Search

APC and Axin are key negative regulators of Wnt signaling in development and oncogenesis. They form a multiprotein complex targeting the key Wnt effector β-catenin for destruction. Essential components of APC and Axin required for their cooperative function are identified, and the data are used to design a minimal β-catenin–destruction machine., Negatively regulating key signaling pathways is critical to development and altered in cancer. Wnt signaling is kept off by the destruction complex, which is assembled around the tumor suppressors APC and Axin and targets β-catenin for destruction. Axin and APC are large proteins with many domains and motifs that bind other partners. We hypothesized that if we identified the essential regions required for APC:Axin cooperative function and used these data to design a minimal β-catenin-destruction machine, we would gain new insights into the core mechanisms of destruction complex function. We identified five key domains/motifs in APC or Axin that are essential for their function in reconstituting Wnt regulation. Strikingly, however, certain APC and Axin mutants that are nonfunctional on their own can complement one another in reducing β-catenin, revealing that the APC:Axin complex is a highly robust machine. We used these insights to design a minimal β-catenin-destruction machine, revealing that a minimized chimeric protein covalently linking the five essential regions of APC and Axin reconstitutes destruction complex internal structure, size, and dynamics, restoring efficient β-catenin destruction in colorectal tumor cells. On the basis of our data, we propose a new model of the mechanistic function of the destruction complex as an integrated machine.

Background Axin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. Methods Using the intestinal epithelial conditional VDR knockout mouse model (VDR ΔIEC ) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. Results We found that VDR deletion led to lower protein and mRNA levels of Axin1 , whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. Conclusion VDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.

Previous meta-analyses reported that the variant T allele of Axin2 rs2240308 is associated with a decreased cancer risk. However, more recent findings have been inconsistent. Therefore, we carried out an updated meta-analysis to examine whether this polymorphism is still associated with a decreased cancer risk. Twelve articles, including 14 case-control studies (2,215 cases and 2,481 controls), were included in our study. Surprisingly, different from previous meta-analyses, no significant association between Axin2 rs2240308 polymorphism and cancer risk was observed (dominant model: OR=0.85; 95% CI=0.68-1.06). In further stratified analyses, rs2240308 was significantly associated with a decreased cancer risk only in Asians (dominant model: OR=0.76; 95% CI=0.66-0.88), while for Caucasians the variant showed no significant association with cancer risk (dominant model: OR=1.09; 95% CI=0.67-1.76). Moreover, the rs2240308 variant exhibited a significant association with a decreased risk of lung cancer and prostate cancer. These findings provided new evidence that differed from previous meta-analyses; Axin2 rs2240308 may not modify general cancer susceptibility. Similar with previous meta-analyses, our analysis indicated that Axin2 rs2240308 may modify cancer susceptibility in an ethnicity- and/or type-specific manner. These findings indicate that further replication studies with large sample sizes are warranted to re-evaluate the relationship between Axin2 rs2240308 and cancer risk, especially in Caucasians.

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

The Wnt-β-catenin signaling pathway regulates embryonic development and tissue homeostasis throughout the animal kingdom. Signaling through this pathway crucially depends on the opposing activities of two cytoplasmic multiprotein complexes: the Axin destruction complex, which destabilizes the downstream effector β-catenin, and the Dishevelled signalosome, which inactivates the Axin complex and thus enables β-catenin to accumulate and operate a transcriptional switch in the nucleus. These complexes are assembled by dynamic head-to-tail polymerization of the DIX domains of Axin or Dishevelled, respectively, which increases their avidity for signaling effectors. Axin also binds to Dishevelled through its DIX domain. Here, we report the crystal structure of the heterodimeric complex between the two DIX domains of Axin and Dishevelled. This heterotypic interface resembles the interfaces observed in the individual homopolymers, albeit exhibiting a slight rearrangement of electrostatic interactions and hydrogen bonds consistent with the heterotypic interaction being favored over the homotypic Axin DIX interaction. Finally, cell-based signaling assays showed that heterologous polymerizing domains functionally substituted for the DIX domain of Dishevelled provided that these Dishevelled chimera retained a DIX head or tail surface capable of binding to Axin. These findings indicate that the interaction between Dishevelled and Axin through their DIX domains is crucial for signaling to β-catenin.

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin. The multiprotein destruction complex binds and phosphorylates β-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin’s RGS domain plays an important role. We find that elevating Dishevelled levels inDrosophilaembryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death worldwide. The named "destruction complex" has a critical function in the Wnt/β-catenin pathway regulating the level of β-catenin in the cytoplasm and nucleus. Alterations in this complex lead to the cellular accumulation of β-catenin, which participates in the development and progression of CRC. This study aims to determine the contribution of polymorphisms in the genes of the β-catenin destruction complex to develop CRC, specifically adenomatous polyposis coli (APC) (rs11954856 GT and rs459552 AT), axis inhibition protein 1 (AXIN1) (rs9921222 CT and rs1805105 CT), AXIN2 (rs7224837 AG), and dishevelled 2 (DVL2) (2074222 GA and rs222836 CT). Genomic DNA from 180 sporadic colorectal cancer patients and 150 healthy blood donors were analyzed. The identification of polymorphisms was made by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) methodology. Association was calculated by the odds ratio (OR) test. Increased susceptibility to CRC was associated with the polymorphic variants rs11954856 (APC), rs222836 (DVL2), and rs9921222 (AXIN1). Decreased susceptibility was associated with the polymorphisms rs459552 (APC) and 2074222 (DVL2). Association was also observed with advanced Tumor-Node-Metastasis (TNM) stages and tumor location. The haplotypes G-T in APC (rs11954856-rs459552) and A-C in DVL2 (rs2074222-rs222836) were associated with decreased risk of CRC, while the G-T haplotype in the DVL2 gene was associated with increased CRC risk. In conclusion, our results suggest that variants in the destruction complex genes may be involved in the promotion or prevention of colorectal cancer.

