CXCL11, while TNF and IFN-γ costimulation results in potent CXCL11 induction. The molecular mechanisms underlying TNF plus IFN-γ-mediated CXCL11 induction are not fully understood. We have previously reported that the protein arginine methyltransferase PRMT5 catalyzes symmetrical dimethylation of the NF-κB subunit p65 in EC at multiple arginine residues. Methylation of Arg30 and Arg35 on p65 is critical for TNF induction of CXCL10 in EC. Here we show that PRMT5-mediated methylation of p65 at Arg174 is required for induction of CXCL11 when EC are costimulated with TNF and IFN-γ. Knockdown of PRMT5 by RNAi reduced CXCL11 mRNA and protein levels in costimulated cells. Reconstitution of p65 Arg174Ala or Arg174Lys mutants into EC that were depleted of endogenous p65 blunted TNF plus IFN-γ-mediated CXCL11 induction. Mass spectrometric analyses showed that p65 Arg174 arginine methylation is enhanced by TNF plus IFN-γ costimulation, and is catalyzed by PRMT5. Chromatin immunoprecipitation assays (ChIP) demonstrated that PRMT5 is necessary for p65 association with the CXCL11 promoter in response to TNF plus IFN-γ. Further, reconstitution of p65 Arg174Lys mutant in EC abrogated this p65 association with the CXCL11 promoter. Finally, ChIP and Re-ChIP assays revealed that symmetrical dimethylarginine-containing proteins complexed with the CXCL11 promoter were diminished in p65 Arg174Lys-reconstituted EC stimulated with TNF and IFN-γ. In total, these results indicate that PRMT5-mediated p65 methylation at Arg174 is essential for TNF plus IFN-γ-mediated CXCL11 gene induction. We therefore suggest that the use of recently developed small molecule inhibitors of PRMT5 may present a therapeutic approach to moderating chronic inflammatory pathologies.Inflammatory agonists differentially activate gene expression of the chemokine family of proteins in endothelial cells (EC). TNF is a weak inducer of the chemokine  VCAM1 and E-selectin, and the chemokines CXCL10 and CX3CL1 in response to TNF [N,N-dimethylarginine . In contrast, type II PRMT are capable of adding a second methyl group to the opposite terminal nitrogen atom of arginine, producing a product with methyl adducts on both terminal nitrogen residues  [Activation of endothelial cells (EC) by inflammatory substances results in stimuli-specific induction of proteins that participate in EC-leukocyte interactions and leukocyte recruitment to inflammatory foci . We havee to TNF , 3. PRMTe, SDMA) –6. MMA, e, SDMA) , 8. The PRMT5 is at type II enzyme is the primary enzyme responsible for formation of SDMA in mammals , 10. AddCXCL10 in response to TNF in EC requires PRMT5-catalyzed arginine methylation of the transcription factor p65 at Arg30 and Arg35 [35 in particular directly interacts with the 3´ subsite of the κB promoter element [Combinations of posttranslational modifications (PTMs) such as arginine methylation, phosphorylation, and acetylation enable a “PTM code” that transduces context-specific information to the nucleus, facilitating nuanced control over transcriptional responses –25. Symm element  residues element , 30.174, a residue accessible to the cytosol that is located in a region of the rel homology domain important for mediating protein-protein interactions [174 methylation is important for mediating stimuli-specific chemokine gene expression. To test this hypothesis, we first suppressed PRMT5 levels in EC using RNAi, and stimulated the cells with TNF, IFN-γ, and TNF plus IFN-γ to identify chemokines requiring PRMT5 for induction. We discovered that PRMT5 is critical for induction of the chemokine CXCL11 when costimulated with TNF plus IFN-γ. CXCL11 is transcribed upon stimulation by EC, astrocytes, monocytes, neutrophils, and keratinocytes [+ and CD8+ cytotoxic T-lymphocytes (CTL), CD4+ and CD8+ memory cells, natural killer (NK), natural killer T cells (NKT), dendritic cells, and some B cells results in recruitment of leukocyte populations to inflamed sites [Our previous study also identified other residues methylated by PRMT5 on p65, such as Argractions . In the inocytes . Ligatioed sites –35. CXCLed sites . CXCL11 ed sites , 37, 38.CXCL11 revealed a requirement for methylation of p65 at Arg174. Together with our previous report [Additional characterization of the role of PRMT5 in the induction of s report , our resHUVEC are isolated from umbilical collected by MetroHealth Hospital and Hillcrest Hospital. The cords are not linked to any patient identification, and isolated EC are pooled. The Cleveland Clinic Foundation Institutional Review Board has confirmed that our use of discarded, de-identified human tissue is exempt from review under National Institutes of Health guidelines.Primary human EC were isolated from human umbilical cords . Cells f174Ala and Arg174Lys cDNA constructs can be found in our previous publication [174 was mutated using the PCR-based GeneArt Mutagenesis System (Life Technologies). We repeated all experiments using two different siRNA sequences for each target to minimize the potential of off-target effects. siRNAs complementary to the PRMT5 coding sequence were sense 5´-GAGGGAGUUCAUUCAGGAAUU-3´, and PRMT5 3´ UTR, sense 5´-GCUCAAGCCACCAAUCUAUUU-3´. siRNAs targeting the NF-κB p65 3´ UTR were sense 5´-GGAUUCAUUACAGCUUAAUUU-3´ and sense 5´-GCUCUUUCUACUCUGAACUUU-3´. All siRNAs were designed using the Whitehead Institute siRNA design tool (http://sirna.wi.mit.edu/) and synthesized by Ambion containing the Silencer Select modifications [Cells were transfected 12–18 h following passage at 70–80% confluency. At this time complete cell media containing 15% FBS was removed, and replaced with a transfection mixture containing Targefect F-2 (5 μl/ml) and peptide enhancer (5 μl/ml) (Targeting Systems) in serum-free Dulbecco’s modified eagle’s medium . siRNA was transfected at 50 nM, whilst cDNA was transfected at 250 ng/ml. The transfection mixture was incubated with the cells at 37°C for 4 h, at which point it was removed and replaced with complete growth media containing serum. Cells were maintained in this media for 24–48 h to allow for protein expression or knockdown. Details of our wild type p65, p65 ArgLysates for immunoblots were prepared by addition of 2X Laemmli buffer. After boiling for 5 min, proteins were resolved on 10% bis-tris acrylamide gels by SDS-PAGE, and transferred to PVDF membranes using a semi-dry transfer apparatus (Bio-Rad). Membranes were blocked with either 5% nonfat milk in 1X TBST or protein-free TBS blocking buffer (Pierce). Primary antibody incubation occurred overnight at 4°C with gentle agitation. Multiple washes were performed with 1X TBST. Secondary antibodies were HRP-conjugated. Chemiluminescence was activated using Western Lightning Plus ECL Substrate (PerkinElmer). All immunoblots were quantified via the Image Studio Lite program (LI-COR Biosciences) and normalized to a loading control.Cells were lysed in 350 μl of Buffer RLT containing 1% 2-mercaptoethanol (Sigma-Aldrich) per each well of a 6-well plate following agonist exposure. Total RNA was isolated with the RNeasy kit (Qiagen). SuperScript First-Strand reagents (Life Technologies) were used to produce cDNA from 1 μg of RNA. The resulting cDNA was diluted 3-fold with water. qRT-PCR was performed using Fast SYBR Green Reagent (Life Technologies) using an ABI StepOnePlus machine (Life Technologies) with 40 cycles of amplification. Primers were separated by an intron on the corresponding gDNA and amplified 100–150 base pairs. Results were normalized to GAPDH according to the –ΔΔCt method. Primer sequences are provided in p65 and PRMT5 siRNAs were transfected into EC. After 40 hours, cells were serum starved and treated with TNF and IFN-γ then lysed in 1X RIPA. FLAG-p65 was immunoprecipitated using anti-FLAG antibodies. Proteins were separated by PAGE and the gel was silver-stained. p65 bands were excised and digested in-gel with trypsin overnight at room temperature. Fragments were assessed using capillary column LC-tandem mass spectrometry using a self-packed 9 cm x 75 μm inner diameter Phenomenex Jupiter C18 reversed-phase capillary chromatography column and Finnigan LTQ (linear trap quadrapole) ion trap mass spectrometer (ThermoFinnigan). Collision-induced dissociation (CID) spectra were analyzed using the NCBI human non-redundant protein database. Targeted experiments were also performed involving selected reaction monitoring (SRM) of specific p65 peptides, including unmodified and arginine methylated forms. Chromatograms derived from these data were assessed in relation to known fragmentation patterns. Peak areas were used to quantitate the extent of methylation.Cells for ChIP were cultured in 10 cm dishes. The ChIP protocol was followed according to the instructions included in the ChIP kit (Millipore 17–295). Crosslinked protein-DNA complexes were formed by incubating the cells with 1% formaldehyde (Sigma-Aldrich) in MCDB media for 10 min at 37°C. Following sonication, immunoprecipitation, and crosslink reversal the immunoprecipitated DNA was purified with the PrepEase DNA Cleanup kit (Affymetrix). In the Re-ChIP experiments the complexes from the initial IP (anti-FLAG) were eluted at 37°C for 30 minutes with 10 mM DTT. Following centrifugation the supernatant was removed to a fresh tube and diluted 20 times in Re-ChIP buffer . The second IP antibody (anti-SDMA) or antiserum was then added, followed by crosslink reversal and DNA purification as outlined above. DNA products from the immunoprecipitation were quantified by qRT-PCR relative to input. Primers were designed to amplify chemokine proximal promoters containing κB site(s) and provided in n = 3–4 and are presented as mean ± standard error of the mean (SEM). One- or two-way analysis of variance (ANOVA) were performed. Pairwise comparisons were assessed with Student’s t-test, and corrected with Bonferroni post hoc tests. A p value of ≤ 0.05 was considered significant.All experiments were repeated with an 30 and Arg35 of p65 is necessary for induction of CXCL10 by TNF in EC [CXCL10 and CX3CL1, and not other pro-inflammatory chemokines, such as CXCL8/IL-8 or CCL2/MCP1. We also discovered methylation at other p65 arginine residues, such as Arg174, and postulated that arginine methylation of this residue may be necessary to induce other pro-inflammatory chemokines in a stimulus-dependent manner. In particular, we explored the effects of EC costimulation by TNF plus IFN-γ as this combination of agonists synergistically induces CXCL10 [CXCL11 [In our previous publication, we identified that symmetrical dimethylarginine (SDMA) formation catalyzed by PRMT5 on Args CXCL10 –42 and C [CXCL11 –45 expre [CXCL11 .p < 0.001). We found that transcription of CXCL11 mRNA was significantly blunted in PRMT5-depleted EC in response to TNF plus IFN-γ costimulation . We observed a significant decrease in CXCL10 mRNA induction when both agonists were presented individually, and in combination (p < 0.05). TNF plus IFN-γ-mediated induction of CCL2  or p65 Arg174Lys  but PRMT5-depletion decreased p65 binding to the CXCL11 promoter significantly  as compared with the unstimulated condition (CXCL10 promoter in PRMT5-depleted EC (CCL2 (MCP1) promoter following costimulation was unaffected by PRMT5 knockdown . When the Arg30Lys and Arg35Lys mutants were expressed, levels of CXCL10 promoter dropped to ~50% of the levels observed with the wild type and Arg175Lys mutant (p < 0.005). These results are consistent with our previous finding that methylation of p65 Arg30 and Arg35 are necessary for CXCL10 induction in EC. Additionally, these results indicate that Arg30 and Arg35 may be methylated independently, as mutation of either Arg30 or Arg35 to lysine does not reduce methylation of the other residue.The ChIP immunoprecipitates were also probed for the presence of the 174 we reconstituted wild type and Arg174Lys mutants in EC, and used the FLAG epitope to immunoprecipitate p65 proteins, which where then probed with anti-SDMA antibody . These results indicate that