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Osteoarthritis (OA) is an age-related chronic joint degenerative disease. Interleukin 1 beta (IL-1β) is considered a marker for the progression of OA. In this study, we found that Ubiquitin-Specific Peptidase 49 (USP49) was significantly less expressed in OA patients compared with healthy individuals. Treating primary rat chondrocytes with different concentrations of IL-1β resulted in decreased Usp49 expression, while Usp49 overexpression could attenuate IL-1β-induced chondrocyte apoptosis by promoting Axin deubiquitination. The deubiquitination of Axin led to the accumulation of the protein, which in turn resulted in β-catenin degradation and Wnt/β-catenin signaling cascade inhibition. Interestingly, we also found that [6]-gingerol, an anti-OA drug, could upregulate the protein level of Usp49 and suppress the Wnt/β-catenin signaling cascade in primary rat chondrocytes. Taken together, our study not only demonstrates that Usp49 can negatively regulate the progression of OA by inhibiting the Wnt/β-catenin signaling cascade, but also elucidates the underlying molecular mechanisms.

The activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres—in cleavage stage embryos—to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical negative regulator of cWnt signaling, during early sea urchin embryogenesis. Sea urchinAxinis ubiquitously expressed in early embryos and by the blastula stage the expression of the gene becomes restricted to the posterior end of the embryo. Strikingly, knockdown of Axin protein levels using antisense Axin morpholinos (MO) led to ectopic nuclearization of β-catenin and activation of endomesoderm gene expression in anterior blastomeres in early embryos. These embryos developed a severely posteriorized phenotype that could be fully rescued by co-injection of Axin MO with wild-type Axin mRNA. Axin is known to negatively regulate cWnt by its role in mediating β-catenin stability within the destruction complex. Consistent with this function overexpression of Axin by mRNA injection led to the downregulation of nuclear β-catenin, inhibition of endomesoderm specification and a strong anteriorization of embryos. Axin has several well-defined domains that regulate its interaction with β-catenin and the key regulators of the destruction complex, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase 3β(GSK-3β), and Dishevelled (Dvl). Using Axin constructs with single deletions of the binding sites for these proteins we showed that only the GSK-3βbinding site on Axin is required for its inhibition of cWnt in the sea urchin embryo. Strikingly, overexpression of the GSK-3β-binding domain alone led to embryos with elevated levels of endomesoderm gene expression and a strongly posteriorized phenotype. These results indicated that Axin has a critical global role in inhibiting cWnt signaling in the early sea urchin embryo, and moreover, that the interaction of Axin with GSK-3βis critical for this inhibition. These results also add to the growing body of evidence that Axin plays a global function in suppressing cWnt signaling in early embryos and indicates that modulation of Axin function may be a critical early step during patterning of the AP axis during bilaterian development

In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically, eLife digest Stem cells can give rise to many types of specialized cells through a process called differentiation, which is partly regulated by changes in the levels of a protein known as β-catenin. On one hand, a ‘destruction complex’ can keep β-catenin levels low; this complex includes a protein called Axin and an enzyme known as GSK-3, which can tag β-catenin for degradation. On the other hand, when β-catenin levels need to increase, another protein called Dishevelled is activated. By binding to Axin, Dishevelled can bring the destruction complex in contact with other proteins, which leads to the deactivation of GSK-3. Dishevelled and Axin interact via a region that is similar in the two proteins, called DIX in Dishevelled and DAX in Axin. Studies of DIX and DAX have shown that both regions can form polymers – that is, a high number of similar units can bind together to form larger structures. However, these experiments were at higher concentrations than would be found in the cell. It was thought that, when combined, DIX and DAX might form these long chains together, preventing Axin from carrying out its role in destroying β-catenin. Kan et al. set out to better understand this process by studying how DIX and DAX behave separately, and how they interact. The proteins were examined using a technique called cryo-electron microscopy, which allows scientists to dissect the structure of large proteins. When there was a high concentration of DIX in the sample, the molecules attached to one another to form long double-stranded helices. Similarly, DAX also formed helices, but these were shorter and only single-stranded. When the two proteins were combined, DAX bound only to the ends of short DIX chains, so that there are not more than four DAX chains attached to each DIX double helix. To see if this behaviour happens naturally, Kan et al. attached fluorescent tags to Dishevelled proteins and followed them in living cells: this showed that Dishevelled forms smaller chains with fewer than ten molecules. Together these results highlight how Dishevelled binds to Axin to deactivate GSK-3, to prevent the enzyme from promoting β-catenin destruction. Mutations in the genes that encode β-catenin or its regulators are associated with cancer. Ultimately, a better understanding of how β-catenin is regulated could help to identify new opportunities for drug development.

Long non-coding RNA (lncRNA) nuclear-enriched assembly transcript 1 (NEAT1) has been reported to be highly expressed in Parkinson's disease (PD). However, the mechanism of NEAT1 in PD progression has not been fully elucidated. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine injection (MPTP) was used to construct PD mouse models in vivo, and 1-methyl-4-phenyl pyridine (MPP+) was used to build PD cell models in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to test the expression of NEAT1, microRNA (miR)-212-3p and axis inhibition protein 1 (AXIN1). The viability, apoptosis and inflammation of cells were determined using cell counting kit 8 (CCK8) assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Then, the protein levels of apoptosis-related markers and AXIN1 were measured by western blot (WB) analysis. Furthermore, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between miR-212-3p and NEAT1 or AXIN1. NEAT1 was upregulated in PD mouse models and cell models. Function experiments confirmed that NEAT1 knockdown could promote the viability, suppress the apoptosis and inflammation of MPP+-stimulated SK-N-SH cells to restrain PD progression. MiR-212-3p was downregulated in PD, and its inhibitor could reverse the suppression effect of NEAT1 knockdown on PD progression. Additionally, AXIN1 was a target of miR-212-3p, and its overexpression could invert the inhibition effect of miR-212-3p mimic on PD progression. Furthermore, AXIN1 expression was inhibited by NEAT1 silencing and promoted by NEAT1 overexpression, while these effect could be recovered by miR-212-3p inhibitor and mimic, respectively. Our results demonstrated that NEAT1 knockdown suppressed PD progression through regulating the miR-212-3p/AXIN1 pathway, indicating that NEAT1 might be a therapeutic target for neuroprotection in PD.

