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1.  New Classes of Mind Bomb-Interacting Proteins Identified from Yeast Two-Hybrid Screens 
PLoS ONE  2014;9(4):e93394.
Notch signaling pathway defines an evolutionarily conserved mechanism in cell-fate determination in a broad spectrum of developmental processes through local cell interactions. mind bomb (mib) encodes an E3 ubiquitin ligase that is involved in Notch activation through Delta ubiquitylation and internalization. To further dissect the function of Mib, two yeast two-hybrid screens for zebrafish Mib/Mib2-binding proteins with different strategies have been performed. 81 putative interesting proteins were discovered and classified into six groups: ubiquitin proteasome pathway, cytoskeleton, trafficking, replication/transcription/translation factors, cell signaling and others. Confirmed by coimmunoprecipitation (Co-IP), Mib interacted with four tested proteins: ubiquitin specific protease 1 (Usp1), ubiquitin specific protease 9 (Usp9), tumor-necrosis-factor-receptor-associated factor (TRAF)-binding domain (Trabid)/zinc finger, RAN-binding domain containing 1 (Zranb1) and hypoxia-inducible factor 1, alpha subunit inhibitor (Hif1an)/factor inhibiting HIF 1 (Fih-1). Usp1, Usp9, Trabid and Fih-1 also bound to zebrafish Mib2, a Mib homolog with similar structural domains and functions. Both Mib and Mib2 can ubiquitylate Trabid and Fih-1, indicating a potential regulating role of Mib and Mib2 on Trabid and Fih-1 and, furthermore, the possible involvement of Notch signaling in hypoxia-regulated differentiation, tumorigenesis and NF-κB pathway. Finally, functions of confirmed Mib/Mib2-interacting proteins are collated, summarized and hypothesized, which depicts a regulating network beyond Notch signaling.
doi:10.1371/journal.pone.0093394
PMCID: PMC3979679  PMID: 24714733
2.  Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner 
PLoS Biology  2010;8(1):e1000296.
Chromatin organizer SATB1 and Wnt transducer β-catenin form a complex and regulate expression of GATA3 and TH2 cytokines in Wnt-dependent manner and orchestrate TH2 lineage commitment.
In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of β-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/β -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with β-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon β-catenin signalling. GATA-3 is a T helper type 2 (TH2) specific transcription factor that regulates production of TH2 cytokines and functions as TH2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells, suggesting that SATB1 influences TH2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature TH2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for TH2 differentiation. Knockdown of β-catenin also produced similar results, confirming the role of Wnt/β-catenin signalling in TH2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits β-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating TH2 cells in a Wnt-dependent manner. SATB1 coordinates TH2 lineage commitment by reprogramming gene expression. The SATB1:β-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates TH2 lineage commitment by mediating Wnt/β-catenin signalling. This report identifies a new global transcription factor involved in β-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.
Author Summary
In vertebrates the canonical Wnt signalling culminates in β-catenin moving into the nucleus where it activates transcription of target genes. Wnt/β-catenin signalling is essential for the thymic maturation and differentiation of naïve T cells. Here we show that SATB1, a T cell lineage-enriched chromatin organizer and global regulator, binds to β-catenin and recruits it to SATB1's genomic binding sites so that genes formerly repressed by SATB1 are upregulated by Wnt signalling. Some of the genes known to be regulated by SATB1 (such as genes encoding cytokines and the transcription factor GATA3) are required for differentiation of Th2 cells, an important subset of helper T cells. Specifically we show that siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells. Inhibiting Wnt signalling led to downregulation of GATA-3 and of signature TH2 cytokines such as IL-4, IL-10, and IL-13. Knockdown of β-catenin also produced similar results, thus together these data confirm the role of Wnt/β-catenin signalling in TH2 differentiation. Our data demonstrate that SATB1 orchestrates TH2 lineage commitment by modulating Wnt/β-catenin signalling.
doi:10.1371/journal.pbio.1000296
PMCID: PMC2811152  PMID: 20126258
3.  Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span 
PLoS Genetics  2014;10(2):e1004117.
