Increased expression of the cyclooxygenase-2 enzyme (COX2) is one of the main characteristics of gastric cancer (GC), which is a leading cause of death in the world, particularly in Asia and South America. Although the Wnt/β-catenin signaling pathway has been involved in the transcriptional activation of the COX2 gene, the precise mechanism modulating this response is still unknown.Here we studied the transcriptional regulation of the COX2 gene in GC cell lines and assessed whether this phenomenon is modulated by Wnt/β-catenin signaling. We first examined the expression of COX2 mRNA in GC cells and found that there is a differential expression pattern consistent with high levels of nuclear-localized β-catenin. Pharmacological treatment with either lithium or valproic acid and molecular induction with purified canonical Wnt3a significantly enhanced COX2 mRNA expression in a dose- and time-dependent manner. Serial deletion of a 1.6 Kbp COX2 promoter fragment and gain- or loss-of-function experiments allowed us to identify a minimal Wnt/β-catenin responsive region consisting of 0.8 Kbp of the COX2 promoter (pCOX2-0.8), which showed maximal response in gene-reporter assays. The activity of this pCOX2-0.8 promoter region was further confirmed by site-directed mutagenesis and DNA-protein binding assays.We conclude that the pCOX2-0.8 minimal promoter contains a novel functional T-cell factor/lymphoid enhancer factor (TCF/LEF)-response element  that responds directly to enhanced Wnt/β-catenin signaling and which may be important for the onset/progression of GC. Helicobacter pylori infection Gastric cancer (GC) is a multifactorial disease, characterized by highly malignant neoplasms in the gastric mucosa, and represents the second leading cause of cancer death worldwide with the highest prevalence in Asia and South America It has been observed that the expression of the COX2 gene is significantly increased in human gastric adenocarcinoma tissues, when compared with paired gastric mucosal specimens devoid of cancer cells Transcriptional control of the COX2 gene depends on the molecular machinery interacting with the COX2 promoter, which seems to be controlled through the activity of various signaling pathways The Wnt/β-catenin signaling pathway is widely acknowledged as playing a major role in human disease, particularly in the onset and development of cancer 2 and saturated humidity.Human cell-lines MKN45 , N87, SNU1, SNU16, KATOIII, AGS, WI38 and HEK293  were used in this study. MKN45, N87, SNU1 and KATOIII cells were grown in RMPI media (Gibco); AGS in F12K medium (Hyclone); WI38 in EMEM (Gibco); and HEK293 in DMEM (Gibco). Culture media was supplemented with 10% FBS (Gibco) (AGS with 20%) and 1% penicillin/streptomycin. Cell-lines were maintained at 37°C in 5% COThe SuperTOPFlash-luciferase and the pRL-TK renilla-luciferase plasmids 2, 2.5 µg BSA, 0.01% Glycerol, 200 µM of dNTPs, 0.3X SYBR Green solution and 0.4 µM of specific primers  and 2 µg of RNA was reverse transcribed with 200 U of SuperScript II Reverse Transcriptase (RT) (Invitrogen) using 500 ng of Oligo(dT) primers. Experimental determination of COX2, c-myc and CCND1 mRNA levels was performed according to mers see . PCR conWnt signaling was pharmacologically induced in MKN45 cells seeded in 6 well culture plates at 80–90% of confluence  either with 10–20 mM of LiCl (Sigma) or 5–10 mM of valproic acid  as previously described Activity of the COX2 promoter was measured in 80–90% confluent MKN45, AGS, WI38 and HEK293 cells seeded in 6 well culture plates. Briefly, cells were co-transfected using FUGENE (Roche) for 24 with the pCOX2-luciferase or the pSuperTOPFlash reporters and either constitutive active β-catenin (S33Y) ChIP studies were performed in MKN45 cells as described earlier 32P-labeled oligonucleotides were incubated in binding buffer with nuclear extracts of MKN-45 cells for 30 min at 30°C. Subsequently, DNA-protein complexes were separated from free oligonucleotides on a 5% non denaturating polyacrylamide gel. After electrophoresis, the gel was dried, and exposed to film for 1 day. The visualization of radioactive bands was analyzed by autoradiography. Binding specificity was checked by incubating the DNA-protein complexes in the presence of an excess of non-labeled wild-type or mutated oligonucleotides EMSA was performed using oligonucleotides containing either the wild-type COX2 TBE-II consensus sequence see  or previP<0.05 was considered significant.Each experiment was repeated at least three times with three replicates. Data are shown as the mean ± SD. Multiple group comparisons were performed by one-way ANOVA using the STATISTICA 9.0 software. We initially determined the expression levels of COX2 mRNA in human GC cell-lines MKN45, N87, SNU1, SNU16, KATOIII and AGS In order to examine if the Wnt cascade is involved in COX2 expression, MKN45 cells were pharmacologically stimulated for different periods of time  with either lithium (LiCl) or valproic acid (VA), since both drugs have been previously shown to modulate Wnt signaling via inhibition of GSK3β activity and thus increasing nuclear levels of β-catenin Previous studies indicated that a Wnt/β-catenin responsive TBE site (consensus core: CTTTG) is located at −1,079/−1,074 bp upstream from the transcriptional start site (TSS) of the human COX2 gene To dissect the contribution of Wnt/β-catenin responsive TBE sites on the transcriptional activity of the COX2 promoter four pCOX2 deletions constructs were generated: pCOX-1.2 ; pCOX-0.8 (−741/+35 bp); pCOX-0.65 (−582/+35 bp); and pCOX-0.4 (−371/+35 bp) . These cWe next examined the effect of Wnt/β-catenin signaling at the novel TBE Site II. We transfected MKN45 cells with the pCOX2-0.8 reporter construct and co-expressed the constitutive active β-catenin S33Y protein observing that there was a significant (2-fold) enhancement of the transcriptional activity of pCOX2-0.8. This β-catenin-mediated increase was specific since the pCOX2-0.4 construct was not stimulated in this β-catenin over-expression condition . To contTo further explore the effects of the Wnt/β-catenin pathway on the activity of the pCOX2-0.8 we carried out loss-of-function experiments with a dominant negative TCF4 (ΔTCF) construct, which codes for a transcription factor lacking 30 residues from its amino-terminus and that is unable to bind β-catenin CTCG). These changes have been shown previously to abolish Wnt/β-catenin-mediated transcription Finally, to directly address the role of the TBE Site II in Wnt/β-catenin-mediated regulation of the COX2 promoter, we introduced by site-directed mutagenesis a change in 2 key nucleotides of the consensus sequence of the TBE Site II core , and two oligonucleotides containing missense mutations in either the core of the TBE Site II (i.e. CTACGAGGA: TBE-MUT1) or in the flanking sequence (i.e. CGCCAAAGA: TBE-MUT2), as reported previously Collectively, these experiments indicate that the TBE Site II is directly involved in β-catenin-mediated transcriptional activation of the COX2 promoter. To further confirm these results we performed electrophoretic mobility shift assays (EMSA) in nuclear extracts of MKN45 cells. For this purpose we prepared 34 based-paired oligonucleotides; one containing the wild-type −689/−684 TBE Site II sequence , one of which has been previously studied by Araki and cols Functionality of this TBE Site II was also confirmed by our mutagenesis studies. Thus, when we mutated the TBE-signature sequence into the pCOX2-0.8 construct (MpCOX2-0.8), it was found that basal COX2 promoter activity was significantly affected in MKN45 cells. In addition our ChIP and EMSA analyses confirmed that β-catenin is preferentially recruited to the −689/−684 COX2 promoter region in GC cells, at a level that resembles that found at the c-myc promoter As our results in GC cells are in agreement with those reported for colon cancer cells, it is tempting to speculate that Wnt/β-catenin signaling could be similarly involved in COX2 regulation in other tumors Figure S1ClustalW multiple sequence alignment of human and murine COX2 promoters. TBE response elements, as well as recognition sequences for SP1 and CRE transcription factors, and the TATA box are enclosed.(PDF)Click here for additional data file.Figure S2Comparison of basal promoter activity of pCOX2-1.6 and pCOX2-1.2 constructs in MKN45 and HEK293 cells. Gene reporter assays in MKN45 (A) and HEK293 (B) cells transiently transfected with increasing concentrations of pCOX2-1.6 or pCOX2-1.2. In all experiments 1 ng of PRL-SV40 Renilla was transfected as an internal control. Promoter activity was normalized as the ratio between firefly luciferase and Renilla luciferase units. RLU: Relative Luciferase Units. Each figure corresponds to at least three independent experiments. Statistical significance was determined through ANOVA test .(PDF)Click here for additional data file.Figure S3Basal promoter activity of pCOX2-deletion constructs examined in WI38 cells. Wi38 cells were transiently transfected with 400 ng pCOX2-deletion constructs (A) or a construct containing the p21 promoter as a control (B) and basal promoter activity was determined. In all experiments 1 ng of PRL-SV40 Renilla was transfected as an internal control. Promoter activity was normalized as the ratio between firefly luciferase and Renilla luciferase units. RLU: Relative Luciferase Units. Each figure corresponds to at least three independent experiments. Statistical significance was determined through ANOVA test .(PDF)Click here for additional data file.Figure S4pCOX2-0.8 activity in response to Wnt/β-catenin signaling in HEK293 cells. (A) Reporter gene assay HEK293 cells, co-transfected transiently with 10 ng pCOX-0, 8 with 5 and 10 ng of β-catenin S33Y and 10 ng of empty vector as control. (B) MKN45 cells was transiently transfected with 10 ng pCOX2-0,4 with 5 and 10 ng of S33Y β-catenin and 10 ng of empty vector as control. (C) As a positive control 10 ng of Super Top Flash (STF) was co-transfected with 10 ng of S33Y β-catenin. (D) Effect of site-directed mutation in TBE site in pCOX2-0,8 reporter gene assays in HEK293 cells transfected with 10 ng of pCOX2-0,8 and mutated pCOX2-0,8 (MpCOX-08) in the presence and absence of 5 and 10 ng of S33Y β-catenin, using equal amounts of empty vector as a control. In all trials 1 ng of PRL-SV40 Renilla was transfected as an internal control. Promoter activity was normalized as the ratio between firefly luciferase and Renilla units (RLU). Each figure corresponds to a representative result of three independent experiments. Statistical significance was determined through ANOVA test .(PDF)Click here for additional data file.Figure S5Binding of β-catenin to TBE sites in the COX-2 promoter. ChIP assays in MKN45 cells using specific antibodies for β-catenin. Quantification was performed by real time PCR using specific primers to TBE IV site (−1079/−1074), TBE III site (−877/872), TBE II (−689/−684) and TBE I (−318/−313) and normalizated by IgG.(PDF)Click here for additional data file.Figure S6COX2 and β-catenin immunoreactivity in gastric cancer tissue. Immunohistochemical images of gastric cancer samples showing moderate to strong levels of COX2 and β-catenin expression in different individuals . COX2 and β-catenin proteins were visualized using the HPA001335 and the CAB000108 antibodies, respectively.(PDF)Click here for additional data file.Table S1Primers used in this study.(PDF)Click here for additional data file.Table S2Gastric cancer samples analysed for COX2 and -catenin levels in the Human Atlas Protein Collection.(PDF)Click here for additional data file.