the symmetrical dimethylation of p65 detected by the anti-SDMA antibody is likely modification of Arg174.To confirm that the SDMA antibody is detecting methylation at p65 Argy SYM10; . We are CXCL11 promoter. We utilized a sequential ChIP (Re-ChIP) approach where we reconstituted wild type and point mutant p65, then immunoprecipitated p65 using the FLAG-tag. After removal of the anti-FLAG antibody, we immunoprecipitated the protein-chromatin complexes a second time using the anti-SDMA antibody. The CXCL11 promoter was present at background levels in the absence of cell costimulation with TNF and IFN-γ following immunoprecipitation of wild type p65 .We next sought to assess whether p65 and SDMA are detected on the same fragments of the type p65 , bar 1. CXCL11 promoter with this control . Crucially, we found that the level of CXCL11 was significantly diminished when Arg174Lys was expressed to ~5% of input . In full, these results indicate that the methylation associated with the CXCL11 promoter is predominately symmetrical dimethylation of p65 at Arg174. Arginine methylation of other p65 arginine residues, such as Arg30 or Arg35, is only a minor component of p65 methylation associated with this promoter under TNF plus IFN-γ costimulated conditions.Enrichment of the CXCL10 promoter in this experiment (174Lys were expressed in stimulated cells and immunoprecipitated with the anti-FLAG antibody (~20% of input). When Arg30Lys and Arg35Lys were present, CXCL10 promoter was enriched to ~7% of input . Only background levels of CXCL10 promoter were found following buffer exchange to remove the anti-FLAG antibody (bars 6–9). Levels of CXCL10 promoter were also at background when rabbit serum was applied in the Re-ChIP. Quantitation of the promoter showed following Re-ChIP with the SYM10 antibody showed that wild type and Arg174Lys p65 pulled down CXCL10 promoter equal to ~20% of input, but that Arg30Lys and Arg35Lys mutant expression was associated with lower quantities of the promoter, ~8–10% of input . These results are consistent with our previous findings that methylation of Arg30 and Arg35 is necessary for CXCL10 induction, but that Arg174 methylation is not required [30 or Arg35 to lysine reduces, but does not eliminate, detection of methylated p65 associated with this promoter, supporting a model where methylation of Arg30 and Arg35 are independent.We also quantitated the presence of the periment . p65 is 174, Arg30 and Arg35, is critical for p65 recruitment to the CXCL11 promoter and the subsequent synergistic induction of CXCL11 in response to TNF plus IFN-γ costimulation. This requirement does not exist for the synergistic induction of CXCL10. Our findings are consistent with a mechanism whereby concurrent stimulation of EC by both TNF and IFN-γ activate PRMT5. PRMT5 is then recruited to p65, where it catalyzes dimethylation of Arg174. Arginine methylation of p65 at Arg174 is necessary for p65 recruitment to the CXCL11 promoter. Given the location of Arg174 on the p65 molecule is probable that modification of this residue facilitates a protein-protein interaction that enhances CXCL11 transcription.Given the sum of our experiments reported here we conclude that PRMT5-catalyzed p65 methylation at Arg174. TNF signaling would then mobilize the p65 methylated at Arg174 to the nucleus, where it associates with transcription cofactors and the CXCL11 promoter. It is not clear whether IFN-γ alone is sufficient to trigger methylation at p65 at Arg174, or whether Arg174 methylation is a product of synergism between the TNF and IFN-γ pathways. We also can not rule out contribution of other PRMT enzymes in the methylation of Arg174, including PRMT1 and PRMT4, both of which are found in EC. PRMT1 and PRMT4 catalyze formation of ADMA, which may differ in transcriptional outcome to the SDMA modification catalyzed by PRMT5 that we report here.We do not currently know the mechanism linking activation of cytokine receptors with p65 methylation by PRMT5. One possible mechanism could involve activation of the JAK kinases by ligation of the IFN-γ receptor by IFN-γ. PRMT5 was first discovered in human cells as a JAK2 binding protein , and it CXCL10 expression is elicited by the widest variety of agonists, including IFN-α/β/γ, and is most sensitive to TNF. In contrast, CXCL11 expression is weakly transcribed by TNF, but is induced by IFN-β/γ [Our findings suggest that the PRMT5-mediated methylation imparts an indexing signal to p65 that enables stimulus-specific chemokine expression. Differential combinations of such modifications enable p65 to interact with DNA or other components of specific transcriptional complexes in a context-specific manner, resulting in unique patterns of gene expression , 52. Suc IFN-β/γ . CXCL11  IFN-β/γ , 58, 59  IFN-β/γ –62 shoulS1 Fig172PLRLPPVLSHPIFDNR185 peptide. This spectra contains several unmodified C-terminal y ions, all of which are consistent with dimethylation at Arg174.MS/MS spectra of the 633 Da triply charged ion identified in the tryptic digestion of NF-κB p65. The mass of this peptide is consistent with the addition of two methyl groups to the (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Y216/β-catenin regulation axis: FAK and PYK2, elevated in adenomas in APCmin/+ mice and in human colorectal cancer tissues, functioned redundantly to promote the Wnt/β-catenin pathway by phosphorylating GSK3βY216 to reinforce pathway output—β-catenin accumulation and intestinal tumorigenesis. We previously showed that Wnt-induced β-catenin accumulation requires Wnt-induced GSK3β/β-TrCP interaction; the current study revealed that phosphorylation of GSK3βY216 was a molecular determinant of GSK3β recruitment of β-TrCP. Pharmacological inhibition of FAK/PYK2 suppressed adenoma formation in APCmin/+ mice accompanied with reduced intestinal levels of phospho-GSK3βY216 and β-catenin, indicating that FAK/PYK2/GSK3βY216 axis is critical for the activation of Wnt/β-catenin signaling in APC driven intestinal tumorigenesis.Aberrant activation of Wnt/β-catenin signaling plays an unequivocal role in colorectal cancer, but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge. New insights into the regulation of this pathway could reveal new therapeutic point of intervention, therefore are greatly needed. Here we report a novel FAK/PYK2/GSK3βDOI:http://dx.doi.org/10.7554/eLife.10072.001 The cells in our body communicate with each other to coordinate many essential processes, including cell division and the formation of tissues and organs. The Wnt signaling pathway is crucial for cell communication across all animal species, but activating this pathway at the wrong time can cause cancer to develop. As part of the investigation into treatments for colon and other intestinal cancers, researchers have tried to identify drugs that inhibit Wnt signaling. This search would be easier if we understood more about how the Wnt signaling pathway is controlled.When the protein GSK3 is active, it can switch the Wnt signaling pathway ‘off’, and inactivating GSK3 can turn the Wnt signaling pathway ‘on’. Enzymes known as kinases can inactivate GKS3 by adding a phosphate group to it, in a process known as phosphorylation. It was unknown which kinases phosphorylate GSK3, and whether this affects how cancerous tumors develop in the colon.Gao, Chen et al. analyzed cells taken from human and mice and observed that two kinases called PYK2 and FAK phosphorylate one form of GSK3, known as GSK3β. Furthermore, both kinases are required to inactivate GSK3β completely, and so turn on the Wnt signaling pathway.During the early stages of colon cancer, cells first develop into polyps, which subsequently become cancerous. Gao, Chen et al. treated mice that had genetic mutations that made them susceptible to colon cancer with a chemical compound that inhibits both FAK and PYK2. These mice grew fewer polyps than untreated mice, and the polyps that did grow tended to be smaller.Tissue samples taken from humans in the early stages of colon cancer—as the polyps progress towards becoming cancerous—had high levels of FAK, PYK2 and phosphorylated GSK3β. Overall, this suggests that drugs that simultaneously inhibit FAK and PYK2 may be an effective treatment for colon cancer, although further studies will be needed to confirm this.DOI:http://dx.doi.org/10.7554/eLife.10072.002 The losstaining , a surroA key feature of the Wnt/β-catenin pathway is the tight control of the abundance of transcription co-activator β-catenin by the β-catenin destruction complex, consisting of GSK3, casein kinase 1 (CK1), APC, scaffold protein Axin and β-catenin. In Wnt-off state, the cytosolic level of β-catenin is kept low by its continuous degradation: β-catenin is phosphorylated by GSK3 on serine 33 and 37 and threonine 41, phosphorylation then triggers β-catenin recruitment of ubiquitin E3 β-TrCP (β-transducin repeats-containing proteins), causing its ubiquitination and proteasomal degradation . In Wnt-β-TrCP ubiquitin ligase supercomplex, recruitment of β-TrCP to its substrate, for example β-catenin, generally requires substrate phosphorylation  restored the interaction, suggesting that it was phosphorylation that determines the recruitment of β-TrCP to GSK3β.β-TrCP binds to its substrates through phosphorylated residues in conserved degradation motifs (DpSGxxpS) , but tyrβ-TrCP1 ubiquitin E3 ligase complex, WT GSK3β and its phosphorylation mimicking mutant Y216E, but not Y216F mutant, were ubiquitinated. Of note, in vitro synthesized GSK3β through Rabbit Reticulocyte Lysate Translation Systems were weakly phosphorylated at Y216 and incubation with ATP further enhances GSK3βY216 phosphorylation through GSK3β autophosphorylation , the loss of GSK3βY216 phosphorylation greatly impaired both basal and Wnt3a-induced GSK3β monoubiquitination , GSK3βY216F and GSK3βY216E phosphorylated β-catenin as efficiently as WT GSK3β in vitro  MEFs (mouse embryonic fibroblasts). GSK3α and GSK3β are functionally redundant in Wnt signaling (S33/S37/T41 (Y216E), but not GSK3βY216F mutant, also restored Wnt-induced accumulation of β-catenin, thus validating the positive role of GSK3βY216 phosphorylation in Wnt signaling. The fact that GSK3βKKKK15/27/32/36RRRR/Y216E mutant failed to restore Wnt-induced β-catenin stabilization  exerted the opposite effect. The results from the reciprocal IP assay confirmed more β-TrCP-bound p-β-cateninS33/S37/T41 in HA-GSK3βY216F cells  are Wnt autocrine cell lines . In SW48Y279 corresponds to GSK3βY216. Given the redundant role of GSK3α and GSK3β in Wnt signaling . A knock-in point mutation of lysine 454 to arginine within the catalytic domain inactivates FAK kinases activity (Y397) but leaves FAK's scaffolding activity intact  and polyp expansion (size)  . The rese)   of colon cancer development. The positive correlation between these elevated expressions also suggested that these inductions were related events.Most colorectal carcinomas develop from adenomas via adenoma-carcinoma-sequence driven by the accumulation of genetic and epigenetic mutations ; Our resgression , demonstY279/Y216 and β-catenin are commonly seen in human CRC tissues, we performed IHC for these markers on CRC tissue microarray containing 34 cases of colorectal adenocarcinoma with 26 matched and 8 unmatched adjacent normal tissues. The results showed that the levels of FAK, PYK2, GSK3β, p-GSK3α/βY279/Y216 and β-catenin were all significantly elevated in tumor tissues, with more than 1 score point difference between normal and tumor tissue on average (Y279/Y216/β-catenin (19 of 26 samples) and PYK2/p-GSK3α/βY279/Y216/β-catenin (18 out of 26 samples) were regulated in the same way, supporting that they are related events in the development of colonic cancer . p values less than 0.05 were considered statistically significant.Data are presented as mean ±SD. The difference between two groups was evaluated using Student's Detailed information for DNA constructs, shRNA, antibodies and reagents, and detailed methods for cell