Xenopus embryos causes a loss of neural crest progenitors, a phenotype that is rescued by expression of Lrp6 or β-catenin. Dkk2 overexpression expands the neural crest territory in a pattern reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes. Mechanistically, we show that Dkk2 mediates its neural crest-inducing activity through Lrp6 and β-catenin, however unlike Wnt8, in a GSK3β independent manner. These findings suggest that Wnt8 and Dkk2 converge on β-catenin using distinct transduction pathways both independently required to activate Wnt/β-catenin signaling and induce neural crest cells.Neural crest progenitors are specified through the modulation of several signaling pathways, among which the activation of Wnt/β-catenin signaling by Wnt8 is especially critical. Glycoproteins of the Dickkopf (Dkk) family are important modulators of Wnt signaling acting primarily as Wnt antagonists. Here we report that Dkk2 is required for neural crest specification functioning as a positive regulator of Wnt/β-catenin signaling. Dkk2 depletion in  The neural crest is a population of cells unique to the vertebrate embryo. They are induced at the neural plate border during gastrulation, and around the time of neural tube closure, leave the neuroepithelium to produce a diverse array of cell types, contributing to multiple tissues, including the heart, the peripheral nervous system, and the craniofacial skeleton.Neural crest cells are generated through a sequence of events orchestrated by the modulation of several signaling pathways and the activation of a complex network of transcription factors . A largeXenopus embryos and promote head formation (Xenopus embryos (The Dickkopf (Dkk) family of secreted glycoproteins acts primarily as negative modulators of Wnt signaling, this is especially true for Dkk1 and Dkk4 . Dkks inormation . Dkk1 in embryos . Similar embryos . During  embryos . The rol embryos , howeverHere we show that Dkk2 knockdown prevents neural crest formation in vivo and in neuralized animal cap explants injected with Wnt8. Furthermore, Dkk2 gain-of-function increases the neural crest progenitor pool, reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes. We demonstrate that Dkk2 mediates its neural crest-inducing activity by activation Wnt/β-catenin signaling in a GSK3β independent manner. We propose that during neural crest formation, Lrp6 mediates two independent signaling events triggered by Wnt8 and Dkk2 converging on β-catenin to specify the neural crest.dkk2 mRNA . A Dkk2MO was designed to specifically interfere with translation of kk2 mRNA . The spees of MO . Unilate embryos . Concomited side . To confjunction , resultijunction . The phexpansion . snai2 ehenotype . Later iype  was significantly repressed in Dkk2-depleted (Dkk2SMO-injected) animal cap explants, while co-injection of a control MO (CoMO) had no effect on snai2 induction  by BMP inhibition or mesoderm (bra) by fibroblast growth factor 8b (FGF8b) in these explants , consistent with a potential role in neural crest induction. This expression increases over time to reach a maximum around stage 20 and then progressively declines  results in axis duplication, while Wnt activation post-MBT expands neural crest progenitors . For thefficient . This redkk2 mRNA (500 pg) or plasmid DNA (25 pg) in one cell at the 2 cell stage. Dkk2 overexpression in both cases resulted in a lateral expansion of snai2 expression domain in the vast majority of the embryos . This phenotype is very reminiscent of Wnt8, Lrp6 and β-catenin gain-of-function phenotypes , Dr. Tatiana Piotrowski  and Dr. Sergei Sokol , respectively. A flagged version of Xenopus Dkk2 was generated by adding a Flag tag (DYKDDDDK) at the C-terminal using the following primers F: GAATTCGCCACCGAGATGAACATGGTTTTGCTGGGGA; R: ctcgagTTACTTGTCGTCGTCGTCCTTGTAGTCTATTTTCTGGCAAATG. The PCR product was cloned into pCS2+ (pCS2+ Dkk2 Flag). Control (CoMO), Dkk2 , Lrp6 , 200 pg (wnt8) or 50 pg  was injected per embryo. MOs, mRNAs and plasmid DNA were injected in one blastomere at the 2 cell stage (NF stage 2) and embryos were analyzed by in situ hybridization at the neurula stage (NF stage 14–17). To identify the injected side, 500 pg of β-galactosidase mRNA was coinjected as a lineage tracer. Only embryos with co-localized expression of the lineage tracer with the cell type marker were considered for analysis. Control and injected embryos were treated in the dark with 10 μM of GSK3 inhibitor  at the gastrula stage (NF stage 11/11.5), and collected at NF stage 14–17. For the axis duplication assay, embryos were injected in the equatorial region in both ventral blastomeres at 4 cell stage (NF stage 3) and analyzed at NF stage 32. For animal cap explant experiments, both blastomeres at the 2 cell stage (NF stage 2) were injected in the animal pole region. Explants were dissected at the blastula stage (NF stage 9) and cultured for 8 hr in NAM 0.5X. In rescue experiments, the injections of mRNAs/plasmid DNA and MOs were performed sequentially. For whole embryo injections and animal cap explant assays each experiment was performed on at least three independent batches of embryos.8 25 pg; , noggin n 10 pg; , dkk2  to visualize the lineage tracer  on the injected side and processed for in situ hybridization. Antisense digoxygenin-labeled probes  were synthesized using template cDNA encoding snai1 , snai2 , containing HaltTM Protease Inhibitor Cocktail . After two consecutive centrifugations to eliminate lipids, the lysate was concentrated on an Amicon Ultra Centrifugal Filter , 5 μl of the concentrated lysate was resolved on a 10% NuPAGE Bis-Tris gel and transferred onto a PVDF membrane using the iBlot system (Invitrogen). Blots were subsequently incubated overnight with one of the following primary antibodies: monoclonal anti-Flag M2 antibody  and anti α-tubulin antibody . The blots were then washed and incubated with anti-mouse IgG coupled to horseradish peroxidase . Peroxidase activity was detected with the Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and imaged on a ChemiDoc MP Biorad gel documentation system . Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) according to the manufacturer recommendations.Embryos were injected at the 4 cell stage with 10 ng of Power SYBR Green PCR Master Mix  on a QuantStudio 3 Real-Time PCR System  with the following primer sets: bra , sox2 , snai2 , luciferase  and odc . The PCR conditions were as follows: denaturation 95°C (15 s), annealing and extension at 60°C (1 min) for 40 cycles.Total RNAs were extracted from embryos or animal cap explants using the RNeasy micro RNA isolation kit . The RNA samples were digested with RNase-free DNase I before RT-PCR. The amount of RNA isolated was quantified by measuring the optical density using a Nanodrop spectrophotometer . Approximately 250 ng of total RNAs from animal caps was reverse transcribed using the Superscript VILO cDNA Synthesis Kit  and 2 μl of 1:1000 dilution of the synthesized cDNA was amplified using  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior and Reviewing Editor.Thank you for submitting your article "Dkk2 promotes neural crest specification by activating Wnt/β-catenin signaling in a GSK3β independent manner" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is an interesting paper that addresses the role of Dkk2 in neural crest formation, showing that it is required for the expression of both Snai2 and Sox10. Loss of Dkk2 can be rescued by β-catenin and Lrp6, as well as Wnt8. Over-expression of Dkk2 leads to an expansion of Snai2. While a GSK3β inhibitor expands Snai2 expression anteriorly, the prospective neural crest territory remains sensitive to Dkk2 MO, suggesting it functions in a GSK3β independent manner.Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or β-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/β-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by β-catenin and Lrp6 injections in Dkk2-depleted embryos?5) In the absence of lineage tracing, the loss of 6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/β-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on β-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of β-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate β-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?4) Better quantification would be appropriate.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio…?6) This statement in the Discussion is debatable:"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/β-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/β-catenin regulated developmental process."Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested? Essential revisions:1) It is critical to provide better expression profile of Dkk2 and it is suggested to move the images to main figures. The published expression pattern for Dkk2 does not show expression in the neural fold or neural crest. Furthermore the RNA seq data available on Xenbase shows a very low expression of Dkk2 during neural crest induction. It is even more striking when the expression data is compared to the Wnt partner namely Wnt8 and Lrp6. It is only in the Discussion that the authors refer to expression data presented in the supplemental material.dkk2 expression data as a main figure of the manuscript . Dkk2 is broadly expressed during embryogenesis and is not distinctly enriched in the neural crest territory. It is likely that the regionalized expression of other components of the pathway, such as Fzd receptors and Wnt ligands, which are expressed at higher levels, provides the spatiotemporal cues to achieve a localized response. The expression pattern of dkk2 is presented and discussed in the Results section of the manuscript .Although the Xenbase RNA-seq data indicates that Wnt8 and Lrp6 are expressed at higher levels than Dkk2 it is difficult to correlate this information to the amount of protein present at the time of neural crest induction. As suggested we have moved the 2) The expression data suggest that Dkk2 is an extremely potent activator of the pathway that despite its minute expression it is essential for proper Wnt signaling. This is not entirely consistent with the fact that loss of Dkk2 can be rescued by overexpression of Wnt8, Lrp6 or β-catenin since all of these mRNA are expressed at 10 to 100 folds excess when compared to Dkk2. Does the Dkk2 protein possess some particular property such as extreme stability or extreme binding affinity to the co-receptors? Following a tagged Dkk2 protein in the embryo (by blot and IF) could provide some clues. Dose response in the embryo or animal cap could be used to determine the effectiveness of Dkk2 compare to Dkk1. What is the minimal dose at which Dkk2 expression can expend the neural crest?Regarding the mRNA expression levels of Dkk2, Wnt8 and Lrp6 see response to point #1. As suggested we have performed Western blot analyses to evaluate the accumulation overtime of a Flag-tagged version of HuDkk2 (see 3) While the results strongly suggest that Dkk2 potentiates WNT signaling towards neural crest formation, the mechanism of action is unresolved. The authors should assess whether Dkk2 triggers expression of known direct targets of WNT/β-cat pathway, or non-canonical WNT pathways. They should also test mesoderm