The development of improved cold-cured resins, to be used as either adhesives or matrices for FRP (fiber reinforced polymer) composites employed in the construction industry, has become the focus of several academic and industrial research projects. It is expected that the use of nano-structured organic–inorganic hybrid materials could represent a realistic alternative to commercial epoxy-based resins due to their superior properties, especially in terms of higher durability against: moisture, temperatures, harsh environments, and fire. In this context, organic–inorganic epoxy hybrids were synthesized by a modified sol–gel method without the addition of water. The experimental formulations were prepared starting from a mixture of a silane-functionalized epoxy resin, alkoxysilane components and a deep eutectic solvent (DES) based on a blend of choline chloride and urea. The latter was added in two different loads in order to analyze in depth its effect as a promoter for an effective dispersion of silica nano-phases, formed through hydrolysis and condensation reactions, into the cross-linked epoxy network. The produced formulations were cold-cured for different time spans in the presence of two hardeners, both suitable for a curing process at ambient temperature. In this first part of a wider experimental program, several analyses were carried out on the liquid  and cold-cured  systems to evaluate and quantify the improvement in properties brought about by the presence of two different phases (organic and inorganic) in the same epoxy-based hybrid system.  Tg) of a few degrees (10–20 °C) higher than the common service (ambient) temperatures. Additionally, the Tg of the cold-cured epoxy resins may even decrease due to plasticization phenomena, i.e., when the resins are exposed to rain or to humid environmental conditions. The mentioned drawbacks make uncertain the durability of the cold-cured adhesives/matrices for fiber-reinforced polymer (FRP) composites, in particular when they are used in outdoor applications [The use of epoxy resins as a matrix for composite materials or adhesives for structural bonding is widespread in the aeronautical and automotive industry. In these fields, the curing reaction of the thermosetting matrix takes place in controlled conditions  where monitoring is a topical issue ,2,3. Morications ,8. Therefore, the development of high performing/durable cold curing resins based on nano-structured epoxy resins represents a growing and attractive area in nano-engineered material science. Nanotechnology supplies opportunities to enhance material performance enabling the development of new polymeric materials with improved mechanical, thermal and functional properties, when compared to the pristine polymers ,10,11.Organic–inorganic (O-I) epoxy-based hybrids, obtained by intermingling at a nanometric scale an organic (epoxy) and an inorganic (mainly silica) components, has recently gained considerable attention as a means to produce high performing nano-materials. In the last years, the traditional approach employed for the production of epoxy-silica nano-composites, i.e., obtained from an epoxy where preformed silica particles are homogeneously dispersed, has been overcome by a technique based on the in-situ generation of siloxane domains by the sol–gel process, through the hydrolysis and condensation of metal alkoxides in aqueous solutions ,19,20,21On the other hand, the authors of the present study have recently developed O-I epoxy-silica hybrids, prepared by a non-aqueous sol–gel process, able to be cold-cured . The modIn the last years, as reported by Donato et al. ,24,25,26More recently, a new class of ionic liquids, known as Deep Eutectic Solvents (DES), has emerged. DESs are defined as a mixture of two or more components that, at a specific composition, are able to form an eutectic with a much lower melting point than those displayed by the individual components ,36. DESsIn this work, an eutectic mixture of choline chloride and urea has been used for the first time for the synthesis of organic–inorganic epoxy hybrids. This type of DES is easy to prepare and is biodegradable; in addition, the starting components are readily available and inexpensive . CholineThe procedure for the synthesis of the epoxy-based organic–inorganic hybrids consists of five steps, as schematized in 1) Silane functionalization of the epoxy matrix: a diglycidyl ether of bisphenol-A (DGEBA) with an epoxy equivalent weight of 184–190 g/mol was selected as an epoxy matrix; it is commercially known as Epikote 828 (Resolution Performance Products). The DGEBA was partially functionalized with a bis-(γ-propyltrimethoxysilane) amine (NPTEOS) obtained from Aldrich  (purity >90%) by stirring for 2 h at 90 °C. The chemical formula of each material used is reported in 2) Pre-hydrolysis of a silane precursor-DES mixture: the alkoxysilane precursors, required for the in-situ silica formation achieved by the sol–gel method, were based on a fixed combination (molar ratio of 1:0.12) of tetraethoxysilane (TEOS) and γ-glycidoxypropyltrimethoxysilane (GOTMS) obtained from Aldrich (purity >97%). In this step, the alkoxysilane precursors were mixed with a deep eutectic solvent (DES) based on a mixture of choline chloride (ChCl) and urea (U), both supplied by Iolitec GmbH  (purity > 97%). The eutectic mixture was produced according to previous literature [terature ,29 by st3) Pre-hydrolyzed alkoxysilane-epoxy oligomer mixture: The two mixtures prepared in steps 1 and 2, i.e., silane-functionalized epoxy resin and TEOS/GOTMS/DES mixture, were mixed and stirred at 90°C for 2 h and then cooled to room temperature. The investigated formulations, whose compositions are summarized in 4) Mixing of alkoxysilane-DES-epoxy mixture with the hardener: A curing agent was, then, mixed to each silane-DES-epoxy mixture in order to induce the cross-linking reactions of the polymeric component of the mixture. To this aim, two different amine hardeners were selected, i.e., an aliphatic amine and a cycloaliphatic one. Triethylenetetramine (TETA), an aliphatic amine supplied by Elantas Italia S.r.l.  with the trade name IG 824-K24, and 4-4’ methylene bis-cyclohexanamine (PACM), a cycloaliphatic amine supplied by Aldrich, are both suitable curing agents for cold-curing of the epoxy resin. Each hardener was, then, added to the mixtures at ambient temperature, in order to avoid premature heat-curing, continuously mixing the liquid formulations for 30 min. The analyzed mixtures were labeled with a final “A” or “B” depending on the hardener used, TETA or PACM, respectively. For comparison purposes, two epoxy controls were also prepared: A neat epoxy system cured with TETA and a neat epoxy system cured with PACM, named Epoxy-A and Epoxy-B, respectively. Two different epoxy/hardener molar ratios were used for A and B systems: For the systems cured with TETA, a stoichiometric molar ratio epoxy:amine = 1:1 was selected; while for the systems cured with PACM, a molar ratio epoxy:amine =1:0.75 was chosen. The last selected value was, in fact, identified as the optimum ratio for a low temperature cure of coating systems ,43.5) Matrix curing and silica condensation: The final step in the synthesis of the organic–inorganic hybrids was the simultaneous cross-linking of the epoxy-based (organic) component of the mixture and the condensation of the siloxane (inorganic) domains for the in-situ production of silica. These two processes occurred both at room temperature. All the fresh  mixtures, prepared as previously described, were immediately subjected to several characterization tests, as detailed in the next paragraph.A part of the liquid mixtures was then deposited on a glassy substrate in the form of thin films (thickness around 0.8 mm) and cured/aged for prolonged times (up to one year) in laboratory-controlled conditions . These cold-cured specimens were employed for thermal analyses.3) and for dynamic-mechanical tests (40 × 10 × 1 mm3). The specimens were cured for 14 days in laboratory conditions and subsequently aged in the same controlled environment (23 ± 2°C and 55% ± 5% R.H.) up to 4 months. The remaining part of liquid mixtures was then poured into two different polytetrafluoroethylene (PTFE) molds, in order to produce rectangular specimens suitable for mechanical flexural tests  were run on three specimens, at least, for each formulation produced, and the results were averaged.A Differential Scanning Calorimeter  was used to monitor the cure advancement and the evolution of -1 with a parallel plate geometry (radius of the plate: 12.5 mm). Three runs were performed on each formulation. The value of the nominal ‘gel-time’ was taken as the time required by each mixture to achieve a viscosity equal to 1000 Pa·s [The evolution of the viscosity of the uncured formulations at 40 °C was monitored over the time, in order to assess their gelation point. The test temperature of 40 °C was chosen to reduce the measuring time, which was several h at room temperature. A strain-controlled rheometer  was employed for this purpose, performing the rheological tests at a shear rate of 0.5 s000 Pa·s ,45. Thermogravimetric analysis  was also performed on cured specimens. The tests were performed from 30 to 800 °C, at a heating rate of 10 °C/min (sample weight about 15 mg). The experiments, at least three for each composition, were performed in air atmosphere on samples taken from films aged at room temperature for 4 months, in order to assess the decomposition temperatures for each system. The latter, in fact, can constitute a useful indication of the fire behavior of the resins. The results of thermogravimetric analyses were also employed to calculate the amount of silica residue in each mixture.3) were subjected to an oscillation in torsion mode at a constant amplitude (1%) and frequency (1 Hz), by heating the specimens from 30 to 150 °C at a constant rate of 2 °C/min.Dynamic-mechanical thermal analysis (DMTA) was carried out on the different samples cold-cured and aged for 4 months at ambient temperature. An ARES rheometer (Rheometric Scientific) was used to this aim, employing the torsion configuration. Samples of all systems (40 × 10 × 1 mm3) aged at room temperature for 4 months. The flexural tests were performed with a LLOYD LR5K machine according to the ASTM D790 standard [fl) and modulus (Efl) were calculated from the following equations:Fmax (N) was the load at failure, L (mm) the span length, F (N) and δ (mm) were the actual load and its displacement below the elasticity limit, respectively; b (mm) and h (mm) were the width and the height of the tested specimen, respectively.Flexural tests were carried out in a three-point bending mode, at a cross-head speed of 1.7 mm/min, with a span/thickness ratio of 16:1, using rectangular specimens . The samples were step-scanned at room temperature from 2θ values of 10 °–50 °.Finally, the morphology of the cured samples, fractured during the flexural tests, was examined by Scanning Electron Microscopy (SEM). A Zeiss EVO 40 Instrument was used at variable pressure and a voltage of 20 KV with the EDS system.onset and Tpeak reported in g, is obtained as the inflection point from a second DSC scan carried out at 10 °C/min.The dynamic DSC thermograms of the two uncured epoxy controls and the relative hybrid systems, containing different DES contents and curing agents, are reported in Tonset, Tpeak and Tg observed for the B-epoxy systems cured with PACM hardener, which is more evident in the case of hybrid systems. Furthermore, both the control and hybrid systems cured with the aliphatic amine (TETA) exhibit higher values of enthalpy of curing compared to the corresponding systems cured with the cycloaliphatic amine (PACM). This difference is clearly evident for the control systems, where the ΔH of the Epoxy-A system is about 18% greater than that measured for Epoxy-B system; this difference is reduced (about 5%) in the case of the hybrid systems. All the investigated hybrid mixtures present a shift of Tonset and Tpeak towards higher temperatures compared to the neat parent epoxy