culture, immunoprecipitation (IP), immunoblotting (IB), transfection, lentivirus and retrovirus production and infection, in vitro kinase assay, ubiquitination assay, soft agar assay, cell proliferation assay, and animal experiments are provided below.2. HEK293T, SW480, and MEF cells were grown in DMEM. DLD-1 cells and HT-29 cells were cultured using RPMI-1640 medium and MoCoy's 5a medium, respectively. All cells were supplemented with 5% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin. Wnt3a-producing L cells and control L cells were obtained from ATCC and used for generating Wnt3a-conditioned medium (CM) and control-CM according to ATCC's instruction. GSK3α+/+ and GSK3β−/− MEFs were generously provided by James Woodgett . FAKR454/R454 knockin MEFs were described previously . Coding sequence for GSK3α was amplified and cloned into pcDNA3-FLAG to genetrate plasmid expressing FLAG-tagged GSK3α. Plasmids expressing ubiquitin- HA-GSK3β fusion protein and GSK3β ubiquitination mutant were previously described . GSK3β, Y216 mutant proteins were synthesized using the TNT T7 quick-coupled transcription/translation kit . To purify in vitro translated HA-GSK3β proteins, 5 μl out of 25 μl of the translation product were used for IP with EZview Red anti-HA affinity gel (Sigma). The immunoprecipitates were subjected to in vitro ubiquitination assay. The assay was carried out in 1× ubiquitination assay buffer  at a final volume of 20 µl containing: 130 ng E1 , 230 ng E2 UbcH5b (Boston Biochem), 300 ng SCFβTrCP1 complex consisting of Rbx1, Cul1, Skp1 and β-TrCP1 , 3 µg FLAG-Ubiquitin (Boston Biochem) and purified HA-GSK3β or its mutants. The reaction mixture was incubated at 37°C for 2 hr, and then subjected to SDS-PAGE followed by IB with anti-GSK3β antibody.Wild type (WT) HA-GSK3β and HA-GSK3βY216.The in vitro kinase assay to assess kinase activity of GSK3β and its mutants towards β-catenin was performed as previously described . To valiGattefosse . MG-132 was obtained from Sigma.Whole cell lysates were prepared in M-PER buffer , resolved by SDS-PAGE and blotted with indicated antibodies. The following antibodies were used in this study: Anti-HA, anti-FLAG, anti-phospho-β-catenin (Ser33/37/Thr41), anti-β-catenin, anti-GSK3α, anti-β-TrCP, anti-α-Tubulin, anti-GSK3β, anti-FAK, anti-phospho-FAK (Tyr397), anti-PYK2 and anti-phospho-PYK2 (Tyr402) ; anti-FLAG M2 (Sigma); anti-ubiquitin and anti-β-catenin ; anti-GSK3, anti-c-Myc, anti-PYK2, anti-β-TrCP, anti-phospho-GSK3β (Tyr216) and anti-β-Actin ; anti-phospho-GSK3 (Tyr279/Tyr216) (Millipore). Anti-GSK3β for IP was purchased from Abcam  and Bethyl Laboratories . SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo) were used to enhance western signal when needed. PF-562271 was purchase from MedKoo Biosciences  and Selleck Chemicals . Gelucire is a gift from Briefly, cells were lysed with M-PER buffer or RIPA buffer (Thermo) supplemented with protease inhibitor and 20 mM N-ethylmaleimide (NEM) (Sigma). The whole cell lysates were pre-cleared with Protein G-sepharose beads at 4°C for 30 min. The cleared lysates were incubated with indicated antibody together with Protein G-sepharose beads or EZview Red anti-HA or anti-FLAG M2 affinity gel (Sigma) at 4°C for 4 hr. The immunoprecipitates were washed three times with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40 and 10% Glycerol, and subjected to SDS-PAGE followed by IB with indicated antibodies. Input corresponded to 5% of the total lysates used for the IPs and was verified by IB.Y142E were also generated by retrovirus-mediated gene transfer.Plasmid transient transfections were performed using PolyJet In Vitro DNA Transfection Reagent  according to the manufacturer's instruction. Lentiviruses encoding the shRNA against target genes were produced in HEK293T cells by transfection of the lentiviral vector expressing shRNA against target gene with the third generation packaging systems (Addgene). The media containing viral particles were filtered through syringe filters and subsequently used to infect target cells. Cell lines stably expressing shRNA were established by selection. To generate FAK/PYK2 double KD cell line, DLD-1 cells were first infected with lentivirus particles encoding the shRNA against PYK2. PYK2 KD DLD-1 cell line was established by puromycin selection and used for second round infection with lentivirus particles encoding the FAK shRNA. Double KD cell line was obtained by puromycin and blasticidin selection. For retrovirus infection, the retroviral vectors expressing shRNA-resistant GSK3β or its mutants were transiently transfected into a potent retrovirus packaging cell line named platinum-A (Plat-A). 48 hr post transfection infectious retrovirus were collected and used to infect SW480 cells. The stable cell lines were established by blasticidin selection and used for cell proliferation assays. The cell lines overexpressing GSK3 shRNA-resistant GSK3β or its mutants were subjected to GSK3 KD by lentivirus-mediated RNA silencing. The resulting cell lines were used for anchorage-independent growth assays and xenograft experiments. The same strategy was used to generate SW480 cells expressing shRNA-resistant GSK3α or its mutants with endogenous GSK3 knocked down for xenograft experiments. HT-29 cells stably expressing FLAG-β-catenin4 cells was plated over the 3 ml bottom agar containing 0.8% LMP agarose in a 60 mm dish. Two independent experiments were performed in triplicates. The dishes were incubated at 37°C with medium replenished every 3 days until colony formed. The colonies were stained with 0.01% crystal violet solution. Pictures of representative field were taken under stereo microscope (Olympus SZX10).The SW480 cells were trypsinized and suspended in DMEM containing 0.4% low melting point (LMP) agarose (Promega). 2 ml of the above medium containing 3 × 103. 24 hr later, the medium was replaced by fresh medium containing 2 µm FAK/PYK2 inhibitor PF-562271. The cell viability was measured by CCK-8 kit (Sigma) according to manufacturer's protocol every day for 4 days. Three independent experiments were performed in triplicates.SW480 parental cells or cells overexpressing HA-tagged GSK3β or its phosphorylation mutants were used for cell proliferation assays. These cells were seeded into 96-well plates at a density of 5 × 10APCmin/+ mice on C57BL/6J background and C57BL/6J female mice (control mice) were obtained from the Jackson Laboratories. At 11 weeks of age, the APCmin/+ were randomized into two groups, receiving either vehicle (5% Gelucire) (n = 8) or PF-562271 (33 mg/kg in vehicle) (n = 8) by oral gavage twice daily for 5 weeks. After the mice were euthanized, the entire small intestinal tracts were carefully dissected, rinsed with ice-cold saline, opened longitudinally on ice. For polyp count experiments (n = 5 for vehicle- or PF-562271-treated group), the excised mice small intestine segments were fixed flat in 10% formalin for 48 hr. The fixed intestine was stained with 0.2% methylene blue (Sigma) for 2 min and then destained in 75% ethanol to reduce background staining. Polyp number and polyp size were counted under a dissecting microscope. For the collection of mice intestine tissue , the mucosal layer of intestine  was harvested by gentle scraping with a glass slide and all procedures were performed on ice. After harvesting, the tissues were collected into tubes and immediately placed on dry ice. The tissue samples were kept a liquid nitrogen tank before processing for western blotting or IP analysis. For these analyses, the tissues were lysed on ice in RIPA buffer (Thermo) supplemented with phosphatase inhibitor (Thermo), protease inhibitor (Thermo) and 20 mM NEM (Sigma). For the indicated experiments, individual polyps in 5-month-old female APCmin/+ mice were isolated for tumor tissue lysates.All animal procedures were carried out according to protocols approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Mice were fed a standard diet . For intestinal tumorigenesis experiments, age-matched female 6 SW480 or HT-29 cells as indicated were injected subcutaneously into both flanks of 4- to 5-week-old athymic nude mice (Charles River), 5 mice in each group. The mice were housed in a sterile environment. Tumor size was determined by caliper measurements twice a week. The tumor volume was calculated using the formula: V = ½ × a × b  patient, archived at the University Of Pittsburgh School Of Medicine, Department of Pathology, were obtained in compliance with a University of Pittsburgh Cancer Institute (UPCI) tissue banking protocol (UPCI 97-130). The immunohistochemical analysis was performed in compliance with the UPCI Institutional Review Board protocol, UPCI 08-026. Immunohistochemistry (IHC) was performed on 4-micron formalin-fixed paraffin-embedded tissue from either tissue microarray or colon cancer resection. Briefly, 4 µm paraffin sections were deparaffinized in xylene solutions and rehydrated in graded alcohol solutions followed by washes in distilled water. Antigen retrieval was performed in the pressure cooker for 15 min in 20 mmole/l Tris-EDTA buffer (pH 9.0). The sections were allowed to cool to room temperature and then incubated overnight in a humidified chamber at room temperature with indicated antibodies. After washing with PBS, the sections were incubated for 1 hr at room temperature with HRP-labeled polymer anti-mouse or anti-rabbit second antibody , depending on the host which individual antibody was prepared. Color visualization was performed with liquid DAB chromogen in imidazole-HCI buffer (pH 7.5) containing hydrogen peroxide until the brown color fully developed. The sections were counterstained with hematoxylin and coverslippped with permanent mounting media. The intensity of TMA staining was score as 0 (negative), 1+ (weak), 2+ (moderate) and 3+ (strong). The following antibodies were used for immunohistochemical staining: anti-FAK , anti-PYK2 , anti-GSK3β , anti-phosphor-GSK3β (Tyr216)  and anti-β-catenin . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and two reviewers, one of whom is a member of our Board of Reviewing Editors. The decision was reached after discussions between the reviewers.Thank you for choosing to send your work entitled “FAK/PYK2 Promotes the Wnt/β-catenin Pathway and Intestinal Tumorigenesis by Phosphorylating GSK3B” for consideration at eLife.One reviewer had detailed comments that would be difficult to address in a timely way, and without which the significance of the work is unclear. The other reviewer had a general comment that the work is preliminary without more in vivo experiments. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Reviewer #1:APC mouse model. This is potentially a second mechanism for FAK to promote β-catenin accumulation (in addition to the phosphorylation of β-catenin by FAK).This paper provides a potential molecular mechanism for the observation that colorectal cancers (CRCs) frequently have high levels of FAK/PYK2 and increased nuclear β-catenin, and that FAK deletion suppresses adenomas in a mutant Y216 stimulates monoubiquitination and binding of β-TrCP. The 216F mutant shows that lack of pY216 and/or lack of ubiquitin increases β-catenin turnover in presence or absence of Wnt, causing low β-catenin levels. The 216E mutant is constitutive. They show that FAK and PYK2 can phosphorylate Y216 , and knockdown of FAK reduces β-catenin level. FAK inhibitor (PF) inhibits tumor formation when GSK3 is wildtype, but only partly inhibits GSK3βY216E mutant tumors. However, the authors do not distinguish effects of pY216 from effects of ubiquitin by use of their KKKK mutant used in their JBC paper. Therefore, the observations could be due to reduced GSK3 auto or trans phosphorylation rather than ubiquitin. It is also remarkable that the low stoichiometry of ubiquitination has a measurable effect, because the non-ubiquitinated molecules should remain active. However, the results would be understandable if the subset of GSK3 that is ubiquitinated is in the destruction complex. Another surprising finding is that GSK3βY216 is not needed to stimulate β-catenin degradation by β-TrCP, which is presumably requires phosphorylation of β-catenin by GSK3 – but they didn't measure phospho-β-catenin with the 216F mutant.In a previous paper, the authors showed that Wnt stimulates monoubiquitination of GSK3 and this monoubiquitination reduces phospho-β-catenin presentation to β-TrCP, thereby stabilizing β-catenin. Here they find that phosphorylation of GSK3βAPC yet only 50% have increased nuclear β-catenin, implying that APC mutation is not sufficient to up-regulate nuclear β-catenin. Their paper provides data in support of a mechanism in which increased FAK/PYK2 activity in CRCs leads to increased GSK3βY216, increases GSK3 ubiquitination, inhibits β-catenin turnover by bTrCP, and raises nuclear β-catenin levels. This predicts that nuclear β-catenin would be detected in CRCs that lack APC mutations, rather than the converse. It isn't clear how many human CRCs have increased nuclear β-catenin but lack APC mutations. Putting that aside, the tumor growth studies with 216F mutant GSK3 support the importance of this site, and the cancer studies with the PF inhibitor and the pathology supports the importance of FAK/PYK2 in CRC. However, the inhibition of tumors by PF does not exclude GSK3-independent effects of FAK.From the significance point of view, the authors introduce the paper by saying that 85% of CRCs have mutant 1) The KKKK mutant should be used to confirm that nuclear β-catenin is increased regardless of FAK and PYK2 activity.2) KKKK mutation of Y216E mutant should suppress activation.S33/37 should be measured with GSK3 YF, YE and KKKK mutants to confirm the inference that pY216 is not needed for phosphorylation of β-catenin.3) Phospho-β-cateninY216 or ubiquitination on the ability of β-TrCP to interact with phospho-β-catenin.4) The mechanism would be much better supported if the authors could show an effect of GSK3β5) Does the PF inhibitor inhibit tumors in which the primary FAK phosphorylation site in β-catenin is mutated?Reviewer #2:Y216/β-catenin signaling axis, through which FAK and PYK2 converge on GSK3βY216 and GSK3αY279, with phosphorylated GSK3βY216 recruiting β-TrCP, which monoubituitinates GSK3β, blocking Wnt-induced degradation of β-catenin. All experiments were done in cell lines only, and a single FAK/PYK dual inhibitor was tested .The authors report a FAK/PYK2/GSK3βY279, suggesting functional redundancy that was not further elucidated experimentally.While experiments shown were convincing, the authors raise two issues not further addressed experimentally. First was that the dual FAK/PYK2 inhibitor also blocked GSK3αSecond, the authors suggest but do not test, that FAK was upstream of PYK2 in vivo but not in vitro. Although the authors claim that use of a dual PYK2/FAK inhibitor might be superior to monospecific inhibitors to block emergent resistance in patients, the relevance of their in vitro experiments to an in vivo setting remain an open question not addressed in experiments presented. In the absence of in vivo validation, the conclusions drawn remain fairly preliminary. [Editors’ note: the author responses to the previous round of peer review follow.]We have made every effort to address the critiques thoroughly in this revision; the results of our additional experiments lend further support for our conclusion. Our response to the reviewers’ specific comments and changes made during the revision are detailed below.Reviewer #1:1) The KKKK mutant should be used to confirm that nuclear β-catenin is increased regardless of FAK and PYK2 activity.As suggested, we now included results from the experiments using the KKKK15/27/32/36 RRRR mutant (S4) and the S4-ubiquitin fusion mutant . As shown in 2) KKKK mutation of Y216E mutant should suppress activation.Y216E/S4 mutant (Y216 phosphorylation mimetic lacking ubiquitination) inhibited Wnt-induced β-catenin accumulation.As predicted by the reviewer, our new data  Phospho-β-cateninS33/37should be measured with GSK3 YF, YE and KKKK mutants to confirm the inference that pY216 is not needed for phosphorylation of β-catenin.As suggested, we have now included new results showing that mutation of GSK3β tyrosine 216  or lysines 15/27/32/36 to arginines did not affect GSK3β activity towards β-catenin phosphorylation at serine S33 and 37 in vitro (4) The mechanism would be much better supported if the authors could show an effect of GSK3βY216or ubiquitination on the ability of β-TrCP to interact with p-β-catenin.S33/S37/T41 was substantially more in GSK3β KO/GSK3α KD MEFs reconstituted with HA-GSK3βY216F than in MEFs reconstituted with WT HA-GSK3β or HA-GSK3βY216E, implying that phosphorylation status of GSK3βY216 impacts phosphorylated β-cateninS33/S37/T41 recruitment of β-TrCP.We conducted new experiments to address this issue. The new results (5) Does the PF inhibitor inhibit tumors in which the primary FAK phosphorylation site in β-catenin is mutated?Y142E partially jeopardized the anti-tumor effect of PF562271, PF562271 treatment significantly inhibited in vivo growth of SW480 cells ectopically expressing Flag-β-cateninY142E. While this result further proved the importance of FAK/PYK2-mediated GSK3 phosphorylation, it also suggested that phosphorylation of β-cateninY142E plays a role in FAK-targeted therapy for CRC.We have conducted a cell line xenograft study to address this important question, and the answer is yes. Our new data  showed tReviewer #2:[…] While experiments shown were convincing, the authors raise two issues not further addressed experimentally. First was that the dual FAK/PYK2 inhibitor also blocked GSK3αY279, suggesting functional redundancy that was not further elucidated experimentally.Y279; (1) in the activation of Wnt/β-catenin pathway and (2) in vivo growth of SW480 cells. Our new results showed that Wnt-induced β-catenin accumulation was inhibited in GSK3β KO/GSK3α KD MEFs reconstituted with GSK3αY279F but not with GSK3αY279E (Y279 phosphorylation in the activation of Wnt signaling. Our new data also showed that in GSK3α/β KD SW480 cells, reconstitution of the cells with WT HA-GSK3α or HA-GSK3αY279E but not HA-GSK3αY279F restored cell growth in a xenograft assay (Y279 phosphorylation in CRC cells.We have performed new experiments to address the role of GSK3αft assay , implyinSecond, the authors suggest but do not test, that FAK was upstream of PYK2 in vivo but not in vitro. Although the authors claim that use of a dual PYK2/FAK inhibitor might be superior to monospecific inhibitors to block emergent resistance in patients, the relevance of their in vitro experiments to an in vivo setting remain an open question, not addressed in experiments presented. In the absence of in vivo validation, the conclusions drawn remain fairly preliminary.R454/R454 knockin MEFs (APC mutated mice.We thank the reviewer for raising this important issue. The fact that PYK2 activity towards GSK3 phosphorylation was intact in FAKkin MEFs  indeed eAPCmin/+ mice, indicating that PYK2 is upregulated during the process of intestinal tumorigenesis. FAK induction, although further enhanced in tumors, however, is triggered by APC mutation – the initiating event for tumorigenesis in the APC-mutant mice, through activated Wnt pathway target c-Myc (APC mutation should occur earlier than PYK2 elevation. If so, it explains why inhibition of FAK by intestinal deletion of FAK was sufficient to suppress tumor development. It also explains why PF562271 treatment inhibited PYK2 elevation in APCmin/+ mice (A prior study  and our et c-Myc . Therefon/+ mice . In the Regardless of the exact mechanisms mediating in vivo PYK2 regulation during intestinal tumorigenesis, both FAK and PYK2 are elevated in already established colon tumors in CRC patients . The cur


AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target  SYT/SS18 gene to one of the SSX family members, most frequently SSX1 and 245Synovial sarcoma (SS) is an aggressive soft tissue malignancy predominantly of young adults that can develop in virtually any organ and is believed to originate from pluripotent mesenchymal cells1β−catenin, adenomateous polyposis coli (APC) and AXIN, is associated with development and progression of diverse cancer types1314151617AXIN2, nuclear localization of ß-catenin is seen in only 30–60% of SS202123β−catenin, APC, AXIN1 and AXIN2 mutationsMounting evidence suggests that the Wnt pathway is strongly implicated in SS pathogenesis78101112Engagement of cell surface receptors by Wnt ligands triggers intracellular signaling that can activate the canonical Wnt/β-catenin and a variety of non-canonical Wnt pathways27AXIN2 in addition to several other Wnt target genes. Because AXIN2 faithfully reflects Wnt pathway activation in neither tissue nor context-dependent manner and is among the most strongly upregulated Wnt target genes in SS929AXIN2 promoter. Our observations suggest that SS18-SSX participates in de-repression of AXIN2 expression by inhibiting HDAC and augmenting Histone H3K9 acetylation at its promoter and propose a mechanistic basis for SS18-SSX-mediated deregulation of Wnt target gene expression in permissive cells.In the present work we addressed possible functionally relevant interactions between SS18-SSX and nuclear components of the Wnt signaling pathway in mouse C3H10T1/2 pluripotent mesenchymal cells. We show that in the absence of exogenous soluble Wnt ligands, SS18-SSX1 forms complexes with TCF/LEF, TLE1 and HDAC that exclude β-catenin and induces a partial Wnt signature that includes elevated expression of AXIN2 expression in 10 synovial sarcoma specimens revealed one to three orders of magnitude higher levels of AXIN2 mRNA than in populations of freshly isolated primary candidate cells of origin of SS, including human mesenchymal stem cells (MSCs), myoblasts and satellite cells  and cells from the two fresh surgical SS samples. We used either a pool of siRNAs directed against the 5′region of human SS18/SYT or a pool of two shRNAs in the lentiviral pLVShRNAmir plasmid targeting the SS18-SSX breakpoint (ShSS18-SSXbp) or an ShRNA already described by Kadoch and CrabtreeSS18-SSX1 and AXIN2 mRNA were depleted respectively by 48% and 40% in SS11, 43% and 54% in SS12, 75% and 50% in HS-SYII, 55% and 34% in FUJI cells , and pluripotent mouse mesenchymal C3H10T1/2 cells that display MSC type plasticity32SS18-SSX1 cells displayed roughly 100 fold induction of AXIN2 that mimicked the expression observed in primary SS samples  through the expression of the CRE recombinase. Q-PCR analysis of SS18-SSX and AXIN2 revealed a very strong reduction of both transcripts in C3H10T1/2SS18-SSX1-V5 cells expressing the CRE recombinase   was constructed in the PGL3 vector. C3H10T1/2pLIVc, C3H10T1/2SS18-SSX1-V5 and C3H10T1/2SS18-SSX1-HA cells were transiently transfected with either the reporter or an empty PGL3 plasmid. STO mouse fibroblasts (where induction of AXIN2 following expression of SS18-SSX1 was not observed) transfected with the same constructs provided a negative control. Luciferase activity was measured 48 hours after transfection and normalized to that of renilla as an internal control. C3H10T1/2wt and C3H10T1/2pLIVc cells as well as STOpLIVc and STOSS18-SSX-V5 containing the reporter plasmid displayed no significant increase in luciferase activity compared to their PGL3 containing counterparts. By contrast, robust induction of luciferase activity was observed in both C3H10T1/2SS18-SSX1-V5 and C3H10T1/2SS18-SSX1-HA cells  and Baird et al.