TRIM28 haploinsufficiency predisposes to Wilms tumor.

Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH.

Chromatin-Remodeling-Factor ARID1B Represses Wnt/ß-Catenin Signaling.

The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/ß-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/ß-catenin target genes are upregulated. Using cellular models of low and high Wnt/ß-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/ß-catenin target genes and Wnt/ß-catenin-dependent transcriptional reporters in a ß-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/ß-catenin signaling downstream of the ß-catenin destruction complex. Both endogenous and exogenous ARID1B associate with ß-catenin and repress Wnt/ß-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with ß-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/ß-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through ß-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/ß-catenin signaling pathway.

Negative-feedback regulation of the Wnt pathway by conductin/axin2 involves insensitivity to upstream signalling.

Axin and conductin (also known as axin2) are structurally related inhibitors of Wnt/ß-catenin signalling that promote degradation of ß-catenin. Whereas axin is constitutively expressed, conductin is a Wnt target gene implicated in Wnt negative-feedback regulation. Here, we show that axin and conductin differ in their functional interaction with the upstream Wnt pathway component Dvl. Conductin shows reduced binding to Dvl2 compared to axin, and degradation of ß-catenin by conductin is only poorly blocked by Dvl2. We propose that insensitivity to Dvl is an important feature of the role of conductin as a negative-feedback regulator of Wnt signalling.

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1γ (CK1γ) is a key step in Wnt/ß-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3ß to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/ß-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

Cell cycle control of Wnt/ß-catenin signalling by conductin/axin2 through CDC20.

Wnt/ß-catenin signalling regulates cell proliferation by modulating the cell cycle and is negatively regulated by conductin/axin2/axil. We show that conductin levels peak at G2/M followed by a rapid decline during return to G1. In line with this, Wnt/ß-catenin target genes are low at G2/M and high at G1/S, and ß-catenin phosphorylation oscillates during the cell cycle in a conductin-dependent manner. Conductin is degraded by the anaphase-promoting complex/cyclosome cofactor CDC20. Knockdown of CDC20 blocks Wnt signalling through conductin. CDC20-resistant conductin inhibits Wnt signalling and attenuates colony formation of colorectal cancer cells. We propose that CDC20-mediated degradation of conductin regulates Wnt/ß-catenin signalling for maximal activity during G1/S.

Amer2 protein interacts with EB1 protein and adenomatous polyposis coli (APC) and controls microtubule stability and cell migration.

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.

Expression and localization of axin 2 in colorectal carcinoma and its clinical implication.

PURPOSE: Aberrant activation of the Wnt/ß-catenin pathway plays a major role in the development of colorectal carcinoma (CRC). Axin 2 is a key protein of this pathway and is upregulated in CRC. Here, we investigated RNA- and protein expression of axin 2 in CRC tissues at the single cell level. Moreover, the association of axin 2 with prognosis and survival was investigated in a large cohort of CRC patients (n = 280). METHODS: Localization and expression of axin 2 and ß-catenin was investigated using in situ hybridization and immunohistochemical staining. The quantitative expression levels of axin 2 were determined using RT-qPCR. The association of axin 2 expression with prognosis and survival of the patients was determined by statistical analysis (logrank test, Kaplan-Meier). RESULTS: Our results confirmed the upregulation of axin 2 in CRC and showed that it is broadly expressed in the cytoplasm of the tumor epithelial cells both, in the tumor center and at the invasion front. Axin 2 was rarely expressed by tumor stromal cells and only weakly by normal colonic epithelial cells. Staining of ß-catenin and axin 2 in consecutive CRC tissue sections revealed that nuclear translocation of ß-catenin in the tumor front was not associated with changes in the cytoplasmic localization of axin 2. Axin 2 did not show any association with proven prognostic factors or survival of the CRC patients. CONCLUSION: The generally increased expression of axin 2 in all tumor stages as compared to normal tissue suggests an initiating pathogenic function in the development of CRC.

Conductin/axin2 and Wnt signalling regulates centrosome cohesion.

