Physalis angulate, acts as an antagonist of Wnt/β-catenin signalling. In vitro, PF decreased Wnt3a-induced TOPFlash reporter activity in HEK293T cells and promoted the formation of the β-catenin destruction complex. Importantly, PF also inhibited Wnt/β-catenin signalling and accelerated the degradation of β-catenin in CRC cells. However, PF did not affect the stabilization of Axin or the interaction of β-catenin with E-cadherin. Interestingly, we further found that PF promoted YAP binding to the β-catenin destruction complex, which facilitated the ubiquitination and degradation of β-catenin. Silencing and pharmacological inhibition of YAP reversed the formation of the β-catenin destruction complex induced by PF, implying that YAP binding to the β-catenin destruction complex was responsible for PF-mediated inhibition of Wnt/β-catenin signalling. Furthermore, PF observably inhibited tumour growth by down-regulating β-catenin in tumour-bearing mice. Collectively, our findings indicated that PF inhibited Wnt/β-catenin signalling by accelerating the ubiquitination and degradation of β-catenin in a YAP-dependent manner and therefore PF could be a novel potential candidate for CRC therapy.Aberrant activation of Wnt/β-catenin signalling is critical in the progression of human cancers, especially colorectal cancer (CRC). Therefore, inhibition of Wnt/β-catenin signalling is a significant potential target for CRC therapy. Here, we identified for the first time that Physalin F (PF), a steroid derivative isolated from  Due to advances in surgery and chemotherapy, patients’ overall survival has increased. However, there have emerged severe toxicities and side effects during long-term administration2. Therefore, it is urgently necessary to develop novel therapeutic agents for CRC therapy.Colorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality worldwide4. Aberrant activation of Wnt/β-catenin signalling is frequently observed in CRC and is considered to be a crucial driver of CRC pathogenesis5. In the absence of the Wnt ligand, cytoplasmic β-catenin is phosphorylated at residues Ser45, Thr41, Ser37, and Ser33 in a destruction complex with adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK-3β) and Axin6. Phosphorylated β-catenin is recognized by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) and subsequently degraded by the ubiquitin-dependent proteasome pathway. Upon Wnt stimulation, the Wnt ligand binds to the Frizzled receptor and the LDL receptor related protein (LRP) complex at the cell surface, which leads to the membrane recruitment and activation of scaffold protein and dishevelled6. Activated dishevelled inactivates the destruction complex in the cytoplasm, thus decreasing the degradation of β-catenin. The stabilized β-catenin translocates to the nucleus and interacts with TCF/LEF transcription factors to activate Wnt target genes and promote the process of cancer7. Therefore, inhibition of β-catenin is a potential strategy for the prevention or treatment of CRC10.The Wnt/β-catenin signalling pathway is crucial in multiple developmental events during embryogenesis and is also involved in tumourigenesis12. In the cytoplasm, YAP interacts with β-catenin directly and restricts nuclear translocation of β-catenin13. Moreover, YAP is essential for β-TrCP recruitment to the destruction complex and facilitates β-catenin degradation14, indicating that YAP is pivotal for the ubiquitination and proteasomal degradation of β-catenin.The transcriptional co-activator Yes-associated protein (YAP), identified as a target of the Hippo pathway has recently been identified as an additional regulatory component of canonical Wnt/β-catenin signalling16. Withanolides, a class of steroid compounds, have attracted attention due to their multiple bioactivities, such as immunosuppressive, anti-inflammation, antimicrobial, antidiabetic and anti-tumour activities18. Our previous studies have also reported the anti-tumour efficacy of some withanolides20. Physalin F (PF), a withanolide derivative extracted from Physalis angulata, has exhibited immunomodulatory and anti-tumour activities23. However, there have been no reports on the therapeutic property of PF towards CRC to date. In the present study, we demonstrated for the first time that PF suppressed Wnt/β-catenin signalling by promoting YAP-mediated ubiquitination and proteasomal degradation of β-catenin in CRC cells.Natural products have recently been gaining more attention because of their multiple biological activities and desirable health benefits, especially in cancer therapyTo investigate whether PF Fig.  could inFurthermore, PF suppressed the Wnt3a-induced expression of β-catenin in the nuclear and cytoplasmic fractions Fig. . ImmunofSince the level of intracellular β-catenin is regulated by the proteasomal degradation pathway, we examined the involvement of the proteasome pathway in PF-mediated inhibition of β-catenin. As shown in Fig. 6. Thus, we assessed whether GSK-3β participated in PF-induced suppression of β-catenin. After incubation with the known GSK-3β inhibitor LiCl, TOPFlash reporter activity was increased, while this stimulation was suppressed by PF 27 increased the expression of Axin1  inhibitors are known to suppress Wnt/β-catenin signalling by stabilizing Axin1 and promoting the formation of the β-catenin destruction complex29. Meanwhile, a deficiency of YAP suppressed the proliferation of SW480 and DLD1 cells  were intraperitoneally administered every three days. The mice were euthanized after four weeks, and their organs were harvested for histopathologic analysis. As shown in Fig. To evaluate the anti-tumour capacity of PF in vivo, we established a xenograft model by subcutaneously injecting SW480 cells in nude mice. After the solid tumours reached 100 mmTo further confirm the tumour inhibition mechanism of PF, we examined the expression of Ki67, PCNA, β-catenin, Cyclin D1 and c-Myc in the tumour tissue. Immunohistochemistry analysis demonstrated that the expression of Ki67 and PCNA was reduced after PF treatment Fig. , indicat5. Increasing evidence strongly suggests that Wnt/β-catenin signalling has emerged as one of the most promising targets for the treatment of CRC9.It is well known that aberrant activation of Wnt/β-catenin signalling is an integral process in the development of most CRC17. Although it has been reported that withanolides can inhibit Wnt/β-catenin signalling, the molecular mechanisms remain poorly understood30. PF, a withanolide isolated from Physalis angulata, exhibited cytotoxicity against a panel of human tumour cell lines23. However, the effect of PF on Wnt/β-catenin signalling had not previously been reported. In this study, we demonstrated that PF decreased TOPFlash reporter activity induced by Wnt3a and promoted the proteasomal degradation of β-catenin by accelerating the formation of the β-catenin destruction complex in HEK293T cells.Withanolides, a class of steroid compounds, exert a wide variety of anti-tumour properties9. Consistently, we observed that PF inhibited the expression of β-catenin and increased the phosphorylation of β-catenin at Ser33/37/Thr41 in CRC cells. Interestingly, the inhibition of β-catenin was markedly restored by MG132, suggesting the degradation of β-catenin induced by PF was dependent on the proteasome pathway. Because the degradation of β-catenin is mainly regulated by the β-catenin destruction complex4, we speculated that PF may affect the formation of the β-catenin destruction complex. Co-immunoprecipitation assays confirmed that PF facilitated the formation of the β-catenin destruction complex and increased the ubiquitination of β-catenin. Moreover, PF had no influence on the interaction of β-catenin and E-cadherin. These data proved for the first time that PF enhanced the degradation of β-catenin through promoting the formation of the β-catenin destruction complex.Previous studies have verified that the β-catenin destruction complex mediates sequential phosphorylation of β-catenin at S45, T41, S37 and S33 for subsequent ubiquitination and proteasomal degradation and thereby maintains low baseline cytosolic levels of the protein in cancer cells31, which facilitated the ubiquitination and degradation of β-catenin. To identify whether YAP was involved in the ubiquitination and degradation of β-catenin mediated by PF, we transfected YAP siRNA into CRC cells. After silencing YAP, inhibition of β-catenin induced by PF was eliminated, suggesting that YAP was involved in the PF-mediated inhibition of β-catenin. Meanwhile, PF promoted YAP binding to β-TrCP and formation of the β-catenin destruction complex. Interestingly, YAP deficiency abolished the increase in β-TrCP binding to the destruction complex mediated by PF, indicating that YAP was responsible for the recruitment of β-TrCP to the destruction complex induced by PF. In addition, it has been reported that TNKS inhibitors inhibit Wnt/β-catenin signalling through stabilization of Axin32. Unlike TNKS inhibitors, PF did not affect the expression of Axin1. Collectively, our data suggested that the PF-induced inhibition of β-catenin was dependent on YAP binding to the β-catenin destruction complex.YAP, a transcriptional co-activator in Hippo signalling, has been reported to bind to Axin and recruit β-TrCP to the destruction complexAdditionally, a xenograft model bearing SW480 cells was used to evaluate the anti-tumour potency of PF in vivo. Our results showed that PF suppressed tumour growth significantly in tumour-bearing nude mice. Compared with the control group, PF had no significant influence on the animals’ body weight. Importantly, no obvious toxicity was observed in the main organs of mice after treatment of PF, while the Oxa treatment group exhibited remarkable injury to the liver and kidney. Immunohistochemistry and western blot analysis revealed that the expression of β-catenin was decreased in tumours from the PF treatment group. These results further confirmed that PF exerted a remarkable anti-tumour effect with high safety in vivo.In conclusion, our findings demonstrated for the first time that PF exhibited potential anti-tumour efficacy against CRC in vitro and in vivo by inhibiting Wnt/β-catenin signalling via accelerating the ubiquitination and degradation of β-catenin in a YAP-dependent manner. Our study suggested that PF could be a potential candidate agent for treatment of CRC.2.HEK293T cells and the human colon cancer cell lines SW480 and DLD1 were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . SW480, DLD1 and HEK293T cells were cultured in DMEM  supplemented with 10% FBS  at 37 °C with 5% CO3/well and incubated overnight. Then, the cells were treated with various concentrations of PF for 24 h. Growth medium was refreshed every three days. After 14 days, the cells were fixed and stained with a crystal violet solution  for 15 min, and images of colonies were taken manually.A 3--2,5-diphenyltetrazolium bromide (MTT) assay was applied to examine cell viability, and relative cell viability was calculated based on the absorbance of untreated cells. Colony formation assays and 5-ethynyl-20-deoxyuridine (EdU) assays were adopted to analyse cell proliferation. For the colony formation assay, cells were seeded into 35 mm plates at a density of 1 × 10EdU labelling and detection was performed according to the manufacturer’s instructions . Cells were fixed with 4% paraformaldehyde for 15 min and then washed with PBS and stained with the anti-EdU working solution at room temperature for 30 min. After a wash with 0.5% Triton X-100 in PBS, the cells were incubated with 4,6-diamino-2-phenyl indole  for 5 min. The cells were observed under ImageXpress® Micro Confocal .33. SW480 and DLD1 cells were seeded at 5000 cells/well into E-Plate 16-well plates and cultured