−6 for induced genes). Thus, SS18/SSX1 induces a SS-related transcriptome in C3H10T1/2 cells . Particularly significant was over-representation of Wnt signaling-associated and Wnt target genes among transcripts induced by SS18-SSX1 in C3H10T1/2 cells  in C3H10T1/2AXIN2 is a direct target of a complex that includes TCF/LEF transcription factors and β-cateninAXIN2 induction by SS18-SSX1, we expressed a dominant negative (ΔN) mouse LEF-1 mutant, which lacks the β-catenin binding domain but retains intact DNA binding activitypLIVc and C3H10T1/2SS18-SSX1-V5 cells. Lentiviral infection efficiency was assessed by red fluorescent protein (RFP) signal intensity co-expressed by the same bicistronic vector . To verify that ΔN LEF-1 inhibits LEF-1/β-catenin complex activity at the AXIN2 promoter in C3H10T1/2 cells, we compared AXIN2 mRNA in C3H10T1/2pLIVc and C3H10T1/2SS18-SSX1-V5 cells expressing or not ΔN LEF-1 stimulated with recombinant mouse Wnt3a , using a polyclonal goat anti-LEF-1 and a mouse anti-β-catenin antibody. Cells stimulated with recombinant mouse Wnt3a for 16 hours provided a positive control. In baseline conditions, the number of fluorescent foci, that represent interactions, in C3H10T1/2SS18-SSX1-V5 was comparable to that in resting C3H10T1/2pLIVc cells , using TOP-FLASH luciferase reporter and corresponding negative control FOP-FLASH plasmids  share the same protein interaction domains, mounting evidence suggests that each member has individual properties that may be responsible for unique functions in defined contextual settings28AXIN2 expression in C3H10T1/2 cells was addressed by their individual depletion or overexpression  and by assessing the effect of the HDAC inhibitor trichostatin A (TSA) on AXIN2 transcripts  using rabbit anti-V5, anti-HDAC and anti-TCF3/4 antibodies  did not result in significant dissociation of HDAC from LEF-1(not shown) whereas it increased association between LEF-1 and β-catenin, as discussed earlier , enhancement of nuclear β-catenin-HDAC1 interactions may be explained at least in part by the moderate SS18-SSX-dependent increase in nuclear β-catenin. As absence of β-catenin-SS18-SSX association suggests that the fusion protein is not part of a complex containing both HDAC and β-catenin, even moderately augmented nuclear β-catenin may help sequester HDAC from LEF-1 and thereby contribute to AXIN2 promoter de-repression. β-catenin-dependent dissociation of the complex may facilitate subsequent SS18-SSX binding to HDAC and TCF/LEF-1. Consistent with this notion, LEF-1-depleted C3H10T1/2SS18-SSX1-V5 cells  on V5 cells . TSA blo1/2pLIVc . The red binding . TSA tre binding  and S8.  binding  and S8, AXIN2 promoter, chromatin immunoprecipitation (ChIP) was performed using anti-histone H3K9Ac antibody and purified precipitated DNA was analyzed by qPCR using several primer pairs that anneal to different promoter regions , they support the notion that HDAC activity at the AXIN2 promoter is reduced upon SS18-SSX expression. Consistent with this notion treatment of C3H10T1/2pLIVc and C3H10T1/2SS18-SSX1-V5 cells with 50 μM anacardic acid or 20 μM curcumin did not alter AXIN2 expression excluding a major role for HATs (not shown). Histone acetylation leads to partial de-condensation of chromosomal domains that augments DNA accessibility and facilitates the action of the transcriptional machinery. To determine whether SS18-SSX1 may affect AXIN2 transcription by altering chromatin conformation, chromatin accessibility assays were performed using methylation-independent restriction enzyme hydrolysis and real-time PCR  and the observation that HDAC-LEF1 interactions are unaltered by SS18-SSX expression. Moreover, the 30% reduction in the number of SS18-SSX-LEF-1 PLA foci following TSA treatment suggests partial dependence of the interaction on intact HDAC availability. The function of LEF-1 as transcriptional repressor in the presence of SS18-SSX also supports ternary complex formation. Nevertheless, SS18-SSX may also engage in binary complexes with LEF-1 and HDAC, as the increase in the number of HDAC-SS18-SSX interactions upon LEF-1 depletion suggests a dynamic equilibrium between complexes containing all three proteins and only SS18-SSX/HDAC or LEF-1/HDAC. Comparison of SS18-SSX/HDAC and LEF-1/HDAC with SS18-SSX/LEF-1 interactions suggests that this is indeed the case as the first two generate a much higher number of PLA foci than the last. The effect of β-catenin depletion on AXIN2 expression raises the possibility that SS18-SSX may fulfill its function subsequent to β-catenin-mediated dissociation of HDAC from TCF/LEF-1. The increased SS18-SSX-HDAC interaction upon LEF-1 depletion suggests that SS18-SSX-HDAC association may precede ternary complex formation.Although SS18-SSX may utilize β-catenin to relieve promoter repression, our observations suggest that SS18-SSX itself participates in AXIN2 promoter which it de-represses by blocking HDAC activity, inducing local chromatin changes and promoting nuclear translocation of available β-catenin to enhance HDAC inactivation/sequestration. If only a fraction of DNA-bound TCF/LEF-1 is occupied by SS18-SSX, stimulation by Wnt ligands can be expected to further augment AXIN2 expression by activating canonical β-catenin-Wnt signaling. However, SS18-SSX interactions with TCF/LEF and TOP flash data that support TCF/LEF implication in SS18-SSX-dependent induction of AXIN2 expression do not exclude the participation of a distinct SS18-SSX-associated transcription factor.Despite binding Wnt transcription factors, SS18-SSX does not prevent Wnt ligand-induced target gene expression but rather appears to have a synergistic effect with canonical Wnt signaling. These observations are consistent with the possibility that SS18/SSX interacts with TCF/LEF at a fraction of their DNA binding sites on the Taken together, our observations suggest that by interacting with TCF/LEF and utilizing β-catenin as a transcriptional de-repressor, SS18-SSX can induce a partial Wnt expression signature in the absence of Wnt ligands without excluding an additive effect of Wnt ligand-dependent stimulation. Synovial sarcoma cells may thus possess a dual mechanism of ensuring Wnt pathway activity that may be essential for defined stages of their development possibly including stemness maintenance and limiting differentiation. The ability of SS18-SSX to engage Wnt signaling without resorting to Wnt ligands may have important therapeutic implications at it suggests that mere impairment of canonical Wnt signaling may not suffice to abrogate SS development.Antibodies used for western blot, immunoprecipitation, immunofluorescence or proximity ligation assay (PLA) were: mouse anti-V5epitope , rabbit polyclonal anti-V5 tag , monoclonal anti-HA (Covance Research Product Inc Geneva Switzerland), mouse anti-Xpress (Invitrogen), mouse anti-tubulin , rabbit anti-α/β tubulin , mouse anti-actin clone AC-40 (SIGMA), anti-TCF4 and anti-LEF-1 (Santa Cruz Biotechnology inc. Germany), rabbit anti-TCF4 , anti-TCF3 + 4 [6F12-3], anti-Histone H3AcK9, anti-HDAC1 Ab7028, anti-HDAC1 Ab46985 and anti-TLE-1 , anti-TLE1,2,3,4 , mouse anti-β-catenin (BD), β-catenin antibody sampler kit . HRP-conjugated secondary antibodies were from Amersham (Goat anti-mouse-HRP) or from Dako, Denmark (goat anti-rabbit and rabbit anti-goat HRP). Fluorocrome-conjugated secondary antibodies were from molecular probes. TSA was from SIGMA, murine recombinant WNT3a from PreproTech, London UK.All experimental protocols were approved by the Ethics Committee of the University of Geneva (protocol 01-172) and of the Canton de Vaud (protocol 131/12) and were carried out in accordance with the approved guidelines. Written informed consent was obtained from all patients from whom tissues were obtained. C3H10T1/2 and STO cells were from ATCC and were cultured in DMEM, 10% FBS; HS-SYII synovial sarcoma cell line were obtained from Riken BioResource Center, Japan, FUJI synovial sarcoma cell line was a gift from Dr Francis Hornicek . Human mesenchymal stem cells were obtained as describedSS18-SSX1 in frame with a V5 or HA tags were obtained using previously described constructsAxin-2 promoter sequence cloned into the pGL3 basic vector, p043 mTcf-4 B and pcDNA3.1/nV5-beta catenin were obtained from Adgene, SS18 and SS18-SSX mutants and HA-tagged mouse LEF-1 were constructed as described in cDNA clones, encoding human Q-PCR analysis was done using a HT-7900 instrument (life technology). Endogenous controls for normalization were mouse GAPDH, mouse and human cyclophilin and 18S. A ΔCT method and protocols for absolute or relative quantification (Applied Biosystems) were used.SS18-1-412-V5, XP-SS18-SSX-161-491, LEF-1-HA was achieved using the self inactivating lentiviral Gene Transfer and Expression system pLIVc which produces a floxed proviral genome (Detailed information on pLIVc is available on demand). SSXfrag.-V5, mouse TCF4 and V5 tagged β-catenin expression were achieved by transfection using Xtreme gene 9 reagent (Roche) using standard transfection protocols. SiRNAs were transfected using Interferin siRNA transfection reagent (Polyplus). siRNAs used were s13510 and s13512 for SS18 (life technology), sc-35805 for mouse LEF1, sc-43526 for mouse TCF4 (Santa Cruz biotechnology), a pool of Mm-Tcf2a, 1,2,3,4 (QUIAGEN) for TCF3 and esiRNA MU-04762-1 (SIGMA)for β-catenin. The shRNA construct for β-catenin knockdown was as describedExpression of SS18-SSX1-V5, SS18-SSX1-HA, SS18-V5, http://www.unil.ch/dafl/). Gene expression levels were obtained with RMARNA extraction and quality assessment were performed as describedAxin-2 promoter constructs along with a pGL4 renilla luciferase construct using X-tremeGENE 9 DNA transfection reagent (Roche) according to manufacturer’s recommendations. Transfection efficiency was ~60% as assessed using a pMAX-GFP control vector (AMAXA). Reporter firefly and renilla luciferase activity were measured in triplicate 48 hours later on cleared cell lysates using the dual luciferase assay system , according to manufacturer recommendations.Cells were transfected with 2 μg of pGL3 vector or the pGL3-renilla luciferase construct, using X-treme gene 9 transfection reagent. Reporter firefly and renilla luciferase activity were measured in triplicate 48 hours later on cleared cell lysates using the dual luciferase assay system .) as instructed by the manufacturer. After normalization of firefly to renilla luciferase activity data were represented as the ratio of TOP-flash:FOP-flash activity.TOP/FOP-flash luciferase constructs were a gift from Dr Huelsken . 70% confluent cells in 6 well plates were transfected with TOP-flash or FOP-flash plasmid DNA along with a pGL4 4 °C were loaded onto a 5% native gel. After transfer to nitrocellulose identical lanes were separated and each protein separately revealed with appropriate primary and secondary antibodies. Proteins were also revealed in the same lanes using sequences of primary and secondary antibodies that guarantee no cross-reactivity, stripping the nitrocellulose after each detection and using secondary antibodies conjugated to different fluorochromes. Signals were revealed using Fusion FX (Vilbert-Loumat).Cell lysates were prepared in lysis buffer  containing proteases and phosphatase inhibitors by performing 6 cycles of freeze/thawing and shearing DNA with a 22G needle. Equal protein amounts from cleared lysates after centrifugation at 14000 rpm for 10 min at 4 °C. After washing, bound proteins were eluted by the addition of excess V5 peptide, subjected to SDS-PAGE and blotted with anti-HDAC1or anti-TCF4 antibody. For LEF-1 immunoprecipitation cells were lysed in RIPA buffer and incubated with 5 μg of antibody overnight at 4 °C, and then with protein G-sepharose beads. After washing, bound proteins were eluted by boiling in sample buffer and analyzed on SDS-PAGE. After transfer to nitrocellulose mouse anti-V5 and HRP-goat anti-mouse antibodies were used.Cell lysates for immunoprecipitation were prepared as above and incubated with anti-V5 conjugated agarose beads (SIGMA) for 3 hrs at 7 cells were cross-linked with 1% folmaldehyde for 10 min. After addition of 0.125 M glycine and washing in PBS, cells were lysed and the chromatin fraction was sheared to roughly 600 bp fragments by sonication. About 1/15 of the lysate was stored as input DNA. Immunoprecipitation was performed using antibody ab7028 (Abcam) or rabbit immunoglobulins as