markers to rule out possible secondary effects.snai2, dkk2 overexpression did not significantly upregulate other known direct targets of Wnt/β-catenin signaling such as pax3, znf703 or kremen2. This result suggests that dkk2 alone is a poor activator of Wnt/β-catenin signaling, consistent with the view that it is acting in concert with a Wnt ligand to activate the pathway. The expression of the mesoderm marker myoD was unaffected in dkk2-depleted embryos ruling out any possible secondary effects .We have performed the suggested experiment and found that with the exception of 4) The authors should test a broader repertoire of neural crest markers (in addition to Sox10 and Snai2). In addition, analysis of early stages (st. 13) is required to test effects on early NC specification markers.pax3, sox8, snai1, twist1 and sox9 . We found that early neural crest markers  such as pax3, sox8 and snai1 were only mildly affected – their expression levels were unchanged but their expression domain appeared to be shifted laterally – while other markers  such as twist1, and sox9 to a lesser extent, were downregulated, consistent with the phenotype observed for sox10 and snai2. This result suggests that dkk2 may not participate in neural plate border specification but rather in the neural crest progenitors formation and/or maintenance.We have performed additional experiments to increase the repertoire of neural crest genes analyzed, which now include 5) In the absence of lineage tracing, the loss of snai2 and sox10 expression is not conclusive evidence for loss of neural crest cells. The authors should examine non-neural ectoderm markers. They should also let embryos grow to later stages, as loss of neural crest should result in complete unilateral absence of craniofacial cartilage and cranial peripheral nervous system. If so, is this rescued by β-catenin and Lrp6 injections in Dkk2-depleted embryos?sox2 expression domain .We have analyzed the expression of keratin, a gene expressed in the non-neural ectoderm, and show that its overall expression is reduced/shifted laterally consistent with the expansion of We also show that neural crest-derived craniofacial cartilages in addition to pigment cells are affected upon Dkk2 depletion , pointing to a loss of neural crest lineages in these embryos.6) While the loss of function phenotype is compelling, the in vitro translation does not allow one to test the effective amount to inject in an embryo. Blocking the translation of a tagged Dkk2 mRNA would help define that dose. In addition, rescuing Dkk2 MO with an insensitive mRNA would confirm the specificity of the phenotype. The splice morpholino need to be more carefully quantified. Using realtime qPCR what is the percentage of properly spliced mRNA with various doses of the MO?snai2 and sox10 expression, associated with an expansion of sox2 expression domain. This phenotype was observed in both whole embryos and animal cap explants. We also used a control MO, which did not affect the expression of these genes. Importantly, this phenotype was efficiently rescued by expression of Lrp6 and β-catenin, two components of Wnt/β-catenin signaling pathway. We believe that this combination of approaches makes a compelling case for a specific requirement of dkk2 in neural crest formation. It is difficult to estimate the efficacy of a translation blocking MO without a good antibody, and we have not been able to identify one that could detect endogenous dkk2 in Xenopus. As suggested, we have performed qRT-PCR to evaluate the proportion of spliced versus unspliced transcripts generated upon injection of increasing doses of MO. These data are presented in Figure 1—figure supplement 1.In this study, we used two MOs interfering with Dkk2 mRNA translation and splicing, respectively. Both gave a similar phenotype resulting in the inhibition of Other suggested revisions:1) The authors should references previous studies that provide molecular insights into how Dkk2 activates Wnt/β-cat. For example, Li et al. (2002) suggested that it is the C2 domain of Dkk2 that is critical in its WNT activating functions, and further provide evidence suggesting that Dkk2 WNT inductive role operates through DVL and GSK3 independent mechanisms. Mao and Niehrs (2003) suggested that Krm2 is responsible for the WNT-antagonistic effect of Dkk2, and that in its absence, Dkk2 operates as a WNT activator.Both studies are now referenced in the text .2) It would be nice to examine biochemical interactions between Dkk2 function with Lrp5/6, Ror, Krm, during neural crest development.We agree with the reviewer, and this is something we are planning to explore in order to elucidate the precise mechanism of Dkk2-mediated signaling during neural crest specification, however we feel that this is beyond the scope of the current study.3) The authors present a model where Dkk2 and Wnt8 appear to function in parallel pathways that converge on β-catenin. What's not clear is why eliminating Dkk2 prevents Wnt8 from its normal activation of β-catenin and neural crest cell formation and also why vice versa, Dkk2 can't activate β-catenin and specify neural crest cells in the absence of Wnt8. Do Dkk2 and Wnt8 somehow impact each other's function?Our model predicts that signaling by Dkk2 and Wnt8 are both independently required to induce neural crest cells, this is specifically supported by the BIO experiment. Further work will address the mechanisms underlying this dual requirement during neural crest formation.4) Better quantification would be appropriate.For all injection experiments we provide a rigorous quantification of the phenotypes, which are summarized in the form of graphs for all figures, supplemented with numerical data provided as “source data” files.5) On BIO experiments, it is puzzling that the Dkk1+BIO does not display expected anterior expansion of Snai2 on the control side, unlike the Dkk2+Bio?We have included a more representative image for Dkk1+BIO .6) This statement in the Discussion is debatable:"While overexpression studies have previously shown that Dkk2 can function as an activator of Wnt/β-catenin signaling when co-expressed with Fzd8 or Lrp6 , this is the first study that provides evidence for its positive role in a Wnt/β-catenin regulated developmental process."We have revised this statement .7) It should be noted that the expanded domain of Sox2 does not correlate with neural crest cell generation. This contrasts with recent studies in Xenopus arguing for such a relationship and maintenance of pluripotency in neural crest cells.sox2 expression does not behave in a way that is compatible with the model of pluripotency retention in the neural crest lineage – a model that is still up for debate. At the neurula stage, we consider sox2 as a marker for neural progenitors and not as much an indicator of pluripotency.We agree with the reviewer, 8) Given the proposed role of Dkk2 one would expect that it could rescue the overexpression of GSK3 to complement the BIO- inhibitor data. Has this been tested?dkk2 and wnt8 are both independently necessary to induce neural crest we did not expect that overexpressing dkk2 would be sufficient to restore neural crest formation in embryos injected with GSK3. We have performed the suggested experiment and found that Dkk2 expression was indeed unable to rescue neural crest formation in embryo overexpressing GSK3 (see Because our model predicts that signaling by GSK3 see .


Radially layered cylindrical piezoceramic/epoxy composite transducers have been designed by integrating the excellent performance of piezoelectric/polymer composites and the radial radiation ability of cylindrical configurations, which are promising in developing novel ultrasonic and underwater sound techniques. Our previous study has explored the effects of the external resistance on the electromechanical characteristics of the transducer, and obtained some valuable findings. To clearly understand the electromechanical characteristics of the transducer and to guide the device design, in this paper, parametric analysis was performed to reveal the effects of multiple key factors on the electromechanical characteristics. These factors include material parameters of epoxy layers, piezoceramic material types, and locations of piezoceramic rings. In order to better analyze the influence of these factors, a modified theoretical model, in which every layer has different geometric and material parameters, was developed based on the model given in the previous work. Furthermore, the reliability of the model was validated by the ANSYS simulation results and the experimental results. The present investigation provides some helpful guidelines to design and optimize the radially layered cylindrical piezoceramic/epoxy composite transducers. Piezoelectric ultrasonic and underwater sound transducers, by virtue of their excellent electromechanical properties, have been widely applied in medical imaging ,2,3, nonOne effective attempt is to add a flexible polymer phase when fabricating the piezoelectric/polymer composites. These composites can overcome the shortcomings of single-phase piezoelectric materials, such as brittleness, high acoustic impedance, and at the same time possess the advantages of both the piezoceramics and the polymers, including low acoustic impedance, high electromechanical coupling coefficient, low mechanical loss and large dielectric constant ,9. ThrouAnother approach is to adopt new structural forms to realize multiple functionalities. The earlier transducers mainly adopted the approximated one-dimensional structures, which can excite one-dimensional longitudinal waves. The representative devices are the longitudinally sandwiched transducers ,27,28,29The above two methods can improve the performance of piezoelectric transducers to meet different requirements. However, both of these methods have certain limitations. In addition, in some special applications, such as endoscopic ultrasound (EUS), it is required that the transducer is able to realize 360° imaging . To obtaThe remainder of this paper is organized as follows. A harmonic form of voltage is used as the excitation source, which is expressed as:i-th piezoelectric layer  are expressed as follows [Under the assumption of plane stress, the harmonic radial displacement  follows ,53:(2)urf1 to f4 can be expressed as following [i-th piezoceramic layer, respectively. In Equations (2)–(5), the functions from ollowing ,53:(6)f1i-th elastic layers  are expressed as follows [Similarly, the harmonic radial displacement  follows ,53,54: to f8 can be expressed as following [In Equations (10) and (11), the functions from ollowing ,53,54:(1As shown in Combining Equations (2), (3), (10), (11), and (16)–(18), 10 coefficients can be derived, which are listed in Equations (A1)–(A5) . FurtherIn Equations (23) and (24), Subsequently, by letting ained as :(26)kd2=R1 = 10 mm, R2 = 15 mm, R3 = 20 mm, R4 = 25 mm, R5 = 30 mm, and h = 5.63 mm. Materials of the piezoceramic layers were selected as piezoceramic material type (PZT-5H), of which material parameters are listed in In this section, an ANSYS numerical simulation and an experimental study were conducted to validate the reliability of the theoretical solution. The geometric dimensions of