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1.  Wilms Tumor Genetics: Mutations in WT1, WTX, and CTNNB1 Account for Only About One-Third of Tumors 
Genes, chromosomes & cancer  2008;47(6):461-470.
Wilms tumor is genetically heterogeneous, and until recently only one Wilms tumor gene was known, WT1 at 11p13. However, WT1 is altered in only ~20% of Wilms tumors. Recently a novel gene, WTX at Xq11.1, was reported to be mutated in Wilms tumors. No overlap between tumors with mutations in WTX and WT1 was noted, suggesting that WT1 and WTX mutations could account for the genetic basis of roughly half of Wilms tumors. To assess the frequency of WTX mutations and their relationship to WT1 mutations in a larger (n = 125) panel of Wilms tumors which had been thoroughly assessed for mutations in WT1, we conducted a complete mutational analysis of WTX that included sequencing of the entire coding region and quantitative PCR to identify deletions of the WTX gene. Twenty-three (18.4%) tumors carried a total of 24 WTX mutations, a lower WTX mutation frequency than that previously observed. Surprisingly, we observed an equivalent frequency of WTX mutations in tumors with mutations in either or both WT1 and CTNNB1 (20.0%) and tumors with no mutation in either WT1 or CTNNB1 (17.5%). WTX has been reported to play a role in the WNT/β-catenin signaling pathway, and, interestingly, WTX deletion/truncation mutations appeared to be rare in tumors carrying exon 3 mutations of CTNNB1, encoding β-catenin. Our findings indicate that WT1 and WTX mutations occur with similar frequency, that they partially overlap in Wilms tumors, and that mutations in WT1, WTX, and CTNNB1 underlie the genetic basis of about one-third of Wilms tumors.
doi:10.1002/gcc.20553
PMCID: PMC4332772  PMID: 18311776
2.  Stratification of Wilms tumor by genetic and epigenetic analysis 
Oncotarget  2012;3(3):327-335.
Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P<0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P<0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P<0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P<0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.
PMCID: PMC3359888  PMID: 22470196
Wilms tumor; WT1; WTX; CTNNB1; TP53; 11p15; somatic genetic mutation; epigenetic
3.  Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H) colorectal cancers 
BMC Cancer  2010;10:413.
Background
Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of β-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (β-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of β-CATENIN. As the WTX-gene harbors a short T6-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of β-CATENIN in human CRCs.
Methods
DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T6-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T5-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE.
Results
In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T6-repeat resulting in a T5 sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes.
Conclusion
Mutations in the WTX-gene might compromise the function of the β-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis.
doi:10.1186/1471-2407-10-413
PMCID: PMC2928794  PMID: 20696052
4.  Wilms Tumor Gene on X Chromosome (WTX) Inhibits Degradation of NRF2 Protein through Competitive Binding to KEAP1 Protein* 
The Journal of Biological Chemistry  2012;287(9):6539-6550.
Background: KEAP1 is a ubiquitin ligase adaptor that promotes the ubiquitination and degradation of NRF2, a transcription factor that drives the antioxidant response.
Results: Wilms tumor gene on the X chromosome (WTX) stabilizes NRF2 by competing with NRF2 for binding to KEAP1.
Conclusion: WTX regulates the antioxidant response.
Significance: This study reveals a novel regulatory mechanism governing the antioxidant response.
WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the β-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor β-catenin, a key control point in the WNT/β-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of β-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response.
doi:10.1074/jbc.M111.316471
PMCID: PMC3307315  PMID: 22215675
Antioxidants; Nrf2; Ubiquitin; Ubiquitination; BTRC; KEAP1; WTX
5.  Clinically Relevant Subsets Identified by Gene Expression Patterns Support a Revised Ontogenic Model of Wilms Tumor: A Children's Oncology Group Study12 
Neoplasia (New York, N.Y.)  2012;14(8):742-756.
