It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity. It has become increasingly apparent that each tissue has a characteristic ‘stiffness phenotype’. All cells in tissues and organs are exposed to ECM stiffness and specifically tuned to the stiffness of the particular tissue in which it resides134567910per se, but caused by the accumulation of β-catenin induced by the activation of integrin/ focal adhesion kinase (FAK) pathway. β-catenin in turn activated the expression of Wnt1 by binding to the promoter region of wnt1 gene and promotes the gene transcription. The integrin-activated β-catenin/Wnt pathway connects with canonical Wnt/β-catenin pathway to form a positive feedback loop, which is crucial to the promotion of Wnt signal by stiff ECM and the regulation of mesenchymal stem cells differentiation and primary chondrocytes phenotype maintenance.Recent reports have shown that Wnt signaling is responsive to matrix rigidityin vitro are sensitive to the elasticity of the substrate coated by type I collagen (ColI), a widely used ECM mimic in the study of the effect of substrate stiffness on many cell types14Cultured chondrocytes for implantation or engineered cartilage on scaffolds are potential therapies to the articular cartilage repair. Previous findings indicated that chondrocytes cultured In vivo chondrocytes were embedded in pericellular matrix (PCM) of which the mechanical property is crucial in the environment of the chondrocytewnt1, wnt3a and canonical Wnt/β-catenin pathway target genes, were up-regulated by the stiff ECM in comparison with the soft ECM, at both mRNA  and matrigel, two frequently used ECM mimics in engineered cartilage, to modify the PAAM gel. The stiffness of the ColII or matrigel-coated substrates also up-regulated the levels of Wnt1 and β-catenin in chondrocytes, as ColI . These rTo evaluate the effect of Wnt signals on cells cultured on substrate with different stiffness, we assayed the β-catenin protein level of the cells treated with WIF-1, sFRP1, or Wnt1. Neither inhibition (with WIF-1 and sFRP1)21wnt1 gene promoter region (wnt1 promoter sequence flanking the putative TRE (wnt1 promoter region on the stiff ECM (Bioinformatics study predicted a β-catenin /TCF-responsive element (TRE) in the mouse r region . LiCl trr region . Chromattive TRE . Quantittiff ECM . These rWe then proceeded to investigate the molecular mechanism by which stiff ECM triggers the Wnt-independent activation of β-catenin.In light of the promoting effects of stiff ECM on cell membrane integrin activityAs an important downstream element of integrin signals, FAK/Akt pathway is well documented as a regulator of GSK3β. Τhe stiff ECM significantly induced the phosphorylation levels of both FAK  and Akt 26Finally, BIO, an inhibitor of GSK3β, significantly diminished the influence of ECM stiffness on Wnt1 expression, as well as β-catenin accumulation . These rWe then explored the effect of the integrin-activated β-catenin/Wnt pathway on the functional responses of primary chondrocytes and bone marrow mesenchymal stem cells (BMMSCs) to ECM stiffness. Chondrocytes, which are surrounded by soft (1–2 kPa) pericelluar matrix ECM, demonstrate compliance-dependent behavior15It is well recognized that changing ECM stiffness has profound impact on the linage specification of stem cellsThe present work demonstrates that the integrin/FAK-activated β-catenin/Wnt pathway plays crucial roles in the mechanical sensing processes of chondrocytes and stem cells. We have previously shown that ECM stiffness modulates stem cell linage commitment through a BMP-dependent pathway, which is known to crosstalk with Wnt signalswnt1, wnt2b, wnt3a, wnt6 were significantly regulated by the substrate stiffness. Previous study demonstrated that Wnt1, Wnt2b and Wnt3a can regulate chondrocyte differentiation and maturation through the canonical β-catenin pathway, while Wnt6 mediated chondrocyte differentiation through Wnt/Ca2+ signaling1334Wnt proteins are a large family of 19 secreted glycoproteins that trigger multiple signaling cascades essential for embryonic development and tissue regenerationwnt1 transcriptional mechanism, in which β-catenin regulates wnt1 expression via binding to the promoter region. These results suggest that the canonical Wnt/β-catenin pathway contribute to stiff ECM inducing β-catenin accumulation, thus synergizing with the β-catenin-induced transcription of wnt1 and form a positive feedback loop.There is substantial knowledge on the signaling cascade downstream to Wnt stimulation; however, the control of production of Wnt proteins has remained an essentially uncharted territory36β-catenin/Wnt has been found to be an auto-regulated pathway at many other levels. The expressions of Wnt canonical receptors, LRP and Frizzleds have been reported to be controlled by β-catenin/TCF complex and contribute to the positive feedback loop of β-catenin/Wnt pathway13in vitro and in situ. Considering that α1β1 and α10β1 were collagen-binding integrins, we used α1β1 and α10β1 siRNA, respectively, to study the role of integrins in the regulation of wnt signaling. Our results showed that integrin is activated on stiff ECM to phosphorylate downstream elements, leading to GSK3β-phosphorylation. As a result, β-catenin is prevented from degradation and translocates into the nucleus to bind with the wnt1-promoter to start the β-catenin/Wnt1 feedback. By enhancing or amplifying Wnt signals, the positive feedback loop initiated by integrin/FAK in response to stiff ECM may provide a key process for the activation of the β-catenin/Wnt pathway.The integrin family is composed of 24 αβ heterodimeric members that involved in processes of cell-cell and cell-extracellular matrix adhesion and signal transduction. Integrin triggers a cascade of signaling pathways, including FAK, integrin-linked kinase, PKC, and YAP/TAZ phosphorylation, which are closely associated with osteogenic cell differentiation, bone formation and repair. The key integrin ligands in osteoblastic or osteocytic cells include αvβ1, αvβ3, α2β1, α4β1, α5β1. Articular chondrocytes were found to predominately express α1β1, α3β1, α5β1, and α10β1 integrins both The downstream regulatory pathways of integrin may branch off at many steps. For instance, we observed that the inhibition of Akt only partially block the effect of ECM stiffness on β-catenin pathway. The up-regulation of ERK on stiff ECM and the co-inhibiting effect of ERK and Akt suggest co-regulatory effects of them on the integrin-initiated β-catenin/Wnt feedback on stiff ECM. However, the blocking experiments on integrin-linked kinase, PKC, and YAP/TAZ showed that these elements have no significant impact on the stiffness-induced difference of β-catenin signaling.Taken together, mechanical stimuli and Wnt signals play significant roles in development, adult tissue homeostasis, and diseases413342. Primary BMMSC culturing was performed according to Tang et al.2.Primary mouse knee chondrocytes were isolated and cultured from one-day-old Kunming mice. In brief, the articular cartilage was isolated and chondrocytes were extracted by collagenase digestion. After configuring the chondrocytes were plated in monolayer culture in DMEM high glucose supplemented with 10% FBS, 1% glutamine, 1% penicillin-streptomycin at 37 °C in 5% CO2.MC3T3-E1 osteoblastic cells were bought from the Cell Center in School of Basic Medicine of Peking Union Medical College and cultured in DMEM medium with the addition of 10% FBS, 2 mmol/L L-glutamine and 1% penicillin-streptomycin, at 37 °C and 5% COAll animal experiments were approved by the guidelines of the Animal Care and Experimentation Committee of Tsinghua University, China and in accordance with the approved guidelines of Tsinghua University.et al.5 Pa, respectively.Polyacrylamide gels with variable Young’s moduli were prepared according to a previously established protocol by Engler ® System . We filtered the spots in which flag = −50 within all arrays and control probes. Probes passed filter were normalized by median scaling to remove systemic effect, then combine the duplicate data by averaging their intensity value. The Matrix value is normalized intensities. The normalized intensities and pairwised comparison were performed by Rosetta Resolver® System error-weighted modeling to find significant diffrential expressed probes (log2 |Fold change| ≥ 1 and P < 0.05). Data have been deposited at the NCBI gene expression omnibus  with GEO accession number GSE63615.Microarray analyses were performed using commercial Mouse cDNA Microarray slides  according to the manufacturer’s instructions. Briefly, cells were collected and total mRNA was isolated with Trizol . The concentration, purity, and integrity (RIN) of the RNA extract were determined by spectrophotometry (Nanodrop 1000) and the Agilent RNA 6000 Nano assay, respectively. aaRNAs were amplified using Amino Allyl MessageAmp™ II aRNA Amplification Kit  from 1 μg total RNA and directed labeled by NHS-CyDye  to uridine (aa-dUTP). The pre-hybridization of MOA v2 arrays were rehydrated by 100% ethanol following with deionized water. After the pre-hybridization, Cy5-labeled aaRNA was hybridize on MOA v2 in duplication (two arrays for each sample) at 50 °C for 16 hours in the presence of the Phalanx OneArray hybridization buffer. The arrays were scanned by Axon 4000B scanner , and the fluoresence intensity was quantified. The data processing was carried out by Rosetta ResolverBriefly, samples of proteins from cytoplasm, nucleus, or whole cell were prepared as previous description® ep realplex  using SYBR® Green Realtime PCR Master Mix . The primers are for mouse wnt1 , mouse GAPDH .Cells were collected and total mRNA was isolated using Trizol . Real-time PCR quantitative mRNA analyses were performed with a MastercylerCells were cultured for 4 d on the stiff or soft gel-coated slides, and