resins, due to the steric hindrance caused by silane-functionalized epoxy oligomers and the presence of alkoxysilane species and DES. As expected, the choice of aliphatic or cycloaliphatic amine has a significant effect on the curing kinetics of the epoxy-based systems. All the epoxy-based systems cured with the aliphatic amine, i.e., TETA, are characterized by an exothermic peak centered at 103–107 °C; the corresponding systems cured with the cycloaliphatic amine, i.e., PACM, exhibit a peak shifted toward higher values, centered between 113 and 122 °C. For the latter systems, furthermore, a shoulder between 140 and 175 °C is observed, which can be attributed to an increase of the reaction rate occurring at very high temperatures. This phenomenon was observed also by Van Assche et al. ,48 for aH, Tonset and Tpeak slightly raise when the content of DES is increased from 1.5 % to 3%. This can be an indication of the occurrence of a reaction between the urea present in the deep eutectic solvent and the oxirane rings present in the epoxy oligomers and also in GOTMS coupling agent. Urea, in fact, can undergo thermal dissociation, generating corresponding amines and ammonium compounds that are able to react with epoxy groups and cause curing according to polyaddition and/or homopolymerization, respectively [The DES content is likely to affect the curing kinetics of the fresh reacting mixtures. As reported in ectively ,49.g of the hardener used to cure the epoxy and a further effect brought about by the hybridization in the presence of DES performed on each resin/hardener couple.As mentioned, the glass transition temperature has been measured on each system completely cured in the first calorimetric scan through a second calorimetric experiment performed on the same specimen, again carried out at 10 °C/min. From the data in g measured only on the hybrid systems cured with TETA with respect to the control Epoxy-A (10–12 °C) could be ascribed to the plasticization of the network structure brought about by the presence of ChCl-U, with a consequent reduction in the effective cross-linking density. This result is coherent with what was previously observed in mixtures of neat epoxy-containing ChCl-U and cured with TETA, where a depression of Tg of 19 °C was found [The significant reduction of the Tas found , and alsas found ,51. g which can be ascribed to a higher cross-linking density achieved, also thanks to the possible reaction between urea and the oxirane rings present in the epoxy oligomers and GOTMS coupling agent, as already underlined.In the hybrid formulations cured with PACM, the increase of the DES content from 1.5% to 3% leads to an increase in the maximum TReferring to the measurements of gel-time obtained in rheological tests, the data reported in the last column of g interval, a broad exothermic peak at higher temperatures can be noticed. It is relative to the completion of the curing reactions, which are often not completed when the cure is performed at ambient temperature [The dynamic DSC thermograms of the systems under analysis after a 7-day curing carried out at ambient temperature are reported in perature . As obseIt should also be noted that, irrespective to DES content and curing agent typology, the endothermic peak associated to the physical aging measured for an hybrid system is smaller compared to the same peak observed for the control resin, demonstrating that the evolution of the short range molecular order within the epoxide network resulting from physical aging has been hindered by the presence of alkoxysilane species. This is confirmed also by monitoring the aging at room temperature for longer times, as reported, as an example, in g for cold-curing resin systems is very important. Thus, in g during the aging taking place at ambient temperature is reported for the epoxy-based systems cured with aliphatic . This trend is maintained after aging at ambient temperature for about 1 year. The content of DES, finally, seems to have a small positive influence on the development of Tg, this latter approaching 70 °C. After 1 year of curing, the curing process in the analyzed systems seems to not yet be stopped: the Tg, in fact, is still increasing.For practical applications, the evolution in time of the Tliphatic  and cyclliphatic  amines, g values much higher (even greater than 70 °C) than those measured for the relative non-hybrid epoxy system, irrespective to aging time and DES content. In this case, the process of cold-curing seems to be stopped after about 9 months, irrespective to the kind (non-hybrid or hybrid) nature of the system. As already mentioned in the Introduction section, the commercial cold-cured epoxy-based resins never achieve glass transition temperatures much higher than 60 °C, even after prolonged cold-curing times [g of 10 °C represents an encouraging result proving the effectiveness of the presence of DES in a cold-curing epoxy resin.Passing to analyze the hybrid systems cured with PACM , both fong times . An incr4+ at 235 °C) [The behavior of the systems at very high temperatures, higher than the typical degradation temperatures, was, then, analyzed in the view to assess their thermal resistance. The thermograms relative to TGA analyses, performed in oxidant atmosphere on hybrid and control systems, are shown in  235 °C) ,56.Referring to the hybrid systems, they undergo a first weight loss between 80 °C and 200 °C, irrespective to the DES content and the curing agent typology, differently from what is observed for both control systems, i.e., Epoxy-A and Epoxy-B. This loss is a measure of the amount of volatiles  that are given out through the condensation reactions within the siloxane domains. This phenomenon has been already reported in literature for other epoxy–silica hybrid systems obtained from the sol–gel process ,22. FurtOn the other hand, the hybrid systems exhibit a higher thermal resistance at very high temperatures with respect to control ones, as seen by the values of T0.5 reported in Referring to the effect of the curing agent, the hybrid systems containing ionic liquid cured with cycloaliphatic amine  show the highest thermal resistance; this could be attributed to the chemical stability of the hardener and to the formation of a more stable interconnected organic–inorganic microstructure. The nature of the particles formed through the hydrolysis and condensation reactions, concurrent with the cross-linking of the organic component of the mixture, was also investigated. To this aim, samples of O-I hybrid systems were burned out at 500 °C for 15 h and then analyzed by X-ray diffraction (XRD) analysis. As an example, the XRD pattern of Hyb-H-B sample is reported in The comparison between the mechanical properties measured in flexural mode  for the four hybrid systems and the respective non-hybrid resins, is shown in Referring to the effect of the curing agent, the use of a cycloaliphatic amine allows to obtain cured epoxy with lower strength and strain in flexural mode, once again attributed to the ring structure of the cycloaliphatic hardener. The values of the modulus of the neat epoxy resin, cured with both curing agents, are in agreement with literature data ,58. The presence of a hybrid structure as well as the content of DES do not seem to influence appreciably the flexural modulus of the hybrid systems cured with TETA , Table 4The positive effects in terms of flexural strength and strain linked to the introduction of ionic liquid in hybrid systems are more evident in systems with the lowest concentration of DES, regardless to the kind of hardener. The silica domains and the presence of a low amount of ionic liquid significantly increase the flexural strength in hybrid systems compared with the epoxy controls, as observed in g along with a reduction of the peak height. These results indicate a hindering of the molecular motion of the organic chains brought about by the presence of nano-structured silica domains. The same results have been previously observed for other hybrid systems where the inorganic phase is produced by the sol–gel method using a coupling agent [The results from dynamic–mechanical thermal analysis (DMTA), performed on the produced cold-cured hybrids, confirm the presence of a strong interconnection of organic and inorganic domains in the investigated materials. In ng agent . Moreoveng agent .The SEM micrographs of the fracture surfaces of neat epoxy and hybrid systems, broken during the flexural tests, are reported in Through EDS analysis, whose results are shown in –inorganic epoxy hybrids have been synthesized in view of producing cold-curing epoxy-based resins able to overcome some of the weaknesses characterizing the commercially available structural adhesives/matrices for fiber-reinforced composites, specifically intended for applications in construction. A modified sol–gel method has been proposed, which relies on a mixture of a silane-functionalized epoxy resin, alkoxysilane components and a deep eutectic solvent (DES), the latter based on a mixture of choline chloride and urea. The formulations have been cold-cured with two hardeners both suitable for the cure at ambient temperature, an aliphatic and a cycloaliphatic amine.OrganicTg with respect to the Epoxy-A system; the elastic modulus, on the other hand, has results that are comparable to that measured on the non-hybrid resin. The weak balance between cross-linking reactions and siloxane condensation reactions is responsible also for the lower residual mass at 800 °C, indicative of a lower content of silica domains.The different results obtained in the hybrid systems produced, hardened with aliphatic (A) or cycloaliphatic (B) amines, can be ascribed to the different cross-linking density and silica-based inorganic structure obtained in the two different systems, as a result of the balance of the kinetics of the two typical processes occurring in the system: Condensation reactions of the siloxane domains and cross-linking reactions of the epoxy oligomers. In the case of systems cured with aliphatic amine TETA, the action of DES seems to not be very effective in the formation of the O-I epoxy hybrid, probably because the cross-linking reaction is much faster than the condensation one, as demonstrated by the reduced gel-time measured for these hybrid systems (labeled with “A”) with respect to the others (“B” type). Furthermore, the addition of DES is likely to result in a plasticization of the network, with a reduction of Tg, in the mechanical characteristics and in thermal resistance in the oxygen atmosphere. Although the role of dispersing agents of imidazolium-based ILs has been recently demonstrated for nanofilled epoxy systems [In contrast, in the case of systems cured with cycloaliphatic amine, i.e., PACM, a favorable balance between the kinetics of condensation and curing reactions is achieved due to a relatively slow cross-linking reaction; the inorganic phase, formed by the condensation process, is able to chemically bind to the growing epoxy network. In this case, the presence of DES in the systems cured with cycloaliphatic amine is beneficial for the dispersion of the in-situ generated silica, this being demonstrated by a general increase in  systems ,60, it iThe work presented in the manuscript is the first part of a wider experimental program aiming at developing cold-cured epoxy resins, to be used as either adhesives or matrices for FRPs employed in the construction industry. The analyzed O-I hybrid formulations’ results are very promising in the view of producing cold-curing epoxy-based resins able to overcome some of the well-known weaknesses characterizing the commercially available structural adhesives/matrices for fiber-reinforced composites specifically intended for civil engineering applications. Further studies will be devoted to a more systematic evaluation of the cold-curing kinetics and the final adhesive properties achieved by the experimented system after different curing times. Finally, the in-service adhesive performance of the hybrid resin if joined with other materials will also be investigated.