Wilms tumors (WT) have provided broad insights into the interface between development and tumorigenesis. Further understanding is confounded by their genetic, histologic, and clinical heterogeneity, the basis of which remains largely unknown. We evaluated 224 WT for global gene expression patterns; WT1, CTNNB1, and WTX mutation; and 11p15 copy number and methylation patterns. Five subsets were identified showing distinct differences in their pathologic and clinical features: these findings were validated in 100 additional WT. The gene expression pattern of each subset was compared with published gene expression profiles during normal renal development. A novel subset of epithelial WT in infants lacked WT1, CTNNB1, and WTX mutations and nephrogenic rests and displayed a gene expression pattern of the postinduction nephron, and none recurred. Three subsets were characterized by a low expression of WT1 and intralobar nephrogenic rests. These differed in their frequency of WT1 and CTNNB1 mutations, in their age, in their relapse rate, and in their expression similarities with the intermediate mesoderm versus the metanephric mesenchyme. The largest subset was characterized by biallelic methylation of the imprint control region 1, a gene expression profile of the metanephric mesenchyme, and both interlunar and perilobar nephrogenic rests. These data provide a biologic explanation for the clinical and pathologic heterogeneity seen within WT and enable the future development of subset-specific therapeutic strategies. Further, these data support a revision of the current model of WT ontogeny, which allows for an interplay between the type of initiating event and the developmental stage in which it occurs.
PMCID: PMC3431181  PMID: 22952427
6.  The WTX/AMER1 gene family: evolution, signature and function 
Background
WTX is a novel gene mutated in a proportion of Wilms' tumors and in patients suffering from sclerosing bone dysplasia. On the molecular level WTX has been shown to act as an antagonist of canonical Wnt/β-catenin signaling in fish and mammals thus linking it to an essential pathway involved in normal development and cancer formation. Interestingly, WTX seems to also localize to an intranuclear component called paraspeckles. In spite of the growing interest of molecular biologists in WTX, little is known about its paralogs and its phylogenetic history.
Results
Using the amino-acid sequence of WTX/AMER1 as a tool for the assignment of orthology and paralogy, we here identify two novel proteins, AMER2 and AMER3, as "WTX" related. This Amer gene family is present in all currently available vertebrate genome sequences, but not invertebrate genomes and is characterized by six conserved blocks of sequences. The phylogenetic analysis suggests that the protoAmer gene originated early in the vertebrate lineage and was then duplicated due to whole genome duplications (WGD) giving rise to the three different Amer genes.
Conclusion
Our study represents the first phylogenetic analysis of Amer genes and reveals a new vertebrate specific gene family that is likely to have played an important role in the evolution of this subphylum. Divergent and conserved molecular functions of Wtx/Amer1, Amer2 and Amer3 are discussed.
doi:10.1186/1471-2148-10-280
PMCID: PMC2949870  PMID: 20843316
7.  WT1 Mutation and 11P15 Loss of Heterozygosity Predict Relapse in Very Low-Risk Wilms Tumors Treated With Surgery Alone: A Children's Oncology Group Study 
Journal of Clinical Oncology  2010;29(6):698-703.
Purpose
Children's Oncology Group defines very low-risk Wilms tumors (VLRWT) as stage I favorable histology Wilms tumors weighing less than 550 g in children younger than 24 months of age. VLRWTs may be treated with nephrectomy alone. However, 10% to 15% of VLRWTs relapse without chemotherapy. Previous studies suggest that VLRWTs with low WT1 expression and/or 11p15 loss of heterozygosity (LOH) may have increased risk of relapse. The current study validates these findings within prospectively identified children with VLRWT who did not receive adjuvant chemotherapy.
Patients and Methods
Fifty-six VLRWTs (10 relapses) were analyzed for mutation of WT1, CTNNB1, and WTX; for 11p15 LOH using microsatellite analysis; and for H19DMR and KvDMR1 methylation.
Results
11p15 LOH was identified in 19 (41%) of 46 evaluable VLRWTs and was significantly associated with relapse (P < .001); 16 of 19 were isodisomic for 11p15. WT1 mutation was identified in nine (20%) of 45 evaluable VLRWTs and was significantly associated with relapse (P = .004); all nine cases also had 11p15 LOH. All evaluable tumors showing LOH by microsatellite analysis also showed LOH by methylation analysis. Retention of the normal imprinting pattern was identified in 24 of 42 evaluable tumors, and none relapsed. Loss of imprinting at 11p15 was identified in one of 42 tumors.