fixed with 4% paraformal- dehyde. After blocking in blocking buffer  for 30 min at room temperature, the slides were incubated with primary antibodies against Sox9 , Aggrecan , ColII , Runx2 (1:100), ColI , MAP2  , Nestin  or NFL , then with anti-mouse IgG conjugated with FITC , anti-rabbit IgG conjugated with TRITC  or anti-goat IgG conjugated with TRITC . Finally, cell nuclei were visualized with DAPI  and viewed under a Leica TCS SP5 confocal microscopy system .The assay was done according to instruction of Upstate ChIP assay kit . Briefly, primary chondrocytes were treated for 24 hr with 20 mM LiCl or NaCl and then cross-linked with 1% formaldehyde at 37 °C for 10 min. After centrifugation at 4 °C for 4 min at 2000 rpm, the cells were suspended with lysis buffer and sonicated three times. The supernatant was collected and diluted with ChIP dilution buffer, followed by immunoclearing with salmon sperm DNA/protein A agarose at 4 °C for 1 hour. Then, anti-β-catenin antibody (Santa Cruz Biotechnology) was added to immunoprecipitate the chromatin overnight. The immunoprecipitation complex was eluted and heated at 65 °C for 4 hours. After digestion with proteinase K, DNA was obtained by phenol/chloroform/isoamyl alcohol extraction and used as PCR template.Negative control pGL3-Basic was purchased from Promega . To construct the plasmid pWNT, a 679-bp fragment containing the putative binding site of β-catenin in WNT promoter  was generated by PCR amplification with the primer pair: forward 5′-GCACTCGAGCAGAGGAGACGGACTTCGAG-3′, and reverse 5′-TACAAGCTTGAGCGGTCAGTGCCAGTAGC-3′, and cloned into the pGL3-Basic. The plasmid pWNTm that contains mutagenesis of the putative binding site in WNT promoter was generated by site-directed mutagenesis PCR using the plasmid pWNT as a substrate. The 6 bp of WNT promoter (bp −352 to −347) was deleted with the following mutagenic primers: 5′-G-GTTAGCCTGTCAGCTTCAGACCGGCAAGA-3′, 5′-TCTTGCCGGTCTGAAGCTGACAGG-CTAACC-3′. Transfection of the reporter constructs into MC-3T3 cells was performed with Lipofectamin 2000  according to the manufacturer’s instructions.2 incubator.At 24 hr before transfection, chondrocytes were plated onto six-well culture plates and transfected with siRNAs at 70–80% confluence. siRNAs for β-catenin was purchased from Santa Cruz Biotechnology (sc-29210). siRNAs for Intigrin β1, Intigrin α1, Intigrin α10, FAK, ILK, YAP/TAZ and control scrambled siRNA were purchased from GenePharma. Chondrocytes were washed in PBS and resuspended in resuspension buffer R included with Neon™ kit (Invitrogen). Transfections of siRNA into chondrocytes with Neon Transfection System (Invitrogen) were performed according to the manufacturer’s protocol with two pulses of 1400 V and 20 ms. Transfection efficiency was assessed using qPCR and western blot (Data not shown). Eletroporated cells were resuspended in culture medium containing serum and supplements without antibiotics and incubated for 24 hr at 37 °C in a humidified 5% COMC-3T3 cells cultured in 24-well plates were transiently transfected with a mixture containing 1 μg of reporter plasmid and 5 ng of pRL-CMV. After 6 hous of transfection, the medium was changed and the cells were treated with 20 mM LiCl or NaCl for 18 hours. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Kit . The firefly luciferase values of each sample were normalized by Renilla luciferase activity and data were reported as relative light units.Wnt1, Wnt3a and WIF were purchased from Abcam . sFRP1 was purchased from Sino Biological Inc. BIO, Akti-1/2, Cardamonin, NaCl, LiCl and DMSO were purchased from Sigma-Aldrich . PMA was purchased from Santa Cruz Biotechnology . PF573228 was purchased from Tocris Bioscience . Anti-integrin β1 (clone HM β1-1) and control IgG (clone HK888) were purchased from Biolegend .t-test was used to compare differences between two experimental groups. ANOVA analysis was used when the comparison was made using any group more than once. When a significant difference was found by ANOVA, the Student– Neumann–Keuls method was used post hoc to determine the significance of differences between groups.Data are presented as mean ± SEM. Student’s How to cite this article: Du, J. et al. Extracellular matrix stiffness dictates Wnt expression through integrin pathway. Sci. Rep.6, 20395; doi: 10.1038/srep20395 (2016).


Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis. Diffuse large B-cell lymphomas (DLBCL) account for the largest number of non-Hodgkin lymphomas, which were classified into two major sub-entities: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) DLBCL.6 Activating upstream mutations have been detected in the BCR adaptors CD79A and CD79B (~21% of ABC cases) or the innate immune adaptor MYD88 (~30% ABC cases).7 Also, inactivating mutations in the tumor suppressor A20, a negative regulator of NF-κB signaling, have been found in ABC DLBCL.8 About 10% of ABC DLBCL and ~4% of GCB DLBCL patients carry gain-of-function mutations in the scaffold protein CARMA1/CARD11.10 Under physiological conditions, CARMA1 undergoes a phosphorylation-induced conformational change to recruit BCL10-MALT1 upon antigen stimulation in B and T cells.11 Oncogenic mutations are all localized within the coiled-coil (CC) domain of CARMA1 and are acting presumably by changing the conformation of the CARMA1 scaffold to allow stimulus-independent recruitment of BCL10-MALT1 and thus permanent CBM assembly.12 Furthermore, CARMA1 mutations render ABC DLBCL cells resistant to inhibition of upstream kinases like SYK, BTK or PKCβ.14 Thus, quite in contrast to CD79A/B mutations, growth of CARMA1 mutated ABC DLBCL does no longer rely on a functional BCR, which underscores the potency of this oncogene.3The key role of constitutive NF-κB activation in ABC DLBCL cells is confirmed by recurrent somatic mutations.in vitro model system to analyze the function of oncogenes.15 We found a robust recruitment of the β-catenin destruction complex and stabilization of β-catenin in oncogenic CARMA1-transduced BJAB as well as in ABC DLBCL cell lines. In most cells, β-catenin is constantly degraded in the cytoplasm, but β-catenin stabilization upon WNT signaling promotes its function as co-activator of TCF/LEF transcription in the nucleus.16 Deregulations in WNT signaling and enhanced β-catenin amounts are found in many human cancers including hematologic malignancies.18 We show here that stabilization of β-catenin by oncogenic CARMA1 engages a novel cross-talk between NF-κB and WNT pathways in DLBCL that can contribute to ABC DLBCL lymphomagenesis.As CC mutations are thought to affect the scaffolding function of CARMA1, we took a mass spectrometry approach to search for novel interaction partners of active CARMA1 in BJAB cell system that provide an 9 Two ABC-derived (L244P and S243P) and two GCB-derived (F123I/K208M and L225LI) CARMA1 mutants were expressed in the GCB DLBCL cell line BJAB that lacks chronic BCR signaling and constitutive NF-κB activation. Lentiviral transduction led to consistent infection rates of >95% as determined by fluorescence-activated cell sorting staining of the co-expressed human ΔCD2 surface marker  and the four oncogenic CARMA1 mutants. Three independent experiments were performed yielding a list of 33 CARMA1 interaction partners that were found consistently and significantly in three independent experiments to associate with at least one CARMA1 construct . Althoug3I/K208M .Pathway analyses revealed one major interaction map that strongly associated with CARMA1 L225LI and more weakly to all other CARMA1 mutants consisting of proteins functionally linked with WNT/β-catenin network . The intUnphosphorylated β-catenin was detected in CARMA1 L225LI precipitations, indicating that active CARMA1 may also exert effects on the WNT/β-catenin pathway . In comp21 However, the recruitment of other WNT signaling components was unexpected and we therefore verified the interaction to CARMA1 L225LI by reciprocal IP of endogenous CK1α, β-catenin and GSK3β  that drives NF-κB signaling through the removal of the negative regulatory linker region .22 Expretivation . To addr1 mutant . In line1 mutant .21 Moreo-catenin .24 Mutation of R35A completely abolished inducible and constitutive BCL10 recruitment and NF-κB activation in the context of CARMA1 WT or CARMA1 L225LI, respectively  DLBCL cell lines showed low β-catenin expression, most ABC DLBCL cell lines showed slightly  to severely  increased β-catenin expression  that are over-represented in ABC DLBCL  was not affected by the inducible β-catenin knockdown after DOX treatment  and the β-catenin destruction complex, which induces in parallel constitutive NF-κB activation and stabilization of β-catenin . Even th21 Our results demonstrate that the function of CARMA1-CK1α binding is not mono-directional and only feeding into the NF-κB pathway, but also recruits and controls β-catenin in cells carrying oncogenic CARMA1 mutations. In fact, CK1α binding and β-catenin stabilization was also detected for CARMA1 WT after P/I stimulation, which is consistent with previous reports showing β-catenin stabilization upon T-cell receptor and BCR engagement in a PKCθ- and PKCβ-dependent manner, respectively.36 In line with BCR-dependent β-catenin stabilization, we found recruitment of the β-catenin destruction complex and elevated β-catenin expression in ABC DLBCL cell lines that display chronic BCR signaling as a result of upstream mutations, for example, in CD79B. At present, it remains unclear how β-catenin is stabilized by CARMA1, but it seems reasonable that CARMA1 recruitment triggers inactivation of the β-catenin destruction complex in a manner that is similar to Frizzled receptor association after WNT stimulation.37 Upon WNT stimulation, the entire destruction complex including β-catenin is recruited to and inactivated at the Frizzled receptor at the membrane to