Developmental signaling pathways control a vast array of biological processes during embryogenesis and in adult life. The WNT pathway was discovered simultaneously in cancer and development. Recent advances have expanded the role of WNT to a wide range of pathologies in humans. Here, we discuss the WNT pathway and its role in human disease and some of the advances in WNT-related treatments. The WNT signaling pathway is an evolutionarily conserved signal transduction pathway that regulates a wide range of cellular functions during development and adulthood. It controls multiple aspects of development, including cell proliferation, cell fate determination, apoptosis, cell migration and cell polarity during development and stem cell maintenance in adults ,2,3. Inaint-1 (mouse mammary tumor virus integration site 1), was isolated from mouse mammary tumors in 1982 [Int-1 turned out to be highly conserved across multiple species and especially similar to the Drosophila gene wingless (Wg) a gene found to be involved in wing development, segmentation and formation of body axis during flight development [WNT comes from a fusion of wg and int [Drosophila and five in Caenorhabditis elegans (C.elegans) [The first WNT gene, then known as  in 1982 . Int-1 telopment ,7,8,9. T and int ,11. Mostelegans) .2+ pathway [The WNT proteins are secreted, lipid-modified glycoproteins, usually 350–400 amino acids in length . WNT pro pathway .Canonical pathway: The stability of cytoplasmic β-catenin is mediated by a multimeric protein complex known as the destruction complex formed by the scaffolding proteins (Axin) [s (Axin) , the tums (Axin) ,19, glycs (Axin) ,20,21,22In the presence of canonical WNT ligands, β-catenin phosphorylation and degradation is inhibited. Gene expression downstream of canonical WNT signaling is regulated by controlling the amount of the transcriptional co-activator β-catenin. When WNT ligands bind to FZD receptors and LRP co-receptors, a cascade of events is initiated, resulting in the disassembly of the destruction complex stabilizing β-catenin . The sigNon-canonical pathways: In contrast to the β-catenin-dependent canonical pathway, WNT proteins are able to activate additional signaling pathways that are independent of β-catenin. These pathways are called non-canonical pathways, which can be further categorized into two distinct branches, the planar cell polarity (PCP) pathway and the WNT/Ca2+ pathway , calcineurin or Protein Kinase C [A second branch of non-canonical WNT signaling is termed the WNT/Caeticulum ,37 and meticulum ,38. Simintration . The relKinase C .Drosophila embryos [Since the identification of the WNT gene more than 30 years ago, various avenues of research have shown roles in myriad processes. The WNT pathway is most famously known for its involvement in hereditary familial adenomatous polyposis (FAP), where a mutated APC tumor suppressor gene fails to regulate β-catenin regulation, allowing tumor cells to progress towards malignancy ,40. Amon embryos , regulat embryos  and many embryos .Beyond embryonic development and cancer progression, the WNT pathway has emerged as a key contributor to Alzheimer’s and metabolic diseases. For example, amyloid-β (Aβ) neurotoxicity in Alzheimer’s disease results in downregulated WNT signaling , which sGiven the vital role that WNT signaling plays in a variety of diseases, this review aims to summarize recent advances in WNT signaling to provide a thorough understanding of WNT signaling in disease.Aging can be defined as the time-related progressive accumulation of detrimental changes that are associated with increasing susceptibility to disease and death . In geneSenescence contributes to tissue aging  through klotho knockout mice that exhibit many age-related disorders [Klotho is a transmembrane protein that acts as an anti-aging hormone [Klotho binds to WNT proteins and reduces β-catenin levels leading to lower levels of WNT signaling which contributes to stem cell depletion and aging [Another example of the detrimental effect of WNT signaling on aging comes from a mouse model of accelerated aging. The authors utilized isorders . Klotho  hormone . Klotho nd aging ,54.Hair graying is another hallmark of the aging process. One mouse study found that β-catenin expression is significantly elevated in the skin of aged mice through WNT10b/β-catenin signaling promoting melanocyte stem cell differentiation. The authors concluded that the increase in WNT signaling is insufficient to induce hair regeneration but may promote melanocyte stem cell differentiation resulting in a decreased number of melanocyte stem cells and eventually hair graying .In contrast, a number of studies have also suggested that WNT signaling has positive effects on aging. In one study, Chen et al. reported that β-catenin plays an important role in the early phase of fracture healing. Several WNTs  were expressed during fracture repair showing activation of WNT/β-catenin signaling through a TCF-dependent transcription reporter in both bone and cartilage formation during fracture repair . AnotherThe WNT pathway plays a complex role in cancer development. Often, mutations of key components are associated with processes like uncontrollable cell proliferation, epithelial–mesenchymal transition (EMT) and metastasis. This section will discuss the main WNT components that have been investigated in oncology and will review the current progress of their roles in cancer development .Frequent APC mutations in colorectal cancer: The APC protein is an important component of the destruction complex in canonical WNT signaling. In addition to this role, APC is also essential for the rapid transition of Axin immediately after WNT stimulation and facilitates the association of Axin and co-receptor LRP6/Arrow in Drosophila [osophila .As APC negatively regulates the canonical WNT pathway, it is likely that it can act as a tumor suppressor. Consistent with this idea, APC mutations, which are present in approximately ~80% of colorectal cancers, are indeed mostly loss of function and/or truncating mutations ,61. ThesAPC mutations in colorectal cancers suggest APC as a powerful target for therapy. TASIN-1 (truncated APC selective inhibitor-1) was identified as a molecule that could specifically kill cells with truncated APC’s. TASIN-1′s administration into xenograft and mouse models has shown its effectiveness in tumor suppression and its specificity in killing cells with truncated APC’s while sparing normal cells in vivo [APC in patients with colorectal cancer, further studies are needed to investigate APC’s molecular mechanism in β-catenin degradation, its use as a potential biomarker and other possible therapeutic targeting approaches.The prevalence of  in vivo . ConsideAXIN1/2: Axin proteins (AXIN1 and AXIN2) are important scaffold proteins that help assemble the β-catenin destruction complex [1 inhibits cell growth in hepatocellular carcinoma (HCC) [Axin1 mutated HCCs do not show a β-catenin activation program. These HCCs might have occurred independently of the WNT/β-catenin pathway and instead involved other pathways like YAP and Notch [ complex . AXIN1/2ma (HCC) . Howevernd Notch .AXIN2 has been shown to behave as both a tumor suppressor and an oncogene. Its mutations are also observed in various cancers. Chapman et al. observed genetic changes (mostly deletions) of AXIN2 in 7% of human adrenocortical adenomas tumor samples and 17% of adrenocortical carcinomas tumor samples [AXIN2 might act as a tumor suppressor, AXIN2 downregulation is associated with poorer overall survival of patients with breast cancer [AXIN2 as tumor-promoting, as it upregulates the transcriptional repressor, SNAI1, leading to increased EMT and metastatic activity in colorectal cancers in mice [ samples . Consistt cancer . However in mice . As thes in mice .LRP5/6: LRP5 and LRP6 in the low-density lipoprotein receptor (LDLR) family are single-pass transmembrane coreceptors in the WNT canonical signaling pathway. The FZD receptor may form a WNT-induced FZD–LRP6 (or LRP5) complex with these coreceptors. After WNT ligands bind to these receptors, the β-catenin signaling is initiated [nitiated ,74.LRP5 failed to block osteosarcoma cell formation, suggesting LRP’s role as a tumor suppressor [LRP5 has contradictory roles in cancer progression as well. When osteosarcoma cells are transfected with dominant negative, soluble LRP5 (sLRP5), epithelial–mesenchymal transition (EMT) is reversed, suggesting wildtype LRP5′s role in promoting EMT and metastasis . Howeverppressor .LRP6 and as a result, increased β-catenin levels, suggesting LRP6 as tumor-promoting [LRP6 is also found in triple negative breast cancers. When LRP6 is knocked-down in triple negative breast cancer cells, tumor growth is suppressed in vivo [LRP5/6 as suppressing metastasis. This study showed that LRP5/6 downregulation is crucial for metastasis in mouse breast cancer models [Similarly, LRP6′s role in cancer progression has been controversial. A total of 45% of human hepatocellular carcinoma cells have overexpressed romoting . Consist in vivo . LRP’s a in vivo . Howeverr models , suggestTherapeutic targeting of LRP5/6 shows promising results. Mesd, a specialized chaperone that binds to LRP5/6 on the cell surface, inhibits LRP5/6 ligands and suppresses downstream WNT/β-catenin signaling in prostate cancer . NiclosaWNT 5A: The WNT5A ligand binds to certain receptors  and activates non-canonical WNT signaling pathways [WNT5A expression induces EMT in a PKC-dependent pathway in human melanoma cell lines [WNT5A in human gastric carcinoma-derived MKN-7 cells promoted cancer cell invasiveness by upregulating a transcription factor involved in EMT, namely SNAI1. This study also suggested that WNT5A may play a role in creating favorable tumor microenvironments and inducing cancer stem cell properties [WNT5A drives pseudo-senescence in melanoma cancer cells. When exposed to stress, pseudo-senescent cells display classical senescence markers, but are highly invasive, metastatic and resistant to therapy. In response to stressors, tumor cells with knocked down WNT5A do not display this pseudo-senescent phenotype, whereas highly expressed WNT5A tumor cells do, suggesting WNT5A’s role in promoting cancer cell adaptiveness under stress [pathways . Mutatiopathways . For exapathways . WNT5A ell lines , suggestoperties . Aside fr stress . WNT5A hr stress . Future RNF43: Ring finger protein 43 (RNF43) is an E3 ubiquitin-protein ligase that degrades WNT receptors such as FZD and LRP5/6. It