cancer tissues compared with corresponding noncancerous mucosae. A reporter gene assay using the 5'-flanking region of APCDD1 indicated that transfection of beta-catenin together with wild-type Tcf4 into HeLa cells increased the reporter activity through two putative Tcf/lymphoid enhancer factor-binding motifs upstream of the transcription start site, indicating that APCDD1 is one of the direct targets of this transcription complex. Exogenous APCDD1 promoted growth of colon cancer cells both in vitro and in vivo, whereas transfection with antisense S-oligodeoxynucleotides decreased cell/tumor growth. These data suggest that APCDD1 is directly regulated by the beta-catenin/Tcf complex and that its elevated expression is likely to contribute to colorectal tumorigenesis. Show less

We present two unrelated patients with various duplications in the caudal region. One patient presented with a duplication of the distal spine from L4, left double ureter, duplication of the vagina and cervix, and duplication of the distal colon. The second patient was diagnosed with a duplication of the colon, bladder, vagina and uterus. The first patient had an unaffected monozygotic twin sister. Dominguez et al. [1993: Am J Dis Child 147:1048-1052] presented six similar cases, and introduced the name "caudal duplication syndrome." The pathogenesis of the caudal duplication anomaly is unclear. The possibility of a polytopic primary developmental field defect or a disruptive sequence are discussed. On the other hand, somatic or germline mutations in certain developmental genes could be involved, as illustrated by the mouse mutations disorganisation and fused. DNA-analysis of the AXIN1 gene, the human homologue of the gene responsible for fused, performed in our first patient, did not show any apparent pathogenic mutation. Show less

To clarify the roles of Wnt pathway in medulloblastoma oncogenesis, immunohistochemical staining of beta-catenin and Wnt-1 and genomic analyses of CTNNB1 (beta-catenin) and AXIN1 (axin 1) were examined in 23 sporadic cases. Accumulation of beta-catenin in tumor cells was immunohistochemically proven in 5 cases; 2 cases showed positive immunoreactivity for Wnt-1 and another 2 showed mutation of either CTNNB1 or AXIN1. AXIN1 mutation was in exon 3, corresponding to GSK-3beta binding site and CTNNB1 mutation was in exon 3, corresponding to its phosphorylation site. Disruption of these proteins could result in upregulation of the Wnt signaling and accumulation of beta-catenin, followed by cell proliferation and medulloblastoma oncogenesis. Show less

Activation of Wnt signaling through beta-catenin mutations contributes to the development of hepatocellular carcinoma (HCC) and hepatoblastoma (HB). To explore the contribution of additional Wnt pathway molecules to hepatocarcinogenesis, we examined beta-catenin, AXIN1 and AXIN2 mutations in 73 HCCs and 27 HBs. beta-catenin mutations were detected in 19.2% (14 out of 73) HCCs and 70.4% (19 out of 27) HBs. beta-catenin mutations in HCCs were primarily point mutations, whereas more than half of the HBs had deletions. AXIN1 mutations occurred in seven (9.6%) HCCs and two (7.4%) HBs. The AXIN1 mutations included seven missense mutations, a 1 bp deletion, and a 12 bp insertion. The predominance of missense mutations found in the AXIN1 gene is different from the small deletions or nonsense mutations described previously. Loss of heterozygosity at the AXIN1 locus was present in four of five informative HCCs with AXIN1 mutations, suggesting a tumor suppressor function of this gene. AXIN2 mutations were found in two (2.7%) HCCs but not in HBs. Two HCCs had both AXIN1 and beta-catenin mutations, and one HCC had both AXIN2 and beta-catenin mutations. About half the HCCs with AXIN1 or AXIN2 mutations showed beta-catenin accumulation in the nucleus, cytoplasm or membrane. Overall, these data indicate that besides the approximately 20% of HCCs and 80% of HBs with beta-catenin mutations contributing to hepatocarcinogenesis, AXIN1 and AXIN2 mutations appear to be important in an additional 10% of HCCs and HBs. Show less

Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3 beta, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/beta-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway, pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/beta-catenin signaling in C. elegans. Show less

Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of beta-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of beta-catenin. An Axam fragment which contains both domains was able to decrease the level of beta-catenin. On substitution of Ser for Cys(547) in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate beta-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, Axam(C547S) showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate beta-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway. Show less

