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/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-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 β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-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/β-catenin signaling pathway.
The paralogous scaffold proteins axin and conductin/axin2 are key factors in the negative regulation of the Wnt pathway transcription factor β-catenin, thereby representing interesting targets for ...signaling regulation. Polymerization of axin proteins is essential for their activity in suppressing Wnt/β-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/β-catenin signaling. Importantly, we identify a short peptide which induces conductin polymerization via masking the aggregon, thereby enhancing β-catenin degradation, inhibiting β-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.
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/β-catenin signaling activation and subsequent transcription of target genes, including conductin/AXIN2. Here we demonstrate that Wnt/β-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.
Axin and conductin (also known as axin2) are structurally related inhibitors of Wnt/β-catenin signalling that promote degradation of β-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 β-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.
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/β-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/β-catenin signaling and we suggest that growth pathways not only control cell cycle progression through G1/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.
Activated Wnt/β‐catenin signalling is a characteristic of many cancers and drives cell‐cycle progression. Here, we report a mechanism linking Wnt/β‐catenin signalling to centrosome separation. We ...show that conductin/axin2, a negative regulator of β‐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 β‐catenin. Conductin promotes phosphorylation of the amino‐terminal serine (Ser 33/37) and threonine (Thr 41) residues of centrosome‐associated β‐catenin. β‐Catenin mutated at these residues causes centrosomal splitting, whereas a phospho‐mimicking mutant of β‐catenin does not. Importantly, β‐catenin‐induced splitting is not inhibited by blocking β‐catenin‐dependent transcription. Treatment with Wnts and inhibition of glycogen synthase kinase 3 block β‐catenin phosphorylation and induce centrosomal splitting. These data indicate that Wnt/β‐catenin signalling and conductin regulate centrosomal cohesion by altering the phosphorylation status of β‐catenin at the centrosomes.
Wnt signalling regulates centrosome cohesion. Work by the Behrens group shows that conductin/axin2, a negative regulator of β‐catenin, localizes to centrosomes by binding to the centriole‐associated component C‐Nap1. Conductin/axin2 promotes centrosome cohesion by phosphorylating β‐catenin at centrosomes and the authors propose a model for the regulation of centrosome separation by conductin and Wnt signalling.
Phosphorylation of the Wnt receptor low‐density lipoprotein receptor‐related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β‐catenin ...signalling, which requires Wnt‐induced formation of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P2‐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)P2. Amer1 translocates to the plasma membrane in a PtdIns(4,5)P2‐dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P2. Amer1 binds CK1γ, recruits Axin and GSK3β 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/β‐catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.
Amer1/WTX has previously been shown to promote β‐catenin degradation. Here, the authors uncover an unexpected role of Amer1/WTX as a positive regulator of Wnt signalling. Amer/WTX acts as a scaffold protein that is recruited to the plasma membrane in response to Wnt stimulation and is needed for the phosphorylation and activation of the LRP6 receptor.
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 β‐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.
Structured digital
AMER3 physically interacts with APC by two hybrid (1, 2).
AMER3 physically interacts with APC by anti tag coimmunoprecipitation (1, 2, 3).
APC physically interacts with AMER3 by anti bait coimmunoprecipitation (View interaction).
AMER3 physically interacts with APC, AMER1 and Conductin by anti bait coimmunoprecipitation (View interaction).
AMER3 physically interacts with AMER1 by anti tag coimmunoprecipitation (1, 2).
AMER3 and APC colocalize by fluorescence microscopy (View interaction).
Conductin physically interacts with AMER3 by anti tag coimmunoprecipitation (View interaction).
APC physically interacts with AMER2 by anti tag coimmunoprecipitation (View interaction).
Conductin physically interacts with AMER3 by anti tag coimmunoprecipitation (1, 2).
AMER1 and AMER3 colocalize by fluorescence microscopy (View interaction).
APC physically interacts with AMER1 by anti tag coimmunoprecipitation (View interaction).
In this study, we functionally characterized Amer3, a new member of the Amer protein family. We show that human Amer3 is a nucleocytoplasmic protein that interacts with the β‐catenin destruction complex proteins APC, Conductin, and Amer1/WTX. In addition, we provide evidence that Amer3 positively influences Wnt signalling in colorectal cancer cells.
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.
Background: Amer2 localizes to the plasma membrane, interacts with adenomatous polyposis coli, and regulates Wnt signaling.
Results: Amer2 recruits the microtubule-associated protein EB1 to the plasma membrane and affects the stabilization of microtubules and cell migration.
Conclusion: Amer2 is a novel regulator of microtubule stability by interacting with EB1.
Significance: A novel membrane-associated regulator of microtubule stabilization at the plasma membrane was identified and shown to affect cell migration.
Purpose
Aberrant activation of the Wnt/β-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 β-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 β-catenin and axin 2 in consecutive CRC tissue sections revealed that nuclear translocation of β-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.
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