serves as a negative regulator of the WNT pathway. The mechanisms of WNT signaling suppression are different between non-canonical and canonical pathways. In non-canonical pathways, the suppression involves the interaction between RNF43′s C-terminal cytoplasmic region and Dsh’s PDZ domain. Suppression of the canonical pathway involves FZD’s extracellular protease-associated domain, cysteine-rich domain and the intracellular ring finger domain of RNF43 [of RNF43 . A recenof RNF43 .RNF43 acts as a tumor suppressor. When 185 human colorectal tumor samples were analyzed, there were RNF43 somatic mutations in over 18% of colorectal adenocarcinomas and endometrial carcinomas. This study also showed that most RNF43 mutations (73%–75% of non-silent mutations) found were truncating, confirming that RNF43 acts as a tumor suppressor [RNF43 mutations were also found in pancreatic cancers [RNF43 in these cancers, so further studies are needed to investigate how certain mutations in RNF43 can possibly lead to the development of these cancers.ppressor . RNF43 m cancers  and muci cancers . HoweverTCF4/TCF7L2: T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors are the downstream effectors of the WNT pathway. When WNT ligands bind to upstream receptors, β-catenin is released from the cytoplasmic destruction complex and moves to the nucleus, where it associates with TCFs to regulate the transcription of target genes [et genes .TCF4, one of the most studied members of the TCL/LEF family, seems to have contradictory roles as both tumor-promoting and tumor-suppressing. TCF4 was originally believed to be tumor-promoting. Van De Wetering et al. demonstrated that dominant-negative TCF4, which fails to bind to β-catenin, halted cell proliferation in colorectal cells . HoweverPTK7: Another gene linking the various types of WNT signaling to cancer is the gene PTK7, an orphan receptor linked to a variety of cancers as a highly upregulated biomarker [Drosophila (known as off-track in flies) [iomarker . PTK7 waiomarker . Its funn flies)  and subsn flies) . The mecn flies) ,107,108,n flies) .WNT pathway as a tumor suppressor: Although WNT is usually referred to as an oncogenic pathway, it can also function in quite the opposite direction depending on cellular context. This has primarily been studied in malignant melanoma, where activation of WNT reduces cell proliferation and can actually function as a tumor suppressor. Overall, the main lesson from these studies is that context is crucial, namely depending on what transforming factor is driving oncogenicity since WNT activation can lead to unexpected consequences [equences ,111,112.PSEN1) and presenilin-2 (PSEN2) genes are known to play causative roles in the disease pathogenesis of Alzheimer’s [Alzheimer’s disease (AD) is an age-associated neurodegenerative disease (ND) characterized by progressive loss of cognitive function, memory and other associated neurobehavioral issues ,114. Extheimer’s ,117,118.heimer’s . Recent heimer’s ,120,121.The WNT signaling pathway is fundamental to the development of the central nervous system and is strongly linked to AD pathogenesis . WNT sigTapia-Rojas and colleagues showed that WNT signaling regulates APP processing by changing the expression of β-secretase (BACE1) enzyme . InhibitPSEN1 promote GSK3 activity and tau phosphorylation [WNT signaling has also been linked to tau microfibrillar phosphorylation, specifically through the finding that mutations in rylation , and GSKrylation ,133.An important clinical consequence of AD is cognitive decline and memory. WNT signaling plays a role in the processes of learning and memory through regulation of synaptic structure and function ,135. ActWNT signaling is an important regulator of development and progression of organ growth and cell fate. Genes encoding WNT ligands are expressed in the pancreas. Many in vivo and in vitro studies have shown that components of the WNT pathway are involved in pancreatic β-cell proliferation, lipid metabolism and glucose-induced insulin secretion ,142,143.TCF7L2/TCF4 as a susceptibility gene for T2D. Polymorphisms of TCF7L2 show a significant effect on the risk of developing T2D [Recent genome-wide association studies have suggested several metabolic connections and the possible underlying signaling pathways in the pathophysiology of several metabolic diseases and neurodegenerative diseases. The major finding of these human genetic studies is identification of ping T2D ,148,149.ping T2D ,151. Sinping T2D . LRP5 enping T2D ,154 and ping T2D . These oping T2D . FOXOs aping T2D .Studies have shown the involvement of the WNT pathway in diabetic neuropathy, one of the long-term complications of diabetes . WNT sigA long-term complication of diabetes is nephropathy, a process linked to WNT/β-catenin signaling . The halWNT signaling in cardiovascular diseases: WNT signaling is essential for heart development, but dysregulation of WNT can cause cardiac and vascular diseases [diseases ,178. Fordiseases , and is diseases . The LRPdiseases . DKK1 isdiseases . The WNTdiseases , was alsdiseases . Moreovediseases ,183.Destabilization of an atherosclerotic plaque over time results in the release of plaque’s contents into circulation which can lead to myocardial infarction. Alternatively, plaque erosion with distal embolization or coronary vasospasm can also lead to myocardial infarction . Either WNT signaling has a role in conferring pathology in several cardiovascular diseases such as cardiac arrhythmias, cardiac hypertrophy and heart failure. For a detailed review of WNT signaling in cardiac and vascular diseases, please refer to Foulquier et al. .WNT signaling in neuronal diseases: Several studies have also reported a causative link between WNT signaling and autism. Mice mutants for Dvl1 and 3 showed reduced β-catenin expression which in turn caused premature deep layer neurogenesis of neural progenitors in specific brain regions during embryonic development. This had an adverse effect on the precise establishment of neural connections in the future prefrontal cortex, which was observed by serious deficits in brain size and social behavior in adults [n adults . This deFmr1 (FMRP gene) knockout mice [Fragile-X syndrome (FXS), a heritable form of autism, is caused by a deficiency of fragile-X linked mental retardation protein (FMRP) that results in various degrees of autism-like behavior. FMRP is a negative regulator of protein translation, which was found to reduce the translation of WNT2 mRNA in the brain of out mice ,195,196.out mice . The conout mice .WNT signaling was linked to the pathogenesis of Parkinson’s disease by the dysregulation of expression of WNT pathway components in brain tissue of Parkinson’s disease patients . FurtherPatients with schizophrenia showed altered GSK3 activity , as wellSurprisingly, a recent study by Paige et al. showed the essential role of the WNT signaling pathway in promoting functional recovery from spinal cord injury in lampreys . These sWNT signaling in liver diseases: A notable feature of the liver, unlike other tissues, is its ability to regenerate post-injury, driven by the WNT/β-catenin signaling pathway [SFRP2, FZD4, SOST, CTNNB1 and CSNK1A1) have also been associated with inflammation and fibrosis in patients with hepatitis C [ pathway ,209,210. pathway ,212,213.atitis C . β-Catenatitis C . Conversatitis C .Mice with mutant LRP6 developed a fatty liver due to increased AKT/mTOR signaling resulting in elevated hepatocyte lipogenesis, which could be reversed by the administration of WNT3a . MoreoveCtnnb1-null mice showed defective bile acid homeostasis with elevated levels of toxic liver compounds in the serum and liver [Accumulation of toxic cholephilic substances such as bile acids due to reduced bile flow can cause liver damage, a condition known as cholestasis. This is characterized by a reduced bile flow, which often results in the accumulation of toxic cholephilic substances such as bile acids that can cause liver damage . Liver-snd liver ,222, sugHypoxia inducible factor-1α (HIF1α), which mediated transcription and promotion of cell survival during ischemia/reperfusion injury in hepatocytes, was also found to be mediated via β-catenin signaling ,224.WNT signaling in kidney diseases: Tubule-specific β-catenin knockout mice showed higher mortality, increased serum creatinine as well as more severe morphologic injury after acute kidney injury as compared to control mice [rol mice . Conversrol mice . WNT7B drol mice . β-Catenrol mice . A WNT arol mice .Continuous activation of WNT/β-catenin results in the progression of acute kidney injury to chronic kidney disease displaying interstitial myofibroblast activation and excessive extracellular matrix deposition . A mouseWNT signaling in lung diseases: Increased levels of matrix metalloproteinases (MMPs) and pro-inflammatory cytokines such as IL1, IL6, IL8 and IL15 are general features of lung inflammation. The canonical WNT signaling pathway has been shown to regulate the expression of both MMPs and pro-inflammatory cytokines. Activation of canonical WNT signaling increased expression of MMP-2, MMP-3, MMP-7, MMP-9 and MT3-MMP in mice [ in mice  suggestiIn addition, the WNT5A-FZD5 signaling cascade may play a role in regulating the levels of pro-inflammatory cytokines. In pathological studies of rheumatoid arthritis, overexpression of WNT5A and FZD5 correlates with increased levels of pro-inflammatory cytokines  . ExperimIn idiopathic pulmonary fibrosis (IPF), immunohistochemical analyses reveal that MMP7 is consistently upregulated . Higher WNT signaling in oral disease: Mutations in WNT signaling have also been associated with oral diseases. In salivary gland tumors, loss-of-function mutations in the WIF1 WNT inhibitor gene are associated with salivary gland oncogenesis [ogenesis . Mutatioogenesis . Mutatioogenesis ,255.WNT signaling in skin disease: A nonsense mutation in WNT10A is responsible for severe hypodontia in odonto-onycho-dermal dysplasia, a rare ectodermal dysplasia characterized by keratoderma, dry hair and hyperkeratosis of the skin [PORCN (porcupine) gene, a regulator of WNT signaling, result in focal dermal hypoplasia, characterized by widespread lesions of dermal hypoplasia [the skin . Mutatiopoplasia . In psorpoplasia , and patWNT signaling in allergic airways: Impaired WNT signaling is also associated with diseases of allergic airways. In a mouse model, expression of WNT suppressed the development of allergen airway disease, while treatment with LiCl  