Conclusion
WT1 mutation and 11p15 LOH are associated with relapse in patients with VLRWTs who do not receive chemotherapy. These may provide meaningful biomarkers to stratify patients for reduced chemotherapy in the future. VLRWTs show a different incidence of WT1 mutation and 11p15 imprinting patterns than has been reported in Wilms tumors of all ages.
doi:10.1200/JCO.2010.31.5192
PMCID: PMC3056654  PMID: 21189373
8.  The WTX tumor suppressor enhances p53 acetylation 9 by CBP/p300 
Molecular cell  2012;45(5):587-597.
WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in β-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53 and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus transformed kidney cells. In other cells without adenovirus expression, the C terminal domain of WTX binds to the DNA binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell cycle arrest, apoptosis, and p53-target gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.
doi:10.1016/j.molcel.2011.12.025
PMCID: PMC3310179  PMID: 22285752
9.  Mutational Analysis of Ctnnb1 and Apc in Tumors from Rats Given 1,2-Dimethylhydrazine or 2-Amino-3-methylimidazo[4,5-f]quinoline: Mutational ‘Hotspots’ and the Relative Expression of β-Catenin and c-jun 
Molecular carcinogenesis  2003;36(4):195-203.
There is growing interest in β-catenin and its role in various human cancers. We recently reported that 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)- and 1,2-dimethylhydrazine (DMH)-induced colon tumors in the rat contain mutations in Ctnnb1, the gene for β-catenin, but the mutation spectrum was influenced by postinitiation exposure to chlorophyllin (CHL) and indole-3-carbinol (I3C) [Blum et al., Carcinogenesis 2001;22:315–320]. The present paper describes a follow-up study in which all of the target organs for IQ- and DMH-induced tumorigenesis were screened; Ctnnb1 mutations were found in 44 of 119 DMH-induced colon tumors, six of 13 IQ-induced colon tumors, 28 of 81 DMH-induced small intestine tumors, none of five IQ-induced small intestine tumors, four of 106 IQ-induced liver tumors, none of 14 DMH-induced Zymbal’s gland tumors, none of 24 IQ-induced Zymbal’s gland tumors, and none of 29 IQ-induced skin tumors. In tumors from rats given carcinogen alone, or carcinogen plus CHL or I3C, Ctnnb1 mutations frequently substituted amino acids adjacent to Ser33, a critical Ser/Thr residue in the glycogen synthase kinase-3β regulatory domain of β-catenin. However, substitution of critical Ser/Thr residues themselves was detected in only three of 24 (12.5%) of the tumors from rats given carcinogen alone, compared with 23 of 58 (40%) of the tumors from rats given carcinogen and treated postinitiation with I3C or CHL (P <0.02). More than 50 of the colon tumors with wild-type β-catenin were examined further for their Apc status; the overall frequency of Apc mutations was <10%, and these genetic changes occurred exclusively in the ‘Mutation Cluster Region’ of Apc. A subset of colon tumors also was examined for expression of β-catenin and c-jun; these proteins were overexpressed in all tumors containing Ctnnb1 mutations, but the expression was highest in tumors with Ctnnb1 mutations affecting Thr41 and Ser45 residues in the glycogen synthase kinase-3β region of β-catenin. Thus, Ctnnb1 mutations occurred more frequently than Apc mutations in colon and small intestine tumors of the rat, and certain mutations upregulated β-catenin/T-cell factor target genes more effectively than others, perhaps influencing the response to phytochemicals administered postinitiation.
doi:10.1002/mc.10112
PMCID: PMC2279233  PMID: 12669311
CTNNB1; APC; Wnt signaling; TCF/LEF target genes; chlorophyllin; indole-3-carbinol
10.  WTX: An Unexpected Regulator for p53 
Molecular cell  2012;45(5):581-582.
The WTX gene is frequently lost or mutated in Wilms’ tumor. In this issue of Molecular Cell, Kim et al., identifies WTX modulation of the p53 tumor suppressor activity through regulating p53 acetylation. Therefore, WTX differentially regulates the oncogenic β-catenin pathway and the tumor suppressing p53 pathway.
doi:10.1016/j.molcel.2012.02.010
PMCID: PMC3632215  PMID: 22405270
11.  Sequential WT1 and CTNNB1 mutations and alterations of β‐catenin localisation in intralobar nephrogenic rests and associated Wilms tumours: two case studies 
Journal of Clinical Pathology  2006;60(9):1013-1016.
