Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with β-catenin to promote cell proliferation.The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/β-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/β-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and β-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of  Axin2, Jun, Myc, Ccnd1, and Sp5 (Specificity protein transcription factor 5) [The Wnt signaling pathway regulates a variety of processes, including cell proliferation and differentiation . Brieflyactor 5) . It has actor 5) , includiactor 5) , breast actor 5) , and HPVactor 5) ,9,10.The persistent infection with high risk human papillomavirus (HR-HPV) is the main risk factor associated to cervical cancer development . HPV-16 The E6–E7 open reading frames (ORFs) contain spliced donor and acceptor sites, highly conserved among the HR-HPV. Those sites are recognized by the spliceosome complex, promoting the removal of a small intron and the generation of a premature stop codon, giving place to short forms of E6, termed E6*. In HPV-16, at least four isoforms of E6* (I–IV) have been identified, whereas in HPV-18, only one has been reported hitherto, termed E6*I . AlthougThe abnormal activation of the Wnt cell signaling pathway has been reported in HPV-related tumors ,19,20. ISp5 gene promoter, in vivo and in vitro. Furthermore, proliferation induced by β-catenin is enhanced by E6 and E6*I proteins. Finally, although E6 intratype variants differentially affected TCF-4 transcriptional activation, no differences appeared in their ability to bind TCF-4 or β-catenin.No interactions of HPV-18 E6 protein with members of the Wnt activation complex  have been identified so far, and the mechanisms by which E6 induces TCF transcriptional activation are poorly understood. Moreover, the effect of E6* proteins in this pathway remains unknown. Therefore, the aim of this study was to investigate the role of HPV-18 E6 and E6*I proteins in the Wnt/β-catenin signaling regulation. Through a TCF-4-dependent luciferase reporter plasmid, we show that E6 and E6*I up-regulate TCF-4 transcriptional activity, which is enhanced with the expression of exogenous β-catenin. Moreover, Wnt target genes are overexpressed in E6 and E6*I transfected cells. We also found that E6 and E6*I increase β-catenin and TCF-4 protein levels, but they do not alter their subcellular distribution. Immunoprecipitation and pull-down assays revealed an interaction of TCF-4 and β-catenin with E6 and E6*I proteins and those interactions impact in TCF-4 stabilization. We found that E6 and E6*I interact with the C33A cells were transiently transfected with a TCF-4-dependent luciferase reporter plasmid (TOPFLASH) and FLAG-tagged versions of 18E6WT, 18E6SM, 18E6*I, or 16E6 expressing plasmids. All experiments were performed co-transfecting the empty vector (p3X) or the β-catenin expressing plasmid, as indicated. After 48 h of transfection, immunoblot assays were performed for each experiment, confirming the expression of FLAG-tagged E6 proteins A. It is Axin2 and Cyclin D1, evaluated by qPCR assay. As observed in Cyclin D1 , while Axin2 reached up to a 1.5-fold increase; although 18E6*I had no effect on Axin2 expression. Taken together, these findings suggest that HPV-18 E6 and E6*I cooperate in the activation of the canonical Wnt/β-catenin cell signaling.Overexpression of E6 proteins also stimulate native promoters containing TCF-4 responsive elements, as evidenced by the expression of the Wnt target genes, To determine the effect of E6 proteins on β-catenin and TCF-4 levels, we performed immunoblot assays using total cell lysates. We observed that both E6 and E6*I proteins significantly increase β-catenin A,B and TPrevious reports demonstrate that HPV-16 E6 interacts with members of the Wnt signaling pathway, such as Dvl2 (Dishevelled Segment Polarity Protein 2) . To inveThese results were further confirmed by GST pull down assays using C33A lysates and GST-E6 fusion proteins C, where To further determine the effect of the interaction of HPV-18 E6 and E6*I with TCF-4, we evaluated the TCF-4 stability through half-life determination assay. C33A cells were transfected with E6 or E6*I expressing plasmids, and 48 h post-transfection, cells were treated with 200 µg/mL of cycloheximide and the TCF-4 degradation rate was evaluated at 0, 6, and 12 h post-treatment. Overtime, it was observed that TCF-4 protein levels decreased considerably in E6 non-transfected cells after 6 and 12 h A,B. InteTo further determine the effect of E6 proteins on nuclear TCF-4 levels, soluble cellular fractionation was performed. As is shown in Sp5 expression was analyzed by qPCR. Sp5 mRNA levels in C33A cells co-transfected with β-catenin. Therefore, we further analyzed whether E6 proteins could interact with the Sp5 promoter, which is TCF-4-dependent. It was previously demonstrated that TCF-4 binds to a conserved sequence, A-C/G-A/T-T-C-A-A-A-G, found in TCF-4/β-catenin-dependent promoters, such as the Sp5 gene promoter [Sp5 promoter sequence flanking the -326 to -129 nucleotides, since this region contains two TCF-4 binding sites located at nucleotides, -303 to -296, and -149 to -142. Those binding sites were confirmed when the fragment was analyzed in silico using the informatics tool, Tfsitescan [Sp5 promoter . The mutation in the 18E6SM plasmid allows the expression of higher amounts of E6 full-length, although some E6*I is still produced since another donor site not yet described could be used during the splicing process. When transfecting this plasmid there was a slight increment in the TCF-4 transcriptional regulation, although non-significant, in relation to the control vector. Interestingly, when the Wnt pathway was over activated with the co-transfection of β-catenin, TCF-4 transcriptional activation was enhanced in the presence of E6 or E6*I proteins, supporting that E6*I and E6 collaborate in Wnt/β-catenin pathway activation. We also found that 16E6 induced a higher response in TCF-4 activation, reaching up to a 2.5-fold induction, and this effect was slightly enhanced in the presence of exogenous β-catenin (3-fold). Our results are in agreement with previous reports, where three-fold-induction of the TCF-4 luciferase reporter was obtained in HEK293T cells ectopically expressing HPV-16 E6, the Wnt receptor HFz1, and the Wnt3a