the transducer are given as: V0 = 1 V was applied. All the voltage DOFs of the negative electrodes were also coupled together, and the electrical condition V0 = 0 V was applied. The harmonic analysis type was selected, and the frequency range was from 10 kHz to 40 kHz. The simulated impedance–frequency curve is plotted in In this section, a finite element analysis based on the software ANSYS R17.1 was performed to compare with the theoretical results. A three-dimensional model of one-twelfth of the transducer was created because of the structural symmetry, as shown in In this section, a test specimen of the radially layered cylindrical piezoceramic/epoxy composite transducer was fabricated by utilizing the mold-filling technique , as showIn this section, the effects of material parameters of epoxy layers, piezoceramic material types, and locations of piezoceramic rings on the electromechanical characteristics will be analyzed and discussed.In the above experiment, the transducer with a sequence of material parameters ①-②-③ for epoxy layers #1, #2 and #3 was fabricated and tested. Here, the sequence ①-②-③ denotes that the material parameters for epoxy layers #1, #2, and #3 are materials ①, ②, and ③, respectively. Keeping the PZT-5H and geometric dimensions of the transducer unchanged, 27 sequences can be formulated according to different material arrangements of these three epoxy layers. Keeping other parameters unchanged, Similarly, 3, 6 N/m2 to 2940 × 106 N/m2. Four cases are presented, i.e., the case of changing all epoxy layers, the case with only epoxy disk #1 changing, the case with only epoxy ring #2 changing, and the case with only epoxy ring #3 changing. It can be seen that the first resonance and anti-resonance frequencies increase with the increase of the Young’s modulus of the epoxy layers. This is because larger Young’s modulus will increase the stiffness of the transducer, which leads to higher resonant frequencies. Furthermore, it can be seen that changing Young’s modulus of epoxy disk #1 and epoxy ring #3 has negligible effects on these two frequencies as compared to the case of changing the Young’s modulus of epoxy ring #2. Therefore, in the transducer design, adjusting the Young’s modulus of epoxy ring #2 can only realize frequency control of the proposed radial layered cylindrical piezoceramic/epoxy composite transducer. From Further, keeping the density of all epoxy layers as 1186 kg/m3, of which material parameters are shown in 3, the transducer has the maximum first resonance and anti-resonance frequencies, but the minimum first electromechanical coupling factor. When the PZT ring #2 are chosen as PZT-5H, PZT-4, EC-64, the transducer has the similar first resonance and anti-resonance frequencies. The reasons are as follows. For a piezoelectric circular ring in radial vibration, when keeping its geometric sizes unchanged, its resonance frequency depends on the radial sound speed Selecting the material parameters of epoxy layers as the sequence ①-①-① and keeping geometric dimensions of the transducer unchanged, =c11E/ρP . The rad1E+s12E) , as showR1 and R3 of the PZT rings #1 and #2 are used to denote their locations, respectively. The corresponding outer radii R2 and R4 of the PZT rings #1 and #2 also need to be changed to maintain the same areas, which are defined as R3 and R4 of the PZT ring #2, and the areas of the epoxy rings #2 and #3 also vary. When fixing the location of PZT ring #2 and varying the location of PZT ring #1, the inner and outer radii R1 and R2 of the PZT ring #1, and the areas of the epoxy disk #1 and ring #2 also vary. From rf = 25.685 kHz, af = 26.578 kHz, dk = 0.26, respectively. The corresponding locations are R1 = 12 mm, 12.5 mm, and 14 mm, respectively. This rule can be used to design the improved transducer that has the maximum first resonance and anti-resonance frequencies as well as the first electromechanical coupling factor.Selecting the material parameters of epoxy layers as the sequence ①-①-①, piezoceramic material types of two PZT rings as PZT-5H, and keeping the area of one pizeoceramic ring unchanged, A parametric analysis was performed to study the effects of multiple key factors, including material parameters of epoxy layers, piezoceramic material types, and locations of piezoceramic rings, on the electromechanical characteristics of the radially layered cylindrical piezoceramic/epoxy composite transducer. The main results can be concluded as follow.(1) Based on the presented three different epoxy materials that have the same Poisson’s ratio, approximately equal density and the certain difference in Young’s modulus, the transducer can present 27 sets of electromechanical characteristics by utilizing different material sequences. Furthermore, these electromechanical characteristics are mainly controlled by the Young’s moduli of the epoxy layers, especially for that of the epoxy ring #2. This result demonstrates that only regulating the Young’s modulus of the epoxy layers can realize the design and optimization of the electromechanical characteristics of the transducer.3), the transducer with the PZT-5A ring #2 has the minimum first resonance and anti-resonance frequencies as well as the maximum first electromechanical coupling factor; the transducer with the BaTiO3 ring #2 has the maximum first resonance and anti-resonance frequencies as well as the minimum first electromechanical coupling factor; the transducer with PZT-5H, PZT-4, EC-64 ring #2 has the similar first resonance and anti-resonance frequencies. That is to say, the selections of piezoceramic material types in the ring #2, the piezoceramic materials with the lager radial sound speed and plane electromechanical coupling factor can optimize the electromechanical characteristics of the transducer.(2) Among five commonly used piezoceramic materials  The locations of piezoceramic rings have great effects on the electromechanical characteristics of the transducer, in particular, an appropriate location can be used to optimize the transducer design, making it have the maximum first resonance and anti-resonance frequencies as well as the first electromechanical coupling factor.


The morphology, porosity, mechanical property, thermal conductivity and adhesive property of foams with different polysilazane content were investigated. Besides, the effect of polyamine curing agent and surfactant on foams was also investigated to optimize the as-prepared epoxy foam.Polysilazane (PSN2) was used as chemical foaming agent to prepare epoxy foam for the first time. Using the new foaming agent, epoxy foams with high strength and low density were successfully prepared. The density of epoxy foam can be tuned from 0.321 to 0.151 g cm Varioxy resin ,26. The  [et al. ,28 carri [et al. –31 used 2, they found some side reactions which complicated the modifying process. The Si–N bonds of some polysilazanes can react with active free hydroxyl groups generated in the oxirane ring opening process to form Si–O–C and ≡Si–NH2. The ≡Si–NH2 can further react with other hydroxyl groups to generate NH3. Such tricky side reactions in that research suggested to us that polysilazane is a potential chemical foaming agent for epoxy resin.Polysilazane is a well-known polymeric precursor for silicon nitride ceramic and it has found various applications in the fields of high temperature-resistant adhesive, coating, water-repellent layer and silicon nitride fibre –37. It a−3 by changing the content of polysilazane from 2.50 to 7.50 wt%, with their compressive strength varying from 7.39 to 1.25 MPa. The influences of polysilazane polyamine hardener and surfactant content on the microstructure, apparent density, porosity and mechanical property of the formed foam were investigated in detail. Furthermore, thermal insulation property and adhesive property of foams were also characterized.In this work, polysilazane was used as a chemical foaming agent to prepare epoxy foam for the first time. The epoxy foams with high strength, low density were successfully prepared, the density of which can be tuned from 0.321 to 0.151 g cm2.2.1.−1, 72.02 mPa s−1), which contains Si–H functional group and synthesized via the aminolysis of chlorosilane according to our previous work, was used as a foaming agent. In addition, emulsifier OP-10  was added into the system as a surfactant to stabilize the foam.Commercial bisphenol-A type epoxy resin  was used as matrix resin. Triethylenetetramine  was used as a hardener. Polysilazane . The Si–H groups in polysilazanes also can react with –NH2 of TETA to produce H2. The release of NH3 and H2 resulted in the formation of porous structure in the foam. In the meantime, the chemical structure unit of PSN2 was introduced into the epoxy–polyamine system effectively, which was quite different from the reported mechanism of other epoxy foaming agents.The chemical formula of PSN2 is shown in 2.3.ρ is the apparent density and m, h and d represent weight, height and diameter, respectively. The porosity of epoxy foam was calculated from apparent density and the density of dense epoxy bulk (1.122 g cm−3) with the following formula (2.2):θ is the porosity and ρ and ρs represent apparent density and the density of dense epoxy bulk, respectively. The mechanical properties, including compressive strength, flexural strength and tensile strength, were characterized using a universal material testing machine  according to GBT8813-2008, GBT8812-2007 and GB9641-1988. The thermal conductivity was tested with a thermal conductivity analyser  at room temperature. The shear strength of epoxy foam adhesive was characterized using a universal material testing machine  according to GJB1480A-2005.The morphology of epoxy foam was observed with a scanning electron microscope . The apparent density was calculated from weight, height and diameter of cylindrical foam sample according to the following formula:3.3.1.a–c exhibits that the homogeneity of microcells is relatively fine as the content of PSN2 increases from 2.50 to 5.00 wt%. However, further content increase in PSN2 to 6.25 and 7.50 wt% resulted in the decrease in size homogeneity and shape regularity as the microcells further expand, especially for the foam with the PSN2 content of 7.50 wt% . Such phenomenon is due to the fast generation of gas when the PSN2 is excessive. The epoxy matrix cannot get enough strength via curing to support foaming, so the microcells deform and merge during expanding.Figure 37.50 wt% d,e. Such−3, while the porosity increases from 71.4 to 86.5%. But as the content of PSN2 increases from 6.25 to 7.50 wt%, the decrease in density and the increase in porosity are both not so obvious while the deterioration of morphology is serious as shown in figure 3e.a, which are quite high compared to those of previously reported epoxy foams with similar density [Figure 5b,c shows the flexural strength and tensile strength of foams with different PSN2 contents. The flexural strength and tensile strength decrease from 5.86 and 3.70 MPa to 1.46 and 1.23 MPa, respectively, along with the density decrease from 0.321 to 0.151 g cm−3 caused by the rise of PSN2 content. There are few reports about these two properties of epoxy foam; however, they are pretty important for practical application. So, the