Cancer is a disease caused by a process that drives the transformation of normal cells into malignant cells. The late diagnosis of cancer has a negative impact on the health care system due to high treatment cost and decreased chances of favorable prognosis. Here, we aimed to identify orofacial conditions that can serve as potential risk markers for cancers by performing a phenome-wide scan (PheWAS). From a pool of 6,100 individuals, both genetic and epidemiological data of 1,671 individuals were selected: 350 because they were previously diagnosed with cancer and 1,321 to match to those individuals that had cancer, based on age, sex, and ethnicity serving as a comparison group. Results of this study showed that when analyzing the individuals affected by cancer separately, tooth loss/edentulism is associated with SNPs in AXIN2 (rs11867417 p = 0.02 and rs2240308 p = 0.02), and leukoplakia of oral mucosa is associated with both AXIN2 (rs2240308 p = 0.03) and RHEB (rs2374261 p = 0.03). These phenotypes did not show the same trends in patients that were not diagnosed with cancer, allowing for the conclusion that these phenotypes are unique to cases with higher cancer risk.

The antagonism between Mdm2 and its close homolog Mdm4 (also known as MdmX) and p53 is vital for embryogenesis and organogenesis. Previously, we demonstrated that targeted disruption of Mdm2 in the Hoxb7+ ureteric bud (Ub) lineage, which gives rise to the renal collecting system, causes renal hypodysplasia culminating in perinatal lethality. In this study, we examine the unique role of Mdm4 in establishing the collecting duct system of the murine kidney. Hoxb7Cre driven loss of Mdm4 in the Ub lineage (Ub(Mdm4−/−)) disrupts branching morphogenesis and triggers UB cell apoptosis. Ub(Mdm4−/−) kidneys exhibit abnormally dilated Ub tips while the medulla is hypoplastic. These structural alterations result in secondary depletion of nephron progenitors and nascent nephrons. As a result, newborn Ub(Mdm4−/−) mice have hypo-dysplastic kidneys. Transcriptional profiling revealed downregulation of the Ret-tyrosine kinase pathway components, Gdnf, Wnt11, Sox8, Etv4 and Cxcr4 in the Ub(Mdm4−/−) mice relative to controls. Moreover, the expression levels of the canonical Wnt signaling members Axin2 and Wnt9b are downregulated. Mdm4 deletion upregulated p53 activity and p53-target gene expression including Cdkn1a (p21), Gdf15, Ccng1, PERP, and Fas. Germline loss of p53 in Ub(Mdm4−/−) mice largely rescues kidney development and terminal differentiation of the collecting duct. We conclude that Mdm4 plays a unique and vital role in Ub branching morphogenesis and collecting system development.

This study was designed to explore the potential therapeutic effects of Mg-based metal organic frameworks (MOFs) in osteoarthritis (OA) and to broaden the application field of MOFs. Mg/HCOOH-MOF was introduced and characterized by using SEM, XRD, TGA, and FTIR. ICP-MS results proved that Mg/HCOOH-MOF is stable in various physiological media and can maintain the release of Mg2+ ions for a long time. In vitro experiments were carried out to evaluate its potential application in attenuating OA. Mg/HCOOH-MOF was successfully synthesized, and results of MTT assays showed that it is biocompatible and can promote cell proliferation. qPCR results suggested that it can significantly regulate the expression of OCN, Axin2, iNOS and IL-1β, which indicated its activity in anti-inflammation and bone protection. We believe that Mg/HCOOH-MOF could benefit OA therapy.

The colonic epithelial turnover is driven by crypt-base stem cells that express the R-spondin receptor Lgr5. Signals that regulate epithelial regeneration upon stem cell injury are largely unknown. Here, we explore the dynamics of Wnt signaling in the colon. We identify two populations of cells with active Wnt signaling: highly proliferative Lgr5+/Axin2+ cells, as well as secretory Lgr5−/Axin2+ cells. Upon Lgr5+ cell depletion, these cells are recruited to contribute to crypt regeneration. Chemical injury induced by DSS leads to a loss of both Lgr5+ cells and Axin2+ cells and epithelial regeneration is driven by Axin2− cells, including differentiated Krt20+ surface enterocytes. Regeneration requires stromal Rspo3, which is present at increased levels upon injury and reprograms Lgr5− but Lgr4+ differentiated cells. In contrast, depletion of stromal Rspo3 impairs crypt regeneration, even upon mild injury. We demonstrate that Rspo3 is essential for epithelial repair via induction of Wnt signaling in differentiated cells., Epithelial turnover in the colon requires stem cells in the crypt that express the R-spondin receptor Lgr5. Here, the authors show that regeneration after colon injury involving loss of Lgr5+ and Axin2+ cells requires stromal derived Rspo3-dependent reprogramming of Lgr4+ differentiated cells, including Krt20+ enterocytes.

Objective Lung cancer is one of the major causes of cancer deaths worldwide, and the five-year survival still remains low despite the improvement of screening, prevention, and treatment methods. Chinese herbal medicines have been widely used for tumor prevention and treatment. Miao-Yi-Ai-Tang (Miao) is a novel herbal formulation and shows a potential anticancer effect. Materials and Methods. Human Small Cell Lung Cancer Cell was used for study in vitro. After treatments by Miao and Cisplatin (DDP), the invasion, migration, proliferation, and apoptosis of cells were detected by transwell, wound healing, CCK-8, and flow cytometry, respectively. The expression of β-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of β-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of Results We found that Miao could inhibit invasion, migration, and proliferation and promote apoptosis of human lung cancer cells. Meanwhile, Miao and DDP presented synergy regulating the proliferation and apoptosis of lung cancer cells. The percentage of lung cancer cells in S and G2 stages was increased markedly by Miao. Besides, the expression of c-myc, AXIN, and β-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of Conclusions Chinese herbal formulas Miao could suppress lung cancer through targeting the β-catenin/AXIN signaling pathway. Therefore, our findings may provide a novel strategy for the prevention and treatment of lung cancer.β-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of

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., Polymerization of axin proteins is essential to suppress Wnt/β-catenin signaling. Here, the authors identify an aggregation site in the conductin/axin2 RGS domain that prevents its polymerization and show that a short peptide masking this aggregon promotes polymerization of conductin/axin2, downregulation of Wnt pathway activity and growth inhibition of colorectal cancer cells.