ligand . AdditioCyclin D1, Axin2, and Sp5 expression, in agreement with a previous report where E6 from HPV-16 was able to activate the Cyclin D1 promoter [Cyclin D1 was also up-regulated [The expression of TCF-4 target genes was evaluated in the presence of E6 and E6*I. In HPV-18, E6WT and E6SM expressing cells increased promoter . Moreoveegulated .Axin2 is considered a tumor suppressor and mutations within this gene are associated to cancer development [Cyclin D1 expression, it failed in promoting an up-regulation of Axin2. It is possible that E6 recruits co-regulators that are distinct to those recruited by E6*I that could be necessary for Axin2 expression.Among the Wnt target genes, elopment ,40. Axinelopment ,42. It iIt has been previously demonstrated that HPV-16 E6 induces TCF-4 transcriptional activation without affecting β-catenin localization . ConsistWe demonstrate that E6 and E6*I from HPV-18 not only interact with TCF-4, but are also able to induce the TCF-4 stabilization. Further studies are needed to elucidate whether the interaction of TCF-4 with E6 proteins is responsible for TCF-4 stabilization. Previous studies have shown that HPV-16 E6 together with the E3 ubiquitin ligase E6AP, and induce stabilization of other members of the canonical Wnt pathway, such as β-catenin, impacting in the activation of the pathway . Sp5 promoter or other TCF-4 response promoters. Additionally, in concordance with previous studies [Furthermore, it is well known that when Wnt signaling is activated, β-catenin complexes with TCF-4 in the nucleus, inducing the TCF-4 transcriptional response . Our fin studies ,45, we fAxin2 and Cyclin D1, with no differences among them. This effect could be due to a distinct capacity of E6 variants to regulate or interact with other untested proteins involved in the activation of the Wnt signaling pathway. However, we observed that both E6 variants interact with TCF-4 and β-catenin in a similar manner, which reveals that aminoacidic changes in E6 variants do not influence at least in such a binding capacity.Since HPV intratype variations have been proposed to affect the HPV biological behavior, we were also interested in determining if HPV-18 E6 variants could differentially affect the Wnt/β-catenin signaling pathway. HPV intratype variants are defined as those containing less than 1% of nucleotide changes in coding regions ,47. Our In this study, we demonstrated that not only E6, but also E6*I from HPV-18 are able to up-regulate Wnt/β-catenin signaling, involving their interaction with the TCF-4 activation complex. Additional effects on members of the Wnt pathway should be analyzed in order to determine the specific contribution of E6 and the spliced isoform E6*I in cell transformation induced by the Wnt/β-catenin pathway.2. Transfections were performed using Lipofectamine 2000 reagent  according to the manufacturer’s instructions.C33A epithelial cells were acquired from ATCC and HaCaT were kindly provided by A. García-Carrancá  and were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) in a humidified incubator with 5% CO18E6WT Open Reading Frame (ORF) was obtained and PCR-amplified from an HPV-18 positive cervical cancer biopsy, 18E6*I ORF was obtained by RT-PCR amplification from HeLa cells (HPV-18 positive), and 16E6 ORF was amplified by PCR from CaSki cells (HPV-16 positive). The HPV-18 E6 spliced mutant (18E6SM) sequence was amplified from the plasmid, pCAHPV18-E6sm . 18E6SM C33A cells were seeded in a 24 well plate and transfected with a mix containing 50 ng of appropriate E6 expressing plasmid, 100 ng of TOPFLASH, and 1 ng of β-galactosidase reporter plasmid, either with 50 ng of empty vector or β-catenin, as indicated. Cell extracts were obtained 48 h post-transfection and assayed for luciferase and β-galactosidase activities  using a Glomax 96-well plate luminometer  The presented data are shown as relative luciferase readouts comparing the E6 expressing cells vs the control vector, where luciferase readouts in empty vector condition were adjusted to 1 after normalizing with β-galactosidase activity. At least three independent experiments were performed, each by triplicate.Cyclin D1 amplification, forward 5′-ACAAACAGATCATCCGCAAACAC-3′ and reverse 5′-TGTTGGGGCTCCTCAGGTTC-3′ primers were used. For Sp5 amplification, forward 5’-TCGGACATAGGGACCCAGTT-3′ and reverse 5′-CTGACGGTGGGAACGGTTTA-3′. As a house keeping control, 18S mRNA was amplified with forward 5’-AACCCGTTGAACCCATT-3′ and reverse 5’-CCATCCAATCGGTAGTAGCG-3′ primers. SYBER select Master Mix  was utilized for qPCR reactions. For Axin2 amplification, Taqman probes were used : Axin2 FAM (Hs00610344_m1) and 18S VIC (Hs99999901_s1) probes, with Taqman Gene Expression Master Mix for qPCR analysis . The results are presented as relative quantification using the CtΔΔ method.C33A cells were seeded in a 60 mm culture dish and transfected with 3 µg of each E6 plasmid. After 48 h post-transfection, cells were collected, and total RNA extraction was performed using the RNeasy mini kit . The isolated RNA was treated with the DNAse Free DNA removal kit  and 400 µg of RNA was reverse-transcribed with random hexamers utilizing the GeneAmp RNA PCR Core Kit  For the 2, 0.5% Nonidet P-40, and protease inhibitor cocktail ). 20 μg of cell protein extracts were analyzed by SDS-PAGE gels (10–12%) and transferred in a 0.22 µm nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% skimmed milk in TBS-0.1% Tween 20 per 1 h at room temperature, followed by incubation with the indicated primary antibody diluted 1:1000: anti-FLAG M2 ; anti-TCF-4 ; anti-β-catenin . After washing three times with TBS-0.1% Tween 20, membranes were incubated with HPR-conjugated secondary anti-mouse antibody in a dilution 1:10000 . Proteins were visualized utilizing the Immobilon Western (Millipore) according to the manufacturer’s instructions. Western blots were performed at least three times each to assure result reproducibility.C33A cells were cultured in 60 mm dishes and transfected with 3 µg of the indicated plasmid. 48 h post-transfection, cells were lysed using 300 µL of RIPA buffer , anti-TCF-4  antibodies, or IgG isotype control  overnight at 4 °C. A total of 20 µL of protein G-agarose beads (Upstate) were added to each sample and incubated at 4 °C, for 3 h. Complexes were washed three times with PBS-0.1% NP-40, resuspended in Laemmli sample buffer, and submitted to immunoblot analysis with anti-FLAG M2 , anti-β-catenin , and anti-TCF-4 antibodies .C33A cells were seeded in 60 mm dishes and transfected with 3 µg of the indicated plasmid. 