The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling. Both the protein kinase A (PKA) and Wnt signaling pathways play important fundamental roles in most tissue types. These signaling cascades play complex roles in differentiation, development, and tumorigenesis, where effects within a specific tissue or developmental stage are highly dependent on context. Proper regulation of these pathways is required for normal formation of both epithelial and mesenchymal tissue, such as bone and muscle The protein kinase A (PKA) pathway has been known to be involved in bone biology since the elucidation of the role of parathyroid hormone (PTH) in bone homeostasis. PTH signals through its 7 transmembrane G protein coupled receptor PTH Receptor 1 (PTHR1) to activate the PKA signaling cascade PRKAR1A, the type 1A regulatory subunit, cause hyperactivation of PKA directly at the level of the PKA holoenzyme in vitro using introduction of activated GNAS1 or shRNA-mediated knockdown of Prkar1a There are also a number of human diseases of bone which are directly related to dysregulated of the PKA signaling pathway. Hyperactivation of the PKA pathway can cause proliferation of undermineralized bone, as can be observed in McCune-Albright Syndrome  and Carney Complex . The former is caused by activating mutations of the stimulatory G protein Gsα, leading to constitutive PKA excess driven through dysregulation of cAMP generation by the mutant G protein β-catenin is a multifunctional protein that serves as a component of the cell-cell adherens junctions as well as a transcriptional regulator of the canonical Wnt signaling pathway Crosstalk between these two pathways has previously been demonstrated by the fact that PKA has been shown to phosphorylate β-catenin in its C-terminus at serines 552 and 675, although the effects of this post-translational modification is unclear Prkar1a as a means to activate PKA signaling In this report, we describe alterations in β-catenin that are observed in bone tumors arising from mice with mutations in All research performed using animals was carried out in accordance with the highest standards of animal care under research protocols (2002-A-0097 and 2008-A-0104) approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC).Primary cultures of mouse tumors and normal bones were prepared and grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum ,10,000 U/liter penicillin G, 10 mg/liter streptomycin, and 1% ascorbic acid (osteoblast medium). as described previously Stable Prkar1a knockdown was achieved as previously described FLAG-tagged β-catenin was obtained from Addgene  and the β-catenin mutants were generated using Site-Directed Mutagenesis . All mutants were sequence verified prior to use. The CFP-PML expression plasmid To measure the Wnt/β-catenin pathway mediated TCF/LEF-1 transcriptional activity, MC3T3-E1 cells were transfected with TCF-luciferase reporter (TOPFlash) or its mutated control reporter (FOPFlash) along with WT or mutants of β-catenin. The luciferase activity was measured using the dual-luciferase assay kit (Promega).Cells were cultured on coverslips, fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Coverslips were incubated with primary antibodies for 2–24 h, washed three times with PBS, and incubated with secondary antibodies for 60 min. Samples were mounted using antifade reagent with or without DAPI  and observed using single-photon Olympus FV1000 confocal microscope. To detect the proteins of interest, we used the following antibodies: phosphoSer133-Creb (pS133-Creb), Creb, pS675-β-catenin, pS552-β-catenin, β-catenin and FLAG were from Cell Signaling Technology; PML was from Millipore; Alexa Fluor conjugated secondary antibodies were from Invitrogen.RNA was isolated from cells using Trizol and RNeasy Mini Columns  and converted to cDNA with the Bio-Rad iScript cDNA SynthesisKit (Bio-Rad Laboratories) according to manufacturer instructions. cDNA was subject to qRT-PCR using the iQ SYBR Green Supermix Kit (Bio-Rad Laboratories) as per the manufacturer's instructions. Reactions were each performed in triplicate. Primers sequences used in this study can be found in Whole-cell lysates were prepared using M-PER protein extraction reagent containing HALT protease inhibitors . For nuclear-cytoplasmic fractionation, cells were lysed in NE-PER extraction reagent (Pierce) and processed according to the manufacturer's instructions. Proteins were resolved by SDS-PAGE gels and transferred to nitrocellulose . Blots were developed with chemiluminescence solution . To detect the proteins of interest, we used the same antibodies described above, with Lamin A and α tubulin antibodies obtained from Santa Cruz Biotechnology  and anti-actin from Sigma.http://www.statcrunch.com). Differences with p<0.05 were considered significant. All values are expressed as the mean ±S.D. of triplicate independent samples.All experiments were repeated at least three times, and all analyses in this manuscript were performed using a two-sided Student's t test, as implemented by StatCrunch . However, analysis of β-catenin localization by immunofluorescence (IF) revealed that β-catenin had undergone an alteration of its subcellular distribution in tumor cells , H2AX (DNA double strand breaks), and PCNA (DNA replication). PML is a scaffold protein forming multiprotein complexes which are thought to represent dynamic structures with many intranuclear functions, including transcriptional activation and repression, protein post-translational modification, and protein storage +/−Prkar1a tumor cells, IF analysis demonstrated that β-catenin exhibited strong co-localization with PML in tumor cells  for 3 or 6 hours. As shown in To verify that β-catenin also underwent relocalization in response to PKA signaling in this model, we analyzed its subcellular localization after treatment as above. As In order to better characterize the role of the PKA phosphorylation events in β-catenin's localization, we took advantage of phospho-specific antibodies that can detect the S675 and S552 phosphorylation events. Using immunofluorescence/confocal microscopy, we examined the subcellular distribution of pS675-β-catenin in response to FSK treatment. We observed that untreated cells had low levels of pSer675-β-catenin, but that the amount significantly increased after FSK; furthermore, the phosphoprotein was observed almost exclusively in the nucleus . EffortsConsistently, western blot analysis after cell fractionation detected an increase in both pS675- and pS552- β-catenin in the nuclei of cells treated with forskolin. Concurrent assessment of cytoplasmic levels of phospho-β-catenin demonstrated no change . To ensuPrkar1a. These cells, which we have previously shown to have elevated PKA activity To test whether the results obtained in forskolin treated cells could be reproduced by other means of PKA activation, we also utilized MC3T3-E1 cells carrying a siRNA-mediated knockdown (KD) of To determine if PKA phosphorylation sites within β-catenin are required for its nuclear translocation, we introduced mutations at S552 and/or S675 into FLAG-tagged β-catenin. We generated serine-to-alanine  mutants to model non-phosphorylatable β-catenin, as well as serine-aspartate  mutants to mimic the phosphorylated isoforms. Use of S>A and S>D mutants produced similar results, suggesting that the S>D mutants did not mimic the inducible phosphorylation of the protein. A similar phenomenon has been observed in the transcription factor CREB, where the S133D mutant behaves similarly to the S133A mutant As confirmation, we obtained YFP-tagged β-catenin and introduced the same phosphosite mutants into this construct. Cells were co-transfected with CFP-tagged PML and co-localization were analyzed after treatment with DMSO or with FSK. Behavior of the fluorescently tagged proteins recapitulated that observed for the native proteins  confirmiPrkar1a knockdown Prkar1a knockdown cells, which have increased PKA activity, showed higher TOPFlash reporter activity compared to the control cells.To understand the functional significance of PKA-dependent relocalization of β-catenin to PML bodies, we first examined how PKA activation affects β-catenin transcriptional activity. We found that forskolin stimulated a significant induction of the Wnt/β-catenin-dependent reporter TOPFlash without altering the control reporter FOPFlash in MC3T3-E1 cells. When cells were stimulated with the canonical Wnt ligand Wnt3a, levels of transcription increased approximately 75-fold, and PKA activation was able to provide an additional increase in transcriptional activity . To ruleCcnd1 (Cyclin D1) and Dkk1 (Dickkopf 1) were increased, whereas the mRNA level for Osteocalcin was decreased in cells treated with forskolin . Use of the β-catenin mutants S552A or S675A showed similar effects, with both basal and FSK-stimulated levels not significantly different from the WT construct. However, mutation of both sites significantly decreased basal (unstimulated) activity, and also reduced the activity of the reporter after FSK induction. Although there was still a statistically significant increase in luciferase activity (p = 0.0.013), the induction was only ∼1.4-fold. Taken together, these results indicated that PKA promotes β-catenin-dependent transcription, at least in part, through phosphorylation of β-catenin at S552 or S675.To test if PKA-Wnt crosstalk flows in both directions, we transfected MC3T3-E1 cells with a cAMP responsive element-luciferase construct and stimulated the cells with vehicle, forskolin or Wnt3a. As shown in +/−Prkar1a bone tumors To assess the functional interaction of PKA and β-catenin targets, we turned to data we had previously generated on mRNA transcripts altered in To predict potential interactions between CREB and TCF/β-catenin in co-regulating gene transcription, we analyzed the promoters of this gene set for CREB or TCF/β-catenin binding sites. For this analysis, the promoter sequences (−5000 bp upstream to +2000 bp downstream of known Transcription Start Site) were retrieved from the MPromDb database Prkar1a knockdown; and Ror2 mRNA expression was also reduced in cells treated with forskolin or with Prkar1a knockdown. Therefore, these data indicate that suppression of Wnt5a/Ror2 pathway occurs concurrently with activation of the Wnt/β-catenin signaling caused by PKA activation.Because it has been shown that