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling., Axin is a scaffolding protein known for its role in Wnt signalling that can be marked with a variety of post-translational modifications. Here, Cong et al. demonstrate that USP7 de-ubiquinates Axin and that canonical Wnt signaling output can be increased with USP7 inhibitors.

To date, attempts to regenerate functional periodontal tissues (including cementum) are largely unsuccessful due to a lack of full understanding about the cellular origin (epithelial or mesenchymal cells) essential for root cementum growth. To address this issue, we first identified a rapid cementum growth window from the ages of postnatal day 28 (P28) to P56. Next, we showed that expression patterns of Axin2 and β-catenin within cementum-forming periodontal ligament (PDL) cells are negatively associated with rapid cementum growth. Furthermore, cell lineage tracing studies revealed that the Axin2(+)-mesenchymal PDL cells and their progeny rapidly expand and directly contribute to postnatal acellular and cellular cementum growth. In contrast, the number of K14(+) epithelial cells, which were initially active at early stages of development, was reduced during rapid cementum formation from P28 to P56. The in vivo cell ablation of these Axin2(+) cells using Axin2(CreERT2/+); R26R(DTA/+) mice led to severe cementum hypoplasia, whereas constitutive activation of β-catenin in the Axin2(+) cells resulted in an acceleration in cellular cementogenesis plus a transition from acellular cementum to cellular cementum. Thus, we conclude that Axin2(+)-mesenchymal PDL cells, instead of K14(+) epithelial cells, significantly contribute to rapid cementum growth.

Objective Lymphoma, a malignant tumor, is mainly characterized by painless lymph node enlargement and hepatosplenomegaly. At present, lymphoma is mainly treated by radiation, chemical drugs, bone marrow transplantation and surgery. However, due to the high degree of heterogeneity, lymphomas are highly different in terms of treatment intensity and prognosis. This study is designed to investigate the function of tripartite motif-containing 11 (TRIM11) in lymphomas. Methods The expression of TRIM11 in lymphoma tissues and multiple lymphoma cell lines was respectively detected by microarray immunohistochemistry, real-time PCR and Western blotting. After TRIM11 knockdown, overexpression, or β-catenin inhibitor XAV939 treatment, proliferation, apoptosis and cell cycle progression, as well as expression of related-genes were detected. Next, Co-Immunoprecipitation (Co-IP) and ubiquitination detection were performed. Results Elevated expression of tripartite motif-containing 11 (TRIM11) was observed in lymphoma tissues and multiple lymphoma cell lines (Raji, Jurkat, U937 and Hut78). Knockdown of TRIM11 in lymphoma cells significantly suppressed cell proliferation and prevented cell cycle progression from entering S or G2 phase. Concurrently, the expression of β-catenin, Cyclin D1 and c-Myc proteins in TRIM11-silenced lymphoma cells was decreased, while Axin1 was increased. In addition, TRIM11 overexpression had an opposite effect to TRIM11 knockdown, and a β-catenin inhibitor, XAV939, potently attenuated the induction of TRIM11 on lymphoma cells. Co-IP assay showed the interaction of TRIM11 and Axin1, and TRIM11 knockdown inhibited Axin1 ubiquitination degradation. Conclusions Together all, the results suggested that TRIM11 may be an oncogene in lymphomas, which involving the activation of the β-catenin signaling and the ubiquitination degradation of Axin1.

Colorectal cancer (CRC) is the leading cause of cancer death, and its 5‐year survival rate remains unsatisfactory. Recent studies have revealed that ubiquitin‐specific protease 44 (USP44) is a cancer suppressor or oncogene depending on the type of neoplasm. However, its role in CRC remains unclear. Here, we found that the USP44 expression level was markedly decreased in CRC, and USP44 overexpression inhibited proliferation while enhancing apoptosis in CRC cells, suggesting that USP44 is a cancer suppressor in CRC. We then investigated if USP44 functioned through regulating the Wnt/β‐catenin pathway. We found that USP44 overexpression increased the Axin1 protein while decreasing β‐catenin, c‐myc, and cyclin D1 proteins, suggesting that USP44 inhibited the activation of the Wnt/β‐catenin pathway. Moreover, we found that two Wnt/β‐catenin activators, LiCl and SKL2001, both attenuated oeUSP44‐mediated proliferation and apoptosis in CRC cells. Collectively, these data points indicated that USP44 inhibited proliferation while promoting apoptosis in CRC cells by inhibiting the Wnt/β‐catenin pathway. Interestingly, we observed that USP44 overexpression did not affect the Axin1 mRNA level. Further study uncovered that USP44 interacted with Axin1 and reduced the ubiquitination of Axin1. Furthermore, Axin1 knock‐down abolished the effects of oeUSP44 on proliferation, apoptosis, and Wnt/β‐catenin activity in CRC cells. Taken together, this study demonstrates that USP44 inhibits proliferation while enhancing apoptosis in CRC cells by inactivating the Wnt/β‐catenin pathway via Axin1 deubiquitination. USP44 is a cancer suppressor in CRC and a potential target for CRC therapy.

Background Cartilage damage is a crucial feature involved in several pathological conditions characterized by joint disorders, such as osteoarthritis and rheumatoid arthritis. Accumulated evidences showed that Wnt/β-catenin pathway plays a role in the pathogenesis of cartilage damage. In addition, it is experimentally documented that lncRNA (long non-coding RNA) HOTAIR plays a key role in the regulation of Wnt/β-catenin pathway based on directly decreased WIF-1 expression. Further, it is reported that Wnt/β-catenin pathway is a potent pathway to regulate the expression of MMP-13, which is responsible for degradation of collagen type II in articular cartilage. It is increasingly recognized that systems modeling approach provides an opportunity to understand the complex relationships and direct quantitative analysis of dynamic network in various diseases. Results A dynamic network of lncRNA HOTAIR-mediated Wnt/β-catenin pathway regulating MMP-13 is developed to investigate the dynamic mechanism of the network involved in the pathogenesis of cartilage damage. Based on the network modeling, the potential therapeutic intervention point Axin is predicted and confirmed by the experimental validation. Conclusions Our study provides a promising strategy for revealing potential dynamic mechanism and assessing potential targets which contribute to the prevention of the pathological conditions related to cartilage damage.