48 h post-transfection, cells were treated with 200 µg/mL of cycloheximide (an inhibitor of protein biosynthesis) . After 0, 6, and 12 h post-treatment protein extracts were isolated using 2× Laemmli sample buffer . Western blot assays were carried out in order to analyze the TCF-4 protein stability.C33A and HaCaT cells were seeded over slides in 6 well plates and transfected with the indicated plasmids. After 48 h post-transfection cells were fixed with 3.7% paraformaldehyde in PBS for 10 min and permeabilized with PBS-0.1% Triton X-100. Then, cells were incubated with anti-FLAG M2  and anti-β-catenin  or anti-TCF-4  antibodies overnight at 4 °C, after blocking with a 0.3% BSA solution. Cells were washed extensively with PBS and later incubated with anti-rabbit or anti-mouse antibodies conjugated to Rhodamine or Alexa-488 , respectively. Slides were washed and mounted with Prolong Diamond Antifade Mounting  and then analyzed with a confocal microscope , with lasers giving excitation lines at 488 and 594 nm. Around twenty fields were observed for each treatment and representative images were acquired. The data of three independent experiments were collected with a 63× objective oil immersion lens.E6 coding sequences were cloned into the pGEX-2T (GE) expression plasmid and the identity of each plasmid was verified by DNA-sequencing. GST-fusion protein production was induced in DH5-α E. coli strain with 10 mM IPTG. After three hours of induction, proteins were purified by lysing the cells using 1% triton/PBS and separating the insoluble fraction by centrifugation. Supernatant was then incubated with glutathione sepharose beads , washed several times, and then re-suspended in 1 mL of 1% triton/PBS and analyzed by SDS-PAGE. Similar amounts of GST-fusion proteins were incubated overnight with 40 µg of C33A cellular protein extract, beads were then washed several times, and bound protein was analyzed by western blot using anti-TCF-4 and β-catenin antibodies.2S2O5, 1% Triton X-100, 10 mM MgCl2, 25 mM Sucrose, and protease inhibitor cocktail) and incubated for 10 min at 4 °C with gentle agitation. The samples were centrifuged and the supernatants were collected (Cytoplasmic fraction). The pellets were resuspended in extraction buffer  and centrifuged through a 0.34 M sucrose gradient. Then, the pellets were resuspended in RIPA buffer ) (Nuclear fraction). The samples were analysed by immunoblot using anti-TCF-4 , anti-Lamin B1 , anti-GAPDH , and anti-FLAG M2  antibodies.C33A cells were seeded into a 60 mm dish and transfected with 3 µg of the indicated plasmid. 48 h post-transfection, cells were pelleted and washed with PBS . Cells were resuspended in 300 µL of lysis buffer  according to the manufacturer’s protocol. C33A cells were seeded and transfected with 10 μg of each plasmid as indicated. 48 h post-transfection cells were lysed using the HKMG buffer , incubated 20 min at 4 °C, and then passed through a 25-gauge needle attached to a 1 mL syringe for 20 times. The lysates were centrifuged, and the supernatant were collected. The protein extracts were pre-cleared with 60 μL of Streptavidin agarose beads  during 30 min at 4 °C and then were centrifuged and the supernatants were collected. For each sample, a total amount of 4 µg of biotin probes and 2.5 µg of Poly dI-dC  were added and incubated overnight. 60 µL of Streptavidin agarose beads were added to each sample and incubated during 30 min at 4 °C. The samples were centrifuged, and the supernatants were discarded. The obtained beads were washed five times with the HKMG buffer and finally resuspended with 2× Laemmli sample buffer . Then, Western blot assays were carried out.A fragment of the 3) and de-crosslinked with 200 mM NaCl for 5 h at 65 °C. All samples were treated with RNAse (200 µg) and Proteinase K (160 µg). DNA fragments were obtained using the phenol/chloroform protocol. Further, qPCR was performed to evaluated proteins interaction with Sp5 promoter using specific primers: Forward 5′-GGGTCTCCAGGCGGCAAG-3′ and Reverse 5′-AGCGAAAGCAAATCCTTTGAATCC-3′. To analyze the data, the fold enrichment method was performed.C33A cells were seeded in a 100 mm plates and co-transfected with 7 µg of each 18E6HA and β-catenin expressing plasmids. 48 h post-transfection, cells were cross-linked with 1% of formaldehyde and quenched with 0.125 M of glycine. Cell lysates were obtained using a Lysis Buffer  and sonicated with a Bioruptor Pico , obtaining DNA fragments ranging from 200 to 500 bp. A total of 20 µg of chromatin per sample was used and diluted 1:5 with a Dilution Buffer . Then, all samples were precleared with 50 µL of protein G agarose/Salmon Sperm DNA beads (Millipore) during 3 h at 4 °C and centrifuged. Supernatants were incubated using anti-HA , anti-TCF-4 , or anti-IgG  rabbit antibodies overnight at 4 °C. Further, 50 µL of protein G agarose/Salmon Sperm DNA (Millipore) was added and incubated during 3 h at 4 °C. Samples were centrifuged, and the beads were washed four times with Wash Buffer I  and once with Wash Buffer II . Immunoprecipitated complexes were eluted with the Elution Buffer  assays were performed using the CellTiter 96 Aqueous One Solution Cell Proliferation kit , according to the manufacturer’s instructions.C33A cells were seeded in a 60 mm dishes and transfected with 3 µg of E6 and β-catenin expressing plasmids, as indicated. After 24 h post-transfection, cells were harvested and seeded in a 96-well plate for 72 h. The MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2For crystal violet assays, cells were fixed with 10% of formol/PBS for 30 min at room temperature while shaking. Cells were then stained with crystal violet/PBS for 15 min. After several washes, cells were treated with acetic acid/PBS and then measured at 490 nm. The data were graphed to determine the percentage of cell proliferation for each assay condition.p was calculated by Student’s t-test or ANOVA Tuckey’s post-hoc analysis. Significance differences were accepted at p ≤ 0.05, as indicated.Data showing the effects of HPV-18 E6 and E6*I and HPV-16 E6 on TCF-4 in the different assays are presented as mean ± SD.


Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa‐B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKβ, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKβ demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKβ induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKβ regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKβ regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKβ as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKβ activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization. The NFκB family of transcription factors consists of 5 members each able to bind DNA, and dimerize with other family members.However, the mechanisms allowing temporal‐specific activation of NFκB in the pulmonary circulation during alveolarization are not known. IKKα and IKKβ have structurally similar catalytic subunits, and both kinases are able to phosphorylate IκB and induce canonical activation of NFκB dimers.Therefore, in this study, we sought to determine the specific roles for IKKα and IKKβ in the pulmonary vasculature during postnatal lung development. We performed microarray analysis of gene expression in neonatal PECs after silencing either IKKα or IKKβ, and found that the 2 kinases regulate unique panels of genes in the pulmonary vasculature. The loss of either kinase resulted in distinct impairments in physiologic functions essential for angiogenesis including, proliferation, survival, adhesion, migration and cytoskeletal remodelling. Although the number of dysregulated genes was higher in response to silencing IKKα, the functional effects on angiogenic function were substantially greater with loss of IKKβ. Taken together, these data suggest that IKKα and IKKβ regulate distinct but complementary patterns of genes important for angiogenic function in the pulmonary circulation and identify IKKβ as the predominant regulator of angiogenesis in the pulmonary endothelium during postnatal lung development.22.1Neonatal mouse  lungs were digested with collagenase IA (0.5 mg/mL) for 30 minutes at 37°C, and resulted cells were incubated with anti‐CD31‐coated magnetic beads . PECs were isolated by magnetic separation and cultivated as previously described.2.2Pulmonary endothelial cells were transfected using either IKKα or IKKβ On‐Target Plus SMART pool siRNA or non‐targeting siRNA as a control  and Lipofectamine 2000 (Invitrogen) for 6 hours. RNA was isolated at 36 hours using the RNeasy kit  for qPCR, and whole cell protein lysate was extracted at 42 hours for Western blot. In separate experiments, additional groups of siRNA‐treated cells were used at 40 hours post‐transfection for functional assays.2.3® PrimeView™ Mouse Gene Expression Array (Affymetrix) according to the manufacturer's protocols. Single‐stranded cDNA was synthesized from total RNA obtained from PEC transfected with NTC, anti‐IKKα or anti‐IKKβ siRNA and hybridized to gene arrays. Differentially expressed genes were determined by Significance Analysis of Microarray (SAM) method and by R software. Drafting principles were applied by fold change ≥2 or ≤0.5, and q‐value ≤5%. Further analysis was then performed with iPathwaysGuide from Advaita Bioinformatics  to assess gene ontology and enriched functional pathways with pathway topologies, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Ontology Based Annotation System (KOBAS 2.0) database.Gene expression patterns were assessed using GeneChip2.4Total RNA was isolated from PEC and whole mouse lung using the RNeasy kit (Qiagen). RNA (2 μg) was reverse‐transcribed using Superscript III (Invitrogen), and qPCR was performed using TaqMan primers (Applied Biosystems) as described previously.2.53 cells were plated into wells of 96‐well plates and starved (0.2% FBS) for 8‐12 hours. The starvation media was then removed and bromodeoxyuridine (BrdU) added to each of the following conditions: complete EGM  + 5% FBS; EGM stripped by all growth factors + 0.2% FBS; EGM stripped by all growth factors + 0.2% FBS + IGF‐1, EGF, VEGF, or FGF2 . Incorporation of BrdU was measured by ELISA at 2‐24 hours per the manufacturer's protocol (Roche Diagnostics).At 40 hours post‐transfection, 6 × 102.64 cells/well) and after reaching 80%‐90% confluence, the media was exchanged for starvation media . Separate groups of cells were exposed to TNF‐α (20 ng/mL) to induce apoptosis for 4, 8 and 24 hours. Apoptosis was measured by determining the amount of active caspase 3/7 using a Luciferase‐Glo system (Promega). The assays were carried out per manufacturer's protocol, and the luciferase reactions read using a Berthold LG&G Lumat LG luminometer as performed previously.At 24 hours post‐transfection, cells were transferred to 96‐well plates  coated culture slides or glass chamber slides pre‐coated with different extracellular matrix proteins (BD variety pack). At 1 hour post‐plating, non‐adherent cells were removed, and adherent cells fixed and F‐actin stained with Phalloidine‐TRITC (ThermoFisher Scientific) and Hoechst reagent. Specimens were imaged with a Leica DM5500B Upright Microscope and Photometrics CoolSNAP HQ2 CCD camera, and Metamorph Image Analysis software was used to manually count the number of adherent cells in serial images of random microscopic fields of each specimen.At 48 hours post‐siRNA transfection, PECs were plated at a density of 2 × 102.8Whole cell lysates were obtained from PEC using a lysis buffer containing 10 mmol/L Tris–HCl and 1% SDS, heated to 100°C. Protein lysates were subjected to SDS‐PAGE. Membranes were incubated with primary antibodies to detect Akt, pAKT, p38, ERK1/2 , tubulin (Abcam), IKKα and IKKβ  and the appropriate HRP‐conjugated secondary antibody, followed by enhanced chemiluminescence detection (GE Life Sciences).2.9At 24 hours post‐siRNA transfection, PECs were plated on BD‐Falcon culture slides coated with 0.2% gelatine. Once confluent, the monolayer was wounded with a fine pipette‐tip creating a wound along the diagonal axis of the well. The wound was permitted to heal by PEC migration for the next 24 hours. Phase contrast images were captured before and after the healing process using a Nikon Eclipse inverted microscope with NIS Elements software. The wound closure area was analysed using Image‐J software.2.10NTC‐, IKKα‐ and IKKβ siRNA‐treated neonatal PECs were plated on glass coverslips 24 hours after transfection and starved for 12 hours. Starvation media (SM) was replaced with either new SM , or SM with VEGF (50 ng/mL). Fixed cells were rehydrated, permeabilized and non‐specific binding diminished using Sea Block Blocking Solution (Thermo Scientific). Cells were then incubated with Phalloidin‐TRITC reagent (ThermoFisher Scientific) and counter‐stained for chromatin with Hoechst reagent (1 μg/mL) (Sigma‐Aldrich). Fluorescent images were captured using a Leica DM5500B Upright Microscope and Photometrics CoolSNAP HQ2 CCD camera, using HC Plan Apo 25‐mm objectives at ×40 magnification.2.114 PECs were plated into 48‐well dishes coated with Reduced Growth Factor Basement Membrane Extract  in the presence of VEGF‐A (50 ng/mL). For all experiments, representative images of tube networks were obtained at 8 hours, and the total tube length and branch points per 4X field was quantified in a blinded fashion by uploading images for evaluation using Wimasis Image Analysis software.Pulmonary endothelial cells were transfected with NTC, IKKβ or VCAM siRNA as described above. At 48 hours after transfection, 3.5 × 102.12t test and between more than 2 groups by either one‐way or two‐way ANOVA, followed by Bonferroni's multiple‐comparison post hoc analysis. A P value of ≤.05 was considered statistically significant. All experiments have been performed with multiple biological and technical replicates, with specific replicate numbers detailed in the figure legends.All data are presented as means ± SE. Statistical differences between 2 groups were determined by Student's 33.1P < .0001 vs NTC siRNA, n = 12) without affecting IKKβ mRNA or protein levels , without altering IKKα mRNA or protein levels , in PEC transfected with NTC, anti‐IKKα or anti‐IKKβ siRNA. Silencing IKKα in PEC dysregulated 511 genes (up‐regulating 217 and down‐regulating 274), while silencing of IKKβ dysregulated 177 genes (up‐regulating 105 and down‐regulating 72). Interestingly, the number of shared targets was particularly small (37 genes) .To evaluate the distinct roles for IKKα vs IKKβ in