protected wild-type mice from development of allergic airway diseases [diseases . In asthdiseases , suggestWNT signaling in skeletal diseases: The notion that WNT is crucial to bone development gained traction in the early 2000s, when several studies reported that an inactivating mutation in LRP5 was associated with osteoporosis-pseudoglioma syndrome (OPPG), characterized by lower bone mass and strength and a higher predisposition to suffer from bone fractures [ractures . Anotherractures ,263. TheThe molecular mechanism by which WNT modulates bone growth and repair is known. WNT regulates the differentiation of osteoblasts and chondrocytes, thereby directly affecting pathologies such as osteoarthritis, a disease characterized by dysregulation of osteoblast formation and bone remodeling . SclerosAs WNT regulation is important to bone development and repair, it is not surprising that dysregulation of the pathway leads to bone disease. As studies that intersect WNT signaling and bone development continue to emerge, novel interactions of WNT mutations that underlie bone-related pathologies will also surface. So far, studies have shown involvement of the WNT pathway in osteoarthritis and osteoporosis ,269,270,x disease” through academic journals and recent publications will almost always yield results. As such, it has become obligatory to invest resources into targeting this pathway, which unfortunately has had little success.The previous sections have highlighted the importance of WNT signaling in various pathological and physiological contexts. A general search of “WNT in The general concept of treating diseases through the WNT pathway is through one of two possibilities: either enhancing or inhibiting the pathway. In diseases where WNT is lacking, the inhibition of naturally occurring WNT antagonists or upregulation of WNT agonists can lead to supplementation of WNT signaling levels to yield therapeutic effects . In contThere are ongoing efforts to find ways of targeting this pathway, mostly for the treatment of various WNT-driven cancers. However, the WNT pathway is a great candidate target for the treatment of pathologies related to musculoskeletal and neurological systems. The WNT pathway has also drawn interest in the field of dermatology. In this section, we present an update on agonists and antagonists of WNT signaling in various clinical contexts.Cancers: Cancers, perhaps, have the highest potential for the development of WNT therapeutics in the clinic. Aberrant activation of WNT through mutations of specific WNT components can be found in colorectal, endometrial, breast and lung cancers, among many others [y others ,276,277.y others ,279. ThePorcupine (PORCN) is a membrane-bound O-acyltransferase involved in the secretion of WNT ligands . There aAnother approach currently in clinical trials is the introduction of WNT signaling inhibitors directly into the blood stream of patients. For example, Vantictumab is an antibody targeting 5 of the 10 human FZD receptors  binding to the extracellular domains of FZD and interfering with signaling ,285. AnoAnother class of WNT inhibitors that has shown efficacy in the treatment of WNT-related cancers are Tankyrase inhibitors. Tankyrase 1 and 2 belong to the poly ADP-ribose polymerase (PARP) family of proteins and are associated with the WNT pathway by facilitating the degradation of Axin ,289. TheThese anti-cancer agents target more peripheral members of the WNT pathway. This is necessary, as the treatment of core WNT components could result in non-selective inhibition of WNT and detrimentally affect WNT-dependent stem cell populations, thereby resulting in unwanted side effects. This is especially true for tissues with high rates of turnover, such as hair follicles and the intestinal crypts, both of which are controlled by WNT signaling. However, there are still several anti-cancer drugs in development which do target components more central to the canonical WNT signaling cascade. For example, the preclinical drug PKF118-310 directly inhibits the β-catenin/TCF transcriptional complex . PRI-724WNT inhibition via cross-talking pathways: As with many other signaling pathways, the WNT pathway is highly intricate and complex. This is reflected in its many interacting partners and involvement in various physiological and pathological contexts. An additional feature of this complexity is the crosstalk that occurs between the WNT pathway and other signaling cascades, especially in cancers [ cancers ,295,296.sFRP-1 is another potential target for the treatment of WNT-driven cancers. It functions as a mediator of crosstalk between two signaling cascades—Hedgehog and WNT . VismodeThere are several ongoing efforts to develop therapies to target the aberrant WNT signaling found in many cancers. With drugs ranging from various antibodies of WNT proteins to small molecule inhibitors, the WNT pathway continues to be an attractive yet elusive target for the treatment of cancers due to its overwhelming complexity and the possible indirect consequences to healthy, WNT-dependent cells. We are certain to see more drugs roll out over the course of the next decade as these WNT anti-cancer agents are further studied and some of the clinical trials move towards FDA approval.Neurological diseases: WNT signaling is necessary for the development of the early brain, as it plays crucial roles in determining cell polarity and migration, as well as neural patterning and synapse development [elopment ,299,300.elopment ,302,303.Antagonists of the WNT pathway, such as DKK1 protect against hippocampal sclerosis, a key characteristic of epilepsy of the temporal lobe . As discAnother neurological disorder where WNT inhibition could potentially be used therapeutically is in cell therapy. More specifically, WNT inhibition could be used to generate cortical motor neurons (CMNs) in order to treat damaged corticospinal tracts, which may occur from various neurodegenerative disorders . The WNTMusculoskeletal diseases: A strategy that has been proposed to treat musculoskeletal diseases via the WNT pathway is to develop inhibitors for WNT antagonists that are relevant to bone development, mainly DKK1 and sclerostin [lerostin . Mouse blerostin ,307. Thelerostin ,309,310.As the use of WNT therapies for musculoskeletal disorders is a relatively new field, there are many drugs that are presently under preclinical and clinical studies. For instance, SM04690 is a small molecule inhibitor of the WNT pathway and is presently undergoing clinical trials for degenerative disc disease . When teOsteoarthritis is another musculoskeletal disease where the WNT pathway is being explored for potential therapy. Fluoxetine and Verapamil, two pre-approved drugs, were able to successfully suppress abnormal WNT activity and slowed cartilage degradation in vitro as well as in rodent models ,314. SM0Dermatological disorders: The interest in WNT-related therapies has also made it to the field of dermatology. Androgenetic alopecia (AGA), commonly known as male pattern baldness, is the most common progressive hair loss disorder in men and is characterized by progressive hair follicular miniaturization [rization . SM04554rization . Psoriasrization .Complexity of treating diseases via WNT modulation: WNT signaling is a highly complex pathway regulating many biological processes. WNT-related therapeutics focus on one of two strategies: they either inhibit aberrant WNT signaling or enhance deficient WNT signaling. However, these two strategies are an all-or-nothing approach, which overlooks the complexity of WNT signaling and its effect on different cells. The various WNT ligands and receptors generate permutations that allow for precise control of signaling in contexts ranging from intestinal crypt cell renewal to hair follicle spacing. The intricacy of WNT signaling in various physiological contexts leads to a complexity of disorders and diseases where the various WNTs and receptor-complexes define diseases [diseases ,318.One approach is to consider the Goldilocks hypothesis (WNT levels being just right) when looking at WNT signaling in diseases. A study found that the level of WNT signaling, the position of the tumor in the colon and the rate of tumorigenesis is highly correlated. For example, APC mutations in the proximal and distal colon vary in their number of β-catenin binding sites required for tumorigenesis ,319,320.


Wnt signaling has been implicated in numerous aspects of development, cell biology, and physiology. When aberrantly activated, Wnt signaling can also lead to the formation of tumors. Thus, Wnt signaling is an attractive target for cancer therapy. Based on our screening program targeting Wnt signaling activity using a cell-based luciferase screening system assessing TCF/β-catenin transcriptional activity, we isolated a series of terpenoids and heterocyclic aromatic compounds that affect the Wnt signaling pathway at different points. Here, we describe our recent results in screening for natural products that inhibit or activate Wnt signaling. In our efforts to identify bioactive natural products , we receWnt signaling is conserved among a variety of species and has been implicated as playing a role in numerous aspects of development, cell biology, and physiology. Wnt signaling has been implicated not only in tumorigenesis, but also diabetes, schizophrenia, and other disorders. Thus, the Wnt signaling pathway is an attractive therapeutic target, and there is significant interest in identifying Wnt signaling inhibitors from libraries of small-molecule natural products. Figure GCC), was provided by Prof R. T. Moon (University of Washington) and used to evaluate and exclude non-selective inhibition of Wnt signaling based on monitoring of luciferase activity (FOP activity). Using this assay system, hundreds of plant extracts were examined by our group, resulting in the selection of several hit samples. The isolation of active constituents from these hit plants is described below.A cell-based reporter luciferase assay system that monitors inhibition of TCF/β-catenin transcriptional activity (TOP activity) was used to screen for compounds that target Wnt signaling. The TCF/β-catenin complex is a downstream transcriptional factor in the Wnt pathway . TOP actCalotropis gigantean (Asclepiadaceae), which was collected in Barisal, Bangladesh, in November 2012, exhibited significant inhibition of TOP activity. After extraction with MeOH and solvent partitioning with hexane, ethyl acetate, and water, the ethyl acetate-soluble