Background Caenorhabditis elegans seam cells serve as a good model to understand how genes and signaling pathways interact to control asymmetric cell fates. The stage-specific pattern of seam cell division is coordinated by a genetic network that includes WNT asymmetry pathway components WRM-1, LIT-1, and POP-1, as well as heterochronic microRNAs (miRNAs) and their downstream targets. Mutations in pry-1, a negative regulator of WNT signaling that belongs to the Axin family, were shown to cause seam cell defects; however, the mechanism of PRY-1 action and its interactions with miRNAs remain unclear. Results We found that pry-1 mutants in C. elegans exhibit seam cell, cuticle, and alae defects. To examine this further, a miRNA transcriptome analysis was carried out, which showed that let-7 (miR-48, miR-84, miR-241) and lin-4 (lin-4, miR-237) family members were upregulated in the absence of pry-1 function. Similar phenotypes and patterns of miRNA overexpression were also observed in C. briggsae pry-1 mutants, a species that is closely related to C. elegans. RNA interference-mediated silencing of wrm-1 and lit-1 in the C. elegans pry-1 mutants rescued the seam cell defect, whereas pop-1 silencing enhanced the phenotype, suggesting that all three proteins are likely important for PRY-1 function in seam cells. We also found that these miRNAs were overexpressed in pop-1 hypomorphic animals, suggesting that PRY-1 may be required for POP-1-mediated miRNA suppression. Analysis of the let-7 and lin-4-family heterochronic targets, lin-28 and hbl-1, showed that both genes were significantly downregulated in pry-1 mutants, and furthermore, lin-28 silencing reduced the number of seam cells in mutant animals. Conclusions Our results show that PRY-1 plays a conserved role to maintain normal expression of heterochronic miRNAs in nematodes. Furthermore, we demonstrated that PRY-1 acts upstream of the WNT asymmetry pathway components WRM-1, LIT-1, and POP-1, and miRNA target genes in seam cell development. Electronic supplementary material The online version of this article (10.1186/s12861-019-0197-5) contains supplementary material, which is available to authorized users.

Cancer stem cells (CSC) constitute heterogeneous cell subpopulations of a tumor. Although targeting CSCs is important for cancer eradication, no clinically approved drugs that target CSCs have been established. Tankyrase poly(ADP-ribosyl)ates and destabilizes AXIN, a negative regulator of β-catenin, and promotes β-catenin signaling. Here, we report that tankyrase inhibitors downregulate c-KIT tyrosine kinase and inhibit the growth of CD44-positive colorectal CSCs. c-KIT expression in CD44-positive subpopulations of colorectal cancer COLO-320DM cells is associated with their tumor-initiating potential in vivo. Tankyrase inhibitors downregulate c-KIT expression in established cell lines, such as COLO-320DM and DLD-1, and colorectal cancer patient–derived cells. These effects of tankyrase inhibitors are caused by reducing the recruitment of SP1 transcription factor to the c-KIT gene promoter and depend on AXIN2 stabilization but not β-catenin downregulation. Whereas c-KIT knockdown inhibits the growth of CD44-positive COLO-320DM cells, c-KIT overexpression in DLD-1 cells confers resistance to tankyrase inhibitors. Combination of a low-dose tankyrase inhibitor and irinotecan significantly inhibited the growth of COLO-320DM tumors in a mouse xenograft model. These observations suggest that tankyrase inhibitors target c-KIT–positive colorectal CSCs and provide a novel therapeutic strategy for cancer.

Cancer stemness drives tumor initiation, progression, metastasis, recurrence, and therapy resistance. However, mechanisms that potentiate the acquisition and maintenance of stemness fate of cancer cells remain incompletely understood. Here, we show that miR-103/107 stimulate multiple stem-like features in colorectal cancer, including expression of stem-like markers, appearance of side-population cells, and capabilities in self-renewal, tumor initiation, recurrence, and chemoresistance. Mechanistically, these stemness-promoting functions are mediated by miR-103/107-dependent repression of Axin2, a negative feedback regulator of Wnt/β-catenin signaling. Through inhibiting Axin2, miR-103/107 trigger a prolonged duration of Wnt/β-catenin signaling and a sustained induction of Wnt responsive genes. In colorectal cancer patients, miR-103/107 expression correlates inversely with Axin2 expression and a signature of miR-103/107 high and Axin2 low expression profile correlates with poor prognosis. Together, our study identifies a novel function of miR-103/107 in promoting colorectal cancer stemness by targeting Axin2 and elucidates the clinical relevance and prognostic value of this axis in colorectal cancer.

The central regulator of the Wnt/β-catenin pathway is the Axin/APC/GSK3β destruction complex (DC), which in unstimulated conditions targets cytoplasmic β-catenin for degradation. How Wnt activation inhibits the DC to permit β-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo. Using Bimolecular Fluorescence Complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila. By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3β interactions that prevent β-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control β-catenin's degradation and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signaling transduction in vivo.