overnight. Then, cells were incubated with or without serial dilutions of PF. Cell proliferation was continuously monitored every 15 min over a time period of 60 h. Data analysis was carried out using RTCA Software 1.2.1 supplied with the instrument.The xCELLigence System  was used to monitor the dynamics of the cytotoxic effects of PF on SW480 and DLD1 cells. The xCELLigence System is a microelectronic biosensor system and allows continuous quantitative monitoring of cellular behaviour, including proliferation by measuring electrical impedanceTCF wild-type (TOPFlash) or mutated control (FOPFlash) luciferase reporter plasmids were purchased from Upstate Biotechnology . Transfection was performed with Lipofectamine 2000  according to the manufacturer’s protocol. Luciferase activity was measured with the Dual Luciferase Reporter Assay System  according to the manufacturer’s manual. The results were normalized to the control renilla activity.Total RNA was isolated using an EASYspin Plus tissue/cell RNA extraction kit . RNA was reverse-transcribed to cDNA using a Transcriptor First Strand cDNA Synthesis Kit . Quantitative PCR was performed on a LightCycler 480 system  using Fast SYBR Green Master Mix . The RT-PCR primers used in this study are listed in Supplementary Table Cells with different treatments were washed twice with PBS, then collected and lysed in RIPA buffer. The cell lysates were separated on SDS polyacrylamide gels and transferred to PVDF membranes . After blocking nonspecific binding with TBS-T (0.1% Tween) containing 5% non-fat milk for 1 h at room temperature, the membranes were immunoblotted with the primary antibodies at 4 °C overnight. Then, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody for 2 h at room temperature. The protein bands were detected using the ChemiDOC™ system . The primary antibodies: Wnt/β-catenin activated targets antibody sampler kit, anti-β-catenin, anti-GSK-3β and anti-YAP were purchased from Cell Signalling Technology . Anti-β-TrCP, anti-ubiquitin and anti-Axin1 were purchased from Abcam . HRP-conjugated goat anti-rabbit secondary antibodies were obtained from Cell Signalling Technology .Targeting YAP siRNA was purchased from Biomics . Cells were seeded in 6-well plates. YAP siRNA was transfected into the cells using Lipofectamine 2000  according to the manufacturer’s instructions.4/well) were cultured on 96-well culture plate . After incubation with different test substances, the cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min. Next, the cells were blocked with 5% BSA for 1 h and incubated with primary antibody overnight at 4 °C. Alexa-conjugated secondary antibodies  were applied and incubated at room temperature for 1 h. Cell nuclei were stained with DAPI  for 10 min. Finally, the cells images were analysed by ImageXpress® Micro Confocal .Cells  for another 2 h. After incubation, the protein A/G beads were washed four times with the lysis buffer. The lysates and IP samples were subjected to SDS-PAGE followed by western blot using the indicated antibodies.6 SW480 cells were suspended in 200 μl PBS and subcutaneously injected into the right flank of the mice34. When the tumours reached approximately 100 mm3, the tumour-bearing mice were randomly divided into four groups. Then, PF (10 or 20 mg/kg) and oxaliplatin  were intraperitoneally administered every three days. After four weeks, all of the mice were euthanized. Then, the tumours and visceral organs of each group were collected and fixed in 4% paraformaldehyde. All animal experimental procedures followed the National Institutes of Health guide for the care and use of laboratory animals and were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of China Pharmaceutical University Experimental Animal Center.Five-week-old male BALB/c-nu/nu mice were purchased from the Model Animal Research Center of Nanjing University . For the tumour xenograft assay, 5 × 10t-test by using GraphPad Prism version 5.0 . The data were presented as the mean ± SD. P < 0.05 was considered significant.Statistical analysis was performed with ANOVA or Student’s Supplementary TablesSupplementary Figures LegendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Author Contribution Form


On the other hand, the impedance spectroscopy capacitances C1 and C2 allow studying the microstructure development of fly ash cement mortars exposed to those conditions, and their results are in accordance with mercury intrusion porosimetry ones. Finally, it has been observed that the combined analysis of the abovementioned capacitances could be very useful for studying shrinkage processes in cement-based materials kept in low relative humidity environments.Today, the characterisation of the microstructure of cement-based materials using non-destructive techniques has become an important topic of study, and among them, the impedance spectroscopy has recently experienced great progress. In this research, mortars with two different contents of fly ash were exposed to four different constant temperature and relative humidity environments during a 180-day period. The evolution of their microstructure was studied using impedance spectroscopy, whose results were contrasted with mercury intrusion porosimetry. The hardening environment has an influence on the microstructure of fly ash cement mortars. On one hand, the impedance resistances R The particular case of fly ash and its effect on the properties of cement-based materials has been the topic of many studies [Firstly, the use of active additions in cement industry has been an important research field ,7,8,9,10 studies ,12,13. T studies ,15,16,17 studies ,18, such studies  and resi studies ,21,22,23 studies ,14,24 du studies , which i studies . This faNowadays, the study of the microstructure of cementitious materials using non-destructive techniques, such as shear wave velocity, SEM/ESEM, and thermal conductivity tests, has become a major topic of study ,31,32,33Real structures are usually exposed to different hardening conditions depending on their geographical location. The different environmental temperatures and relative humidities can affect the development of clinker hydration ,40,41,42On the other hand, regarding the microstructure characterisation using impedance spectroscopy of cement-based materials with active additions, hardened in non-optimal conditions, there is only a previous recent authors’ work  in whichCement mortars were studied in this research. They were prepared using two different commercial fly ash cements, a Portland cement with fly ash, CEM II/B-V 42.5 R   . The meaThe causal, linear, and stable nature of the data recorded was validated using the Kramers-Kronig (K-K) relations  see Figa. MoreovFor each cement type, condition and w:c ratio four different samples were tested. The evolution of impedance parameters with time is reported to over a 180 hardening day period.The results of total porosity for CEM II mortars are depicted in For those mortars, the high total porosity decrease and the progressive pore network refinement with time observed for condition A could be related to the very high RH present in the environment, combined with a high enough temperature. The unlimited availability of water would make easier the development of clinker hydration ,39, so mThe slower decrease of porosity  and the The similar values of total porosity obtained for conditions A and C in the short-term see  could beThe lower total porosities see  and the Regarding the MIP results for CEM IV mortars, the total porosity ones are depicted in The evolution of total porosity and pore size distribution of CEM IV mortars kept in condition B followed a similar tendency than for condition A until 90 days; although, in general, the porous network was less refined for environment B. In the previous discussion of MIP results for CEM II samples (with a lower content of fly ash) exposed to condition B, it was explained that in an environment with a lower temperature, the clinker hydration is slowed down , and as The relatively low values of total porosity for CEM IV mortars hardened in condition C, and the strong similarity to those obtained for condition A at early ages, could be related to the high enough temperature, as has been already explained for CEM II results. However, in the long-term, the low RH of condition C and the consequent limited availability of water would make the hydration  and pozzThe tendencies shown by MIP results for CEM IV mortars exposed to condition D were similar than those described for condition C. At 7 days, the total porosities were the lowest of all conditions studied. This could be due to the high temperature, which would speed up the clinker hydration and would advance the starting of pozzolanic reactions, as happened for CEM II mortars. Despite that, after 7 days, the total porosity hardly changed or even increased and the microstructure became less refined. On the one hand, this could probably be related to the lack of water in condition D, which would block the development of hydration and pozzolanic reactions in the long-term, as happened for CEM II samples kept in low RH conditions. On the other hand, the previously mentioned formation of shrinkage microcracks induced by the very low RH could also explain these results.The Hg retained at the end of the experiment is related to the tortuosity of the pore network of the samples . With reHereafter, the discussion will be focused on impedance spectroscopy parameters results.1 and R2, they are related to the electrolyte which fills the pores of the sample [Regarding impedance spectroscopy resistances Re sample . Then, i1 and R2 and the condition A the lowest ones. These results agree with those obtained in a previous research [1 and R2 were not a reliable parameter for characterising the changes in the microstructure of cement-based materials in non-saturated samples, like those exposed to non-optimum conditions [Nevertheless, this did not happen see  and the research , in whicresearch , the resnditions . This ar1 is associated to the solid fraction in the samples [1 are depicted in The capacitance C samples . Then, i1 value observed for CEM IV mortars at 180 days compared to CEM II ones, especially for w:c ratio 0.4, would be also in keeping with the more refined porous network of CEM IV samples; see In general, for both cements studied, this parameter was higher and its rise with time was more important for specimens exposed to environment A. This would imply that in this condition, there is a faster and more continuous formation of solid phases, which is in agreement with MIP results. This result could be related to the higher RH of condition A, which could facilitate the hydration  and pozz1 for condition B was slower, mainly for CEM IV mortars. However, this parameter rose progressively, and at 180 days, for w:c ratio 0.4 specimens, it reached similar values than those observed for condition A. These results show that the solid phase formation was slower for environment B, which coincides with the MIP results. This behavior could be due to the lower temperature of condition B, which would slow down the development of clinker hydration [The increase of capacitance Cydration ,54, entaydration .1 at early ages was higher and increased faster for both types of cement compared to condition B. These results would mean that in the short-term the formation of solids would be produced relatively quick, which would be also in accordance with MIP results (effect of the relatively high temperature). Nevertheless, in the long-term, the solid phases formation would be produced more slowly, as suggested by the slower rise of capacitance C1 observed for condition C compared to condition A. This could be related to the lower RH of condition C, which would make the development of hydration and pozzolanic reactions at greater ages difficult. This result is in agreement with that observed for plain ordinary Portland cement mortars exposed to the same condition [For condition C, generally the capacitance Condition .1 for mortars exposed to environment D showed relatively high values at early ages. This would indicate that a higher solid formation is produced in the short-term, which would again coincide with MIP results . 