Background
Intralobar nephrogenic rests (ILNRs) are precursor lesions for Wilms tumours and are associated with WT1 gene mutations. ILNR‐associated Wilms tumours have a co‐clustering of WT1 and β‐catenin (CTNNB1) mutations and unique histological features characterised by a stromal‐predominant histology.
Aim
To determine the order in which WT1 and CTNNB1 mutations occur to understand the ILNR–Wilms tumour sequence.
Methods
Of nine Wilms tumours with WT1 and CTNNB1 mutations, three ILNRs lesions in two Wilms tumours were available for analysis of WT1 and CTNNB1 mutations using microdissection. Immunohistochemistry was also performed to investigate how the mutations in β‐catenin alter the localisation in Wilms tumour development.
Results
WT1 mutations were present in the ILNRs, however CTNNB1 mutations were absent. Immunohistochemistry for WT1 confirmed inactivation of WT1 in both ILNRs and Wilms tumours. Both the ILNRs and the associated Wilms tumours had similar immunostaining patterns for β‐catenin in the blastemal and epithelial components. Although rhabdomyoblasts were not included in ILNRs, the associated Wilms tumours showed rhabdomyogenic differentiation with a positive β‐catenin nuclear staining.
Conclusions
The results suggest that CTNNB1 mutation is a later event in Wilms tumourigenesis. CTNNB1 mutations might be associated with rhabdomyogenesis.
doi:10.1136/jcp.2006.043083
PMCID: PMC1972432  PMID: 17172473
nephroblastoma; beta‐catenin; Wnt sinalling pathway; microdissection
12.  The WTX Tumor Suppressor Regulates Mesenchymal Progenitor Cell Fate Specification 
Developmental cell  2011;20(5):583-596.
SUMMARY
WTX is an X-linked tumor suppressor targeted by somatic mutations in Wilms tumor, a pediatric kidney cancer, and by germline inactivation in osteopathia striata with cranial sclerosis, a bone overgrowth syndrome. Here, we show that Wtx deletion in mice causes neonatal lethality, somatic overgrowth, and malformation of multiple mesenchyme-derived tissues, including bone, fat, kidney, heart, and spleen. Inactivation of Wtx at different developmental stages and in primary mesenchymal progenitor cells (MPCs) reveals that bone mass increase and adipose tissue deficiency are due to altered lineage fate decisions coupled with delayed terminal differentiation. Specification defects in MPCs result from aberrant β-catenin activation, whereas alternative pathways contribute to the subsequently delayed differentiation of lineage-restricted cells. Thus, Wtx is a regulator of MPC commitment and differentiation with stage-specific functions in inhibiting canonical Wnt signaling. Furthermore, the constellation of anomalies in Wtx null mice suggests that this tumor suppressor broadly regulates MPCs in multiple tissues.
doi:10.1016/j.devcel.2011.03.013
PMCID: PMC4052985  PMID: 21571217
13.  Wilms’ tumours: about tumour suppressor genes, an oncogene and a chameleon gene 
Nature reviews. Cancer  2011;11(2):111-121.
Genes identified as being mutated in Wilms’ tumour include TP53, a classic tumour suppressor gene (TSG); CTNNB1 (encoding β-catenin), a classic oncogene; WTX, which accumulating data indicate is a TSG; and WT1, which is inactivated in some Wilms’ tumours, similar to a TSG. However, WT1 does not always conform to the TSG label, and some data indicate that WT1 enhances cell survival and proliferation, like an oncogene. Is WT1 a chameleon, functioning as either a TSG or an oncogene, depending on cellular context? Are these labels even appropriate for describing and understanding the function of WT1?
doi:10.1038/nrc3002
PMCID: PMC4332715  PMID: 21248786
14.  β-Catenin Mutation Status and Outcomes in Sporadic Desmoid Tumors 
The Oncologist  2013;18(9):1043-1049.
Mutations in the gene-encoding β-catenin, CTNNB1, are highly prevalent in sporadic desmoid tumors and may predict the risk for recurrence. We sought to determine the prevalence of CTNNB1 mutations and to determine whether the CTNNB1 mutation status correlates with disease outcome.