fraction proved to be active. Further fractionation of the ethyl acetate fraction using silica gel, Sephadex LH20, and ODS chromatography and guided by the same assay yielded six compounds, all of which were identified as cardenolides based on spectral data, as shown in Fig. 4, 5, and 6 in particular did not significantly decrease cell viability. Therefore, these compounds were revealed to be potent TCF/β-catenin transcriptional inhibitors. All six isolated compounds exhibited cytotoxicity against three Wnt-dependent colon cancer cell lines , with IC50 values ranging from 1.8 to 7.0 nM, but the compounds were not significantly cytotoxic to Wnt-independent RKO colon cancer cells (IC50 > 10 nM).A methanol (MeOH) extract of the exudate of C. gigantea constituents was carried out using compound 4  and SW480 colon cancer cells. Treatment of SW480 cells with calotropin (4) led to a significant dose-dependent decrease in both nuclear and cytosolic β-catenin protein expression. Calotropin (4) also decreased protein levels of c-myc, which is a target of Wnt, indicating that calotropin inhibits the Wnt signaling pathway. It was reported that the level of β-catenin protein is regulated by a proteasomal degradation system. The decrease in β-catenin expression mediated by calotropin (4) was abolished by MG-132, a proteasome inhibitor; calotropin (4) did not decrease β-catenin expression in the presence of MG-132. This result suggests that calotropin (4) accelerates the proteasomal degradation of β-catenin.Further studies on the Wnt signaling inhibitory activity of 4) induced β-catenin degradation, which was recovered using the GSK3β inhibitor LiCl; (2) calotropin (4) induced phosphorylation of β-catenin at the CK1α site (S45), which was not affected by LiCl; and (3) calotropin (4) induced phosphorylation of β-catenin at the GSK3β sites , which was inhibited by LiCl. It is known that β-catenin is phosphorylated first at the CK1α site, followed by phosphorylation at the GSK3β sites. These results thus indicated that calotropin (4) induces phosphorylation of the CK1α site of β-catenin.Proteasomal degradation takes place after ubiquitination of the target protein to be degraded, and ubiquitination of the protein takes place after its phosphorylation by GSK3β (glycogen synthase kinase 3β) and CK1α (casein kinase 1α). We therefore examined the level of β-catenin phosphorylation in SW480 cells as follows: (1) calotropin (4) increased the CK1α protein level, which was not affected by CKI-7; (2) calotropin (4) increased β-catenin phosphorylation at the CK1α and GSK3β sites, which was inhibited by CKI-7; and (3) calotropin (4) induced β-catenin degradation, which was recovered by CKI-7. CKI-7, a kinase inhibitor, inhibited phosphorylation by CK1α without affecting the level of CK1α protein. These results indicated that the level of CK1α protein increases as a result of treatment with calotropin (4).The phosphorylation of β-catenin was then examined using the CK1α inhibitor CKI7, with the following results: (1) calotropin (4) was further examined by knockdown of CK1α expression using siRNA. Following CK1α knockdown by siRNA, calotropin (4) did not induce phosphorylation of β-catenin. Subsequently, no degradation of β-catenin was observed, even in the presence of calotropin (4), under conditions of siRNA knockdown. These results indicated that the effect of calotropin (4) is dependent on CK1α. Furthermore, real-time PCR experiments revealed that calotropin (4) increases the expression of CK1α mRNA. From these observations, the effect of calotropin (4) can be explained as follows. Calotropin (4) induces an increase in CK1α protein levels and then induces degradation of β-catenin, followed by a decrease in the expression of genes such as c-myc. Thus, calotropin (4) inhibits the Wnt signaling pathway  inhibition differs from that of the other two compounds. Pyrvinium activates CK1α enzyme activity, but has no effect on CK1α protein level  was was9) (FEurycoma longifolia (Simaroubaceae), collected from Khon Kaen, Thailand, was partitioned with hexane, ethyl acetate, and 1-butanol. Activity-guided fractionation of the hexane and ethyl acetate layers using silica gel and ODS column chromatography, followed by preparative HPLC, led to the isolation of a compound that inhibited TOP activity with an IC50 of 6.3 μM. This active compound was identified as a β-carboline alkaloid, 9-hydroxycanthin-6-one (10)  led to a significant decrease in the level of β-catenin protein and increased levels of phosphorylated β-catenin (p-β-catenin) , without any effect on levels of GSK3β, CK1α, and p-β-catenin (Ser45). Intracellular β-catenin levels are regulated by the ubiquitin–proteasome system. β-Catenin is first phosphorylated on Ser45 by CK1α, followed by phosphorylation on Thr 41, Ser37, and Ser33 by GSK3β. Phosphorylation of these residues is essential for the recognition of β-catenin by the F-box β-transducin repeat-containing protein (β-TrCP), leading to ubiquitination and degradation by the proteasome pathway.The MeOH extract of air-dried roots of 10) was suppressed by GSK3β-siRNA but not CK1α-siRNA. These results suggest that 9-hydroxycanthin-6-one (10) inhibits Wnt signaling via activation of GSK3β enzyme activity independent of CK1α. The effect of 9-hydroxycanthin-6-one (10) on zebrafish was also investigated. 9-Hydroxycanthin-6-one (10) decreased the expression of the Wnt target genes mitf and zic2a in zebrafish embryos and induced malformation of the MHB (midbrain-hindbrain boundary) and formation of a curled tail in zebrafish embryos. 9-Hydroxycanthin-6-one (10) also decreased TOPdGFP activity in TOPdGFP-transgenic zebrafish embryos. Although treatment of zebrafish embryos with BIO  alone induced an eyeless phenotype, additional treatment with 9-hydroxycanthin-6-one (10) rescued the eyeless phenotype caused by BIO. These observations indicate that 9-hydroxycanthin-6-one (10) inhibits Wnt signaling in vivo.The degradation of β-catenin by 9-hydroxycanthin-6-one (11) , collected in Bangladesh in 2011. Coronaridine (11) inhibited TOP activity with an IC50 of 5.8 μM. Coronaridine (11) also decreased β-catenin protein levels in SW480 colon cancer cells, and this decrease was not affected by co-treatment with MG132 (a proteasome inhibitor). In addition, treatment of SW480 cells with coronaridine (11) caused a decrease in β-catenin mRNA expression. These results suggest that coronaridine (11) inhibits the Wnt signaling pathway by decreasing the expression of β-catenin mRNA.Coronaridine (11) Fig.  28] was was11)  specimens collected in Bangladesh in 2010 led to the isolation of four lignans exhibiting dose-dependent TOP-inhibition activity without significant effects on FOP activity. Of these four lignans, boehmenan (12)  decreased the levels of both cytosolic and nuclear β-catenin in STF/293 cells and decreased levels of c-myc protein, a TCF/β-catenin target. In addition, the decrease in β-catenin levels mediated by boehmenan (12) was abolished by co-treatment with the proteasome inhibitor MG132. Thus, the downregulation of β-catenin levels by boehmenan (12) may be related to increased proteasomal degradation.Activity-guided separation based on TOP-inhibition activity in the MeOH extract of stems of 12) Fig.  29] exh exhHibis3 extracts of dried roots of Polyalthia cerasoides (Annonaceae) specimens collected in the Khon Kaen area of Thailand exhibited significant TOP-inhibition activity. Activity-guided fractionation using an ODS column and preparative HPLC resulted in the isolation of a new as well as a previously described bis-aporphine alkaloid . The structure of the new compound, which was designated cerasoidine (13), was determined by X-ray analysis as a novel dimeric structure with a unique ureido bond. Cerasoidine (13) was revealed as a 57:43 enantiomeric mixture by chiral HPLC analysis. The known bis-aporphine alkaloid, bidebiline E (14), markedly inhibited Wnt signaling by suppressing the accumulation of β-catenin in the nucleus. Liriodenine (15), an aporphine alkaloid with a monomeric structure, was also obtained from P. cerasoides. Studies suggested that liriodenine (15) inhibits Wnt signaling via up-regulation of the proteasome system to increase β-catenin degradation  and andRicinNocardia sp. as unutilized resources for the isolation of bioactive natural products [Nocardia sp. with animal cells to isolate compounds that are not produced in Nocardia sp. in mono-culture [Nocardia tenerifensis IFM 10554T with mouse macrophage-like J774.1 cells in modified Czapek-Dox medium resulted in the isolation of a new cyclic nonapeptide, nocarjamide (17)  possesses nine amino acid residues connected by eight amide bonds, while the N-terminal threonine residue was connected with 3-methylbutanoic acid moiety and the C-terminal N-methylvaline residue with an ester bond. Of nine amino acids, two residues  prove to be D. In addition, the X-ray analysis of nocarjamide (17) revealed that two conformers are present in a 1:1 ratio in its crystal state. Although nocarjamide (17) exhibited almost no cytotoxicity against several cancer cell lines, TOP activity in the presence of 20 and 40 μM nocarjamide (17) was 1.8- and 2.2-fold higher than that of the control, respectively. At both of these concentrations, the TOP activity was 1.5-fold higher than the FOP activity. Western blot analyses of HEK293 cells showed that nocarjamide (17) increased protein levels of β-catenin and c-myc, suggesting that nocarjamide (17) activates Wnt signaling.We recently initiated a study of pathogenic actinomycetes products , 33 and -culture . Co-cult17) Fig. , the strScreening studies of extracts of various medicinal plants and actinomycetes using a cell-based luciferase assay assessing TCF/β-catenin transcriptional activity (TOP activity) resulted in the isolation of a number of bioactive constituents that affect the Wnt signaling pathway. These active constituents affect Wnt signaling via different mechanisms, including increasing CK1α protein  levels, activating GSK3β enzyme activity (9-hydroxycantin-6-one), decreasing nuclear β-catenin levels (xylogranin B), decreasing β-catenin mRNA expression (coronaridine), and enhancing β-catenin degradation . Our screening also identified a Wnt signaling activator that functions via inhibition of CK1α activity.Further natural products-based screening studies are in progress aimed at discovering compounds exhibiting unique effects on various signaling pathways that are expected to be useful in basic biological research and disease therapy.