2 is related to the pore surface in contact with electrolyte present in the material and gives information about the formation of CSH gel layers, which occupy the pores [2 results are shown in The capacitance Che pores . This pahe pores ,38. The 2 have been observed for condition A. Moreover, it continuously increased during the studied period. This result is in accordance with capacitance C1 and MIP ones, and it showed the greater formation of solid phases in this condition, which would increase the pore surface.In general, the highest values and rise with time of capacitance C2 values for condition B were the lowest of all studied environments. However, at later ages, they were similar or even higher than those observed for conditions C and D, but lower than environment A ones. These results would show that the increase of pore surface in condition B, due to solids formation, was produced more slowly, probably by the effect of the lower temperature of this environment, as has been already explained for MIP and capacitance C1 results.The initial C2 values relatively highly compared to the rest of environments. However, the rise with time of this parameter was not as noticeable as observed for condition A. The evolution of capacitance C2 in condition C suggests that at early hardening ages, there was a moderately fast formation of solid fraction, which was slowed down in the long-term. Again, this result coincides with previous ones, and it could be related to the low RH in the environment, which would hinder the development of hydration and pozzolanic reactions at greater ages.The CEM II and IV mortars hardened under condition C had C2 also showed high values compared to the rest of conditionsin the short-term. Nevertheless, since then this parameter hardly rose with time. In view of that, it seems that in condition D, new solids would be formed quickly at early ages, which would increase the pore surface, although in the long-term this formation would decrease considerably. In general, C2 results are in agreement with capacitance C1 and MIP ones, so their discussion is also valid for capacitance C2, and they show the effect of combined high temperature and very low RH.With respect to condition D, the capacitance C2 for condition D did not change at higher ages, while total porosity increased and capacitance C1 decreased. This could confirm the hypothesis of the appearance of shrinkage microcracks in dry conditions. This result has been also observed for plain ordinary Portland cement and slag cement mortars exposed to the same condition D in a previous research [2 [1 and a rise of total porosity, as has been observed. Then, the present results obtained for fly ash cement mortars, in addition to those previously obtained for OPC and slag cement mortars [1 and C2 could be very useful for analyzing the effects of shrinkage in the long-term in cement-based materials hardened in conditions with low RH.Finally, it is important to emphasize the fact that the capacitance Cresearch . This coearch [2 . On the  mortars , would sThe influence of the environmental condition on microstructure of fly ash cement mortars can be studied with impedance spectroscopy, using the equivalent circuits already defined for ordinary Portland cement.A high environmental relative humidity entails a more refined microstructure of CEM II and IV mortars, because the high availability of water facilitates the clinker hydration, producing a quicker formation of portlandite, which permits an earlier beginning of fly ash pozzolanic reactions.Conditions with high temperature bring a more refined microstructure and a lower porosity of CEM II and IV mortars in the short-term, because the clinker hydration is accelerated, causing the subsequent earlier development of fly ash pozzolanic reactions.1 and R2 obtained for fly ash mortars hardened in conditions with relative humidities lower than 100% are more affected by the drying of the materials than by the microstructure development. This result coincided with those obtained in a previous research for OPC and slag cement mortars exposed to the same conditions, so they would confirm that impedance resistances R1 and R2 are not suitable for following the microstructural evolution of cement-based materials hardened under non-optimum conditions.The impedance spectroscopy resistances R1 and C2 allow following the microstructure development of fly ash cement mortars, and their results are in accordance with MIP ones for all conditions studied in this research.The impedance capacitances CThe rise of total porosity and the pore refinement loss observed in the long-term for the mortars exposed to drier conditions could be due to the appearance of shrinkage microcracks as a consequence of the low environmental relative humidity.1 and C2 evolution could justify the abovementioned formation of shrinkage microcracks for CEM II and IV mortars exposed to the drier environments. This result is also in agreement with that obtained in a previous research for OPC and slag cement mortars exposed to the same conditions, which would corroborate the usefulness of both capacitances for studying shrinkage processes in cement-based materials.The combined analysis of capacitances CThe main conclusions that can be drawn from the results previously discussed can be summarized as follows:


PLOD2, HADH, LCOR and REEP1 as non-canonical target genes in various colon cancer cells. Moreover, non-canonical Wnt target genes are regulated via RoR2, Dvl2, ATF2 and ATF4. Furthermore, we show that the ligands Wnt5a/b are upstream regulators of the non-canonical signature and moreover regulate proliferation of cancer cells in a β-catenin-independent manner. Our experiments indicate that colon cancer cells are dependent on both β-catenin-dependent and –independent Wnt signaling routes for growth and proliferation.Wnt signaling is an evolutionarily conserved signaling route required for development and homeostasis. While canonical, β-catenin-dependent Wnt signaling is well studied and has been linked to many forms of cancer, much less is known about the role of non-canonical, β-catenin-independent Wnt signaling. Here, we aimed at identifying a β-catenin-independent Wnt target gene signature in order to understand the functional significance of non-canonical signaling in colon cancer cells. Gene expression profiling was performed after silencing of key components of Wnt signaling pathway and an iterative signature algorithm was applied to predict pathway-dependent gene signatures. Independent experiments confirmed several target genes, including  Canonical Wnt signaling has been associated with breast5, colon6 and gastric cancer development8. Non-canonical, β-catenin-independent Wnt signaling is proposed to have a multifunctional role in tumorigenesis, being either tumor supportive or suppressive depending on tumorstage and tumortype9.Wnt signaling pathways control a wide range of processes during embryonic development and tissue homeostasis. Deregulation of these pathways has been linked to many human diseases, including cancer11. Cells secrete Wnts in autocrine or paracrine manner with the help of two Wnt-secretion specific proteins: Porcupine (Porcn), an O-acyltransferase located in the endoplasmic reticulum (ER) responsible for Wnt palmitoylation13, and Evi/Wls/GRP177 which is as a cargo-receptor required for transport of palmitoylated Wnt proteins ER to Golgi and then to the extracellular space17.In mammals, the family of Wnt ligands consists of 19 members, which trigger multiple intracellular signaling cascades and orchestrate complex, context-dependent responses. Expression of Wnt ligands is tissue- and cell type specific18 and the presence or absence of enhancers or inhibitors of signaling, such as members of the R-spondin, SFRP and Dickkopf (DKK) families20. While Wnt1, Wnt3 and Wnt3a mostly activate a β-catenin-dependent signaling pathway21, Wnt5a/b and Wnt11 primarily induce non-canonical, β-catenin-independent pathways23. However, predicting the signaling outcome of different Wnt ligands has remained difficult.Wnt ligands can induce multiple pathways depending on the available receptors24. Recruitment of additional factors, including kinases, leads to phosphorylation of LRP6. In the absence of Wnt ligands β-catenin is constantly degraded by a destruction complex containing adenomatous polyposis coli (APC)25, a tumorsuppressor and negative regulator of Wnt signaling, the scaffold protein Axin1 and the kinases GSK3β and CK1α27. These kinases phosphorylate β-catenin, leading to its ubiquitylation and proteasomal degradation. Axin1 and GSK3β are recruited to the receptor complex upon Wnt stimulation and this leads to a breakdown of the destruction complex and β-catenin stabilization. β-catenin then translocates to the nucleus where it forms a complex with TCF/LEF transcription factors and activates Wnt target gene expression29.Binding of canonical Wnt proteins to Frizzled (FZD) receptors and the LRP5/6 co-receptors leads to the relocation of Dishevelled (Dvl), a multi-domain scaffolding protein to the plasma membrane31, Wnt/Ca2+32, RoR2/JNK signaling33, are less understood34. In mouse, RoR1/2 and Wnt5a knockouts show comparable phenotypes during development10 and blastocyst attachment and implantation35. Dvl, an adaptor protein composed of a DIX, PDZ and DEP domain, is required for both non-canonical and canonical Wnt signaling36. Its DIX domain is required for canonical Wnt signaling, the DEP domain links receptor activation to non-canonical pathways39. FZDs, RoR1/241, Ryk42 and PTK743 transmembrane proteins have all been proposed as non-canonical Wnt receptors depending on the model or cellular system.Non-canonical, β-catenin independent pathways, such as planar cell polarity (PCP)47. Rac1 and RhoA were reported to activate JNK after pathway stimulation by Wnt5a48. Wnt5a and Wnt11 binding to FZD and/or RoR2 also can lead to intracellular Ca2+ release, which activates Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC)32.Downstream effectors of the Wnt/PCP pathway are Rac, RhoA and JNK, linking signaling at the membrane to AP1 and ATF2 transcription factorsvia a β-catenin-independent signaling pathway. Taken together, these results indicate that survival of colon cancer cells is dependent on both canonical and non-canonical Wnt signaling.To date, the functional role and potential transcriptional targets of non-canonical, β-catenin-independent Wnt signaling in colon cancer cells is not well understood. In order to identify targets, we first performed RNAseq analysis of colon cancer cells upon silencing of several key Wnt signaling components. We then applied an iterative signature algorithm to identify genes, which are regulated by a Wnt cargo receptor Evi/Wls, independent of β-catenin and APC. Several identified genes were confirmed by independent methods and shown to be targets of a non-canonical RoR2/Dvl2/ATF signaling. Additionally, we found that these genes are regulated by auto- and paracrine Wnt5a/b secretion in colon cancer cells. We further demonstrate that Wnt5a/b is required for proliferation and viability of colon cancer cells 49. After siRNA silencing of APC, β-catenin/CTNNB1 and Evi/Wls, changes in the transcriptome of HCT116 colon cancer cells were analyzed by RNA sequencing (RNAseq)  to the transcriptome data .In order to characterize the role of β-catenin-independent signaling in colon cancer, we devised perturbation experiments to identify gene sets that are dependent on auto-paracrine Wnt secretion, but unaffected by depletion of β-catenin or APCa priori knowledge  by homologous recombination. The data confirmed a decrease in lls Fig. . Treatmeion Fig. .PLOD2, HADH, and LCOR as non-canonical Wnt targets that depend on Wnt secretion but not on β-catenin in colon cancer cell lines as well as primary colon cancer cells and MEFs.Taken together, these experiments confirmed In order to identify Wnt ligands required for regulation of β-catenin-independent target gene expression in HCT116 cells we determined expression patterns of all 19 Wnt genes in these cells. Among others the non-canonical Wnt5b, Wnt11 and Wnt16, as well as the canonical ligands Wnt3, Wnt3a, Wnt9a and Wnt10b were detected by RNAseq experiments  in carcinoma compared to normal colon/rectum   in the Evi/Wls deficient cohort  Fig. . SamplesGA) Fig.  