Learning Objectives
Describe the frequency of CTNNB1 mutations in sporadic desmoid tumors.Summarize findings regarding CTNNB1 mutation status and disease outcome.
Background.
Mutations in the gene-encoding β-catenin, CTNNB1, are highly prevalent in sporadic desmoid tumors and may predict the risk for recurrence. We sought to determine the prevalence of CTNNB1 mutations in a large cohort of sporadic desmoid tumors and to determine whether CTNNB1 mutation status correlates with disease outcome.
Methods.
Single-base extension genotyping of the CTNNB1 gene was performed on 145 sporadic, paraffin-embedded desmoid tumor specimens. Correlation of mutation status with outcome was performed on a subset of 115 patients who underwent macroscopically complete surgical resection.
Results.
CTNNB1 mutations were detected in 106 of 145 (73%) tumor specimens and in 86 of 115 (75%) specimens from patients who underwent curative-intent surgical resection, including discrete mutations in the following codons of CTNNB1 exon 3: T41A (46%), S45F (25%), S45P (1.7%), and S45C (0.9%). Desmoid tumors of the superficial trunk were significantly less likely to harbor CTNNB1 mutations than tumors located elsewhere, but none of the other examined clinicopathologic factors were found to be associated with CTNNB1 mutation status. At a median follow-up of 31 months, 5-year recurrence-free survival was slightly, although not statistically significantly, worse for patients with β-catenin-mutated tumors than for those with wild-type tumors (58% vs. 74%, respectively). The specific CTNNB1 codon mutation did not correlate with the risk for recurrence.
Conclusion.
CTNNB1 mutations are indeed common in sporadic desmoid tumors. However, our study did not detect a statistically significant difference in recurrence risk according to either the CTNNB1 mutation status or the specific CTNNB1 mutation.
doi:10.1634/theoncologist.2012-0449
PMCID: PMC3780636  PMID: 23960186
Desmoid tumor; Beta-catenin; Mutation status; CTNNB1; Fibromatosis
15.  Integrative analysis of aberrant Wnt signaling in hepatitis B virus-related hepatocellular carcinoma 
AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma (HCC).
METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer (COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function (GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of CpG islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nested-reverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ2 test as appropriate.
RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC (P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies (P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates that the mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC.
CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.
doi:10.3748/wjg.v21.i20.6317
PMCID: PMC4445110  PMID: 26034368
β-catenin; CTNNB1; Hepatitis B virus; Hepatocellular carcinoma; TP53; Wnt signaling
16.  Nuclear Accumulation of β-Catenin Protein in Chemically Induced Rat Nephroblastomas 
Wnt-signalling plays an important role in Wilms tumorigenesis. Upon activation, intracellular signal transduction results in stabilization, accumulation and nuclear translocation of β-catenin. Nuclear β-Catenin then acts in conjunction with members of the TCF/Lef family to cause transcriptional upregulation of specific proliferation-associated target genes such as c-myc or cyclin D1. Constitutive activation of β-Catenin through mutations in CTNNB1 has been found in about 15% of Wilms tumors. Nuclear β-catenin protein has been detected by immunohistochemistry in an even higher proportion of Wilms tumors suggesting alternative genetic pathways leading to β-Catenin activation.
Nephroblastomas induced in rats by either N-ethylnitrosourea or methyl(methoxymethyl)nitrosamine are histologically similar to Wilms tumors and provide a valuable rodent model. To study the involvement of the wnt-signalling pathway in rat nephroblastomas we examined 25 chemically induced rat nephroblastomas for nuclear accumulation of β-Catenin protein and for mutations in Ctnnb1. 16 of 25 tumors showed nuclear accumulation of immunoreactive β-catenin protein although no mutation was found in any of the tumors analyzed. These findings support the idea that active wnt-signalling contributes to tumorigenesis in carcinogen-induced nephroblastomas.
doi:10.2350/08-03-0443.1
PMCID: PMC2990985  PMID: 19348510
17.  CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants 
Clinics  2013;68(2):167-172.
OBJECTIVES:
We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor.
METHODS:
Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information.
RESULTS:
CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants.
CONCLUSIONS:
A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.
doi:10.6061/clinics/2013(02)OA08
PMCID: PMC3584274  PMID: 23525311
β-catenin; Gene Expression; Immunohistochemistry; Medulloblastoma; Wnt Pathway
18.  Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination 
Cancer research  2013;73(7):2199-2210.
