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.
Wilms tumor; WT1; WTX; CTNNB1; TP53; 11p15; somatic genetic mutation; epigenetic
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.
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.
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.
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.
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.
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.
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.
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.
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.
Antioxidants; Nrf2; Ubiquitin; Ubiquitination; BTRC; KEAP1; WTX
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.
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.
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.
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.
To determine the order in which WT1 and CTNNB1 mutations occur to understand the ILNR–Wilms tumour sequence.
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.
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.
The results suggest that CTNNB1 mutation is a later event in Wilms tumourigenesis. CTNNB1 mutations might be associated with rhabdomyogenesis.
nephroblastoma; beta‐catenin; Wnt sinalling pathway; microdissection
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.
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.
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.
CNS PNET; sPNET; medulloblastoma; WNT pathway; β-catenin; cyclin D1
The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics.
In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation.
Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation.
CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.
This review describes treatment options and management strategies for patients with desmoid tumors with a focus on advanced disease.
Desmoid tumors describe a rare monoclonal, fibroblastic proliferation characterized by a variable and often unpredictable clinical course. Although histologically benign, desmoids are locally invasive and associated with a high local recurrence rate, but lack metastatic potential. On the molecular level, desmoids are characterized by mutations in the β-catenin gene, CTNNB1, or the adenomatous polyposis coli gene, APC. Proof of a CTNNB1 mutation may be useful when the pathological differential diagnosis is difficult and location might be predictive for disease recurrence.
Many issues regarding the optimal treatment of patients with desmoids remain controversial; however, surgery is the therapeutic mainstay, except if mutilating and associated with considerable function loss. Postoperative radiotherapy reduces the local recurrence rate, in cases of involved surgical margins. Because of the heterogeneity of the biological behavior of desmoids, including long periods of stable disease or even spontaneous regression, treatment needs to be individualized to optimize local tumor control and preserve patients' quality of life. Therefore, the application of a multidisciplinary assessment with multimodality treatment forms the basis of care for these patients. Watchful waiting may be the most appropriate management in selected asymptomatic patients. Patients with desmoids located at the mesentery or in the head and neck region could present with life-threatening complications and often need more aggressive treatment. This review describes treatment options and management strategies for patients with desmoid tumors with a focus on advanced disease.
Aggressive fibromatosis; Desmoid tumor; Advanced disease; β-catenin; Individualized treatment
An unselected series of 310 colorectal carcinomas, stratified according to microsatellite instability (MSI) and DNA ploidy, was examined for mutations and/or promoter hypermethylation of five components of the WNT signaling cascade [APC, CTNNB1 (encoding β-catenin), AXIN2, TCF4, and WISP3] and three genes indirectly affecting this pathway [CDH1 (encoding E-cadherin), PTEN, and TP53]. APC and TP53 mutations were each present more often in microsatellite-stable (MSS) tumors than in those with MSI (P < .001 for both). We confirmed that the aneuploid MSS tumors frequently contained TP53 mutations (P < .001), whereas tumors with APC mutations and/or promoter hypermethylation revealed no associations to ploidy. Mutations in APC upstream of codons 1020 to 1169, encoding the β-catenin binding site, were found in 15/144 mutated tumors and these patients seemed to have poor clinical outcome (P = .096). Frameshift mutations in AXIN2, PTEN, TCF4, and WISP3 were found in 20%, 17%, 46%, and 28% of the MSI tumors, respectively. More than half of the tumors with heterozygote mutations in AXIN2 were concurrently mutated in APC. The present study showed that more than 90% of all samples had alteration in one or more of the genes investigated, adding further evidence to the vital importance of activated WNT signaling in colorectal carcinogenesis.
WNT signaling; colorectal cancer; genomic instability; mutation; hypermethylation
Reports on common mutations in neuroendocrine tumors (NET) are rare and clonality of NET metastases has not been investigated in this tumor entity yet. We selected one NET and the corresponding lymph node and liver metastases as well as the derivative cell lines to screen for somatic mutations in the primary NET and to track the fate of genetic changes during metastasis and in vitro progression.
Applying microarray based sequence capture resequencing including 4,935 Exons from of 203 cancer-associated genes and high-resolution copy number and genotype analysis identified multiple somatic mutations in the primary NET, affecting BRCA2, CTNNB1, ERCC5, HNF1A, KIT, MLL, RB1, ROS1, SMAD4, and TP53. All mutations were confirmed in the patients’ lymph node and liver metastasis tissue as well as early cell line passages. In contrast to the tumor derived cell line, higher passages of the metastases derived cell lines lacked somatic mutations and chromosomal alterations, while expression of the classical NET marker serotonin was maintained.