Evi/Wls AXIN259, CCND1/cyclin D160, BRACHYURY61, PITX261 and MYC62 greatly accelerated the analysis of Wnt pathways during development and disease. To date, comparable target gene sets for non-canonical Wnt signaling are not known and their discovery opens new possibilities to investigate non-canonical signaling. Additionally, new non-canonical target genes might facilitate the identification of small molecule antagonists and agonists in β-catenin-independent Wnt signaling.Non-canonical Wnt signaling and its role in tumorigenesis still remains poorly understood. The signaling routes downstream of the receptors are complex; there is still a lack of cellular markers to assess the activity of this pathway. For canonical, β-catenin-dependent signaling, the identification of PLOD2, HADH, LCOR and REEP1. Our experiments showed that these target genes also depend on Dvl2 and RoR2 which is in line with current knowledge of the role of RoR2/Dvl2 in non-canonical Wnt signaling39.In this study, we used a perturbation and profiling approach to identify target genes that are directly or indirectly controlled by non-canonical, β-catenin-independent Wnt signaling in colon cancer cells. We used HCT116 cells as a model system that expressed endogenous canonical and non-canonical Wnt ligands to perform perturbations inhibiting either the secretion of Wnts or β-catenin signaling followed by expression profiling. Using an iterative signature algorithm, we identified several candidate genes that are dependent on autocrine, non-canonical Wnt signaling and validated their expression response in multiple independent cell models using both genetic and pharmacological approaches. Computational analysis revealed that several of the predicted non-canonical Wnt target genes contain ATF2 binding motifs in their promoter regions. In line with this, we showed that ATF2 as well as ATF4 led to downregulation of identified non-canonical target genes, including 64, which are required for multiple biological processes. Stabilization of GSK3β-target proteins could lead to a secondary effect on our identified target genes, as they are independent of β-catenin-mediated transcription. However, we found that GSK3β-inhibition did not affect the predicted non-canonical target genes thereby excluding Wnt-STOP dependent regulation.Other canonical but non-transcriptional pathways induced by Wnt ligands were recently discovered. For example, Wnt/STOP signaling stabilizes GSK3β-target proteins66. Wnt5a expression has also been shown to enhance resistance to BRAF inhibitors35. Recently, it was shown that in breast cancer Wnt5a inhibits proliferation of tumour-initiating cells via TGFβ/SMAD signaling. Furthermore, heterozygous loss of WNT5A correlated with shorter survival of breast cancer patients67. In addition, it was recently shown Wnt5a ability to promote breast cancer progression is also dependent on the cellular context68. In colon cancer, the current model suggests that Wnt5a inhibits canonical β-catenin dependent Wnt signaling via RoRα69. However, we showed that proliferation of colon cancer cells depends on Wnt5a/b independent from the canonical Wnt pathway. We further showed that Wnt5a/b as non-canonical ligands were responsible for regulation of these target genes in colon cancer cells. Although we identified Wnt5b as the main regulator of non-canonical Wnt target genes in HCT116 cells, we cannot exclude that other non-canonical Wnt ligands can substitute Wnt5a/b in other cellular systems.The role of Wnt5a in tumorigenesis is tissue specific. In melanoma, Wnt5a correlates with tumor progression and has been shown to play an important role in epithelial mesenchymal transition (EMT) and cell migrationet al.70 and Gujral, T. S. et al.71, which showed that Wnt5a is overexpressed in colon cancer.Since our experiments are based on low, endogenous levels of Wnt5a/b, we cannot exclude that high expression of Wnt5a/b could have an opposite effect. When Wnt5a is overexpressed, it can substitute canonical Wnt proteins by blocking their FZDs. The requirement of Wnt5a for proliferation also correlates with an upregulation of Wnt5a as shown by TCGA. This result is also consistent with observations made by Yoshida, N. In conclusion, we demonstrate how a systematic approach can be applied for the identification of target gene clusters, which expression is dependent on specific downstream signaling routes. Iterative signature algorithm predicted specific non-canonical, β-catenin independent target genes. Several of them were validated in different cellular models. Future studies on Wnt non-canonical pathway regulators and their interplay with the canonical pathway will be necessary to improve our knowledge on Wnt signaling in colon cancer. Our data may open new methods for the analysis of non-canonical Wnt signaling in cancer cells.All cell lines, except MEFs, were obtained from ATCC. HCT116, HT29 , L cells, HEK293T, MDA-MB231, MEF , DLD1  cells were cultured without antibiotics unless they were stably transduced. Media were supplemented with 10% fetal bovine serum (PAA). Primary colon cancer cell lines HROC40 and HROC69 were used on passage 28–34 and treated with 20 uM LGK974 for 120–144 h before analysis of Evi/Wls target gene expression. Cell lines were culture in DMEM supplemented with 20% fetal bovine serum (PAA). All cells were regularly tested for the mycoplasma contamination.cDNA was prepared from 0.5–1 µg total RNA, using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific) according to the manufacturer’s protocol (Thermo Fischer Scientific). cDNA was diluted to 5ng/µl and used for qPCR on the Lightcycler480 (Roche), using the universal probe library system (Roche) in a 384-well format. UBC was used as a reference gene for relative quantification. Oligonucleotide sequences are listed in Supplementary Table siRNAs were obtained from GE Dharmacon  or Ambion (Thermo Fischer Scientific). Cells were transfected using a total of 0.1% Lipofectamine RNAiMAX Transfection Reagent (Thermo Fischer Scientific). Sequences of siRNAs used in this paper are shown in Supplementary Table et al.49. The data set is available at the ArrayExpress database with accession number E-MTAB-651 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-651).The normalized RNAseq data of siRNA knockdowns  and normal state (Ctrl) in HCT116 cells are provided in Moffa c and tg, which determine how closely related are the genes (corresponds to tg) or conditions (corresponds to tc) within the transcription module. We used tc = 0.45 and tg = 1.75. Before starting the algorithm an initial set of genes has to be provided. The ISA will stepwise refine this input by finding the driving conditions for these genes and including/removing genes that are well/poorly co-expressed with the input set. The iterative manner of the algorithm will find a set of co-regulated genes by its self-consistency. Therefore we used 300 randomly generated gene sets as input and filtered duplicated outputs with a total gain of 23 unique transcription modules73. The script can be accessed at https://github.com/boutroslab/Supplemental-Material.The eisa package (v 1.12.0), part of the Bioconductor package suite, was employed to perform the biclustering. First, the RNAseq data was combined to a matrix, where the columns represent the conditions (siRNA knockdowns) and the rows the genes. Subsequently, genes were filtered with a variance greater than 0.5 and expression higher than 3 RPKM in at least 2 conditions. In the next step two normalized matrices were generated. Gene expression values were normalized to an average of 0 and a variance of 1, in one matrix across the columns (conditions), in the other across the rows (genes). The algorithm uses two user-defined thresholds, t74 colorectal carcinoma data set TCGA-COAD . Samples were divided into tumor samples and healthy samples according to their barcode as described in the TCGA Wiki . FPKM-normalized RNA sequence samples were log2-scaled. Statistical significance of gene expression differences between healthy tissue samples and tumor samples was determined using a two-sample Student’s t-test.The data from TCGAEVI/WLS mRNA expression over all samples were defined as high/low EVI/WLS expression. Differential gene expression between those samples was calculated with the multtest package (v 2.16.0) part of the Bioconductor suit. t-test was applied to determine p-values, which were corrected for multiple testing 75. The script can be accessed at https://github.com/boutroslab/Supplemental-Material.Samples with a greater/lower expression than at least one standard deviation (SD) from the median Supplementary information.Table S1Table S2Table S3


This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM.n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR.ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (p < 0.05).Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM. Wif1 is a member of the families of secreted molecules known to inhibit Wnt signalling activity. Wif1 was first identified as an expressed sequence tag from the human retina, and highly conserved orthologues have been isolated from mouse, Xenopus and zebrafish  and hypospadias-like phenotypes  are very common surgical disorders frequently encountered in pediatric surgery practice. The incidence is approximately one in 5,000 live births. There is a wide spectrum of ARM phenotypes, ranging from stenotic anus to cloacal malformation . Surgicaebrafish . β-catenebrafish . Previouenotypes . Both β-enotypes . These rMature Wistar rats  were provided by the Medical Animal Center, Shengjing Hospital of the China Medical University . Ethical approval was obtained from the China Medical University Animal Ethics (no. 200(7) PS14) prior to the study. Procedures for generating ARMs in fetal rats are described in earlier study . Seventyhttps://www.protocols.io/view/immunohistochemical-staining-kujcwun).The slides were treated and incubated with primary Anti-Wif1  and horseradish peroxidase (HRP)-conjugated secondary antibody . Antibody incubations were performed in phosphate-buffered saline (PBS) supplemented with 10% goat serum. Incubation with the secondary antibody was performed for 20 min at room temperature, and signals were visualized by using 3’3Pdiaminobenzidine . Two pathologists independently reviewed the immunohistochemical stained slides and agreed on results by consensus .Protein preparation was performed as described previously : the cloGAPDH was used as the reference gene as reported previously  according to the manufacturer’s protocol. RNA (1 µg) was reverse transcribed by using the Prime Script RT reagent kit  following the manufacturer’s instructions. Quantitative real-time RT-PCR was accomplished with SYBR Premix Ex Tap  on the 7900HT fast real-time PCR system  under the following conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 60 s. A dissociation procedure was performed to generate a melting curve for confirmation of amplification specificity. eviously . The relWif1 forward: 5′-AGCCATTCCCGTCAATATCCAC-3′; reverse: 5′-TGCCATGATGCCTTTATCCAG-3′.