Activated Wnt/beta-catenin signalling is a characteristic of many cancers and drives cell-cycle progression. Here, we report a mechanism linking Wnt/beta-catenin signalling to centrosome separation. We show that conductin/axin2, a negative regulator of beta-catenin, localizes at the centrosomes by binding to the centriole-associated component C-Nap1. Knockout or knockdown of conductin leads to premature centrosome separation--that is, splitting--which is abolished by knockdown of beta-catenin. Conductin promotes phosphorylation of the amino-terminal serine (Ser 33/37) and threonine (Thr 41) residues of centrosome-associated beta-catenin. Beta-catenin mutated at these residues causes centrosomal splitting, whereas a phospho-mimicking mutant of beta-catenin does not. Importantly, beta-catenin-induced splitting is not inhibited by blocking beta-catenin-dependent transcription. Treatment with Wnts and inhibition of glycogen synthase kinase 3 block beta-catenin phosphorylation and induce centrosomal splitting. These data indicate that Wnt/beta-catenin signalling and conductin regulate centrosomal cohesion by altering the phosphorylation status of beta-catenin at the centrosomes.

An aggregon in conductin/axin2 regulates Wnt/ß-catenin signaling and holds potential for cancer therapy.

The paralogous scaffold proteins axin and conductin/axin2 are key factors in the negative regulation of the Wnt pathway transcription factor ß-catenin, thereby representing interesting targets for signaling regulation. Polymerization of axin proteins is essential for their activity in suppressing Wnt/ß-catenin signaling. Notably, conductin shows less polymerization and lower activity than axin. By domain swapping between axin and conductin we here identify an aggregation site in the conductin RGS domain which prevents conductin polymerization. Induction of conductin polymerization by point mutations of this aggregon results in enhanced inhibition of Wnt/ß-catenin signaling. Importantly, we identify a short peptide which induces conductin polymerization via masking the aggregon, thereby enhancing ß-catenin degradation, inhibiting ß-catenin-dependent transcription and repressing growth of colorectal cancer cells. Our study reveals a mechanism for regulating signaling pathways via the polymerization status of scaffold proteins and suggests a strategy for targeted colorectal cancer therapy.

Restricted expression of mouse GCMa/Gcm1 in kidney and thymus.

Mouse Glial Cells Missing a (mGCMa) belongs to a small family of transcription factors named after the prototypical GCM from Drosophila. mGCMa was expected to regulate gliogenesis, but instead found to be primarily expressed during development in trophoblasts of chorion and labyrinth. Its deletion causes abnormal placental labyrinth formation and results in embryonic lethality. Here we identify kidney and thymus as sites of mGCMa expression. In thymus, mGCMa is restricted to few small clusters of cells, in kidney to the S3 segment of proximal tubules. mGCMa expression is primarily postnatal, arguing for a role in organ function rather than organ development.

Adenomatous polyposis coli (APC) membrane recruitment 3, a member of the APC membrane recruitment family of APC-binding proteins, is a positive regulator of Wnt-ß-catenin signalling.

The adenomatous polyposis coli (APC) membrane recruitment (Amer) family proteins Amer1/Wilms tumour gene on the X chromosome and Amer2 are binding partners of the APC tumour suppressor protein, and act as negative regulators in the Wnt signalling cascade. So far, nothing has been known about the third member of the family, Amer3. Here we show that Amer3 binds to the armadillo repeat domain of APC, similarly to Amer1 and Amer2. Amer3 also binds to the Wnt pathway regulator conductin/axin2. Furthermore, we identified Amer1 as binding partner of Amer3. Whereas Amer1 and Amer2 are linked to the plasma membrane by an N-terminal membrane localization domain, Amer3 lacks this domain. Amer3 localizes to the cytoplasm and nucleus of epithelial cells, and this is dependent on specific nuclear import and export sequences. Functionally, exogenous Amer3 enhances the expression of a ß-catenin/T-cell factor-dependent reporter gene, and knockdown of endogenous Amer3 reduces Wnt target gene expression in colorectal cancer cells. Thus, Amer3 acts as an activator of Wnt signalling, in contrast to Amer1 and Amer2, which are inhibitors, suggesting a nonredundant role of Amer proteins in the regulation of this pathway. Our data, together with those of previous studies, provide a comprehensive picture of similarities and differences within the Amer protein family.