Our study reveals that both metastases have evolved from the same pair of genetically differing NET cell clones. In both metastases, the in vivo dominating “mutant” tumor cell clone has undergone negative selection in vitro being replaced by the “non-mutant” tumor cell population. This is the first report of a bi-clonal origin of NET derived metastases, indicating selective advantage of interclonal cooperation during metastasis. In addition, this study underscores the importance to monitor cell line integrity using high-resolution genome analysis tools.
Neuroendocrine tumors; Clonality of metastases; Somatic mutations
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.
CTNNB1; APC; Wnt signaling; TCF/LEF target genes; chlorophyllin; indole-3-carbinol
The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in Europe. To evaluate whether sporadic CRCs in Czech patients have specific mutational profiles we analysed somatic genetic changes in known CRC genes (APC, KRAS, TP53, CTNNB1, MUTYH and BRAF, loss of heterozygosity (LOH) at the APC locus, microsatellite instability (MSI), and methylation of the MLH1 promoter) in 103 tumours from 102 individuals. The most frequently mutated gene was APC (68.9% of tumours), followed by KRAS (31.1%), TP53 (27.2%), BRAF (8.7%) and CTNNB1 (1.9%). Heterozygous germline MUTYH mutations in 2 patients were unlikely to contribute to the development of their CRCs. LOH at the APC locus was found in 34.3% of tumours, MSI in 24.3% and MLH1 methylation in 12.7%. Seven tumours (6.9%) were without any changes in the genes tested. The analysis yielded several findings possibly specific for the Czech cohort. Somatic APC mutations did not cluster in the mutation cluster region (MCR). Tumours with MSI but no MLH1 methylation showed earlier onset and more severe mutational profiles compared to MSI tumours with MLH1 methylation. TP53 mutations were predominantly located outside the hot spots, and transitions were underrepresented. Our analysis supports the observation that germline MUTYH mutations are rare in Czech individuals with sporadic CRCs. Our findings suggest the influence of specific ethnic genetic factors and/or lifestyle and dietary habits typical for the Czech population on the development of these cancers.
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.
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.
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.
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.
In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.
We investigated whether one of the Wnt receptors, frizzled-7 (FZD7), functions in the canonical Wnt signaling pathway of colorectal cancer (CRC) cells harboring an APC or CTNNB1 mutation and may be a potential therapeutic target for sporadic CRCs. The expression level of FZD gene family members in colon cancer cells and primary CRC tissues were determined by real-time PCR. Activation of the Wnt signaling pathway was evaluated by TOPflash assay. The expression level of Wnt target genes was determined by real-time polymerase chain reaction and/or Western blot analysis. Cell growth and cell invasion were assessed by MTS and matrigel assays, respectively. Among 10 FZD gene family members, FZD7 mRNA was predominantly expressed in six colon cancer cell lines with APC or CTNNB1 mutation. These six cell lines were transfected with FZD7 cDNA together with a TOPflash reporter plasmid, resulting in a 1.5- to 24.3-fold increase of Tcf transcriptional activity. The mRNA expression levels of seven known Wnt target genes were also increased by 1.5- to 3.4-fold after transfection of FZD7 cDNA into HCT-116 cells. The six cell lines were then cotransfected with FZD7-siRNA and a TOPflash reporter plasmid, which reduced Tcf transcriptional activity to 20% to 80%. FZD7-siRNA was shown to significantly decrease cell viability and in vitro invasion activity after transfection into HCT-116 cells. Our present data demonstrated that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA may be used as a therapeutic reagent for CRCs.
An epithelial sheet, the epicardium, lines the surface of the heart. In the developing embryo, the epicardium expresses the transcriptional regulator Wilm’s Tumor Gene 1 (Wt1). Through incompletely understood mechanisms, Wt1 inactivation derails normal heart development. We investigated mechanisms by which Wt1 regulates heart development and epicardial epithelial to mesenchymal transition (EMT). We used genetic lineage tracing approaches to track and isolate epicardium and epicardium derivatives in hearts lacking Wt1 (Wt1KO). Wt1KO hearts had diminished proliferation of compact myocardium and impaired coronary plexus formation. Wt1KO epicardium failed to undergo EMT. Wt1KO epicardium expressed reduced Lef1 and Ctnnb1 (β-catenin), key components of the canonical Wnt/β-catenin signaling pathway. Wt1KO epicardium expressed decreased levels of canonical Wnt downstream targets Axin2, Cyclin D1, and Cyclin D2 and exhibited decreased activity of the Batgal Wnt/b-catenin reporter transgene, suggestive of diminished canonical Wnt signaling. Hearts with epicardium-restricted Ctnnb1 loss of function resembled Wt1KO hearts and also failed to undergo epicardial EMT. However, Ctnnb1 inactivation did not alter WT1 expression, positioning Wt1 upstream of canonical Wnt/β-catenin signaling. Wnt5a, a prototypic non-canonical Wnt with enriched epicardial expression, and Raldh2, a key regulator of retinoic acid signaling confined to the epicardium, were also markedly downregulated in Wt1KO epicardium. Hearts lacking Wnt5a or Raldh2 shared phenotypic features with Wt1KO. Although Wt1 has been proposed to regulate EMT by repressing E-cadherin, we detected no change in E-cadherin in Wt1KO epicardium. Collectively, our study shows that Wt1 regulates epicardial EMT and heart development through canonical Wnt, non-canonical Wnt, and retinoic acid signaling pathways.