prevent ubiquitin-dependent β-catenin degradation.37 Thus, CARMA1 activation through BCR signaling or oncogenic mutations seems to stabilize β-catenin by a mechanism that is analogous to the WNT-Frizzled pathway.We defined CK1α as the bridging factor that connects the β-catenin destruction complex to CARMA1. Interaction of CK1α and CARMA1 has already been shown to contribute to NF-κB activation in T cells and ABC DLBCL.38 However, Ge et al. found β-catenin largely in the nucleus of DLBCL cells and we detected predominantly cytosolic staining in the ABC DLBCL cell lines and DLBCL biopsies. The positive β-catenin staining in DLBCL resembled the perinuclear or diffuse localization observed in lymphocytes or chronic lymphocytic leukemia cells.39 We observed a significant over-representation of β-catenin positivity in non-GCB (34%) versus GCB (17%) DLBCL samples. Even though the status of CARMA1 and other oncogenes was not analyzed in the respective patient samples, high β-catenin amounts are more frequent than CARMA1 mutations and may be in the range of patients that carry either CARMA1 or CD79B mutations .9 Clearly, further analyses will be required to correlate the mutation status and β-catenin levels in DLBCL subtypes. The observation that a considerable number of GCB and not all non-GCB/ABC DLBCL cells were β-catenin positive indicates that alternative processes besides chronic BCR signaling may contribute to β-catenin stabilization. Indeed, WNT3a secretion by a subpopulation of cells in a DLBCL population was shown to be able to induce WNT signaling, indicating that especially in primary samples the tumor microenvironment may contribute to increasing β-catenin amounts.40 Thus, the increased β-catenin levels could be at least partially a result of autocrine or paracrine WNT signals within the tumor microenvironment.As β-catenin was predominantly found in the cytosol, increased β-catenin in oncogenic CARMA1 cells was apparently not sufficient to drive strong TCF/LEF transcriptional activation or induce a WNT target gene signature. Nevertheless, we suppose that a small fraction of stabilized β-catenin is localizing in the nucleus, as we detected slightly augmented TCF/LEF reporter activation in CARMA1 L225LI cells. Importantly, elevation of β-catenin by all oncogenic CARMA1 mutants promoted TCF/LEF reporter activity after WNT pathway activation. In line with a previous report, we could detect enhanced β-catenin protein expression in a subset of DLBCL biopsies.33 Binding of β-catenin and p65 were shown to enhance tumorigenesis also in intestinal epithelial cells.34 Co-regulation of target genes may either rely on the direct binding of β-catenin and p65, but it can also function more indirectly at the level of transcriptional activation. β-catenin-TCF/LEF complexes were shown to induce IL-10 transcription through binding sites in the promoter.41Stabilized β-catenin in oncogenic CARMA1-expressing BJAB cells is interacting with NF-κB p65 and β-catenin regulates the expression of distinct NF-κB target genes. An NF-κB—β-catenin cross-talk that modulates gene expression through physical interaction has already been observed in colon and breast cancer cells.26 As IL-6 expression was not affected by β-catenin knockdown, this interplay is most likely not severely affected. However, tumor-induced β-catenin activation and IL-10 secretion in dendritic cells can suppress CD8 T-cell-dependent antitumor immunity.42 Also, β-catenin-driven IL-10 production in melanoma cells impaired dendritic cell maturation and inhibited an antitumor immune response.41 Further, high expression of CCL3 (MIP-1α) and other chemokines in melanomas was associated with improved recruitment of CD8 effector T cells.43 CCL3 is a candidate for gene therapy, because its heterologous expression can enhance antitumor immune responses.45 Recently, melanoma WNT/β-catenin signaling was shown to prevent antitumor immunity and CCL3 and CCL4 expression were repressed by high β-catenin expression.46 Even though we did not see effects on CCL4 production by oncogenic CARMA1 in the absence of β-catenin, these data point out that β-catenin is able to repress the expression of chemokines that promote T-cell infiltration. Thus, increased IL-10 and decreased CCL3 production in ABC DLBCL that display augmented β-catenin activation may add to an immunosuppressive tumor microenvironment. Of note, mature B-cells switch upon expression of oncogenic CARMA1 from self-antigen-induced cell death to proliferation and IκB kinase β/NF-κB signaling is not sufficient to induce this shift.47 It will be interesting in how far augmented β-catenin may be required in this process.Functionally, β-catenin knockdown did not affect CARMA1-triggered survival of different ABC DLBCL cell lines and the decreased viability upon β-catenin knockdown in BJAB cells was also not connected to oncogenic CARMA1 or the NF-κB activation status. In line with this we did not observe β-catenin co-regulation of critical antiapoptotic NF-κB target genes. Thus, β-catenin is not involved in controlling cell-intrinsic survival of DLBCL cell lines, but this may change in a setting when WNT signaling is active, for example, in a tumor microenvironment. Interestingly, NF-κB target genes that are also regulated by β-catenin are the cytokine IL-10 and the chemokine CCL3 that both have been associated with regulating cell communication and antitumor immunity. NF-κB-dependent IL-10 and IL-6 secretion can activate STAT3 signaling in an autocrine manner.Our data using oncogenic CARMA1 mutants unravel a novel cross-talk between the tumorigenic NF-κB and β-catenin pathways. Functionally, we demonstrated that β-catenin activates or suppresses certain NF-κB-dependent cytokines and chemokines that control the tumor microenvironment and especially have been linked with preventing antitumor immunity in cancers. It will be interesting to evaluate the effects of β-catenin for tumor immunity in DLBCL. Our data suggest that the critical cross-talk of NF-κB and β-catenin could be promising for future therapeutic approaches.48 contained Flag-Strep-Strep-tagged49 CARMA1; Lentiviral infection, shRNA expression and TCF/LEF luciferase reporter constructs were described before.32 shRNA sequences: CARMA1 5′-GGGAGAATGTGGAGTGTAA-3′, β-catenin , CK1α 5′-GAGCAAGCTCTATAAGATTCT-3′, MSMO1 5′-CTCTCAACCCTTTAAATCTGA-3′, Myc 5′-CCTATGAACTTGTTTCAAATG-3′.Following antibodies were used: β-catenin (clone 14), e-Cadherin (610405) ; AXIN1 (S20), BCL10 , β-Actin (I-19), p50 (NLS), p65 (C-20), cRel (N), CK1α (C-19) ; CARMA1 (1D12), GSK3β (27C10), GAPDH-HRP (14C10) ; unphosphorylated β-catenin ; StrepTagII , hCD2-APC , Tubulin ; horseradish peroxidase-conjugated secondary antibodies . Following lentiviral constructs were used: pHAGE-PGK-hCD2-T2A backbone2, except OCI-Ly19 that were maintained in IMDM (Iscove's Modified Dulbecco's Medium) and OCI-Ly10 in IMDM containing 20% human serum instead. DLBCL cell lines have been obtained by DSMZ  or cooperation partners. The cells are authenticated by sequencing known mutations and are checked for mycoplasms on a regular basis. For stimulations 200 ng/ml PMA and 300 ng/ml Ionomycin  were applied. Lentivirus and retrovirus production and transductions were described previously.51 Cells were treated with DOX (0.02 μg/ml) for 3 days to induce shRNA expression. Flow cytometry analysis was conducted using Attune Acoustic Focusing Cytometer system  and FlowJo  software. Cell viability was determined by counting cell numbers after trypan blue exclusion. TCF/LEF luciferase reporter activity was measured in a luminometer by adding Luciferase Assay ReagentII  to cell lysates that were adjusted using Bradford method.Unless otherwise stated, DLBCL lines were cultured in 15% fetal bovine serum and 100 U/ml penicillin/streptomycin containing Roswell Park Memorial Institute medium at 37 °C and 5% COM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.5, 150 mM NaCl, 0.2% NP-40, 10% glycerol, phosphatase inhibitors 1 mM dithiothreitol, 10 mM sodium fluoride, 8 mM β-glycerophosphate, 300 μM sodium vanadate and protease inhibitors ) followed by antibody incubation as described previously.52 For disruption of nuclei, cells were additionally passed through a 26-gauge needle. StrepTactin-PD were conducted in co-IP buffer using Strep Tactin-Sepharose (IBA). Harsh cell lysis was conducted in radioimmunoprecipitation assay buffer  supplied with phosphatase and protease inhibitors. Mass spectrometry was carried out as described in the supplement. High-salt buffer lysates were analyzed in electrophoretic mobility shift assay as previously described.53For immunoprecipitation studies cells were lysed in co-IP buffer .RNA was extracted using RNeasy Mini Kit  and duplicates of each sample were analyzed in genome wide expression analyses on an Illumina HT12 v4 1-color chip  according to the manufacture protocol. Details are given in the supplement. The gene expression data have been deposited in the Gene Expression Omnibus database50 mRNA was normalized by RNA polymerase II mRNA. Primer sequences are available in Supplement. IL-10 and CCL3 secretion was measured 20 h after accumulation using human ready-set-go ELISA kit (eBioscience).Quantitative real-time-PCR was conducted as previously described.25 β-catenin staining was done on a semi-automated LabVision tissue stainer using the monoclonal antibody clone 14  at a dilution of 1:25. Cases showing unequivocal staining in >30% of cells were regarded as positive. Use of archival formalin-fixed DLBCL samples was approved by the ethics committee of the University of Würzburg.Tumors from 103 untreated DLBCL patients were immunostained for CD10, BCL6 and IRF4/MUM1 on a tissue microarray format to establish GCB and non-GCB types according to the Hans classificator.t-test as further specified in the supplement. Sample size is mentioned for each experiment and data are expressed as mean±s.e.m. Unless specified otherwise statistical significance was determined using unpaired Student's t-test with *P⩽0.05, **P⩽0.01 and ***P⩽0.001.Mass spectrometry was analyzed using analysis of variance test and microarrays were analyzed using Student's


Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-β-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33ΔN-Bcat mice showed an increase in the constitutive Wnt/β-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33ΔN-Bcat mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7  and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33ΔN-Bcat mice, but those Wnt target genes associated with excessive proliferation  were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33ΔN-Bcat mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-β-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/β-catenin pathway to facilitate and promote tumorigenesis.Activation of the Wnt/β-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant β-catenin ( Summary: The gpA33ΔN-Bcat mice provide a model that better mimics some aspects of sporadic colon cancer induction in humans in terms of tumor multiplicity and latency and site-specific (i.e. colon) tumor occurrence that coincides with upregulation of markers for human colorectal cancer progression. APC (adenomatous polyposis coli) gene are the most common form of genetic alteration in CRC and represent the earliest detectable genetic change in tumorigenesis -dependent transcription and associated neoplastic transformation and intestinal adenoma formation. Besides its role in the canonical Wnt/β-catenin pathway, APC also regulates cell migration, adhesion, chromosome segregation, spindle assembly and apoptosis . The canonical Wnt/β-catenin signaling pathway is aberrantly activated in the majority of colorectal cancers. Mutations of the igenesis . Most of pathway , 2000. Ailencing  or aminopoptosis . Meanwhipoptosis .APC truncation mutations identified suggest molecular complexity of the mechanism(s) by which deregulated Wnt/β-catenin signaling drives intestinal tumor formation. For instance, Apc1638N mice express undetectable levels of C-terminal truncated Apc protein and carry five or six tumors by 10 months of age, whereas ApcMin mice develop more tumors with a shorter latency period, which is preceded by the obligatory loss of Apc heterozygosity β-catenin was introduced as a knock-in transgene into the intestinal gene-specific Implications and future directionsgpA33ΔN-Bcat mice provide a model that better mimics some aspects of sporadic colon cancer induction in humans, including tumor multiplicity and latency and site-specific (i.e. colon) tumor occurrence; in addition, these mice show upregulation of markers of colorectal cancer progression in humans. Therefore, the gpA33ΔN-Bcat mice are likely to be useful for functional assessment and identification of mutations that co-operate with canonical Wnt/β-catenin signaling during the initiation of adenoma formation. In addition, this approach is likely to identify components that might constitute new pharmacological targets for the treatment and prevention of sporadic colon cancer in humans.The The various zygosity . Likewiszygosity . Howeverzygosity . A sharerategies .TRANSLAgpA33 gene locus to drive intestine-specific expression of an N-terminal deletion mutant of β-catenin (ΔN-Bcat) encoded by a bicistronic gpA33-IRES-ΔN-βcatenin RNA . For this, a cDNA encoding ΔN(1-131)β-catenin, in which a FLAG epitope replaced the first 131 amino terminal amino acids, was inserted in the 3′ untranslated region (UTR) of the gpA33 antigen gene locus -flanked neo cassette in the corresponding gpA33Neo (henceforth referred to as A33Neo) mouse strain. Transcriptional activation of the silent ΔN-Bcat transgene was achieved by excising the lox(P)-flanked neo cassette in the germline to yield the gpA33ΔN-Bcat (henceforth referred to as Bcat) strain .The gpA33 antigen is a glycosylated transmembrane protein that is expressed specifically in the intestinal epithelium . We explne locus . As gpA3ΔN-Bcat, we performed RT-qPCR analysis on different tissues in A33Neo and Bcat mice. We detected ΔN-βcatenin mRNA in the small and large intestines of Bcat mice but not in liver or in intestines of A33Neo mice . Transcript levels of the truncated β-catenin were higher in the large intestine as predicted from the rostrocaudal expression gradient of endogenous gpA33 . We confirmed by northern blot and anti-FLAG immunoprecipitation analysis that ΔN-Bcat transgene expression occurred in a gene dose-dependent manner  and that it did not affect expression of endogenous β-catenin in Bcat mice . We also ascertained nuclear accumulation of ΔN-βcatenin by subcellular fractionation of intestinal lysates from Bcat mice . As predicted from the introduced truncation mutations, immunoprecipitation with an anti-E-cadherin antibody confirmed that ΔN-βcatenin retained the capacity to bind to E-cadherin at the membrane . The expression level of the mutant ΔN-βcatenin was approximately 50% compared with endogenous β-catenin in total lysate and further reduced to about 30% in the nuclear compartment .To verify tissue-specific expression of myc, cyclinD1 and CD44, expression of these genes remained unaffected in Bcat mice  and Bcat mice. Although expression of the constitutive active Δ-exon 3 β-catenin mutant results in elevated expression of the prototypical Wnt target genes cat mice A-C. In acat mice D,E. Howe. We detected increased β-catenin expression in the nucleus, cytosol and membrane fraction of isolated intestinal epithelium  for 16 h (Expression of (ΔN28-134)β-catenin results in loss of intercellular adhesiveness  in the hithelium B that waithelium C-E, suggfor 16 h F. Followfor 16 h G alongsifor 16 h C-E, presfor 16 h , possiblTable S1). Meanwhile, none of the age-matched Wt littermates (n=18) showed any sign of intestinal lesions. Most of the detected lesions in homozygous Bcat mice were relative small colonic adenomas (<1.0 mm); however, in old mice (>22 months) we also occasionally detected larger adenomas (1.9-2.7 mm diameter). Histopathological analysis revealed that all tumors in Bcat mice corresponded to hyperproliferative tubular adenomas with equally sized cell nuclei and a prominent desmoplastic reaction . Consistent with the expression of MMP7 and Tenascin-C being highly sensitive to Wnt-pathway activation, we also detected upregulation of these genes within the tumors of Apc mutant mice . Mice were then analysed 5 and 12 weeks after the last of six consecutive AOM injections, and colons were stained in methylene blue to identify aberrant crypt foci  carried missense mutations in the ctnnb1 codons that substituted the regulatory serine residues at positions 37 and 38 (Table S2) to result in a stabilized, more active form of β-catenin. In order to determine whether AOM also introduced functionally equivalent nonsense mutations in the Apc gene that result in a truncated, less stable or non-functional Apc protein, we stained tumor sections with an antibody that specifically recognizes a C-terminal epitope of the Apc protein. We observed weaker Apc staining in a much larger proportion of adenomas from Bcat than from Wt mice (Table S2 and data not shown). Collectively, these results suggested that AOM-induced somatic mutations that further activate the Wnt/β-catenin pathway , rather than mutations in the Ras-Erk signaling cascade, co-operate with ΔN-Bcat to trigger intestinal tumorigenesis.In order to gain insights into the molecular events underpinning the co-operation between the Apc and Kras increased intestinal tumorigenesis and mortality of compound pVillin-KrasV12G;Apc1638N mice, where the Apc1638N allele alone, upon loss of heterozygosity of the remaining wild-type Apc allele, confers development of three or four tumors per mouse , Apc1638N;gpA33ΔN-Bcat mice (referred to as 1638N/Bcat) and ApcMin;gpA33ΔN-Bcat mice (referred to as Min/Bcat). Consistent with our results from the AOM challenge, we observed that the homozygous ΔN-Bcat allele conferred a small additive effect in the compound Ras/Bcat mice, yielding an average of four tumors per mouse compared with 1.7 and 2.3 tumors observed in the corresponding single mutants . Likewise, overall tumor incidence remained independent of the presence of the ΔN-Bcat allele   C,D, whicenase 2) . We confterparts A,B. Stricat mice C-F. CollgpA33ΔN-Bcat knock-in mouse model to study intestinal tumor susceptibility by inserting the 1-131 amino-terminal truncation mutant of β-catenin into the 3′ UTR of the endogenous gpA33 antigen locus. Expression of this ΔN-Bcat transgene per se resulted in the formation of primarily colonic tumors in 37% of all Bcat mice of at least 8 months of age, akin to the long latency and low penetrance observed in human sporadic CRC. Our observation that ΔN-Bcat expression co-operates functionally with loss-of-function mutations in Apc rather than gain-of-function Kras mutations implies the existence of a minimal threshold level for canonical Wnt/β-catenin signaling to trigger tumor formation , which also regulate cell fate decisions in the intestine. Likewise, in Bcat mice, we did not observe the increase in progenitor cell proliferation characteristically observed when the Wnt/β-catenin pathway is maximally stimulated following biallelic Apc inactivation  of the endogenous ctnnb1 gene throughout the intestinal mucosa, thereby deleting all the regulatory serine and threonine residues that control the turnover of the β-catenin protein .We speculate that this might result from the impaired interaction of ΔN-Bcat with α-catenin, which is likely to weaken the interaction of the actin cytoskeleton with the plasma membrane in intestinal epithelial cells. Our observations suggest that oncogenic activation of the canonical Wnt/β-catenin