Gastrulation is a uniquely metazoan character, and its genesis was arguably the key step that enabled the remarkable diversification within this clade. The process of gastrulation involves two tightly coupled events during embryogenesis of most metazoans. Morphogenesis produces a distinct internal epithelial layer in the embryo, and this epithelium becomes segregated as an endoderm/endomesodermal germ layer through the activation of a specific gene regulatory program. The developmental mechanisms that induced archenteron formation and led to the segregation of germ layers during metazoan evolution are unknown. But an increased understanding of development in early diverging taxa at the base of the metazoan tree may provide insights into the origins of these developmental mechanisms.Nematostella vectensis, initial archenteron formation begins with bottle cell-induced buckling of the blastula epithelium at the animal pole. Here, we show that bottle cell formation and initial gut invagination in Nematostella requires NvStrabismus (NvStbm), a maternally-expressed core component of the Wnt/Planar Cell Polarity (PCP) pathway. The NvStbm protein is localized to the animal pole of the zygote, remains asymmetrically expressed through the cleavage stages, and becomes restricted to the apical side of invaginating bottle cells at the blastopore. Antisense morpholino-mediated NvStbm-knockdown blocks bottle cell formation and initial archenteron invagination, but it has no effect on Wnt/ß-catenin signaling-mediated endoderm cell fate specification. Conversely, selectively blocking Wnt/ß-catenin signaling inhibits endoderm cell fate specification but does not affect bottle cell formation and initial archenteron invagination.In the anthozoan cnidarian Nematostella, and provides evidence that these two processes could have evolved independently during metazoan evolution. We propose a two-step model for the evolution of an archenteron and the evolution of endodermal germ layer segregation. Asymmetric accumulation and activation of Wnt/PCP components at the animal pole of the last common ancestor to the eumetazoa may have induced the cell shape changes that led to the initial formation of an archenteron. Activation of Wnt/ß-catenin signaling at the animal pole may have led to the activation of a gene regulatory network that specified an endodermal cell fate in the archenteron.Our results demonstrate that Wnt/PCP-mediated initial archenteron invagination can be uncoupled from Wnt/ß-catenin-mediated endoderm cell fate specification in  The origin of metazoans from a choanoflagellate-like protist and the vast diversification of this clade is a remarkable evolutionary chronology in the history of life on Earth. From relatively simple origins, metazoans have radiated to produce organisms with levels of physiological and morphological complexity unmatched in other multicellular forms that have emerged in several clades ,2. The uOne ancient polarity that is present in most metazoan eggs is the animal-vegetal (AV) or primary axis of the egg ,6. The aNematostella vectensis has emerged as an important model organism for comparative molecular studies of embryonic development. Recent studies have identified several key cellular and molecular processes involved in initial germ layer segregation and archenteron formation in this animal . Th. ThNemats Figure . These rNematostella requires Wnt/ß-catenin signaling [Nvß-catenin::RFP mRNA (0.3 μg/μl) and NvStbm-MO or Control-MO to determine if NvStbm-knockdown affected the nuclearization of ß-catenin in blastomeres at the animal pole [In bilaterians endoderm specification and morphogenesis are tightly coupled, and usually cell fate specification precedes morphogenesis -32. Endoignaling ,14, and ignaling . This ramal pole ,14. AnalNvStbm is maternally expressed and hence its early expression would be independent of Wnt/ß-catenin signaling and endoderm specification. The maternal expression of NvStbm raised the possibility that if signaling by this protein through the Wnt/PCP pathway was independent of Wnt/ß-catenin signaling, then selectively blocking this branch of Wnt signaling would have no effect on bottle cell formation and initial archenteron invagination. To test this possibility we examined archenteron formation in embryos inhibited in Wnt/ß-catenin signaling. Previous studies blocking Wnt/ß-catenin signaling by overexpressing Cadherin or NvDsh-DIX, a Dsh dominant-negative in the Wnt/ß-catenin pathway, have produced somewhat different outcomes [Nematostella embryos overexpressing Cadherin did not express endodermal markers, and did not gastrulate [GFP RNA-injected control embryos, NvDsh-DIX::GFP RNA-injected embryos at the early gastrula stage were able to undergo normal initial archenteron invagination  constructs have been described previously [PCR was used to amplify full-length NvStbm and partial fragments of NvFz10 and NvFz5 from cDNA prepared from mixed-stage nin::GFP  with RFPeviously ,18,35. CSynthesis of mRNA was done as previously described ,14. AntiAnti-NvStbm polyclonal rabbit antibodies were raised against a selected sequence corresponding to amino acids 308 to 322 of NvStbm (NH2-TASSQGRRNPGRNDR-COOH), and affinity-purified using the immunizing peptide . Western blots were performed as previously described ,14. AntiImmunochemistry and WMISH was performed as previously described . Anti-NvLytechinus variegatus; MO: Morpholino; Nv: Nematostella vectensis; PCR: Polymerase chain reaction; RFP: Red fluorescent protein; Sp: Strongylocentrotus purpuratus; Stbm: Strabismus; WMISH: Whole mount in situ hybridization; Wnt/PCP pathway: Wnt/Planar Cell Polarity pathway; Xß-catEn: Xenopus ß-catenin engrailed;AV: Animal-vegetal; CE: Convergence and extension; Dsh: Dishevelled; Fz10: Frizzled 10; Fz5: Frizzled 5; GFP: Green fluorescent protein; Hpf: Hours post fertilization; Lv: The authors declare that they have no conflict of interest.SK, NW and AHW designed the research. SK, NW and RX performed the research. SK, NW and AHW analyzed the data and wrote the paper.


Cordyceps sinensis, regulates β-catenin expression in leukemia cells.Leukemia stem cells (LSCs) are a limitless cell source for the initiation and maintenance of leukemia. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs. Therefore, targeting β-catenin is considered a therapeutic strategy for the treatment of leukemia. The goal of this study was to explore whether cordycepin, an active component of the traditional medicine In this study, we found that cordycepin significantly suppressed cell proliferation in all malignant cancer cells, including U937, K562, A549, HepG2, SK-Hep1 and MCF7 in a dose-dependent manner. However, cordycepin reduced β-catenin levels in U937, K562 and THP1 leukemia cells and had no effect on other solid cancer cells. In addition, treatment with cordycepin significantly suppressed leukemia colony formation in soft agar assay. Cordycepin enhanced proteasome-dependent degradation and inhibited nuclear translocation of β-catenin in leukemia cells. Cordycepin-reduced β-catenin stability was restored by the addition of a pharmacological inhibitor of GSK-3β, indicating that cordycepin-suppressed β-catenin stability is mediated by the activation of GSK-3β. Furthermore, cordycepin abolished the effect of Wnt3a-induced β-catenin in leukemia cells. In addition, cordycepin-impaired β-catenin is regulated by Akt activation but is not significantly influenced by AMPK or mTOR signal pathways.via elimination of LSCs.Our findings show for the first time that codycepin selectively reduces β-catenin stability in leukemia but not in other solid tumor cells. This suppressive effect is mediated by regulating GSK-3β. A synergistic combination of cordycepin with other treatments should be used as a novel strategy to eradicate leukemia  Our results suggest that cordycepin can synergize with other anti-leukemia reagents by targeting LSCs to treat leukemia.Although cordycepin was shown to have anti-leukemia properties 2 humidified incubator. Cordycepin, adenosine, SB216763 and rapamycin were purchased from Sigma-Aldrich . Compound c and MG-132 were from Merck KGaA, . Ly-294002 was obtained from Enzo Life Sciences  and Wnt-3a was from R&D Systems .U937, K562 and THP1 cells were maintained in RPMI 1640 medium  and A549, HepG2, SK-HEP-1 and MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, 100 µg/ml streptomycin  at 37°C in a 5% COCell proliferation of adherent cells  was determined using a 3--2,5-diphenyltetrazolium bromide (MTT) assay described previously g at 4°C for 20 minutes. 20 µg of proteins from each sample were applied to the gradient SDS-PAGE gel and immunoblotted onto PVDF membranes. The membranes were blocked, incubated with primary antibodies against Akt1, Phospho-Akt (Ser-473), GSK-3β, Phospho-GSK-3β (Ser9) and Cyclin D1 , β-catenin , β-actin  or Lamin A/C , followed by an incubation with a secondary antibody conjugated horseradish-peroxidase. Protein levels were determined by the use of enhanced chemiluminescence reagents.Protein expression was determined by Western blot analysis described previously Immunofluorescence staining was performed as described previously A colony forming assay was performed using a soft-agar assay U397 cells treated with indicated reagents were harvested and nuclear as well as cytosolic proteins were extracted and prepared by using the ProteoExtract® Subcellular Proteome Extraction Kit  as described previously t-test was used to analyze differences between 2 experimental groups. A p value less than 0.05 was considered statistically significant.The Student’s To explore the potential role of cordycepin in modulating tumor growth, several solid and suspension cancer cells including U937, K562, THP1, A549, HepG2, SK-Hep1 and MCF-7 cells were treated with cordycepin (50 to 200 µM) for 24–72 hrs. Cell proliferation rates were determined by MTS or MTT assays for suspension and adherent cells, respectively. Cordycepin significantly inhibited cell proliferation of all cell lines in a dose- and time-dependent manner . We nextβ-catenin is considered an important regulator of cell proliferation in various cell types including malignant cancer cells. We examined whether cordycepin affects β-catenin expression. Solid and suspension cancer cells were treated with 50 to 200 µM of cordycepin for 4 to 24 hrs and the expression of β-catenin was determined by Western blotting. Cordycepin dramatically reduced β-catenin expression in a dose-dependent manner in K562, U937 and THP1 cells . HoweverThe accumulation of β-catenin results in nuclear translocation, thereby inducing the expression of downstream genes. We determined the effect of cordycepin on the nuclear translocation of β-catenin by Western blots in subcellular fractions of U937 cells. β-catenin levels were eliminated in total cell lysates, cytosolic and nuclear fractions in cordycepin-treated U937 cells . As the vs. lane 1). However, Wnt3a-induced β-catenin was significantly abolished by treatment of cordycepin (vs. lane 2). These results reveal that cordycepin suppresses expression of β-catenin in leukemia and this effect is mediated through modulating Wnt/GSK-3β signaling.Stability of β-catenin is tightly controlled by GSK-3β. GSK-3β phosphorylates β-catenin and subsequently facilitates the ubiquitination and degradation of β-catenin. We assumed that cordycepin-induced β-catenin degradation is mediated by regulating GSK-3β. To investigate whether cordycepin influences GSK-3β activity, U937 and A549 cells were treated with cordycepin alone or in combination with SB216763, a pharmacological inhibitor of GSK-3β, for 4 or 8 hrs. Cordycepin-reduced β-catenin was significantly restored by SB216763. No such effect was found in A549 cells treated with either cordycepin alone or combined with the GSK-3β inhibitor . In addivia the inactivating of mTOR, AMPK or Akt. To test this hypothesis, U937 cells were treated with inhibitors of AMPK (compound c), mTOR (rapamycin) and PI3-K (Ly-294002), alone or in combination with cordycepin. Inhibition of AMPK or mTOR had no significant effect on the suppression of β-catenin  for 24 and 48 hrs. Cell proliferation was determined by an MTT assay. These data are from three independent experiments. Each bar denotes mean ± S.E.M.(TIF)Click here for additional data file.Figure S2Effect of adenosine on β-catenin expression. U937, K562 and A549 cells were treated with cordycepin (100 µM) or adenosine (50 to 200 µM) for 4 hrs and the expression of β-catenin was determined by Western blot analysis. Actin was used as a loading control.(TIF)Click here for additional data file.Figure S3Efect of cordycepin on cyclin D expression. A549 and U937 cells were treated with 100 µM cordycepin for 2 or 4 hrs. The expression of β-catenin and cyclin D1 was determined by Western blot analysis. Actin was used as a loading control.(TIF)Click here for additional data file.