non-canonical Wnt signaling mediated by Wnt5a/Ror2 interactions can inhibit canonical Wnt/β-catenin signaling at the level of TCF-mediated transcription +/−Prakr1a mice or the well-established MC3T3-E1 cell line. Our data indicates that in this system, β-catenin activation occurs without an overall increase in levels of this protein. However, dissection of the mechanism by which this occurred revealed the striking finding that β-catenin undergoes PKA- dependent relocalization to nuclear PML bodies. We propose that localization at this intranuclear structure enables β-catenin to interact with other transcriptional components (including phosphoCREB) and enables enhanced β-catenin-dependent transcription.Crosstalk between the PKA and Wnt/β-catenin signaling pathways has been known since the mid 2000's, when it was reported that β-catenin was a substrate for PKA PML is a multifunctional protein with multiple splice isoforms that play roles in a variety of intranuclear functions, including DNA damage response, apoptosis, senescence, and transcriptional activation. One of the roles of PML is to affect gene transcription, and the studies described here indicate that the ability to modulate transcription of the luciferase reporter construct is strongly correlated with the ability to bind PML in the nucleus. Unlike prior studies, we found that mutation of one of the PKA sites within β-catenin was insufficient to suppress its transcriptional activity or its entry into a PML complex. However, when both PKA sites (S552 and S675) were mutated, both abilities were lost.in situ crosslinking experiments would not identify a complex between and S552A/S675A-β-catenin and PML because these proteins are not localized within the same cellular compartment. These experiments, however, have not been performed.In terms of a mechanism, the most likely explanation for these observations is that PKA-mediated phosphorylation of β-catenin promotes its transport into the nucleus, where it is then available to bind to PML and PML-associated complexes. PKA also may potentiate intranuclear β-catenin by interfering with the ability of nuclear APC to chaperone it out of the nucleus Alternative explanations might include β-catenin binding to other proteins in the PML complexes, and that these collaborative binding interactions are disrupted by the S>A mutations.+/−Prkar1a mice also suggest the cooperation of the cAMP-responsive transcription factor CREB and β-catenin, as binding sites for CREB and TCF strongly tended to occur together in the promoters of genes with altered transcription in the tumors. As TCF sites were more commonly observed in isolation in gene promoters whose transcripts were reduced in tumors, we propose that TCF/β-catenin primarily acts as a transcription suppressor in osteoblasts, whereas CREB primarily promotes gene transcription. However, when both TCF and CREB sites are found (which presumably happens in the context of a PML body), transcriptional effects are much more variable. Interestingly, one of the actions of recruitment of β-catenin to PML bodies appears to be a suppression of non-canonical Wnt signaling including Wnt5a and Ror2. Although we have not formally investigated whether suppression of Wnt/Ror2 signaling causes enhancement of the Wnt/β-catenin pathway, this association has been reported in other systems Analysis of the genes altered in bone tumors from in vivo and in vitro approach to study the interaction of PKA with Wnt/β-catenin signaling. We make the surprising observation that PKA drives β-catenin into the nucleus, where it binds to PML bodies to exert transcriptional effects on Wnt/β-catenin target genes. There is also secondary inhibition of competing non-canonical Wnt/Ror2 signaling. Although some of our data confirms prior studies, the fact that this data was generated in a more physiologic system in a tissue type where both PKA and Wnt signaling are known to play significant roles has enabled us to shed new light on intranuclear β-catenin function and add important information to the understanding of the modes of crosstalk between these central signaling cascades.Overall, we have used a combined Figure S1Leptomycin does not altered β-catenin localization. WT osteoblasts were treated with the nuclear export inhibitor Leptomycin B with or without Forskolin (FSK) and probed for β-catenin localization by immunofluorescence. Nuclei are counterstained with DAPI. Note that FSK induced nuclear accumulation of β-catenin but that leptomycin B did not produce further alterations. Scale bar: 10 µm.(TIF)Click here for additional data file.Figure S2PKA activation promotes β-catenin to co-localize with PML bodies. A. Primary osteoblasts were co-transfected with WT or mutant YFP-tagged β-catenin and CFP-PML and treated with forskolin. Proteins were visualized by immunofluorescence confocal microscopy and counted to determine nuclear localization of β-catenin as well as co-localization with PML. B. Quantitation of data on transfected cells. At least 100 transfected cells were counted in each assay. Note that the S>A and S>D mutants behave in the same fashion, and that these results closely parallel those shown in (TIF)Click here for additional data file.Figure S3β-catenin co-immunoprecipiates with PML. A. MC3T3-E1 pre-osteoblasts were treated with Forkolin (Fsk) or vehicle (Ctl) and subject to immuneprecipitation (IP) with primary antibodies against β-catenin or PML. After SDS-PAGE, the blots were probed with anti-β-catenin or anti-PML, as indicated. The arrows at the left of each panel indicate the respective proteins. Note that Fsk does not alter the interaction between β-catenin (94 kDa) and PML (78/97 kDa). The vertical white line in the right panel indicates that different exposure times for this blot are shown for the two images. B. MC3T3-E1 cells were transfected with CFP-PML and Flag-tagged β-catenin (WT or S552A/S675A double mutant) and treated with vehicle (Ctl) or Fsk. Lysates were immunoprecipitated with anti-Flag or anti-CFP and immunoblotted with the other antibody as indicated. As in panel (A), no differences were detected irrespective of the use of the β-catenin mutants or Fsk treatment.(TIF)Click here for additional data file.Table S1Primer sequences for real-time PCR of mouse RNA targets.(DOCX)Click here for additional data file.


Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the normal adrenal (NA) samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 with intracellular β-catenin accumulation showed β-catenin mutations. Thus, genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation. Adrenocortical (AC) tumors can be classified as either a benign AC adenoma or AC carcinoma. The former is a common disorder of the adrenal glands with a prevalence of 3-10% of the general population, whereas AC carcinomas are extremely rare, with an estimated annual incidence of 1-2 cases per 1 million individuals in the United States .2-terminal residues, in which a glycogen synthase kinase 3β (GSK-3β) consensus motif is present, with the aid of a scaffolding complex composed of axin and adenomatous polyposis proteins (APCs). Subsequently, phosphorylated β-catenin is subject to degradation by the ubiquitin-proteasome system. Under activation of Wnt signaling with the cooperation of Wnt proteins bound to their frizzled transmembrane receptors, the functional loss of GSK-3β is a common event, namely, failure to phosphorylate β-catenin. Thus, non-phosphorylated forms of β-catenin can accumulate in the cytoplasm, enter the nucleus, and finally activate the Wnt target genes c-myc and cyclin D1 as a transcriptional activator with the aid of TCF/LEF family proteins [The canonical Wnt/β-catenin pathway has been shown to play an important role in organ development, while dysregulation of Wnt signaling has been implicated as causative factor in some genitourinary malignancy cases -8. The cproteins -11.Active involvement of β-catenin has been postulated in the pathogenesis of AC tumors by a number of gene profiling studies -16. TheyThe secreted frizzled-related protein (sFRP), known to be a Wnt antagonist, inhibits the Wnt signaling pathway by direct binding to Wnt molecules instead of frizzled molecules . In a reWnt inhibitory factor-1 (Wif-1), a sFRP family protein initially identified in the human retina , has beeA representative Wif-1 immunostaining pattern is shown in Figure β-catenin gene was found in only 2 of the 20 AC tumors that showed intracellular β-catenin accumulation , and unmethylation-specific PCR (USP), as well as typical bisulfite DNA sequencing of the NA samples and AC tumors in relation to primer location are shown in Figure Representative MSP and USP bands are shown in Figure The central constituent of Wnt signaling is β-catenin, thus the relationships of β-catenin with other Wnt-related genes were evaluated. In the total series, samples with intracellular β-catenin accumulation showed lower levels of Wif-1 mRNA transcription as compared to those with negative accumulation (p<0.05) Fig. . LikewisWnts are a family of secreted glycoproteins that regulate a wide variety of biological processes, including embryonic development, cell proliferation, cell migration, tumor suppression, and oncogenesis. Previous studies have shown that activation of Wnt signaling can promote the pathogenesis of various human cancers, including AC tumor development through a canonical pathway, which is also called the Wnt/β-catenin signaling pathway -16, 21. Among AC tumors, it has been reported that approximately 24-40% of AC adenomas and 30-80% of AC carcinomas have abnormal intracellular accumulation of β-catenin -16. In tActivation of a β-catenin exon 3 mutation is one of the most frequent genetic alterations seen in association with activated Wnt signaling. However, the majority of AC tumors are known to accumulate intracellular β-catenin despite the lack of genetic mutations of β-catenin -16. IndeWif-1 gene expression in AC tumors. Furthermore, we found an inverse correlation between mRNA transcription levels of β-catenin and Wif-1 in AC tumors, which indicates that intracellular β-catenin accumulation accelerating Wnt signaling is related to epigenetics-associated down-regulation of Wif-1, but not to posttranslational modification of β-catenin. Therefore, it is quite possible that epigenetics-related functional impairment of Wif-1 may lead to constitutive β-catenin