A relatively unexplored nexus in Drosophila Immune deficiency (IMD) pathway is TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and apoptosis. In a cell culture screen, we identified that Lysine at position 142 was a K63-linked Ubiquitin acceptor site for TAK1, required for signalling. Moreover, Lysine at position 156 functioned as a K48-linked Ubiquitin acceptor site, also necessary for TAK1 activity. The deubiquitinase Trabid interacted with TAK1, reducing immune signalling output and K63-linked ubiquitination. The three tandem Npl4 Zinc Fingers and the catalytic Cysteine at position 518 were required for Trabid activity. Flies deficient for Trabid had a reduced life span due to chronic activation of IMD both systemically as well as in their gut where homeostasis was disrupted. The TAK1-associated Binding Protein 2 (TAB2) was linked with the TAK1-Trabid interaction through its Zinc finger domain that pacified the TAK1 signal. These results indicate an elaborate and multi-tiered mechanism for regulating TAK1 activity and modulating its immune signal.
Author Summary
Chronic activation of immune responses results in health problems including gastrointestinal infections, metabolic imbalances and inflammatory bowel diseases that may lead to colorectal cancer. Central to this, is the balance of activation/restriction of nuclear factor-κB (NF-κB) during innate immune responses. To study signaling through NF-κB, we use the fruit fly Drosophila melanogaster as a genetically tractable model system that reflects human biology (due to the evolutionary conservation between innate immunity in flies and mammals), while reducing the complexity of the human disease of interest. We have found a new negative regulator of the Drosophila NF-κB pathway named Trabid. Its loss released the pathway and resulted in constitutive immune activation both in the gut as well as in the whole fly. This spontaneous immune activation reduced life span in the absence of infection, especially when it was combined with loss of another known negative regulator of the same pathway, a protein named Pirk. Stem cell activity in the gut in a pirk;trabid double mutant was found to be significantly increased, as the gut was trying to balance enterocyte loss. Trabid was acting at the level of TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and cell death.
doi:10.1371/journal.pgen.1004117
PMCID: PMC3930493  PMID: 24586180
4.  WNT7B mediates autocrine Wnt/β-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma 
Oncogene  2013;33(7):899-908.
Developmental and cancer models show Wnt/β-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/β-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/β-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression based assays revealed wide heterogeneity in Wnt/β-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally-generated, pancreas-specific gene expression signature of Wnt/β-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early stage PDAC tumors (N=41). In this cohort, higher Wnt/β-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease specific survival (median survival time 20.3 versus 43.9 months, log rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/β-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/β-catenin activity. Gene knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B mediated cell autonomous Wnt/β-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/β-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/β-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.
doi:10.1038/onc.2013.23
PMCID: PMC3923845  PMID: 23416978
pancreatic cancer; WNT7B; Wnt/β-catenin signaling
5.  Beta-Catenin Signaling Plays a Disparate Role in Different Phases of Fracture Repair: Implications for Therapy to Improve Bone Healing 
PLoS Medicine  2007;4(7):e249.
Background
Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The β-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of β-catenin signaling in bone repair.
Methods and Findings
Western blot analysis showed significant up-regulation of β-catenin during the bone healing process. Using a β-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that β-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/β-catenin pathway) inhibited β-catenin signaling and the healing process, suggesting that WNT ligands regulate β-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized β-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific β-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of β-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated β-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture.
Conclusions
These results demonstrate that β-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of β-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, β-catenin positively regulates osteoblasts. This is a different function for β-catenin than has previously been reported during development. Activation of β-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage.
In a study in mice Benjamin Alman and colleagues show that β-catenin functions differently in different stages of fracture repair; moreover, activation of β-catenin by lithium improves fracture healing when used in later phases of repair.
Editors' Summary
Background.
Most people break at least one bone during their life. If the damaged bone is immobilized with a plaster cast or with metal plates and pins, most fractures heal naturally and quickly. Soon after a bone is damaged, cells called pluripotent mesenchymal cells collect at the injury site. Here, they multiply and change (differentiate) into osteoblasts (cells that make bone) and chondrocytes (cells that make cartilage, the dense connective tissue that covers joints). Osteoblasts and chondrocytes mend the fracture by making new bone, a process called ossification. Bone healing involves two types of ossification. In intramembranous ossification, mesenchymal cells and osteoblast progenitor cells make bone directly, forming a hard “callus” within the fracture. In endochondral ossification, mesenchymal cells differentiate into chondrocytes and make cartilage at the fracture site, which osteoblasts turn into bone. Finally, the bone made by both types of ossification is remodeled so that it closely resembles the damaged bone's original shape and strength.