Objective- The Wnt/β-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/β-catenin signaling by induced overexpression of Axin1, an inhibitor of β-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/β-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, β-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific β-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific β-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/β-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-β-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article., SCOPUS: ar.j, info:eu-repo/semantics/published

Parkinson's disease (PD) is a progressive neurodegenerative disorder. A common and disabling disease of the elderly, the standard dopamine replacement therapies do not arrest the ongoing neurodegeneration, thus calling for new treatment strategies. The present study aimed to clarify the functional relevance of the hypoxia inducible factor-1α (HIF-1α)/microRNA-128-3p (miR-128-3p) axis in hippocampal neurodegeneration in a PD mouse model obtained by intraperitoneal injection of MPTP. Targeting relationship between miR-128-3p and Axin1 was verified, so we probed the roles of Hif1a, miR-128-3p, and Axin1 in apoptosis of hippocampal neurons with gain- and loss-of function experiments using flow cytometry and TUNEL staining. We found that Axin1 was upregulated in hippocampal tissues and cells of the MPTP-lesioned mouse model of PD, while Hif1a and miR-128-3p were downregulated. Elevation of HIF-1α/miR-128-3p inhibited apoptosis of hippocampal neurons via Wnt/β-catenin signaling pathway activation due to the suppression of Axin1 in PD. In addition, forced overexpression of Hif1a could ameliorate motor dysfunction and pathological changes in the model. Collectively, activation of the HIF-1α/miR-128-3p axis could repress hippocampal neurodegeneration in MPTP-lesioned mice through an activated Wnt/β-catenin pathway due to Axin1 downregulation.

Micro-RNAs regulate multiple biological behaviors of cancers, making them potential targets of new cancer therapies. MiR-1181 has been demonstrated to perform oncogenic or tumor-suppressing function in a tissue-dependent way, but its role in hepatocellular carcinoma (HCC) was unclear. Here, we showed that miR-1181 was significantly overexpressed in HCC tissues when compared with tumor-adjacent normal ones or normal liver tissues from donated organ, and that inhibition of miR-1181 could repress the growth of HCC cells. Through bioinformatics analysis and luciferase reporter assays, we found that axis inhibition protein 1 (AXIN1) was a direct target of miR-1181, and the expression of AXIN1 showed a negative correlation with that of miR-1181 in HCC. Therefore, these data indicated an oncogenic function of miRNA-1181 in the development of HCC and a potential target for the clinical treatment of HCC.

It is well known that exposure of double-stranded RNA (dsRNA) to intestine immediately induces villus damage with severe diarrhea, which is mediated by toll-like receptor 3 signaling activation. However, the role of intestinal stem cells (ISCs) remains obscure during the pathology. In the present study, polyinosinic-polycytidylic acid (poly[I:C]), mimicking viral dsRNA, was used to establish intestinal damage model. Mice were acutely and chronically exposed to poly(I:C), and ISCs in jejunum were analyzed. The results showed that the height of villus was shorter 48 hr after acute poly(I:C) exposure compared with that of controls, while chronic poly(I:C) treatment increased both villus height and crypt depth in jejunum compared with control animals. The numbers of ISCs in jejunum were significantly increased after acute and chronic poly(I:C) exposure. Poly (I:C)-stimulated ISCs have stronger capacities to differentiate into intestine endocrine cells. Mechanistically, poly(I:C) treatment increased expression of Stat1 and Axin2 in the intestinal crypt, which was along with increased expression of Myc, Bcl2, and ISC proliferation. These findings suggest that dsRNA exposure could induce ISC proliferation to ameliorate dsRNA-induced intestinal injury.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer’s disease and the ‘Tc1’ DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer’s disease treatment.

Background The role and mechanism of the nicotinamide adenine dinucleotide (NAD+) salvage pathway in cancer cell proliferation is poorly understood. Nicotinamide phosphoribosyltransferase (NAMPT), which converts nicotinamide into NAD+, is the rate-limiting enzyme in the NAD+ salvage pathway. Here, we assessed the role of NAMPT in the proliferation of colorectal cancer. Methods Real-time PCR, immunohistochemistry, western blotting, and analyses of datasets from Oncomine and Gene Expression Omnibus were conducted to assess the expression of NAMPT at the mRNA and protein levels in colorectal cancer. The Kaplan Meier plotter online tool was used to evaluate the prognostic role of NAMPT. Knockdown of NAMPT was performed to assess the role of NAMPT in colorectal cancer cell proliferation and tumorigenesis both in vitro and in vivo. Overexpression of NAMPT was used to evaluate impact of NAMPT on colorectal cancer cell proliferation in vitro. NAD+ quantitation, immunofluorescence, dual luciferase assay and western blot were used to explore the mechanism of colorectal cancer proliferation. Transwell migration and invasion assays were conducted to assess the role of NAMPT in cell migration and invasion abilities of colorectal cancer cells. Results Our study indicated that the inhibition of NAMPT decreased proliferation capacity of colorectal cancer cells both in vitro and in vivo. Conversely, overexpression of NAMPT could promote cell proliferation in vitro. NAMPT inhibition induced β-catenin degradation by increasing Axin expression levels; this resulted in the inhibition of Wnt/β-catenin signaling and cell proliferation in colorectal cancer. The addition of nicotinamide mononucleotide, the enzymatic product of NAMPT, effectively reversed β-catenin protein degradation and inhibited growth. Similarly, the knockdown of Axin also decreased the cell death induced by the inhibition of NAMPT. In addition, we showed that colorectal cancer tissues harbored significantly higher levels of NAMPT than the levels harbored by paired normal tissues, especially in colorectal cancer stages I and II. And the overexpression of NAMPT was associated with unfavorable survival results. Conclusions Our findings reveal that NAMPT plays an important role in colorectal cancer proliferation via Wnt/β-catenin pathway, which could have vital implications for the diagnosis, prognosis and treatment of colorectal cancer.