negative control and herring sperm DNA blocked protein A-sepharose beads. Cross-linkage was reversed using proteinase K and DNA purified by phenol/chloroform extraction and ethanol precipitation. Quantitative PCR on the immunoprecipitate and on input DNA was performed on a ABI Prism 7700 instrument (Applied Biosystems). Primers complementary to several regions of the mouse Axin-2 promoter within residues –1731 to +3268 were designed using Assay Design Center ProbeFinder (Roche). Primer sequences are reported in ChIP for Histone H3 AcK9 was performed according to Abcam protocols . Briefly 102 dishes, fixed in 4% paraformaldehyde in PIPES buffer for 13 minutes at RT and permeabilized with 0.3% triton in PBS for 3 minutes. Primary antibodies were used at the following dilutions: 1:2000 for mouse anti-V5, rabbit anti V5, monoclonal anti-HA, rabbit anti-HDAC1 Ab7028, mouse anti-β-catenin and rabbit anti-β-catenin. Anti-HDAC1 Ab46985 1:100, anti LEF-1 1:400, anti TLE-1and anti TLE1,2,3,4 1:100, anti TCF4:1:1000. PLA amplification was labeled with Alexa Fluor 594 . Slides were counterstained with DAPI, mounted and imaged using the Zeiss Confocal Fluorescent Microscope LSM710, with oil immersion objective 63X, NA 1.4. For each channel the pin hole was set to 0.9 AU. For each sample the Z-stack was acquired with a line averaging of 2 passages and with a sampling in the XYZ according to the optimal Nyquist criteria. Before analysis the Z-stack was converted with maximum intensity projection. The resulting images were analyzed using ImageJ software (http://rsbweb.nih.gov/ij/) with a script that defines as region of interest (ROI) the DAPI stained nuclei and counts the included PLA fluorescent foci  as instructed by the manufacturer. Cells were seeded at 70% confluence in 0.2 cmChART-PCR was performed as previously described with slight modificationsHow to cite this article: Cironi, L. et al. The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma. Sci. Rep.6, 22113; doi: 10.1038/srep22113 (2016).


Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos.Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated ‘flash’ of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.We used fluorescent probes expressed Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.The methods developed in this manuscript provide an alternative protocol to investigate The online version of this article (doi:10.1186/s12860-014-0044-2) contains supplementary material, which is available to authorized users. Aequorea [Nematostella vectensis embryonic and larval/adult cell morphology have mainly been studied using Electron Microscopy (EM) or classic labeling techniques such as in situ hybridization or immunohistochemistry. While these traditional techniques have revealed many important aspects about the cell biology of these animals, they rely on fixed specimens and developmental processes must be inferred by imaging many embryos at specific time points of interest. Imaging using probes fused to fluorescent proteins during developmental processes is a robust technique to visualize dynamic processes in living cells and multicellular structures, with minimal introduction of artifacts created by fixation techniques [Cnidarians and fluorescent proteins have been interconnected since the discovery of green fluorescent protein (GFP) in the jellyfish Aequorea . Today fAequorea -4. CnidaAequorea , gene-knAequorea ,7 througchniques ,9. The uActin and tubulin proteins are essential for a wide range of physiological functions in many organisms, including plants and multicellular animals ,12. Actiin vivo visualization studies [In principal, the broad role of actin and tubulin in a variety of biological processes make them interesting candidates for  studies -19. Howe studies ,20,21 an studies ,23. Give studies ,23-27.in vivo that Lifeact does not inhibit the normal function of actin or associated cellular processes [Lifeact is a 17 amino acid peptide, originally isolated from yeast, which binds with low affinity to filamentous actin (F-actin), associating with a number of actin rich cell structures such as lamellipodia, cilia and flagella . Furtherrocesses . End Binrocesses  and has rocesses ,31.in vivo studies.In the following study, we used targeted probes against F-actin (Lifeact-mTurquoise2), microtubules (EB1-venus), the nucleus (tdTomato-NLS) in comparison to classical histological staining techniques to determine their versatility as structural markers for Nematostella vectensis homologue of the End-binding 1 (EB1) gene (NCBI accession #XP_001641989.1) (cloned using the forward primer – CACCATGGCCGTAA-ACGTATTTTCCACA and reverse primer - GTATTCTTCTTGTTCGAAGTCCCCG). The coding region was then sub-cloned into pSPE3-mVenus, providing the fluorescent tag at the C-terminal of the protein, using the Gateway system [Coding DNA for Lifeact-mTurquoise2 was PCR-amplified from the plasmid pLifeact-mTurquoise2 N1, which was a kind gift from Joachim Goedhart. The PCR product was then cloned in pSPE3-mVenus using the Gateway system . We devey system . FinallymRNA encoding the fusion protein of interest was injected into unfertilized eggs prior to fertilization as previously described  with the4 0.005 M, Dextrose 0.4 M, Triton x-100 0.2%, PBS 1× and distilled water for 1 hour at room temperature. Fixed embryos were rinsed 5× in PBT (PBS buffer plus 0.1% triton X-100). To visualize F-actin, embryos were incubated in Bodipy-FL phallacidin  diluted 1:200 in PBT. Nuclei were visualized by incubation in Hoechst  diluted 1:100 in PBT. Tubulin was visualized by incubation in anti-alpha tubulin (Sigma T6793) in 5% normal goat serum (in PBT). After incubation of the primary alpha tubulin antibody, animals were washed with PBT (5×) for ten minutes each wash. A secondary antibody  was used at 1:500 to allow for visualization. All incubations were conducted over night at 4°C. In experiments where all three probes were visualized, animals were first incubated with the tubulin antibody, washed and incubated with phallacidin, Hoechst and Alexa 594 at the same time. Stained embryos were rinsed again in PBS (5×) and then dehydrated into isopropanol gradient 50%, 75%, 90% and 100% and mounted in Murray’s Mounting Media , for visualization.Cleavage stage embryos were fixed approximately 3 h post fertilization at room temperature in fresh 3.4% paraformaldehyde, HEPES 0.1 M (pH 6.9), EGTA 0.05 M (pH 8–9), MgSOImages of embryos injected with Lifeact-mTurquoise2 mRNA were taken using a confocal Zeiss LSM 510 microscope using a Zeiss Plan-Neofluor, 100× oil immersion objective (N.A. 1.30). The samples were excited at 458 nm for mTurquoise2. Light was guided via D458/514 dichroich mirror to the sample. Fluorescence passed through a ~30 μm pinhole and a 475-505-nm band-pass filter for detection.Images of embryos injected with Lifeact-mTurquoise2 and tdTomatoNLS mRNA were obtained using a confocal Zeiss 700 microscope using a Zeiss Plan-Apochromat, 63× oil immersion DIC objective (N.A. 1.40). The samples were excited at 458 nm for mTurquoise2 and 543 nm for tdTomato. Light was guided via D 405/488/555/639 dichroich mirror to the sample. Fluorescence passed through a ~30 μm pinhole and a 475-525-nm band-pass filter for mTurquoise2 detection and a 585-nm long-pass filter for tdTomato detection, in sequential mode.Images of embryos injected with Lifeact-mTurquoise2 and EB1-mVenus or mVenus (untagged) mRNA were obtained using a confocal Zeiss 700 microscope using a Zeiss Plan-Apochromat, 63× oil immersion objective DIC (N.A. 1.40). The samples were excited at 458 nm for mTurquoise2 and 514 nm for mVenus. Light was guided via D458/514 dichroich mirror to the sample. Fluorescence passed through a ~30 μm pinhole and a 475-525-nm band-pass filter for mTurquoise2 detection and a 520-555-nm band-pass filter for mVenus detection in sequential mode.Images of stained embryos were taken using a confocal Zeiss LSM 510 microscope equipped with a Zeiss Plan-Neofluar, 100× oil immersion objective (N.A. 1.30). The samples were excited at 561 nm for Alexa 594, 514.5 nm for phallacidin and 351 nm for Hoechst. Light was guided via a D UV/488/543/633 dichroic mirror (Chroma). Fluorescence passed through a ~30 μm pinhole and a 585-nm long-pass filter for Alexa-594 detection and a 520-555-nm band-pass filter for phallacidin detection and 385–470 band-pass filter for Hoechst detection in sequential mode.2 before being pipetted directly onto a pre-wetted 0.2um GTTP filter  mounted in a Swinnex filter holder . MgCl2 solution was then replaced with standard EM fixative  and samples were fixed overnight at 4°C. Fixative was removed with three washes in 0.1 M Cacodylate buffer (with 15% sucrose) and samples were post-fixed for 1 h at 25°C in 2% Osmium tetraoxide (in 0.2 M cacodylate buffer) and dehydrated through a graded ethanol series. Filters were then dried in a Samdri critical point dryer  following the manufacturer’s protocol. Dried filters were mounted on stubs using carbon tape and stored under desiccant prior to imaging with a Hitachi S-4800-I field-emission scanning electron microscope at the University of Hawaii’s Biological Electron Microscopy Facility (UH BEMF). For transmission EM, samples were collected in 1.5 ml sterile microcentrifuge tubes and were otherwise fixed as described above. Following the ethanol dehydration series, samples were washed three times (30 mins each) in propylene oxide and infiltrated in a 50:50 mix of propylene oxide and Lx-112 resin  overnight at 25°C, following the manufacturer’s protocol. Polypropylene/resin mix was then replaced with a fresh sample of resin three times the following day  before samples were transferred to silicone molds and polymerized at 60°C for approximately 72 h. Thin sections (~60 nm) were cut using a PowerTomeXL ultramicrotome  and mounted on formvar-coated copper slot grids. For visualization, grids were counter-stained in 5% uranyl acetate and 0.3% lead citrate using standard procedures and imaged on a Hitachi HT7700 (UH BEMF).For scanning EM, embryos or juvenile polyps were relaxed in 7.5% MgClin vivo, we co-injected different combinations of three chimeric constructs .During early development in la stage , which cN. vectensis cell cycle. Figure To better characterize cellular dynamics of Lifeact-mTurquoise2 during a cleavage event, we concentrated on visualizing individual cells with sufficient zoom and time resolution  near the nuclear boundary, we constructed a contour around the nuclear edge for each time point. The average intensity profile perpendicular to the nuclear boundary, normalized with respect to the nucleoplasm intensity was then calculated. The profiles from the cell shown in Figure The ‘flash’ of Lifeact at the nuclear boundary doubles in intensity with respect to background fluorescence in the nucleoplasm and is distinctly visible during a relatively short time window of approximately two minutes. To further validate and corroborate the results found with Lifeact-mTurqoise2 we used classical staining techniques in preserved embryos. Similar to Lifeact-mTurquoise2 labeled embryos, phallacidin staining labels F-actin near the nuclear boundary Figure D,E at a N. vectensis, these structures can be visualized by scanning electron microscopy during early embryonic development  were obtained to determine to what extent long-term visualization would have an effect on normal development of the animal , with little photo-bleaching (<10%) and without any detectable photo-toxicity. We attempted similar long-exposure studies with tdTomato-NLS and Eb1-mVenus but bleaching of the fluorescence protein limited the length of time over which data could be collected , expresses detectable levels of F-actin labeled with Lifeact-mTurquoise2. While the centrosomes form and position themselves adjacent to the nucleus a distinct and robust F-actin “flash” occurs and then the presumptive NE begins to deform and break down. When the spindle fibres connect between the two asters, the nuclear