Dapper was isolated in a screen for proteins interacting with Dishevelled, a key factor in Wnt signaling. Dapper and Dishevelled colocalize intracellularly and form a complex with Axin, GSK-3, CKI, and beta-catenin. Overexpression of Dapper increases Axin and GSK-3 in this complex, resulting in decreased soluble beta-catenin and decreased activation of beta-catenin-responsive genes. Dapper also inhibits activation by Dishevelled of c-Jun N-terminal kinase (JNK), a component of beta-catenin-independent Frizzled signaling. Inhibition of Dapper activates both beta-catenin-responsive genes and an AP1-responsive promoter, demonstrating that Dapper is a general Dishevelled antagonist. Depletion of maternal Dapper RNA from Xenopus embryos results in loss of notochord and head structures, demonstrating that Dapper is required for normal vertebrate development. Show less

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes. Show less

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3 beta and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration. Show less

Glycogen synthase kinase-3 beta (GSK-3) is a key downstream target of Wnt signaling and is regulated by its interactions with activating and inhibitory proteins. We and others have shown that GSK-3 activity toward non-primed substrates is regulated in part through a competition between its activating (Axin) and inhibitory (GBP/FRAT) binding partners. Here we use a reverse two-hybrid screen to identify mutations in GSK-3 that alter binding to GBP and Axin. We find that these mutations overlap and propose that GBP and Axin compete for binding to the same region of GSK-3. We use these mutations to examine the ability of GSK-3 to block eye development in Xenopus embryos and suggest that GSK-3 regulates eye development through a non-Wnt pathway. Show less

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal. Show less

It is generally accepted that both dysfunction of the Wnt signaling pathway, including mutations in the adenomatous polyposis coli (APC) and beta-catenin genes, and genetic instability play important roles in colorectal carcinogenesis. However, alteration of the components in the Wnt signaling pathway in colorectal cancer (CRC) with microsatellite instability (MSI) has not been elucidated. In order to assess the status of the Wnt signaling components in CRC with MSI, mutational analyses of the beta-catenin, APC, Axin 1, and T cell factor 4 (TCF4) genes were performed. Three of 33 samples had mutations in exon 3 of the beta-catenin gene and two in the APC gene. Eight mutations in seven samples were detected by single-strand conformation polymorphism and subsequent direct sequence analysis of the entire coding region of the Axin 1 gene. Furthermore, TCF4, which is one of the transcriptional factors in the Wnt signaling pathway and has a mononucleotide repeat sequence (a nine- adenine repeat, (A)9) in its C-terminal region, was mutated in 13 of the 33 samples. Thus, alteration in the Wnt signaling pathway is frequently observed in CRC with MSI, including hereditary nonpolyposis colorectal cancer, as well as in familial adenomatous polyposis and sporadic CRC without MSI. Show less

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression. Show less

Clinical and molecular findings suggest that the four major histological subtypes of ovarian carcinoma (serous, clear cell, mucinous, and endometrioid) likely represent distinct disease entities. Prior studies have shown that ovarian endometrioid adenocarcinomas (OEAs) often carry mutations in the CTNNB1 gene, which encodes beta-catenin, a critical component of the Wnt signaling pathway. However, the nature of other defects in the Wnt signaling pathway in ovarian carcinomas remains largely unknown. Thus, in 45 primary OEAs and two OEA-derived cell lines, we sought to comprehensively address the prevalence of and mechanisms underlying beta-catenin and Wnt pathway deregulation. CTNNB1 missense mutations were detected in 14 primary tumors. All mutations affected the NH(2)-terminal regulatory domain of beta-catenin, presumably rendering the mutant proteins resistant to degradation. Immunohistochemical studies revealed nuclear accumulation of beta-catenin in all but two tumors with CTNNB1 mutations. Two primary tumors lacking CTNNBI mutations showed strong nuclear immunoreactivity for beta-catenin. In one of the two tumors, biallelic inactivation of the APC gene was found. In the remaining 29 primary OEAs, unequivocal nuclear beta-catenin immunoreactivity was not observed, though a nonsense mutation in AXIN1 was observed in one tumor and a truncating frameshift mutation in AXIN2 was seen in another case. Both OEA-derived cell lines studied (TOV-112D and MDAH-2774) had elevated constitutive T-cell factor/lymphoid enhancer factor transcriptional activity. TOV-112D cells were shown to harbor mutant beta-catenin, whereas a missense AXIN1 sequence alteration was identified in MDAH-2774 cells. Collectively, our findings demonstrate frequent defects of the Wnt signaling pathway in a particular subtype of ovarian carcinomas, i.e., OEAs. Although mutations in the CTNNB1 gene are the most common mechanism of beta-catenin deregulation in OEAs, beta-catenin deregulation may also result from mutations in the APC, AXIN1, and AXIN2 genes. Show less