Dilated cardiomyopathy (DCM) is a common type of cardiomyopathy. The pathogenesis of DCM remains unclear and involves varied genes.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

Background. Previous evidence has indicated that the reduction of axis inhibition protein 1 (AXIN1) expression is related with the poor differentiation of non-small-cell lung cancer (NSCLC). However, the potential association between AXIN1 and bladder cancer (BC) is unknown. We aimed to initially explore the relevance of AXIN1 gene polymorphisms (rs12921862 C/A, rs1805105 T/C, and rs370681 C/T) and BC. Methods. Three hundred and sixteen BC patients and 419 healthy controls had been enrolled. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for genotyping three tag single-nucleotide polymorphisms (SNPs) of AXIN1. The SNPstats online analysis software and SPSS software were used for statistical analysis. Results. Our data revealed that three tag SNPs were associated with an increased risk of BC (rs12921862: P<0.001, OR 95%CI=4.61 (3.13-6.81); rs1805105: P=0.046, OR 95%CI=1.35 (1.00-1.82); and rs370681: P=0.004, OR 95%CI=1.56 (1.15-2.10)). For rs12921862, A allele was an independently protective factor which correlated with a better prognosis in non-muscle-invasive bladder cancer (NMIBC) patients (P=0.03, OR 95%CI=0.10 (0.01-0.84)). Stratification analysis demonstrated that rs370681 polymorphism was related with high-grade bladder cancer (P=0.04, OR 95%CI=1.85 (1.04-3.23)). Conclusion. The AXIN1 gene polymorphisms might implicate in BC risk, and rs12921862 could be a potential forecasting factor for prognosis of BC patients.

Objective: Activation of endothelial β-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/β-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial β-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial β-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial β-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial β-catenin. Importantly, impaired endothelial β-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium. Conclusions: Our study identifies molecular crosstalk between the Wnt/β-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial β-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.

Summary Endothelial and fibroblast niches are crucial for epithelial organs. How these heterotypic cells interact is of great interest. In this study, we reveal an axis of signaling in which fibroblasts relay Wnt signals from the endothelial niche to organize epithelial patterning. We generate an Axin2-membrane GFP (mGFP) reporter mouse and observe robust Wnt/β-catenin signaling activities in fibroblasts surrounding the mammary epithelium. To enable cell-type-specific gene manipulation in vitro, we establish an organoid system via coculture of endothelial cells (ECs), fibroblasts, and mammary epithelial cells. Deletion of β-catenin in fibroblasts impedes epithelium branching, and ECs are responsible for the activation of Wnt/β-catenin signaling in fibroblasts. In vivo, EC deletion of Wntless inhibits Wnt/β-catenin signaling activity in fibroblasts, rendering a reduction in epithelial branches. These findings highlight the significance of the endothelial niche in tissue patterning, shedding light on the interactive mechanisms in which distinct niche components orchestrate epithelial organogenesis and tissue homeostasis.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by remodeling of the pulmonary vasculature, including marked proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Members of the nuclear receptor 4A (NR4A) subfamily are involved in a variety of biological events, such as cell apoptosis, proliferation, inflammation, and metabolism. Activation of Nur77 (an orphan nuclear receptor that belongs to NR4A subfamily) has recently been reported to be as a beneficial agent in the treatment of cardiovascular and metabolic diseases. In the present study, we investigated the effects of NR4A on human PASMCs function in vitro and determined the underlying mechanisms. We found a robust expression of NR4A receptors in lung tissues of PAH patients and hypoxic mice but a highly significant downregulation within pulmonary arteries (PAs) as assessed by quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. In vitro, NR4A receptors were found significantly decreased in PASMCs derived from PAH patients. To explore the pathological effects of decreased Nur77 in PASMCs, PASMCs were transduced with siRNA against Nur77. The siRNA-mediated knockdown of Nur77 significantly augmented PASMCs proliferation and migration. In contrast, Nur77 overexpression prevented PASMCs from proliferation and migration. Mechanistically, overexpression of Axis inhibition protein 2 (Axin2) or inhibition of β-catenin signaling was shown to be responsible for Nur77 knockdown-induced proliferation of PASMCs. Following hypoxia-induced angiogenesis of the pulmonary artery in C57BL/6 mice, small-molecule Nur77 agonists-Octaketide Cytosporone B (Csn-B) can significantly decreased thickness of vascular wall and markedly attenuated the development of chronic hypoxia-induced PAH in vivo. Therefore, reconstitution of Nur77 levels represents a promising therapeutic option to prevent vascular remodeling processes.

Pygopus 2 (Pygo2) is a chromatin effector that plays an essential role in canonical Wnt signaling associated with development and stem cell growth. Its function is to facilitate histone acetylation by recruitment of histone acetyltransferases (HATs) at active sites of β-catenin-mediated transcription. In the present study, we report that Pygo2 itself is transiently acetylated when bound to the activated TCF/β-catenin transcription complex, which correlated with β-catenin binding and Axin2 gene activation. The HAT CBP/p300, but not GCN5/PCAF, targeted specific lysine residues of the N-terminal homology domain of Pygo2 for acetylation. Functional analyses revealed that the presence of CBP and p300 increased the association of Pygo2 with GCN5, independent of Pygo2 acetylation status. Finally, while acetylation of Pygo2 had little effect on active β-catenin complex formation, p300-mediated Pygo2 acetylation resulted in the displacement of Pygo2 from the nucleus to the cytoplasm by targeting specific lysine residues in the Pygo2 nuclear localization sequence. Taken together, these findings are consistent with a model in which acetylation of Pygo2 by CBP/p300 family members in the active TCF/β-catenin complex occurs coincident with histone acetylation and may be required for the recycling of Pygo2 away from the complex subsequent to target gene activation.