β-catenin forward: 5′-CGCTTGGCTGAACCGTCACA-3′; reverse:5′-TGGTCCTCGTCATTTAGCAGT-3′.GAPDH forward: 5′-GCTGGTCATCAACGGGAAA-3′; reverse:5′ -CGCCAGTAGACTCCACGACAT-3′.p < 0.05 was considered statistical significance.The Statistical Program for Social Sciences, version 13.0  was used for statistical analysis. The two-way ANOVA with Post-hoc test was used to compare the Wif1 and β-catenin protein and mRNA levels between the ARM and normal groups. All numerical data were presented a mean ± standard deviation. A value of In this study, no malformations were observed in the 288 embryos of the normal rats. Among the ETU-treated embryos, all 378 embryos had short or no tail and 19 of embryos died in utero. The incidence of ARMs in ETU-treated embryos was 81.0% (306/378). The embryos for immunohistochemistry staining, Western blot, and real time RT-PCR in each group are shown in On GD13, the cloaca was divided into urogenital sinus (UGS) ventrally and primitive hindgut dorsally by the L-shaped URS. Wif1-immunopositive cells were extensively expressed on the epithelium and mesenchyme of the cloaca , 1B.On GD14, a potential canal located between the tip of the URS and the cloacal membrane (CM). Wif1-immunopositive cells were detected on the hindgut, URS, urethra and CM , 2B.On GD15, the epithelium on the tip of the URS fused with the dorsal CM, leading to separation of the hindgut and UGS. The anal membrane (AM) was nearly ruptured. Wif1-immunopositive cells were mainly detected on the very thin AM , 3B.On GD16, the AM ruptured and the rectum separated from the UGS completely. The anorectum communicated with the outside. Wif1-immunolabeled cells were observed on the epithelium of the distal anorectum , 4B.On GD13, comparing with normal embryos, the distance between the URS and the CM was long, and the CM was shorter and thicker. Wif1-labeled cells were extensively expressed on the epithelium and mesenchyme of the cloaca , 1D.On GD14, the URS was high in the cloacal cavity, and the distance between URS and CM was relatively long. Wif1 was faintly expressed on the epithelium of the hindgut, URS and the urethra , 2D.On GD15, the distance between the URS and CM shortened, but the URS did not fused with the CM. The fistula between the rectum and urethra was evident, and the hindgut did not separate from UGS. Positive cells were sparsely located on the epithelium of the hindgut, fistula and the urethra , 3D.On GD16, the fistula between the rectum and urethra was existing, and rectal terminus was still not opened to the outside. Wif1 demonstrated low expression on the epithelium of the rectum, fistula and the urethra , 4D.p < 0.05).Western blot specific for Wif1 was performed to quantify protein expression in the anorectal development  . Wif1 anWif1 and β-catenin mRNA expression was calculated in the normal and ARM groups  . ± 0.09) . On GD14ETU-induced ARMs in rat embryos has been previously employed to study the morphological changes of ARMs by several groups, including our laboratory . In thisWif1 and β-catenin expression shows time-dependent changes during anorectal development. Western blot analysis and real time RT-PCR shown that, in the normal embryos, Wif1 and β-catenin expression was at its highest level at the key time-point of anorectal development (GD14 and GD15), suggesting that it may play an role in the development of the anorectum. However, Wif1 and β-catenin expression levels on GD14 and GD15 were significantly lower in the ARM group compared with the normal group, implying that downregulation of Wif1 and β-catenin expression during the critical period of anorectal development may contribute to the ARMs. Additionally, when the anus opened on GD16, the expression of Wif1 and β-catenin protein decreased. This suggest that Wif1 may play an essential role during initial morphogenesis of the anorectum, but its role during subsequent development of the anorectum may be less important.Wif1 is one member of Wnt antagonists, which bind to Wnt directly and inhibit the link with their receptors, and as a result, the accumulation of β-catenin is reduced and canonal and noncanonal pathway are inhibited . Ng et aETU is known to disturb the expression of the shh signaling pathway during the development of the hindgut . RC-L NgIn summary, the expression pattern of Wif1 and β-catenin was impaired during development of anorectum in fetal rats with ETU-induced ARMs. This indicates that Wif1 and β-catenin might play an important role in morphogenesis of the anorectum. Decreased Wif1 and β-catenin expression might be related to the development of ARMs. Further studies are needed to define the specific roles of Wif1 and β-catenin during anorectal development, and thus improve our understanding of the pathogenesis of ARMs.10.7717/peerj.4445/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4445/supp-2Supplemental Information 2Click here for additional data file.


We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3′-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes.Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs)  Primordial germ cells (PGCs), the precursors of sperm or oocytes, undergo numerous spatially and temporally specific regulatory pathways during early developmentPreviously, several studies reported a list of PGC specific genes by transcriptome analysis containing Wnt/β-catenin signaling-related genes4Drosophila815The Wnt pathway is a well-known signaling cascade that is involved in PGC development including specification, migration, and proliferation7891113in vitro.Several inhibitors, such as 6-bromoindirubin-3′-oxime (BIO) and JW74, have been utilized to activate or block such signaling. BIO, an inhibitor of the negative regulator GSK3β, leads to target gene expression by stabilizing β-catenin from the degradation complex11et al.et al.in vitro PGC proliferation24et al.27282931Another growth factor family involved in cell proliferation, migration, and differentiation during embryonic development is bFGF, also known as FGF219in vitro proliferation assay was performed in the presence of a Wnt ligand, BIO, JW74, or bFGF to verify the signaling mechanism underlying PGC proliferation in chickens. Lastly, to verify the crosstalk between bFGF and Wnt/β-catenin signaling in PGC proliferation, Western blotting and immunocytochemistry were used to examine the translocation of β-catenin into the nucleus, which was stimulated by bFGF after JW74 treatment.In this study, the expression of Wnt/β-catenin signaling-related genes was examined to determine whether PGCs are capable of activating this signaling pathway. BIO and JW74 were applied to modulate Wnt/β-catenin signaling in cultured chicken PGCs. Subsequently, an β-catenin, GSK3β, and TCFs (except for TCF3) were also expressed in gonadal stromal cells and chicken embryonic fibroblasts as well as gonadal PGCs and cultured PGCs, indicating that chicken PGCs might be capable of activating Wnt/β-catenin signaling , BIO or JW74, were applied at different concentrations, after which a cell proliferation assay was performed , indicating that bFGF helps PGCs to proliferate through direct activation of the downstream Wnt/β-catenin signaling pathway. Previous studies on the crosstalk between β-catenin and bFGF support our results. For example, in the absence of bFGF, β-catenin is only located in the membrane with E-cadherin, whereas it accumulates and is stably translocated into the nucleus to activate downstream genes in the presence of bFGF14272829In our study, the role of the Wnt/β-catenin signaling pathway was investigated for the first time in chicken PGCs, and the relationship between Wnt/β-catenin and bFGF was revealed. We concluded that Wnt/β-catenin can enhance the proliferation of chicken PGCs, and act as a factor downstream of bFGF signaling. Interestingly, in mouse, Wnt/β-catenin signaling is suppressed and deleterious for PGC proliferationThe care and experimental use of chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-150827-1). The chickens were maintained in accordance with a standard management program at the University Animal Farm, Seoul National University, South Korea. The procedures for animal management, reproduction, and embryo manipulation adhered to the standard operating protocols of our laboratory.in vitro culture, SNUhp26 male line cultured PGCs2 and 60–70% relative humidity. The cultured PGCs were subcultured onto mitomycin-inactivated murine embryonic fibroblasts in 5 to 6-day intervals by gentle pipetting without any enzyme treatment.To conduct gene expression analysis, PGCs from White Leghorn embryonic gonads at day 6 (stage 28) were isolated by magnetic activated cell sorting as previously reported® Safe (Invitrogen) and loaded for gel electrophoresis. Confirmation of the results was performed using the ChemiDoc XRS System . Downregulation of cell cycle- or germ cell-related genes at the mRNA level was analyzed using quantitative RT-PCR (qRT-PCR). Relative expression was calculated after the threshold cycle had been normalized to that of chicken glyceraldehyde 3-phosphate dehydrogenase.Total RNA was extracted from each sample with Trizol reagent (Invitrogen), in accordance with the manufacturer’s protocol. The RNA quantity was determined by spectrophotometry at 260 nm, and 1 μg of each RNA sample was reverse-transcribed with the Superscript III First-Strand Synthesis System (Invitrogen). The cDNA was diluted five-fold and quantitatively equalized for PCR amplification. RT-PCR was conducted to examine the change in expression of candidate genes in cultured chicken PGCs using specific primer sets . PCR ampTM Protein Extraction Reagent , separated on a 10% polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane (Millipore). The primary antibodies were mouse anti-β-actin  and rabbit anti-non-phospho (Active) β-catenin (Ser33/37.Thr41) . Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (Santa Cruz) were used as the secondary antibodies. The blots were treated with the enhanced chemiluminescence substrate solutions and exposed to a ChemiDoc XRS System (Bio-Rad) to detect chemiluminescence.Total protein was extracted with the CytoBusterSNUhp26 male line cultured PGCs, expressing GFP, were used for evaluating the rate of proliferation® adhesive microscope slides  for approximately 30 min. Then they were fixed in 100% methanol and prechilled for 3 min at −20 °C. After washing the cells three times for 10 min each in phosphate-buffered saline containing 1% normal goat serum, they were incubated in diluted anti-β-catenin antibody (mouse IgG) or anti-SSEA-1 (mouse IgM) antibody as a primary antibody at 4 °C for 24 h, and then incubated again in diluted Alexa-IgG-568 antibody or fluorescein isothiocyanate -conjugated goat anti-mouse IgM, respectively, as a secondary antibody for 1 h at room temperature. Samples were then mounted with Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen) and visualized using fluorescence microscopy.Cells were moved from the culture plate and dried on HistoBondP < 0.05.Statistical analysis was performed using SAS version 9.4 software . When a significant main effect was detected by analysis of variance using SAS, the least-squares analysis was used to compare the different treatments. Differences between control and treatment groups were deemed to be significant when How to cite this article: Lee, H. C. et al. Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro. Sci. Rep.6, 34510; doi: 10.1038/srep34510 (2016).


Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis. Chlamydia trachomatis genital infections represent the most common notifiable infectious disease in the United States with 1.5 million cases reported in 2015. In fact, C. trachomatis has been one of the most prevalent sexually transmitted pathogens in the United States for over two decades . Upon transmission to a new host, EB infect genital luminal and glandular epithelial cells, where they establish a modified endocytic vesicle called an inclusion. After inclusion formation, EB decondense to form larger, metabolically active, non-infectious forms called reticulate bodies (RB). RB undergo several rounds of division within the inclusion before converting into EB, which are released from the infected cell and disseminate to new host cells  and GSK-3β, potentially increasing β-catenin accumulation in the infected cell and expression of β-catenin-regulated anti-apoptotic genes  is that they are stimulated to proliferate, differentiate, and are shed in response to hormone-induced signaling events during the menstrual cycle. Interestingly, modulation of Wnt signaling pathways by sex hormones plays a role in this cyclic remodeling of the endometrium. .Ishikawa (IK), Hec-1B and an endometrial stromal cell line (SHT-290) were maintained as described previously  was overlaid on the IK monolayers. A low MOI of 0.03 IFU/cell was used for all studies so that the percentage of infected cells could be accurately determined. SHT-290 monolayers were overlaid with an equal volume of mock inoculum (2SPG) to prevent drying. The cultures were incubated for 1 h at 35°C before the inoculum was removed. The cultures were refed with the appropriate experimental medium containing DMSO or IWP2 and incubated for 48 h at 35°C before collection for various analyses. Alternatively, replicate Hec-1B and/or IK nonpolarized monolayers were exposed to IWP2 or 25 uM IWR-1 (Selleckchem) as described above or at 0, 24, or 36 h post Chlamydia infection (hpi). The chlamydia-infected, DMSO, IWP2 or IWR-1-exposed Hec-1B and/or IK monolayers were collected at 44 hpi. In duplicate experiments, samples were washed and refed with culture medium containing neither DMSO nor IWP2 at 44 hpi. These cultures were incubated for an additional 72 h at 35°C before collection. Inhibitor concentrations were chosen based upon the manufacture's and published literature in combination with preliminary dose response curves in which the viability of host cells was determined by monitoring cell growth and Live/Dead assay  cells were cultured on Matrigel-coated filters (Corning) until they reached 75% confluency. The IK cultures were placed into wells containing SHT-290 monolayers growing on the bottom of the well so that the IK and SHT-290 monolayers were in close proximity to each other, but not touching. Co-cultures were replenished with SHT-290 medium containing DMSO or 5 uM IWP2 (Tocris) and incubated at 37°C for 48 h. At this time, the medium was removed from the cultures and an inoculum of Infected IK or Hec-1B samples were fixed with methanol and stained for chlamydial inclusions using Pathfinder anti-chlamydia MOMP stain (BioRad). Cell nuclei were stained with DAPI (ThermoFisher). Stained coverslips and filters were mounted on glass slides. Chlamydial inclusions and nuclei were visualized on a Ziess Axiovert Discovery Microscope at 400x magnification. In each experiment, the number of inclusions and nuclei were counted in 15 random fields from triplicate samples and the average percentage of infected cells was calculated per condition.Chlamydia trachomatis-infected monolayers were scraped into the culture medium and frozen at −80°C. EB released from the cells by freeze/thaw and sonication were diluted in culture medium and used to infect HeLa 229 monolayers grown on coverslips by spin infection . Triplicate infected monolayers were incubated for 48 h at 35°C before methanol fixation and staining with Pathfinder anti-chlamydia MOMP stain (BioRad). Total inclusions were counted on each coverslip using a Ziess Axiovert Discovery Microscope at 200x magnification. The average number of inclusion forming units (IFU)/ml was calculated per sample and experimental condition.Triplicate chlamydia-infected IK and Hec-1B nonpolarized monolayers grown on coverslips or polarized IK monolayers from IK/SHT-290 co-cultures were stained with Pathfinder anti-chlamydia MOMP stain (BioRad). Five images of random fields were taken of each sample at 630x magnification using a Ziess Axiovert Discovery Microscope. The area of 10–12 inclusions/sample was measured using Zen Professional Imaging Software (Ziess). The average inclusion size was determined for each experimental condition.Ishikawa (IK) cultures were exposed to DMSO or IWP2 for a period of 1–4 days at 35°C. Cell death in the cultures was assessed using the Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit (Molecular Probes) according to the manufacturer's instructions.Infected IK monolayers from IK/SHT-290 co-cultures exposed to DMSO or IWP2 were stained for percent infectivity analysis as described above. Alternatively, IK and Hec-1B nonpolarized monolayers were stained with anti-β-catenin , and anti-chlamydial MOMP (Meridian LifeSciences) primary antibodies followed by staining with rabbit-anti-mouse Alexa Fluor 488 (Molecular Probes), rabbit-anti-goat Alexa Fluor 594 (Molecular Probes) secondary antibodies, and DAPI (ThermoFisher) stain. Confocal images of chlamydial inclusions were captured on a Leica TCS SP8 with Leica LASX software at 630x or 1,000x magnification. Imaris imaging and Leica LASX software were used to analyze the images. All images used in intensity measurements were taken at a standard exposure. Digital slices representing the center depth of 24 inclusions/condition were used to determine the intensity of β-catenin staining inside the inclusion. The intensity of β-catenin staining within a region of the host cell equivalent to the area of each inclusion was determined to assess the ratio of β-catenin staining inside the inclusion compared to the cell cytoplasm and/or nucleus.Infected IK monolayers from IK/SHT-290 co-cultures exposed to DMSO or IWP2 were fixed with glutaraldehyde in 0.1 M Cacodylate buffer (EM Sciences) for 24 h. The monolayers were then collected, agar enrobed, and stained with osmium tetroxide and uranyl acetate before alcohol and propylene oxide dehydration. Dehydrated samples were embedded in EPON  embedding medium  staining and Western Blot analysis as previously described . Means were compared by Analysis of Variance (ANOVA) and independent two-sample C. trachomatis infection. Replicate samples were harvested at 44 hpi for chlamydial infectivity, EB titer analysis, inclusion size measurements and Western blot analysis of β-catenin accumulation. In a separate study, Live/Dead assays revealed that prolonged exposure to 5 uM IWP2 did not increase IK cell death significantly compared to the DMSO control (data not shown). Chlamydial infectivity was lowered in the presence of Wnt inhibition compared to DMSO exposure, although not to a significant degree . Inhibition of Wnt by IWP2 significantly decreased the production of infectious EB during IK/SHT-290 co-culture . Inclusion size was also significantly decreased in cultures exposed to IWP2 compared to DMSO . Accumulation of β-catenin was significantly decreased in SHT-290 cells (p = 0.04), but not in IK cells (p = 0.60) from infected IK/SHT-290 co-cultures /stromal (SHT-290) cell co-culture model. This physiologically relevant co-culture model incorporates paracrine regulatory signaling events that occur between endometrial stromal and epithelial cells  before collection for EB titer analysis. Compared to DMSO-exposed controls, chlamydial infectivity was significantly decreased in IK cultures when exposed to IWP2 before infection (p < 0.001). However, addition of the Wnt inhibitor at times after infection did not significantly impact chlamydial inclusion development in IK cells  chlamydial infection; however, Wnt inhibition had no significant effect on continued inclusion development when IWP2 was added to Hec-1B cultures at 24 or 36 hpi . Inclusion size in Hec-1B cells was also decreased in response to Wnt inhibition, but not to a significant degree . EB production was suppressed in both IK . Inclusion size in IWP2-exposed samples was increased at 72 h R compared to 44 hpi . Overall, these data indicate that inhibition of Wnt signaling in nonpolarized endometrial epithelial cells severely inhibits chlamydial inclusion development and infectious progeny production. Importantly, the detrimental effects of Wnt inhibition on C. trachomatis was evident no matter if the inhibition occurred before infection, or during early, mid, or late infection, indicating that Wnt signaling is important at every stage of the chlamydial intracellular development cycle.Confocal microscopy examination of fluorescently labeled β-catenin in DMSO or IWP2-exposed, chlamydia-infected IK or Hec-1B cultures revealed that β-catenin localized to chlamydial inclusions under both experimental conditions Figures . AlthougC. trachomatis infection. Samples were harvested for analysis of infectivity and EB production at 44 hpi. As shown in Figures C. trachomatis percent infectivity (p = 0.01) and the production of infectious EB (p = 0.001) from the cultures. These data confirm the importance of Wnt/β-catenin signaling on chlamydial infection in endometrial cells.To confirm that the observed effects of Wnt inhibition on chlamydial infection were not limited to IWP2 exposure, we examined the effect of another Wnt inhibitor, IWR-1, on chlamydial infection in endometrial epithelial cells. IWR-1 is a small molecule that inhibits canonical Wnt activity via stabilization of the β-catenin destruction complexes (Chen et al., C. trachomatis may enter