Background Triple‐negative breast cancer (TNBC) is one subtype of breast cancer, which is characterized by an aggressive disease. It is commonly accompanied with extremely poor prognosis because of no available molecularly targeted therapy. Thus, understanding the detailed molecular mechanisms of TNBC is urgently needed. Methods The levels of Axis inhibition protein 1 (Axin1), Cyclin D1, c‐Myc, and miR‐124‐3p.1 were measured by quantitative real‐time PCR (qRT‐PCR). Furthermore, the breast cancer cell proliferation was measured by CCK‐8 assay, colony formation assays, and EdU staining. Xenograft model was used to show the tumor genesis of breast cancer cells. The regulatory function of miR‐124‐3p.1 on Wnt/β‐catenin signaling activation through directly targeting Axin1 was proven using qRT‐PCR, Western blot analysis, and dual‐luciferase reporter assay. To further assess the clinical significance of miR‐124‐3p.1 in the prognosis of breast cancer patients, we performed Kaplan‐Meier survival analysis and log‐rank tests. Results miR‐124‐3p.1 expression was elevated in advanced TNBC patients, and high miR‐124‐3p.1 predicts poor overall survival in TNBC patients. Further data showed that miR‐124‐3p.1 downregulation diminished, while miR‐124‐3p.1 upregulation increased the growth of TNBC cells in vitro and in vivo. Finally, we proved that miR‐124‐3p.1 exerted its function via targeting tumor suppressor gene Axin1 and activating the Wnt signaling pathway. Conclusion In summary, all the results demonstrate that miR‐124‐3p.1 promotes TNBC cell growth by controlling Axin1, suggesting that targeting miR‐124‐3p.1 might offer an effective therapeutic strategy for TNBC in the future.

Wnt/β-catenin signaling is essential for intestinal homeostasis and is aberrantly activated in most colorectal cancers (CRC) through mutation of the tumor suppressor Adenomatous Polyposis Coli (APC). APC is an essential component of a cytoplasmic protein complex that targets β-catenin for destruction. Following Wnt ligand presentation, this complex is inhibited. However, a role for APC in this inhibition has not been shown. Here, we utilized Wnt3a-beads to locally activate Wnt co-receptors. In response, the endogenous β-catenin destruction complex reoriented toward the local Wnt cue in CRC cells with full-length APC, but not if APC was truncated or depleted. Non-transformed human colon epithelial cells displayed similar Wnt-induced destruction complex localization which appeared to be dependent on APC and less so on Axin. Our results expand the current model of Wnt/β-catenin signaling such that in response to Wnt, the β-catenin destruction complex: (1) maintains composition and binding to β-catenin, (2) moves toward the plasma membrane, and (3) requires full-length APC for this relocalization.

The dysregulation of ubiquitin ligase is the cause of many human diseases. Tripartite motif protein 32 (TRIM32) is an E3 ubiquitin ligase whose role in nucleus pulposus (NP) cell apoptosis is unclear. The expression of TRIM family protein and β-catenin in 40 NP tissue samples was detected by RT-PCR. Interleukin (IL)-1β or tumor necrosis factor (TNF)-α was used to treat rat NP cells. Knockdown and overexpression of Trim32 were achieved using specific siRNA and recombinant plasmids. Western blotting, RT-PCR, and flow cytometry were used to assess the expression of TRIM32/β-catenin and the apoptosis rate of NP cells. Coimmunoprecipitation was adopted to analyze the possible interactions between AXIN1 and TRIM32. In clinical samples, TRIM32 expression was of positive relevance with the expression of CTNNB1 (β-catenin). In vitro, apoptosis of IL-1β- or TNF-α-treated rat NP cells was induced through upregulated Trim32 expression and activated β-catenin signaling, whereas Trim32 siRNA and inhibition of β-catenin reversed the induction effect of cytokines. Further studies indicated that TRIM32 activated the β-catenin signaling pathway through ubiquitination of AXIN1, thereby regulating apoptosis. Collectively, this study reveals that TRIM32 promotes inflammatory factor-induced apoptosis of rat NP cells, in part by direct degradation of AXIN1 to trigger β-catenin signaling.

Background: Systemic sclerosis is an autoimmune skin disease which is associated with inflammation and resulting skin fibrosis. Myofibroblasts are the key cell type associated with the fibrosis but how they are differentiated is not clear. DKK‐1 is a Wnt antagonist that blocks Wnt‐mediated fibrosis and is reduced in fibrotic conditions. Thus DKK‐1 is a clear negative regulator of fibrosis in systemic sclerosis and its regulation is unknown.The aim of this work is to determine the levels of DKK‐1 in serum and tissues of SSc and its regulation. Methods: Skin biopsies were taken from early diffuse systemic sclerosis patients and healthy controls and DKK‐1 measured by ELISA; serum was also isolated and DKK‐1 quantified. DKK‐1 was also measured by qRT‐PCR. MicroRNA33a‐3p was measured by Taqman PCR. miR mimics and controls were transfected into dermal fibroblasts. Bleomycin mouse model was employed and compared to vehicle control treated mice and gene expression was employed for DKK‐1 and various extracellular matrix genes. Results: DKK‐1 is reduced in SSc skin and fibroblasts but is not reduced in the circulation in patients. microRNA33a‐3p regulates DKK‐1 levels epigenetically and is significantly reduced in SSc cells and whole tissue. DKK‐1 is also reduced in the bleomycin mouse modelandpro‐fibrotic genes elevated. Conclusion: DKK‐1 is reduced in SSc cells and is regulated by miR33a‐3p and restoring DKK‐1 levels through epigenetic means could be a therapeutic target in systemic sclerosis.

Salivary glands exert exocrine secretory function to provide saliva for lubrication and protection of the oral cavity. Its epithelium consists of several differentiated cell types including acinar, ductal and myoepithelial cells that are maintained in a lineage-restricted manner during homeostasis or after mild injuries. Glandular regeneration following a near complete loss of secretory cells, however, may involve cellular plasticity, although the mechanism and extent of such plasticity remain unclear. Here, by combining lineage-tracing experiments with a model of severe glandular injury in the mouse submandibular gland, we show that de novo formation of acini involves induction of cellular plasticity in multiple non-acinar cell populations. Fate-mapping analysis revealed that although ductal stem cells marked by cytokeratin K14 and Axin2 undergo a multipotency switch, they do not make a significant contribution to acinar regeneration. Intriguingly, more than 80% of regenerated acini derive from differentiated cells including myoepithelial and ductal cells that appear to dedifferentiate to a progenitor-like state before re-differentiation to acinar cells. The potential of diverse cell populations serving as a reserve source for acini widens the therapeutic options for hyposalivation.