Beta-catenin (β-catenin) is a multifunction protein with a central role in physiological homeostasis. Its abnormal expression leads to various diseases including cancer. In normal physiology, β-catenin either maintains integrity of epithelial tissues or controls transcription of various genes on extracellular instigations. In epithelial tissues, β-catenin functions as a component of the cadherin protein complex and regulates epithelial cell growth and intracellular adhesion. In Wnt signalling, β-catenin is a major transcriptional modulator and plays a crucial role in embryogenesis, stem cell renewal and organ regeneration. Aberrant expression of β-catenin can induce malignant pathways in normal cells and its abnormal activity is also exploited by existing malignant programmes. It acts as an oncogene and modulates transcription of genes to drive cancer initiation, progression, survival and relapse. Abnormal expression and function of β-catenin in cancer makes it a putative drug target. In the past decade, various attempts have been made to identify and characterize various pharmacological inhibitors of β-catenin. Many of these inhibitors are currently being investigated for their anticancer activities in a variety of cancers. The first half of this review will focus on the role of β-catenin in cancer initiation, maintenance, progression and relapse whereas the second half will briefly summarize the recent progress in development of agents for the pharmacological modulation of β-catenin activity in cancer therapeutics. IntroductionRole of β-catenin in cancerβ-catenin inhibitorsPlant-derived β-catenin modulatorsConclusion and prospectivevia the proteolytic pathway, β-catenin accumulates in the perinuclear region and forms a cytoplasmic pool of free signalling molecules Beta-catenin (β-catenin) is the mammalian homologue of the drosophila armadillo gene Various extracellular signals regulate the localization of β-catenin either on the membrane or in the cytoplasm et al. while studying hepatocyte differentiation in mesenchymal stem cells have found that the activation of Wnt signalling and β-catenin nuclear localization results in a tumoural phenotype et al. observed rapid formation of lipogenic liver tumours in mice on AKT and β-catenin co-activation et al. and Shtutman et al. in colon cancers revealed that β-catenin activates transcription from the cyclin D1 (CCND1) promoter, and consensus TCF/LEF-binding sites are necessary for this activity Accumulating evidence indicates that β-catenin has a central role in the malignant transformation of normal cells Metastasis is an important cancer hallmark and it is often supported by abnormal β-catenin expression or localization et al. explored the role of β-catenin in haematopoietic malignancies Abnormal activity of β-catenin is further associated with cancer drug resistance and cancer stem cell state et al. for the first time demonstrated that specific inhibition of the oncogenic form of β-catenin is sufficient to reverse the transformed properties of human cancer cells et al. made initial attempts to screen and identify compounds capable of disrupting TCF/β-catenin complexes et al. and Gandhirajan et al. investigated the effects of CGP049090 (IC50 ≤ 1 μM) and PFK115-584 (IC50 ≤ 1 μM) in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) cells respectively e.g. c-myc, cyclin D1 and survivin). Down-regulation of these survival-related genes resulted in the induction of cell death in AML cell lines and cells derived from AML patients et al. found that PKF118-310 (IC50 ≤ 1 μM) was effective against breast cancer initiating cells (BTIC) where it inhibited tumour growth and proliferation in vivo and viable tumour cells harvested from PKF118-310 treated mice were unable to induce the growth of secondary tumours after transplantation et al. identified a novel inhibitor  of beta-catenin/CREB-binding protein transcription activity. ICG-001 induced apoptosis in transformed cells selectively and also reduced in vitro and in vivo growth of colon carcinoma cells et al. screened a chemical library against a transcription factor reporter cell line in which the activity of the pathway was induced at the level of dishevelled (dsh) protein et al. identified nine potent β-catenin inhibitors (IC50 ≤ 2.5 μM) in vitro to identify less toxic and highly selective inhibitors against the Wnt/β-catenin signalling pathway el al. identified a novel inhibitor (XAV939) which antagonized Wnt/β-catenin pathway by inhibiting tankyrase et al. employed a high-throughput screen to identify inhibitors of Wnt/β-catenin signalling et al. that iron can induce Wnt/β-catenin signalling et al. used a cell-based assay system as well as transgenic MMTV-Wnt1 and MMTV-PyMT mice models to screen Wnt/β-catenin inhibitors Inhibition of β-catenin using small molecule inhibitors or siRNA abrogates tumour growth bitor XAV9 which abitor XAV9 which abitor XAV9 which abitor XAV9 which aFurthermore, inhibition of β-catenin can also be employed against cancer stem cells and chemo-resistant cancer cells. The Rosen laboratory evaluated radiation resistance in CSCs isolated from p53-null mouse mammary tumours Stephania tetrandra exhibited significant anticancer activity by inhibiting β-catenin/Tcf transcriptional activity  Silybum marianum, inhibits melanoma cell migration (IC50 ≤ 20 μM) by inhibiting β-catenin nuclear localization via its ability to enhance E-cadherin-mediated adhesion and inhibition of β-catenin tyrosine phosphorylation Aplinia katsumadai inhibits 65–70 per cent of β-catenin activity at a dose ≤10 μM, without compromising cell viability Various plant-derived compounds with anticancer activities are also known to inhibit or modulate the Wnt/β-catenin signalling pathway. Tetrandrine (TET), a bis-benzylisoquinoline alkaloid purified from the root of i.e. initiation, progression and chemoresistance in conjunction with the traditional chemotherapy. However, the long-term effects of the pharmacological manipulation of β-catenin remain still unclear. The overall regulation of β-catenin involves multiple signalling pathways and therefore pharmacological modulation could be counterbalanced through the activation of compensatory signalling pathways. The possibility of adverse side effects of β-catenin inhibition cannot be ruled out at this juncture and more detailed studies will be required to address this key issue. To date, use of various small molecule inhibitors of β-catenin targeting cancer have provided some encouraging results. Further efforts can be directed towards evaluating the efficacy of the existing inhibitors in variety of cancer types, stages and especially against cancer initiating cells/cancer stem cells and chemo-resistant cancers. As it is evident that microenvironmental regulation of the β-catenin activity plays a central role in the malignant transformation and induction of metastasis; these inhibitors can also be used in combination with inhibitors of cancer survival pathways and modulators of tumour microenvironment. Some of the phytochemicals that seem to modulate β-catenin activity can also be used as lead compounds for developing β-catenin-targeted therapeutics. Targeting Wnt–β-catenin activity could open new avenues for novel and tailor-made cancer therapeutic approaches.The potential of the pharmacological modulation of β-catenin in cancer therapeutics is paramount. This may possibly provide an attractive option of targeting various aspects of the carcinogenic process


We have recently unravelled a novel function for CD82 in E-cadherin-mediated cellular adhesion. CD82 inhibits β-catenin tyrosine phosphorylation and stabilizes E-cadherin-β-catenin complexes at the cell membrane. This function inhibits cancer cell dissociation from the primary cancer nest and limits metastasis. In this study, we focused on the effect of CD82 on the Wnt/β-catenin  pathway, which controls the cellular distribution of β-catenin. CD82 had no effect on the expression of Wnt proteins but led to significant downregulation of Frizzled (Fzd) 2, 3, 5, 7 and 9, suggesting downregulation of the Wnt/β-catenin pathway. CD82 also inhibited phosphorylation of β-catenin at Ser45, Ser33, Ser37 and Thr41 by downregulation of glycogen synthase kinase-3β (GSK-3β) and kinase casein kinase 1α (CK1α). Downregulation of GSK-3β and CK1α also led to accumulation of β-catenin in the cytoplasm or at the cell membrane. CD82 translocated β-catenin to the cell membrane, suggesting that CD82 strengthens the interaction between E-cadherin and β-catenin. We concluded that CD82 attenuates Wnt signalling by controlling β-catenin cellular distribution at multiple levels: i) inhibition of β-catenin nuclear translocation by downregulation of Fzd receptor proteins; ii) accumulation of β-catenin at the cell membrane by downregulation of GSK-3β and CK1α; and iii) stabilization of the E-cadherin-β-catenin complex. Metastasis is a multi-step cascade involving the migration of tumour cells