Why Was This Study Done?
Unfortunately, fractures do not always heal efficiently. If healing is delayed, additional surgery may be needed to repair the break. But surgery can be risky, so drug-based ways of encouraging bone repair would be very useful. To develop such treatments, researchers need to understand what controls the differentiation and activity of osteoblasts and chondrocytes during normal healing. In this study, the researchers have investigated the role of the β-catenin signaling pathway in bone repair. This pathway regulates bone formation during embryonic development, a process that closely resembles bone healing. β-catenin is usually degraded rapidly in cells. However, if a member of a particular family of proteins known as the WNT family binds to a WNT receptor on the surface of a cell, β-catenin moves into the cell's nucleus where it interacts with a protein called T cell factor (TCF). This interaction activates the transcription (the copying of DNA into messenger RNA, which is used to make proteins) of numerous genes and alters the behavior of the cell.
What Did the Researchers Do and Find?
The researchers first measured β-catenin levels in mouse and human bones. In both species, much more β-catenin was made in bones undergoing repair than in intact bones. Then they studied TCF reporter mice—animals in which TCF controls the expression of a marker gene. β-catenin-mediated TCF-dependent transcription, they report, was activated during both bone and cartilage formation after a fracture in these mice. Next, the researchers made mice that could be induced to express an inactive form of β-catenin or a stabilized (permanently active) form of β-catenin in all the cells in a bone fracture. Expression of inactive β-catenin slowed the rate of healing but, unexpectedly, so did expression of stabilized β-catenin. Osteoblast-specific expression of inactive β-catenin also delayed bone healing, whereas osteoblast-specific expression of stabilized β-catenin enhanced the process. Finally, treatment of wild-type mice with lithium (which prevents the degradation of β-catenin) enhanced bone healing if given after a fracture, but interfered with it if given before.
What Do These Findings Mean?
These findings indicate that β-catenin signaling (which, the researchers show, is mainly activated by WNT signaling) has different effects at different stages of bone repair. Early in the process, it controls the ratio of osteoblasts and chondrocytes made from the pluripotent mesenchymal cells. Consequently, too much or too little β-catenin interferes with bone healing at this stage. Later on, β-catenin promotes the differentiation of osteoblasts and enhances their ability to make bone, and so too little β-catenin at this stage prevents healing, whereas increased β-catenin levels stimulate healing. These findings need to be confirmed in people before testing agents that affect β-catenin signaling for their effects on human bone healing. Nevertheless, the researchers' final discovery that lithium improves bone healing if given at the right time is particularly encouraging; lithium is widely used to treat one form of depression so could be readily tested in clinical trials.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040249.
MedlinePlus encyclopedia contains pages on broken bones and on bone fracture repair (in English and Spanish)
Wikipedia has pages on bone fracture and on bone healing (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The UK National Health Service Direct encyclopedia provides pages on broken bones
Animations of intramembranous and endochondral ossification are available from the Ministry of Advanced Education, Training and Technology, Province of British Columbia, Canada
The American Academy of Orthopedic Surgeons has an informative discussion of fractures
The Hospital for Sick Children in Toronto (where the authors of this study are affiliated) has a Web site called SickKids, which contains a page on child physiology, including diagrams of bone development
doi:10.1371/journal.pmed.0040249
PMCID: PMC1950214  PMID: 17676991
6.  p300 Influences Butyrate-Mediated WNT Hyperactivation In Colorectal Cancer Cells 
Journal of Cancer  2013;4(6):491-501.