In adult hippocampal neurogenesis, stem/progenitor cells generate dentate granule neurons that contribute to hippocampal plasticity. The establishment of a morphologically defined dendritic arbor is central to the functional integration of adult‐born neurons. We investigated the role of canonical Wnt/β‐catenin signaling in dendritogenesis of adult‐born neurons. We show that canonical Wnt signaling follows a biphasic pattern, with high activity in stem/progenitor cells, attenuation in immature neurons, and reactivation during maturation, and demonstrate that this activity pattern is required for proper dendrite development. Increasing β‐catenin signaling in maturing neurons of young adult mice transiently accelerated dendritic growth, but eventually produced dendritic defects and excessive spine numbers. In middle‐aged mice, in which protracted dendrite and spine development were paralleled by lower canonical Wnt signaling activity, enhancement of β‐catenin signaling restored dendritic growth and spine formation to levels observed in young adult animals. Our data indicate that precise timing and strength of β‐catenin signaling are essential for the correct functional integration of adult‐born neurons and suggest Wnt/β‐catenin signaling as a pathway to ameliorate deficits in adult neurogenesis during aging., Precise timing and strength of β‐catenin signaling determines correct functional integration of adult‐born neurons, implicating Wnt/β‐catenin activity as protective pathway against aging‐related deficits in adult neurogenesis.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Adenomatous Polyposis Coli (APC) gene mutations are responsible for the onset of familial adenomatous polyposis (FAP) and sporadic colorectal cancer and have been associated with miRNAs dysregulation. The capacity of miR-155, a cancer-related miRNA, to target components of the WNT/β-CATENIN pathway suggests that APC gene mutations, controlling miRNAs expression, may critically regulate WNT/β-CATENIN signaling. To this end, APC gene target sequencing was performed on colonic adenomatous polyps and paired normal mucosa clinical specimens from FAP patients (n = 9) to elucidate the role of miR-155-5p in APC-mutant setting. The expression of selected miRNAs and WNT/β-CATENIN signaling components was characterized in FAP patients and non-FAP control subjects (n = 5). miR-155-5p expression and functional effects on WNT cascade, cell survival, growth, and apoptosis were investigated in different colorectal cancer cell lines. A somatic second hit in the APC gene was found in adenomatous polyps from 6 of 9 FAP patients. Heterozygous APC gene mutations in FAP patients were associated with altered expression of candidate miRNAs and increased levels of AXIN1 and AXIN2 mRNAs. miR-155-5p was downregulated in FAP patients and in the APC and β-CATENIN–mutant colorectal cancer cell lines, and critically regulates WNT/β-CATENIN cascade by targeting both AXIN1 and TCF4. Importantly, miR-155-5p may sustain long-term WNT/β-CATENIN activation in colorectal cancer cells upon WNT3A stimulation. Implications: This study supports a key role of miR-155-5p in modulating WNT/β-CATENIN signaling in colorectal cancer and unravels a new mechanism for AXIN1 regulation which represents a potential therapeutic target in specific tumor subtypes.

Although increasing long noncoding RNAs (lncRNAs) have been identified by high-throughput sequencing, their functions in human cancer remain largely unknown. The function of lncRNA miR143HG has not been explored before. In the present study, we found that miR143HG expression was significantly downregulated in bladder cancer tissues (BCa) compared with normal tissues. We showed that miR143HG high expression was associated with a high survival rate in BCa patients. Gain-of-function assays demonstrated that miR143HG overexpression suppressed the proliferation, arrested cell cycle progression, and attenuated migration and invasion of BCa cells in vitro. In vivo assay illustrated that ectopic expression of miR143HG inhibited BCa growth in vivo. Mechanistically, miR143HG was identified to inhibit the level of miR-1275, whereas miR-1275 directly targeted AXIN2, a negative regulator of the Wnt/β-catenin pathway. Restoration of miR-1275 or knockdown of AXIN2 significantly rescued the proliferation, migration, and invasion abilities of BCa cells. In summary, our findings demonstrated that miR143HG/miR-1275/AXIN2 axis regulates BCa development by modulating the Wnt/β-catenin pathway.

Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down‐regulation of Zbed3 inhibited β‐catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/TPC/glycogen synthase kinase 3β (Gsk‐3β) complex to the response. Coimmunoprecipitation assays showed that wild‐type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including β‐catenin, cyclin D1 and MMP7 (P 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild‐type Zbed3 (P 0.05). Similarly, treating the cells with a GSK‐3β inhibitor abolished Zbed3's ability to increase β‐catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK‐3β‐mediated negative regulation of β‐catenin levels.

Wnt-β-catenin signaling is essential for homeostasis of intestinal stem cells in mice and is thought to promote intestinal crypt fission. The aim of this study was to investigate Wnt-β-catenin signaling in intestinal crypts of human infants. Duodenal biopsies from nine infants (mean, range 0.9 years, 0.3–2 years) and 11 adults (mean, range 43 years, 34–71 years) were collected endoscopically. Active β-catenin signaling was assessed by cytoplasmic and nuclear β-catenin, nuclear c-Myc, and cytoplasmic Axin-2 expression in the base of crypts. Tissues were stained by an immunoperoxidase staining technique and quantified as pixel energy using cumulative signal analysis. Data were expressed as mean ± SD and significance assessed by Student’s t test. Crypt fission was significantly higher in infants compared to adults (16 ± 8.6% versus 0.7 ± 0.6%, respectively, p

Wnt/β-catenin signaling plays a key role in regulating adipogenesis through indirectly inhibiting the expression of C/EBPα and peroxisome proliferator–activated receptor γ (PPARγ); however, the detailed molecular mechanism remains poorly understood. Moreover, the factor(s) that determines the Wnt/β-catenin output level during adipogenesis is also not completely defined. In this study, we showed that Pygo2 exhibited a declined expression pattern during adipocyte differentiation, resulting in an attenuated Wnt/β-catenin output level. The mechanism study indicated that Pygo2 inhibition led to the downregulation of Axin2, a constitutive Wnt target, in the cytoplasm. Consequently, Axin2-bound GSK3β was released and translocated into the nucleus to phosphorylate C/EBPβ and Snail, resulting in an increase in the DNA binding activity of C/EBPβ and decreased protein stability of Snail, which subsequently activated the expression of C/EBPα and PPARγ. Consistent with this, embryonic fibroblasts from Pygo2−/− mice exhibited spontaneous adipocyte differentiation, and adipocyte precursor–specific Pygo2-deficient mice exhibited increased adiposity with decreased energy expenditure. We further showed impaired glucose tolerance and decreased systemic insulin sensitivity in Pygo2-deficient mice. Our study revealed an association between Pygo2 function and obesity or diabetes.

Axin1, a concentration-limiting component of the β-catenin destruction complex, negatively regulates the Wnt/β-catenin pathway. Axin1 concentration is reported to be regulated by proteasomal degradation; however, its transcriptional regulation has not yet been reported. Here, we demonstrated that CCAAT/enhancer-binding protein-β (C/EBP-β) activates axis inhibition protein 1 (AXIN1) gene expression, thereby attenuating Wnt/β-catenin signaling. C/EBP-β interacted with cis-regulatory element for C/EBP-β in the 5′-upstream sequences of the AXIN1 gene and increased AXIN1 promoter activity. Functional analysis using Drosophila and zebrafish models established that C/EBP-β negatively regulates the Wnt/β-catenin pathway. Small-molecule-based up-regulation of C/EBP-β induces AXIN1 gene expression and down-regulates the intracellular β-catenin level, thereby inhibiting hepatoma cell growth. Thus, our findings provide a unique mechanistic insight into the regulation of Axin homeostasis and present a novel strategy for the development of anticancer therapeutics targeting Wnt/β-catenin signaling.