pathway might help to set up a pro-tumorigenic microenvironment that precedes subsequent neoplastic transformation of the epithelium. This might be further exaggerated once tumors are established, given that the leakiness in r stroma . Interesr stroma . Meanwhir stroma , and Coxtastasis . ConversIn conclusion, the Bcat mice provide a model that mimics some of the critical aspects of sporadic CRC induction in humans in terms of tumor multiplicity, tumor latency and tumor site specificity, and coincides with upregulation of the earliest markers for emerging CRC in humans, including Tenascin-C, MMP-7, osteopontin and COX2.ad libitum. The ApcMin, Apc1638N and pVillin-KrasG12V models have been published previously  was generated from a knock-in gene targeting vector comprising a lox(P)-flanked IRES-neo cassette and a cDNA in which the FLAG-epitope tag replaced the most amino terminal 131 amino acids of mouse β-catenin. The IRES sequence used is a modified version of the 5′ UTR from encephalomyocarditis virus (EMCV) mRNA β-catenin (The CV) mRNA . The vecCV) mRNA  also conithelium . A lox, and RNA integrity was confirmed on a denaturing formaldehyde-agarose gel. Preparation of cDNA was performed according to standard procedures, using RevertAid H-minus M-Mulv Reverse Transcriptase, random primer and oligo dT primers .Quantitative real-time PCR was performed with the ABI PRISM 7300 detection system  using SYBRGreen dye. Relative RNA abundance was calculated using the ΔΔCT formula and normalized to the transcript levels of the housekeeping gene β-actin with the help of the Sequence Detection Software v.1.4 (Applied Biosystems). Assays were performed in duplicate. TaqMan primers for IL23 and IL17 were purchased from Life Technologies . Primer sequences used for SybrGreen RT-qPCR were as follows: β-actin/for, 5′-AGCCAGGTCCAGACGCAGG-3′; β-actin/rev, 5′-ACCCACACTGTGCCCATCTAC-3′; β-catenin/for, 5′-GCTGACCTGATGGAGTTGGA-3′; β-catenin/rev, 5′-GCTACTTGCTCTTGCGTGAA-3′; CD44/for, 5′-GTCTGCATCGCGGTCAATAG-3′; CD44/rev, 5′-GGTCTCTGATGGTTCCTTGTTC-3′; Cmyc/for, 5′-TAGTGCTGCATGAGGAGACA-3′; Cmyc/rev, 5′-GGTTTGCCTCTTCTCCACAG-3′; Cox2/for, 5′-ACACACTCTATCACTGGCACC-3′; Cox2/rev, 5′-TTCAGGGAGAAGCGTTTGC-3′; Cryptdin1/for, 5′-AAGAGACTAAAACTGAGGAGCAGC-3′; Cryptdin1/rev, 5′-CGACAGCAGAGCGTGTA-3′; CyclinD1/for, 5′-GCACAACGCACTTTCTTTCCA-3′; CyclinD1/rev, 5′-CGCAGGCTTGACTCCAGAAG-3′; ΔN-Bcat/for, 5′-GGATTACAAAGACGATGATGACAAGTTG-3′; ΔN-Bcat/rev, 5′-GTCAGCTCAGGAATTGCACGTG-3′; MMP7/for, 5′-GAGATGTGAGCGCACATCAGTG-3′; and MMP7/rev, 5′-GATGTAGGGGGAGAGTTTTCCAGT-3′.n=8) and from benign adenomas (n=9) were also analyzed. None of the individuals received neoadjuvant treatment or suffered from a known second neoplastic disease. Tumors were classified according to the UICC/TNM system (7th edition): UICC stage I (n=11 cases), stage II (n=10), stage III (n=8) and stage IV (n=13). The median of histologically reviewed lymph nodes per case was 21 . Tissues from all 42 patients who underwent surgical resection between 1987 and 2006 at the Klinikum rechts der Isar were obtained immediately after surgical resection. Specimens were transferred into liquid nitrogen and stored at −80°C.All samples were collected after prior informed written consent as part of a study (no. 1926/7) approved by the human ethics committee of the Klinikum rechts der Isar. Samples of histologically confirmed normal colonic mucosa from resected specimens , anti-β-catenin , anti-E-cadherin , anti-Cox2 , anti-CD31 , anti-Ki67, anti-Lysozym  and the dyes 4,6-diamidino-2-phenyl indol , TRITC-Phalloidin (Sigma) and TRITC-UEA1 (Sigma). Immunofluorescent staining of mouse tissue sections was detected with a Zeiss Axiovert 200M microscope with an AxiocamMR3 camera, with the following objectives: LD A-Plan 10×/NA:0.25, LD A-Plan 20×/NA:0.30 and Plan Neofluoar 40×/NA:0.75 lenses . The tissues were imaged using standard filter sets and laser lines, acquiring single-labeled images. DAPI, FITC and Cy3 fluorescence were excited with a HXP120C lamp with filters at excitation wavelengths of 360, 490 and 550 nm, respectively, and the emission was measured at wavelengths 460, 520 and 562 nm, respectively. The images were captured using Zeiss Axiovision software (version 4.8.2). The Zeiss image files (.zvi) were imported into the Adobe Photoshop version 12.0.4 software for processing and display.Cryosections of mouse tissues embedded in acetate buffer  were cut at 7 µm thickness, air dried and processed by routine hematoxylin and eosin staining. Some tissue sections were fixed in 10% normal buffered formalin overnight, processed and embedded in paraffin. For immunofluorescence, sections were fixed with either 3% paraformaldehyde at room temperature for 20 min or with methanol at −20°C for 10 min. The paraformaldehyde-fixed sections were treated with 50 mM NHFor immunofluorescent staining of paraffin-embedded tissue, sections were dewaxed in xylene and rehydrated. Antigen retrieval was in boiling 10 mM citrate buffer pH 6.0 for 15 min. Primary antibodies were diluted in 5% normal goat serum/0.5% Triton X-100 in PBS and incubated overnight at 4°C. Secondary antibodies used were goat anti-mouse IgG, goat anti-rat IgG and goat anti-rabbit IgG coupled to Alexa488, Alexa546 or Cy3 . Tissues were mounted in ProLong Antifade plus DAPI (Life Technologies). Immunofluorescent staining of the distal colon was detected with a Leica SP8 Confocal microscope with Resonant Scanner (C4.50) on a 10× water immersion (NA 0.40) lens. The tissues were imaged using standard filter sets and laser lines, acquiring single-labeled images. DAPI and α-catenin fluorescence were excited with the 405 and 488 nm laser lines, respectively, and the emission was measured at wavelengths of 405 and 473 nm, respectively. The images were captured using Leica LAS-AF software. The Leica image files (lif) were imported into the ImageJ/Fiji software  for procImmunofluorescent staining of crypts was detected with an Olympus FV1000 Spectral Confocal attachment to an Olympus IX-81 microscope on a 60× water immersion lens (NA 1.2). The crypts were imaged using standard filter sets and laser lines, acquiring single-labeled images. DAPI, β-catenin and E-cadherin fluorescence were excited with the 405, 488 and 546 nm laser lines, respectively, and the emission was measured at wavelengths of 405, 473 and 559 nm, respectively. The images were captured using Olympus FluoroView software (version 1.7c). Three-dimensional (3D) image stacks were acquired, which encompassed the entire depth of the crypt(s) in the field of view. The entire depth of the sample was acquired as 3D image stacks at approximately 20 µm thickness for each optical section. For quantitative spatial analysis of key proteins in isolated crypts, cubic voxels were acquired for each image stack. The output analog signal, representing the fluorescence intensities, was digitized to 16 bits resolution at 65,536 levels of gray and saved as an Olympus Image Binary (OIB) image. The OIB image files from the fluorescently stained individual whole-mount crypts were imported into the ImageJ/Fiji software  for processing and display. 3D image stacks were extracted and imported as ‘tiff’ files into MATLAB  for analysis. Quantification of β-catenin and E-cadherin was conducted as described by g centrifugation for 15 min at 4°C.Protein lysates were prepared from tissue samples stored at −80°C. Samples were homogenized in a Dounce Homogenizer  with RIPA buffer . Soluble proteins were extracted after a 15,000 g for 30 s at 4°C and washed three times with RIPA buffer.Protein G sepharose beads were incubated with 2 µg of anti-β-catenin, anti-FLAG or anti-E-cadherin antibody for 1 h at 4°C, and 40 µl of the Sepharose-antibody mixture was added to 400 µl cell lysate . After 2 h incubation, the samples were centrifuged at 15,000 For immunoblotting, protein lysates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to a nitrocellulose membrane as previously described . Immunor3, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM EDTA and 1 mM Pefablock and protease inhibitor cocktail) on ice for 5 min, homogenized in a Dounce Homogenizer (Wheaton) and centrifuged at 1000 g (4°C) for 5 min. The cytosolic fraction (supernatant) was harvested after a centrifugation at 39,000 g for 15 min. The nuclear fraction (pellet) was resuspended in TSE buffer , homogenized and centrifuged at 1000 g (4°C) for 5 min. The pellet was resuspended in 100 µl RIPA buffer , and the samples were prepared for immunoblotting or immunoprecipitation.Tissue samples were resuspended in ice-cold CLB buffer  and processing for cryosections. Some freshly isolated tumors were snap-frozen in liquid nitrogen and stored at −80°C for subsequent DNA/RNA extraction or protein analysis. Aberrant crypt foci in the colonic mucosa of longitudinally opened, pinned out and fixed tissues  were detected after staining for 1 min in 0.2% methylene blue (Sigma) in PBS and rinsed in fresh phosphate buffer at 4°C for 2 h. The tissue segments were placed with the luminal side up on microscope slides and observed with a low-magnification lens. All aberrant crypt foci were at least three times larger in diameter than normal crypts, and their lumina were mostly oval or elongated rather than circular  and β-catenin . Loss of heterozygosity was implicated by immunohistochemical absence of Apc protein staining using an antibody raised against the C-terminus of APC .Intestinal permeability was assessed by gastric gavage of TRITC-dextran  as a non-metabolizable macromolecule . Prior tt-test was performed and data were expressed as means±s.e.m.Unless otherwise indicated, Student's