neonatal PECs, we disrupted IKK signalling in a subunit‐specific manner by RNA interference. Transfection of PEC with siRNA to IKKα decreased IKKα mRNA levels by 82% ,3.3P < .0001 for NTC siRNA, IKKα siRNA and IKKβ siRNA‐treated cells). The IKKα depleted cells also demonstrated slightly increased apoptosis at 4 hours, and significantly higher levels at 24 hours as compared to NTC cells. In the IKKβ depleted cells, levels of apoptosis were similar to controls at 4 and 8 hours, but significantly increased by 24 hours compared to NTC or IKKα siRNA‐treated cells.The NFκB/IKK signalling pathway can have divergent effects on cell survival, inducing either pro‐ or anti‐apoptotic targets.P < .0001 for NTC siRNA, IKKα siRNA and IKKβ siRNA‐treated cells). Loss of IKKα worsened cell survival in response to TNF‐α at 8 hours, with over 50% greater levels of active caspase 3/7 as compared to NTC siRNA‐treated cells (P < .0001). However, by 24 hours, the levels of apoptosis became similar to controls. Loss of IKKβ caused a similar, but more sustained increase in apoptosis in response to TNF‐α compared to NTC‐cells at 8 hours, and significantly higher levels compared to both NTC and IKKα transfected cells at 24 hours.Stimulation with TNF‐α, a cytokine that induces apoptosis in PECs,3.4In addition to proliferation and survival, effective angiogenesis requires coordinated steps of cell motility, including the attachment of cellular protrusions to the extracellular matrix.We also evaluated the impact of silencing IKKα, and IKKβ on PEC migration by wound‐healing assays in the NTC, IKKα and IKKβ siRNA‐treated neonatal PEC.3.5Cell migration requires coordinated changes in the remodelling of the actin cytoskeleton to form filopodia, lamellipodia and stress fibres.3.6Vascular endothelial growth factor‐mediated angiogenesis is associated with the activation of additional intracellular signalling pathways that promote angiogenic function, including activation of the MAPK signalling. Specifically, activation of ERK1/2 promotes endothelial cell proliferation, and activation of p38 promotes actin reorganization and cell migration.3.7We next sought to identify downstream effector molecules regulated by the NFκB pathway that were promoting angiogenesis in the PEC. Given that silencing of either IKKα or IKKβ induced either mild or severe impairments in angiogenic functions, we focused on the small group of shared genes dysregulated by silencing either IKKα or IKKβ, such as vascular cell adhesion molecule (VCAM). Microarray analysis found VCAM to be down‐regulated 1.9‐fold in both the IKKα and IKKβ silenced cells. qPCR to validate these results identified a small, but statistically significant decrease in VCAM gene expression in the IKKα‐silenced cells, and a more marked decrease in the IKKβ‐silenced cells Figure A. DespitTo confirm the importance of VCAM in modifying angiogenic function in PECs, we silenced VCAM expression using siRNA and assessed PEC adhesion, migration and in vitro tube formation. By Western blot, we confirmed almost complete loss of VCAM protein in the VCAM siRNA‐treated PEC Figure C. Loss o4We previously identified the NFκB as an essential regulator of pulmonary angiogenesis during postnatal lung growth and alveolarization.In the canonical pathway of NFκB activation, IKKα and IKKβ work in concert to release NFκB dimers from the inhibitory IκBs, and allow their translocation to the nucleus to regulate gene expression. Our prior studies found that NFκB is endogenously active in neonatal PECs during early alveolarization, but the mechanism allowing for this developmental activation is not yet clear. Constitutive activity of NFκB is normally restricted to cells from the hematopoietic lineage.Despite significant sequence homology, silencing IKKα and IKKβ in PEC resulted in the dysregulation of both shared and unique genes. Both IKKα and IKKβ can exert distinct NFκB‐dependent and independent effects that vary in a cell stimulus‐specific manner.These data add to the work of others associating the IKK/NFκB pathway with physiologic and pathologic angiogenesis. Tie2‐mediated −/− IKKβ phenotype of hematologic and endothelial cells disrupts liver and placental vascular development inducing embryonic lethality, while the ± deletion impairs post‐ischaemia neovascularization in adult mice.In our study, silencing either IKKα or IKKβ impaired PEC survival. Anti‐apoptotic functions of the IKK/NFκB pathway have been well described. Deletions of either IKKβ or the NFκB subunit RelA result in early embryonic lethality as a result of extensive liver apoptosis.We found that IKKβ was clearly the predominant regulator of angiogenesis with more profound defects in proliferation, adhesion, migration and cytoskeletal remodelling. In the IKKβ depleted cells, proliferation was impaired in response to EGM, VEGF and FGF‐2. Both VEGF and FGF‐2 are important growth factors that regulate angiogenesis during development and disease.Vascular endothelial growth factor signalling through the VEGFR2 receptor is an essential pathway mediating postnatal pulmonary angiogenesis during alveolarization.We further identified VCAM as one IKKβ regulated target that appears to play a significant role in promoting normal PEC adhesion, motility and in vitro angiogenesis Figure F,G. VCAMIn addition to VCAM, we found that IKKα and IKKβ regulate over 700 genes in the PEC. Although the scope of this study did not permit a comprehensive investigation of all the putative downstream targets that may have a role in mediating angiogenic function in the PEC, there were numerous candidates identified by microarray that could play critical roles. These include targets that are important in regulating discrete steps in cell motility such as Rac3,There are a number of limitations to our study. First, given that the loss of both IKKα and IKKβ also induced impairments in cell survival it is impossible to fully separate the effects of cell death on other measures of angiogic function, including adhesion, migration and in vitro angiogenesis. However, the greater degree of cell death was observed in response to TNF‐α stimulation, or in response to prolonged serum starvation. In attempt to limit these confounders as much as possible, we performed these function assays with either no or short periods of starvation, and with assay times that were also short in nature. Second, although our prior work demonstrated that pharmacologic inhibition of both IKKα and IKKβ effectively impairs pulmonary angiogenesis and disrupts alveolarizartion in neonatal mice in vivo,In summary, we explored the specific roles of IKKα and IKKβ in the developing pulmonary endothelium. Silencing either IKKα and IKKβ disrupted hundreds of genes, but the panels of genes regulated by the 2 kinases were distinct, suggesting that each kinase has separate functions in the pulmonary endothelium of developing vessels. Additional studies to assess the functional roles of IKKα and IKKβ in the pulmonary endothelium demonstrated that IKKβ plays a predominant role in promoting a broad array of cellular functions including survival, proliferation, adhesion, migration and actin reorganization. These angiogenic defects were more exaggerated in cells stimulated with VEGF alone, suggesting a specific impairment in VEGF‐mediated angiogenesis. VCAM was identified as an IKKβ‐regulated downstream target, and silencing of VCAM alone resulted in significant impairments in cell adhesion, migration and tube formation, identifying an important, but previously undescribed role for VCAM in pulmonary vascular development. Angiogenesis is essential for alveolarization, the stage of lung development that exponentially increases gas exchange surface area. Disrupted angiogenesis contributes to many paediatric diseases, including bronchopulmonary dysplasia, and therapeutic strategies to enhance angiogenesis may have potential benefit to these and other lung diseases marked by impaired angiogenesis. Our prior study showed that disrupting IKK‐mediated signalling disrupts both angiogenesis and alveolarization. Data from this study identify IKKβ as the predominant regulator of angiogenic function in developing pulmonary endothelial cells and suggest that strategies aimed to specifically preserve and enhance IKKβ activity may be an important therapeutic strategy to preserve late lung growth.The authors confirm that there are no conflicts of interest.