activation and subsequent adrenal oncogenesis via cyclin D1 activation.Dysregulation of Wnt antagonists such as Wif-1 comprises an alternative mechanism underlying aberrant activation of the Wnt signaling pathway. In bladder and kidney cancer, epigenetic promoter CpG hypermethylation of Wif-1 is attributable to its down-regulation , 20. In As for the other Wnt target genes of c-myc, there were no significant differences in regard to c-myc mRNA transcription between the AC tumors and NA samples (data not shown). Previous studies have suggested that these tumors might represent a unique class characterized by low c-myc expression , 25, whiRecent studies of gene regulation at the posttranslational level have investigated microRNAs, which contribute to oncogenesis in various carcinomas via activation of the Wnt signaling pathway -29. In pA major limitation of the present study is the relatively small number of specimens examined, especially AC carcinomas. Dysregulation of the canonical Wnt/β-catenin signaling pathway may be a minimum requirement in the initial step of adrenal tumorigenesis, though that appears to be insufficient for development of malignant transformation . Other gWif-1 gene via epigenetic pathways in AC tumors. In addition to genetic alterations of β-catenin, the epigenetics of promoter CpG hypermethylation of Wif-1 may be an important mechanism associated with the initial process of adrenal oncogenesis via aberrant canonical Wnt/β-catenin signaling activation. Although further research is required to verify our findings, our report is expected to contribute to a better understanding of the pathogenesis of AC tumors.In conclusion, this is the first clinical study of inactivation of the Tissue samples were obtained from 42 patients with various types of AC lesions, including 39 AC adenomas  and 3 AC carcinomas. Clinical and pathological features are described in Table Genomic DNA and total RNA were isolated from all formalin-fixed paraffin-embedded AC sample tissues using a QIAamp Tissue kit  and RecoverAll™ Total Nucleic Acid Isolation kit , respectively. RNA pellets obtained after isopropanol and ethanol precipitation were dried, re-suspended in 25 μL of RNase-free water, and stored in aliquots at -80°C until reverse transcription was performed. The concentrations of DNA and RNA were determined with a spectrophotometer, and the integrity of those measurements was checked by gel electrophoresis.Using 1 μg of RNA, 0.5 μg of oligo-dT primer, and 0.5 units of RNase inhibitor, cDNA was constructed using reverse transcriptase (Promega). The mRNA transcript levels of Wif-1, β-catenin, c-myc, and cyclin D1 were measured by a StepOne RT-PCR system (Applied BioSystem), with GAPDH utilized as the reference. The reaction protocol recommended by the manufacturer (Applied BioSystem) was strictly followed. For each run, a standard curve was generated using a serial dilution of the external standard. The expression level was calculated as the ratio of the target gene to that of GAPDH.http://rsb.info.nih.gov/ij).Immunostaining of Wif-1, β-catenin, and cyclin D1 was performed using 5-μm-thick consecutive sections obtained from paraffin-embedded materials. Slides were prepared with antigen retrieval using citrate buffer , then incubated for 12 hours with mouse monoclonal antibody for Wif-1 , β-catenin , and cyclin D1 . For a negative control, the primary antibody was replaced with non-immune serum. DAB (Sigma-Aldrich) was used as the chromogen and counterstaining was performed with hematoxylin. For β-catenin expression, cytoplasmic/nuclear staining was considered to indicate a positive reaction and staining intensity was not scored, according to a previously reported method . For Wifβ-catenin gene encompassing the region for phosphorylation sites, which is considered to be the hotspot of β-catenin mutation. The primer sequences and PCR conditions are shown in Table. The coding region of exon 3 of β-catenin was analyzed, using a previously reported technique . Brieflyhttp://itsa.ucsf.edu/~urolab/methprimer). As shown in Table Genomic DNA (100 ng) was modified with sodium bisulfite using a commercial kit . Based on the functional promoter sequence of the Wif1 gene , methylaAll data were analyzed using the StatView V statistical package . Statistical analysis was done using the Mann-Whitney test. Non-parametric data were analyzed by a chi-square test. Correlations between 2 continuous variables were analyzed by Spearman rank correlation. A P value of less than 0.05 was considered to be statistically significant.


Tgm2−/− MEFs) display increased and accelerated lipid accumulation due to increased expression of major adipogenic transcription factors, PPARγ and C/EBPα. Examination of Pref-1/Dlk1, an early negative regulator of adipogenesis, showed that the Pref-1/Dlk1 protein was completely absent in Tgm2−/− MEFs during early differentiation. Similarly, Tgm2−/− MEFs displayed defective canonical Wnt/β-catenin signaling with reduced β-catenin nuclear translocation. TG2 deficiency also resulted in reduced ROCK kinase activity, actin stress fiber formation and increased Akt phosphorylation in MEFs, but did not alter fibronectin matrix levels or solubility. TG2 protein levels were unaltered during adipogenic differentiation, and was found predominantly in the extracellular compartment of MEFs and mouse WAT. Addition of exogenous TG2 to Tgm2+/+ and Tgm2−/− MEFs significantly inhibited lipid accumulation, reduced expression of PPARγ and C/EBPα, promoted the nuclear accumulation of β-catenin, and recovered Pref-1/Dlk1 protein levels. Our study identifies TG2 as a novel negative regulator of adipogenesis.Differentiation of preadipocytes to lipid storing adipocytes involves extracellular signaling pathways, matrix remodeling and cytoskeletal changes. A number of factors have been implicated in maintaining the preadipocyte state and preventing their differentiation to adipocytes. We have previously reported that a multifunctional and protein crosslinking enzyme, transglutaminase 2 (TG2) is present in white adipose tissue. In this study, we have investigated TG2 function during adipocyte differentiation. We show that TG2 deficient mouse embryonic fibroblasts ( This expansion initially occurs via an increase in the size of existing adipocytes (hypertrophy) which is followed by an increase in adipocyte number via proliferation of preadipocytes (hyperplasia) and their differentiation into mature adipocytes (adipogenesis).3 Impaired adipogenesis and adipose tissue function are associated with the development of metabolic complications in obesity, such as the development of type 2 diabetes.5The prevalence of obesity is steadily increasing globally and is recognized as a major risk factor for diabetes, heart disease and certain cancers.7 which is associated with remodeling of extracellular matrix (ECM) fibronectin (FN) matrix to laminin-rich matrix. Adipogenesis is regulated by various factors that can either promote or inhibit adipogenesis. Many of these factors regulate ECM components and cytoskeletal tension, some of the factors or proteins which maintain preadipocyte state and act as inhibitors during early phase of adipogenesis include Wnt/β-catenin signaling, Pref-1/Dlk1, RhoA and ROCK kinases. These factors are amongst those which determine whether preadipocytes will be in quiescence, or undergo proliferation and differentiate to adipocytes.9Adipogenesis involves conversion of spindle-shaped preadipocytes to round lipid filled adipocytes, this morphological change requires conversion of filamentous actin to cortical actin10 TGs are enzymes with ability to form isopeptide bonds between glutamine residue of one protein to a lysine residue of another protein by transamidation reaction.13 TGs can also have functions that do not involve their transamidase activity.11 In our recent work, we have shown that FXIII-A is responsible for the transamidase/crosslinking activity during adipocyte differentiation. In 3T3-L1 adipocyte and mouse embryonic fibroblasts (MEFs) cultures, FXIII-A crosslinking activity increased plasma FN assembly into preadipocyte matrix which promoted preadipocyte proliferation. Inhibition of TG activity of FXIII-A in these cultures resulted in increased adipocyte differentiation.10 The role of TG2 in adipogenesis remained unaddressed.In our previous work we have identified two members of transglutaminase (TG) enzyme family, Factor XIII-A (FXIII-A) and transglutaminase 2 (TG2), in white adipose tissue (WAT).16 TG2 is expressed by many cell types such as osteoblasts,17 chondrocytes,19 mesenchymal stem cells (MSCs),20 neuronal and glial cells,21 phagocytes, monocytes, neutrophils and T-cells23 and pancreatic β-cells.24 TG2 has been implicated in various biological functions including cell differentiation and maturation, cell morphology and adhesion, ECM stabilization, cell death, inflammation, cell migration and wound healing.13 TG2 is present in both extracellular and intracellular compartments of the cell. In the extracellular compartment, TG2 can be found on the cell surface and in the ECM. In the intracellular compartment, TG2 is mostly cytosolic but also found on the plasma membrane, in the nuclear membrane and in mitochondria.25 Dysregulation of TG2 function(s) has been implicated in pathogenesis of celiac disease,26 diabetes,24 neurodegenerative disorders such as Huntington's, Alzheimers's and Parkinson's disease21 as well as inflammatory disorders and cancer.11TG2 is the most ubiquitous of the TG family members and expressed in many tissues such as bone, cartilage, kidney, colon, liver, heart, lung, spleen, blood and nervous tissue.Tgm2+/+) and TG2 deficient mice (Tgm2−/−) to address the potential role of TG2 during adipocyte differentiation. We report that TG2 deficiency results in accelerated and increased adipogenesis in MEFs due to increased expression of adipogenic transcription factors PPARγ and C/EBPα. We further examined the role of TG2 in several anti-adipogenic pathways and demonstrate that TG2 regulates adipogenesis via multiple factors – these include Pref-1/Dlk1 expression and modulation of Wnt/β-catenin signaling, ROCK kinase activity and Akt signaling.In this study, we have used MEFs from TG2 wild-type pentylamine, showed no significant change in Tgm2−/− MEFs compared with Tgm2+/+ MEFs during early adipocyte differentiation, suggesting that the lack of