Following incubation of the ADSCs with Ex-4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT-qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell-derived factor-1α (SDF-1α) were evaluated in the NRVC-CM treated with Ex-4 by ELISA, RT-qPCR and western blot analysis. The migration of the ADSCs to the NRVC-CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p−)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex-4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF-1α in the cardiomyocytes and increased the expression levels of p-Akt in both cells. However, the alterations to the SDF-1α/C XC R4 cascade in the cells were abrogated following pretreatment with LY-294002, a phosphoinositide 3-kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC-CM, following treatment with Ex-4. However, these effects were reduced significantly by pretreatment of the cells with the SDF-1α/CXCR4 cascade antagonist, AMD3100, and the PI3K inhibitor, LY-294002. These results indicated that Ex-4 augmented the SDF-1α/CXCR4 cascade by activating the PI3K/Akt pathways in the ADSCs and NRVCs. Furthermore, enhancement of the PI3K/Akt-SDF-1α/CXCR4 pathway may be important in the migratory response of ADSCs to NRVC-CM in vitro.Adipose-derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex-4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte-derived conditioned medium (NRVC-CM). The ADSCs and cardiomyocytes were cultured  AMI is mic area . Of notemic area . These pmic area –14.in vitro scratch wound assays (Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor agonist, which has cell protective effects, including the inhibition of cardiomyocyte apoptosis , promotid assays . HoweverThe present study aimed to investigate whether Ex-4 affected the migration of ADSCs to conditioned medium, derived from neonatal rat ventricular cardiomyocytes (NRVC-CM), via the SDF-1α/CXCR4 cascade.The present study was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethical Committee of the Chinese People’s Liberty Army (PLA) General Hospital .70-μm filters , followed by centrifugation for 10 min at 400 × g and resuspended in L-Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). The cell fraction was cultured at 37°C in 5% CO2 and the medium was refreshed every three days. The subsequent experiments were performed with ADSCs between the fourth and fifth passages.The ADSCs used in the present study were obtained from a total of five male Sprague-Dawley rats (60–80 g) obtained from the Laboratory Animal Center, Chinese PLA General Hospital, as described previously, with minor modifications, including the adjustment of collagenase I concentration to 0.1% (v/v) and the dose of trypsin to 0.05% (v/v) . Rats we5 cells/ml in DMEM supplemented with 15% (v/v) fetal calf serum  at 37°C and 5% (v/v) CO2, and cultured according to a standard procedure, as previously described (−1 penicillin and 100 mg/ml−1 streptomycin) at a density of 2×104 cells/cm−2 in a humidified incubator. After 48 h incubation, the cardiomyocytes were confluent and were beating spontaneously. The morphological characteristics of ADSCs and NRVCs were evaluated by inverted microscope .The NRVCs were isolated by enzymatic dissociation, where the heart tissue was treated with 0.1% (w/v) collagenase for 20 min at 37°C, and then incubated with 0.25% (w/v) trypsin overnight at 4°C. The cells were subsequently plated at a density of 5×10escribed . The NRVThe ADSCs were harvested in the fourth passage to detect surface antigens. Prior to immunostaining, the cells were washed twice with PBS. The cells were then incubated with anti-rat fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies CD29 , CD31 , CD34 , CD45  and CD90  at concentrations specified by the manufacturer. FITC-labeled immunoglobulin(Ig)G-stained cells were used as negative controls.Following treatment with various concentrations of Ex-4  for 24 h, the ADSCs at passage 4 were used to detect the expression of CXCR4. The cells were washed with PBS and each group of cells was stained with either FITC-labeled CXCR4  or FITC-labeled IgG for 30 min at room temperature. Flow cytometric analyses were performed using a BD FACSCalibur™ flow cytometer (BD Biosciences), as previously described (3 cells/well and treated with various concentrations of Ex-4 (0–20 nm/l) in triplicate for seven days at 37°C, with 5% CO2. Each day, 20 μl MTT  was added to the cells for 4 h. The supernatants were then discarded and 100 μl dimethyl sulfoxide (Sigma-Aldrich) was added to each well for 10 min. The optical density (OD) of the samples was measured at an absorbance of 490 nm . The assay was repeated three times.An MTT assay was performed to examine cell proliferation. ADSCs were seeded in a 96-well plate at a density of 1×10® 20  with 5% non-fat milk for 90 min at room temperature. Following blocking, the membranes were incubated with the following primary antibodies: Polyclonal CXCR4 , total Akt, , phosphorylated (p-)Akt , SDF-1α,  and β-actin  in TBST containing 5% non-fat milk at 4°C overnight. The membranes were then washed with TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies. The blots were visualized using enhanced chemiluminescence reagents . The mean densities of the bands were represented as the OD in units per square millimeter and normalized to that of β-actin .The cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay lysis buffer  supplemented with protease inhibitor phenylmethanesulfonyl fluoride . The proteins (60–80 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes . The membranes were then blocked with tris-buffered saline plus 0.1% Tweenμl culture supernatant) was added into 50 μl dilution buffer  in each well and it was washed twice. Subsequently, the detection antibody  was added and it was incubated for 1 h at room temperature. The substrate and stop solutions were then added into each well. Immediately following this, the optical density of each well was determined using the microplate reader set to a wavelength of 450 nm .The NRVCs were cultured with various concentrations of Ex-4 (0–20 nm/l) for 24 h at 37°C and the expression of SDF-1α in the culture supernatant was measured using an ELISA kit . The sample (50 μl (60 ng/μl) cDNA using a One-Step RT-PCR kit , according to the manufacturer’s instructions. Quantification of gene expression was performed using an ABI PRISM 7500 Sequence Detection system  with SYBR® Green . The relative mRNA expression levels of SDF-1α and CXCR4 were normalized to that of β-actin using the 2−ΔΔCT method  and was reverse transcribed into a total of 1 T method . The folT method ; rat SDFT method  and rat μm pore size . Briefly, 2 ml normal NRVC-CM (Nor-CM) and 2 ml NRVC-CM induced with Ex-4 (Ex-4-CM) were collected and placed in the lower chamber of the plate and 105 ADSCs, with or without the aforementioned treatment of Ex-4, were added to the upper chamber in serum-free DMEM. The chemotaxis chambers were then incubated for 12 h at 37°C, followed by removal of the non-migrating cells from the upper chamber and fixing of the migrated cells in methanol for 15 min at room temperature prior to staining with 0.05% crystal violet for 15 min at room temperature in the dark. To quantify the levels of chemotaxis, the number of cells that had migrated through to the underside of the insert membranes were counted in at least five randomly selected fields using a BX51 microscope . The data were expressed as the ratio of the experimental samples to the control samples × 100.The migration of the ADSCs was evaluated using a 24-well Transwell plate with an 8 μm/l; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h prior to treatment with Ex-4 (20 nm/l) for 24 h. Following treatment, the conditioned medium was collected and used for subsequent experiments. In addition, prior to western blotting and migration assay, the ADSCs were incubated with or without either LY294002 (30 μm/l) for 2 h or an SDF-1α/CXCR4 cascade antagonist    for 1 h,SPSS for Windows version 15.0  was used for statistical analyses. The data are expressed as the mean ± standard deviation. One-way analysis of variance was used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.The ADSCs cultured in medium exhibited a spindle-shaped or fibroblast-like morphology . After 4A dose-dependent increase in the proliferative capacity of the ADSCs was observed following treatment with Ex-4 . The optSince no significant difference was observed in the proliferative ability of the cells following treatment with or without Ex-4 for 24 h, the cells were treated with Ex-4 for 24 h in the subsequent experiments to exclude the effects of proliferation on the results. The ADSCs were treated with various concentrations (0–20 nm/l) of Ex-4 for 24 h, following which the mRNA expression levels of CXCR4 were analyzed by RT-qPCR. Treatment with Ex-4 caused a dose-dependent increase in the mRNA expression levels of CXCR4, which was highest in the cells treated with 20 nm Ex-4 . Further4 ADSCs compared with the control group, peaking at >6-fold higher  for 24 h and the mRNA and protein expression levels of SDF-1α were then analyzed by RT-qPCR, western blot analysis and ELISA. The mRNA expression levels of SDF-1α, relative to the levels of a constitutively expressed control gene, were ~three times higher in the 20 nm group compared with the normal group . The proal group  and incral group . The conμg/ml), a SDF-1α/CXCR4 cascade antagonist, which inhibits the binding of SDF-1α to CXCR4 As expected, the increased chemotactic response of the Ex-4-ADSCs to the Ex-4-CM was markedly inhibited by pretreatment with AMD3100  and then treated with Ex-4 (20 nm/l) for 24 h. The NRVC-CM were collected and a Transwell assay was performed. Fewer ADSCs translocated through the membrane following pretreatment of the NRVCs with LY294002 (μm/ml) and, as expected, the number of migratory ADSCs decreased. When the NRVCs and ADSCs were pretreated with LY294002 for 2 h, followed by treatment with Ex-4 for 24 h, the migratory response of the ADSCs to the NRVC-CM was minimal. These results indicated that inhibition of the