The E3 ubiquitin ligase ring finger protein 146 (RNF146) has been implicated in tumor development. However, the role and clinical significance of RNF146 in colorectal cancer (CRC) remain unknown. In this study, we reported for the first time that RNF146 was upregulated in CRC tissues as well as in cell lines. Further, RNF146 expression was independent prognostic factor for poor outcome of CRC patients. RNF146 knockdown in cell lines inhibited cell growth, promoted cell apoptosis in vitro and suppressed colorectal tumor growth in vivo. Mechanistic investigations revealed that RNF146 exerted oncogenic role through ubiquitination of Axin1 to activate β-catenin signaling. In addition, RNF146 expression was positively correlated with β-catenin expression in CRC tissues. Collectively, our data suggest that RNF146 might function as a oncogene in human CRC, and represent a promising prognostic factor and a valuable therapeutic target for CRC.

Despite significant progress in the treatment of breast cancer due to advances in surgery, cytotoxic agents, and endocrine therapy, the prognosis for patients has not improved much. Accumulated evidence indicates that heterogeneous nuclear ribonucleoprotein M (hnRNPM) and Wnt/β-catenin function as tumor oncogenes in the progression of many cancers. The present study aimed to explore whether HnRNPM/β-catenin signaling molecules might serve as a genetic target for breast cancer treatment. To shed light on this issue, quantitative real-time polymerase chain reaction (qRT-PCR) detection, Western blotting, and immunohistochemical staining were performed. The hnRNPM is expressed at a much higher level in breast cancer tissues and cell lines than in noncancerous tissues and cell lines. In vitro studies revealed that overexpressed hnRNPM promoted cell proliferation and colony formation but inhibited cell apoptosis. In vivo results demonstrated that upregulation of hnRNPM dramatically increased breast cancer xenograft tumor growth. Western blotting and immunofluorescence studies revealed that hnRNPM markedly activated the Wnt/β-catenin pathway and catalyzed its translocation from the cytoplasm to the nucleus by targeting axin, a negative regulator of Wnt/β-catenin signaling in MCF-7 and KPL-4 cells. Elevated levels of c-Myc and cyclin D1 were observed when MCF-7 and KPL-4 cells were transfected with a hnRNPM vector. These findings indicate that the hnRNPM/axin/β-catenin signaling pathway acts as an oncogenic promoter in the progression of breast cancer, suggesting that hnRNPM may be a potential target for the treatment of this disease.

FAM83G/Fam83g genetic variants have been described in dogs, mice and recently also in humans. They are associated with palmoplantar keratoderma and altered hair or coat phenotype, reported as wooly phenotype in mice. FAM83G/Fam83g is an unexplored effector of temporally and spatially coordinated Wnt and BMP signalling which are key pathways in pre- and postnatal hair follicle morphogenesis and differentiation. The aim of this study was to unravel phenotypic consequences of FAM83G/Fam83g variants on hair coat formation in dogs and mice. Our results show differences in hair types and hair shaft structures in both species. Additionally, mice exhibit deregulated hair cycle progression which timely correlates with defective Wnt signalling (Axin2) and Bmp2/4 expression. These results affirm the involvement of FAM83G in hair morphogenesis, hair follicle differentiation and cycling.

The epithelial disintegration and the mesenchymal bridging are critical steps in the fusion of facial prominences during the upper lip development. These processes of epithelial-mesenchymal transition and programmed cell death are mainly influenced by Wnt signals. Axis inhibition protein2 (Axin2), a major component of the Wnt pathway, has been reported to be involved in lip development and cleft pathogenesis. We wanted to study the involvement of Axin2 in the lip development, especially during the epithelial disintegration of facial prominences. Our results show that Axin2 was expressed mainly in the epithelium of facial prominences and decreased when the prominences were about to contact each other between Hamburger-Hamilton stages 27 and 28 of chicken embryos. The epithelial integrity was destructed or kept intact by the local gain or loss of Axin2 expression, resulting in morphological changes in the facial processes and their skeletal derivatives including the maxilla, nasal, premaxilla bone, and their junctions without cleft formation. These changes were related to expression changes in nuclear β-catenin, pGSK3β, Slug, Smad3, E-cadherin, and p63. All these data indicate that Axin2 participates in the regulation of epithelial integrity and fusion by promoting epithelial disassociation, basement membrane breakdown, and seam loss during the fusion of facial prominences in lip development.

Tooth agenesis (TA) is one of the most common developmental anomalies that affects the number of teeth. An extensive analysis of publicly accessible databases revealed 15 causative genes responsible for nonsyndromic TA, along with their signaling pathways in Wnt/β-catenin, TGF-β/BMP, and Eda/Edar/NF-κB. However, genotype-phenotype correlation analysis showed that most of the causal genes are also responsible for syndromic TA or other conditions. In a total of 198 different mutations of the 15 genes responsible for nonsyndromic TA, 182 mutations (91.9%) are derived from seven genes (AXIN2, EDA, LRP6, MSX1, PAX9, WNT10A, and WNT10B) compared with the remaining 16 mutations (8.1%) identified in the remaining eight genes (BMP4, DKK1, EDAR, EDARADD, GREM2, KREMEN1, LTBP3, and SMOC2). Furthermore, specificity analysis in terms of the ratio of nonsyndromic TA mutations versus syndromic mutations in each of the aforementioned seven genes showed a 98.2% specificity rate in PAX9, 58.9% in WNT10A, 56.6% in MSX1, 41.2% in WNT10B, 31.4% in LRP6, 23.8% in AXIN2%, and 8.4% in EDA. These findings underscore an important role of the Wnt and Wnt-associated pathways in the genetic etiology of this heterozygous disease and shed new lights on the discovery of novel molecular mechanisms associated with tooth agenesis.

Colorectal cancer (CRC) is the most common malignant disease worldwide due to its metastasis via the epithelial-mesenchymal transition (EMT) process. E-cadherin and Wnt signaling are emerging as potential targets for suppressing the EMT. In this context, Axin2 has been recognized as a negative regulator that inhibits glycogen synthase kinase 3β (GSK3β)-mediated degradation of Snail1, a transcriptional repressor of E-cadherin. However, Axin2 can also impede Wnt signaling via β-catenin degradation. Therefore, Axin2 may serve as either a promoter or suppressor of tumors, and the effects of its inhibition on the cell proliferation and metastasis of CRC require further elucidation. Here, esculetin (ES), a coumarin, was found to have the most potential effects on both β-catenin-responsive transcriptional and E-cadherin promoter activities. ES also showed anti-proliferative and anti-invasive activities in CRC cells. Mechanistically, Axin2 suppression by ES contributed to E-cadherin-mediated Wnt signaling inhibition. Moreover, the ability of ES to inhibit tumor growth and metastasis via Axin2 suppression was further supported in an HCT116-implanted orthotopic mouse model. Collectively, these findings suggest that targeting the Axin2/E-cadherin axis by ES may be an attractive therapeutic strategy for the treatment of metastatic CRC.