Wnt signaling inhibits Forkhead box O3a-induced transcription and apoptosis through up-regulation of serum- and glucocorticoid-inducible kinase 1.

In human cancers, mutations in components of the Wnt signaling pathway lead to beta-catenin stabilization and result in augmented gene transcription. HCT116 colon cancer cells carry stabilizing mutations in beta-catenin and exhibit an elevated activation of Wnt signaling. To clarify the role of an overactive Wnt signaling, we used DNA microarray analysis to search for genes whose expression is up-regulated after knockdown of the wild type adenomatous polyposis coli (APC) tumor suppressor in HCT116 cells, which further enhances Wnt signaling activation. Serum and glucocorticoid-inducible kinase 1 (SGK1) was among the most up-regulated genes following APC knockdown through small interfering RNA. Up-regulation of SGK1 in response to small interfering RNA against APC was inhibited by concomitant knockdown of beta-catenin. Quantitative real time reverse transcription-PCR, Western blot, and chromatin immunoprecipitation analyses confirmed that SGK1 is a direct beta-catenin target gene. SGK1 negatively regulates the pro-apoptotic transcription factor Forkhead box O3a (FoxO3a) via phosphorylation and exclusion from the nucleus. We show that Wnt signaling activation results in FoxO3a exclusion from the nucleus and inhibits expression of FoxO3a target genes. Importantly, FoxO3a mutants that fail to be phosphorylated and therefore are regulated by SGK1 are not influenced by activation of Wnt signaling. In line, knockdown of SGK1 relieves the effects of Wnt signaling on FoxO3a localization and FoxO3a-dependent transcription. Finally, we show that induction of Wnt signaling inhibits FoxO3a-induced apoptosis. Collectively our results indicate that evasion of apoptosis is another feature employed by an overactive Wnt signaling.

CIN By WNT: growth pathways, mitotic control and chromosomal instability in cancer.

There is mounting evidence suggesting that an instable genome is directly involved in the development of cancer. The predominant form of genomic instability in most cancers presents itself as an increased rate of loss or gain in chromosome number and parts, referred to as chromosomal instability (CIN). Indeed, mutations in components of mitotic checkpoints have been described in human cancers, albeit in low numbers, suggesting that although CIN principally arises due to defective surveillance of mitosis, its molecular causes remain largely unclear. We have recently shown that the Wnt/beta-catenin signaling pathway, whose aberrant activation has been established as the driving force of tumorigenesis in many cancers particularly colorectal cancer, can generate CIN through the transcriptional target gene conductin/axin2. Here we propose a model for the generation of CIN by aberrant Wnt/beta-catenin signaling and we suggest that growth pathways not only control cell cycle progression through G(1)/S transition but have also evolved cross talks to regulate mitosis. We speculate that aberrant activation of these pathways, as observed in cancer can result in chromosomal instability thus explaining the widespread appearance of CIN in human cancers.

Aberrant Wnt/beta-catenin signaling can induce chromosomal instability in colon cancer.

Chromosomal instability (CIN), a hallmark of most colon tumors, may promote tumor progression by increasing the rate of genetic aberrations. CIN is thought to arise as a consequence of improper mitosis and spindle checkpoint activity, but its molecular basis remains largely elusive. The majority of colon tumors develop because of mutations in the tumor suppressor APC that lead to Wnt/beta-catenin signaling activation and subsequent transcription of target genes, including conductin/AXIN2. Here we demonstrate that Wnt/beta-catenin signaling causes CIN via up-regulation of conductin. Human colon tumor samples with CIN show significantly higher expression of conductin than those without. Conductin is up-regulated during mitosis, localizes along the mitotic spindles of colon cancer cells, and binds to polo-like kinase 1. Ectopic expression of conductin or its up-regulation through small interfering RNA-mediated knock-down of APC leads to CIN in chromosomally stable colon cancer cells. High conductin expression compromises the spindle checkpoint, and this requires localized polo-like kinase 1 activity. Knock-down of conductin by small interfering RNA in colon carcinoma cells or gene ablation in mouse embryo fibroblasts enforces the checkpoint.