Deregulated WNT/catenin pathway, usually resulting from mutations in the adenomatous polyposis coli and beta-catenin genes, drives colorectal tumorigenesis. Dietary fiber has been shown to have a protective role against colorectal cancer (CRC). We have previously demonstrated that the histone deacetylase inhibitor (HDACi) butyrate, a fermentation product of dietary fiber, induces WNT/catenin hyperactivation, which promotes CRC cell apoptosis. Therefore, the ability of butyrate to induce WNT hyperactivation and thus promote CRC cell apoptosis may in part explain the preventive function of fiber against CRC. The association between beta-catenin and the transcriptional coactivator p300 may influence WNT/catenin signaling and, therefore, colonic cell physiology. p300 functions as a histone acetylase (HAT); therefore, the modulation of WNT/catenin activity by p300 may influence the ability of the HDACi butyrate to hyperinduce WNT signaling and apoptosis in CRC cells. Our findings indicate that p300 affects the hyperinduction of WNT activity by butyrate. Knockdown of p300 levels represses butyrate-mediated WNT/catenin activity; but still allows for butyrate-mediated apoptosis. Overexpression of p300 stimulates basal and butyrate-induced WNT signaling in some, but not all, CRC cell lines. We also evaluate the role of p300 in therapeutic approaches that target CBP. The small molecule ICG-001, in clinical trial, is a specific inhibitor of CBP-mediated WNT signaling, and previous studies have suggested that p300 is required for the activity of ICG-001. However, we report that ICG-001 maintains full activity against CBP-mediated WNT signaling in p300-deficient cell lines, including the butyrate-resistance line HCT-R. In addition, our findings evaluating combinatorial treatment of ICG-001 and butyrate in HCT-R cells may have important therapeutic implications for the treatment of butyrate-resistant CRCs.
doi:10.7150/jca.6582
PMCID: PMC3726711  PMID: 23901349
p300; colorectal cancer; WNT; butyrate; fiber; ICG-001.
7.  Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling Pathway 
PLoS ONE  2012;7(3):e33021.
While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway.
doi:10.1371/journal.pone.0033021
PMCID: PMC3302780  PMID: 22427932
8.  Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells 
Journal of periodontal research  2011;47(3):10.1111/j.1600-0765.2011.01433.x.
Background and Objectives
Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway.
Materials and Methods
SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin.
Results
Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling.
Conclusions
These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.
doi:10.1111/j.1600-0765.2011.01433.x
PMCID: PMC3865600  PMID: 22150562
dental follicle cells; Wnt; cementoblast; maturation; BMP
9.  Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells 
Background
Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.
Results
Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.
Conclusions
These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.
doi:10.1186/1750-2187-6-12
PMCID: PMC3198762  PMID: 21970630
10.  The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis 
PLoS Biology  2010;8(11):e1000539.
The leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l are identified as Tcf4/β-catenin co-activators and shown to be essential for Wnt-driven endogenous gene expression, intestinal development and homeostasis.
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
Author Summary
The canonical Wnt pathway is a key regulatory pathway controlling intestinal cell proliferation, differentiation, and stem cell maintenance, and its deregulation leads to malignancies in the mammalian gut. A decade has passed since the discovery of the transcription factors TCF4-β-catenin as the downstream intestinal molecular effectors of Wnt, but few transcriptional activators essential and unique to the regulation of this transcription program have been found. In this study, using a proteomics approach, we identify the leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l as interactors with Tcf4/β-catenin in the mouse small intestinal epithelium. We demonstrate that Mllt10/Af10–Dot1l are recruited to Wnt target genes in intestinal epithelial cells and are essential to regulate expression of these targets. We also show a genetic link between the Wnt pathway and Mllt10/Af10-Dot1l in zebrafish and delineate their essential role in Wnt-driven endogenous gene expression. Finally, we demonstrate the physiological role of Mllt10/Af10-Dot1l in Wnt-driven intestinal development and homeostasis; depletion of Mllt10/Af10-Dot1l in zebrafish embryos mimics the Tcf4-depleted phenotype in which significant intestinal proliferation defects accompany a decrease in total number of intestinal cells. We conclude that the enzyme Dot1l may present an attractive candidate for drug targeting in colorectal cancer.
doi:10.1371/journal.pbio.1000539
PMCID: PMC2982801  PMID: 21103407
11.  Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response 
PLoS Pathogens  2012;8(7):e1002783.
Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.
Author Summary
Deubiquitinases (DUBs) are enzymes, which are implicated in many cellular processes but their functions during virus infection are not well understood. We used WP1130, a small molecule inhibitor of a subset of DUBs, as a probe to unravel the functions of DUBs during norovirus infections. We identified USP14 as a cellular DUB target of WP1130 that is required for optimal norovirus infection. Furthermore, we demonstrated that chemical induction of the unfolded protein response can significantly inhibit viral progeny production of several RNA viruses, including noroviruses. These results suggest that chemical inhibition of cellular DUBs and/or modulation of the unfolded protein response could represent novel targets for therapy against a variety of viral pathogens.
doi:10.1371/journal.ppat.1002783
PMCID: PMC3390402  PMID: 22792064
12.  A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis 
Cell Research  2011;21(5):730-740.