TG-activity is not the cause of inhibitory effects seen in Tgm2−/− MEFs  was added to −/− MEFs . Lipid a−/− MEFs ,B. The t−/− MEFs , but sig−/− MEFs . Pref-1 −/− MEFs . However−/− MEFs . These r34 and the role of ROCK as an inhibitor of adipogenesis35 we analyzed ROCK activity during early differentiation. Data shows a moderate but significant downregulation of ROCK activity in Tgm2−/− MEFs on days 1 and 2 , and their differentiation into mature adipocytes (adipogenesis).To see how an increase in adipogenesis in +/+ mice . The inc10 In this study, we have examined the role of TG2 in adipogenesis and report for the first time that TG2 also acts as an inhibitor of adipocyte differentiation. We show that Tgm2−/− MEFs display accelerated and enhanced adipogenesis which is associated with downregulation of multiple anti-adipogenic signaling pathways that jointly lead to increased expression and activation of master transcription factors of adipogenesis, Pparγ and Cebpα, and increased lipid accumulation in the cells. The pathways identified in this study are; regulation of Pref-1 protein levels, β-catenin signaling and modulation of ROCK-mediated cytoskeletal tension and Akt signaling.Our previous work identified two members of TG family, FXIII-A and TG2, in WAT and demonstrated that FXIII-A can regulate adipocyte proliferation via promoting plasma FN matrix assembly which inhibits adipogenesis.11 Our report for the first time show that in MEFs Tgm2 deficiency dramatically reduces Pref-1 protein expression. Pref-1 is an major inhibitor of adipocyte differentiation and whose expression is highest during early differentiation and then gradually disappears as the cells differentiate into adipocytes.27 Pref-1 protein mediates its effects on adipocyte differentiation by directly binding to FN, which activates integrin signaling to engage the MAPK/ERK pathway. This induces Sox9 expression which inhibits adipocyte differentiation.40Pref-1 knockout mice have increased adipose tissue mass, pre- and postnatal growth retardation and skeletal abnormalities41 and conversely Pref-1 overexpressing mice have reduced adipose tissue mass, impaired glucose tolerance and reduced insulin sensitivity.43 Other effects of Pref-1 protein are also mediated by Sox9 which promotes chondrogenic commitment of MSCs, but inhibits chondrocyte maturation and osteoblast differentiation.44 Linked with Pref-1 function in chondrocyte maturation, TG2 has been shown to regulate the transition into the prehypertrophic stage during chondrocyte maturation. Premature, forced expression of TG2 accelerated progression toward prehypertrophy and it was shown that extracellular TG2 can increase Sox9 expression.19 Based on our work, it is highly possible that the effects of TG2 on chondrocytes may also be mediated via Pref-1. TG2 is also expressed by osteoblasts where it is located on the cell surface.17 TG2 knockout mice do not show any chondrogenic or osteogenic abnormalities during development or postnatally,45 which is likely due to compensatory function from upregulation of FXIII-A and TGFβ1.46 It is possible that TG2, jointly with other TG enzymes and Pref-1 may act as an upstream regulators of mesenchymal stem cell differentiation into the different lineages, particularly into chondrocytes, osteoblasts and adipocytes.The identification of TG2 in as an anti-adipogenic factor is not entirely unexpected as TG2 has been implicated in regulation of number of pathways in multiple location in cells.32 During early phase of adipogenesis PPARγ suppresses Wnt signaling by increasing β-catenin degradation and PPARγ upregulation coincides with decreased total and nuclear β-catenin levels, suggesting a reciprocal relation between Wnt and PPARγ.49 Here we report that Tgm2−/− cells display increase in Pparγ and Cebpα mRNA expression and reduced β-catenin nuclear accumulation during the early phase of adipogenesis. When exogenous TG2 was added, a significant decrease in lipid accumulation was seen and this was associated with an increase in nuclear accumulation of β-catenin as well as decreased Pparγ and Cebpα mRNA expression. Interestingly, Pref-1 was shown to be a Wnt target gene and was, in fact, shown to be downregulated by TCF/β-catenin complex in fetal lung epithelial cells and MEFs.52 Furthermore, Pref-1 can act as a noncanonical Notch ligand and inhibit Notch signaling and, in turn, Wnt/β-catenin signaling is negatively regulated by Notch.53 This crosstalk between Notch and Wnt signaling may be one of the regulatory mechanism for Pref-1 production. We are currently exploring the mechanisms how TG2 affects the Pref-1 protein regulation.In addition to Pref-1 we reported here that TG2 regulates β-catenin nuclear translocation in preadipocytes. Canonical Wnt signaling is a crucial pathway that regulates lineage determination of MSCs. In preadipocytes, Wnt signaling maintains preadipocytes in undifferentiated state by inhibiting PPARγ and C/EBPα.54 Canonical Wnt signaling can also inhibit adipogenesis in lineage committed preadipocytes.32 Furthermore, canonical Wnt receptor LRP6 knockout MEFs show increased adipogenesis.55 In smooth muscle cells, extracellular TG2 regulates canonical Wnt signaling and β-catenin nuclear translocation which promotes calcification of the cell culture system. This effect in smooth muscle cells is mediated by extracellular TG2 binding to LRP5/6 on the smooth muscle cell surface and this reported interaction does not require transamidation activity.30 In this study, we show that extracellular TG2 activates canonical Wnt signaling contributing to the inhibitory effect on adipogenesis. While we did not address the exact mechanism how exogenous extracellular TG2 here regulates β-catenin nuclear translocation, it is plausible that the mechanism is the same as in smooth muscle cells. It is also possible that the exogenous TG2 promotes β-catenin release from the plasma membrane E-cadherin to the cytosol and from there to nucleus. This concept is supported by the observation that exogenous TG2 addition caused an increase in cytosolic and nuclear pools of β-catenin without affecting the total β-catenin levels in protein extracts.It is well documented that canonical Wnt signaling inhibits adipogenesis and promotes osteogenesis in MSCs.Tgm2-deficient MEFs have decreased ROCK kinase activity and decreased actin stress fibers, which also contributes to increased adipogenesis. Adipogenesis is characterized by change in cell shape, from spindle-shaped preadipocytes to round adipocytes and this transition is partly determined by the cytoskeletal tension. During adipogenesis filamentous actin from stress fibers is rearranged to cortical pattern and down regulation of ROCK kinase disrupts actin stress fibers.56 Inhibition of ROCK kinase was shown to promote adipogenesis and Akt signaling57 and conversely activating ROCK kinase inhibits adipogenesis.56 TG2 was reported to activate ROCK via two pathways - retinoic acid-induced TG2 enzymatic activity was reported to activate ROCK kinase by intracellular TG258 and cell surface TG2 was reported to amplify integrin mediated signaling to activate ROCK kinase in a non-enzymatic manner.34 ECM quantity and quality is a major regulator of cytoskeleton59 and it is known that cell surface TG2 cooperates with α5β1 integrins to enhance FN-integrin binding which is required for FN assembly. Furthermore, TG2 has been suggested to stabilize ECM in a number of studies and factors such as TGFβ—an inhibitor of adipogenesis—was shown to increase cell surface expression of TG2 and increase FN assembly.60 FN matrix itself is a major inhibitor of adipogenesis and must be decreased for the preadipocytes to allow differentiation toward mature adipocytes. However, in our study, we show that the absence of TG2 does not affect FN matrix levels or solubility in preadipocytes which strongly suggests that TG2 is not involved in FN matrix assembly and that cytoskeletal alterations in Tgm2−/− MEFs are not mediated by ECM itself, but likely via cell surface TG2 and the manner cells adhere to ECM. Indeed, Tgm2−/− deficient fibroblasts have been demonstrated to have an adhesion defect.62 Moreover, the data strongly suggest that TG2 is not involved in MEF matrix assembly and does not appear to participate in MEF extracellular transamidation/crosslinking events. This is also supported by the fact that Tgm2−/− and Tgm2+/+ MEFs had similar levels of TG activity – this activity likely deriving from FXIII-A. Interestingly, both TG2 and FXIII-A act as negative regulators of adipogenesis and thus they may have a complementary effect on adipogenesis.Consistent with previous work on the role of TG2 in maintaining cytoskeletal tension, we also report here that 3 Defects in this expansion are linked to obesity-linked comorbidities such as development of type 2 diabetes.5 In this work, we have identified a new factor, TG2 that maintains preadipocyte state and thus acts as a negative regulator of adipogenesis. It is thus likely that regulation of TG2 in preadipocytes is tightly controlled to balance proliferation and differentiation. Further understanding of TG2 and its role and regulation in metabolic disorders would aid the development of new therapies to maintain healthy energy metabolism.Increased fat mass in obesity is associated with an increase in adipocyte cell size and/or adipocyte number which are reactions to expand adipose tissue upon need to increase energy storage.Tgm2−/− mice were described before.45 Wild-type (WT) mice were purchased from Jackson Laboratories . Mice were kept under a normal diurnal cycle in a temperature-controlled room and fed with standard chow. Animal procedures (WAT extraction and MEF isolation) and study protocols were approved by the McGill University Animal Care Committee.473)(D9E), rabbit anti-PPARγ, rabbit anti-histone, rabbit anti-Pref-1 were purchased from Cell Signaling Technology Inc . Rabbit anti-actin, mouse anti-tubulin antibodies were obtained from Sigma-Aldrich . Rabbit anti-fibronectin antibody, human recombinant MYPT1 (654–880) and rabbit anti-phospho-MYPT1 (Thr696) were from EMD Millipore . Rabbit anti-C/EBPα purchased from Santa Cruz Biotechnology . Rabbit anti-β-catenin purchased from Abcam . Mouse monoclonal TG2 Ab-3 antibody (Clones CUB 7402+TG100) was from Fisher Scientific . Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Cell Signaling Technology Inc. Horseradish peroxidase-conjugated goat anti-rabbit IgM was purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated anti-mouse, and anti-rabbit IgG were from Jackson ImmunoResearch Inc. Alexa Fluor 488 and 596, Alexa Fluor 568-phalloidin and Bodipy 493/503 were from Life Technologies .Antibodies against rabbit anti-Akt (pan), rabbit anti- phospho-Akt (SerDulbecco's modified Eagle's medium (DMEM) and 0.5 mg/ml trypsin and 0.2 mg/ml EDTA from ATCC . Fetal bovine serum (FBS) and penicillin-streptomycin were from Gibco . Oil Red O, IGEPAL CA-630, dexamethasone, insulin, 3-Isobutyl-1-methylxanthine (IBMX), 3,3′,5,5′-Tetramethylbenzidine (TMB) were from Sigma-Aldrich. Troglitazone were purchased from Santa Cruz Biotechnology. Sulfo-NHS-LC-biotin and 5-(biotinamido)pentylamine were purchased from Pierce . ECL kit was from Zmtech Scientifique . All other reagents unless otherwise specified were purchased from Sigma-Aldrich or Fisher Scientific.Tgm2+/+ and Tgm2−/− mouse embryos, MEFs isolation, culture and staining with Oil Red O was done according to previously published protocol.10 MEFs were differentiated into adipocytes with 10% FBS, 1μM dexamethasone, 0.5 mM isobutyl-1-methylzanthine, 1 μg/ml insulin, and 10μM troglitazone for 2 days. On day 2, media were replaced with maintenance medium which includes 10% FBS and 1 μg/ml insulin and 10μM troglitazone. On day 4, maintenance media was replaced with medium containing 10% FBS and cells were cultured in this until the end of the experiment, that is, day 8. Intracellular triglyceride was stained with Oil Red O and quantified; cells were counter stained with haematoxylin and photographed with a light microscope.Mouse embryonic fibroblasts (MEFs) were prepared from 13.5 days Tgm2+/+ mice was fixed in 10% neutral-buffered formalin. Fixed tissue was cut with a scalpel to 5 mm × 5 mm sections, and blocked with 3% BSA, 0.3% Triton X-100, in PBS for 12-24 h at 4 °C. Tissue pieces were incubated with primary antibodies overnight at 4 °C which was followed by incubation with Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI. Antibody omission and isotype specific immunoglobulins were used as controls.10 For histology, mouse epididymal fat pad from Tgm2+/+ and Tgm2−/− mice were fixed in 10% neutral-buffered formalin and paraffin embedded and stained with eosin and hematoxylin.10 For immunofluorescence, cells were grown in 8-well Nunc Lab-Tek II glass chamber slides (Fisher Scientific). Cells were fixed with 10% neutral-buffered formalin for 30 min at room temperature or with 1% formaldehyde for 15 min at room temperature . Staining was done as previously described.10 Quantification was done with Image J .For whole-mount staining, mouse WAT from g at 4 °C. Nuclear and cytosolic fractions were prepared as previously described.63 Deoxycholate (DOC)-soluble and DOC-insoluble FN matrix extracts were prepared as described previously.64 Western blotting and quantification of bands with Image J  was done as previously described.10Total cell lysate was prepared with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% DOC, 0.5% Triton X-100, 1 mM PMSF and 1 mM orthovanadate and protease inhibitor cocktail (Sigma). Cell lysates were incubated on ice for 30 min with occasional vortexing and then centrifuged for 15 min at 15,000xTgm2+/+ MEFs as previously described.10Cell surface biotinylation was done for Pref-1,65 EDA, EDB,66Fn, Gapdh,17Pparγ2 and Cebpα.67 Real-time PCR was performed on a ABIHT7900 RT-PCR machine using the comparative CT method in triplicate using the TaqMan Universal Master Mix II. Expression levels of Pparγ2  and normalized to Rn18S (Mm 03928990_g1).mRNA was isolated using Trizol method. RNA was treated with DNase , and PCR was performed with SuperScriptIII One-Step RT-PCR System with Platinum Taq DNA Polymerase . PCR products were analyzed by 1.5% agarose gel electrophoresis. Primers used were previously described In situ transglutaminase activity assay was done by giving 2 mM 5-(biotinamido)pentylamine to the cells during differentiation. At the indicated time point, total cell extracts were prepared with 50 mM Tris-HCl (pH 8.0), 135 mM NaCl, and 1% Triton X-100, 1 mM EDTA, 1 mM sodium orthovanadate and EDTA-free protease inhibitor cocktail. To see the basal level of TG activity under these conditions, biotinamidopentylamine (BPA) was added to the cultures on day -1 . The total cell lysate was extracted on day 0, that is, 24 hr after the BPA was added, but before adipogenic treatment was started. The value obtained by microplate TG-activity assay from cell lysate without BPA was subtracted from the value of cell lysate containing BPA, and the resulting value was considered TG-activity on day 0. Microplate assay to detect biotin was done as previously described.10in vitro kinase assay as previously described.10 Cell lysate was prepared with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM 2-glycerophosphate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4 and EDTA-free protease inhibitors cocktail.ROCK kinase activity was determined by using microplate P-values are as follows: *P>0.05, **P>0.01, ***P>0.001.All values are expressed as standard error of the mean (SEM) of three independent experiments. Statistical significance was assessed by student's T-test.


The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling. The blocked membranes were then incubated with a mouse specific anti-SIRT1 antibody , or a rabbit polyclonal anti-PPARγ , or a rabbit polyclonal anti-FABP4 , or a rabbit monoclonal anti-adiponectin , α-Tubulin , and β-actin  for overnight at 4 °C. Secondary antibodies were used according to the manufacturer’s instructions. Secondary-antibody binding was detected using an enhanced chemiluminescence detection kit  according to the manufacturer’s instructions. Protein levels were normalized to β-actin using Image J analysis software.Western blotting was performed as previously described. Whole-cell protein lysates were extracted with a solution containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 % Triton X-100, 10 mM Na4 cells/well 24 h before transfection. The cells were transiently transfected with plasmids at 80 % confluence using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The DNA/reagent ratio was 1 μg/2 μL. After 24 h transfection, the cells were harvested for subsequent analysis. The RNA interfering (RNAi) plasmid pSIREN-RetroQ-ZsGreen (pSIREN-SIRT1) (siSIRT1) 5′-GATGAAGTTGACCTCCTCA-3′, synthesized according to the literature . pCDNA The TOPflash plasmid (Millipore) was used to monitor the Wnt/β-catenin signaling. The TOP/FOP Flash assays were performed according to the manufacturer’s instructions. The cells were treated as indicated, and luciferase activity was measured with the Dual-Luciferase reporter assay system (Promega).Results are presented as the means ± standard errors. Statistical analysis was performed with SAS. Version 8 . Data were analyzed by Duncan’s multiple-range test was performed if differences were identified among the groups at P < 0.05.


Key signalling pathways, such as canonical Wnt/β-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates β-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/β-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated β-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts. Highlighted article: Dual ChIP-seq and RNA-seq in vivo experiments show that the context-specific events that occur subsequent to β-catenin binding enable gene-specific regulation, rather than β-catenin recruitment per se. Key signalling mechanisms are deployed repeatedly during embryonic development to regulate differential gene expression, often in combination with each other and with other regulatory mechanisms. Wnt/β-catenin signalling  is an important, evolutionarily conserved cell-to-cell signalling mechanism that regulates the transcription of specific target genes : maternally activated Wnt signalling before the general onset of zygotic transcription at the mid-blastula transition (MBT)   regulateon (MBT) ; but, onon (MBT) ; yet, boon (MBT) . This rabed e.g. ; howeverXenopus embryo shows β-catenin occupancy at many genomic loci, our analysis reveals that transcriptional expression is Wnt regulated at only a subset of these loci. Thus, Wnt-regulated β-catenin recruitment to gene loci is required, but not sufficient, for Wnt target gene expression. We find instead that the tissue- and stage-specific context can regulate Wnt target gene expression subsequent to β-catenin recruitment to cis-regulatory modules at these loci.Here, we report genome-wide identification of such stage-specific Wnt target genes through β-catenin chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) combined with RNA sequencing (RNA-seq) analysis of the relevant Wnt-regulated transcriptome. Although the early Xenopus tropicalis embryos would identify early gastrula-specific Wnt target genes.Nuclear localisation of β-catenin is the hallmark of canonical Wnt signalling . In the Fig. S2) by optimising first chromatin shearing conditions for fragments of ∼200 bp , then the immunoprecipitation of chromatin-associated β-catenin protein with two different β-catenin antibodies, as well as IgG as a negative control . Specific binding of β-catenin by the antibodies was validated by western blotting and also by β-catenin ChIP-qPCR . In the ChIP-qPCR validation, we analysed known WREs in genes known to be Wnt regulated at this stage . Heat mn system . We carrX. tropicalis genome JGI 4.2 with gsnap. Aligned reads were counted using HTSeq  or a derivative vector pβ-actin-luc carrying a heterologous basal promoter. Embryos were injected with 40 pg reporter plasmid DNA together with 40 pg pRL-CMV (Promega) at the two- to four-cell stage, collected at the early gastrula stage, and assayed for luciferase activity. For cloning into luciferase reporter constructs, see the


Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p = 0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased ( Metastasis is a highly selective process conditioned by the genetic profile of the original tumor as well as by organ environment. The susceptibility of the brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. Reports indicate that specific set of genes mediate metastasis to the brain according to discrete changes ,2.etc. [The work presented in this paper focused on the analysis of different expression levels of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), beta-catenin (CTNNB1) and E-cadherin (CDH1) in brain metastases that originated from primary lung carcinomas. All the molecules studied are key players in the classical Wnt signaling pathway which has today been established as one of the basic cellular pathways and whose misregulation plays an important role in tumorigenesis. Initiation of the Wnt signaling cascade depends on the presence of Wnts, glycoproteins that bind to receptors called frizzled. Thus, activated, the receptors recruit Dishevelleds at cytoplasmic membrane and this interaction also pulls axin from the beta-catenin destruction complex to the plasma membrane. In this fashion the complex is inactivated [etc. ,7,8,9,10etc. ,5,11. HoThe cellular function of human Dishevelled genes has not been completely elucidated yet. Nevertheless, Dishevelleds (DSH or DVL) are considered to be the central hub of Wnt signaling and are all multifunctional phosphoproteins that have been shown to shuttle between the cytoplasm and the nucleus ,13,14. TOf particular interest is a process very much involved in invasion and metastasis of tumors—the so called Epithelial-to-Mesenchymal Transition (EMT), where noninvasive tumor cells acquire motility and ultimately disseminate to places distant from the primary site. It has been shown that the Wnt signaling has a particularly tight link with EMT. For example, the nuclear translocation of beta-catenin can induce EMT ,20,21 byBesides being the main signaling effector molecule of the pathway, beta-catenin is also involved in the cellular architecture. It is bound to E-cadherin and is an essential component of adherens junctions. In this respect, it is important to remember that the most prominent feature of EMT is the destruction of adherens junctions and the loss of expression of the cell-cell adhesion molecule E-cadherin.Our hypothesis is that molecular components of the Wnt pathway, Dishevelleds, E-cadherin and beta-catenin, play important roles in metastasis of lung cancer to the brain. We believe that their altered levels of expression contribute to the outline of molecular profile of distant brain metastases.DVL1 and DVL3 showed over expression in brain metastasis tissues in 87.1% and 90.3% of samples respectively. The staining was observed mainly in the cytoplasm and was diffusely or granularly distributed. Nevertheless, we also observed nuclear staining of DVL1 in 54.8% of cases and DVL3 in 53.3% of cases.2 = 9.375; df = 8; p = 0.312). In contrast to this, the expression levels of DVL3 were statistically significantly different between the same pathological diagnoses .At the level of the complete brain metastasis sample, our analyses showed that there were 12.9% of samples demonstrating weak expression of DVL1; 45.2% moderate expression and 41.9% strong expression. Considering the expression levels of DVL3 in our total sample we observed 9.7% of samples with weak expression; 51.6% with moderate expression and 38.7% with strong expression. The normal levels of expression in healthy tissues were very weak. The expression levels of the two proteins in metastases, according to the starting points of the specific types of primary lung cancers are shown in p = 0.856 for DVL1; p = 0.310 for DVL3).Normal levels of E-cadherin staining were scored as +++, and the protein was located along the cell membrane or inside the cytoplasm. The investigation on E-cadherin showed that overall 80% of metastases had down-regulation of E-cadherin. Intense down-regulation was noticed in 52% of cases. The majority of samples (88.9%) with E-cadherin’s gross deletions (LOH) were accompanied with down-regulation of E-cadherin protein. All metastases originated from small cell lung cancer (SCLC), adenocarcinoma and carcinosarcoma showed E-cadherin down-regulation, while in large cell carcinoma and squamous cell carcinomas E-cadherin was down-regulated in 75% and 60% cases respectively. The expression levels of Dishevelleds were not associated to E-cadherin down-regulation (p = 0.0001). The same trend was noticed when DVL3 was up-regulated .Our next step was to detect whether the expression and cellular localization of beta-catenin, correlated with the expression levels of Dishevelleds and E-cadherin. We noticed that beta-catenin was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. It is interesting to note that 40.9% of cases with up-regulated DVL1 expression had beta-catenin transferred to the nucleus. The percentage of metastases with up-regulated DVL3 that also exhibited nuclear staining of beta-catenin was 32%. When DVL1 was up-regulated the number of cases with nuclear beta-catenin significantly increased , but no correlation between nuclear DVL3 and beta-catenin (p = 0.261).Approximately 25% of metastases showed simultaneous nuclear localization of Dishevelleds and beta-catenin. Statistical analysis showed a significant correlation between the nuclear localization of beta-catenin and DVL1  gene when assessed with 3 markers. The highest frequency of genetic changes was observed in metastases originating from primary sites with the diagnosis of lung adenocarcinoma and SCLC with 83.3% and 75% of changes, respectively, while squamous cell carcinoma (SCC) exhibited 25%, and large cell carcinoma (LCC) 12.5% of LOHs. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and SCLC were significantly associated to E-cadherin genetic changes with χ2 = 10.364; df = 1; p = 0.001. A type of genomic instability—microsatellite instability (MSI) was detected in one metastasis originated from adenocarcinoma. The results of heteroduplex analysis of exon 3 did not show samples with additional bands which indicated that mutational hot-spot of beta-catenin, was not targeted in our metastasis sample.The results of our genetic analysis indicate a total of 36% of the sample with LOH of the et al. [Our results on DVL1, DVL3, beta-catenin and E-cadherin bring novel insights on the Wnt signaling role in brain metastases that originated from primary lung carcinomas. We investigated the combination of Wnt key molecules in our collected tumor sample because Wnt signaling is involved in EMT and metastasis formation . The reset al.  report oet al. [Li et al.  discoveret al. [et al. [et al. [The findings that our investigation builds on are brought by Zhao et al.  and Wei  [et al.  who foun [et al.  found poet al. [et al.’s [Wei et al.  showed tet al.’s  data on et al. [DVL1 gene are involved in breast carcinogenesis, especially in the acceleration of tumor growth. The involvement of DVLs in invasive ductal carcinoma of the breast was also reported by Prasad et al. [et al. [et al. [et al. [DVL genes may play important role in the development of a portion of human cancers through derangement of the Wnt signaling pathway.So far it has been reported that DVLs are over expressed in various tumors types. The results from Nagahata et al.  suggest d et al. . Mizutan [et al.  got simi [et al.  showed t [et al.  demonstrAlthough reports indicate DVLs location in the nucleus is only sporadic , our stuet al. [et al. [The majority of metastases had down-regulation of E-cadherin expression with intense down-regulation in 52% of cases. Similar results were reported by McDonald et al.  who also [et al.  demonstr [et al. ,42,43 co [et al. , and in  [et al. . In 2 in [et al. .et al. [Recent studies  also shoet al.  indicatep = 0.0001). Additionally, when DVL3 was up-regulated, there was a significant number of cases with beta-catenin nuclear staining (p = 0.0001). A study by Guldur et al. [et al. [Nuclear location of beta-catenin is an indicator of its acquisition of oncogenic activity. In our study, beta-catenin was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. It is interesting to note that when DVL1 was up-regulated the number of cases with nuclear beta-catenin significantly increased . Annealing of mutant DNA to wild type probe gives duplexes with one or more mismatched bases (heteroduplexes). Mismatching causes the double helix to take on a conformation which retards its mobility during electrophoresis [CTNNB1 gene are not associated to brain metastasis process. This finding is not unusual since many investigations [APC, AXIN2 or beta-catenin are rarely seen in lung cancer [et al. [In heteroduplex analysis, the conformational properties of the double stranded molecules are used to distinguish different base pairing  metastasis lesions were found in different cerebral regions, most frequently in parietal and frontal regions and cerebellum, with the surrounding zone of perifocal oedema. During the operative procedure the metastasis was maximally reduced using a microneurosurgical technique. The patients had no family history of brain tumors and did not receive radio- or chemotherapy before metastasis excision. All metastases were studied by pathologists and classified according to the WHO criteria . There wTwenty-two patients were male, and 9 were female. The age of patients varied from 46 to 81 . The mean age at diagnosis for males was 59.95, and for females 61.4 years.The local Ethical Committee approved our study, grant 1.2.1.19., and patients gave their informed consent.2O2. Non-specific binding was blocked by incubating samples with goat serum for 30 min at 4 °C. Next, the primary antibodies were applied for 30 min at room temperature. The antibodies used for Dishevelled protein detection were: polyclonal rabbit anti-human DVL1 (diluted 1:50), Abcam, Cambridge, UK and monoclonal mouse anti-human DVL3 (diluted 1:50), Santa Cruz Biotehnology, Dallas, TX, USA. For E-cadherin protein detection monoclonal mouse anti-human E-cadherin NCH-38 (diluted 1:100), and for beta-catenin monoclonal mouse anti-human antibody (diluted 1:200), both Dako Corporation, Carpinteria, CA, USA, were used.Immunohistochemistry was performed in order to establish the levels of expression and cellular localization of Dishevelled-1, Dishevelled-3, beta-catenin and E-cadherin proteins. The samples were fixed in formalin, embedded in paraffin, sliced into 4-μm thick sections, and then fixed onto capillary gap microscope slides . Sections were immunostained using streptavidin horseradish peroxidase/DAB  . Briefly, sections were deparaffinized and rehydrated and then microwaved twice for 3 min at 700 W in citrate buffer and once for 4 min at 350 W to unmask epitopes. To block endogenous