from their site of origin, evasion from host defence systems, subsequent seeding at distant organs and growth of secondary tumours. The first step of metastasis is migration of the tumour cells from the primary tumour nest. In this process, tumour cells are required to loosen their homophilic cell adhesion, enabling tumour cells to escape from the tumour nest. Classic cadherins interact homophilically with cadherins of neighbouring cells to form adherens junctions, which serve both as mechanical linkages between cells and signalling hubs that relay information from the extracellular environment –5. EpithThe cellular distribution of β-catenin has a major influence on the control of the malignant phenotype of cancer cells. Accumulation of β-catenin in the nucleus correlates with poor prognosis in many types of cancer. Upon translocation into the nucleus, β-catenin forms complexes with members of the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors –14, leadTetraspanins, or TM4SF (transmembrane 4 superfamily) proteins, compose a large group of cell-surface transmembrane proteins, some of which can form complexes with integrins. Several tetraspanins appear to be particularly relevant to tumour cell metastasis ,26. CD82In this study, we investigated the effect of CD82 on the canonical Wnt pathway  and showed that CD82 inhibits Wnt signalling in a multifunctional way.Mouse monoclonal antibodies against KAI-1 (G-2), E-cadherin (H-106) and rabbit polyclonal antibodies against KAI-1 (C-16) were purchased from Santa Cruz Biotechnology . Rabbit polyclonal and mouse monoclonal antibodies against β-catenin were purchased from Upstate Laboratories . The rabbit monoclonal antibody against phospho-β-catenin (pSer33/pSer37) was purchased from Upstate Laboratories. Mouse monoclonal antibodies against phospho-β-catenin (pThr41 and pSer45) were purchased from Sigma-Aldrich . The sheep polyclonal antibody against CK1α was purchased from R&D Systems  and the rabbit monoclonal antibody against GSK-3β, from Cell Signalling Technology .The anti-Wnt protein antibodies used in this study were as follows: anti-Wnt1 , anti-Wnt2 , anti-Wnt2B , anti-Wnt3 , anti-Wnt3a , anti-Wnt4 , anti-Wnt5a , anti-Wnt5b , anti-Wnt6 , anti-Wnt7a and anti-Wnt7b , anti-Wnt8a , anti-Wnt8b , anti-Wnt9a , anti-Wnt9b , anti-Wnt10a  and anti-Wnt10b (Wnt12) .The anti-Fzd protein antibodies used in this study were as follows: rabbit polyclonal antibodies against Fzd1, Fzd3, Fzd4, Fzd6, Fzd7, Fzd8, Fzd9 and Fzd10  and rabbit polyclonal antibodies against Fzd2 and Fzd5 .2.The human cell line h1299  and its transfectant derivatives (h1299/zeo and h1299/CD82) were established in our laboratory by transfection of a control vector or CD82 cDNA and cell sorting-based clone selection, as described previously . Furtherμg/ml puromycin.The h1299/CD82-sh.control and h1299/CD82-sh.CD82 cell lines were generated by Lipofectamine  transfection of h1299/CD82 cells with pLKO.1-puro Control Vector (Sigma) and pLKO.1-puro/sh.CD82 , respectively. Colonies that showed resistance to puromycin (Sigma) were pooled from the individual transfection experiments. The expression levels of CD82 in shRNA-transfected h1299 cells were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting. The h1299/CD82-sh.control and h1299/CD82-sh.CD82 cells were maintained in DMEM containing 10% FBS and 2 2, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF, 50 mM Tris-HCl (pH 7.2)]. Subcellular fractionation was performed using the ProteoExtract® Subcellular Proteome Extraction kit from Merck  according to the manufacturer’s instructions.Cell lysates for immunoblotting were prepared in cell lysis buffer [1% Triton X-100, 2 mM sodium orthovanadate, 500 mM NaCl, 10 mM MgClThe samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane  and incubated with specific primary antibodies. Protein bands were visualized using horseradish peroxidise (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence Reagent . The bands were scanned by computer-assisted densitometry  and analysed using the Quantity One software (Bio-Rad).Total RNA was extracted from h1299 cells by using TRIzol  and used for first-strand cDNA synthesis. The mRNA levels were measured in triplicate using a real-time PCR system with the Brilliant SYBR Green qPCR kit . Specific primers for GSK-3β and CK1α were as follows: GSK-3β (F: 5′-GG TCTATCTTAATCTGGTGCTGG-3′ and R: 5′-AGGTTCTGC GGTTTAATATCCC-3′) and CK1α (F: 5′-F:GGAAAAGAAGC ATGACTGTTAG-3′ and R: 5′-TCTGTATGGTATGTGTTGCC TT-3′). PCR cycling conditions were 10 min at 95°C for 1 cycle followed by 45 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 60 sec. Dissociation curve analyses confirmed that signals corresponded to unique amplicons. Expression levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level of each sample, obtained from parallel assays.The cellular distribution of β-catenin is regulated by the Wnt/β-catenin  signalling pathway. This pathway is initiated by binding of Wnt ligands to their Fzd receptor proteins. To evaluate the effect of CD82 on Wnt signalling, we first analysed the protein expression levels of Wnt ligands on h1299 cells by immunoblotting. As shown in Next, we examined the expression of Fzd proteins, which are the transmembrane receptors for Wnt ligands . BindingFzd1-Fzd10 were expressed on h1299 cells. In our model, the expression of Fzd1, Fzd4, Fzd6, Fzd8 and Fzd10 was not markedly affected by CD82. In contrast, CD82 significantly downregulated the expressions of Fzd2, Fzd3, Fzd5, Fzd7 and Fzd9. Knocking down of CD82 mRNA expression by shRNA in h1299/CD82 cells resulted in recovery of the expression of those downregulated Fzd proteins, suggesting a specific effect of CD82 on Fzd2, Fzd3, Fzd5, Fzd7 and Fzd9 expression .This result suggests that CD82 reduces receptor binding of Wnt ligands by inhibiting Fzd expression, thereby reducing Wnt signalling and β-catenin translocation to the nucleus.In the absence of Wnt ligands or in the event of impaired Wnt signalling, cytosolic β-catenin is phosphorylated at Ser45 by CK1α. Consequently, GSK-3β phosphorylates β-catenin at Thr41, Ser37 and Ser33 . Ser33/STherefore, we next examined the effect of CD82 on the cellular distribution and phosphorylation of β-catenin. We performed subcellular fractionation and determined the amount of total and phosphorylated β-catenin in h1299 cells by immunoblotting.2+ treatment enhances E-cadherin internalization (which is independent of tyrosine phosphorylation and ubiquitination) and E-cadherin is recycled back to the plasma membrane  at the cell membrane. Moreover, CD82 reduces nuclear translocation of β-catenin even when E-cadherin is destabilized.Inhibition of β-catenin phosphorylation  by CD82 also suggests that β-catenin may be differently phosphorylated by GSK-3β and CK1α.In a first step, we examined the protein expression levels of GSK-3β and CK1α by immunoblotting. The protein levels of GSK-3β and CK1α were downregulated by CD82 and this effect was reverted by knocking down CD82. These results indicate a specific effect of CD82 on GSK-3β and CK1α .Next, we examined the mRNA levels of GSK-3β and CK1α by real-time RT-PCR. GSK-3β mRNA levels were significantly downregulated by CD82 and this downregulation reverted by knocking down CD82 . In contIn our previous research, we showed that CD82 inhibits tyrosine phosphorylation of β-catenin and stabilizes E-cadherin-β-catenin complexes at the cell membrane . CD82 inAll members of the Wnt protein family are extracellular secreted proteins. Binding of a Wnt ligand to its co-receptors Fzd and LRP5/6 results in the activation of Dvl, which in turn inhibits GSK-3β-mediated phosphorylation of β-catenin . We coulAccumulated cytoplasmic β-catenin is phosphorylated at Ser45 by CK1α and incorporated into a cytosolic protein complex containing Axin, APC and GSK-3β. GSK-3β phosphorylates β-catenin at residues Ser33, Ser37 and Thr41 , therebyFurthermore, this result also serves as evidence to show that CD82 inhibits the nuclear translocation of β-catenin. Accumulation of β-catenin in the cytoplasm will result in nuclear translocation of β-catenin, as observed in the EDTA-treated h1299/zeo cells . In contRecently, a novel inhibitory mechanism of Wnt signalling by CD82 was described. CD82 and other tetraspanins are organized in a signalling complex with E-cadherin at the plasma membrane. This signalling complex, including tetraspanins, E-cadherin and β-catenin, is internalized and delivered to early endosomes . ExosomeAltogether, CD82 attenuates Wnt signalling in multiple ways : i) inhiIn conlusion, CD82 suppresses cancer metastasis through the canonical Wnt pathway via multifunctional ways. Down-regulation of Fzd receptors also suggests inhibition of the non-canonical Wnt pathway. However, this mechanism remains to be elucidated. Overall, accumulating evidence of the anti-metastatic effect of CD82 suggests CD82 as a novel therapeutic target for anti-metastasis cancer therapy.