boundary is no longer visible by Lifeact-mTurquoise2 or phallacidin, suggesting it has been dissembled. As the two daughter cells form, F-actin is localized to the cell boundaries of each cell and F-actin returns to the nuclear boundary after the two cells separate.By using a combination of Lifeact and EB1 we were able to observe cleavage events in real time. It is our current hypothesis that the process of cell division in ian taxa . During Lifeact-mTurqoise2 as well as the untagged mVenus used as a control appears to be associated with the cytoplasmic position of centrosomes and mitotic spindles, however this phenomenon was not observed when using phallacidin. Although the levels of free cytosolic Lifeact-mtq2 and/or untagged mVenus are expected to be uniformly distributed in the cytoplasm, one potential explanation could be that the presence of yolk platelets reduces the effective volume for proteins. This effect will occur both for transparent and opaque yolk particles. When formation of tubulin fibres locally pushes yolk platelets away the effective volume that is accessible for fluorescent probes increases, and hence locally increases fluorescence that comes from free Lifeact-mTurqoise2 or free FP (untagged mVenus).Caernorhabditis elegans and Drosophila melanogaster which have genome size of respectively 100 and 180 Mb [N. vectensis (450 Mb) [N. vectensis may support this hypothesis.Compartmentalized F-actin along the nuclear boundary is not always clearly detectable by actin staining using common fixation methods. With the methods developed here, the rapid accumulation of F-actin prior to nuclear disassembly could be clearly and robustly observed. To date, this phenomena has been described in the sand dollar (Echinodermata)  and polyd 180 Mb , conside(450 Mb) . If openin vivo cell cleavage in embryos, this technique also allows for visualization of F-actin throughout all stages of development. We observed F-actin in specific cellular structures such as microvilli, cnidocytes, ciliary cones in ciliated cells, and the plasma membrane. Furthermore, lifeact-mTurqoise2 allows for observation of real-time cell boundary dynamics and muscular contractions during animal movements in the adult polyps. Having the ability to trace cells in vivo will help validate modeling studies of cell mechanics [Beyond the advantages of using Lifeact-mTurquoise2 to study echanics .in vivo technique to follow cell cleavage during embryogenesis in Nematostella vectensis. The fluorescent probe, Lifeact-mTurquoise2, utilized in this manuscript provide a means to visualize specific cell types and structures that contain F-actin during development. One striking observation present during cellular cleavage was a ‘flash’ of Lifeact prior to nuclear disassembly. The presence of nuclear disassembly suggests that cell division in Nematostella vectensis goes through open mitosis, similar to many bilaterian taxa. The techniques developed in this study make Nematostella a more attractive model system for comparative developmental and cellular biology.Methods developed in this manuscript provide an


Upon implantation of the cells in the mammary fat pad of immunocompromised mice, we found that β-catenin shRNA HCC38 cells formed markedly smaller tumors than control cells and grew much more slowly. In in vitro assays, β-catenin silencing significantly reduced the percentage of Aldefluor-positive cells, a read-out of the stem-like cell population, as well as the expression of stem cell-related target genes including Bmi-1 and c-Myc. β-catenin-knockdown cells were also significantly impaired in their ability to migrate in wound-filling assays and form anchorage-independent colonies in soft agar. β-catenin-knockdown cells were more sensitive to chemotherapeutic agents doxorubicin and cisplatin. Collectively, these data suggest that β-catenin is required for triple-negative breast cancer development by controlling numerous tumor-associated properties, such as migration, stemness, anchorage-independent growth and chemosensitivity.Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether β-catenin was necessary for tumorigenic behaviors  Triple-negative breast cancer (TNBC), including basal-like breast tumors, is an aggressive tumor subtype that is distinguished from other breast cancer subtypes by its gene expression profiles, histopathological features, and clinical characteristics –3. Thesecyclin D1 [c-Myc [Bmi-1 [Mmp-7 [Axin2 [in vitro and in vivo. In the present study, our group analyzed the role of β-catenin in the tumorigenic properties of TNBC cell lines to address its potential utility as a therapeutic target in this tumor type.The canonical Wnt signal transduction pathway is activated in several tumor types, including colorectal and breast cancer –8. Amongyclin D1  and c-My1 [c-Myc , stem cec [Bmi-1 , matrix 1 [Mmp-7 , and Wnt7 [Axin2 . Our lab7 [Axin2 –17. More7 [Axin2 . We sougAll procedures were performed with prior approval (protocol #71828) from the University of Chicago Institutional Animal Care and Use Committee, which has full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care.MDA-MB-231 and HCC38 cell lines were from ATCC and cultured in RPMI 1640 media  supplemented with 10% FBS and 1% penicillin/streptomycin. The stable knock down lines of MDA-MB-231 and HCC38 were established by infection of MISSION shRNA Lentiviral Transduction Particles against CTNNB1  and selected with puromycin (1ug/ml).2, 5 mM EDTA and protease inhibitors and sonicated. 25 µg of lysate from each cell line was separated on a 10% SDS-PAGE gel and transferred to Immobilon P membrane (Millipore). Blots were probed with 1:1000 anti-β-catenin monoclonal antibody (BD Biosciences), 1:1000 anti-active (unphosphorylated) β-catenin (EMD Millipore) and 1:1000 anti-actin monoclonal antibody (Sigma) in blocking buffer (5% nonfat dried milk in TBST).Total cell lysates were extracted with 50 mM Tris pH7.5, 0.1% IGEPAL, 100 mM NaCl, 1 mM MgCl4 cells/well and transfected using Optifect (Invitrogen) with 2.6 μg of pTOPflash or pFOPflash (obtained from H. Clevers at the Hubrecht Insitute) along with pRL-TK (Promega) for determination of transfection efficiency. Lysates were harvested after 48 hrs and analyzed using the Dual Luciferase Assay System kit (Promega). Luciferase activity was normalized for transfection efficiency and graphed as ratio of TOPflash/FOPflash activity. Mutant β-cateninS37A was from Stephen Byers at Georgetown University. The breast cancer lines were transfected with stabilized mutant β-cateninS37A as described previously [Cells were plated in a 24-well plate at a density of 1x10eviously . Express3 cells/well. Cell viability was assessed by adding 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to the culture medium and incubated for 4 h at 37 °C. The optical density was measured at 490 nm using Synergy 2 plate reader . Cell migration was measured by wound healing assays as described [5 cells per well in a 6-well plate were grown to confluence, at which time they were scratched with a micropipette tip. Phase-contrast images were captured with a Zeiss Axiovert 35 inverted microscope using AxioVision REL 4.6 imaging software (Zeiss) every 12 hr. The area of three representative wounds for each cell line was quantified at each time point using Image J software (NIH). Anchorage-independent growth soft agar assays were performed by seeding 4 x 103 cells in 0.3% agarose as described [Cell growth was analyzed with MTS assay using CellTiter 96 AQueous One Solution Cell Proliferation Assay  following the protocol. Cells were seeded in 96-well culture plates at a density of 5×10escribed . Brieflyescribed . The col5 cells were stained with ALDEFLOUR reagent or ALDEFLOUR reagent plus ALDEFLOUR inhibitor. Stained cells were sorted by the University of Chicago Flow Cytometry Core Facility.ALDEFLOUR assays (Stemcell) were used. 5x10c-Myc: Forward 5’-CAG CTG CTT AGA CGC TGG ATT T-3’; Reverse 5’-ACC GAG TCG TAG TCG AGG TCA T-3’.The primer sequences for Axin2: Forward 5’-CTG GCT TTG GTG AAC TGT TG-3’; Reverse 5’- AGT TGC TCA CAG CCA AGA CA-3’. The primer sequences for Bmi-1: Forward 5’-CTC CCA ACT GGT TCG ACC TT-3’; Reverse 5’-CGG TTT CCA TAT TTC TCA GT-3’. The primer sequences for ribosomal RNA 18S has been reported [-∆∆Ct method.Gene expression changes were analyzed by real-time RT-PCR as described  except r6 parental, scrambled control shRNA or β-catenin-knockdown cells were injected with Matrigel (BD Biosciences) into the mammary fat pad of Nu/Nu female mice (n = 10 per group). Tumors were measured biweekly with calipers after implantation, and the animals were sacrificed 11 weeks later. Tumor volume was calculated as described previously [5 x 10eviously . After eβ-catenin-knockdown and control HCC38 cells were treated with 0.5 μM doxorubicin or 3.0 μM cisplatin for 24 hrs and subjected to an Caspase 3/7 Glo assay . Relative sensitivity was shown as β-catenin-knockdown over control.All experiments were performed at least in triplicate, and statistical significance was defined as a p value of <0.05 using a one-way ANOVA with a Student’s t-test post-test, unless noted otherwise. For quantification, means are shown with standard deviation (SD).in vitro models of TNBC that recapitulate some of the features of human tumors with respect to Wnt signaling, several breast cancer cell lines were analyzed for total and “activated”, or non-phosphorylated, β-catenin by Western blotting. Total and “activated” β-catenin were expressed at high levels in HCC1937, UACC3199 and HCC38 TNBC cell lines compared to the ER-positive MCF-7 and another TNBC line MDA-MB-231 . As shown in in vivo.We next tested the requirement for β-catenin in the Finally, the impact of Wnt/β-catenin signaling on the chemotherapeutic response of TNBC cells was investigated. Specifically, the sensitivity of β-catenin-knockdown cells was compared to that of control cells each treated with 0.5 μM doxorubicin or 3.0 μM cisplatin, and we found that the β-catenin-knockdown cells were more sensitive to doxorubicin or cisplatin-induced cell death after 24-hour treatment compared to control shRNA cells . The effin vitro and tumorigenesis in immunocompromised mice. In addition, the responsiveness of TNBC cells to chemotherapeutic agents, such as doxorubicin and cisplatin, is dependent on β-catenin and Wnt pathway status.We have previously shown that β-catenin-dependent Wnt signaling is enriched in TNBCs and is associated with reduced overall survival in breast cancer patients . In the Apc-driven cancer. In a mouse model, Apc1638N, with reduced β-catenin levels, female mice developed mammary tumors instead of gastrointestinal tumors [et al. reported that Wnt signaling was not required for the proliferation of SUM1315 cells in culture as cells overexpressing the Wnt inhibitors SFRP1, DKK1, or shRNA against LRP6, grew at similar rates in vitro compared with SUM1315 control cells [et al. demonstrated that ectopic SFRP1 expression caused a reduction of motility in MDA-MB-231 cells [It has been reported that β-catenin signaling dosage dictates tissue-specific tumor predisposition in l tumors . We founol cells . Consisthigh/CD24low [high/CD24low stem cell population in primary TNBC/basal-like breast cancers [Bmi-1 and c-Myc, were down-regulated in β-catenin-knockdown cells compared to control cells. The epithelial-mesenchymal transition (EMT) promoter and Wnt target gene Axin2 [Wnt/β-catenin pathway activation has been implicated in stem cell self-renewal, maintenance, and differentiation , 24–26. /CD24low  and ALDE/CD24low . We and  cancers , 29. In ne Axin2 , 30 was Because TNBCs lack treatment targets due to its triple-negative nature, chemotherapy is the standard treatment. Doxorubicin has been widely used to treat breast cancer especially TNBC , 32. In S1 FigMDA-MB-231 (A) and HCC38 (B) cells were seeded in 96-well plates and cell proliferation was measured with MTS assay for 96 hrs. Bar represents means ± SD.(TIF)Click here for additional data file.