The Wnt/β-catenin signaling pathway is a highly conserved pathway in organism evolution and regulates many biological processes. Aberrant activation of the Wnt/β-catenin signaling pathway is closely related to tumorigenesis. In order to identify potent small molecules to treat the over-activated Wnt signaling-mediated cancer, such as colon cancer, we established a mammalian cell line-based reporter gene screening system. The screen revealed a diterpenoid derivative, 15-oxospiramilactone (NC043) that inhibits Wnt3a or LiCl-stimulated Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and Caco-2. Treatment of SW480 cells with NC043 led to decreases in the mRNA and/or protein expression of Wnt target genes Axin2, Cyclin D1 and Survivin , as well as decreases in the protein levels of Cdc25c and Cdc2. NC043 did not affect the cytosol-nuclear distribution and protein level of soluble β-catenin, but decreased β-catenin/TCF4 association in SW480 cells. Moreover, NC043 inhibited anchorage-independent growth and xenograft tumorigenesis of SW480 cells. Collectively these results demonstrate that NC043 is a novel small molecule that inhibits canonical Wnt signaling downstream of β-catenin stability and may be a potential compound for treating colorectal cancer.
doi:10.1038/cr.2011.30
PMCID: PMC3203668  PMID: 21321609
Wnt; small molecule; inhibitor; tumorigenesis
13.  Deubiquitinases Regulate the Activity of Caspase-1 and Interleukin-1β Secretion via Assembly of the Inflammasome* 
The Journal of Biological Chemistry  2012;288(4):2721-2733.
Background: The inflammasome is a multimolecular complex that regulates the processing of the pro-inflammatory cytokine interleukin-1β.
Results: Inhibitors of deubiquitinase (DUB) enzymes inhibited the release of interleukin-1β.
Conclusion: DUBs regulate assembly of the inflammasome.
Significance: DUBs may represent new anti-inflammatory drug targets for the treatment of inflammatory disease.
IL-1β is a potent pro-inflammatory cytokine produced in response to infection or injury. It is synthesized as an inactive precursor that is activated by the protease caspase-1 within a cytosolic molecular complex called the inflammasome. Assembly of this complex is triggered by a range of structurally diverse damage or pathogen associated stimuli, and the signaling pathways through which these act are poorly understood. Ubiquitination is a post-translational modification essential for maintaining cellular homeostasis. It can be reversed by deubiquitinase enzymes (DUBs) that remove ubiquitin moieties from the protein thus modifying its fate. DUBs present specificity toward different ubiquitin chain topologies and are crucial for recycling ubiquitin molecules before protein degradation as well as regulating key cellular processes such as protein trafficking, gene transcription, and signaling. We report here that small molecule inhibitors of DUB activity inhibit inflammasome activation. Inhibition of DUBs blocked the processing and release of IL-1β in both mouse and human macrophages. DUB activity was necessary for inflammasome association as DUB inhibition also impaired ASC oligomerization and caspase-1 activation without directly blocking caspase-1 activity. These data reveal the requirement for DUB activity in a key reaction of the innate immune response and highlight the therapeutic potential of DUB inhibitors for chronic auto-inflammatory diseases.
doi:10.1074/jbc.M112.422238
PMCID: PMC3554938  PMID: 23209292
Caspase; Deubiquitination; Inflammation; Interleukin; Macrophages; Inflammasome
14.  Aesculetin suppresses proliferation of human colon cancer cells by directly targeting β-catenin 
Cancer prevention research (Philadelphia, Pa.)  2013;6(12):10.1158/1940-6207.CAPR-13-0241.