Aims Myxomatous valve disease (MVD) is the most common aetiology of primary mitral regurgitation. Recent studies suggest that defects in heart valve development can lead to heart valve disease in adults. Wnt/β-catenin signalling is active during heart valve development and has been reported in human MVD. The consequences of increased Wnt/β-catenin signalling due to Axin2 deficiency in postnatal valve remodelling and pathogenesis of MVD were determined. Methods and results To investigate the role of Wnt/β-catenin signalling, we analysed heart valves from mice deficient in Axin2 (KO), a negative regulator of Wnt/β-catenin signalling. Axin2 KO mice display enlarged mitral and aortic valves (AoV) after birth with increased Wnt/β-catenin signalling and cell proliferation, whereas Sox9 expression and collagen deposition are decreased. At 2 months in Axin2 KO mice, the valve extracellular matrix (ECM) is stratified but distal AoV leaflets remain thickened and develop aortic insufficiency. Progressive myxomatous degeneration is apparent at 4 months with extensive ECM remodelling and focal aggrecan-rich areas, along with increased BMP signalling. Infiltration of inflammatory cells is also observed in Axin2 KO AoV prior to ECM remodelling. Overall, these features are consistent with the progression of human MVD. Finally, Axin2 expression is decreased and Wnt/β-catenin signalling is increased in myxomatous mitral valves in a murine model of Marfan syndrome, supporting the importance of Wnt/β-catenin signalling in the development of MVD. Conclusions Altogether, these data indicate that Axin2 limits Wnt/β-catenin signalling after birth and allows proper heart valve maturation. Moreover, dysregulation of Wnt/β-catenin signalling resulting from loss of Axin2 leads to progressive MVD.

// Claudia Paret 1, * , Johanna Theruvath 1, * , Alexandra Russo 1 , Bettina Kron 1 , Khalifa El Malki 1 , Nadine Lehmann 1 , Arthur Wingerter 1 , Marie A. Neu 1 , Aslihan Gerhold-Ay 2 , Wolfgang Wagner 3 , Clemens Sommer 4 , Torsten Pietsch 5 , Larissa Seidmann 6 , Jorg Faber 1 1 Section of Pediatric Oncology, Children's Hospital, University Medical Center of The Johannes Gutenberg University Mainz, Germany 2 Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), University Medical Center of The Johannes Gutenberg University Mainz, Germany 3 Section of Pediatric Neurosurgery, Department of Neurosurgery, University Medical Center of The Johannes Gutenberg University Mainz, Germany 4 Institute of Neuropathology, University Medical Center of The Johannes Gutenberg University Mainz, Germany 5 Department of Neuropathology, University of Bonn, Germany 6 Institute of Pathology, University Medical Center of The Johannes Gutenberg University Mainz, Germany * These authors have contributed equally to the work Correspondence to: Claudia Paret, email: Claudia.paret@unimedizin-mainz.de Keywords: HGNET-BCOR, BCOR, WNT, GLI, AXIN Received: June 13, 2016 Accepted: October 19, 2016 Published: November 04, 2016 ABSTRACT High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches. We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO , PTCH1 and SUFU , three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC 50 of 1.3 μM. In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.

Alveologenesis is the culmination of lung development and involves the correct temporal and spatial signals to generate the delicate gas exchange interface required for respiration. Using a Wnt-signaling reporter system, we demonstrate the emergence of a Wnt-responsive alveolar epithelial cell sublineage, which arises during alveologenesis, called the axin2+ alveolar type 2 cell, or AT2Axin2. The number of AT2Axin2 cells increases substantially during late lung development, correlating with a wave of Wnt signaling during alveologenesis. Transcriptome analysis, invivo clonal analysis, and exvivo lung organoid assays reveal that AT2sAxin2 promote enhanced AT2 cell growth during generation of the alveolus. Activating Wnt signaling results in the expansion of AT2s, whereas inhibition of Wnt signaling inhibits AT2 cell development and shunts alveolar epithelial development toward the alveolar type 1 cell lineage. These findings reveal a wave of Wnt-dependent AT2 expansion required for lung alveologenesis and maturation.

The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification.

Hereditary syndromes causing colorectal cancer include both polyposis and non-polyposis syndromes. Overlapping phenotypes between the syndromes have been recognized and this make targeted molecular testing for single genes less favorable, instead there is a gaining interest for multi-gene panel-based approaches detecting both SNVs, indels and CNVs in the same assay. We applied a panel including 19 CRC susceptibility genes to 91 individuals of six phenotypic subgroups. Targeted NGS-based sequencing of the whole gene regions including introns of the 19 genes was used. The individuals had a family history of CRC or had a phenotype consistent with a known CRC syndrome. The purpose of the study was to demonstrate the diagnostic difficulties linked to genotype-phenotype diversity and the benefits of using a gene panel. Pathogenicity classification was carried out on 46 detected variants. In total we detected sixteen pathogenic or likely pathogenic variants and 30 variants of unknown clinical significance. Four of the pathogenic or likely pathogenic variants were found in BMPR1A in patients with unexplained familial adenomatous polyposis or atypical adenomatous polyposis, which extends the genotype-phenotype spectrum for this gene. Nine patients had more than one variant remaining after the filtration, including three with truncating mutations in BMPR1A, PMS2 and AXIN2. CNVs were found in three patients, in upstream regions of SMAD4, MSH3 and CTNNB1, and one additional individual harbored a 24.2 kb duplication in CDH1 intron1. Electronic supplementary material The online version of this article (doi:10.1007/s10689-016-9934-0) contains supplementary material, which is available to authorized users.

Summary The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-β-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active β-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive β-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling., Graphical Abstract, Highlights • SAM domain crystal structures reveal mechanism of Tankyrase polymerization • Catalysis-independent Tankyrase scaffolding drives Wnt-β-catenin signaling • Tankyrase polymerization supports PARP activity and AXIN binding, Catalysis-independent scaffolding by Tankyrase supports Wnt-β-catenin signaling. Scaffolding is mediated by AXIN-binding ankyrin repeat clusters and a polymerizing SAM domain. In a structure-function approach, Mariotti et al. show that polymerization supports Tankyrase PARP activity and enables avidity-dependent AXIN binding.

Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T-cell factor/lymphoid enhancer factor (TCF/LEF) and β-catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/β-catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits β-catenin and the coactivator GRIP-1, and induces local H3K-acetylation. Barx2 also promotes nuclear localization of β-catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2-mediated H3K-acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation-competent and -incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/β-catenin complex.

Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein “destruction complex,” assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.