In ovarian carcinoma, abnormal expression of β-catenin, E-cadherin and WNT-1 was observed, but their prognostic and predictive role is unclear. The aim of this study was to clarify the prognostic and predictive role of E-cadherin, β-catenin and WNT-1 in advanced epithelial ovarian carcinoma (AEOC).The expression of E-cadherin, β-catenin and WNT-1 was determined by immunohistochemistry in AEOC. The correlation between expression of these proteins and progression-free survival (PFS) and overall survival (OS) was evaluated. Statistical analyses included Kaplan-Meier estimation, log-rank test, Spearman correlation and Cox proportional-hazards model.In ovarian cancer, intense expression of E-cadherin, β-catenin and WNT-1 was found. In multivariate analysis, strong membrane β-catenin expression was an independent unfavourable predictor for PFS , while in univariate analysis, strong membrane β-catenin expression was a prognostic factor for OS in patients with AOC (p = 0.039). In multivariate analysis, only resistance to first-line chemotherapy was an adverse independent prognostic factor for OS . Additionally, strong membranous β-catenin expression was associated with resistance to platinum-based chemotherapy (p = 0.027).These findings support that WNT/β-catenin pathway and E-cadherin are important factors in advanced epithelial ovarian cancer. Epithelial ovarian cancer (EOC) is the most common ovarian malignancy and accounts for 90% of cases of ovarian tumours . In 2008The most important prognostic factors for EOC include: age, performance status, histology type, International Federation of Gynaecology and Obstetrics (FIGO) stage and tumour grade ,5. HowevRecently, more attention has been focused on the molecular markers of the WNT/β-catenin pathway, which is widely studied in EOC -14. The Deregulation of WNT/β-catenin pathway or altered expression of E-cadherin was found in many cancers as well as in EOC ,18. One The aim of this study was to determine the expression of β-catenin, E-cadherin and WNT-1 in advanced epithelial ovarian cancers and to assess the correlation of expression of the studied proteins with patient survival and response to platinum-based chemotherapy. We found changes in the expression of the studied proteins in ovarian cancer cells. Moreover, strong membrane β-catenin expression was identified as an unfavourable predictor for PFS and was associated with resistance to platinum-based chemotherapy for EOC patients.2) with cisplatin (75 mg/m2) or paclitaxel (175 mg/m2) with carboplatin (AUC6), administered every 3 weeks for 6 cycles; (3) accessibility of primary tumour specimens and full medical data. Response to first-line chemotherapy according to RECIST criteria (version 1.0), PFS and OS were obtained from medical records and analysed retrospectively. This study was approved by the institutional review board of the Military Institute of Medicine in Warsaw (46/WIM/2009).We analysed medical records of all consecutive EOC patients treated in the Department of Oncology at the Military Institute of Medicine in Warsaw, Poland between March 2001 and December 2007. The inclusion criteria were as follows: (1) histologically confirmed advanced epithelial ovarian cancer in FIGO stage III-IV; (2) history of debulking surgery followed by first-line chemotherapy regimen: paclitaxel  and subjected to antigen retrieval in Target Retrieval Solution, pH 9 (DAKO) with PT Link (DAKO). Tissues were incubated with mouse monoclonal anti-β-catenin antibody , mouse monoclonal anti-E-cadherin antibody  or rabbit polyclonal anti-WNT-1 antibody . Negative controls were incubated with mouse or rabbit IgGs (DAKO). Subsequently, sections were incubated with peroxidase-based EnVision™ + system (DAKO). Colorectal epithelium was used as an external positive control showing strong membranous expression of E-cadherin and β-catenin.The expression was scored by three independent observers  without knowledge of the clinical data. All membranous, cytoplasmic and nuclear staining was evaluated in cancer cells. The intensity of membrane staining was categorized as follows: strongly positive when intensity was equal to the intensity of cell membrane in positive control and weakly positive corresponding to the intensities between strong and negative. The presence of cytoplasmic and nuclear staining was graded into two groups: negative and positive if 10% of tumour cells showed immunoreactivity.Statistical analyses included descriptive statistics with determination of minimal and maximal values, means and medians, with 95% confidence interval (CI) for particular variables. OS was defined as time elapsed between the date of diagnosis and date of death or the date of last follow-up. PFS was defined as the time from diagnosis until disease recurrence or death or date of last follow-up. A Spearman test for non-parametric variables was used to assess correlation between histoclinical data and the expression of studied proteins. A Mann–Whitney U test for non-parametric variables was used to assess if the expression of studied proteins had any predictive value to response to chemotherapy according to RECIST criteria (version 1.0). Univariate analyses of variables influencing PFS or OS were performed by log-rank test, which identified a preliminary list of significant factors. All variables found to be significant and factors that showed a trend towards significance (p < 0.1) in the univariate analysis were included in the multivariate analysis. Multivariate analyses of PFS and OS were performed by Cox proportional-hazards regression using the forward stepwise method. Median and life tables were computed using the product-limit estimate by the Kaplan and Meier method and the log-rank test was employed to assess the statistical significance, p values less than 0.05 were considered as significant. Statistical calculation was performed using the STATISTICA for Windows Version 7.0 software.Among 132 patients with epithelial ovarian cancer in our database, 46 were eligible. The main reasons for the exclusion of patients from the study were lack of access to tumour samples (71 patients) and early stage of EOC (15 patients). Patient characteristics are summarized in Table In ovarian cancer cells, membrane E-cadherin expression was strongly positive in 39 (84.8%) patients. Moreover the presence of cytoplasmic E-cadherin was observed in tumours of almost all patients . Expression of β-catenin was strong in almost half of all patients . Moreover, β-catenin was present in the cytoplasm of EOC cells in 19 patients (41.3%), while it was absent in the cell nuclei in all EOC tumours. In ovarian tumour cells, WNT-1 expression was present in 31 (67.4%) women . There was a difference observed in complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) distribution according to membrane β-catenin expression p = 0.044). Eight (61.5%) EOC patients with tumours demonstrating strong β-catenin expression in the cell membrane had SD or PD, while most of the patients  with tumours displaying weak membrane β-catenin expression had an objective response (CR + PR) that was statistically significant (p = 0.027; Table 4. Eight In the present study, we analysed the expression of E-cadherin, β-catenin and WNT-1 in advanced ovarian carcinoma. We demonstrated the presence of E-cadherin expression both in the cell membrane and cytoplasm of ovarian tumour cells. The expression of E-cadherin has been investigated in ovarian carcinoma, but in most of the studies, patients in different stages of the disease were examined ,28. AccoIn primary and well-differentiated cancers E-cadherin expression is high, while in advanced EOC, E-cadherin expression is moderate, although complete loss of E-cadherin is rare ,28-30,33Furthermore, we detected intense expression of membrane β-catenin in advanced EOC. Our results are concordant with earlier reports showing positive β-catenin expression in ovarian tumours, but they differ from the results reported by other groups where the expression of β-catenin was decreased ,25. Addiβ-catenin is also a key component of the signalling pathway that is activated by WNT ligands. We showed that in most of the ovarian tumours, there was a strong WNT-1 expression; however, no nuclear β-catenin was found. It might suggest that the WNT/β-catenin signalling pathway was not activated despite the presence of WNT-1 ligand. WNT-1 expression has been detected in ovarian carcinomas ,44; howeIn our study, we found prognostic factors for OS and PFS of EOC patients and predictors for chemotherapy response. We revealed that strong membrane β-catenin expression was an independent adverse prognostic factor for PFS, while only resistance to first-line chemotherapy was an unfavourable prognostic factor for OS of advanced ovarian cancer patients. Davidson et al. also investigated the influence of β-catenin expression on advanced EOC patient survival, but they did not find any statistically significant correlation . HoweverFinally, we found that strong membranous β-catenin expression was associated with the lack of response to chemotherapy, which corroborates in vitro studies. Silencing of β-catenin leads to the increased sensitivity of A2780 ovarian cancer cells to cisplatin, paclitaxel and vincristine . MoreoveIn conclusion, our study showed the presence of β-catenin and E-cadherin expression in advanced ovarian cancers. Our results imply that β-catenin and E-cadherin expression may be required for ovarian carcinogenesis because these proteins are involved in signalling pathways that control cell proliferation. Additionally, β-catenin could be responsible for resistance to chemotherapy, because some cell adhesion proteins are associated with resistance of tumour cells to chemotherapy. Moreover, we speculate that canonical and non-canonical WNT pathways could control ovarian carcinogenesis, because β-catenin is absent in cancer cell nuclei, despite a strong WNT-1 expression. These findings support that WNT/β-catenin pathway, as well E-cadherin, are important in advanced epithelial ovarian cancer.APC: Anaphase promoting complex; βTrCP: Ubiquitin ligase protein; CA-125: Cancer antigen 125; CAM-DR: Cell-adhesion-mediated drug resistance; CI: Confidence interval; CKI: Casein kinase I; CR: Complete response; EOC: Epithelial ovarian cancer; FIGO: International Federation of Gynaecology and Obstetrics; FZD/LRP: Frizzled/lipoprotein receptor-related protein; GSK3β: Glycogen synthase kinase 3β; OS: Overall survival; PD: Progressive disease; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; TCF/LEF: T-cell factor/lymphoid enhancer factor.All authors declare that they have no competing interests.LB participated in acquisition of data, performed the statistical analysis and wrote the manuscript. AS carried out immunohistochemical analyses and evaluation of the staining, and wrote the manuscript. SC carried out evaluation of the staining. MS participated in acquisition of data. MC carried out immunohistochemical analyses. WK carried out evaluation of the staining. CS conceived of the study and participated in its design and coordination. MW conceived of the study and participated in its design and coordination and helped to draft the manuscript. MLP conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.This work was supported in part by a grant from Military Institute of Medicine in Warsaw and by Celon Pharma Inc.