characterization of them is a good reference for future research and application.The mechanical property of epoxy foams with different PSN2 content is shown in figure 5 density ,29,44–46−1, respectively, which are all much lower than that of dense epoxy block (0.133 W (m K)−1). Such phenomenon can be explained by the special heat transfer mechanism of foam materials [The thermal insulation performance of such epoxy foam was also studied. aterials . There aAs we can know from the above, the foams with 2.50, 3.75 and 5.00 wt% PSN2 exhibited relatively good morphology and mechanical property. So, their corresponding unfoamed precursors were further used as foam adhesive in the adhesion of aluminium alloy concentric tubes. The shear strengths of the epoxy foam adhesive were tested at different temperatures and the result is shown in 3.2.The effect of curing agent content on the morphology and mechanical property of the foams was also investigated. As can be seen from −3 due to the acceleration of curing. The porosity decreases but still over 76%. The mechanical property of epoxy foams with different TETA content is shown in −3 when the content of TETA is higher than 15.00 wt%. Besides, when the content of TETA is 8.75 wt% or less, the epoxy cannot be cured effectively.Apparent density and porosity of epoxy foams with different TETA content are shown in 3.3.a with figure 10b,c, it can be found that the size of microcells becomes more homogeneous and the shape of microcells becomes more regular after the addition of OP-10. But when the OP-10 content is up to 3 and 4 wt%, the microcells begin to deform as figure 10c,d shows. When the OP-10 content is further raised to 5 wt%, the homogeneity of microcells begins to decline. Such phenomenon may be attributed to the strength reduction in bubble wall caused by the addition of OP-10 during the curing and foaming process. Therefore, judging from the morphology of foams, the optimum content of OP-10 is 1–2 wt%. Both the microcell homogeneity and regularity of foam using optimized content of OP-10 are better than those of the others.OP-10 is used as a surfactant in the preparation of the epoxy foam. Though the content of OP-10 is quite low, it had a significant effect on epoxy foam. The surfactant can affect epoxy foam in three ways. First, it can homogenize the size of microcells via increasing the compatibility among polar PSN2, polar epoxy matrix and non-polar curing agent. Second, it can facilitate the formation of microcells via lowering the surface tension of the mixture. Finally, it can increase the surface viscosity and elasticity of the microcells thus can prevent the microcells from bursting. Therefore, the effect of OP-10 on the morphology and mechanical property of the epoxy foam was also investigated in this research. The content of OP-10 in foam varied from 0 to 5 wt% as the content of PSN2 (2.50 wt%) and TETA (10.00 wt%) remained unchanged. figure 104.−3 and 71.4% to 0.151 g cm−3 and 86.5%, respectively, by changing the content of PSN2 from 2.50 to 7.50 wt%. As the result exhibits, the morphology, mechanical property, thermal conductivity and adhesive property of epoxy foam change along with the density and porosity change caused by the change of PSN2 content. Especially, the compressive strength of the as-prepared foam is quite good compared with other epoxy foams that have a similar density, which demonstrates that PSN2 is not only a foaming agent but also a modifier of the epoxy foam. In addition, both the morphology and mechanical property of the foam can be optimized by choosing proper content of TETA and OP-10. The most suitable contents of TETA and OP-10 are 15.00 and 1 wt%, respectively.Epoxy foams were successfully prepared using PSN2 as a chemical foaming agent for the first time. The density and porosity of epoxy foam can be tuned from 0.321 g cm


Odontogenic tumours are a heterogeneous group of lesions that develop in the oral cavity region and are characterized by the formation of tumoural structures that differentiate as teeth. Due to the diversity of their histopathological characteristics and clinical behaviour, the classification of these tumours is still under debate. Alterations in morphogenesis pathways such as the Hedgehog, MAPK and WNT/β-catenin pathways are implicated in the formation of odontogenic lesions, but the molecular bases of many of these lesions are still unknown. In this study, we used genetically modified mice to study the role of IKKβ  in oral epithelial cells and odontogenic tissues. Transgenic mice overexpressing IKKβ in oral epithelial cells show a significant increase in immune cells in both the oral epithelia and oral submucosa. They also show changes in the expression of several proteins and miRNAs that are important for cancer development. Interestingly, we found that overactivity of IKKβ in oral epithelia and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins , leads to the appearance of odontogenic tumours that can be classified as ameloblastic odontomas, sometimes accompanied by foci of secondary ameloblastic carcinomas. These tumours show NF-κB activation and increased β-catenin activity. These findings may help to elucidate the molecular determinants of odontogenic tumourigenesis and the role of IKKβ in the homoeostasis and tumoural transformation of oral and odontogenic epithelia. Interestingly, IKKβ can either promote or prevent tumour development, depending on the cell type and other circumstances that are not yet well understood, probably due to the large number of proteins regulated by this kinase.4The IKK complex is responsible for the regulation of the NF-κB family of transcription factors, which regulates genes associated with cell survival and increased proliferation. Deregulated NF-κB activation underlies disease states in many organs, including chronic inflammation and cancer. The IKK complex is formed by two catalytic subunits with kinase activity (IKKα and IKKβ) and one regulatory subunit (IKKγ or NEMO).+ haematopoietic cells as well as granulocytes (Gr-1+), macrophages (F4/80+) and B cells (B220+) in the forestomach epithelium5 and to the development of supernumerary teeth due to reduced apoptosis and upregulation of the WNT signalling pathway in the embryonic incisor bud epithelium of K5-IKKβ mice.6 Carcinogenesis experiments performed in a genetic background prone to tumour development through the expression of an active form of RAS revealed that K5-IKKβ mice are resistant to skin cancer,7 but they develop more malignant tumours than control littermates in the forestomach and the palate.5 In the course of these experiments, we crossed the K5-IKKβ transgene into backgrounds lacking p53 in epithelial cells (p53EKO/K5-IKKβ mice) or lacking p16 and p19 in every cell (Ink4a/ArfKO/K5-IKKβ). Surprisingly, a high percentage of these mice developed spontaneous odontogenic tumours.Previously, we generated transgenic mice overexpressing IKKβ in basal cells of the stratified epithelia and in exocrine glands. In addition to other phenotypes, IKKβ overexpression in these mice led to greater numbers of CD458 These lesions are currently classified as odontogenic cysts and odontogenic tumours , and each of these categories is in turn classified into a number of subcategories. Although the aetiology of these tumours is unknown in many cases, activating mutations in the Hedgehog (mutations in SMO and PTCH1) and mitogen-activated protein kinase  pathways have been identified in benign and malignant odontogenic tumours. Other genetic alterations found in odontogenic tumours are associated with the WNT pathway  and other genes that are commonly mutated in cancer .8 In addition, epigenetic alterations have been described in these tumours.9Odontogenic tumours are a heterogeneous group of lesions of the oral cavity that result in the formation of tumoural structures that differentiate as teeth. Their classification is still under debate and has been recently modified by the World Health Organization.11 Although these animal models do not survive postnatally, precluding the study of tumour formation in adulthood, the alterations that they develop resemble odontogenic tumours. Postnatal activation of the WNT pathway in dental stem cells results in odontoma formation.12 However, other determinants of odontogenic tumours are still unknown, and there is a need for additional animal models to reveal the genetic diversity underlying odontogenic tumours.Transgenic mice in which WNT signalling is activated through the expression of stabilized forms of β-catenin in oral epithelia resulted in the development of supernumerary teeth.Here, we analyse the effect of IKKβ overexpression in epithelial cells of the gingiva and palate and the inflammatory disease caused by IKKβ in the oral epithelia as well as the associated changes in the expression of several key proteins and miRNAs that are important for cancer development. We demonstrate that the overactivity of IKKβ in oral epithelial cells and odontogenic tissues, in conjunction with the loss of tumour suppressor proteins , leads to the appearance of odontogenic tumours. Molecular characterization of these tumours indicates that they show NF-κB activation and increased β-catenin activity. These findings shed some light on the role of IKKβ in the homoeostasis and tumoural transformation of oral and odontogenic epithelia.5 With the aim of understanding the role played by IKKβ in the oral epithelia, we studied these organs in K5-IKKβ mice in depth. In in vitro studies with cultured oral keratinocytes, K5-IKKβ cells showed more proliferation (measured as BrdU incorporation) and fewer senescent cells  than cultured wild-type (wt) oral keratinocytes  cells. Almost half of these cells could be classified as either macrophages or T-lymphocytes based on the labelling obtained with CD11b  and CD3 antibodies, and some of these T-cells were of the γδ subtype, similar to the dendritic epidermal T cells residing in the skin. Interestingly, epithelial IKKβ overexpression in transgenic (tg) mice led to a large  increase in haematopoietic CD45+ cells in the palate epithelia, where haematopoietic infiltrating cells represented more than half of the total cell number  that are important for tumour development in cultured oral epithelial cells from wt and tg mice with IKKβ overexpression. K5-IKKβ oral keratinocytes showed increased phosphorylation of Serion Fig. . Interesins Fig. , suggestins Fig. . Immunohins Fig. . In summ16 In summary, K5-IKKβ oral epithelial cells presented lower levels of a number of miRNAs that are usually downregulated in transformed states. In contrast, we did not observe a decrease in miR-203, which is another microRNA whose downregulation is usually associated with tumoural transformation, nor a significant difference for miR-205  or p16 and p19 in every cell (Ink4a/ArfKO/K5-IKKβ mice). Interestingly, in addition to an Ink4a/Arf-dependent antitumoural role of IKKβ in skin cancer,7 we observed that overexpression of IKKβ in the absence of either epithelial p53 or both p16 and p19 led to the appearance of maxillary tumours  that caused facial distortion and swelling in some mice . We did not observe differences between the sexes in the incidence of odontogenic tumours in