Differential diagnosis among basal cell adenoma (BCA), basal cell adenocarcinoma (BCAC), adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA) of the salivary gland can be challenging due to their similar histological appearance. Although frequent nuclear β-catenin expression and CTNNB1 mutations have been reported in BCA, further details of the Wnt/β-catenin signal alterations are unclear. The aim of this study was to assess the diagnostic utility of Wnt/β-catenin signal alteration in BCA and morphological mimics. We performed immunohistochemical staining for β-catenin and mutation analysis for Wnt/β-catenin-related genes (CTNNB1, APC, AXIN1 and AXIN2) in BCA (n = 34), BCAC (n = 3), ACC (n = 67) and PA (n = 31). We also analyzed ACC-specific MYB and MYBL1 gene rearrangements by fluorescence in situ hybridization (FISH). Nuclear β-catenin expression (≥3%) was present in 32/34 cases (94.1%) of BCA, and the nuclear β-catenin labeling index was significantly higher than in other tumor types (p = 0.0001). In BCA, we found mutations in CTNNB1, APC and AXIN1 genes (41.1%, 2.9% and 8.8%, respectively). In BCAC, nuclear β-catenin expression with CTNNB1 mutation was present in 1/3 cases (33.3%). As for ACC, nuclear β-catenin expression was observed in 3/67 cases (4.4%), but all 3 cases harbored either MYB or MYBL1 gene rearrangement. The results suggest that nuclear β-catenin immunoreactivity with appropriate criteria may be helpful to distinguish BCA from histologically similar tumors. However, a minor subset of ACCs with nuclear β-catenin expression require careful diagnosis. In addition, Wnt/β-catenin signal alteration may play a role in the pathogenesis of BCA and BCAC.

Dysregulation of human trophoblast invasion and differentiation with placental hypoxia can result in preeclampsia, a hypertensive disorder of pregnancy. Herein, we characterized the role and regulation of miR-1246, which is markedly induced during human syncytiotrophoblast differentiation. miR-1246 targets GSK3β and AXIN2, inhibitors of WNT/β-catenin signaling, which is crucial for placental development, and is predicted to target JARID2, which promotes silencing of developmentally regulated genes. Human cytotrophoblasts cultured in 20% O(2) spontaneously differentiate to syncytiotrophoblast with induction of hCYP191A/aromatase, a marker of differentiation. miR-1246 was induced >150-fold during syncytiotrophoblast differentiation in 20% O(2), whereas targets—GSK3β, AXIN2, and JARID2—were significantly decreased. However, when cytotrophoblasts were cultured in 2% O(2), miR-1246 and aromatase induction were prevented. miR-1246 was significantly decreased in placentas of women with severe preeclampsia, whereas AXIN2, GSK3β, and JARID2 were increased, compared with normotensive subjects. To identify factors that regulate miR-1246, we investigated the redox-regulated transcription factor NRF2, which has predicted binding sites in the miR-1246 promoter. Intriguingly, NRF2 messenger RNA was upregulated during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in preeclamptic placentas. Moreover, NRF2 knockdown in cytotrophoblasts inhibited induction of miR-1246 and hCYP19A1, as well as transcription factors C/EBPβ and PPARγ, which are implicated in placental differentiation. Using chromatin immunoprecipitation–quantitative polymerase chain reaction, we found that binding of endogenous NRF2 to the miR-1246 and hCYP191A promoters increased during syncytiotrophoblast differentiation. Thus, NRF2 promotes syncytiotrophoblast differentiation by inducing C/EBPβ, PPARγ, hCYP19A1, and miR-1246, which targets WNT inhibitors and JARID2 and is dysregulated in preeclampsia.

Wnt/β-catenin signaling activity is maintained in homeostasis by an expanding list of molecular determinants. However, the molecular components and the regulatory mechanisms involved in its fine-tuning remain to be determined. Here, we identified C9orf140, a tumor-specific protein, as a novel Axin1-interacting protein by tandem-affinity purification and mass spectrometry. We further showed that C9orf140 is a negative regulator of Wnt/β-catenin signaling in cultured cells as well as in zebrafish embryos. It functions upstream of β-catenin, outcompetes PP2A for binding to Axin1, influences the balance between phosphorylation and de-phosphorylation of β-catenin, and ultimately compromises Wnt3A-induced β-catenin accumulation. Interestingly, Wnt-induced C9orf140 expression via β-catenin. We propose that C9orf140 mediates a negative feedback loop of Wnt/β-catenin signaling by interacting with Axin1. Our results advance the current understanding of the exquisite control of Wnt/β-catenin signaling cascade, and provide evidence of the new role of C9orf140.

Mitochondrial stress induces PARL-mediated cleavage and cytosolic release of the mitochondrial phosphatase Pgam5. In the cytosol, Pgam5 interacts with the Wnt pathway component axin and dephosphorylates axin-bound β-catenin, thereby cell-intrinsically activating Wnt/β-catenin signaling to induce mitochondrial biogenesis., Mitochondrial abundance is dynamically regulated and was previously shown to be increased by Wnt/β-catenin signaling. Pgam5 is a mitochondrial phosphatase which is cleaved by the rhomboid protease presenilin-associated rhomboid-like protein (PARL) and released from membranes after mitochondrial stress. In this study, we show that Pgam5 interacts with the Wnt pathway component axin in the cytosol, blocks axin-mediated β-catenin degradation, and increases β-catenin levels and β-catenin–dependent transcription. Pgam5 stabilized β-catenin by inducing its dephosphorylation in an axin-dependent manner. Mitochondrial stress triggered by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment led to cytosolic release of endogenous Pgam5 and subsequent dephosphorylation of β-catenin, which was strongly diminished in Pgam5 and PARL knockout cells. Similarly, hypoxic stress generated cytosolic Pgam5 and led to stabilization of β-catenin, which was abolished by Pgam5 knockout. Cells stably expressing cytosolic Pgam5 exhibit elevated β-catenin levels and increased mitochondrial numbers. Our study reveals a novel mechanism by which damaged mitochondria might induce replenishment of the mitochondrial pool by cell-intrinsic activation of Wnt signaling via the Pgam5–β-catenin axis.

The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non‐pigmented ciliary epithelium (NPCE)–derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose–response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE‐derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β‐Catenin, Axin2 and LEF1) and protein (β‐Catenin, GSK‐3β) expression using real‐time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β‐Catenin, GSK‐3β, as opposed to exposure to high exosomal concentrations. Pro‐MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE‐ or RPE‐derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration‐dependent at their target site. Specifically in the present study, we described a general dose–response at the gene and MMPs activity and a different dose–response regarding key canonical Wnt proteins expression.

It is well known that colon cancer stemness and invasiveness are the main reasons for tumor recurrence and metastasis. MicroRNAs dysregulation can disrupt the balance of cell signaling and growth processes, resulting in cancer proliferation, invasion and metastasis, chemoresistance and so on. In this study, we used colon cancer cell lines HCT-116 and SW-480 to investigate the effects of miR-3120-5p on stemness and invasiveness of colon cancer. We found that the population of CD133 + and Lgr5+ stem cells in both cell lines expressed miR-3120-5p highly, and introducing miR-3120-5p into both cell lines increased the population of cancer stem cells, as measured by flow cytometry, qRT-PCR and sphere formation assays. Transwell assay, Gelatin zymography assay and Western blot assays further revealed that miR-3120-5p promotes colon cancer cells invasive ability. By the target prediction algorithm TargetScan, we found Axin2 is a potential target for miR-3120-5p, and luciferase reporter assay demonstrated that miR-3120-5p reduces Axin2 expression. Transfection of siRNA against Axin2 into colon cancer cells promoted the stemness and invasion of colon cancer cells. Furthermore, Axin2 overexpression partially reversed the promotion of stemness and invasiveness caused by miR-3120-5p in colon cancer cells. Together, all the results demonstrated miR-3120-5p promotes stemness and invasiveness of colon cancer cells through direct targeting of Axin2. They suggest that antago-miR-3120-5p plays important roles on treatment strategy for colon cancer.