Lithium chloride (LiCl) which mimics canonical Wnt/β-catenin signaling was added to cells transfected with BMP4 expressing plasmid. Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation. Gene expression analysis exhibited upregulation of cathepsin H by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 protein accumulation from BMP4 expressing cells. These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.The effect of canonical Wnt/ From observations of mutant mice, deletion of canonical Wnt/β-catenin pathway leads to the defect of osteoblastogenesis both in endochondral and membranous ossification [β-catenin pathway prevents osteoblast from differentiation into chondrocyte and is required for osteogenesis. Interestingly, stabilized β-catenin negatively affected both osteogenesis and chondrogenesis in gain-of-function mutant study [ Bone morphogenetic proteins (BMPs) play critical roles in chondrogenic and osteogenic differentiation of mesenchymal multipotent cells . The usefication , 4. Thesβ-catenin signaling on the BMP4 mediated chondrogenic differentiation. LiCl inhibits glycogen synthase kinase-3β (GSK-3β) and leads to accumulation of β-catenin. Therefore, LiCl is known to mimic the activity of Wnt/β-catenin signaling [ Gene transfer of BMP expressing plasmid drives recipient cells to produce BMP continuously . High deignaling .From our findings in this study, we focused on cathepsin H. Cathepsin H is a member of cathepsins, which are proteases ubiquitously expressed in animal tissues. Cathepsin family includes the serine proteases cathepsins A and G, the aspartic proteases cathepsins D and E, and the lysosomal cysteine cathepsins B, C, F, H, K, L, O, S, V, X, and W . CathepsA 1.6 kb mouse BMP-4 cDNA is a gift from Hogan et al. . It was 7 cells/mL. A 10 μL drop of this cell suspension was placed in the center of a well in 12-well tissue culture dish . The cells were allowed to adhere for 60 min and, then, the wells were filled with 1 mL of DMEM/F12 (1 : 1) supplemented with 1% fetal bovine serum (Invitrogen), 0.2% BSA , 50 μg/mL ascorbic acid 2 phosphate (Sigma), Penstrep, and ITS+ , that is, differentiation medium. This medium was changed every 3 days. C3H10T1/2 clone 8 cells were obtained from RIKEN cell bank  and maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fatal bovine serum (Invitrogen), 50 U/mL penicillin, and 50 mg/mL streptomycin (Penstrep: Invitrogen), that is, growth medium. The micromass culture technique was modified from Ahrens et al. . Trypsin2, 10 mM K2HPO4 pH7.6, 5 mM MgCl2; 25% Opti-MEM1) at a concentration of 2.5 × 106 cells/mL. Plasmid solution was added into 600 μL of cell suspension (1.5 × 106 cells). Electric pulses were generated by CUY-21 in vitro  and applied through 4 mm gap cuvette. Pulse settings were 480 V, 2.5 ms, 2 pulses at the interval of 1 second. After electroporation, the cells were recovered in growth medium overnight. On the next day of electroporation, dead cells were removed by washing with phosphate buffered saline (PBS: Invitrogen) and micromass culture was started as mentioned above. The timeline of experiments was counted from the start of micromass culture in this study.Before electroporation, cells were lifted with 0.25% Trypsin EDTA (Invitrogen) and dissolved in electroporation buffer  . For quantification, alcian blue stain was solubilized in 4 M guanidine HCl, 50 mM Tris HCl (pH7.4), 0.1% CHAPS, and its absorbance was measured by a spectrophotometer at a wavelength of 595 nm. For immunostaining, micromass was fixed in −20°C cold ethanol. Primary antibody for BMP4  was added and incubated overnight at 4°C. Anti-mouse IgG conjugated with Alexa Fluoro 555 was used as secondary antibody (1 : 500). Fluorescent signal was checked by fluorescent microscope .−ΔΔCt. Primers for RT-PCR were listed on For quantitative reverse transcription polymerase chain reaction (qRT-PCR), total RNA was isolated using RNeasy Mini kit and RNase-free DNase set  according to the manufacturer's protocol. On-column DNA digestion was routinely done. Single stranded cDNA was synthesized using High-Capacity cDNA Archive Kit . qRT-PCR analyses were carried out with StepOnePlus and Power SYBR green master mix (Applied Biosystems). The fractional cycle number at which the fluorescence passes the threshold  was used for quantification by using a comparative Ct method. Values of gene-of-interest (GOI) were normalized to the threshold value for GAPDH: ΔCt = Ct  (GOI) − Ct (GAPDH). The Ct value of control was used as a reference. ΔΔCt = ΔCt  (experiment) − ΔCt (control). The fold change in mRNA expression was calculated by the following formula: 2 Culture medium was subjected to SDS-PAGE with 10% acrylamide gel. Samples were blotted to a polyvinylidene difluoride membrane . The membranes were blocked with 2% nonfat dry milk for 1 hour at room temperature and incubated with anti-BMP4 primary antibody (1 : 400 Santa Cruz) at 4°C overnight. Then HRP-conjugated secondary anti-mouse IgG  was applied for 2 hours at room temperature. Signals were visualized by chemiluminescence using ECL plus western blotting detection reagents  with a digital luminescent image analyzer LAS-1000 . Band intensities were analyzed by ImageJ 1.37v software program .t-test was used for the analysis in the gene-silencing experiment.Data were presented as means ± standard deviation of independent experiments. One-way analysis of variance (ANOVA) was performed using GraphPad Prism version 5.0 program . Statistical analyses of differences between experimental groups and control were performed using Dunnett's post hoc test. Student's To confirm the chondrogenesis, pCAGGS-BMP4 transfected C3H10T1/2 cells were cultured in micromass. From day 3, the cells metamorphosed into round shape. Alcian blue staining at day 6 exhibited positive stain. The intensity of Alcian blue staining showed dose-dependent increase depending on the amount of plasmid used at electroporation . Gene exLiCl was added to the micromass culture of pCAGGS-BMP4 transfected C3H10T1/2 cells. To correct the osmolality of medium, NaCl was added up to 25 mM together with LiCl . Alcian blue staining of micromass at day 6 was inhibited by the addition of LiCl in dose-dependent manner. The supplementation of wnt3a protein also inhibited Alcian blue staining of micromass at the concentration of 50 mM significantly Figures . To testμg/cuvette), pRL-SV40 (20 μg) and pCAGGS-BMP4 (20 μg), was cotransfected by electroporation. After a 3-day culture in micromass with or without LiCl, luciferase activity of cell lysate was assessed. The promoter activity of TOPFLASH was enhanced by addition of LiCl  were analyzed at day 3  by electroporation, cells were cultured in micromass. qRT-PCR at day 3 showed suppression of cathepsin H mRNA expression. Cathepsin H gene silencing slightly increased the expression level of BMP4 by 1.5 ± 0.4-folds  with pCAGGS-BMP4 . Intracellular β-catenin undergoes digestion via ubiquitin proteasome system when the receptor frizzled and coreceptor LRP were not bound with Wnt ligand. The inhibition of GSK-3β blocks this digestion and increases the accumulation of intracellular β-catenin [β-catenin signaling directly inhibits chondrogenesis. In our experiments, Wnt/β-catenin signaling synergistically inhibits chondrogenesis of C3H10T1/2 cells through both the degradation of BMP4 and direct inhibition by Wnt/β-catenin signaling. In the current study, LiCl was used to mimic the action of canonical Wnt/-catenin . LiCl in-catenin . This daβ-catenin signaling may suggest that catalytic processes through cathepsin H are active at a certain level in C3H10T1/2. Although this activity could vary according to types of cells and conditions, the inhibition of cathepsin H has a potential to enhance the effects of BMPs in the gene-based tissue engineering of bone and cartilage. Gene silencing of cathepsin H in BMP4 expressing cells exhibited greater accumulation of BMP4 protein in the absence of LiCl. This result without mimicking Wnt/β-catenin signaling mimicked by LiCl caused discrepancy between mRNA and protein expression of BMP4. Cathepsin H, a cysteine protease which degrades BMP4 mRNA expression, was also upregulated by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 accumulation from BMP4 expressing cells. Wnt/β-catenin signaling may regulate the biological activity of BMP4 through the regulation of cathepsin H. In conclusion, upregulation of canonical wnt/


Aberrant Wnt/β-catenin signaling is associated with breast cancer even though genetic mutations in Wnt signaling components are rare. We have previously demonstrated that Rad6B, an ubiquitin conjugating enzyme, stabilizes β-catenin via polyubiqutin modifications that render β-catenin insensitive to proteasomal degradation. Rad6B is a transcriptional target of β-catenin, creating a positive feedback loop between Rad6B expression and β-catenin activation.high and ZsGreenlow subpopulations, reflective of high and low Rad6B promoter activity, respectively, were isolated by FACS. To determine the relevance of Wnt signaling in Rad6B-mediated β-catenin stabilization/activation, the ZsGreenhigh cells were transfected with signaling-defective Wnt coreceptor LRP6Δ173. Rad6B expression and promoter activity were determined by RT-PCR, Western blot and Rad6B promoter-mediated luciferase assays. β-catenin levels and transcriptional activity were determined by Western blot and TOP/FOP Flash reporter assays. Tumor formation and morphologies of ZsGreenlow, ZsGreenhigh, and ZsGreenhigh/LRP6Δ173 cells compared to unsorted vector controls were evaluated in nude mice. Expression of Wnt signaling related genes was profiled using the Wnt signaling pathway RT2 Profiler PCR arrays.To isolate subpopulations expressing high or low Rad6B levels, we transfected MDA-MB-231 or WS-15 human breast cancer cells with ZsGreen fluorescent reporter vector in which the expression of ZsGreen was placed under the control of Rad6B promoter. ZsGreenhigh subpopulations showed high Rad6B expression and Rad6B promoter activity as compared to ZsGreenlow cells. ZsGreenhigh (high Rad6B expressors) also showed elevated β-catenin levels and TOP/Flash activity. Inhibiting Wnt signaling in the high Rad6B expressors decreased ZsGreen fluorescence, Rad6B gene expression, β-catenin levels and TOP/Flash activity. Tumors derived from high Rad6B expressors were predominantly composed of cells with epithelial mesenchymal transition (EMT) phenotype as compared to control tumors that were composed of both cuboidal and EMT-type cells. Tumors derived from low Rad6B expressors lacked EMT phenotype. Inhibition of LRP6 function in the high Rad6B expressors abrogated the EMT phenotype. Gene expression profiling showed upregulation of several Wnt signaling pathway regulators in high Rad6B expressors that were downregulated by interference of Wnt signaling with mutant LRP6 or by Rad6B silencing.ZsGreenThese data reveal a functional link between the canonical Wnt pathway and Rad6B in β-catenin activation and breast cancer progression. The canonical Wnt signaling pathway regulates several processes including early neoplasia. Activation of the canonical Wnt pathway involves stabilization of β-catenin through the binding of Wnt ligands to the cell surface Frizzled (Fz) family receptors and low density lipoprotein receptor (LDLR)-related protein 5 (LRP5) and LRP6. The major output of this pathway is nuclear translocation of β-catenin, which stimulates expression of β-catenin responsive target genes that promote cell proliferation, differentiation, and invasion -4. In thHere we determined if Rad6B mediated β-catenin stabilization in breast cancer cells requires intact Wnt signaling. Using the human Rad6B promoter to direct expression of ZsGreen reporter protein, we isolated breast cancer subpopulations expressing high and low levels of Rad6B, and demonstrated β-catenin activation in high Rad6B subpopulations. Further, the Rad6B-mediated β-catenin activation requires intact Wnt signaling since disruption of Wnt signaling in high Rad6B expressors with a signaling defective LRP6, decreases β-catenin levels and activity, and Rad6B promoter-directed reporter and Rad6B gene expression. Breast cancer subpopulations selected for high Rad6B produce tumors with the EMT phenotype, which is suppressed by blocking LRP6 function. These data suggest that Rad6B functions downstream of Wnt signaling in β-catenin stabilization and breast cancer progression.MDA-MB-231 (American Type Culture Collection), WS-15 (Cell Core Facility of Karmanos Cancer Institute), pLKO-Rad6BshRNA and empty pLKO WS-15 human breast cancer  cells weNM_003337 forward and reverse primers, respectively [XM_006959] primers. The reaction conditions that yielded a detectable product with the minimum number of cycles were employed: 95°C, 1 min/55°C, 1 min/65°C, 2 min for 21 cycles.Total RNA was prepared from control, R6B-Zsectively . GAPDH e2 Profiler PCR arrays (SuperArray Bioscience) were used to profile the expression of 84 genes related to Wnt signaling. Total RNA was