either p53EKO/K5-IKKβ or Ink4a/ArfKO/K5-IKKβ mice.In the context of our study on the role of IKKβ in skin cancer, we generated cohorts of K5-IKKβ transgenic mice that also lacked either p53 in stratified epithelial tissues  that agreed with the international classification of dental tumours in mice.our Fig. . The harour Fig.  to maturses Fig. , inset; We next immunohistochemically studied the expression of epidermal and mesenchymal markers in odontogenic tumours as well as the expression of proteins that are important in the transduction of prosurvival and proliferation signals that are frequently activated in cancer. The ameloblastic odontomas of mice overexpressing IKKβ maintained the epithelial differentiation of the soft ameloblastic epithelium, as it was mainly positive for staining with an antibody specific for keratin K5 Fig. . The ameInk4a/ArfKO/K5-IKKβ and two from the p53EKO/K5-IKKβ background as well as non-tumoural oral and odontogenic tissues from the genotypes indicated in Fig. Ink4a/Arf locus (lanes 4–5).These results were confirmed by western blot analysis of protein extracts from several odontogenic tumours. We analysed three ameloblastic odontomas from In summary, the analyses of odontogenic tumours and non-tumoural samples by immunohistochemistry and western blotting indicated activation of the NF-κB, AKT, STAT3 and WNT/β-catenin pathways in tumoural samples, independent of their genetic background.17 By contrast, the tumoural lesions observed in K5-IKKβ mice simultaneously lacking p53 or p16 and p19 were able to metastasize . In addition, IKKβ overexpression triggers a strong oral inflammatory response, as the percentage of immune cells increases up to more than 50% of the total cells in the oral epithelia. Accordingly, in the underlying submucosa, there is also a marked inflammatory response, although it is not as strong as in the oral epithelium. Some ameloblastic carcinomas in humans have been proposed to develop from ameloblastomas.19 Our results suggest that increased IKKβ expression provokes NF-κB activation and a strong inflammatory response that could contribute to the tumoural transformation of the odontogenic epithelium through the uncontrolled release of inflammatory cytokines and facilitate the development of a secondary ameloblastic carcinoma in a pre-existing ameloblastic odontoma, although the existence of other driving factors cannot be ruled out.NF-κB drives a proinflammatory pathway that is constitutively activated and promotes invasion in oral squamous cell carcinomas (SCC).15 The miRNAs that were downregulated by IKKβ overexpression included let-7b, let-7c, miR-125b, miR-21 and miR-34c; interestingly, all of these miRNAs are functionally related to tumoural transformation.miRNAs are small non-coding RNAs that regulate gene expression by modifying the stability and transcription rate of target messenger RNAs. Each miRNA usually regulates a number of different targets and, hence, shows pleiotropic effects. Not surprisingly, miRNA deregulation is an important player in cancer pathogenesis, including oral SCC and head and neck cancer.RAS, MYC, HMGA2 and STAT3,21 and a negative feed-back loop between let-7 members and β-catenin has been described.22 Therefore, the low levels observed for let-7b and let-7c could be important in the development of ameloblastic odontomas and secondary ameloblastic carcinomas in K5-IKKβ mice or, alternatively, could be secondary to β-catenin overactivity.miRNAs of the let-7 family promote cellular differentiation and are considered tumour suppressors. These miRNAs are frequently expressed at low levels in human cancers and cancer stem cells. Among their targets, there are several cancer genes, such as 23 miR-125b is downregulated in oral SCC, and low expression of miR-125b contributes to head and neck cancer development;14 moreover, the restoration of miR-125b expression in oral SCC cell lines reduces the proliferation of these cells.13Although the function of the miR-125 family is somewhat controversial, its members are known to target several oncogenes, transcription factors and growth factors, and its function is tumour suppressive in a number of organs and tissues, including ectodermal derivatives.24 This miRNA also regulates multiple components of the WNT pathway, including β-catenin, and acts as a tumour suppressor miRNA in many cell types.25 Restoration of miR-34c expression leads to a decrease in the migration, invasion and metastatic potential of nasopharyngeal carcinoma cell lines.26 Notably, miR-34c is expressed in oral keratinocytes at much higher levels than in skin keratinocytes . This difference indicates that the functions exerted by this specific microRNA could be more important in oral epithelia than in skin. More studies are needed to determine whether the different effects of IKKβ on tumoural transformation in oral and skin keratinocytes are mediated by miR-34c.miR-34c belongs to a family of microRNAs that are induced by p53 and cause cell cycle arrest and apoptosis, partially by targeting the expression of c-MYC.27 Thus, the overexpression of miR-203 found in K5-IKKβ oral keratinocytes could contribute to the development of ameloblastic odontomas and secondary ameloblastic carcinomas observed in p53EKO/K5-IKKβ and Ink4a/ArfKO/K5-IKKβ mice.The reduced level of miR-21 found in K5-IKKβ oral keratinocytes is surprising, as this miRNA is considered to be oncogenic and is overexpressed in many types of tumours. miR-203 is differentially expressed in the epithelium and mesenchyme of dental primordia, and hence, it could be important in tooth organogenesis.5 Interestingly, these mice do not develop odontogenic tumours. By contrast, the cooperation of IKKβ overexpression with the lack of tumour suppressor proteins (p53 or p16 and p19) leads to the appearance of spontaneous ameloblastic odontomas in ~25% of animals, indicating that although the overexpression of IKKβ makes odontogenic epithelia prone to tumoural transformation, a concomitant lack of tumour suppressor proteins is needed for odontogenic tumour formation.Under treatment with oral carcinogens, IKKβ promotes the appearance of more oral tumoural lesions  in the palate and forestomach in a genetic background prone to the development of epithelial cancers (TgAC mice).TP53 (71.5%) and CDKN2A . The implication of CDKN2A in the pathogenesis of odontogenic and other head and neck cancers is reinforced by the characterization of CDKN2A as a susceptibility locus for nasopharyngeal carcinoma in a genome-wide association study performed in a Chinese population;28 in addition, it has been suggested that the methylation of the CDKN2A locus is an important mechanism of odontogenic tumourigenesis,30 and loss of heterozygosity is observed for both TP53 and 9p22-p21 (the genomic region where the CDKN2A locus occurs) in odontogenic tumours.31There are no published data regarding the genomic alterations present in ameloblastic tumours in repositories of genomic data for human cancers such as TCGA. Nevertheless, data available at cBioportal indicate that the most mutated genes in head and neck SCC include CDKN2A locus needs to be lost to cooperate with IKKβ overexpression in odontogenic tumour formation. Mice lacking both p16 and p19 develop tumours, mainly sarcomas and lymphomas, but not odontogenic tumours. Mice that are null for p19 (but not for p16), generated by deleting exon E1β,32 develop tumours similar to those observed in p16 and p19 double-null mice, although a lack of p19 in association with Tax oncogene expression has been implicated in osteosarcoma development.33 Wild-type keratinocytes of the oral epithelia express p16 at higher levels than skin keratinocytes, suggesting that p16 loss could be important in oral tumourigenesis. Nevertheless, the deletion of either p16 or p19 individually in an animal model overexpressing IKKβ would allow the specific role of these proteins in the development of odontogenic tumours to be discerned.At present, it is uncertain which of the proteins encoded by the 11 and mice with increased WNT activity in the oral epithelium develop odontomas.12 In addition, β-catenin expression appears to increase with the aggressiveness of odontogenic lesions, which also increases its nuclear localization.34 Accordingly, through western blotting and immunohistochemical analyses, we detected increased β-catenin levels and/or activation in the ameloblastic odontomas of our animal models, especially in the areas of secondary ameloblastic carcinoma. Therefore, p53EKO/K5-IKKβ mice and Ink4a/ArfKO/K5-IKKβ mice represent good models for studying the pathogenesis of the progression to secondary ameloblastic carcinoma from pre-existing ameloblastic odontoma and are also very useful for testing the efficacy of IKKβ or WNT/β-catenin inhibitors in preventing odontogenic tumour development.β-catenin deserves particular attention as a possible driver of odontoma formation, as WNT overactivity causes the development of supernumerary teeth,In summary, our results show that IKKβ overactivity in oral and odontogenic epithelia leads to changes in both the cellular composition of the epithelia and the expression of several tumour suppressor proteins and miRNAs that result in a preneoplastic state of the cells. IKKβ overactivity in transgenic mice in conjunction with the absence of tumour suppressor proteins  leads to the appearance of odontogenic tumours, which show activation of the WNT/β-catenin pathway. Therefore, these results indicate the possibility of treating ameloblastic odontomas with pharmacologic inhibitors of IKKβ or WNT/β-catenin to prevent their evolution to ameloblastic carcinoma.All mouse husbandry and experimental procedures were performed according to European and Spanish laws and regulations and were approved by the local Animal Ethical Committee and the competent authority (code PROEX 086/15).EKO/K5-IKKβ and Ink4a/ArfKO/K5-IKKβ mice was performed by PCR analysis of tail DNA as previously described.38 K5-IKKβ mice are available from the European Mouse Mutant Archive (code EM:09179). The expected genotypes were obtained in roughly Mendelian rates in the different crosses.The genotyping of K5-IKKβ, p53Mouse tissues were dissected, immediately fixed in 10% buffered formalin or 70% ethanol, and embedded in paraffin. Five micron thick sections were used for H&E staining or immunohistochemical preparations. The antibodies used are listed in Supplementary Table 39 with minor modifications. The samples were incubated overnight at 4 °C with 1 unit per mL dispase ; then, the oral epithelia were separated and digested with trypsin (Gibco) for 15 min at 37 °C. Keratinocytes were seeded in high-calcium medium [Ca2+-free and Mg2+-free EMEM  supplemented with 4% Chelex-treated  foetal bovine serum  and 0.2 mmol·L−1 Ca2+]. After overnight culture for cell attachment, the dishes were washed three times with cold PBS, and growth medium  was added. Then, the keratinocytes were allowed to grow to confluence. The keratinocytes were subjected to consecutive passages at low dilution using high-calcium medium for seeding. Cells that had undergone fewer than five passages were used for western blot analysis.Epithelial and submucosal cells were harvested from the oral tissue  of 2-day-old K5-IKKβ and