peroxidase activity, cells were fixed in methanol with 3% HSlides were then washed three times in phosphate-buffered saline (PBS)/goat serum, and secondary LINK antibody was applied for 1 h at 4 °C. Slides were again washed three times in PBS/goat serum and were incubated with substrate chromogen solution  for 30 s.Negative controls underwent the same staining procedure but without incubating samples with the primary antibodies. The frontal cortex of a normal brain, normal human placenta and normal bronchial epithelia served as positive controls. Antibody labeling was analyzed by three independent and blinded observers using an Olympus BH-2 microscope. No expression or very weak expression was labeled as 0/+, moderate expression as ++, and strong expression as +++. Two hundred cells of each sample were analyzed.g). The pellet was then processed as for DNA extraction from the tissue samples.For the genomic DNA extraction approximately 0.5 g of metastasis tissue was homogenized with 1 mL extraction buffer  and incubated with proteinase K . Phenol chloroform extraction and ethanol precipitation followed. Blood was used to extract leukocyte DNA. Five milliliter of blood was lysed with 7 mL distilled water and centrifuged (15 min/5000× CDH1 (E-cadherin) gene was used to test loss of heterozygosity (LOH) in brain metastases. Allelic loss at the CDH1 locus was assessed using 3 highly polymorphic microsatellite markers—D16S265, D16S398 and D16S752, which map to 16q21–22.1 [2, 2.5 mM of each dNTP, 0.5 U Taq . PCR conditions for D16S265, D16S398: initial denaturation 1 min/95 °C; denaturation, 30 s/95 °C; annealing, 1 min/55 °C; extension, 30 s/72 °C; final extension, 72 °C/7 min; 30 cycles. PCR conditions for D16S752: initial denaturation 3 min/96 °C; denaturation, 30 s/96 °C; annealing, 35 s/55 °C; extension, 30 + 1 s/72 °C; final extension, 72 °C/10 min; 30 cycles.PCR amplification of microsatellite markers for the q21–22.1 . D16S265Primers for D16S752: 5'-AATTGACGGTATATCTATCTGTCTG-3'; and 5'-GATTGGAGGAGGGTGATTCT-3'; for D16S265: 5'-CCAGACATGGCAGTCTCTA-3' and 5'-AGTCCTCTGTGCACTTTGT-3'; and for D16S398: 5'-CTTGCTCTTTCTAAACTCCA-3' and 5'-GAAACCAAGTGGGTTAGGGTC-3'.PCR products were analyzed on 2% agarose gels, length of the D16S752 repeat was 102–126 bp, length of the D16S265 repeat was 89–117 bp, and length of the D16S398 repeat was 180–196 bp.CDH1 gene. The samples were electrophoresed on Spreadex gels EL 300 in the SEA 2000 submarine electrophoresis apparatus  at 120 V. Temperature of the running buffer was kept constant at 55 °C. The samples were stained with SyberGold . All the PCR experiments were repeated twice and the LOHs were confirmed. The samples with LOHs were additionally electrophoresed on 15% polyacrylamide gels stained with silver.Absence or significant decrease in the intensity of one polymorphic allele in metastasis compared to the heterozygous autologous blood sample was considered as LOH of MSI is a type of genomic instability indicating impaired cellular mismatch repair. Samples demonstrating MSI have bands on different positions in comparison to bands of autologous blood tissue due to a defect in the replication/repair machinery in tumor cells.CTNNB1 (beta-catenin) gene was screened for mutations. Heteroduplexes were formed by heating 3 µL of PCR products  at 95 °C for 3 min, followed by incubation on ice for 20 min. About 3 µL of each sample was mixed with 7 µL of mixture of formamide and 10 mM NaOH (1:100) prior to loading to a gel. The electrophoresis was performed on the GMA  gels in the SEA 2000 submarine electrophoresis apparatus . The temperature of the running buffer was kept constant at 9 °C.Exon 3 of the CDH1 genetic changes, E-cadherin, Dishevelled 1, 3 and beta-catenin protein expression levels and localizations. Differences in the frequencies of the analyzed features were tested with the Pearson χ2 test employing Yates correction when appropriate and McNamar’s test. All statistical evaluations were performed using the SPSS statistical package, version 14.0 .All individuals were analyzed for the following features: pathohistological diagnosis (PHD) status, sex, age, Our findings suggest that altered expressions of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases from lung cancer which indicates that Wnt signaling may be essential for the progression of lung cancer.


Importantly, upregulating miR-1207 promoted, while silencing miR-1207 inhibited, the tumorigenicity of ovarian cancer cells. Hence, our results suggest that miR-1207 plays a vital role in promoting the cancer stem cell-like phenotype in ovarian cancer and might represent a potential target for anti-ovarian cancer therapy.Wnt/β-catenin signaling pathway is strictly controlled by multiple negative regulators. However, how tumor cells override the negative regulatory effects to maintain constitutive activation of Wnt/β-catenin signaling, which is commonly observed in various cancers, remains puzzling. In current study, we reported that overexpression of miR-1207 in ovarian cancer activated Wnt/β-catenin signaling by directly targeting and suppressing secreted Frizzled-related protein 1 (SFRP1), AXIN2 and inhibitor of β-catenin and TCF-4 (ICAT), which are vital negative regulators of the Wnt/β-catenin pathway. We found that the expression of miR-1207 was ubiquitously upregulated in both ovarian cancer tissues and cells, which inversely correlated with patient overall survival. Furthermore, overexpression of miR-1207 enhanced, while silencing miR-1207 reduced, stem cell-like traits of ovarian cancer cells  Ovarian cancer, one of the most lethal gynecologic malignancies, constitutes 4% of all cancers diagnosed in women, with 6.6 new cases per 100,000 women occurring worldwide each year . The 5-yThe Wnt/β-catenin signaling pathway, one of the most relevant pathways involved with CSCs, has been reported to be aberrantly activated in various types of cancers, including ovarian cancer –13. PrevIn the canonical Wnt/β-catenin signaling pathway, when ligand Wnt engages to the Frizzled receptor (Fz) and the low-density lipoprotein receptor-related protein-5/6 (LRP-5/6), Dishevelled (Dvl) is phosphorylated and consequently releases β-catenin from the destruction complex, formed by Axin, adenomatous polyposis coli (APC), casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK-3β) , 17. TheOn the other hand, there are multiple levels of negative modulators involved in the Wnt/β-catenin signaling pathway for fine tuning the signaling. , 20. ForMicroRNAs (miRNAs), endogenous non-coding small RNAs, can simultaneously interact with multiple targets at the post-transcriptional level . In the By analyzing published miRNA expression profiles obtained from 183 patients of ovarian cancer (E-MTAB-1067), we found that miR-1207 was significantly upregulated in 183 ovarian cancer tissues compared to eight normal human ovarian surface epithelial (HOSE) cell lines Figure . FurtherP < 0.001), lymph node metastasis (P < 0.001) in patients with ovarian cancer  mice . As shows Figure . Importad Figure . These rbona fide targets of miR-1207.By employing publicly available algorithms (TargetScan6.2 and miRanda), we found that three negative regulators of Wnt/β-catenin signaling, including SFRP1, AXIN2 and ICAT, might be miR-1207 targeting genes Figure . WesternSince Wnt/β-catenin signaling pathway has been demonstrated to play important roles in the ovarian cancer progression and regulation of ovarian CSC-like traits , we therSilencing SFRP1, AXIN2, and ICAT potently antagonized the inhibitory effect of miR-1207-mediated Wnt/ β-catenin activity , demonstr = −0.74, P < 0.05), AXIN2  and ICAT  but was strongly associated with nuclear β-catenin   was purchased from ScienCell Research Laboratories and cultured in ovarian epithelial cell medium. The ovarian cancer cell lines, including COV362, SKOV3, OV56, A2780, EFO-21, TOV-21G, TOV-112D and OV90 were grown in the DMEM medium  supplemented with 10% fetal bovine serum .A total of 140 paraffin-embedded and archived ovarian cancer samples, which were histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center from 2001 to 2006, were examined in this study. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained for the use of these clinical materials for research purposes. Clinical information on the samples is summarized in The miR-1207 gene was PCR-amplified from genomic DNA and cloned into a pMSCV- puro retroviral vector. The miR-1207 anti-sense was cloned into miRZip plasmid purchased from System Biosciences and used according to previous report.  pMSCV-miCloning miR-1207: 5′- GCCAGATCTTGATTG ACTTACAG CCCAGTT-3′ and 5′-GCCGAA TTCCA CCTGTCTTTATTCCACCC-3′; Cloning SFRP1- 3′UTR-luci: 5′-GCCCCGCGG CTTGCCCTAACAACTCA-3′ and 5′-GCCCTGCAGTTT CCT TTGGGCGTTG-3′; Cloning AXIN2–3′UTR-luci: 5′-GCCCCGCGGATGTTTG CCGTGAGG A-3′ and 5′-GCCCTGCAGCCTGGGTCC ACATATTTAC-3′; Cloning ICAT-3′UTR-luci: 5′-G CCCC GCGGC GTTTATAGTTGAATTGG-3′ and 5′-GCCCTGC AGTTATTGGTACTAAAGCC T-3′. For depletion of SFRP1, AXIN2 and ICAT siRNA was synthesized and purified by RIBOBIO Company .4) were seeded in triplicate in 24-well plates. 24-hours later, indicated luciferase reporter plasmids plus 3 ng pRL-TK Renilla plasmid were transfected into the cells using Lipofectamine 2000 Reagent . 