extracted from R6B-Zshigh, R6B-Zshigh/LRP6Δ173, Rad6BshRNA MDA-MB-231 or WS-15 cells and their controls with TRIzol. Single stranded cDNA was synthesized from 2 μg of total RNA by using the SuperArray reaction ready first strand cDNA synthesis kit. The cDNAs were mixed with SuperArray RT2 Real time SYBR Green/ROX PCR master mix and real time PCR performed in accordance with the manufacturer's instructions. Thermal cycling and fluorescence detection were performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems), and expression of Wnt regulated transcripts were compared between the groups.The human Wnt signaling pathway RT6 or 5 × 106 MDA-MB-231 or WS-15 derivatives, respectively, in 0.1 ml serum-free media or Matrigel subcutaneously near the nipple of gland #5 of female nude mice. Xenografts were removed at 50 days and fixed in buffered-formalin. In vivo experiments were approved by the Institutional Animal Care and Use Committee, and conformed to the NIH regulatory standards.Xenografts of MDA-MB-231 or WS-15 derived subpopulations were generated by injecting 1 × 10TUNEL staining was performed as previously described . ApoptotData were analyzed with GraphPad software using either Student's t test or ANOVA. P < 0.05 was considered significant.high) and low (R6B-Zslow) promoter activities were isolated by cell sorting, and expanded for further analysis , but were significantly larger (P = 0.03) than those produced by R6B-Zslow cells. To determine the contribution of Wnt signaling in Rad6B mediated breast cancer progression, R6B-Zshigh/LRP6Δ173 cells were implanted into the mammary fatpads of female nude mice. Inhibition of LRP6 function in Rad6B-overexpressing subpopulations resulted in a significant decrease of tumor growth (P = 0.01) as compared to R6B-Zshigh cells , which was inhibited by ectopic expression of LRP6Δ173 , which was dramatically decreased by expression of mutant LRP6  as compared to vector control or R6B-Zshigh WS-15 cells  or Rad6B silencing (Rad6BshRNA WS-15 cells), confirming our previous report that Rad6B induced β-catenin activation does not involve transcriptional events [To analyze the link between Rad6B and Wnt signaling pathway, we used RTNA WS-15  breast cs Figure . Wnt poss Figure . Interess Figure . Similar Figures  and 6B. l events . LRP5 anhigh) require intact Wnt signaling since disruption of Wnt signaling in R6B-Zshigh cells with a signaling defective LRP6 lacking 173 amino acids in the c-tail [high breast cancer cells induces a reduction in Rad6B promoter activity as evidenced by decrease in Rad6B promoter-mediated ZsGreen and luciferase reporter expressions and Rad6B gene expression.In this study, we provide novel evidence for a functional link between Wnt signaling and Rad6B in β-catenin activation, and the potential value of using Rad6B to target the Wnt/β-catenin pathway in human breast cancer. The data from this paper show that elevated β-catenin levels and activity observed in Rad6B-overexpressing breast cancer subpopulations  and lymph node (2 out of 8) of nude mice. Epithelial to mesenchymal transition plays a critical role in regulating cell migration during neoplastic invasion [high MDA-MB-231 tumors show strong Snail1 expression, which is downregulated by mutant LRP6, whereas R6B-Zslow MDA-MB-231 tumors lack the spindle shaped cells and express low or negligible Snail1. EMT is also frequently associated with loss of E-cadherin [Rad6B is overexpressed in human breast tumors ,24. To dinvasion . The Snainvasion . Wnt siginvasion . R6B-Zshcadherin . Howevercadherin . Thus, ihigh MDA-MB-231 cells as compared to R6B-Zshigh/LRP6Δ173 derived tumors that are growth inhibited and lack the EMT phenotype. These findings are consistent with the data from Liu et al [in vivo assays with WS-15 breast cancer cells further confirm the role of Rad6B in breast cancer progression since R6B-Zshigh WS-15 cells produced very angiogenic carcinomas as compared to controls, whereas Rad6B silencing inhibited progression to carcinoma with resultant benign hyperplastic tumors.The Wnt/β-catenin is frequently activated in human breast cancer. 40-60% of human breast cancers exhibit nuclear/cytoplasmic β-catenin and aberrant activation of the pathway at the receptor level is common ,24,43-45iu et al  that inhhigh cells, the net outcome is one of increased β-catenin activity and tumor progression.Our data from profiling of Wnt related genes show upregulation of several Wnt signaling pathway regulators in Rad6B overexpressing breast cancer cells, which are downregulated either by mutant LRP6 or Rad6B silencing. Wnt ligands are upregulated in subset of human breast cancers ,48. SincThe MDA-MB-231 cell line belongs to a subset of triple negative breast cancers that also express Vimentin . This suTaken together that similar outcomes on β-catenin activity, EMT suppression, or breast cancer progression are achieved by inhibiting LRP6 or Rad6B , illustrThe authors declare that they have no competing interests.BG participated in the generation of vector constructs and acquisition of data; PN participated in the in vivo assays; LT acquired, analyzed and interpreted immunohistochemistry and immunofluorescence data; MS conceived, designed and coordinated the study, acquired, analyzed and interpreted data, and drafted the manuscript. All authors read and approved the final manuscript.This work was funded by grants from the United States Department of Defense W81XWH07-1-0562 and W81XWH-09-1-0608 (MPS).


By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of  The vertebrate eye develops through a series of co-ordinated interactions between tissues of different embryonic origin. The eye field is specified in the anterior neural plate immediately following gastrulation Microphthalmia-associated transcription factor (Mitf) is essential for RPE cell development but is expressed throughout the early optic vesicle where it marks undifferentiated bipotential neuroepithelial precursor cells and following cellular commitment becomes restricted to RPE cells Orthodenticle homolog 2 (Otx2) transcription factor is expressed in the eye field before RPE specification but later becomes restricted to the RPE cells Otx2 is required for Mitf expression and activates genes important for pigmentation in co-operation with Mitf Specification of different cell types in the eye is mediated by a number of key paracrine signalling molecules. Early neural retina specification is mediated by fibroblast growth factor (FGF) emanating from the surface ectoderm in the prospective lens placode leading to expression of the transcription factor Vsx2  in the lateral part of the optic vesicle Bmp4 gene is expressed in the dorsal part of the optic vesicle and is involved in dorsal patterning Tbx5 and Vax2, expressed dorsally and ventrally, respectively During the transformation of the optic vesicle into the optic cup, the opposing actions of bone morphogenetic protein (Bmp) and hedgehog signalling are thought to generate dorsoventral patterning. Hedgehog signalling has been implicated in the specification of ventral structures such as the optic stalk In addition to dorsoventral patterning the neural retina is composed of functionally distinct cell types organised into a laminar structure. The different cellular layers are referred to as follows (from outside-in), the outer nuclear layer, the inner nuclear layer and the ganglion cell layer. Photoreceptor proteins (opsins) are activated by light (photons) in the rods and cones that are in the outer nuclear layer. The inner nuclear layer consists of amacrine cells, bipolar cells and horizontal cells that essentially transfer and modulate information from the outer nuclear layer to the ganglion cell layer. The ganglion cell layer consists of retinal ganglion cells which are projection neurons and convey visual input information from the retina along the optic nerve to the brain Lrp6, a co-receptor of canonical Wnt signalling, causes a defect in dorsal retinal patterning leading to a coloboma β-catenin in early eye progenitor cells prior to cellular commitment in the optic vesicle. This approach revealed that β-catenin is essential for eye development since the optic vesicle is unable to transform into the optic cup. This phenotype is most likely due to that the RPE layer is not specified at the correct time during eye development.β-catenin has been shown to be involved in different aspects of eye development. The β-catenin molecule has two different cellular functions; to regulate cellular adhesion by interacting with cadherins, and to mediate the canonical Wnt signalling pathway The mice were maintained at the animal facility at Umeå University and all experiments involving animals were approved by the Animal Review Board at the Court of Appeal of Northern Norrland in Umeå.Lhx2-Cre transgenic mouse, flox/floxβ-catenin mice (exons 2–6 were flanked by loxp sites) and ROSA26R mice has been described previously β-catenin germ-line null allele was generated by crossing flox/+β-catenin with Zp3-Cre transgenic mice which inactivate genes in the female germ-line flox/floxLhx2-Cre:β-catenin and the flox/Lhx2-Cre:β-catenin− mice are born but die prior to postnatal day 2 (P2) of unknown causes. The genotypes of the control mice in these crosses were; +/Lhx2-Cre:β-catenin−, flox/+Lhx2-Cre:β-catenin, flox/floxβ-catenin, flox/β-catenin−, flox/+β-catenin and +/β-catenin−. Genotyping was performed by PCR analysis of genomic DNA extracted from tail biopsies using the following primer pairs: Lhx2-Cre transgene: Lhx2CreF: 5′-TTCCACAGTCTGTCGGGC-3′ and Lhx2CreR: 5′-CCTGGCGATCCCTGAACATGT-3′. floxβ-catenin and the β-catenin− alleles, IMR1512: 5′-AAGGTAGAGTGATGAAAGTTGTT-3′, IMR1513: 5′-CACCATGTCCTCTGTCTATTC-3′ and RM43: 5′-TACACTATTGAATCACAGGGACTT- 3′. ROSA26R mice, IMR8052: 5′-GCGAAGAGTTTGTCCTCAACC-3′, IMR8545: 5′-AAAGTCGCTCTGAGTTGTTAT-3′ and IMR8546: 5′-GGAGCGGGAGAAATGGATATG-3′. The morning of the vaginal plug was considered as embryonic day 0.5 (E0.5).The in situ hybridisation were fixed between 30 minutes to 2 hours. After fixation the embryos were transferred to 30% (w/v) sucrose in PBS for 24 hours at 4°C, mounted in Tissue-tek (Sakura) and stored at −80°C. Sectioning (8–10 µm) was performed on a cryostat (Microm HM505E) and collected on superfrost plus slides (Menzel-Gläser). In situ hybridisation using DIG labelled probes were performed essentially as previously described β-catenin is inactivated), Lhx2 , BMP4 , Pax6 , Pax2 (IMAGE clone: 40142573), Rx (IMAGE clone: 5366450), Sox2 (IMAGE clone: 6413283), Otx2 , Six6 , Six3 , Vsx2 (IMAGE clone: 6492679), Mitf (IMAGE clone: 40047440), Axin2 , Tbx5 , Raldh1 , Vax2 (IMAGE clone: 40101825), Nr2f1 (IMAGE clone: 6511382), Nr2f2 (IMAGE clone: 6829692), Nrl , Crx , Trβ2 (IMAGE clone: 40057540) and Wnt2b (IMAGE clone: 8734027).Embryos were isolated and fixed in 4% (w/v) paraformaldehyde (PFA) in PBS at 4°C. Embryos used for For hematoxylin-eosin staining, tissue sections were incubated sequentially in the following solutions; Mayer’s hematoxylin solution for 2 minutes, water for 15 minutes, eosin solution for 2 minutes, 95% ethanol for 2×1 minutes, in 99% ethanol for 2×1 minutes and in xylene for 5 minutes. The slides were mounted with DPX mounting media (VWR). To get an approximate value of the relative eye size for the different genotypes, the largest diameter of the control eyes and the mutant optic rudiment at different ages were measured and compared.Immunohistochemistry was performed essentially as previously described + cells (e.g. BrdU+ cell/DAPI+ cell) was counted in comparable areas in the prospective neural retina in control and flox/floxLhx2-Cre:β-catenin embryos. The absolute numbers of activated Casp-3+ cells was counted in prospective neural retina in control and flox/floxLhx2-Cre:β-catenin embryos. The data presented are based on two sections from both optic vesicles in three control and three mutant embryos. Data are presented as mean ± standard deviation (SD). p values were calculated using Student’s t test.Pregnant mice were injected intraperitoneally with 5-bromo-2′-deoxyuridine  at 50 µg/g body weight 1 hour before sacrifice and collection of embryos. Embryos were fixed for 1 hour in 4% (w/v) PFA. After