control mice as described previously−1 BrdU for 1 h, and BrdU incorporation was detected by immunohistochemistry. The detection of senescence was carried out by using a senescence β-galactosidase staining kit . In colony-forming assays, growing colonies were fixed and stained with Coomassie blue, and the number of colonies was counted.For proliferation studies, cells were incubated with 10 µmol·L7Epithelial keratinocytes and submucosal cells were harvested from the oral tissue  of 9-week-old K5-IKKβ and control mice and analysed as previously described for skin cells.Total protein extracts (30 μg) from cultured mouse oral keratinocytes, tumours or tissues were prepared following standard techniques. The antibodies used are listed in Supplementary Table 7 For miRNA, TaqMan probes were used. TBP was used for the normalization of mRNA expression, and SnoRNA202 and SnoRNA234 were used for the normalization of miRNA expression.Total RNA, including miRNA, was isolated from cell cultures from oral tissue by using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The reverse transcription reaction was performed by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for mRNA and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) for miRNA. Quantitative qRT-PCR was performed in a 7500 Fast Real-Time PCR System using GoTaq qPCR Master Mix (Promega) for mRNA and TaqMan Universal PCR Master Mix (Applied Biosystems) for miRNA. The sequences of the oligonucleotides used are provided in.CT studies were performed in a small-animal Argus PET-CT scanner  in mice that were anaesthetized by the inhalation of 2%–2.5% isoflurane in 100% oxygen, with the following acquisition parameters: voltage 45 kV, current 150 μA, 8 shots, 360 projections and standard resolution.Images were analysed using the image analysis software ITK-SNAP.p-values were determined by using the unpaired, two-tailed Student’s t-test. For flow cytometry assays, the Mann-Whitney test was used. All experiments were performed at least three times. P < 0.05 were considered significant, and the data were expressed as the mean ± SEM. For miRNA expression, we employed REST 2009 Software (Qiagen), which calculates statistical significance using bootstrapping and randomization algorithms.40For mRNA expression, BrdU incorporation, colony formation and SA-β-galactosidase assays, Supplementary Figures S1-S3Supplementary Table S1


Using human CRC cells harboring different Wnt/β-catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic β-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear β-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that β-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon β-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single β-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/β-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with β-catenin knockdown alone. Collectively, our data unveil novel β-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining β-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/β-catenin-targeted therapeutics.Nuclear activated  Speβ-catenin depletion  or scramble shRNAs as control (shCtr) and then cultured with or without Dox (±Dox) to modulate concomitantly stable siβ-catenin expression. As shown in Fig. β-catenin annulled the hyper-activation of ERK1/2 and the accumulation of the autophagy markers Beclin-1 (BECN1) and Rab7 elicited by single-β-catenin depletion  and DLD1shβ-Cat/shNHERF1 (82 ± 8%) cells, compared with <5% in controls (shβ-Cat/siCtr+Dox or shβ-Cat/shNHERF1−Dox)  Fig. , as furtox) Fig.  and Caspox) Fig. . Interesays Fig. , in keepshNHERF1 2 ± 8% ceβ-catenin-silenced CRC cells, in which NHERF1 is highly/ectopically re-expressed, is mandatory to promote an autophagy-to-apoptosis switch in CRC cells refractory to single-β-catenin targeting.Collectively, these data indicate that combining NHERF1 inhibition in settings of Previous studies focused on small molecule ligands of the PDZ1-domain of NHERF1 to prevent its ectopic cytoplasmic/nuclear mislocalization and oncogenic function –23. ThesThe chemical structure of RS5517, namely 3-benzyl-5-chloro-N-(4-(hydroxymethyl)phenyl)-1H-indole-2-carboxamide, is depicted in Fig. To address the inhibitory effect of RS5517, we resorted to test the effect of this molecule on NHERF1/PDZ1 domain in vitro. A construct encoding for NHERF1 PDZ1 was expressed and challenged with a dansylated peptide corresponding to the C-terminal sequence of β2-AR (D-NDSLL). In order to measure binding, a fluorescent pseudo-wild type was produced by replacing Tyr38 of PDZ1 with a Trp. Binding was then measured by Forster Resonance energy transfer between the fluorescent donor (Trp) and acceptor (Dansyl). Binding experiments were performed in the presence and in the absence of a constant concentration of RS5517 Fig. . It is eβ-Cat, or DLD1shβ-Cat cells were exposed to escalating doses (0.1–100 μM) of the compound in the presence or absence of Dox for 4 days and then analyzed by MTT assays. As shown in Fig. 50) for RS5517 in Dox-untreated Ls174Tshβ-Cat or DLD1shβ-Cat cells expressing low levels of NHERF1 was 52 ± 6 μM and 65 ± 11 μM, respectively. Conversely, the equivalent IC50 value of RS5517 was reduced to 8 ± 2 μM in Ls174Tshβ-Cat cells and 11 ± 5 μM in DLD1shβ-Cat cells maintained under concomitant Dox exposure. Our mechanistic investigations suggest that the enhanced cytotoxic effects of RS5517 against Dox-treated CRC cells can be explained by inhibition of a NHERF1-mediated survival response restricted to cells that silence β-catenin   Fig. , by alsolls Fig. . Intereslls Fig. , while plls Fig. .Fig. 7RSβ-catenin protein content regardless of Dox treatment  indicating that the estimated half-life of β-catenin was longer than 8 h in RS5517-treated samples over DMSO control samples (−). The occurrence of apoptotic cell death in Ls174shβ-Cat cells exposed to Dox and RS5517 . Intriguingly, the RS5517-mediated targeting of NHERF1 did not affect the overall Rab7 expression neither at baseline or during β-catenin knockdown (Dox setting), in contrast to what herein reported for shRNA-mediated NHERF1 knockdown  or somatic intragenic missense mutations occur in the majority of human ovarian and breast cancers but not other diseases examined to date [NHERF1 gene contains estrogen-responsive elements [Loss of heterozygosity at the  to date . The proelements , and NHEelements .β-catenin signaling [β-catenin signaling in CRC cell lines harboring different Wnt/β-catenin pathway mutations. Intriguingly, ChIP assays demonstrated that nuclear activated β-catenin can regulate NHERF1 via TCF4 directly, while the interaction between TCF1 and the nherf1 promoter increases upon β-catenin knockdown. These findings provide novel insight into the β-catenin/TCF-dependent mechanisms of CRC carcinogenesis, suggesting that the relative amounts of β-catenin and those of different TCFs may ultimately dictate or relieve a default repression of nherf1 gene, in keeping with the notion that TCFs function as powerful transcriptional activators or repressors [α (hypoxia-inducible factor 1α) [β-catenin under hypoxia [β-catenin cascade, to modulate NHERF1 expression, as well as the phenotypic heterogeneity observed in primary human CRC [Regarding CRC, a recent study stated the tumor suppressor activity of NHERF1 , 8. NHERpressors . NHERF1 pressors , and wasctor 1α) , 31 that hypoxia . Thus, iuman CRC .β-catenin-depleted CRC cells which adopt a cytostatic viable phenotype and a unique proteomic signature centred around autophagy and energy metabolism processes . Accordingly, recent evidence earmark NHERF1 as an autophagy regulator in breast cancers via its influence on the ubiquitin-dependent degradation of BECN1, a critical component of the autophagic core lipid kinase complex [To our knowledge, this is the first report disclosing NHERF1 as a major survival driver in settings of  complex . Moreove complex .β-catenin knockdown. These data were remarkably consistent across two human CRC cell lines harboring different Wnt/β-catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, thus excluding any clonal effects. Mechanistically, combined targeting of NHERF1 and β-catenin triggered the activation of Caspase-3 and the cleavage of PARP also abrogating the hyper-activation of ERK1/2, the accumulation of autophagy-related proteins like Beclin-1 (BECN1), Rab7 or its effector RILP elicited by single β-catenin knockdown. These molecular changes may relatively explain why a double β-catenin/NHERF1-inhibitory strategy may be a fruitful mechanism-based strategy to augment apoptotic death of CRC cells refractory to Wnt/β-catenin-targeted therapeutics [In our hands, genetic or pharmacological targeting of NHERF1 was sufficient to prime an ‘autophagy-to-apoptosis switch’ that strikingly impacted the fate of CRC cells during apeutics , 6.β-catenin-depleted CRC cells which highly re-express NHERF1.However, as physiological or pathological roles of NHERF1 strictly depend on its subcellular localization, targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve the normal functions of this versatile protein. By far, particular attention has been paid to the NHERF1/PDZ1-domain that governs its membrane recruitment/displacement through a transient phosphorylation switch , 21–23. β-catenin signaling negatively regulates NHERF1 in CRC tumorigenesis will trigger the refinement of Wnt-targeted approaches to overcome the therapeutic resistance occurring at early disease stages.Although, further studies will exploit the safety profile of RS5517 in vivo, understanding that β-catenin (Ls174Tshβ-Cat and DLD1shβ-Cat) or a scramble control shRNA (shCtr), as previously described [β-Cat and DLD1shβ-Cat were transfected with 250 nM of NHERF1 shRNA (shNHERF1) or a scramble control shRNA (shCtr) (sc-156113) , according to the manufacturer’s instructions.Colorectal cancer (CRC) cell lines Ls174T and DLD-1 were purchased from the American Type Culture Collection  and routinely assessed to check their identity . Cells wescribed , 45. To FH535  and pyrvinium pamoate (Sigma-Aldrich) were resuspended in dimethyl-sulfoxide .