We report solid-state Nuclear Magnetic Resonance (ssNMR) studies on amyloid-like protein complexes formed by DIX domains that mediate key protein interactions in the Wnt signalling pathway. Our results provide insight into the 3D fold of the self-associated Axin-DIX domain and identify a potential lipid cofactor.

Summary The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFβ-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling.

Summary: Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3−/− iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo. : Using an eye organoid culture system, Takata et al. identified the Wnt signaling partner R-spondin 2 as a critical player in neuroretina fate determination in vivo and in vitro. They also validated these findings using newly derived conditional null Six3delta/f;Rax-Cre ESCs from mouse blastocysts. Keywords: eye, optic vesicles, Six3, pluripotent stem cells, organoids, neuroretina, R-spondins, Wnt, mouse, Six3 conditional knockout ESCs

There is a growing body of evidence supporting the involvement of the Wnt signaling pathway in various aspects of skeletal and joint development; however, it is unclear whether it is involved in the process of temporomandibular joint development. In order to clarify this issue, we examined the spatio-temporal distribution of mRNAs and proteins of the Wnt family during the formation of the mandibular condylar cartilage at the prenatal and postnatal stages. An in situ hybridization test revealed no mRNAs of β-catenin and Axin2 during early mesenchymal condensation; the ligands surveyed in this study (including Wnt-4, 5a, and 9a) were clearly detected at various ranges of expression, mainly in the condylar blastema and later distinct cartilaginous layers. Apart from β-catenin and Axin2, the Wnt family members surveyed in this study, including Lef-1, were found to be immunopositive during early chondrogenesis in the condylar cartilage at E14.5. After distinct chondrocyte layers were identified within the cartilage at E16.5, the expression of the Wnt signaling members was different and mainly restricted to proliferating cells and mineralized hypertrophic chondrocytes. In the adult mandibular condylar cartilage, the Wnt-4 mRNA, as well as the Wnt-4 and Wnt-9a proteins, was not observed. Our findings demonstrated that the Wnt signaling pathway was associated with the development of mandibular condylar cartilage.

Aim: The purpose of the present study is to investigate the association between the polymorphisms in AXIN1 with susceptibility to clear cell renal cell carcinoma (ccRCC). Materials & methods: A total of 284 ccRCC patients and 439 healthy volunteers were enrolled. Totally three tag single nucleotide polymorphisms in AXIN1 gene were genotyped using PCR & restriction fragment length polymorphism. Results: Significantly increased ccRCC risk was observed to be associated with the CT/CC genotypes of rs1805105 and AA genotype of rs12921862. Patients carrying the rs1805105 CT genotype had a 1.92-fold increased risk to developing clinical stage III and IV cancer. Conclusion: Our results suggested the rs1805105 CT/CC genotypes and rs12921862 AA genotype may relate to ccRCC development.

The RING finger protein Rnf146 encodes an E3 ubiquitin ligase capable of targeting poly-ADP-ribosylated substrates for proteasomal degradation. Rnf146 has been identified as a critical regulator of Axin1 and thus of Wnt/β-catenin signaling. However its physiological significance in vertebrate embryonic development remains to be demonstrated. In this study, we take advantages of early Xenopus embryos to demonstrate that Rnf146 is essential for embryonic pattern formation. Depletion of zygotic Rnf146 using a translation blocking morpholino oligo (MO) results in anteriorized development and increased expression the anterior marker gene Otx2, consistent the notion that Rnf146 is a positive regulator of Wnt/β-catenin signaling through negatively regulating Axin1 expression. This notion is further supported by examination of the role of maternal Rnf146 in the context of Spemann organizer formation and dorsal axis development. Depletion of maternal Rnf146 using an antisense oligodeoxynucleic acid (ODN) leads to ventralized development and diminished expression of organizer genes. Together, we have provided evidence for the first time that Rnf146 is a critical regulator of embryonic pattern formation in vertebrates.

The skin surface area varies flexibly in response to body shape changes. Skin homeostasis is maintained by stem cells residing in the basal layer of the interfollicular epidermis. However, how the interfollicular epidermal stem cells response to physiological body shape changes remains elusive. Here, we identify a highly proliferative interfollicular epidermal basal cell population in the rapidly expanding abdominal skin of pregnant mice. These cells express Tbx3 that is necessary for their propagation to drive skin expansion. The Tbx3+ basal cells are generated from Axin2+ interfollicular epidermal stem cells through planar-oriented asymmetric or symmetric cell divisions, and express transit-amplifying cell marker CD71. This biased division of Axin2+ interfollicular epidermal stem cells is induced by Sfrp1 and Igfbp2 proteins secreted from dermal cells. The Tbx3+ basal cells promote wound repair, which is enhanced by Sfrp1 and Igfbp2. This study elucidates the interfollicular epidermal stem cell/progeny organisation during pregnancy and suggests its application in regenerative medicine., The abdominal skin expands rapidly during pregnancy. Here the authors show that a population of highly proliferative stem cell progenies expressing the transcription factor Tbx3 is required for abdominal skin expansion in pregnant mice.

Lichens are a source of secondary metabolites which possess important biological activities, including antioxidant, antibacterial, anti-inflammatory, and cytotoxic effects. The anticancer activity of lichens was shown in many types of tumors, including colorectal cancers (CRC). Several studies revealed that the application of lichen extracts diminished the proliferation of CRC cells and induced apoptosis. Colon carcinogenesis is associated with aberrations in Wnt signaling. Elevated transcriptional activity of β-catenin induces cell survival, proliferation, and migration. Thus, the inhibition of Wnt signaling is a promising therapeutic strategy in colorectal cancer. The aim of this study was the evaluation of the effects of lichen-derived depsides (atranorin, lecanoric acid, squamatic acid) and depsidones (physodic acid, salazinic acid) and a poly-carboxylic fatty acid-caperatic acid, on Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines. HCT116 cells were more sensitive to the modulatory effects of the compounds. PKF118-310, which was used as a reference β-catenin inhibitor, dose-dependently reduced the expression of the classical β-catenin target gene-Axin2 in both cell lines. Lecanoric acid slightly reduced Axin2 expression in HCT116 cells while caperatic acid tended to reduce Axin2 expression in both cell lines. Physodic acid much more potently decreased Axin2 expression in HCT116 cells than in DLD-1 cells. Physodic acid and caperatic acid also diminished the expression of survivin and MMP7 in a cell line and time-dependent manner. None of the compounds affected the nuclear translocation of β-catenin. This is the first report showing the ability of caperatic acid and physodic acid to modulate β-catenin-dependent transcription.