sectioning, the slides were washed in PBS and then denatured in 2 M HCl for 30 minutes at 37°C. The slides were subsequently neutralised with 0,1 M Borate buffer , followed by a standard immunostaining as described above using mouse anti-BrdU . The relative number of BrdUβ-catenin in the early optic vesicle we crossed mouse lines harbouring a null allele or floxed alleles of the β-catenin gene +/β-catenin− or flox/floxβ-catenin mice) to Lhx2-Cre transgenic mice. In the Lhx2-Cre transgenic mouse embryos Cre recombinase is expressed within the first progenitor cells committed to eye development in the anterior neural plate β-catenin inactivation in the early eye and not secondary effects resulting from defects in forebrain development flox/floxLhx2-Cre:β-catenin and flox/Lhx2-Cre:β-catenin− will both result in inactivation of β-catenin in the early eye-committed progenitor cells, but since the severity of their respective phenotypes differ they will be referred to by their full genotype. The phenotype observed in embryos of other possible genotypes generated in these crosses was indistinguishable from wild type and will be referred to as “control mice” .Previous studies have shown that β-catenin is important for maintaining RPE cell identity in the mouse and to indirectly maintain dorsal identity of the zebrafish retina flox/floxLhx2-Cre:β-catenin (n = 30) and flox/Lhx2-Cre:β-catenin− (n = 9) embryos  (data not shown). The optic rudiment was smaller in the flox/Lhx2-Cre:β-catenin− compared with the flox/floxLhx2-Cre:β-catenin embryos at all developmental stages analysed  in the optic vesicle  in the control optic vesicle, we found that expression of Mitf was rapidly down-regulated in the dorsal/distal part of the mutant optic vesicle in the area where β-catenin was inactivated , whose expression domain appeared to replace that of Mitf and Otx2 expression  , a marker for apoptotic cells, in the control and mutant embryos at E10.5. There was a slight increase in the number of cells containing activated Casp-3 cells in the mutant neural retina  compared to control embryos  (p<0.001)  in the prospective neural retina in the control and mutant embryos at a stage when optic cup formation was initiated. At this stage 57% (±6) of the cells in the control embryos and 34% (±4) of the cells in the mutant embryos were BrdU0.00001) , revealip<0.001) . Thus, tIt has been suggested that the transition of the optic vesicle to the optic cup is due to a self-organizing mechanism where the RPE layer imposes a structural constraint on the neural retina leading to an apically convex invagination of the neural retina into the RPE layer Bmp4, Tbx5 and Raldh1 are normally expressed in the dorsal domain of the optic vesicle at E9.5 and in the optic cup at E10.5  have recently been shown to be involved in dorsoventral patterning of the optic vesicle Nr2f1 is preferentially expressed in the ventral part and Nr2f2 is preferentially expressed in the prospective RPE cells in the optic cup  in the cytoskeleton + and β-catenin− (Lhx2-Cre mouse strain was crossed to the ROSA26 reporter (ROSA26R) mouse β-catenin inactivation were derived from the progenitor cells defined by Cre expression and not from a separate progenitor cell population in the anterior neural plate. To distinguish between these two possibilities, we crossed the Lhx2-Cre, flox/floxβ-catenin and ROSA26R mouse lines to generate flox/flox:ROSA26RLhx2-Cre:β-catenin mouse embryos. Since all lineage-traced cells express β-Galactosidase  we analysed the mutant embryos for β-Gal and β-catenin expression. All cells in the mutant optic rudiment, including both the β-catenin+ and the β-catenin− cells, were β-Gal+   to generate β-catenin− cells  . These dn− cells . Further+)     [55] rod+, Crx+)  [56], , [5+, Crx+) . Thus, mrudiment –G, furthβ-catenin gene prior to the cellular commitment into prospective neural retinal cells and RPE cells in the optic vesicle, we have demonstrated that this gene is essential for optic cup formation. β-catenin has at least two different cellular functions; i) activating canonical Wnt signalling by translocation into the nucleus. ii) regulating cell adhesion by binding to the cadherin family of adhesion molecules β-catenin mutant embryos have significantly down-regulated canonical Wnt signalling in the dorsal optic vesicle. This reduction in canonical Wnt signalling in the dorsal optic vesicle causes a severely compromised commitment to RPE cells in the mutant embryos as evidenced by a lack of Mitf and Otx2 expression. However, a small number of RPE cells could develop in the mutant embryos leading to the formation of an optic rudiment. While we demonstrate that the RPE cells are derived from the Cre+ eye committed progenitor cells in the anterior neural plate, we clearly demonstrate that these cells have escaped β-catenin inactivation and are hence β-catenin+. Moreover, the significantly reduced expression levels of dorsal markers led to an almost complete ventralisation of the non-RPE cells in the optic rudiment. Moreover, active Wnt/β-catenin signalling in RPE cells at an early stage of optic vesicle development is evolutionary conserved in fish, chicken and frogs β-catenin mutant embryos is due to the lack of canonical Wnt signalling resulting in blockade of RPE cell commitment in the optic vesicle. RPE cells have been suggested to be specified by the TGFβ family member Activin A which is secreted by the extraocular mesenchyme By inactivating the Mitf or Otx2 are inactivated, or when both Nr2f1 and Nr2f2 are inactivated Mitf and Otx2 enhancers + neural retina domain at the expense of the Mitf+/Otx2+ RPE domain. This change in fate is further supported by the expression of the retina-specific genes Six6, Six3 and Rx that appears to replace expression of the RPE-specific genes Otx2, Wnt2b and Nr2f2. Although delayed, the subsequent transition of the optic vesicle to the optic cup is initiated in the mutant embryos as the optic vesicle invaginates, leading to the formation of a two-layered structure and a simultaneous invagination of the lens placode. At this stage eye morphogenesis is developmentally arrested in the mutant embryos and an optic cup is never formed. This is most likely due to that the mutant optic vesicle is unable to form an RPE layer at the correct time. Thus, the eye phenotype in the flox/Lhx2-Cre:β-catenin− and flox/floxLhx2-Cre:β-catenin embryos we observe here is much more severe compared with the earlier studies on β-catenin inactivation during eye development. The results presented in this study are reminiscent of the eye phenotype observed when the development of RPE cells are severely compromised using another approach In previous studies β-catenin was inactivated during eye development at a stage when the optic cup had been formed + and had thus escaped β-catenin inactivation, emphasising that β-catenin is essential for RPE cell development. It has been suggested that dorsal and ventral RPE cells develop by different mechanisms, and that development of ventral RPE cells might be independent of β-catenin + or β-catenin−. These non-RPE cells did not form an organised retina although various neurons developed suggesting that commitment of neuronal cell types is not solely dependent on β-catenin and tissue organisation. Moreover, our data implies that in the mutant, non-RPE cells are not maintained in the absence of RPE cells, further arguing for a central role of the RPE cells in the development of the eye.Although our data strongly suggests that β-catenin is essential for the formation of the RPE layer, a small number of RPE cells were still present in all optic rudiments analysed in the mutants at later developmental stages. However, all the RPE cells that developed in the mutant were β-cateninWnt2b null mice have no reported eye defects The data presented here suggest that Wnt/β-catenin signalling is important for maintaining dorsal identity of the retina. Previous studies in zebrafish have also suggested that Wnt/β-catenin signalling is important for maintaining dorsal identity of the retina in a cell nonautonomous manner since Wnt/β-catenin signalling is restricted to the RPE cells in zebrafish Lrp6/−− mice do share some of the phenotypes with the β-catenin mutants presented here, but these are less severe. Although the Lrp6/−− mice have microphthalmia with coloboma, they develop the basic structures of the eye: neural retina, RPE cell layer and the lens β-catenin mutants presented in this work. However, in agreement with our data the neural retina in the Lrp6/−− mice lose or down-regulate expression of dorsal markers such as Tbx5, Raldh1 and BMP4 and the expression domain of the ventral marker Vax2 extends dorsally Lrp6/−− mice and hence the phenotype will not be as severe as in the β-catenin mutant which presumably block all canonical Wnt signalling. Other mediators of Wnt signalling that have been studied during eye development are the Fz receptors Fz5 and Fz8. The Fz5/−−+/:Fz8− compound mutants show a severe retinal coloboma and microphthalmia although the major eye structures, neural retina, RPE cells and lens, developed in this mutant β-catenin mutant mice presented in our study.Another mediator of canonical Wnt signalling is the Fz co-receptor Lrp6. In conclusion, β-catenin is important for eye development since it is essential for the transition of the optic vesicle to the optic cup. This defect is due to the fact that commitment of RPE cells depends on β-catenin. Moreover, β-catenin is also important for the dorsoventral patterning of the retina whereas differentiation of the various cells in the retina is independent of β-catenin.Figure S1Maintenance but not induction of dorsal identity of the optic vesicle is dependent on β-catenin. (A–C) In situ hybridization analyses on coronal sections of E9.5 (ss25–26) optic vesicles from control (left panels) and flox/floxLhx2-Cre:β-catenin embryos (right panels). (D–F) In situ hybridization analyses on coronal sections of E10.5 (ss32–34) optic cups from control (left panels) and flox/floxLhx2-Cre:β-catenin embryos (right panels).  In situ hybridization analyses on coronal sections of E9.25 (ss21–23) optic vesicles from control embryos (left panels) and flox/floxLhx2-Cre:β-catenin embryos (right panels). Arrow heads indicate the boundaries where β-catenin has been inactivated in the optic vesicle. Arrows indicate Bmp4 expression. Dorsal-ventral (D–V) orientation for all panels is indicated in A. Scale bars:  100 µm.(TIF)Click here for additional data file.Figure S2β-catenin protein remains associated with N-cadherin and F-actin in the mutant optic vesicle at E10.5. (A–D) Immunohistochemical analyses for cellular localisation of the indicated proteins on coronal sections of E10.5 (ss 33–35) optic vesicles from control embryos (left panels) and flox/floxLhx2-Cre:β-catenin embryos (right panels). (A) and (B) are merged in (C) to show co-localisation (yellow) on the apical side of the cells in the optic vesicle. (D) Serial section following those in (A–C). Note that while the RPE and the neural retina have been specified in the control embryo (left panels), the corresponding structure in the mutant embryo contains almost exclusively cells of neural retinal fate at this developmental stage Click here for additional data file.Figure S3Retinal cell classes can develop independent of β-catenin. (A) Hematoxylin/eosin staining of coronal sections of an E18.5 control embryo (left panel) and an flox/floxLhx2-Cre:β-catenin mutant embryo (right panel). Arrow indicates the optic rudiment that can develop in mutant embryos (right panel).  Immunohistochemical analyses for the presence of the indicated proteins on coronal sections of control (left panels) and mutant embryos (right panels). (C–E) In situ hybridization analyses for gene expression analysis of the indicated genes on coronal sections of control (left panels) and mutant embryos (right panels). All the sections from the mutant embryo have been analysed for β-catenin expression on a consecutive section to ensure that the distribution of β-catenin+ and β-catenin− cells shown in panel (B), is maintained in all panels. Scale bars: (A) 500 µm.  100 µm.(TIF)Click here for additional data file.