β-Cat and DLD1shβ-Cat cells was isolated using Trizol reagent . The Nherf1 mRNA levels were determined using an RT-PCR kit  and the following primers: forward 5′-CCCAGTGGCTATGGCTTCAA-3′ and reverse 5′-GAAGTCTAGGATGGGGTCGG-3′. The primers for β-actin were: forward 5′-CCACGGCTGCTTCCAGCTCC-3′ and reverse 5′-GGAGGGCCGACTCGTCAT-3′. The relative Nherf1 mRNA abundance versus β-actin mRNA was quantified by Image J analysis.Total RNA of Ls174TshGapdh promoter primers was from Millipore (#17-10109). An anti-TCF1 antibody (clone 7H3) was also from Millipore. TCF-associated DNA immunoprecipitates were verified by qPCR using SYBR Green Mix (TaKaRa) and Nherf1 promoter primers as follows: Nherf1 5′-CCTCCGTCTTAATTCTCGAG-3′ (forward) and 5′-CCTTCACCTTCACAAACAAT-3′ (reverse). Data are reported as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample.A CHIP-KIP, including an anti-TCF4 antibody, a mouse IgG control and β-catenin primary antibody  followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG-FITC or a goat anti-rabbit IgG-TRITC  secondary antibodies, and then analyzed using a confocal laser scanning microscope , equipped with a 60 oil-immersion objective and a spatial resolution of 200 nm in x–y and 100 nm in z.Cells were assayed using an anti-NHERF1  or Cells were stained with 0.05 mM MDC in PBS at 37 °C for 10 min, washed and assayed by fluorescence microscope . Images were captured using Image Pro-plus software.5–505 cells/well) were treated with serial dilutions of RS5517 or DMSO in the absence (−Dox) or presence (+Dox) of Dox in a volume of 200 μl. Cell growth was quantified by MTT  (Sigma-Aldrich) according to manufacturer’s recommendations. For apoptosis assays, the cells were stained with Annexin V-PE  or Caspase-Glo assay kit (Promega) and analyzed by flow cytometry.Cells  were added at time of use. Lysates were then put on a tube roller (30–50 r.p.m.) at 4 °C for 30 min and centrifuged at 720×g at 4 °C for 5 min. Pellet was washed with 500 μl of SF buffer, centrifuged at 720×g at 4 °C for 10 min, and dissolved for 15 min in nuclear lysis buffer (NL buffer): 50 mM Tris-HCl (pH 8), 150 Mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, to which 10% glycerol and protease/phosphatase inhibitors were added at time of use. To obtain cytosolic fraction, the supernatant was centrifuged at 10,000×g at 4 °C for 10 min and ultracentrifuged at 100,000×g at 4 °C for 1 h. To obtain the membrane fraction, the ultracentrifuged pellet was washed with SF buffer and ultracentrifuged at 100,000×g at 4 °C for 1 h. Last pellet was dissolved in NL buffer and sonicated on ice.Cells were collected in the subcellular fractionation buffer (SF buffer): 250 mM sucrose, 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgClβ-catenin was from BD Transduction Laboratories, total NHERF1 from ThermoFisher, total c-Myc, cyclin D1, p27, Rab7, RILP, V1G1, Cathepsin D, and α-Tubulin were from Santa Cruz Biotechnology Inc. , total and phosphorylated pERK1/2 (pThr202/Tyr204) MAPK, total AKT, LC3, Beclin-1 (BECN1), and PARP were from Millipore.Cells were assayed as previously described . Primaryg for 15 min. Samples were then further diluted in 8 M urea, centrifuged again, reduced in 10 mM DTT for 30 min, and then alkylated in 50 mM IAM for 20 min. After four washes (2 in 8 M urea and 2 in 50 mM NH4HCO3), trypsin solution was added in an enzyme-to-protein ratio of 1:100 w/w, and samples were maintained at 37 °C for 16 h. Peptides were centrifuged and acidified by trifluoroacetic acid, desalted-concentrated on C-18 ZipTip (Millipore), dried under vacuum and then resuspended in 20 µl of ACN/H2O (FA 0.1%) . Separation was obtained using an EASY-nLC 1000 UPLC (Thermo Scientific) through 75 mm × 2 cm pre-column with nanoViper fittings  and 50 mm ID × 150 mm analytical column with nanoViper fittings . Elution was carried out over 120 min by using a 2-h gradient of ACN. The Q-Exactive instrument (Thermo Scientific) was set up to a spray voltage of 1.6 kV and the survey scans were taken at 70,000 FWHM (at m/z 400) resolving power in positive ion mode with a scan range from 300 to 1600 m/z.Quantity of 20 μg of whole-protein extracts prepared using Illustra TriplePrep kit  were diluted 10-fold in 8 M urea in 0.1 M Tris-HCl, pH 8.5, filtered into the Microcon Ultracel YM-30 devices (Millipore), and centrifuged at 14,000×Q-Exactive spectra were processed using the MaxQuant proteomics software (version 1.5.3.8) and matched using the Andromeda algorithm . TrypsinRS5517, namely 3-benzyl-5-chloro-N-(4-(hydroxymethyl)phenyl)-1H-indole-2-carboxamide, was synthesized by coupling reaction of 3-benzyl-5-chloro-1H-indole-2-carboxylic acid with protected (4-(hydroxymethyl)aniline in the presence of PyBOP reagent and triethylamine in DMF. The product was deprotected by reaction with tetrabutylammonium fluoride solution (1 M in anhydrous THF).cpptraj modules [The NHERF1 PDZ1 domain was retrieved from the Protein Data Bank, under the PDB accession code 21GQ4 and the substrates (DSLL and ETVM) were drawn by Maestro  and analysed by molecular dynamics with Amber 12 . The min modules . This le modules . An in-h modules . StructuD-NDSLL, was from JPT Peptide Technologies  and purified using HPLC. Equilibrium binding experiments were carried out in the presence of 50 mM Na phosphate buffer, pH 7.2, 300 mM NaCl at 25 °C by monitoring fluorescence upon exciting the sample at 280 nm and measuring emission on a Fluoromax single-photon spectrofluorometer  [A NHERF1 PDZ1 subcloned in pETG41 plasmid was kindly obtained from Dr. Nicolas Wolff . A site-directed mutagenesis by Quickchange Lighting mutagenesis kit was confirmed by DNA sequencing. A dansylated peptide relative to the C-terminal portion of the β2-adrenergic receptor, t-test. Proteomic statistical analysis was perfomed with the Perseus software (version 1.5.2.4). Multiple-samples tests were performed using ANOVA test by using a false discovery rate threshold of 0.05 and preserving grouping in randomization. Hierarchical clustering of proteins was performed in Perseus on logarithmized intensities after z-score normalization of the data, using Euclidean distances. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of our protein dataset was performed by STRING version 10 (http://string-db.org) :D447-52. doi: 10.1093/nar/gku1003).Data are reported as the mean ± SEM. Significance levels were determined with the Student's Supplementary Figure S1, S2, S3(PDF 6310 kb)


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with limited treatment options. Mutation of β-catenin is one of the most frequent genetic events along hepatocarcinogenesis. β-catenin mutations can be in the form of point mutation or large N-terminal deletion. Studies suggested that different β-catenin mutations might have distinct oncogenic potential.We tested the oncogenic activity of β-cateninS45Y, one of the most frequent point mutations of β-catenin, and ∆N90-β-catenin, a form of β-catenin with a large N-terminal deletion, in promoting HCC development in mice. Thus, we co-expressed β-cateninS45Y or ∆N90-β-catenin together with c-Met into the mouse liver using hydrodynamic injection.We found that both β-catenin mutations were able to induce HCC formation in combination with c-Met at the same latency and efficiency. Tumors showed similar histological features and proliferation rates. However, immunohistochemistry showed predominantly nuclear staining of β-catenin in c-Met/∆N90-β-catenin HCC, but membrane immunoreactivity in c-Met/β-cateninS45Y HCC. qRT-PCR analysis demonstrated that both ∆N90-β-catenin and β-cateninS45Y induced the same effectors, although at somewhat different levels. In cultured cells, both ∆N90-β-catenin and β-cateninS45Y were capable of inducing TCF/LEF reporter expression, promoting proliferation, and inhibiting apoptosis.Our study suggests that β-cateninS45Y and ∆N90-β-catenin, in combination with the c-Met proto-oncogene, have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity. Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, with up to 1 million cases diagnosed and almost 800,000 deaths globally each year , 3. ThusTERT) promoter region, TP53, ARID1A, ARID2, and CTNNB1 genes. Among them, mutations of CTNNB1, which encodes β-catenin, were found in 13–43% of HCCs [Recent genomic studies provide a comprehensive genetic landscape of human HCCs. The most frequent genetic alterations found in this tumor type include mutations of telomerase reverse transcriptase . Activation of c-Met is frequently found in human HCCs . RecentlIn the present study, we investigated the oncogenic potential of the weak β-catenin mutation (β-cateninS45Y) and the strong β-catenin mutation (∆N90-β-catenin) in inducing HCC formation in mice. Our investigation demonstrates that both alterations can equally promote HCC development in combination with the c-Met protooncogene.Two mouse HCC cell lines, HCC3–4 and HCC4–4, which were derived from c-Myc transgenic mice , were cuThe constructs used for mouse injection including pT3-EF1α, pT3-EF1α-c-Met, pT3-EF1α-β-cateninS45Y , pT3-EF1α-∆N90-β-catenin , and pCMV/sleeping beauty transposase (pCMV/SB) have been described previously , 17. AllWild-type (WT) FVB/N mice were obtained from The Jackson Laboratory . Hydrodynamic injection was performed as described previously . In brieMouse liver lesions were fixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraffin. Hematoxylin & Eosin (H&E) staining on 4 μm liver sections was performed to determine the time of appearance and characteristics of neoplastic foci. For immunohistochemistry, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by placement in a microwave oven on high for 10 min, followed by a 20-min cool down at room temperature. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit , the slides were incubated with the following primary antibodies: mouse anti-β-catenin , mouse anti-Glutamine Synthetase , β-catenin (D10A8) XP® Rabbit mAb , anti-Myc tag , Ki-67  overnight at 4 °C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and subsequently the biotin conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Signal was detected using the Vectastain ABC Elite kit (Vector Laboratories Inc.) and developed using DAB . Sections were counterstained with hematoxylin solution  and passed through the dehydration process and covered-slipped.Tissue were homogenized with a Polytron and cells were washed in PBS and lysed in M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific) containing the Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Protein concentration was quantified using the Pierce™ Microplate BCA Protein Assay Kit (ThermoFisher Scientific). Membranes were blocked in in 5% non-fat milk for 1 h and incubated with the following primary antibodies: β-catenin , p-β-catenin , Myc-tag , Glutamine Synthetase , c-Met , p-Met , cyclin D1 , β-actin  and GAPDH . Each primary antibody was followed by incubation with secondary antibody  for 1 h. After appropriate washing, bands were revealed with the Super Signal West Dura Kit (ThermoFisher Scientific).-ΔCt, wherein the ΔCt value of each sample was calculated by subtracting the maximum Ct value of the target gene from the average Ct value of the rRNA gene.Total RNAs were isolated from frozen mouse tissue samples using the Quick-RNA™ MiniPrep . Reverse transcription was conducted according to the manufacturer’s instructions . The mouse primers used for PCR analysis were synthesized by Integrated DNA Technologies . The sequences of the primers were as follows: ribosomal RNA (rRNA):5’-CGGCTACCACATCCAAGGAA-3′ (Forward) and 5’-GCTGGAATTACCGCGGCT-3′ (Reverse); Alpha Fetoprotein (AFP): 5’-TCTGCTGGCACGCAAGAAG-3′ (Forward) and 5’-TCGGCAGGTTCTGGAAACTG-3′ (Reverse); Glypican 3 (GPC3): 5’-CAGCCCGGACTCAAATGGG-3′ (Forward) and 5’-CAGCCGTGCTGTTAGTTGGTA-3′ (Reverse); axis inhibition protein 2 (AXIN2): 5’-GCTCCAGAAGATCACAAAGAGC-3′ (Forward) and 5’-AGCTTTGAGCCTTCAGCATC-3′ (Reverse); G-protein coupled receptor 5 (LGR5): 5’-ACCGAGCCTTACAGAGCCT-3′ (Forward) and 5’-GCCGTCGTCTTTATTCCATTGG-3′ (Reverse); MYC proto-oncogene (c-Myc): 5’-TGTACCTCGTCCGATTCC-3′ (Forward) and 5’-CATCTTCTTGCTCTTCTTCAG-3′ (Reverse); Cyclin D1 (CCND1): 5’-CGTGGCCTCTAAGATGAAGGA-3′ (Forward) and 5’-CCTCGGGCCGGATAGAGTAG-3′ (Reverse); Glutamine Synthetase (GS): 5’-CAGGCTGCCATACCAACTTCA-3′ (Forward) and 5’-TCCTCAATGCACTTCAGACCAT-3′ (Reverse); T-Box3 (TBX3): 5’-CAGGCAGCCTTCAACTGCTT-3′ (Forward) and 5’-GGACACAGATCTTTGAGGTTGGA-3′ (Reverse); Ornithine Aminotransferase (OAT): 5’-GGAGTCCACACCTCAGTCG-3′ (Forward) and 5’-CCACATCCCACATATAAATGCCT-3′ (Reverse); Leukocyte cell-derived chemotaxin-2 (LECT2): 5’-CCCACAACAATCCTCATTTCAGC-3′ (Forward) and 5’-ACACCTGGGTGATGCCTTTG-3′ (Reverse). Quantitative real-time polymerase chain reaction was performed with 100 ng of cDNA, using an ABI Prism 7000 Sequence Detection System and TaqMan Universal PCR Master Mix (ThermoFisher Scientific). Cycling conditions were: 10 min of denaturation at 95 °C and 40 cycles at 95 °C for 15 s and at 52 °C for 1 min. Quantitative values were calculated by using the PE Biosystems Analysis software and expressed as N target (NT). NT = 2HCC3–4 and HCC4–4 cells were plated in triplicate in 24-well plate at 80–90% confluency. Cells were transfected using the Lipofectamine 2000 reagents (Invitrogen). In brief, each well was transfected with 600 ng of pT3-EF1α (empty vector control) or pT3-EF1α-β-cateninS45Y or pT3-EF1α-∆N90-β-catenin, together with 200 ng of TOPFlash plasmid DNA or the negative control FOPFlash, as well as 8 ng of pRL-CMV. Cells were harvested 48 h post transfection. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s protocol. Experiments were repeated at least three times in triplicate.HCC3–4 and HCC4–4 cells were plated in triplicate in 24-well plate at 80–90% confluency. Cells were transfected using the Lipofectamine 2000 reagents (Invitrogen). Proliferation and apoptosis were assessed using the BrdU Cell Proliferation Reagent  and the Cell Death Detection Elisa Plus Kit , respectively, following the manufacturers’ protocol. Experiments were repeated at least three times in triplicate.P < 0.05 was considered significant.Statistical analysis was performed using Student’s t-test and Tukey-Kramer test. The data were expressed as the mean ± SD of at least three independent experiments. First, we investigated the oncogenic potential of “weak” β-catenin mutant (S45Y) and “strong” β-catenin mutant (∆N90-β-catenin) proto-oncogenes. However, since overexpression of β-cateninS45Y or ∆N90-β-catenin alone does not lead to tumor formation , 17, 19,Grossly, numerous tumor nodules could be found throughout the liver parenchyma of c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mice Fig. . HistoloCollectively, our results suggest that β-cateninS45Y and ∆N90-β-catenin cooperate with c-Met to promote HCC formation in vivo at similar latency and efficiency.Next, we performed IHC of β-catenin in normal liver as well as HCCs from c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mice. Using a widely used mouse monoclonal antibody against β-catenin, we found that, in normal liver, β-catenin staining could only be detected at the hepatocyte membrane Fig.  and b. IAt the molecular level, using Western blotting, we found that both c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin HCC lesions showed expression of ectopically injected human c-Met (Fig. Next, we investigated whether c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs showed similar expression patterns of Wnt/β-catenin canonical target genes. A total of 8 genes were analyzed via qRT-PCR, including the pan-β-catenin targets Axin2, LGR5, c-Myc, and Cyclin D1 Fig. , as wellIn summary, our study shows that β-catenin displays distinct staining patterns in c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs. However, the staining patterns were not associated with β-catenin activity, as both c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs demonstrated high and similar expression of β-catenin target genes.Subsequently, we evaluated the transcriptional activity of β-cateninS45Y versus ∆N90-β-catenin with the widely used TCF/LEF reporter constructs. Specifically, two mouse HCC cell lines isolated from c-Myc transgenic mice, HCC3–4 and HCC4–4 cells , were usFinally, we assessed the consequence of the transfection of β-cateninS45Y and ∆N90-β-catenin constructs in terms of proliferation and apoptosis in HCC3–4 and HCC4–4 cells. Noticeably, both mutant forms of β-catenin were able to induce higher proliferation and lower apoptosis than untreated and empty vector transfected cells Fig. . Once agTERT promoter mutation and TP53 mutation, in HCCs [Somatic mutations of β-catenin are one of the most frequent genetic alterations along hepatocarcinogenesis. Specifically, in the most recently released TCGA dataset based on whole-exome sequencing of 363 HCC cases, β-catenin mutation was identified in 27% cases . Survey  in HCCs . Trunk m in HCCs  and are  in HCCs . Thus, iIn this study, we compared the oncogenic potential of β-cateninS45Y and ∆N90-β-catenin mutations in combination with c-Met proto-oncogene in promoting HCC development using mouse modeling. In a previous study, β-cateninS45Y was characterized as a “weak” mutant, whereas ∆N90-β-catenin was characterized as a “strong” mutant . We founPerhaps the most intriguing results from our current study is that ∆N90-β-catenin/c-Met mouse HCC demonstrated strong nuclear β-catenin staining while β-cateninS45Y/c-Met showed predominantly membranous β-catenin immunolabeling Fig. . It is wPrevious studies showed that approximately 10% of human HCCs have concomitant β-catenin mutations along with c-Met overexpression . β-catenOur study suggests that β-cateninS45Y and ∆N90-β-catenin have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity.


Notably, these changes in glycan structure do not occur as a random consequence of disorder biology. Only a limited subset of oligosaccharides is found frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, altered sialylation has long been associated with metastatic cell behaviors such as invasion and enhanced cell survival and accumulating evidence points to the alteration occurring in the sialic acid linkage to other sugars, which normally exists in three main configurations: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three configurations has been described in cancer progression. However, the increased α2,6 sialylation catalyzed by β-galactoside α2,6 sialyltranferase 1  is frequently observed in many types of the cancers. In this review, we describe the findings on the role of ST6Gal I in cancer progression, and highlight in particular the knowledge of how ST6Gal I-mediated α2,6 sialylated glycans or sialylated carrier proteins regulate cell signaling to promote the malignant phenotype of human carcinoma.Altered glycosylation is a common feature of cancer cells. It takes a variety of forms, which includes loss of expression or excessive expression of some structures, the accumulation of precursors, the appearance of novel structures,  N-glycans, O-glycans and glycosphingolipids [i-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), although only the former is present in human cells [N-glycans, because the altered expression of ST6Gal I are observed in many types of cancers Gal/GalNAc sequence. The earliest identified is the lectin named Sambucus nigra agglutinin (SNA) ,59. The in vitro and in vivo [The up-regulated expression of ST6Gal I was first described in colon cancer, but successively confirmed in other carcinomas of breast, liver, cervix, choriocarcinomas, acute myeloid leukemias and some malignancies of the brain as well ,38,39,40 in vivo . Given t in vivo , it is rin vitro studies show that the effects of ST6Gal I on cell migratory response are mediated, at least in part, by the α2,6 sialylation of the β1 integrin because cells deficient in β1 integrin do not exhibit differential invasion upon forced expression of ST6Gal I expression [In contrast to the elusive role in the tumor growth, increasing evidence indicates that ST6Gal I is critical for the tumor malignancy including metastasis and invasion. It has been shown in colon cancer that metastatic tumor growth is accompanied by a significant increase of α2,6 sialylated carbohydrate sequences produced by ST6Gal I . Knockdopression ,74,75.On the other hand, the selective enrichment of α2,6 sialic acids produced by ST6Gal I on tumor cells has also been shown to render the cells resistant to apoptosis. One striking example is the effect of α2,6 sialylation on the apoptosis signaling mediated by cell galectins. Galectins are a family of animal lectins with affinity for β-galactosides ,77,78. ACancer stem cells (CSCs) refer to a minority population of cancer cells that are capable of self-renewal and generation of differentiated progeny. Eradication of this rare population is a new insight in cancer treatment. Intriguingly, high expression of ST6Gal I has been correlated with human induced pluripotent stem cells and CSCs, indicating that ST6Gal I activity may be involved in maintaining some aspect of stem like cell behavior . ConsideThe functions of ST6Gal I in cancer progression could be more complicated than discussed above. The pro-migratory and anti-apoptotic roles have been challenged by several reports that: (1) forced expression of ST6Gal I suppressed the cell migration and enhanced the cell death induced by chemotherapeutic agents in glioma cells ,100; (2)N-glycans masks the galectin recognition sites for binding of β-galactoses, which in turn switches off the galectin functions including adhesion, migration and apoptosis. In contrast to the inhibitory effect on the glycan–galectin binding, α2,6 sialic acids have also been reported to bind specifically to the siglec-2 family of lectins [While not completely understood, the functions of α2,6 sialylation described above may be exerted by affecting the structures of attached glycans or carrier proteins. In the first case, as mentioned earlier, the addition of α2,6 sialic acid to the terminal galactose of  lectins . Since s lectins , cluster lectins , EGFR [1 lectins  and PECA lectins , cell su lectins  and Fas  lectins . There i lectins . Given tO-glycans) clearly demonstrated the difference in their glycan acceptor binding domain [Given the accumulating evidence for the importance of ST6Gal I mediated α2,6 sialylation in cancer progression, much attention has been paid to elucidating the regulatory mechanisms of its expression. It has been shown that the expression of α2,6 sialylation on tumor cell surface can be modulated at different levels . The mosg domain ,126. Theg domain ,128. Alsg domain . Better Given increasing evidence implicating ST6Gal I in multiple cancers, much effort has been undertaken to clarify the functions of this enzyme in cancer progression during the last decade. Furthermore, it is becoming clear that ST6Gal I plays important roles in regulating tumor metastatic behaviors including migration and enhanced cell survival. In contrast, there is a marked dearth of information on how tumor cells regulate the expression of ST6Gal I and α2,6 sialylation. Defining regulatory mechanisms and specific ST6Gal I substrates will be necessary for a complete understanding of the roles of ST6Gal I during cancer progression and may provide new insights for targeting aggressiveness and drug resistance of the cancer cell by manipulating the level of cell surface α2,6 sialylation. In addition, although ST6Gal I is mainly localized in the Golgi apparatus, this protein can also be proteolytically processed to a soluble form and secreted into the serum ,131. The