PI3K/Akt pathway partially impaired the effects of Ex-4 on the migration of the ADSCs to the NRVC-CM.The present study also aimed to identify the potential role of the PI3K/Akt pathway on the migration of the ADSCs to the NRVC-CM. The NRVCs were pretreated with or without the LY294002 PI3K/Akt inhibitor (20 LY294002 . In addi+ ADSCs. Furthermore, an increased number of ADSCs migrated to the NRVC-CM following treatment with Ex-4. These results demonstrated that Ex-4 was capable of upregulating the SDF-1α/CXCR4 cascade and the chemotaxis of ADSCs to NRVC-CM. Therefore, Ex-4 may be a mediator with an important role in advancing the migration of ADSCs. It was previously demonstrated that SDF-1α alone has cardioprotective effects without the involvement of stem cells (in vivo, SDF-1α activates the reperfusion injury signaling kinase pathway in cardiomyocytes, resulting in the recruitment of the anti-apoptotic kinases, extracellular signal-regulated kinase (in vivo is sufficient to ameliorate cardiac function following AMI.Stem cell transplantation has recently emerged as a promising tool for the treatment of AMI and ADSCs appear to be a suitable candidate for stem cell therapy. However, despite the improved cardiac function and reduced infarct size observed following injection of ADSCs, the clinical benefits and long-term outcomes remain under debate –29. The em cells ,45. Folld kinase , Akt (44d kinase . The preet al (0 ADSCs and 2.54% in P3 ADSCs in vitro, which may affect the homing and reparative potential of ADSCs, impairing the efficacy of ADSC-based therapy on ischemic or injured tissue (4 ADSCs with a weak migratory response contained only 2.73% CXCR4+ cells, which was consistent with the results of the previous study (4 ADSCs increased in a dose-dependent manner, resulting in a higher number of ADSCs in the lower Transwell chamber. These results indicated that exposure of ADSCs to Ex-4 may increase the expression of CXCR4, functionally contributing to the increased migration of ADSCs. This suggested that Ex-4 may be used as an adjuvant to optimize the phenotype and subsequent function of ADSCs in vitro, however, whether the change in phenotype and function of ADSCs is associated with the increased efficiency of cell-based therapy requires further investigation.Li et al  previousd tissue . The resus study . HoweverIt may be hypothesized that increased expression of the SDF-1α/CXCR4 cascade may explain the modulatory role of Ex-4 in the migration of ADSCs; and the increased expression of SDF-1α/CXCR4 may be responsible for upregulating the migration of ADSCs to NRVC-CM. To assess this hypothesis, the ADSCs were pretreated with AMD3100, a specific antagonist of the SDF-1α/CXCR4 cascade. As expected, treatment with AMD3100 inhibited the upregulation of Ex-4-induced ADSC migration. These results indicated that the Ex-4-mediated improvement in the chemotaxis of the ADSCs to the NRVC-CM was due to enhancement of the SDF-1α/CXCR4 cascade. These findings were concordant with those of previous studies, which reported that the SDF-1α/CXCR4 cascade contributed to cell mobilization and the homing of hematopoietic and mesenchymal stem cells –50. NumeAkt is an important mediator of stem cell survival , growth in vitro is associated with improved recruitment and homing of transplanted cells in vivo requires further investigation.In conclusion, the present study provided novel insight into potential methods to improve the migration of ADSCs and identified several key signaling molecules involved in this process, including PI3K/Akt and SDF-1α/CXCR4. The PI3K/Akt pathway was found to be responsible for the Ex-4-induced upregulation of the SDF-1α/CXCR4 cascade, which represents the final mediator of ADSC migration and, therefore, offers a potential mechanism to maximize the effectiveness of ADSC-based therapy. However, the detailed genetic changes underlying Ex-4, and whether the increased migratory capacity of the ADSCs observed


DDX3, a subunit of CK1ε, phosphorylates Dvl2 to promote β-catenin activation. Overexpression of the Dvl2 protein results in potent activation of β-catenin/TCF signaling in colorectal cancer. Therefore, we hypothesized that DDX3 might promote tumor invasion via the CK1ε/Dvl2 axis due to β-catenin/TCF activation. Western blotting showed that β-catenin expression was decreased by DDX3 knockdown and increased by DDX3 overexpression in colorectal cancer cells. The TCF promoter activity and invasion capability were concomitantly increased and decreased by DDX3 manipulation in these cells. The invasion capability in colon cancer cells and xenograft lung tumor nodules induced by a DDX3-overexpressing T84 stable clone in tail-vein injection model were nearly suppressed by inhibitors of CK1ε (PF4800567) and β-catenin/TCF signaling (XAV939). Among colorectal cancer patients, DDX3 expression was positively correlated with the expression of pDvl2 and nuclear β-catenin in tumor tissues. The expression of pDvl2 occurred more frequently in high-nuclear than in low-nuclear β-catenin tumors. A prognostic significance of DDX3, pDvl2, and nuclear β-catenin on overall survival and relapse free survival was observed in this study population. We therefore suggest CK1ε or β-catenin/TCF signaling as potential targets for improving tumor regression and outcomes in colorectal cancer, particularly tumors with high-DDX3/high-nuclear β-catenin or high-DDX3/high-pDvl2/high-nuclear β-catenin expression. Wnt/β-catenin signaling plays a critical role in embryogenesis as well as in tumorigenesis489Dysregulation of Wnt/β-catenin signaling is therefore an initiating event underlying colon adenoma formation following the loss of APC1111314151617DDX3, a DEAD-box RNA helicase, has been identified as a regulator of the β-catenin/TCF signaling that acts as a regulatory subunit of CK1ε to promote phosphorylation of disheveled segment polarity protein 2 (Dvl2). A requirement for DDX3 has been suggested for β-catenin activation during the development of mammalian cellsWe examined whether DDX3 could promote β-catenin activation and cell invasiveness using high-DDX3-expressing CCM2 and HCT116 cells and low-DDX3-expressing T84 and HCT15 cells to knock down and overexpress DDX3 using two shRNAs and its expression vector. As expected, DDX3 expression was significantly decreased by both shRNA transfections in CCM2 and HCT116 cells. Interestingly, expression of β-catenin and its downstream genes, cyclin D1 and c-Myc, were significantly decreased by DDX3 knockdown in CCM2 and HCT116 cells . MoreoveWe further examined whether β-catenin could be responsible for DDX3-mediated cell invasion by transfecting CCM2, HCT116, T84, and HCT15 cells with shDDX3 and its expression vector and/or with shβ-catenin. The expression levels of DDX3 and β-catenin in CCM2 and HCT116 cells with DDX3 manipulation and/or β-catenin silencing were confirmed by western blotting . The invWe examined the possibility that DDX3 could activate β-catenin/TCF signaling via the CK1ε/Dvl2 axis through increases in β-catenin protein stability. The CCM2 and T84 cells were transfected with shDDX3 and its expression vector, respectively, with or without treatment with a proteasome inhibitor MG132. Western blotting showed that the disappearance of β-catenin expression in DDX3-knockdown CCM2 and T84 NC cells was reversed by MG132 treatment . The rolImmunoprecipitation (IP) analysis further showed that the interaction between PP2A and β-catenin was observed in CCM2 cells with shDDX3, shCK1ε, or shDvl2 transfections in the presence of MG132 . We therMechanistically, an increase in the interaction of PP2A with β-catenin, due to silencing of DDX3, CK1ε, or Dvl2, would promote β-catenin phosphorylation through the activation of GSK3β due to GSK3β dephosphorylation at Ser9 . These rWe next examined whether an inhibitor of CK1ε or of β-catenin/TCF signaling could inhibit the cell invasion capability induced by DDX3 overexpression in T84 cells. Western blotting showed that pDvl2 expression was markedly decreased by treatment of the DDX3-overexpressing T84 cells with the CK1ε inhibitor PF4800567. β-catenin expression was markedly decreased by treatment with PF4800567 or with a β-catenin/TCF signaling inhibitor (XAV939) . The taiWe examined the possibility that DDX3 could be associated with phosphorylation of Dvl2 and nuclear β-catenin expression in colorectal cancer. In total, 75 tumors surgically resected from colorectal cancer patients were enrolled to evaluate DDX3, pDvl2, and β-catenin expression by immunohistochemistry. The representative immunostaining results of DDX3, pDvl2, and β-catenin are shown in We next examined whether DDX3, pDvl2, and nuclear β-catenin expressions could be associated with clinical outcomes in colorectal cancer patients. Kaplan-Meier analysis showed that patients with high-DDX3, high-pDvl2, and high-nuclear β-catenin tumors had shorter OS and RFS periods when compared to patients with low-DDX3, low-pDvl2, and low-nuclear β-catenin tumors . Cox regThe evidence provided here to demonstrate that β-catenin/TCF activation due to increased β-catenin protein stability may be responsible for DDX3-mediated cell invasion and xenograft lung tumor nodules formation via the CK1ε/Dvl2 axis. Consistent with a previous study, the interaction between PP2A and β-catenin can be blocked by Dvl2 phosphorylation, which destabilizes the β-catenin degradation complexMin mouse modelDvl2 promotes self-renewal and tumorigenesis in human gliomas via activation of Wnt/β-catenin signalingThe decreases in the TCF promoter activity and invasion capability were lower following DDX3 knockdown than following CK1ε and Dvl2 silencing in colon cancer cells . These r4In summary, we have provided evidence that DDX3 promotes cell invasiveness and xenograft lung tumor nodules formation via the CK1ε/Dvl2 axis due to activation of β-catenin/TCF signaling. The