The osteogenesis differentiation of human bone marrow stem cells (BMSCs) is essential for bone formation and bone homeostasis. In this study, we aim to elucidate novel molecular targets for bone metabolism diseases.https://www.ncbi.nlm.nih.gov/geo/). The osteogenic differentiation of BMSCs was measured by ALP staining, AR staining and expression of osteogenic markers in vitro. For in vivo assay, we seeded BMSCs onto beta‐tricalcium phosphate (β‐TCP) and transplanted them into muscle pockets of nude mice. Luciferase assay, co‐immunoprecipitation assay and in vitro ubiquitination assay were carried out to investigate the molecular mechanism.The dataset GSE80614 which includes mRNA expression profile during BMSCs osteogenic differentiation was obtained from the GEO database  expression was elevated during the process of BMSCs osteogenic differentiation. Further studies showed that upregulation of CRYAB significantly enhanced the osteogenic differentiation, while downregulation of CRYAB suppressed it. CRYAB regulated BMSCs osteogenic differentiation mainly through the canonical Wnt/β‐catenin signalling. In addition, we found that CRYAB could physically interact with β‐catenin and protect it from ubiquitination and degradation, which stabilized β‐catenin and promoted the Wnt signalling.The present study provides evidences that CRYAB is an important regulator of BMSCs osteogenic differentiation by protecting β‐catenin from ubiquitination and degradation and promoting the Wnt signalling. It may serve as a potential therapeutic target for diseases related to bone metabolism. Commonly, targeted proteins are labelled by ubiquitin molecules through a sequence of enzymatic reactions, including E1 (ubiquitin‐activating enzymes), E2 (ubiquitin‐conjugation enzymes) and E3 (ubiquitin ligases), and are eventually degraded into peptides by proteasomes. Evidences have verified that β‐catenin, a key factor of Wnt/β‐catenin signalling pathway, is also a target for the ubiquitin‐proteasome system, and that the degradation of β‐catenin directed by ubiquitin‐proteasome system plays important roles in tumour suppression,α‐B‐crystallin (CRYAB) has been reported as a major structural protein of the eye lens and is abundant in kidney, central nervous system, skeletal muscle and heart.22.1https://www.ncbi.nlm.nih.gov/geo/) and analysed the differentially expressed mRNAs at different time points (3 and 4 days vs 1 and 2 hours). |logFC| > 2 and P‐value < .01 was set as threshold.The mRNA expression profile during osteogenic differentiation process of human bone marrow stem cells was included in the dataset GSE80614. We obtained the dataset from the GEO database (2.22 humidified incubator.Human bone marrow stem cells (BMSCs) were got from a 43‐year‐old donor who suffered an severe trauma and underwent amputation and were maintained in α‐modified essential medium  that contained 10% foetal bovine serum (FBS) (Gibco) and 100 µg/mL penicillin‐streptomycin sulphate (Sigma‐Aldrich) at 37°C in a 5% CO2.3For the in vitro adipogenic and osteogenic differentiation assays, BMSCs were treated with adipogenic differentiation medium (Cyagen) or osteogenic differentiation medium (Cyagen) follow the operating instruction in 24‐well plates for 7 or 14 days. The differentiation medium was refreshed every 2 days. Oil Red O staining, Western blot and qRT‐PCR were used to measure the adipogenic differentiation efficacy. Alizarin red (AR) staining, alkaline phosphatase (ALP) staining, Western blotting and qRT‐PCR were used to measure the osteogenic differentiation efficacy.2.4The isolation of total RNA from BMSCs was performed using TRIzol reagents (Invitrogen) and isolated the total RNA was then quantified using Nanodrop 2000 (Thermo). The PrimerScriptRT Reagent (TaKaRa) was used according to the operating instruction for reverse‐transcription of cDNA. The reactions of real‐time PCR were performed with the aid of SYBR Green kit (TaKaRa) using ABI HT7900 . The gene expression levels were normalized to β‐actin. Primer sequences are listed in Table 2.5Cells were lysed in Cell Lysis Buffer (Sigma‐Aldrich) on ice and denatured by heating at 95°C for 15 minutes with the addition of loading buffer (BioTNT). The samples were separated using a 10% SDS‐PAGE gel (EpiAyme) by electrophoresis at 120 V for 75 minutes. Then, the proteins were transferred through electroblotting at 280 mA for 70 minutes to a PVDF membrane (Millipore). After blocking in 5% non‐fat milk for 60 minutes, the membrane was incubated with primary antibodies at 4°C for 12 hours, followed by incubation with the secondary antibody at room temperature for 1 hour. Image Quant LAS 4000  was used to confirm the expression levels of proteins. The expression of β‐actin was used as control.An immunoprecipitation kit (Abcam) was used for co‐immunoprecipitation assay according to the operating instruction. Briefly, lysis buffer was pre‐cooled on ice and the Protease Inhibitor Cocktail was added for the preparation of cell lysis. The supernatant of the cell lysis was collected after centrifuged at 1000 g for 15 minutes, and the primary antibodies were added and incubated at 4°C for one night. Then, the Protein A/G Sepharose beads were added and incubated at 4°C for 1 hour and then separated by centrifugation at low speed. The beads were washed for three times and the precipitated proteins were eluted followed by Western blotting analysis.The antibodies are listed in Table 2.6For siRNA transfection, Lipofectamine 2000  was used according to the manufacturer's instructions and the cells were collected to assess the efficacy of transfection by qRT‐PCR or Western blotting 24 hours later. For virus productions, 293T cells were transfected with 3.6 µg envelope plasmid, 9 µg packaging plasmid and 12 µg targeting plasmids using Lipofectamine 2000, and the viruses were harvested and filtered 48 hours later. The infection of BMSCs was performed with the addition of polybrene follow the operating instruction. For TOPFlash reporter assay, 293T cells were transfected with β‐galactosidase, CRYAB‐specific siRNA or corresponding control siRNA and Super (8×) TOPFlash plasmid using Lipofectamine 2000. Serum starvation was performed for overnight 24 hours after the transfection, and the osteogenic differentiation medium was then used for cell culture. The cells were lysed 48 hours later and a luciferase assay system (Promega) was used to measure the luciferase activity.2.7Cells were lysed using cell lysis buffer after treated with a proteasome inhibitor, MG 132, for 8 hours, and the lysis was incubated with antibodies for 3 hours at 4°C. Then the Sepharose beads of protein A/G were added to the lysis and rotated gently for 10 hours at 4°C. The beads were collected and washed three times with centrifugation at low speed. The SDS‐loading buffer was used to elute the immunoprecipitated proteins at 95°C for 3 hours, and Western blotting assay was applied for analysis.2.86) seeded on was transplanted and buried into muscle pockets on the posterior limb of the mice. Forty‐two mice were divided into seven groups, namely sh‐Control group, sh‐CRYAB group, Vector group, CRYAB group, sh‐Control + Vector group, sh‐CRYAB + Vectro group and sh‐CRYAB + β‐catenin group. Corresponding kind of BMSCs was used in each group. The mice were put to death 8 weeks later, and the transplants were collected from the posterior limbs for histological analysis. 