a persistent state or be killed when Wnt signals are inhibited in the host cell. Overall these data lead us to conclude that Wnt signaling is a key host pathway for the proper development of C. trachomatis in the endometrium. Our observations support previous work that indicate activation of Wnt signaling and/or β-catenin is important for chlamydial development in other areas of the FGT, including the fallopian tubes and cervical cells (Prozialeck et al., C. trachomatis infections throughout the entire tract.Here, we have demonstrated that Wnt signaling is an essential pathway for chlamydial development in endometrial epithelial cells. Inhibition of Wnt significantly decreases chlamydial inclusion development, causing decreased percent infectivity and inclusion size. Furthermore, RB that formed during Wnt inhibition were aberrant in morphology. Inhibition of Wnt also caused a dramatic decrease in EB formation regardless of the time that Wnt was inhibited during the developmental cycle. Recovery of progeny formation following the removal of the Wnt inhibitor was only observed when the Wnt inhibitor was added late during chlamydial development. These data indicate that C. trachomatis infection, we have demonstrated that estrogen increases chlamydial development whereas progesterone negatively impacts C. trachomatis infection by unknown molecular mechanisms. These studies revealed the paracrine stromal cell signaling molecules released in response to hormones play a role in hormone-induced effects on chlamydial development (Hall et al., Previous research has demonstrated that female sex hormones, estrogen and progesterone, can influence chlamydial development (Rank et al., C. trachomatis infection. The demonstrated effects of Wnt/β-catenin signaling on chlamydial development could be attributed to hormonal modulation of Wnt signaling. For example, Kessler et al. proposed that Chlamydia-activated Wnt/β-catenin signaling in fallopian tube epithelia altered host cell adhesion and polarity (Kessler et al., C. trachomatis-infected endometrial epithelial cells will further illuminate the importance of this intricate and complex system to chlamydial growth and pathogenesis.Given that Wnt signaling is important to chlamydial development in the endometrium and that hormones naturally regulate Wnt signaling as a part of the menstrual cycle, it is intriguing to hypothesize that hormonal regulation of Wnt signaling may contribute to the impact of estrogen and progesterone on JK, CM, JW, MB, and JH contributed substantially to the design, execution, and/or data collection/analysis of the experiments within this study. JH drafted the manuscript. All authors contributed to revision and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. Wnt signalling governs cell fate and proliferation during embryonic development and plays important roles in adult tissue homeostasis1Aberrant Wnt/β-catenin signalling drives oncogenesis in several human cancers with mutations in core signalling components, such as APC and CTNNB1 (β-catenin), driving constitutive pathway activation44KIF3A belongs to the kinesin family of proteins that function as a molecular motor transporting cargo along microtubules. KIF3A is best known for its role in molecule transport along the axoneme of cilia, and loss of KIF3A causes defects in cilium biogenesis7Further complexity in the relationship between KIF3A and Wnt signalling comes from the finding that primary cilia are involved in the regulation of the Wnt response. Ablation of primary cilia via deletion of ciliary or basal body genes other than KIF3A causes hyperactivation of Wnt/β-catenin signalling in response to Wnt3a stimulation1012In our current study, we reveal a tumour suppressor role for KIF3A as an inhibitor of the Wnt/β-catenin pathway in NSCLC cells. We provide evidence that KIF3A inhibits Wnt signalling through interaction with β-arrestin, as a non-ciliary mechanism. Importantly, KIF3A loss was correlated with increased expression of both β-catenin and cyclin D1 in clinical NSCLC samples and also associated with worse patient survival. Our results indicate that KIF3A is a class of tumour suppressor in NSCLC.To investigate the role of KIF3A in Wnt pathway regulation in lung cancer, we established A549 and SW900 NSCLC cell lines stably expressing small hairpin RNAs (shRNAs) specific for KIF3A. Two distinct KIF3A shRNAs were used to minimize sequence-dependent off-target effects, and cell lines harbouring non-target shRNA (NT-sh) were also generated. Our real-time RT-PCR and western blot analyses showed that both KIF3A shRNA1 and shRNA2 decreased the level of KIF3A mRNAs and proteins . Next, wNext to test if KIF3A knockdown affects β-catenin–dependent transcriptional activity, we performed a SuperTOPFlash reporter assay. As expected, KIF3A knockdown increased the transcriptional activity of β-catenin . Upregul16Cancer stem-like cells (CSCs), also called tumour-initiating cells, are a subset of tumour cells characterized by a capacity for self-renewal and high tumourigenic potential. NANOG, OCT4, and SOX2, which are transcription factors essential for maintaining the pluripotency of normal stem cells, control tumour initiation and malignancy in several types of cancers, including lung adenocarcinoma18Next, to demonstrate the tumourigenicity of KIF3A-depleted cells, we implanted the cells into nude mice through subcutaneous injection, and measured the tumour volume. Although injected control SW900 cells formed a tumour mass, the tumours did not grow in size for at least 1 month . In contActivation of the Wnt/β-catenin pathway is likely to underlie the CSC-like malignant properties observed in KIF3A-depleted cells. To confirm this idea, we investigated the effect of the Wnt pathway inhibitor ICG-001 on cell proliferation and stemness marker expression. ICG-001 blocks the interaction of CREB-binding protein with β-catenin and thus inhibits the transcriptional co-activator function of β-cateninAlthough there are some contradictory results in the literature121314222325To further demonstrate cilium-independent regulation of Wnt signalling in lung cancer cells, we examined the effect of HPI4, a small molecule inhibitor of ciliogenesisAutocrine signalling has been shown to contribute to the activation of the Wnt/β-catenin pathway in NSCLCWe next investigated the mechanism of KIF3A-mediated inhibition of Wnt signal transduction. Previous proteomic analyses revealed that KIF3A physically interacts with β-arrestin30β-arrestin forms a ternary complex with DVL and axin and thus plays a role in the inactivation of the β-catenin destruction complex30in vitro data, there was an inverse correlation between KIF3A and β-catenin expression in the NSCLC samples  according to the estimated KIF3A expression levels in the tumour cells compared with the expression in normal bronchial epithelial cells . Next, w samples . We examsamples 4. These rsamples 4. Next, wsamples 4. This dasamples 4. Taken tDeregulated expression of Wnt pathway components are frequently observed in NSCLCs32Growing evidence supports the involvement of stemness pathways in tumour progression, metastasis, and drug resistance2034428Primary cilia can influence multiple signalling pathways that are implicated in cancer, including the Wnt13393943444610The arrestin family of proteins has been identified as mediators of internalization and downregulation of GPCRs. In addition, arrestins can interact with other classes of cell surface receptors and also function as intracellular scaffolds and signalling intermediatesA previous study using genetic modification of β-catenin showed that stabilization of β-catenin alone in the bronchiolar epithelium of the adult mouse lung does not promote tumour development, but dramatically accelerates lung tumorigenesis induced by a constitutively active Kras mutationIn conclusion, the KIF3A/β-arrestin complex restrains Wnt/β-catenin pathway activation. Moreover, KIF3A is a potential tumour suppressor in NSCLC, suppressing both autocrine Wnt/β-catenin signalling and stem cell-like properties. Importantly, the correlation between low KIF3A expression levels and a reduced overall survival of NSCLC patients indicates that the use of KIF3A as a prognostic marker would be worth exploring in the future.The lung adenocarcinoma cell line A549 and lung squamous cell carcinoma cell line SW900 were purchased from American Type Culture Collection . Cells were cultured as previously describedKIF3A and non-target (NT) shRNA constructs were generated by Openbiosystem . The lentiviral particles expressing NT-shRNA, KIF3A shRNA1, and KIF3A shRNA2 were produced in HEK293T cells using Lenti-X™ Lentiviral Expression Systems . A549 and SW900 were transduced with Lenti-X Viruses following the manufacturer’s instructions. Knockdown efficiency was determined by quantitative RT-PCR as well as western blotting 48 h after transduction. Cells harboring NT-sh or KIF3A shRNAs were selected in the media containing 2 μg/ml puromycin .Total RNA was extracted by Trizol  and the RNA samples were treated with DNase . The samples were used to generate cDNA using a High Capacity cDNA Reverse Transcription Kit . Target gene-expression levels were quantified using target specific primers. The sequence information used for RT-PCR and quantitative RT-PCR is presented in Harvested cultured cells were lysed in lysis buffer  containing phosphatase inhibitor cocktail C . The concentration of proteins in cell lysates was quantified by the Enhanced BCA Protein Assay Kit  and 25 μg of proteins was loaded in each lane. Western blotting was performed as previously described4 cells per well for 24 h. β-catenin was detected by anti-β-catenin . For analysis of primary cilia, cells were cultured in 8-well Lab-Tek II chamber slides. Primary cilia were detected by anti-ARL13B  and poly-glutamylated Tubulin .The samples were fixed in 4% PFA for 15 min, permeated with 0.5% TritonX-100/PBS for 5 min, and blocked with 3% bovine serum albumin/PBS. They were incubated with primary antibodies overnight at 4 °C. Primary antibodies were detected with Alexa Fluor 488- and 594-conjugated secondary antibodies  for 1 h at room temperature. Nuclei were counterstained with DAPI  for 15 min and imaging was visualized on Carl Zeiss fluorescence microscope . To investigate the localization of β-catenin, cells were grown on 8-well Leb-tek II chamber slides  at 2 × 104 cells per well. The cells were incubated for 24 h, and lysed to measure luciferase reporter gene expression by Luciferase Assay System . Firefly luciferase activity was normalized to Renilla luciferase activity. All results are expressed as a mean ± SEM of independent quintuplicate cultures. To test the secretion of WNTs from KIF3A shRNA cells, HEK293T cells were seed in 96-well plates at 2 × 104 cells per well. Cells were transfected with either TOPFlash or FOPFlash (100 ng) and the internal control plasmid pRL-TK  using ViaFect™ Transfection Reagent (Promega). The next day, the medium was changed to growth medium or conditioned medium from KIF3A knockdown cells treated without or with IWP-2. After 48 hr, WNT3A was treated. The next day, luciferase reporter gene expression was measured by Luciferase Assay System. All results are expressed as a mean ± SEM of independent quintuplicate cultures.Wnt signalling activation was measured by transfection with SuperTOPFlash reporter plasmids . Cells were transfected with either TOPFlash or FOPFlash (100 ng) and the internal control plasmid pRL–TK , using ViaFect™ Transfection Reagent (Promega) in 96-well plates at 2 × 103 cells per well. CCK-8 was added to each well, and cells were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm.Cell proliferation was monitored every 24 h for 4 days using a Cell Counting Kit-8 (CCK-8) assay . Cells were plated in 96-well plates at 3 × 10Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan Medical Center, Korea. To generate xenograft tumour models, 100 μl of SW900 cells transduced with either NT-sh or KIF3A sh2 (6 × 106 cells/100 μl) mixed in Matrigel  were subcutaneously injected into BALB/cA-nu nude mice (6 weeks old). After 12 days, tumour growth was measured using calipers. The estimated volume was calculated according to the formula: Tumour volume (mm3) = 0.5 × length × width2.The entire experimental protocol was conducted in compliance with the institutional guidelines and approved by 4 cells/ml in serum-free medium  supplemented with 20 ng/ml of basic fibroblast growth factor , 50 ng/ml human epidermal growth factor , N2 (Invitrogen), B27 (Invitrogen), 10 μM Rock inhibitor (Enzo life science) and 1% penicillin/streptomycin (Gibco). Culture medium was replaced twice a week.To obtain tumour spheres in culture, cells were plated in ultra-low attachment dishes (Corning) at a density of 1 × 104 cells/200 μl, SW900: 1 × 105 cells/200 μl) were pipetted into the top chambers containing RPMI 1640 without FBS. After 24 h incubation, non-migrating cells were removed from the upper face of the filter using cotton swabs and cells on the lower filter surface were fixed in 4% PFA for 15 min. After washed using PBS, the cells were stained with haematoxylin (Sigma). The number of cells per microscopic field was counted light microscopically. Migration capacity was measured by OD value and the average OD value of migrating cells within five random fields was calculated.Transwell chambers (Corning) were used according to the manufacturer’s instructions. Briefly, RPMI 1640 containing 10% FBS was added to each bottom chamber. Then, the top chambers, which have uncoated transwell membrane filter inserts , were placed in each well of a 24 well culture plate (Thermo Fisher Scientific). Cells  containing phosphatase inhibitor cocktail C (Santa Cruz). The concentration of proteins in cell lysates was quantified by the Enhanced BCA Protein Assay Kit . Each 5 ul antibody for β-arrestin  or axin  per 1 mg lysate was incubated overnight at 4 °C with constant rotation. The next day, a protein G sepharose  bead was added to each lysate for 4 h at 4 °C with constant rotation. Unbound proteins were removed by four washes using lysis buffer and proteins were eluted from the beads by the addition of 40 μl sample buffer. SDS-PAGE was used to analyze 20 μl samples.Samples were formalin-fixed, paraffin-embedded, sectioned, and stained with following primary antibodies as previously describedthe Ethics Committee of Asan Medical Center, Korea. Surgical samples were collected from consenting patients at the Asan Medical Center. All subjects approved the “informed consent” to the study. All patients were first diagnosed with primary NSCLC and showed metastasis. Samples were shipped to the laboratory in cold Hank’s Balanced Salt Solution (HBSS) with antibiotics (Lonza) within 1 h of removal from patients. Samples were washed with cold HBSS with antibiotics three times, chopped with a sterile blade, and incubated in 0.001% DNase (Sigma-Aldrich), 1 mg/ml collagenase/dispase , 200 U/ml penicillin, 200 mg/ml streptomycin, and 0.5 mg/ml amphotericin B  in DMEM/F12 medium (Lonza) at 37 °C for 2 h with intermittent shaking. After incubation, the suspensions were repeatedly triturated and passed through 70-μm cell strainers . The strained cells were centrifuged at 1000 rpm for 3 min. Cells were seeded in Collagen-I coated dishes (Corning) and cultured in serum-free medium  supplemented with 20 ng/ml of basic fibroblast growth factor (Invitrogen), 50 ng/ml human epidermal growth factor (Invitrogen), N2 (Invitrogen), B27 (Invitrogen), 10 μM ROCK inhibitor (Enzo Life Sciences), and 1% penicillin/streptomycin (Gibco).The entire experimental protocol was conducted in compliance with the institutional guidelines. The study protocol was approved by P < 0.05 was considered to indicate a statistically significant difference.Data are mainly expressed as a mean ± SEM from at least three experiments. Statistical analysis was performed with SPSS and graphs were designed using Graph Pad Prism 5. How to cite this article: Kim, M. et al. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer. Sci. Rep.6, 32770; doi: 10.1038/srep32770 (2016).


The arginine methyltransferase PRMT5 has been increasingly associated with cancer development. Here we describe our recent findings that PRMT5 is a critical regulator of breast cancer stem cell survival via the epigenetic regulation of FOXP1. Consequently, PRMT5 inhibitors could potentially eradicate cancer stem cells thereby preventing tumour relapse. Consequently, targeting enzymes that catalyse and remove protein modifications are attractive strategies enabling rational drug design. Arginine methylation, catalysed by protein arginine methyltransferases (PRMTs), was identified over 45 years ago, however the significance of this modification for oncogenesis and malignant progression is only just becoming apparent.,We have recently extended our understanding into the oncogenic nature of PRMT5 through the study of breast cancer stem cells (BCSCs).in vivo, implying that PRMT5 is crucial for driving function. Importantly, to mimic a patient presenting with disease, we depleted PRMT5 in an established tumour xenograft which led to a substantial 12-fold decrease in stem cell numbers as assessed by limiting dilution. Furthermore, we found that treatment of BCSCs isolated from resected primary tumours with a pre-clinical PRMT5 inhibitor significantly reduced BCSC numbers. Collectively, this demonstrates a functional requirement for PRMT5 for the generation and maintenance of the BCSC population and, more importantly, the potential clinical impact of targeting PRMT5.In our study, we found that PRMT5 levels were significantly elevated in BCSCs compared to bulk differentiated cells, and depletion of PRMT5 reduced the number, proliferative and self-renewal potential of BCSCs. Conversely, overexpression of PRMT5 and its essential cofactor MEP50 increased stem cell frequency FOXP1, a gene which is associated with cancer stem cell function,,PRMT5 is a major epigenetic regulator of gene expression, therefore, to gain mechanistic insight we compared the transcriptome of BCSCs before and after PRMT5 depletion. Interestingly, Wnt/β-catenin genes that had previously been reported as PRMT5 targets in leukaemic stem cellsThe success of current therapies for breast cancer remains somewhat marred by poor long-term survival rates due to relapse and metastasis. One major goal for researchers is to prevent relapse by designing therapies to target the hard-to-eliminate cancer stem cells either through eradication of BCSCs or by rendering them more chemosensitive through inducing differentiation. Our study demonstrating that PRMT5 depletion in an established tumour can reduce stem cell frequency is highly significant for the patient, as targeting this enzyme in conjunction with standard chemotherapies, could lead to eradication of all tumours cells, thus preventing relapse. However, further questions remain. We need to fully understand the roles of PRMT5 in this cell type to fully exploit the clinical potential of drug targeting. Although here we describe a role for PRMT5 in the epigenetic regulation of FOXP1, given the pleiotropic roles of this enzyme, it is unlikely to be the only way in which PRMT5 regulates BCSCs. For example, PRMT5 regulates the DNA damage response,There is a lot of promise and excitement surrounding epigenetic therapies, and clinical PRMT5 inhibitors have entered phase one trials for leukaemia treatment. Our findings provide compelling evidence that drug targeting PRMT5 could have significant clinical benefit in solid cancers enabling the complete eradication of the tumour-initiating population to improve long-term patient survival.