Axin, an important regulator of beta-catenin, is frequently mutated in human hepatocellular carcinomas (HCCs), and transduction of the wild-type Axin gene (AXIN1) induces apoptosis in HCC cells as well as in colon cancer cells. To investigate the detailed biological function of Axin, we searched on a cDNA microarray for genes whose expression was altered by transfer of wild-type AXIN1 into colon-cancer cell line LoVo. Among the genes showing altered expression, we focused on one, termed AXUD1 (AXIN1 up-regulated), that revealed enhanced expression in response to exogenously expressed AXIN1 but not to LacZ, a control gene. The AXUD1 gene consists of five exons and encodes a transcript with an open reading frame of 1767 bp. A 3.2-kb transcript of AXUD1 was expressed in all human tissues examined, most abundantly in lung, placenta, skeletal muscle, pancreas and leukocyte. By radiation-hybrid mapping we assigned its chromosomal location at 3p22, a region where frequent loss of heterozygosity has been reported in lung, renal, prostate, breast and cervical cancers. AXUD1 was frequently down-regulated in lung, kidney, liver and colon cancers compared with their corresponding normal tissues, suggesting that AXUD1 may have a tumor-suppressor function in those organs. Show less

The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces. Show less

Regulation of the stability of beta catenin protein is a critical role of Wnt signaling cascades. In early Xenopus development, dorsal axis specification depends on regulation of beta catenin by both cytoplasmic and nuclear mechanisms. While the cytoplasmic protein axin is known as a key component of the cytoplasmic beta catenin degradation complex, loss-of-function studies are needed to establish whether it is required for dorso-ventral patterning in the embryo, and to test where in the embryo it carries out its function. Here, we show that embryos lacking maternal axin protein have increased levels of soluble beta catenin protein and increased nuclear localization of beta catenin in ventral nuclei at the blastula stage. These embryos gastrulate abnormally and develop with excessive notochord and head structures, and reduced tail and ventral components. They show increased expression of dorsal markers, including siamois, Xnr3, chordin, gsc, Xhex, and Otx2, decreased expression of Xwnt 8 and Xbra, and little alteration of BMP4 and Xvent1 and -2 mRNA levels. The ventral halves of axin-depleted embryos at the gastrula stage have dramatically increased levels of chordin expression, and severely decreased levels of Xwnt 8 mRNA expression, while BMP4 transcript levels are only slightly reduced. This dorso-anterior phenotype is rescued by axin mRNA injected into the vegetal pole of axin-depleted oocytes before fertilization. Interestingly, the phenotype was rescued by ventral but not dorsal injection of axin mRNA, at the 4-cell stage, although dorsal injection into wild-type embryos does cause ventralization. These results show directly that the localized ventral activity of maternal axin is critical for the correct patterning of the early Xenopus embryo. Show less

Wnt signaling increases beta-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, beta-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3beta and axin but upstream of beta-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the beta-catenin degradation complex. PP2A appears to be essential for beta-catenin degradation, since beta-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis. Show less

To clarify the molecular mechanisms of human carcinogenesis associated with abnormal Wnt/wingless signaling, we searched for genes the expression of which was significantly altered by introduction of wild-type AXIN1 into LoVo colon cancer cells. By means of a cDNA microarray, we compared expression profiles of LoVo cells infected with either adenoviruses expressing wild-type AXIN1 (Ad-Axin) or those expressing a control gene (Ad-LacZ). Among the genes showing altered expression, the ectodermal-neural cortex 1 (ENC1) gene was down-regulated in response to Ad-Axin. The promoter activity of ENC1 was elevated approximately 3-fold by transfection of an activated form of beta-catenin together with wild-type T-cell factor (Tcf)4 in HeLa cells. Semiquantitative reverse transcription-PCR experiments revealed that expression of ENC1 was increased in more than two-thirds of 24 primary colon cancer tissues that we examined compared with corresponding noncancerous mucosae. Introduction of exogenous ENC1 increased the growth rate of HCT116 colon cancer cells in serum-depleted medium. In other experiments, overexpression of ENC1 in HT-29 colon cancer cells suppressed the usual increase of two differentiation markers, in response to treatment with sodium butyrate, a differentiation-inducible agent. These data suggest that ENC1 is regulated by the beta-catenin/Tcf pathway and that its altered expression may contribute to colorectal carcinogenesis by suppressing differentiation of colonic cells. Show less