10% EDTA was used to decalcify the transplants after fixation in 10% formalin for 3 days. The samples were then sliced using Leica RM2235 for H&E (Haematoxylin and Eosin) staining. BIOQUANT OSTEO software (BIOQUANT) was used to collect the images. All experiments were subject to approval by the Ethics Committee of Shanghai Sixth People's Hospital.Eight‐week‐old male nude mice were employed for animal studies in this experiment, and all of these mice were kept in a specific‐pathogen‐free condition. Beta‐tricalcium phosphate  with BMSCs  was used to evaluate the statistical differences among more than two groups. Values of P < .05 were considered statistically significant. All values are presented as means ± SD (standard deviation).All results were replicated independently for three times. SPSS 16.0 (IBM Corporation) was used to complete the statistical analysis. Two‐tailed Student's 33.1In the present study, the dataset GSE80614 was analysed to explore the differentially expressed genes at different time points during BMSCs osteogenic differentiation process. A total of 109 differentially expressed genes (including 52 downregulated genes and 57 upregulated genes) were identified when comparing the differentiation time of 3 or 4 days to that of 1 or 2 hours , followed by transplantation into muscle pockets on the posterior limb of nude mice. The mice were put to death 8 weeks later, and the transplants were collected from the posterior limbs for histological analysis. We found that less bone tissue was formed in the CRYAB‐downregulated group when compared with the control group Figure A and B, To further verify our hypothesis, we assessed the adipogenic differentiation efficacy of BMSCs using Oil Red O staining and qRT‐PCR assays after the cells were treated with adipogenic induction medium. It was demonstrated that the adipogenesis of BMSCs was enhanced by downregulating CRYAB expression Figure E, while 3.4The canonical Wnt signalling has been proven to be vital for human bone homeostasis and human BMSCs osteogenic differentiation. To explore whether CRYAB influenced the process of osteogenesis through the Wnt signalling, we firstly measured the expression of β‐catenin, a core element of the Wnt/β‐catenin pathway. We found that the downregulation of CRYAB expression significantly decreased β‐catenin protein level as measured by Western blotting, and upregulation of CRYAB expression increased it Figure A and C. 3.5The ubiquitin‐proteasome system has been verified to play essential roles in regulating the stability, subcellular localization and activity of proteins. Since the alteration of CRYAB expression significantly influenced the protein level of β‐catenin without changing its mRNA level Figure A‐D, we sBesides, CRYAB‐downregulated BMSCs were transfected with β‐catenin‐lentivirus or the corresponding control lentivirus, and the cells were loaded on β‐TCP and transplanted into muscle pockets on the posterior limb of nude mice. Eight weeks later, the mice were put to death and the transplants were harvested for histological analysis. As shown in Figure Additionally, the regulatory network of CRYAB on the osteogenic differentiation of BMSCs is shown in Figure 4Bone is a metabolically active organ of human and is continuously remodelled via the formation of new bone by osteoblasts and the resorption of old bone by osteoclasts.Total mRNA expression of human BMSCs cultured in osteogenic differentiation medium was measured at different time points by van de Peppel J. and his colleagues, and the mRNA expression profile was included in the dataset GSE80614.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80614.As a member of the small heat‐shock protein family, CRYAB possesses several important roles. In cardiomyocytes and vascular endothelial cells, CRYAB has been shown to exhibit a protective role against apoptosis.The canonical Wnt/β‐catenin signalling has been verified to exhibit a vital effect on BMSCs osteogenic differentiation and bone metabolism.In conclusion, we found that the expression of CRYAB is upregulated in the osteogenic differentiation process of BMSCs. CRYAB acts as a regulator of BMSCs osteogenic differentiation, and the regulatory effect is achieved by stabilizing β‐catenin and regulating the Wnt signalling. These findings illustrate that CRYAB might be a new regulator of BMSCs osteogenic differentiation and may serve as an underlying therapeutic target for diseases related to bone homeostasis.The authors declare no conflicts of interest. Click here for additional data file.


Aspirin inhibits BCCMI by attenuating Wnt/β-catenin signaling and suppressing FMOD expression via inhibiting deacetylation of β-catenin by histone deacetylase 6 (HDAC6) leading to β-catenin phosphorylation and cytoplasmic degradation. Moreover, expression of the transcriptional complex components β-catenin/TCF4/LEF1 is upregulated by the Wnt/β-catenin pathway, constituting positive feedback loops that amplify its signal output. Our findings identify a critical role of FMOD in cancer metastasis, reveal a mechanism regulating FMOD transcription and impacting tumor metastasis, uncover action targets and mechanism for the anticancer activity of Aspirin, and expand the understanding of the Wnt/β-catenin pathway and tumor metastasis, which are valuable for development of cancer therapeutics.Emerging evidence suggests that fibromodulin (FMOD), an extracellular matrix protein, is associated with cancer, and yet little is known about the regulation of FMOD expression and its role in cancer metastasis. Aspirin, a classic anti-inflammatory drug, has been indicated to offer anticancer benefits, but its action targets and mechanisms remain obscure. In the present study using cell lines, animal model and database analysis, we show that FMOD is crucial for breast cancer cell migration and invasion (BCCMI)  Breast cancer (BC) occurs commonly in women and is amongst the deadliest cancers  as aggreFibromodulin (FMOD) is a small leucine-rich proteoglycan ECM protein, and is a member of a family of secreted proteoglycans that play important roles in signaling, collagen fibrillogenesis, cell migration, adhesion, growth, differentiation, and apoptosis , while i2+ signaling pathway  and is aberrantly activated in many human cancers and important for tumor initiation, growth and metastasis . The Wnt pathway . In the  pathway . The β-c pathway .Aspirin, also known as acetylsalicylic acid (ASA), is one of the best known nonsteroidal anti-inflammatory drugs (NSAIDs) which has long been widely used for the treatment of pain, fever, and inflammatory conditions. The regular use of Aspirin has been shown to reduce the risk of developing colorectal, breast, esophageal, lung, stomach, and ovarian cancers . Aspirinin vivo mouse model of human breast