Wnt/β-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, β-catenin is degraded by a multiprotein "destruction complex" that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of β-catenin destruction complexes by absolute protein quantification using LC-MS/MS. Similar amounts of destruction complex-constituting proteins and β-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the β-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in β-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of β-catenin destruction complexes.

Autophagy is a pathophysiological phenomenon in liver cirrhosis that can further progress into hepatocarcinoma. Liver cancer stem cells (CSCs) are believed to initiate hepatocarcinogenesis. To investigate the precise mechanism related to the origin of CSCs in liver cirrhosis and hepatocarcinogenesis, we labeled Axin2+ hepatic cells with EGFP in Axin2Cre;Rosa26EGFP transgenic rats, and then stratified clinical and rat liver cirrhosis samples by autophagy flux. Clinical follow-up and lineage tracing in transgenic rat liver cirrhosis revealed that while Axin2/EGFP+ hepatic cells were present in normal livers and cirrhotic livers without aberrant autophagy, hepatic Axin2/EGFP+CD90+ cells were generated exclusively in cirrhotic livers with aberrant autophagy and promoted hepatocarcinogenesis. Aberrant autophagy in liver cirrhosis resulted in hepatocyte growth factor (HGF) expression, leading to activation of Met/JNK and Met/STAT3 signaling in sorted hepatic Axin2/EGFP+ cells and their transition into Axin2/EGFP+CD90+ cells that possess CSC properties. In a transgenic rat liver cirrhosis model, induction or inhibition of autophagy in cirrhotic livers by systemic administration of rapamycin or chloroquine or transfection with Atg3- and Atg7-shRNAs significantly induced or suppressed HGF expression, which in turn increased or reduced generation of EGFP+CD90+ hepatic cells by activating or inactivating Met/JNK and Met/STAT3 signaling, thereby promoting or preventing hepatocarcinogenesis. Systemic treatment with HGF-shRNA, SP600125 or stattic also reduced generation of EGFP(Axin2)+ hepatic cell-originated CD90+ CSCs in aberrant autophagic cirrhotic livers by inactivating HGF/Met/JNK or HGF/Met/STAT3 signaling, further preventing hepatocarcinogenesis. These data suggest that activation of Met/JNK and Met/STAT3 signaling in Axin2+ hepatic cells via autophagy-dependent HGF expression and the resultant generation of Axin2+CD90+ CSCs is a major mechanism of hepatocarcinogenesis in cirrhotic livers.

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

Adenomatous polyposis coli (APC) and Axin interactions serve an important role in colorectal cancer (CRC) pathogenesis. The aim of the present study was to assess the combined effects of Axin and APC co‑expression in CRC cells, and to determine the underlying mechanisms involved. SW480 cells were divided into the following groups: Untransfected (SW480 group), transfected with pEGFP‑N3plus pCS2‑MT (SW480/vector‑vector), transfected with pEGFP‑N3‑APC5 (SW480/APC5), and transfected with pEGFP‑N3‑APC5 pluspCS2‑MT‑Axin (SW480/APC5‑Axin). APC5 and Axin mRNA levels were determined by reverse transcription‑polymerase chain reaction. MTT assays and flow cytometry analysis were performed to assess cell growth and cell cycle distribution, respectively. Quantitative PCR and western blot analyses were conducted to evaluate the mRNA and protein levels, respectively, of Wnt signaling effectors, including β‑catenin, c‑myc and survivin. Successful transfection of SW480 cells was determined with APC and APC‑Axin plasmids as indicated by the green fluorescence signals. Notably, SW480/APC5 cell growth was inhibited by 40.33%, and cells co‑expressing APC5 and Axin demonstrated 61.27% inhibition of cell growth compared with SW480 control cells. The results demonstrate that APC5 may induce G1/S arrest in SW480 cells, and Axin may enhance cell growth arrest induced by APC5. The mRNA and protein levels of β‑catenin, c‑myc and survivin were significantly reduced in SW480/APC‑Axin cells when compared with the SW480/APC group. In conclusion, co‑expression of APC5 and Axin genes significantly downregulated Wnt signaling in human SW480 CRC cells and inhibited cell growth, when compared with cells transfected with APC5 alone. These results may provide experimental evidence to support combined gene therapy in CRC.

Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of β-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of β-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/β-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/β-catenin pathway.

// Jeetendra Kumar Nag 1 , Arun Kancharla 1 , Myriam Maoz 1 , Hagit Turm 1 , Daniel Agranovich 1 , Chhedi Lal Gupta 2 , Beatrice Uziely 1 and Rachel Bar-Shavit 1 1 Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel 2 Department of Biosciences, Integral University, Lucknow, Uttar Pradesh 226026, India Correspondence to: Rachel Bar-Shavit, email: Rachelbar@ekmd.huji.ac.il Keywords: protease-activated receptors (PARs), protease, G-protein coupled receptors (GPCRs), cancer, β-catenin stabilization Received: April 14, 2016 Accepted: February 17, 2017 Published: March 16, 2017 ABSTRACT Protease-activated receptor-2 (PAR 2 ) plays a central role in cancer; however, the molecular machinery of PAR 2 -instigated tumors remains to be elucidated. We show that PAR 2 is a potent inducer of β-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds ( Fz ), with PAR 2 takes place following PAR 2 activation. The association between PAR 2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR 2 -LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR 2 -driven mammary gland tumors in vivo , as well as in vitro confirms the association between PAR 2 and LRP6. Indeed, sh RNA silencing of LRP6 potently inhibits PAR 2 -induced β-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR 1 ) and β-catenin stabilization, both in a transgenic ( tg ) mouse model with overexpression of human PAR 1 ( hPar1 ) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR 1 -G α13 axis, both G α12 and G α13 are equally involved in PAR 2 -induced β-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR 2 -LRP6-Axin interaction as a key axis of PAR 2 -induced β-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.

The Wnt/ $$\beta $$ -catenin pathway plays a crucial role in stem cell renewal and differentiation in the normal human colonic crypt. The balance between $$\beta $$ -catenin and APC along the crypt axis determines its normal functionality. The mechanism that deregulates this balance may give insight into the initiation of colorectal cancer. This is significant because the spatial dysregulation of $$\beta $$ -catenin by the mutated tumor suppressor gene/protein APC in human colonic crypts is responsible for the initiation and growth of colorectal cancer. We consider a regulatory function that promotes APC synthesis within the cell and its effect on the accumulation of the Wnt target protein, $$\beta $$ -catenin. It is evident that an APC gradient exists along the crypt axis; however, the mechanism by which APC expression is regulated within the cell is not well known. We investigate the dynamics of an APC regulatory mechanism with an increased level of Axin at the subcellular level. Model output shows an increase of APC for a diminished Wnt signal, which explains the APC gradient along the crypt. We find that the dynamic interplay between $$\beta $$ -catenin, APC, and Axin produces oscillatory behavior, which is controlled by the Wnt stimulus. In the presence of reduced functional APC, the oscillations are amplified, which suggests that the cell remains in a more proliferative state for longer periods of time. Increased Axin levels (typical of mammalian cells) reduce oscillatory behavior and minimize the levels of $$\beta $$ -catenin within the cell while raising the levels of APC.