48 hours after transfection, Dual Luciferase Reporter Assay  was performed according to the manufacturer's instructions.Cells ; anti-AXIN2 , anti-ICAT ; anti-β-catenin . To control sample loading, the blotting membranes were stripped and re-probed with an anti–α-tubulin or anti-p84 antibody . Nuclear extracts were prepared using the Nuclear Extraction Kit (Active Motif), according to the manufacturer's instructions. Briefly, cells were collected in the PBS/phosphatase inhibitors solution and lysed in the Lysis Buffer, and then centrifuged for 5 minutes. Suspending nuclear pellet in 50 ul Complete Lysis Buffer, then incubating for 30 minutes on ice. Vortex the mixture for 30 seconds followed by centrifuge for 10 minutes at 14,000 g at 4°C, the collected nuclear extraction were store at −80°C for further examination. The expression levels of SFRP1, AXIN2, ICAT and β-catenin were determined by densitometry. The ratio of first sample  was considered as 1.0.n = 5 per group). Cells  were inoculated subcutaneously together with Matrigel  into the inguinal folds of the nude mice. Tumor volume was determined using an external caliper and calculated using the equation (L × W2)/2. The mice were sacrificed 35 days after inoculation and the tumors were excised and subjected to pathologic examination.All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University. The 6-week-old BALB/c-nu mice were randomly divided into three groups  supplemented with 2% B27 , 20 ng/ml EGF, 20 ng/ml bFGF, and 5 μg/ml insulin .6 cells/mL in DMEM containing 2% FBS, and then incubated at 37°C for 30 minutes with or without 100 μM verapamil (Sigma-Aldrich) to inhibit ATP-binding cassette (ABC) transporters. The cells were subsequently incubated for 90 minutes at 37°C with 5 μg/mL Hoechst 33342 (Sigma-Aldrich). Lastly, cells were incubated on ice for 10 minutes and washed with ice-cold phosphate-buffered saline (PBS) prior to flow cytometry analysis. Flow cytometric analysis or flow cytometric cell sorting was conducted using fluorescein isothiocyanate (FITC)-conjugated monocloncal mouse anti-human CD133 . Samples were analyzed and sorted on BD FACSCanto II and FACSAria I, respectively  with data analyzed using FlowJo software .Cells were dissociated with trypsin and resuspended at 1 × 10http://www.targetscan.org/); and miRanda (http://www.microrna.org/microrna/getGeneForm.do).The following on-line software programs were used for bioinformatics analysis: TargetScan 6.2 (t-test. In all cases, P < 0.05 was considered statistically significant.All statistical analyses were carried out using SPSS 18.0 statistical software. The Kaplan-Meier method was used to establish survival curves, and the survival differences were compared using the log-rank test. Continuous data were compared using Student's 2-tailed


CTNNB1 or APC. We showed that the quinazoline leads suppressed Wnt/β-catenin signaling without altering the level of β-catenin protein in colorectal cancer cells, suggesting that they act on the downstream elements of the pathway. Moreover, the quinazoline leads displayed potent anticancer activities with IC50 values between 4.9 and 17.4 μM in colorectal cancer cells. Importantly, we also found that a structurally related quinazoline lacking inhibitory effect on Wnt/β-catenin signaling was unable to suppress colorectal cancer cell proliferation. Together, these results suggest that the quinazoline lead compounds identified in this study have therapeutic potential for the prevention and treatment of colorectal cancer.The Wnt/β-catenin signaling pathway is critical for the initiation and progression of most colon cancers, and has emerged as one of the most promising targets for colorectal cancer chemoprevention and treatment. In this study, we have discovered a structurally related series of quinazolines as potent inhibitors of Wnt/β-catenin signaling in colorectal cancer cells harboring mutations in  CTNNB1 (β-catenin encoding gene) are present in approximately 10% of the colorectal cancers. The consequence of either APC inactivation or CTNNB1 activation mutations is the failure of proper β-catenin degradation leading to its cytosolic accumulation. The β-catenin then translocates into the nucleus where it interacts with T-cell factor/lymphoid enhancing factor (TCF/LEF) to induce the expression of downstream target genes [Wnt/β-catenin signaling is important for cancer progression, including tumor initiation, tumor growth, cell death, and metastasis -5. β-Catet genes , 7. It iet genes -8, and tet genes , 9-11.Among heterocyclic compounds, libraries of compounds possessing the quinazoline template in particular have attracted considerable attention in lead discovery efforts. Contributing factors for their desirability include: (a) facile synthetic access, (b) lead-like and drug-like attributes and (c) opportunities that the template provides for molecular manipulations for lead optimizations. The introduction of quinazolines such as lapatinib and gefitinib as kinase inhibitory drugs for cancer treatment exemplifies recent successes in optimization of quinazoline lead compounds into drugs -15.CTNNB1 or APC. Although a few quinazoline analogs have been reported in the literature as inhibitors of the Wnt/β-catenin signaling pathway [50 values between 4.9 and 17.4 μM for colorectal cancer cells, and the IC50 values are comparable to their inhibitory activities on Wnt/β-catenin signaling in colorectal cancer cells.In our efforts to discover novel inhibitors of Wnt/β-catenin signaling, we have identified a structurally related series of quinazolines as potent inhibitors in colorectal cancer cells harboring mutations in  pathway -19, the CTNNB1 mutation) and SW480 cells (harboring an APC mutation)  Figure , and dis50 values between 5.9 and 6.7 μM for HCT116, SW480 and SW620 cells  can translocate to the cell nucleus to activate the transcription factors of the TCF/LEF family, leading to the transcription of Wnt target genes. To identify the site of action of the quinazoline leads in Wnt/β-catenin signaling, we determined the β-catenin levels after the treatment with the lead compounds. It was found that the levels of cytosolic β-catenin and total cellular β-catenin were not significantly changed in response to treatment with the six quinazoline leads at 20 μM for 24 h Figure  or SRI-3Compound SRI-20039 is a 2-phenylquinazoline derivative that was ineffective in blocking Wnt/β-catenin signaling in colorectal cancer cells. As a negative control, SRI-20039 displayed very weak activity against colorectal cancer cell proliferation Figure . These rAPC and CTNNB1. Recent studies suggest that such targeting of downstream signaling could provide therapeutic advantages over targeting upstream signaling components [Aberrant activation of the Wnt/β-catenin signaling pathway is a necessary initiating event in the genesis of most colorectal cancers -8. Accummponents , 31.CTNNB1 or APC, suggesting that they act on the downstream elements of the pathway. Importantly, we also found that a structurally related quinazoline lacking inhibitory effect on Wnt/β-catenin signaling was unable to suppress colorectal cancer cell proliferation, suggesting that the anticancer activity of the quinazoline lead compounds is associated with their inhibitory effect on Wnt/β-catenin signaling. However, additional studies are needed to dissect molecular mechanisms underlying the quinazoline-mediated inhibition of Wnt/β-catenin signaling in detail.Among heterocyclic compounds, libraries of compounds possessing the quinazoline template in particular have attracted considerable attention in lead discovery efforts -15. In tIn summary, we have discovered a structurally related series of quinazolines as potent inhibitors of Wnt/β-catenin signaling in colorectal cancer cells. Of interest is the fact that structure–activity relationships (SAR) are discernible in the profile of this initial set of compounds, thus providing an opportunity to follow-up the SAR trend in a systematic way to enhance potency, selectivity, and drug-like characteristics in the future. The Wnt/β-catenin pathway is critical to uncontrolled cell proliferation in a number of tumor types, particularly colorectal cancer. Therefore, the quinazoline leads identified in the current effort are potentially promising candidates for development as novel chemotherapeutic agents for colorectal cancer.All of the test compounds were synthesized at Southern Research Institute. Details of the chemical synthesis will be published elsewhere. Plasmid pGST-E-cadherin was provided by Dr. Gail Johnson . The Super8XTOPFlash luciferase construct was provided by Dr. Randall T. Moon . The β-galactosidase-expressing vector was obtained from Promega. Rabbit monoclonal anti-axin2 (#2151) was purchased from Cell Signaling Technology. Rabbit polyclonal anti-cyclin D1 (#04-221) was from EMD Millipore. Mouse monoclonal anti-survivin (D-8) (sc-17779) was from Santa Cruz Biotechnology. Mouse monoclonal anti-β-catenin (#61054) was from BD Biosciences. Mouse monoclonal anti-actin (#A2228) was from Sigma. Peroxidase labeled anti-mouse and anti-rabbit secondary antibodies and ECL system were purchased from Amersham Life Science. The luciferase and β-galactosidase assay systems were from Promega. Tissue culture media, fetal bovine serum (FBS), and plastic-ware were obtained from Life Technologies, Inc. Proteinase inhibitor cocktail Complete™ was obtained from Boehringer Mannheim.2. The cells were cultured in DMEM medium containing 10% of FBS, 2 mM of L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin.All cell lines were obtained from ATCC and grown under standard cell culture conditions at 37°C in a humidified atmosphere with 5% COColorectal cancer cells in 6-well plates were lysed in 0.5 ml of lysis buffer  at 4°C for 10 min. Equal quantities of protein were subjected to SDS-PAGE under reducing conditions. Following transfer to immobilon-P transfer membrane, successive incubations with a primary antibody, and a horseradish peroxidase-conjugated secondary antibody were carried out for 60-120 min at room temperature. The immunoreactive proteins were then detected using the ECL system. Films showing immunoreactive bands were scanned by HP Scanjet 5590. Blots were quantitated by densitometry using Image J Software  and normalized to a housekeeper marker β-actin.The GST-E-cadherin binding assay was carried out exactly as previously described , 33. BriColorectal cancer cells were plated into 24-well plates. After overnight culture, the cells were transiently transfected with the Super8XTOPFlash luciferase construct and β-galactosidase-expressing vector. After 24 h incubation, cells were treated with the test compounds at the indicated concentrations. Cells were then lysed 24 h later and both luciferase and β-galactosidase activities were determined. The luciferase activity was normalized to the β-galactosidase activity.Colorectal cancer cells were seeded into 96-well tissue culture treated microtiter plates at a density of 5000 cells/well. DMEM containing 10% FBS was used as assay media. After 24 h incubation, the cells were treated with the test compounds at the indicated concentrations for 72 h. Cell viability was measured by the Cell Titer Glo Assay, which is a luminescent assay that is an indicator of live cells as a function of metabolic activity and ATP content.Statistical analyses were performed using Student's unpaired t-test. Data were presented as mean ± SD. Differences at P < 0.05 were considered statistically significant.