formation of xenograft lung tumor nodules was significantly suppressed by an inhibitor of CK1ε (PF4800567) or β-catenin/TCF signaling (XAV939). The expression of pDvl2 and nuclear β-catenin mediated by DDX3 was associated with poor outcomes in colorectal cancer patients. Therefore, we suggest that CK1ε or the β-catenin/TCF signaling might be potential targets for improvement of tumor regression and outcomes, particularly in patients who harbor high-DDX3/high-nuclear β-catenin or high-DDX3/high-pDvl2/high-nuclear β-catenin tumors.This study consisted of 75 patients with colorectal cancer. All patients were unrelated ethnic Chinese and residents of Central Taiwan. The inclusion criteria for patients were: primary diagnosis with colorectal carcinoma; no metastatic disease at diagnosis; no previous diagnosis of carcinoma; no neoadjuvant treatment before primary surgery; no evidence of disease within one month of primary surgery. Tumor specimens collected from surgically resected colorectal cancer patients were stored at 80°C at the Division of Colon and Rectum, Chung Shan Medical University Hospital , between 1994 and 2006. The present experiments were conducted in accordance with the Declaration of HelsinkiCCM2 cells were primary cultures from Taiwanese colorectal cancer patients and were kindly provided by Dr. Wun-Shaing Wayne Chang . The T84, HCT116, and HCT15 cells were obtained from the American Type Culture Collection (ATCC) and were cultured under conditions described elsewhere and stored according to the suppliers’ instructions. The cells were used at passages 5 to 20. Once resuscitated, cell lines were routinely authenticated  through cell morphology monitoring, growth curve analysis, species verification by isoenzymology and karyotyping, identity verification using short tandem repeat profiling analysis, and contamination checks. The CCM2, HCT15, and T84 cell lines harbored KRAS, APC, and p53 mutations, but possessed a wild-type β-catenin gene. The HCT116 cells harbored KRAS and β-catenin mutations, but possessed wild-type APC and p53 genes. .XAV939 was obtained from Selleckchem . PF4800567 was purchased from Sigma Chemical . Anti-PP2A, Anti-CK1εand anti-Dvl2 antibodies were obtained from GeneTex . Phosphorylated Dvl2 (S143) antibody were purchased from Abcam . Phosphorylated β-catenin , GSK-3β, and phosphorylated GSK-3β (S9) antibodies were purchased from Cell Signaling . All other antibodies were purchased from Santa Cruz Biotechnology .× 106) using the Turbofect reagent . After 48 hours (h), the cells were harvested and whole cell extracts were assayed in subsequent experiments.DDX3 , β-catenin (TRCN0000314991), CK1ε (TRCN0000009965), and Dvl2 (TRCN0000033340) shRNAs were purchased from the National shRNA Core Facility, Academia Sinica, Taiwan, ROC. DDX3 overexpression plasmids were provided from Addgene . The TOP flash plasmid (a reporter plasmid containing multiple copies of wild-type TCF-binding sites) was purchased from Millipore . Different concentrations of expression plasmids were transiently transfected into lung cancer cells  24 h after transfection. Normalized luciferase activity was reported as the ratio of luciferase activity/β-galactosidase activity.For immunoblotting, cell lysates were prepared as described previouslyThe immunohistochemical procedures and quantification methods were as described previouslyin vitro cell invasion assay. Cells (1 × 104) in culture medium  containing 0.5% serum were plated in the upper chamber and 10% fetal bovine serum was added to culture medium in the lower chamber as a chemoattractant. The upper side of the filter was covered with 0.2% Matrigel  diluted in RPMI-1640. After 16 h, cells on the upper side of the filter were removed and cells that adhered to the underside of membrane were fixed in 95% ethanol and stained with 10% Giemsa dye. The number of invasive cells was counted by examining 10 contiguous fields of each sample to obtain a representative number of cells that had invaded across the membrane.A Boyden chamber with a pore size of 8 μm was used for the 2. After the indicated incubation, the in vitro effects of these treatments were determined by the MTT assay (at 570 nm).The cell lines were cultured in 96-well flat-bottomed microtiter plates supplemented with RPMI 1640 or DMEM  at 37 °C in a humidified incubator in an atmosphere of 95% air and 5% CO-vein injection animal model was conducted as previously reported2930325 cells in 0.1 mL of PBS) at day 7. Six weeks after injections, the mice were euthanized and the lungs were dissected and examined for the development of visible lung tumor nodules. Tissues were processed for hematoxylin and eosin staining. Animal care and experimental procedures were performed with approval from the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-2013-0205) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .The tailStatistical analysis was performed using the SPSS statistical software program . The association between DDX3, pDvl2, and β-catenin protein expression was analyzed by the chi-square test. Survival plots were generated using the Kaplan-Meier method and differences between patient groups were determined by the log-rank test. Multivariate Cox regression analysis was performed to determine OS and RFS. The analysis was stratified for all known variables  and protein expression.How to cite this article: He, T.-Y. et al. DDX3 promotes tumor invasion in colorectal cancer via the CK1ε/Dvl2 axis. Sci. Rep.6, 21483; doi: 10.1038/srep21483 (2016).


Xenopus, was shown to augment Wnt/β-catenin signalling, and also interacts with those Wnt ligands that act on β-catenin-independent Wnt pathways.Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus Fz4-v1 can activate and inhibit the β-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/β-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced β-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin.Here we show that For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators. Xenopus embryos demonstrated that low levels of SFRPs enhance the Wnt/β-catenin pathway, but high levels of SFRPs suppress Wnt signalling [SFRPs consist of two domains, an N-terminal cysteine-rich domain (CRD) and a C-terminal Netrin-like domain -3. Basedgnalling ,8.Xenopus Fz4-v1 is an SFRP-type protein which is generated from the Frizzled-4 receptor mRNA by intron retention. In contrast to classical SFRPs Fz4-v1 includes a CRD domain but lacks a Netrin-like domain [Xenopus Frizzled-4 receptor and were previously named Fz4S [Xenopus Fz4-v1 is a secreted protein and can modulate Wnt/β-catenin signalling in a non-cell autonomous manner  were injected, which alone were not sufficient to induce a secondary axis, co-injection of 1 ng fz4-v1 induced ectopic axes in more than 50% of the embryos, consistent with previous studies [To investigate the dual function of Fz4-v1, we took advantage of the Xenopus embryos in order to quantitatively measure Wnt/β-catenin signalling strength  was enhanced by co-injection of 5 and 50 pg of fz4-v1, but repressed by 1000 pg. In contrast, strong Wnt/β-catenin signals triggered by high doses of wnt3a (5 pg) were inhibited by both, high (1000 pg) and low (5 and 50 pg) amounts of fz4-v1  in the mesoderm and the neuroectoderm. Since Fz4-v1 can interact with Wnt5a and Wnt11, we hypothesized that it could interfere with non-canonical Wnt signalling [Xenopus embryos caused malformations such as shortened body axes and spina bifida, indicating that β-catenin-independent Wnt signalling was perturbed  cells was inhibited  event essential for dorsal fin induction is controlled by Wnt11-R , which cXenopus laevis frogs were obtained from Nasco and all experiments complied with local and international guidelines for the use of experimental animals. Xenopus eggs were obtained from females injected with 500 IU human chorionic gonadotropin (Sigma), and were fertilized in vitro. Embryos were dejellied with 2% cysteine hydrochloride (pH8) and embryos were microinjected in 1x MBSH 2, 10 mM HEPES (pH7.4), 10 μg/ml penicillin). The embryos were cultured in 0.1x MBSH and staged according to Nieuwkoop and Faber [nd Faber .For the animal cap elongation assay, 4-cell-stage embryos were injected animally into two opposing blastomeres with synthetic RNAs. Animal caps were excised at stage 9 and cultivated in 1x MBSH together with 10 ng/μl gentamycine overnight.Capped RNAs were synthesized form linearized plasmids using the mMessage mMachine Kit (Ambion).Xenopus) were linearized with Not1 and pSP64T-BVg1 (Xenopus) was linearized with EcoRI. Sense RNA was transcribed by SP6 polymerase.pCS2-Wnt3a (mouse), pCS2-Wnt8b, pCS2-Wnt11, pCS2-Fz4, pCS2-Fz4-v1, pCS2-Fz4-myc, pCS2-Fz4-v1-myc and pCS2-JNK-APF  or control morpholino (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was injected.in situ hybridization was performed as described [in situ hybridization for fz4-v1 was performed using a double (5′ and 3′) DIG-labelled LNA probe (5′-AGTATAGAAAGTAAACCCCCTGTG-3′) from Exiqon, according to manufacturer’s instructions.Embryos were fixed in MEMFA and whole-mount escribed . pBluescescribed  was lineescribed  was lineescribed  was lineFor reporter assays 4-cell stage embryos were injected animally with 80 pg M50 Super 8x Topflash  or 50 pgMaterials and Methods used for experiments in Additional files The authors declare that they have no competing interests.AKG and HS designed the experiments. AKG, LTK and RKS performed the experiments and contributed to the writing of the manuscript. HS supervised the work and wrote the manuscript. All authors read and approved the final manuscript.Xenopus development.Fz4-v1 is a secreted protein during Click here for fileSpecificity of Fz4/Fz4-v1 morpholino oligonucleotide mediated knock-down of Fz4 and Fz4-v1.Click here for fileMaterials and methods [. methods .Click here for file