// Sung Yong Ahn 1, * , Nam Hee Kim 1, * , Kyungro Lee 2, 3 , Yong Hoon Cha 1 , Ji Hye Yang 1 , So Young Cha 1 , Eunae Sandra Cho 1 , Yoonmi Lee 1 , Jeong Seok Cha 3 , Hyun Soo Cho 3 , Yoon Jeon 4 , Young-Su Yuk 1 , Suebean Cho 1 , Kyoung Tai No 2, 3 , Hyun Sil Kim 1 , Ho Lee 4 , Jiwon Choi 2 , Jong In Yook 1 1 Department of Oral Pathology, Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul 03722, Korea 2 Bioinformatics and Molecular Design Research Center, Yonsei University, Seoul 03722, Korea 3 Department of Systems Biology and Division of Life Science, Yonsei University, Seoul 03722, Korea 4 Graduate School of Cancer Science and Policy, Research Institute, National Cancer Center, Goyang 10408, Korea * These authors have contributed equally to this work Correspondence to: Jong In Yook, email: jiyook@yuhs.ac Jiwon Choi, email: edccjw@bmdrc.org Ho Lee, email: ho25lee@ncc.re.kr Keywords: niclosamide, epithelial-mesenchymal transition (EMT), Axin-GSK3 interaction, Wnt signaling, familial adenomatosis polyposis (FAP) Received: November 22, 2016 Accepted: February 20, 2017 Published: March 16, 2017 ABSTRACT The epithelial-mesenchymal transition (EMT) is implicated in tumorigenesis and cancer progression, and canonical Wnt signaling tightly controls Snail, a key transcriptional repressor of EMT. While the suppression of canonical Wnt signaling and EMT comprises an attractive therapeutic strategy, molecular targets for small molecules reverting Wnt and EMT have not been widely studied. Meanwhile, the anti-helminthic niclosamide has been identified as a potent inhibitor of many oncogenic signaling pathways although its molecular targets have not yet been clearly identified. In this study, we show that niclosamide directly targets Axin-GSK3 interaction, at least in part, resulting in suppression of Wnt/Snail-mediated EMT. In vitro and in vivo , disruption of Axin-GSK3 complex by niclosamide induces mesenchymal to epithelial reversion at nM concentrations, accompanied with suppression of the tumorigenic potential of colon cancer. Niclosamide treatment successfully attenuates Snail abundance while increasing E-cadherin abundance in xenograft tumor. Notably, oral administration of niclosamide significantly suppressed adenoma formation in an APC-MIN mice model, indicating that niclosamide is an effective therapeutic for familial adenomatosis polyposis (FAP) patients. In this study, we identified a novel target to control the canonical Wnt pathway and Snail-mediated EMT program, and discovered a repositioned therapeutics for FAP patients.

Pigment epithelium-derived factor (PEDF) expression is down- regulated in the kidneys of diabetic rats and delivery of PEDF suppressed renal fibrotic factors in these animals. PEDF has multiple functions including anti-angiogenic, anti-inflammatory and anti-fibrotic activities. Since the mechanism underlying its anti-fibrotic effect remains unclear, we studied this in several murine models of renal disease. Renal PEDF levels were significantly reduced in genetic models of type 1 and type 2 diabetes (Akita and db/db, respectively), negatively correlating with Wnt signaling activity in the kidneys. In unilateral ureteral obstruction, an acute renal injury model, there were significant decreases of renal PEDF levels. The kidneys of PEDF knock-out mice with ureteral obstruction displayed exacerbated expression of fibrotic and inflammatory factors, oxidative stress, tubulointerstitial fibrosis and tubule epithelial cell apoptosis, compared to the kidneys of wild-type mice with obstruction. PEDF knock-out enhanced Wnt signaling activation induced by obstruction, while PEDF inhibited the Wnt pathway-mediated fibrosis in primary renal proximal tubule epithelial cells. Additionally, oxidative stress was aggravated in renal proximal tubule epithelial cells isolated from knock-out mice, and suppressed by PEDF treatment of renal proximal tubule epithelial cells. PEDF also reduced oxidation-induced apoptosis in renal proximal tubule epithelial cells. Thus, the renoprotective effects of PEDF are mediated, at least partially, by inhibition of the Wnt pathway. Hence, restoration of renal PEDF levels may have therapeutic potential for renal fibrosis.

Epidemiological studies have convincingly suggested that obesity is an important risk factor for postmenopausal breast cancer, but the mechanisms responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low-grade inflammatory microenvironment, which stimulates Wnt-signaling and thereby promotes the development of breast cancer. To test this hypothesis, we evaluated the correlations between expression of multiple inflammatory cytokines and Wnt pathway downstream genes in mammary tissues from women (age ≥ 50) undergoing reduction mammoplasty. Moreover, we specifically examined the role of tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine associated with obesity and a possible modulator of the Wnt pathway. The regulatory effects of TNF-α on Wnt pathway targets were measured in an ex vivo culture of breast tissue treated with anti-TNF-α antibody or TNF-α recombinant protein. We found that BMI was positively associated with the secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α, all of which were negatively correlated with the expression of SFRP1. The transcriptional expression of Wnt-signaling targets, AXIN2 and CYCLIN D1, were higher in mammary tissue from women with BMI ≥ 30 compared to those with BMI < 30. Our ex vivo work confirmed that TNF-α is causally linked to the up-regulation of active β-CATENIN, a key component in the Wnt pathway, and several Wnt-signaling target genes (i.e. CYCLIN D1, AXIN2, P53 and COX-2). Collectively, these findings indicate that obesity-driven inflammation elevates Wnt-signaling in mammary tissue and thereby creates a microenvironment conducive to the development of breast cancer.

RUNX1 plays opposing roles in breast cancer: a tumor suppressor in estrogen receptor-positive (ER+) disease and an oncogenic role in ER-negative (ER−) tumors. Potentially mediating the former, we have recently reported that RUNX1 prevents estrogen-driven suppression of the mRNA encoding the tumor suppressor AXIN1. Accordingly, AXIN1 protein expression was diminished upon RUNX1 silencing in ER+ breast cancer cells and was positively correlated with AXIN1 protein expression across tumors with high levels of ER. Here we report the surprising observation that RUNX1 and AXIN1 proteins are strongly correlated in ER− tumors as well. However, this correlation is not attributable to regulation of AXIN1 by RUNX1 or vice versa. The unexpected correlation between RUNX1, playing an oncogenic role in ER− breast cancer, and AXIN1, a well-established tumor suppressor hub, may be related to a high ratio between the expression of variant 2 and variant 1 (v2/v1) of AXIN1 in ER− compared with ER+ breast cancer. Although both isoforms are similarly regulated by RUNX1 in estrogen-stimulated ER+ breast cancer cells, the higher v2/v1 ratio in ER− disease is expected to weaken the tumor suppressor activity of AXIN1 in these tumors.