The Wnt pathway is a promising therapeutic and preventive target in various human cancers. The transcriptional complex of β-catenin/Tcf, a key mediator of canonical Wnt signaling, has been implicated in human colon cancer development. Current treatment of colon cancer depends on traditional cytotoxic agents with limited effects. Therefore, the identification of natural compounds that can disrupt the β–catenin/TcF complex to suppress cancer cell growth with fewer adverse side effects is needed. To identify compounds that inhibit the association between β-catenin and Tcf, we used computer docking to screen a natural compound library. Aesculetin, also known as 6,7-dihydroxycoumarin, is a derivative of coumarin and was identified as a potential small molecule inhibitor of the Wnt/β-catenin pathway. We then evaluated the effect of aesculetin on the growth of various human colon cancer cell lines and its effect on Wnt/β-catenin signaling in cells and in an embryonic model. Aesculetin disrupted the formation of the β-catenin/Tcf complex through direct binding with the Lys312, Gly307, Lys345 and Asn387 residues of β-catenin in colon cancer cells. Additionally, aesculetin effectively decreased viability and inhibited anchorage-independent growth of colon cancer cells. Aesculetin potently antagonized the cellular effects of β-catenin-dependent activity and in vivo treatment with aesculetin suppressed tumor growth in a colon cancer xenograft mouse model. Our data indicate that the interaction between aesculetin and β-catenin inhibits the formation of the β-catenin/Tcf complex, which could contribute to aesculetin’s positive therapeutic and preventive effects against colon carcinogenesis.
doi:10.1158/1940-6207.CAPR-13-0241
PMCID: PMC3885338  PMID: 24104353
aesculetin; human colon cancer; Wnt; β-catenin; Tcf
15.  Galectin-3 Mediates Nuclear β-Catenin Accumulation and Wnt Signaling in Human Colon Cancer Cells by Regulation of GSK-3β Activity 
Cancer research  2009;69(4):1343-1349.
Wnt/β-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a β-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/β-catenin pathway in colon cancer cells, and the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, shRNA or full length galectin-3 cDNA, and effects on β-catenin levels, subcellular distribution, and Wnt signaling determined. Galectin-3 levels correlated with β-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased β-catenin protein levels but no change in β-catenin mRNA levels, suggesting that galectin-3 modulates β-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear β-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased β-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase (GSK)-3β dephosphorylation and increased GSK activity, increasing β-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT suppressed TCF4 transcriptional activity induced by galectin-3, while LiCl, a GSK-3β inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3β phosphorylation and activity via the PI3K/AKT pathway, and thus the degradation of β-catenin in colon cancer cells.
doi:10.1158/0008-5472.CAN-08-4153
PMCID: PMC2990400  PMID: 19190323
Galectin-3; β-catenin; TCF4; GSK-3β activity; PI3K/AKT pathway
16.  MSX2 IS AN ONCOGENIC DOWNSTREAM TARGET OF ACTIVATED WNT SIGNALING IN OVARIAN ENDOMETRIOID ADENOCARCINOMA 
Oncogene  2011;30(40):4152-4162.
Ovarian endometrioid adenocarcinomas (OEAs) frequently exhibit constitutive activation of canonical WNT signaling, usually as a result of oncogenic mutations that stabilize and dysregulate the β-catenin protein. In prior work, we used microarray-based methods to compare gene expression in OEAs with and without dysregulated β-catenin as a strategy for identifying novel β-catenin/TCF target genes with important roles in ovarian cancer pathogenesis. Among the genes highlighted by the microarray studies was MSX2, which encodes a homeobox transcription factor. We found MSX2 expression was markedly increased in primary human and murine OEAs with dysregulated β-catenin compared to OEAs with intact β-catenin regulation. WNT pathway activation by WNT3a ligand or GSK3β inhibitor treatment potently induced MSX2, and ectopic expression of a dominant negative form of TCF4 inhibited MSX2 expression in ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated that β-catenin/TCF directly regulates MSX2 expression via binding to TCF binding elements in multiple regions of the MSX2 gene. Notably, ectopic MSX2 expression was found to promote neoplastic transformation of the rodent RK3E model epithelial cell line and to enhance the invasiveness of immortalized human ovarian epithelial cells in vitro and ovarian carcinoma cells in vivo. Inhibition of endogenous MSX2 expression in ovarian endometrioid cancer cells carrying a β-catenin mutation using shRNA approaches inhibited neoplastic properties of the cells in vitro and in vivo. Expression of MSX2 in selected ovarian carcinoma cells induced changes suggestive of epithelial-mesenchymal transition (EMT), but based on analysis of ovarian cell lines and primary tumor tissues, effects of MSX2 on EMT appear to be complex and context-dependent. Our findings indicate MSX2 is a direct downstream transcriptional target of β-catenin/TCF and has a key contributing role in the cancer phenotype of OEAs carrying WNT/β-catenin pathway defects.