Objective Pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, altered vasoreactivity and vascular remodeling induced by smooth muscle cell proliferation. Similarities exist between cancer and PAH. Aberrant expression of the tumor suppressor protein is closely associated with PAH. Here, we tested the hypothesis that a tumor suppressor-axis inhibition protein 2 (Axin2) deficiency leads to PAH. Methods and results We measured right ventricular systolic pressure in Axin2 knockout mice and assessed the expression of Axin2 in patients. We found that Axin2 expression level was decreased in both mice exposed to chronic hypoxia and patients with PAH in remodeled pulmonary arterioles. Axin2 knockout mice showed elevated mean right ventricular systolic pressure and enhanced contraction in response to phenylephrine. An increase in the cross-sectional area of the vessels was occupied by the vessel wall, indicating pulmonary vascular remodeling. Furthermore, knocking down Axin2 with small interfering RNA inhibited apoptosis of pulmonary arterial smooth muscle cells (PASMCs). This inhibition was significantly abolished by β-catenin inhibitors, indicating that Axin2 through β-catenin increased vascular wall by inhibiting the apoptosis of PASMCs. Importantly, overexpression of Axin2 attenuates the development of hypoxia-induced PAH in mice. Conclusion Taken together, our study, for the first time, established that Axin2 plays a key role in the progression of PAH. We identified Axin2 as a novel mediator of pulmonary vasoconstriction and PASMC growth in hypoxia-mediated PAH. Our results suggest that downregulation of Axin2 in the pulmonary vasculature may be an underlying mechanism in the development of hypoxia-induced PAH.

The stability of β‐catenin is very important for canonical Wnt signaling. A protein complex including Axin/APC/GSK3β phosphorylates β‐catenin to be degraded by ubiquitination with β‐TrCP. In the recent study, we isolated WDR26, a protein that binds to Axin. Here, we found that WDR26 is a negative regulator of the canonical Wnt signaling pathway, and that WDR26 affected β‐catenin levels. In addition, WDR26/Axin binding is involved in the ubiquitination of β‐catenin. These results suggest that WDR26 plays a negative role in β‐catenin degradation in the Wnt signaling pathway.

Tooth agenesis is a congenital anomaly frequently seen in humans. Several genes have been associated with non-syndromic tooth agenesis, including msh homeobox 1 (MSX1), paired box 9 (PAX9), axis inhibition protein 2 (AXIN2), ectodysplasin A (EDA), and wingless-type MMTV integration site family member 10A (WNT10A). In this study, we investigated a Chinese family with non-syndromic tooth agenesis. A novel missense mutation (c.C1978T) in AXIN2 was identified in affected members. The mutation results in a His660Tyr substitution located between the Axin beta-catenin binding domain and the DIX domain of the axis inhibition protein 2 (AXIN2). We analysed this novel AXIN2 mutant, together with two reported AXIN2 mutants [c.1966C>T (p.Arg656Stop) and c.1994delG (p.Leu688Stop)] that cause colorectal cancer with and without oligodontia, to study the effect of the mutant p.His660Tyr on the Wnt/β-catenin signaling pathway and to compare the molecular pathogenesis of different AXIN2 mutants in tooth agenesis and carcinogenesis. Further in vitro experiments indicated that the mutant p.His660Tyr caused inhibition of the Wnt/β-catenin pathway, and the mutants p.Arg656Stop and p.Leu688Stop resulted in over-activation of the Wnt/β-catenin pathway. In line with previous AXIN2 mutation studies, we suggest that AXIN2 mutations with different levels of severity may have distinct effects on the Wnt pathway and the phenotype of disease. Our study provides functional evidence supporting the notion that both inhibition and over-activation of the Wnt pathway may lead to tooth agenesis.

// Kevin S. Quackenbush 1 , Stacey Bagby 1 , Wai Meng Tai 1, 2 , Wells A. Messersmith 1 , Anna Schreiber 1 , Justin Greene 1 , Jihye Kim 1 , Guoliang Wang 1 , Alicia Purkey 1 , Todd M. Pitts 1 , Anna Nguyen 1 , Dexiang Gao 1 , Patrick Blatchford 1 , Anna Capasso 1 , Alwin G. Schuller 3 , S. Gail Eckhardt 1 , John J. Arcaroli 1 1 Division of Medical Oncology, University of Colorado Denver and University of Colorado Cancer Center, Denver, CO, USA 2 Division of Medical Oncology, National Cancer Centre Singapore, Singapore 3 AstraZeneca R & D Boston, Massachusetts, Waltham, Massachusetts, MA, USA Correspondence to: John J. Arcaroli, email: john.arcaroli@ucdenver.edu Keywords: CRC, WNT, IRN, NuMA, PDTX Received: December 30, 2015 Accepted: February 28, 2016 Published: April 07, 2016 ABSTRACT Background: Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. In this study, we investigated the antitumor effects of AZ1366 (a novel tankyrase inhibitor) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Results: Six out of 18 CRC explants displayed a significant growth reduction to AZ1366. There was one CRC explant (CRC040) that reached the threshold of sensitivity (TGII ≤ 20%) in this study. In addition, the combination of AZ1366 + irinotecan demonstrated efficacy in 4 out of 18 CRC explants. Treatment effects on the WNT pathway revealed that tankyrase inhibition was ineffective at reducing WNT dependent signaling. However, the anti-tumor effects observed in this study were likely a result of alternative tankyrase effects whereby tankyrase inhibition reduced NuMA levels. Materials and Methods: Eighteen CRC explants were treated with AZ1366 single agent or in combination for 28 days and treatment responses were assessed. Pharmacokinetic (AZ1366 drug concentrations) and pharmacodynamic effects (Axin2 levels) were investigated over 48 hours. Immunohistochemistry of nuclear β-catenin levels as well as western blot was employed to examine the treatment effects on the WNT pathway as well as NuMA. Conclusions: Combination AZ1366 and irinotecan achieved greater anti-tumor effects compared to monotherapy. Activity was limited to CRC explants that displayed irinotecan resistance and increased protein levels of tankyrase and NuMA.