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.
doi:10.1158/0008-5472.CAN-12-4400
PMCID: PMC3618590  PMID: 23382044
19.  Comprehensive Mutation Analysis in Colorectal Flat Adenomas 
PLoS ONE  2012;7(7):e41963.
Background
Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes.
Methods
A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in “hot spot” regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (β-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing.
Results
APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes.
Conclusion
The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway.
doi:10.1371/journal.pone.0041963
PMCID: PMC3407043  PMID: 22848674
20.  An investigation of WNT pathway activation and association with survival in central nervous system primitive neuroectodermal tumours (CNS PNET) 
British Journal of Cancer  2009;100(8):1292-1302.
Central nervous system primitive neuroectodermal tumours (CNS PNET) are high-grade, predominantly paediatric, brain tumours. Previously they have been grouped with medulloblastomas owing to their histological similarities. The WNT/β-catenin pathway has been implicated in many tumour types, including medulloblastoma. On pathway activation β-catenin (CTNNB1) translocates to the nucleus, where it induces transcription of target genes. It is commonly upregulated in tumours by mutations in the key pathway components APC and CTNNB1. WNT/β-catenin pathway status was investigated by immunohistochemical analysis of CTNNB1 and the pathway target cyclin D1 (CCND1) in 49 CNS PNETs and 46 medulloblastomas. The mutational status of APC and CTNNB1 (β-catenin) was investigated in 33 CNS PNETs and 22 medulloblastomas. CTNNB1 nuclear localisation was seen in 36% of CNS PNETs and 27% of medulloblastomas. A significant correlation was found between CTNNB1 nuclear localisation and CCND1 levels. Mutations in CTNNB1 were identified in 4% of CNS PNETs and 20% of medulloblastomas. No mutations were identified in APC. A potential link between the level of nuclear staining and a better prognosis was identified in the CNS PNETs, suggesting that the extent of pathway activation is linked to outcome. The results suggest that the WNT/β-catenin pathway plays an important role in the pathogenesis of CNS PNETs. However, activation is not caused by mutations in CTNNB1 or APC in the majority of CNS PNET cases.
doi:10.1038/sj.bjc.6604979
PMCID: PMC2676550  PMID: 19293793
CNS PNET; sPNET; medulloblastoma; WNT pathway; β-catenin; cyclin D1
21.  Inheritance of susceptibility to induction of nephroblastomas in the Noble rat 
Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53 percent of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28 and 30 percent, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46 percent, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16 percent). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.
doi:10.1016/j.diff.2008.12.003
PMCID: PMC2696123  PMID: 19281789
22.  Integrative ChIP-seq/Microarray Analysis Identifies a CTNNB1 Target Signature Enriched in Intestinal Stem Cells and Colon Cancer 
PLoS ONE  2014;9(3):e92317.
Background
Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells.
Results
We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis.
Conclusion
Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.
doi:10.1371/journal.pone.0092317
PMCID: PMC3961325  PMID: 24651522
23.  Abnormality in Wnt Signaling is Causatively Associated with Oxidative Stress-Induced Intestinal Tumorigenesis in MUTYH-Null Mice 
MUTYH is a DNA glycosylase that excises adenine paired with 8-oxoguanine to prevent mutagenesis in mammals. Biallelic germline mutations of MUTYH have been found in patients predisposed to a recessive form of familial adenomatous polyposis (MAP: MUTYH-associated polyposis). We previously reported that Mutyh-deficient mice showed a high susceptibility to spontaneous and oxidative stress-induced intestinal adenoma/carcinoma. Here, we performed mutation analysis of the tumor-associated genes including Apc, Ctnnb1, Kras and Trp53 in the intestinal tumors of Mutyh-deficient mice. In the 62 tumors, we identified 25 mutations in Apc of 18 tumors and 36 mutations in Ctnnb1 of 36 tumors. Altogether, 54 out of the 62 tumors (87.1%) had a mutation in either Apc or Ctnnb1; no tumor displayed mutations simultaneously in the both genes. Similar to MAP, 60 out of 61 mutations (98.3%) were identified as G:C to T:A transversions of which 85% occurred at either AGAA or TGAA sequences. Immunohistochemical analyses revealed the accumulation of β-catenin in the nuclei of tumors. No mutation was found in either Kras or Trp53 in the tumors. These results indicate that the uncontrolled activation of Wnt signaling pathway is causatively associated with oxidative stress-induced intestinal tumorigenesis in the Mutyh-deficient mice.