Mesothelium; Epicardium; Epithelial to Mesenchymal Transition; WT1; Wnt/β-catenin signaling; Retinoic acid signaling
MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3′-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3′-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3′-untranslated regions are thus uncommon in ovarian cancer.
Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35–7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09–3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05–0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.
The colorectal cancer paradigm explains how genetic and histological changes lead normal epithelial cell to transform into pre-malignant adenomas then progress to malignant carcinomas. Using the Genetic Alterations in Cancer Knowledge System intragenic allele loss and gene mutation data from approximately 9000 colorectal tumors were compared to the model of colorectal tumor development. The distribution of mutations along the TP53 codons as a function of tumorigenesis also was analyzed. Alterations of APC, KRAS and TP53 were observed in a higher percentage of adenocarcinomas compared to adenomas (P<0.05) indicating that the alterations accumulated with malignancy. Alterations in BRAF, CTNNB, HRAS and NRAS were infrequent regardless of morphology. Differences were observed in the distribution of TP53 mutations with tumorigenesis. Mutations (single base substitutions) occurred most frequently at codons 175 and 273 in both tumor types; however, in adenocarcinomas the mutation incidence at codon 248 was approximately three times that reported in adenomas. It is proposed that the higher incidence of mutation at codon 248 is a later event in colorectal tumorigenesis that occurs as the tumors become malignant.
Wnt/β-catenin signaling plays an important role in liver development and regeneration. Its aberrant activation, however, is observed in a subset of primary hepatocellular cancers (HCCs). In the present study we compare and contrast the tumor characteristics of HCC in the presence or absence of mutations in the β-catenin gene (CTNNB1). Frozen HCC (n=32) including five fibrolamellar (FL) variants, and control livers (n=3) from Health Sciences Tissue Bank and Department of Surgery at the University of Pittsburgh Medical Center, were examined for mutations in CTNNB1, protein levels of β-catenin, tyrosine-654-phosphorylated-β-catenin (Y654-β-catenin) and glutamine synthetase (GS). Missense mutations in the exon-3 of CTNNB1 were identified in 9/32 HCCs. Total β-catenin levels were higher than controls in most tumors, however GS was exclusively increased in HCC with mutations. Phenotypically, greater percentages of mutated HCCs showed macro- and micro-vascular invasion. Also the tumor size was greater than double in mutated HCCs. High levels of total β-catenin protein were observed in multinodular tumors independent of β-catenin mutations. In addition, significant cases with mutations showed absence of cirrhosis. Finally, highest levels of Y654-β-catenin were exclusively observed in FL-HCC cases.
Thus, HCCs that harbor missense mutations in exon-3 of CTNNB1 exhibit histologically, a more aggressive phenotype. Also, CTNNB1 mutations might lead to HCC, in absence of cirrhosis. Finally, FL-HCC cases display a unique upregulation of tyrosine-phosphorylated-β-catenin suggesting robust receptor tyrosine kinase signaling in this tumor type.
We describe a patient with a novel WT1 pS50X germ line mutation, who developed bilateral Wilms tumours, both with stromal‐type histology. Both tumours showed loss of the wild type WT1 allele (loss of heterozygosity (LOH)) and a tumour specific mutation in catenin beta1 (CTNNB1), S45P in the left and Δ45S in the right tumour. Molecular analysis of microdissected cells from the left tumour revealed the same S45P CTNNB1 mutation in blastema, tubuli, stroma and muscle, and a different CTNNB1 mutation (T41A) in stromal cells isolated from another area of the same slide. Microdissection of two areas of muscle cells from the right tumour revealed the same Δ45S mutation and no CTNNB1 mutation nor LOH of WT1 in normal kidney cells. One year later, the patient developed a new set of bilateral tumours. Both tumours showed LOH of the wild type WT1 allele, but different CTNNB1 mutations as in the first tumours: S45C on the right and S45F on the left side, demonstrating that these developed independently and are not relapses. This case demonstrates the high risk for the development of Wilms tumours in patients with germ line truncation mutations.
bilateral Wilms' tumour;
wt1 germ line mutation;
ctnnb1 mutation; multiple independent tumours