Bone sarcomas such as osteosarcoma and chondrosarcoma are frequently refractory to conventional chemotherapy and radiotherapy that exhibit poor prognosis. The Wnt signaling are evolutionarily conserved and implicated in cell proliferation and sarcomagenesis. However, the potential role of the Wnt signaling in bone sarcomas is still unclear. Here we demonstrate aberrant activation of Wnt/β-catenin signaling in bone sarcoma cells, involving an autocrine Wnt signaling loop with upregulation of specific Wnt ligands and receptors. Activation of Wnt/β-catenin signaling with Wnt3a or GSK-3β inhibitor drives the proliferation of bone sarcoma cells, whereas downregulation of activated Wnt signaling with dnTCF4 or siLEF1 suppresses bone sarcoma proliferation and induces cell cycle arrest. Taken together, our findings establish the evidence that aberrant activation of Wnt/β-catenin pathway involving an autocrine Wnt singaling drives the proliferation of bone sarcoma cells, and identify the autocrine activation of the Wnt/β-catenin signaling as a potential novel therapeutic target for bone sarcomas. Osteosarcomas and chondrosarcomas are recognized as the most common primary bone sarcomas , which i2+ pathway [The WNT signaling pathways are evolutionarily conserved and implicated in multiple crucial physiological and pathophysiological events . To date pathway , 8. In t pathway . Emergin pathway . Intrigu pathway , and fee pathway , 11. DKK pathway , disrupt pathway , 13. Ano pathway . Further pathway , 16. AllAbnormalities in Wnt signaling are implicated in a variety of human tumors by mechanisms such as inactivating aberrations of APC, CTNNB1, or Axin1 (deletion) , 18, 19,The Wnt signaling is mediated through the β-catenin dependent canonical pathway and the β-catenin independent pathway -8. To idNext, we analyzed the expression of the Wnt receptors and co-receptors in hMSC and bone sarcoma cell lines by Real-time PCR. We found that FZD3, FZD5, FZD9 and FZD10 were prominently expressed in all bone sarcoma cells as compared to hMSC Figure . LRP6, LPreviously, we have presented the evidence that DKK1 levels were remarkably elevated in chondrosarcoma specimens and DKK1 suppressed canonical Wnt/β-catenin signaling in human chondrosarcoma SW1353 cells . Thus, tIn an effort to independently confirm the existence of an autocrine Wnt loop in bone sarcoma cells, we generated siRNAs directed against LRP6 that was markedly higher in bone sarcoma cells as shown in Figure Having identified bone sarcoma cells with autocrine Wnt signaling activation, we analyzed the functional effects of autocrine Wnt signaling inhibition on the phenotypes of sarcoma cells. As shown in Figure In order to assess whether human recombinant Wnt3a treatment affects β-catenin, in particular its intracellular redistribution, we performed western blot analysis and found that Wnt3a stimulation resulted in nuclear translocation of a fraction of β-catenin with fractioned extracts, while the amount of control nuclear protein hnRNP remained unchanged Figure . After WThe phosphorylation of β-catenin by GSK-3β marks it for ubiquitination and degradation . Thus, iTo further address the biological effects of upregulated canonical Wnt signaling in Wnt autocrine SW1353 and U2OS cells, we transduced sarcoma cells with dnTCF4 or siLEF targeting TCF/LEF signaling. Using this strategy, we observed approximate 50% inhibition of TCF reporter activity Figure  associatOur present findings have established that constitutive activation of the canonical Wnt/β-catenin signaling in human bone sarcoma cells, by a novel mechanism involving an autocrine Wnt signaling loop, drives the proliferation of bone sarcoma cells. Stimulation of Wnt/β-catenin signaling with Wnt3a or GSK-3β inhibitor enhanced the proliferation and survival of bone sarcoma cells, whereas antagonism of this pathway with dnTCF4 or siLEF1 had the exact opposite effect. These data suggest that aberrant activation of Wnt/β-catenin signaling involving an autocrine Wnt signaling loop is frequent event in bone sarcomas and drives cell proliferation. Thus, disruption of autocrine activation of Wnt/β-catenin signaling could be a novel attractive therapeutic target for bone sarcomas.Previous studies have documented that hMSCs exhibit a low level of endogenous Wnt signaling and can differentiate into osteogenic and chondrogenic lineages , 25. HowSeveral studies have illuminated the antagonism and cross-talk between the canonical and noncanonical Wnt pathways , 37. HowOur previous studies have documented that β-catenin was accumulated and increased in chondrosarcoma tissue and SW1353 cells without the exploration of potential mechanisms . In thisOur current study indicates that aberrant Wnt signaling drives the proliferation of bone sarcoma cells and could represent an important step in the pathogenesis of bone sarcomas. As shown in Figures. in vitro and in vivo [Accumulated data suggest that the Wnt signaling exerts its oncogenic functions via upregulation of target genes involved in cell proliferation, exemplified by the β-catenin/TCF transcriptional effects on c-Myc expression in carcinomas , 21, and in vivo . TherefoIn summary, the data presented in this study implicate the aberrant activation of canonical Wnt/β-catenin signaling pathway involving an autocrine Wnt signaling loop in the pathogenesis of bone sarcomas. Autocrine activation of the canonical Wnt/β-catenin signaling drives the proliferation of bone sarcoma cells, whereas antagonism of this pathway decreased the proliferation and induced cell cycle arrest. Our findings have implied that aberrant activation of the Wnt/β-catenin pathway drives the proliferation of bone sarcomas and identify the autocrine Wnt/β-catenin signaling as a potential novel therapeutic target in bone sarcomas.2 at 37°C.Human osteosarcoma U2OS and Saos-2 cell lines, and chondrosarcoma SW1353 cell line were obtained from the American Type Culture Collection  and grown according to ATCC recommended media and conditions. Human mesenchymal stem cells (hMSCs) were obtained from Lonza and expanded in basal medium according to the manufacturer's instructions. All cells were maintained in a humidified cell incubator with 5% CORecombinant human Wnt3a protein and DKK1 protein were purchased from R&D Systems . The GSK-3β inhibitor SB-216763 was obtained from Sigma. Antibodies for detection of the following targets were purchased as indicated: total β-catenin and GSK-3β from BD Biosciences, active β-catenin  dephosphorylated on Ser37 or Thr41 from Millipore Corporation , Cyclin D1 and c-Myc from Santa Cruz Technology, Axin2, TCF4 and LEF1 from Cell Signaling Technology, LRP6 and hnRNP from Abcam, and β-actin from Sigma.Total RNA was isolated using Trizol reagent . Total RNA (2 μg) was used for the synthesis of first-strand cDNA using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using the SYBR green mix (Applied Biosystems). The reactions were performed with a 7500 Fast Real-Time PCR System (Applied Biosystems). The data were displayed as 2−ΔΔCt values and were representative of at least three independent experiments. Sequences of the qRT-PCR primers were presented in Table Detailed experimental procedures for protein extraction and western blot analysis have been previously described . Briefly5 per well in 6-well plates were cotransfected with 0.2 μg of either the TOP-flash or FOP-flash plasmids (Upstate Biotechnology) and 0.02 μg of the Renilla control plasmid (pRL-TK) utilizing Lipofectamine 2000 (Invitrogen). Cells were harvested at 48 h after transfection, and were lysed and analyzed using the Dual Luciferase Reporter Assay System  according to the manufacturer's protocol. Luciferase reporter activity was calculated by dividing the ratio TOP/RL by the FOP/RL ratio.After treatment, cells plated at 3×10Cells were seeded in triplicate on 96-well plates and incubated with complete medium for 24 h. Quiescent cultures were treated with daily supplemented medium. After treatment, cell proliferation was determined by a Cell Proliferation ELISA, BrdU kit  as per the manufacturer's instructions. Each point represents a mean value and standard of three experiments with three replicates.SW1353 and U2OS cells were incubated with 200ng/ml Wnt3a for different time points . Cells were then added to the MTS solution according to the manufacturer's protocols for growth assay using the MTS kit (Promega) and the absorption was read at 490 nm. Cell survival was calculated as follows: cell survival = absorbance of test group/absorbance of control group.5 cells per well) and cultured overnight. After treatment, cells were trypsinized, washed with cold PBS, and resuspended in 100 μl of binding buffer containing 5 μl of FITC-conjugated AnnexinV and 5 μl of PI according to the manufacturer's instructions. After 20 min at room temperature in the dark, 400 μl of binding buffer was added, and samples were immediately analyzed on a FACS Calibur flow cytometer  and analyzed with Cell Quest Software . The results shown are representative of at least three separate experiments.SW1353 and U2OS cells were stained with fluorescein isothiocyanate (FITC) labeled AnnexinV that binds to membrane phosphatidylserine and with propidium iodide (PI) that binds to cellular DNA according to the manufacturer's instructions . Flow cytometry was used to discriminate between intact and apoptotic cells. Briefly, SW1353 and U2OS cells were seeded in 24-well plates (1×105 cells per well) and cultured overnight. After treatment, cells were trypsined, then fixed with 75% ethanol at −20°C for 3 hours. After fixing, cells washed with PBS, then stained with 0.5 ml PI/RNase staining buffer  for 30 minutes at room temperature in the dark. Cell cycle distribution was analyzed by FACS Calibur flow cytometer  and analyzed for cell cycle phases with ModFit LT 3.2 Software . The results shown are representative of at least three separate experiments.The percentage of cells in G0/G1, S and G2/M phases was determined by DNA flow cytometry. Briefly, SW1353 and U2OS cells were seeded in 24-well plates . Transient transfection of cells was performed using the Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions.t-test. Statistical analysis was performed using SPSS  and the difference is considered significant when a P value is less than 0.05.All experiments were performed independently at least three times, and the results were expressed as the Mean ± SD. Differences between groups were analyzed using the unpaired two-tailed Student's