doi:10.7150/ijbs.9241
PMCID: PMC4147226  PMID: 25170306
MAP; DNA repair; oxidative DNA damage; Wnt signaling pathway; mutagenesis
24.  FAM123A Binds Microtubules and Inhibits the Guanine Nucleotide Exchange Factor ARHGEF2 to Decrease Actomyosin Contractility*** 
Science signaling  2012;5(240):ra64.
The FAM123 gene family comprises three members, FAM123A, the tumor suppressor WTX(FAM123B) and FAM123C. WTX is required for normal development and causally contributes to human disease, in part through its regulation of β-catenin-dependent WNT signaling. The roles of FAM123A and FAM123C in signaling, cell behavior and human disease remain less understood. We defined and compared the protein-protein interaction networks for each member of the FAM123 family by affinity purification and mass spectrometry. Protein localization and functional studies suggest that the FAM123 family members have conserved and divergent cellular roles. In contrast to WTX and FAM123C, we found that microtubule-associated proteins were enriched in the FAM123A protein interaction network. FAM123A interacted with and tracked dynamic microtubules in a plus-end direction. Domain interaction experiments revealed a ‘SKIP’ amino acid motif in FAM123A that mediated interaction with the microtubule tip tracking proteins EB1 and EB3, and therefore with microtubules. Cells depleted of FAM123A showed compartment-specific effects on microtubule dynamics, increased actomyosin contractility, larger focal adhesions and decreased cell migration. These effects required binding of FAM123A to and inhibition of the guanine nucleotide exchange factor ARHGEF2, a microtubule-associated activator of RhoA. Together, these data suggest that the ‘family-unique’ SKIP motif enables FAM123A to bind EB proteins, localize to microtubules and coordinate microtubule dynamics and actomyosin contractility.
doi:10.1126/scisignal.2002871
PMCID: PMC3733483  PMID: 22949735
25.  Genetic Changes of Wnt Pathway Genes are Common Events in Metaplastic Carcinomas of the Breast 
Purpose
Metaplastic carcinomas are distinct invasive breast carcinomas with aberrant non-glandular differentiation, which may be spindle, squamous, or chondroid. The limited effective treatments result from the lack of knowledge of its molecular etiology. Given the role of the Wnt pathway in cell fate and in the development of breast cancer, we hypothesized that defects in this pathway may contribute to the development of metaplastic carcinomas.
Design
In 36 primary metaplastic carcinomas we comprehensively determined the prevalence of and mechanism underlying β-catenin and Wnt pathway deregulation using immunohistochemistry (IHC) for β-catenin expression and localization, and mutational analysis for CTNNB1 (encoding β-catenin), APC, WISP3, AXIN1, and AXIN2 genes. By IHC, normal β-catenin was seen as membrane staining, and it was aberrant when > 5% of tumor cells had nuclear or cytoplasmic accumulation or reduced membrane staining.
Results
By IHC aberrant β-catenin was present in 33 of 36 (92%) cases, revealing deregulation of the Wnt pathway. CTNNB1 missense mutations were detected in 7 of 27 (25.9 %) tumors available for mutation analyses. All mutations affected the NH2-terminal domain of β-catenin, presumably rendering the mutant protein resistant to degradation. Two of 27 tumors (7.4 %) had mutations of APC, and 5 (18.5 %) carried a frame shift mutation of WISP3. No AXIN1 or AXIN2 mutations were found.
Conclusions
Activation of the Wnt signaling pathway is common in this specific subtype of breast carcinoma. The discovery of CTNNB1, APC, and WISP3 mutations may result in new treatments for patients with metaplastic carcinomas of the breast.
doi:10.1158/1078-0432.CCR-07-4379
PMCID: PMC3060761  PMID: 18593979
metaplastic carcinoma; breast cancer; epithelial to mesenchymal transition; Wnt; β-catenin; WISP3; CCN6; stem cells; differentiation

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