doi:10.1038/onc.2011.123
PMCID: PMC3140605  PMID: 21499300
MSX2; ovarian cancer; endometrioid; WNT signaling; β-catenin
17.  Transcriptional Regulation of SM22α by Wnt3a: Convergence with TGFβ1/Smad Signaling at a Novel Regulatory Element 
The role of canonical Wnt signaling in myofibroblast biology has not been fully investigated. The C3H10T1/2 mesenchymal cell line recapitulates myofibroblast differentiation in vitro and in vivo, including SM22α expression. Using this model, we find that Wnt3a upregulates SM22α in concert with TGFβ1. Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFβ1 signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22α histone H3 acetylation and β-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22α promoter –luciferase (LUC) reporter constructs, we mapped Wnt3a-regulated DNA transcriptional activation to nucleotides −213 to −192 relative to the transcription initiation site. In gel shift assays, DNA-protein complexes assembled on this element were disrupted with antibodies to β-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFβ transcriptional mediators. Mutation of a CAGAG motif within this region abrogated recognition by these DNA binding proteins. Wnt3a treatment increased Smad2/3 binding to this element. Mutation of the cognate within the context of the native 0.44 kb SM22α promoter resulted in a 70% decrease in transcription, and reduced Wnt3a + TGFβ1 induction. A concatamer of SM22α [−213 to −192] conveyed Wnt3a + TGFβ1 activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22α [−213 to −192]×6 RSVLUC activation. Moreover, ICAT (inhibitor of β-catenin and TCF) decreased while TCF7L2 and β-catenin enhanced 0.44 kb SM22α promoter induction by Wnt3a + TGFβ1. RNAi “knockdown” of β-catenin inhibited Wnt3a induction of SM22α. Thus, Wnt/β-catenin signaling interacts with TGFβ/Smad pathways to control SM22α gene transcription.
doi:10.1016/j.yjmcc.2009.01.005
PMCID: PMC2666882  PMID: 19344627
Msx2; Wnt; β-catenin; type II diabetes; myofibroblast; vascular calcification
18.  Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism 
Developmental biology  2011;352(1):58-69.
Wnt4 and β-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of β-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of β-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of β-catenin’s helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of β-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca2+-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca2+ and caused phosphorylation of CaMKII. Moreover, Ionomycin, a Ca2+-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney.
doi:10.1016/j.ydbio.2011.01.012
PMCID: PMC3049843  PMID: 21256838
Wnt; β-catenin; mesenchymal-epithelial transition; kidney; tubulogenesis; peptidomimetic
19.  CK1ε Is Required for Breast Cancers Dependent on β-Catenin Activity 
PLoS ONE  2010;5(2):e8979.
Background
Aberrant β-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate β-catenin activity for therapeutic purposes have proven elusive to date.
Methodology
To uncover genetic dependencies in breast cancer cells that harbor active β-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized β-catenin activity. Here we identify CSNK1E, the gene encoding casein kinase 1 epsilon (CK1ε) as required specifically for the proliferation of breast cancer cells with activated β-catenin and confirm its role as a positive regulator of β-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated β-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/β-catenin signaling. We also find that expression of CK1ε is able to promote oncogenic transformation of human cells in a β-catenin-dependent manner.
Conclusions/Significance
These studies identify CK1ε as a critical contributor to activated β-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active β-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors.
doi:10.1371/journal.pone.0008979
PMCID: PMC2813871  PMID: 20126544
20.  Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir 
Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.
doi:10.1016/j.bbrc.2011.11.089
PMCID: PMC3259256  PMID: 22142846
β-catenin; human immunodeficiency virus; osteoclast; pathogenesis; protease inhibitor; Wnt signaling
21.  Selective Small Molecule Targeting β-Catenin Function Discovered by In Vivo Chemical Genetic Screen 
Cell reports  2013;4(5):898-904.