FBXW2 overexpression reduces β-catenin levels and protein half-life, whereas FBXW2 knockdown increases β-catenin levels, protein half-life and transcriptional activity. Functionally, FBXW2 overexpression inhibits migration and invasion by blocking transactivation of MMPs driven by β-catenin, whereas FXBW2 knockdown promotes migration, invasion and metastasis both in vitro and in vivo lung cancer models. In human lung cancer specimens, while FBXW2 levels are inversely correlated with β-catenin levels and lymph-node metastasis, lower FBXW2 coupled with higher β-catenin, predict a worse patient survival. Collectively, our study demonstrates that FBXW2 inhibits tumor migration, invasion and metastasis in lung cancer cells by targeting β-catenin for degradation.FBXW2 inhibits proliferation of lung cancer cells by targeting SKP2 for degradation. Whether and how FBXW2 regulates tumor invasion and metastasis is previously unknown. Here, we report that FBXW2 is an E3 ligase for β-catenin. FBXW2 binds to β-catenin upon EGF-AKT1-mediated phosphorylation on Ser FBXW2 is an F-box protein that target substrates for degradation through SCF E3 ligase. Here, the authors show that FBXW2 suppresses lung cancer migration and invasion by promoting degradation of β-catenin and this is dependent on EGF-AKT1. Due to recurrence, extensive invasion and metastasis, the overall 5-year survival rate of NSCLC is lower than 15% after initial diagnosis2. Although multiple gene mutations, including EGFR, KRAS, p53, and PTEN, have been extensively reported in NSCLC3 comprehensive molecular mechanisms that underlie the initiation, progression, and metastasis of NSCLC remain elusive. To pursue additional involving genes, we recently reported that FBXW2 (F-box and WD-repeat domain-containing 2), a poorly characterized F-box protein, acts as a tumor suppressor to inhibit growth and survival of lung cancer cells4.Lung cancer, ~80% of which are non-small-cell lung cancer (NSCLC), is the leading cause of cancer-related deaths in the world5. The SCF ubiquitin ligases, also known as CRL1 (Cullin-RING ligase 1), consist of adapter protein SKP1, scaffold protein Cullin-1, Ring box protein-1 (RBX1)/ROC1, and an F-box protein. While Cullin and RING protein are required for ligase activity, the F-box protein determines the substrate specificity. As the largest family of ubiquitin ligases, SCF ubiquitin ligases promotes timely ubiquitylation and degradation of diverse regulatory proteins to control many biological processes7. FBXW2 is originally identified as an ubiquitin ligase for polyubiquitination and degradation of GCM1 , which suppresses placental cell migration and invasion10. Our recent study identified FBXW2 as a tumor suppressor via promoting ubiquitylation of SKP2 (S phase kinase-associated protein 2) for targeted degradation to inhibit growth and survival of lung cancer cells4. However, whether FBXW2 targets other substrates to regulate the migration and invasion of lung cancer cells is totally unknown.FBXW2, one of the 69 F-box proteins, functions as a substrate recognition receptor in the SCF (SKP1-Cullin1-F-box protein) ubiquitin ligase complexes11. β-catenin, an Armadillo protein, acts both as a component of cell–cell adhesion structure by interacting with the cytoplasmic domain of E-cadherin and as a cellular signaling molecule involved in the regulation of gene expression following Wnt pathway activation12. In the absence of Wnt (Wnt-off phase), cytoplasmic β-catenin forms a complex with axin/conductin, casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK-3β), and the adenomatous polyposis coli protein (APC). CK1 and GSK-3β sequentially phosphorylate β-catenin at N-terminal Ser and Thr residues , resulting in its ubiquitylation and proteasomal degradation by SCFβ-TrCP ubiquitin ligase, in which F-box protein β-TrCP (β-transducin repeats-containing protein) acts as the substrate recognition receptor13. Upon exposure to the Wnt ligand (Wnt-on phase), the Axin complex is inactivated and hypophosphorylated β-catenin is stabilized by escaping from degradation, and then translocates to the nucleus, where it interacts with transcription factors, the TCF/LEF-1 family to transactivate the expression of Wnt response genes14 including MYC15, CCND116, MMP217, MMP718, and MMP917. In various cancers, including colorectal, gastric, hepatocellular carcinoma, and NSCLC19, β-catenin was found to be over-activated genetically or transiently, and accumulated in the nucleus of tumor cells. In addition, β-catenin can also be activated by Wnt-independent pathways, such as by growth factors including epidermal growth factor (EGF)21, hepatocyte growth factor/scatter factor (HGF/SF)22, and fibroblast growth factor (FGF)23. For example, EGF or HGF induced β-catenin activation to stimulate cell motility25. Upon EGF stimulation, β-catenin escaped from the degradation in cytoplasm and translocated into the nucleus to transactivate expression of genes involving in tumor invasion in a mechanism distinct from Wnt-dependent canonical signaling26. However, the ubiquitin ligase responsible for β-catenin degradation under this condition is little known.The Wnt/β-catenin pathway plays pivotal roles in development, cell proliferation, and differentiation552, which facilitates FBXW2 binding and subsequent ubiquitylation and degradation. Upon FBXW2 knockdown, accumulated β-cateninpS552 enters the nucleus to transactivate expression of MMPs to promote migration, invasion and metastasis of lung cancer cells. Finally, we found that in human lung cancer tissues, FBXW2 levels are inversely correlated with β-catenin levels and lymph-node metastasis, and lower FBXW2, coupled with higher β-catenin, predicts a worse patient survival. Thus, FBXW2 suppresses migration, invasion, and metastasis of lung cancer cells via promoting ubiquitylation and degradation of β-catenin.Herein, we reported that the tumor suppressor FBXW2 is an ubiquitin ligase responsible for β-catenin degradation following EGF stimulation. Specifically, AKT1  activation following EGF exposure phosphorylates β-catenin on Ser4. To further elucidate the mechanism of its anticancer activity, we used affinity purification-mass spectrometry method to identify FBXW2-binding proteins in H1299 cells with ectopic expression of tagged wild-type FBXW2. Among 196 putative binding proteins, we searched for consensus FBXW2 degron sequence TSXXXS, recently defined by us4, and identified oncogenic protein β-catenin as a potential candidate, which contains such a motif (codons 551-556) in an evolutionarily conserved manner  and phosphorylation-dead (S552A) single mutants, or triple mutants with all three S/T residues on the binding motif mutated (designated as β-catenin-3D or β-catenin-3A). Transfection followed by IP/IB assay revealed that compared to WT β-catenin constitutively active forms of β-catenin-S552D and β-catenin-3D bound more effectively to exogenous or endogenous FBXW2, whereas inactive forms of β-catenin-S552A and β-catenin-3A failed to do so . We then determined whether β-catenin level was negatively regulated by FBXW2. Indeed, while FBXW2 knockdown increased the levels of total β-catenin as well as phosphorylated β-catenin-Ser552, FBXW2 overexpression reduced them in a dose-dependent manner in various lung cancer cell lines. However, the levels of phosphorylated β-catenin-Ser33/37 and total β-catenin mRNA were not affected by FBXW2 manipulations  and FOP-Flash control luciferase reporters and found that β-catenin transactivation activity was increased upon FBXW2 knockdown, but reduced upon FBXW2 overexpression in multiple lines of lung cancer cells Fig. . Further4 Fig. . Finally4 Fig. . Taken tFbxw2 depletion in MEF cells remarkably extended the protein half-lives of both phospho-β-cateninSer552 and total β-catenin  to block new protein synthesis. FBXW2 knockdown in lung cancer cells or We next investigated, using classic in vivo and in vitro ubiquitylation assays, whether FBXW2 indeed promoted the ubiquitylation of β-catenin for enhanced degradation. The in vivo ubiquitylation assay showed that like β-TrCP, wild-type FBXW2, but not FBXW2-ΔF, significantly promoted polyubiquitylation of exogenous β-catenin . Wild-type FBXW2 also promoted polyubiquitylation of endogenous β-catenin and this activity was remarkably reduced when FBXW2-ΔF mutant was used Fig. . Similar552 to promote its accumulation in the nucleus21. To investigate whether AKT1 is responsible for FBXW2-mediated β-catenin degradation, we first examined whether the FBXW2-β-catenin binding is affected by AKT1 manipulation. Indeed, the FBXW2-β-catenin binding was abrogated by siRNA-based AKT1 knockdown . Furthermore, while FBXW2 failed to bind to two β-catenin mutants , β-TrCP1 bound to them equally well as to WT β-catenin , suggesting that the Wnt-β-TrCP1-β-catenin axis, while mainly controls proliferation, also contributes to migration and invasion, whereas the AKT1-FBXW2-β-catenin axis is more specific for migration and invasion.To determine the biological significance of FBXW2-mediated β-catenin degradation, we measured effect of FBXW2 on migration and invasion of lung cancer cells, since it was reported that phosphorylation of β-catenin by AKT1 increased its transcriptional activity and promoted cancer cell invasionFinally, we evaluated FBXW2 effect on in vivo lung metastasis by mouse tail vein injection of H1299 or H358 cells with stable FBXW2 knockdown alone or in combination with stable β-catenin knockdown. Six or eight weeks post inoculation, mice were killed and the lungs were collected. Compared with the control groups, the number of lung metastasis nodules in FBXW2 knockdown group was significantly increased, which is fully rescued by simultaneous β-catenin knockdown , stem cell fate determination (via ASCL235), migration , and angiogenesis (via VEGF36). These processes were abnormally activated due to β-catenin deregulation seen in multiple human cancers37. To further elucidate how the FBXW2-β-catenin axis regulates migration and invasion, and its mechanism of action, we transfected various β-catenin mutants into H1299 cells and found that β-catenin-S552D, a constitutive active form upon AKT1 activation, is the most effective in promoting migration and invasion  and renal cell carcinoma (RCC)42. We, therefore, performed the β-catenin pull-down assay after transfection of wild-type β-catenin and its few phospho-mutants to identify their TCF binding partners. Interestingly, we found that β-catenin-S33Y preferentially binds to TCF isoform TCF4E, whereas β-catenin-S552D preferred the isoform TCF4M/S . Taken together, these results demonstrate that FBXW2 inhibition of migration and invasion in lung cancer cells is mediated via promoting β-catenin degradation and consequent downregulation of MMP expression, which is preferentially regulated by the β-catenin-S552D-TCF4M/S transcription factors.As a transcription co-activator, β-catenin regulates a variety of cellular processes, including cell proliferation 4. To determine whether the inverse expression pattern between FBXW2 and β-catenin is correlated with patient overall survival, we performed the Kaplan–Meier survival analysis. Among 88 cancer samples  with inverse correlation between FBXW2 and β-catenin, we found that high FBXW2/low β-catenin (n = 20) predicts a better patient survival, whereas low FBXW2/high β-catenin (n = 68) was associated with poor survival of lung cancer patients . These association studies strongly suggested that the FBXW2-β-catenin axis could play a critical role in regulation of migration and invasion/metastasis of human lung cancer.Finally, we performed immunostaining of β-catenin in xenograft tumors derived from H1299 stable clones with FBXW2 knockdown or overexpression14. Given biological significance of β-catenin, it is not surprising that multiple ubiquitin ligases were found to regulate its stability, including β-TrCP30, Siah-143, Jade-144, c-Cb145, and TRIM3346. However, ubiquitin ligase responsible for targeted ubiquitylation and degradation of β-catenin following EGF-AKT1 activation, leading to enhanced cell motility25, is previously unknown.The Wnt/Wingless-dependent or -independent signals stabilizes β-catenin and increases its nuclear translocation to act as a transcription coactivator to drive expression of genes controlling proliferation and invasion, while constitutive activation of β-catenin leads to tumorigenesispS552), and β-catenin transcriptional activity, respectively  by SCFβ-TrCP to regulate cell growth30, or in response to activation of EGF signal via AKT1-mediated phosphorylation at the C-terminus (Ser552) by SCFFBXW2 to regulate cell motility (this study and see below).Here we report that FBXW2 is the responsible E3 in this role. Our conclusion is supported by the following lines of evidence: 1) FBXW2 binds to β-catenin under physiological conditions, and FBXW2-β-catenin binding is dependent on an evolutionarily conserved FBXW2 consensus binding motif (TSMGGT) on β-catenin  and metastasis (e.g. MMPs) through partnering with different TCF4 isoforms  cells, lung cancer H1299 and A549 cells, cervical carcinoma HeLa cells, and breast cancer MBA-MD-231 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). Lung cancer H358 cells were maintained in RPMI 1640 Medium with 10% FBS. ALL cell lines were maintained in Yi Sun lab. All cell lines were routinely tested to be negative for mycoplasma contamination.The plasmid constructs expressing HA-FBXW2, FLAG-FBXW2, HA-FBXW7, HA-β-TrCP1, HA-SKP2, FLAG-FBXW4, FLAG-FBXW5, FLAG-FBXW8, FLAG-β-TrCP1, FLAG-FBXL3, and FLAG-FBXO4 were maintained in Yi Sun lab. TOP-Flash, FOP-Flash, and pRL-TK were obtained from Dr. Li Ma . HA-AKT1 and HA-CA-AKT1 were obtained from Dr. Wenyi Wei . Human β-catenin and β-catenin mutants were generated by PCR and using the QuikChange Site-Directed mutagenesus kit , according to the manufacturer’s instructions. The primers used to generate the β-catenin mutants are shown in Supplementary Table 1.The pooled siRNA oligos targeting for β-catenin, AKT1, and GSK-3β were obtained from Santa Cruz. The sequences of siRNA oligos and siRNA oligos used for construction of lentivirus silencing vector are shown in Supplementary Table Recombinant human Wnt3a and recombinant human EGF were purchased from R&D Systems. AKT1 inhibitors MK2206 and MMPs inhibitors GM6001 were purchased from Selleck. Wnt3a inhibitor LGK974 was purchased from MedChemExpress.Cells were transfected with a variety of plasmids or siRNA oligos, using Lipofectamine 2000, according to the manufacturer’s protocols, followed by various assays 48 h post transfection. For gene knockdown, cells were infected with the lentivirus expressing sh-RNAs, produced by University of Michigan Vector Core, and then selected with 1.5 μg/ml puromycin for stable cell lines, or followed by various assays 96 h post infection.Cells were lysed in an IP lysis buffer , supplemented with complete protease inhibitor cocktail . For immunoprecipitation, 1–2 mg lysates were incubated with bead-conjugated FLAG or the appropriate antibody (2 μg) in a rotating incubator overnight at 4 °C, followed by 2 h incubation with Protein-A Sepharose beads . Immuno-complexes were washed four times with IP lysis buffer before resolved by SDS-PAGE, and analyzed by immunoblot. For direct IB analysis, cells were lysed in lysis buffer . Proteins were resolved on SDS polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% (w/v) milk, the membrane was stained with the corresponding primary antibodies.The antibodies were purchased as follows: β-catenin , FBXW2 . The following antibodies were used for immunoblotting only: phospho-β-catenin (Ser552) , SKP2 , β-TrCP1 , HA , FLAG , β-Actin , FBXL11 , GSK-3β , AKT1 , phospho-AKT1 (Ser473) , PARP , and caspase3 , phospho-β-catenin (Ser33/37Thr41) , TCF4 .g for 5 min at 4 °C. The supernatant was transferred (cytoplasmic extract) to a clean pre-chilled tube. The pellet was sequentially washed 3 times with lysis buffer A and then lysed in buffer B  for 30 min on ice, followed by centrifugation at 15,000 × g at 4 °C for 15 min. The supernatants were collected as the nuclear fractions.For nuclear and cytoplasmic fractionations, cells were washed twice with cold PBS and collected by scraping, followed by the addition of lysis buffer A . Cell lysates were incubated on ice for 10 min, followed by centrifugation at 400 × Cells were transfected with the TOP-FLASH or FOP-FLASH reporter plasmids together with pRL-TK. Luciferase activity was measured with the Promega Dual-Luciferases Reporter Assay kit (Promega E1980) according to manufacturer’s instructions. The relative firefly luciferase activity was normalized to renilla luciferase activity.ChIP assay was performed using the Simple ChIP Enzymatic Chromatin IP Kit  according to the manufacturer’s protocol. The primers used to amplify the β-catenin target genes’ promoter are shown in Supplementary Table 20 primer. Quantitative real-time reverse-transcription PCR were performed using the SYBR green reagent on an 7900HT Real-Time PCR System (Applied Biosystems). The housekeeping gene, β-Actin, was used as a loading control. The sequences of primer sets are shown in Supplementary Table Total RNA was isolated using RNeasy reagents  and then transcribed into complementary DNA using SuperScript III reagents (Invitrogen) with an oligo(dT)H1299 or HEK293 cells were co-transfected with FBXW2, His-HA-Ub, and β-catenin, β-catenin-3A or β-catenin-3D, along with mock vector or FBXW2-ΔF controls. Cells were lysed in buffer A  and sonicated. The lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN) for 4 h at room temperature. The beads were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and once with buffer TI . The beads were boiled and the pull-down proteins were resolved by SDS-PAGE for immunoblotting using anti-β-catenin or anti-FLAG Ab.2) in the presence of E1, E2 and purified E3s. Poly-ubiquitinated β-catenin was resolved by SDS-PAGE, followed by IB with anti-FLAG Ab.HA-FBXW2 or HA- FBXW2-ΔF was respectively purified from HEK293 cells transfected with either plasmid using HA beads (Sigma), and then eluted with 3 × HA peptide (Sigma), serving as the source of E3. FLAG-tagged β-catenin, β-catenin-3A, or β-catenin-3D was respectively pulled down by FLAG beads (Sigma) from HEK293 cells transfected with according plasmid, serving as the substrate. The reaction was carried out at 37 °C for 1 h in 30 μl reaction buffer  was added to the cell medium. At the indicated time points, cells were harvested, lysed and subjected to western blotting analysis. The densitometry quantification was performed using Image J processing software.The monolayer cells in 6-well plate were scraped in a straight line with a 10-μl pipette tip to produce a wound. Plate was then washed with PBS to remove detached cells and the cells were incubated in serum free medium. Photographs of the scratch were taken at 0 h, 24 h, or 48 h after wounding using Nikon inverted microscope. Gap width at 0 h was set to 1. Gap width analysis was performed with Image-Pro Plus 6.0 software. Multiple defined sites along the scratch were measured. Each scratch was given an average of all measurements. Data are shown as the average of three independent experiments.4 cells in 100 μl serum free medium were plated in an 8.0-mm, 24-well plate chamber insert , with medium containing 10% fetal bovine serum (FBS) at the bottom of the insert. Cells were incubated for 12–24 h, and then fixed with 4% paraformaldehyde for 5 min. After washing with PBS for three times, cells were stained with 0.5% crystal violet blue for 5 min, and then washed with double-distilled H2O. Cells on the upper surface of the insert were removed with a cotton swab. The positively stained cells were examined under the microscope. For the cell invasion assay, Matrigel-coated chambers  were used instead of the chamber inserts used in migration assay.For cell migration, 2–5×106) or H358 cells (4×106) stably expressing indicated sh-RNA were injected via tail-vein. Five mice were used in each experimental group. After 6 or 8 weeks, mice were killed and all the lung tissues were collected, fixed and sectioned, followed by hematoxylin and eosin staining. Macroscopic metastases were quantified by counting lesions in all five lobes of the lung per mouse.Five- to six-week-old BALB/c athymic nude mice  were purchased from Shanghai SLAC Laboratory Animal Center. Mice were fed with a regular diet (RD) and had free access to water and food. All mice procedures were approved by the Zhejiang University Committee on Use and Care of Animals. H1299 cells  or anti-FBXW2  antibody. Following several washes, the sections were treated with HRP conjugated secondary antibody for 30 min at room temperature, and stained with 0.05% 3, 3-diaminobenzidine tetrahydrochloride (DAB). Slides were photographed with Virtual slide microscope . The photographs were analyzed with the Image-Pro Plus 6.0 software .Human lung tumor tissue microarrays with lymph nodes metastasis data were purchased from Shanghai Outdo Biotech, China. For immune-histochemical staining, the sections were deparaffinized in xylene and rehydrated through graded ethanol. Antigen retrieval was performed for 20 min at 95 °C with 0.1% sodium citrate buffer (pH 6.0). Following quenching of endogenous peroxidase activity with 3% HQuantification of the immune-histochemical staining was conducted based on the ratio and intensity of the staining. Three random fields in each section were calculated. The measurement parameters collected by Image-Pro Plus 6.0 included density mean, area sum, and IOD. The optical density was counted and the area of interest was set through: hue, 0–30; intensity, 0–255, saturation, 0–255; next the image was converted to gray scale image, and the values were counted. The mean of density, equal to (IOD SUM)/area, represented the levels of specific protein per unit area.t-test, and multiple group comparisons were analyzed by one-way ANOVA. All statistical analyses were two-sided, and different cut off values, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), were considered significant.The significance of the data between two experimental groups was determined by Student’s Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting Summary