SUMMARY
Canonical Wnt signaling pathway, mediated by the transcription factor β-catenin, plays critical roles in embryonic development, and represents an important therapeutic target. In a zebrafish-based in vivo screen for small molecules that specifically perturb embryonic dorsoventral patterning, we discovered a novel compound, named windorphen, which selectively blocks the Wnt signal required for ventral development. Windorphen exhibits remarkable specificity toward β-catenin-1 function, indicating that the two β-catenin isoforms found in zebrafish are not functionally redundant. We show that windorphen is a selective inhibitor of p300 histone acetyl transferase, a co-activator that associates with β-catenin. Lastly, windorphen robustly and selectively kills cancer cells that harbor Wnt-activating mutations, supporting the therapeutic potential of this novel Wnt inhibitor class.
doi:10.1016/j.celrep.2013.07.047
PMCID: PMC3923627  PMID: 24012757
22.  β-Catenin-Independent Activation of TCF1/LEF1 in Human Hematopoietic Tumor Cells through Interaction with ATF2 Transcription Factors 
PLoS Genetics  2013;9(8):e1003603.
The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.
Author Summary
The Wnt signaling pathway plays a crucial role during embryonic development and in the maintenance of stem cell populations in various organs and tissues. Aberrant activation of this pathway through different mechanisms participates in the onset and progression of several types of human cancers. In the presence of Wnt ligands, stabilized β-catenin acts as a transcriptional activator to induce the expression of target genes through binding to the TCF/LEF family of transcription factors. Using in vitro and in vivo models, we show that TCF/LEF proteins can be activated independently of β-catenin through cooperation with members of the ATF2 subfamily of transcription factors. This novel alternative mechanism of TCF/LEF activation is constitutively up-regulated in certain hematopoietic tumor cells, where it regulates the expression of TCF/LEF target genes and promotes cell growth.
doi:10.1371/journal.pgen.1003603
PMCID: PMC3744423  PMID: 23966864
23.  Beta-catenin is required for Ron receptor induced mammary tumorigenesis 
Oncogene  2011;30(34):3694-3704.
Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in β-catenin expression and tyrosine phosphorylation. β-catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer, and Fyn. However, the molecular and physiological roles of β-catenin and β-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and β-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of β-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL mediated Ron activation induces both β-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of β-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced β-catenin-dependent transcriptional activation and cell growth which can rescued by activation of canonical Wnt/β-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the β-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or β-catenin. Finally, we show that genetic ablation of β-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis following orthotopic transplantation into the mammary fat pad. Together, our data suggest that β-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.
doi:10.1038/onc.2011.86
PMCID: PMC3134560  PMID: 21423209
beta-catenin; hepatocyte growth factor like protein; Ron receptor; breast cancer; receptor tyrosine kinase
24.  Role of GAC63 in transcriptional activation mediated by β-catenin 
Nucleic Acids Research  2007;35(6):2084-2092.
β-Catenin is a key mediator in the canonical Wnt signaling pathway, which plays important roles in multiple developmental processes. Inappropriate activation of this pathway leads to developmental defects and development of certain cancers. Upon Wnt signaling, β-catenin binds TCF/LEF transcription factors. The TCF/LEF-β-catenin complex then recruits a variety of transcriptional coactivators to the promoter/enhancer region of Wnt-responsive genes and activates target gene transcription. In this article, we demonstrate that GRIP1-associated coactivator 63 (GAC63), a recently identified nuclear receptor (NR) coactivator, interacts with β-catenin. The N-terminus of GAC63 is the binding site for β-catenin, whereas a C-terminal fragment of β-catenin including armadillo repeats 10–12 binds to GAC63. Over-expression of GAC63 enhanced the transcriptional activity of β-catenin, and also greatly enhanced TCF/LEF-regulated reporter gene activity in a β-catenin-dependent manner. Endogenous GAC63 was recruited to TCF/LEF-responsive enhancer elements when β-catenin levels were induced by LiCl. In addition, reduction of endogenous GAC63 level by small interfering RNA (siRNA) inhibited TCF/LEF-mediated gene transcription. Our findings reveal a new function of GAC63 in transcriptional activation of Wnt-responsive genes.
doi:10.1093/nar/gkm095
PMCID: PMC1874623  PMID: 17344318
25.  Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells 
Molecular Cancer  2008;7:73.
Background
β-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex.
Results
We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.
Conclusion
Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.
doi:10.1186/1476-4598-7-73
PMCID: PMC2565678  PMID: 18826597

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