tumor xenografts and analysis of clinical data from databases, in conjunction with various approaches and technical methods.How FMOD expression is regulated, what role FMOD plays in cancer metastasis, how Aspirin exerts anticancer effects, and what mechanistic parts of the Wnt/β-catenin signaling pathway are involved remain as important outstanding issues in biology and cancer therapy. In the current study, we have sought to address these questions in the context of breast cancer. Our investigations are focused on molecular and cellular analysis using highly metastatic human and mouse breast cancer cell lines and a non-tumor cell line, and also extended to an via promoting ERK activation, and thus FMOD, as a transcriptional target gene of the Wnt/β-catenin pathway, mediates the promotive effects of the pathway on BCCMI. Furthermore, we find that Aspirin inhibits BCCMI by suppressing FMOD expression through hampering Wnt/β-catenin signaling via inhibiting HDAC6 to enhance acetylation of β-catenin, causing its phosphorylation and cytoplasmic degradation. Thus Aspirin modulates the Wnt/β-catenin pathway, with HDAC6 as a direct target protein, and FMOD as a downstream transcriptional target gene in cancer metastasis, which reveals a significant link between regulation of FMOD with Aspirin action. In addition, expression of TCF4, LEF1 and β-catenin is upregulated by the Wnt/β-catenin pathway, constituting positive feedback loops.We find that the expression of FMOD is regulated positively by the Wnt/β-catenin pathway, in which nuclear β-catenin in complex with TCF4/LEF1 mediates transcription of FMOD, while β-catenin phosphorylation and subcellular localization is regulated by HDAC6 acting on β-catenin, wherein HDAC6 deacetylates β-catenin causing its dephosphorylation and nuclear translocation. Meanwhile, we find that FMOD plays an essential role in breast cancer cell migration and invasion (BCCMI) 2 at 37°C. Mice breast cancer cell line 4T1 cells were maintained in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin in 5% CO2 at 37°C. Human embryonic kidney HEK 293T cells were grown in DMEM supplemented with 10%FBS, 100U/ml penicillin, and 100 mg/ml streptomycin in 5% CO2 at 37°C. Transfection of the plasmids was performed with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol.Human breast cancer MDA-MB-231 cells, mouse breast cancer 4T1 cells, and human embryonic kidney HEK 293T cells were obtained from the American type culture collection (ATCC). MDA-MB-231 (Triple negative highly invasive human breast cancer cell line) cells were cultured in Leibovitz L-15 Medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin without COInvitrogen. HRP conjugated, fluorescently-labeled secondary antibodies and the ECL detection system for western blot were purchased from KPL .Aspirin , Lithium Chloride , and B.D Matrigel were purchased from Sigma-Aldrich. Dynabeads Protein G magnetic beads for ChIP assay were purchased from To generate the expression plasmid for human FMOD, cDNAs were PCR amplified, then cloned into a pflag CMV2 vector. For constitutive expression, FMOD was subcloned into pBoBi lentivirus expression plasmids to produce the lentivirus, then recombinant pBoBi FMOD was cotransfected with pMDLg/pRRE, pRSV-Rev, pVSV-G into HEK 293T cells, and culture supernatants containing the virus were collected 48 h and 72 h after transfection , and a total of 1 µg of RNA was reverse-transcribed into cDNA using the Primescript RT Reagent Kit (Takara) according to the manufacturer’s instructions. Constructed cDNA was then subjected to quantitative PCR analysis using Fast Start Universal SYBR Green Master . The PCR setting used were as follows: 95 °C for 5 min for Hot-Start DNA Polymerase Activation, 40 cycles at 95 °C for 15 s (denaturing) and 60 °C for 1 min , and one cycle at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. All of the target gene expression levels were normalized using GAPDH, and the data was expressed as student t-test Two-way ANOVA test using means ± SD of 3 to 8 independent samples. The sequences of primers used is listed in Constructs expressing shRNA against target proteins FMOD, β-catenin, TCF4, and LEF1 were generated by subcloning the following oligonucleotides into a pSilencer 2.1-U 6 hygro vector, with a BamHI/HindIII restriction site, and then shuttled into the FG12 vector. Primers are listed in Following lentiviral or Aspirin treatment, MDA-MB-231 or 4T1 cells were fixed onto slides with 4% paraformaldehyde for 30 min and permeabilized with 0.4% Triton X-100 and blocked with 3% BSA for 1 h. The cells were then incubated with primary antibody (dilution 1:200) against β-catenin overnight at 4 °C, following which, FITC- or Cy3-conjugated goat anti-rabbit IgG antibodies were used as the secondary antibody. The cells were incubated with the FITC-Conjugation secondary antibody (dilution 1:50) for 2 h. To visualize the cell’s nucleus, DAPI  was used. Sections were observed using an Olympus Laser Scanning Confocal Microscope with imaging software .Cells or tumor tissues were lysed in a buffer containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 2 µg/ml leupeptin, and 1 mM PMSF. The proteins were fractionalized by 10% and 12% polyacrylamide SDS page and electrophoretically transferred to a PVDF (Millipore) membrane. After blocking the membrane with 5% non-fat dry milk for 1 h at room temperature, the membrane was washed 3 times for 7 min intervals with 1XTBST and then incubated with primary antibodies against FMOD , GSK-3β (27C10) Rabbit Mab, Phospho GSK-3β (Ser 9) (5b3) Rabbit Mab, β-catenin Rabbit primary antibody (D10A8), Phospho β-catenin (Ser 33/37/Thr 41) antibody, anti-ERK 1/2, phospho-ERK1/2 (Thr202/Tyr204) ; TCF4 (CST#2569), LEF1 (CST), Acetyl-β-catenin (Lys 49) (CST), GAPDH (Santa Cruz), Lamin B (Santa Cruz), and anti-Ubiquitin (Santa Cruz) overnight at 4 °C. After which, the blots were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Pierce) at room temperature for 1 h. Detection was performed using a Chemiluminescent Western Blot detection kit  . The proFor nuclear and cytoplasmic protein extraction, treated cells were initially washed and scraped with cold PBS pH 7.4 in 1 ml per 100-mm dishes and subsequently transferred into 1.5 ml Eppendorf tubes. Cell were centrifuged at 3,000 rpm for 3 min at 4 °C, suspended and incubated in 400 ul of buffer A  on ice for 10 min, and then added to 10 ul of 10% NP-40 to lyse the cells. The supernatant was collected by centrifugation  to obtain the cytoplasmic fraction. The pellet was resuspended in 50 ul buffer B  and incubated on ice for 15 min to dissolve the nuclear proteins. The isolate was identified by western blot using Lamin B and GAPDH as controls.Homo sapiens chromosome 1, GRCh38.p7 Primary Assembly) which was obtained from NCBI as previously described. All the transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cell lysates were used for luciferase assay using a luciferase assay kit .MDA-MB-231 and HEK 293T cells were seeded onto 12-well plates. When cells grew to 60–70% confluence, the cells were transfected with luciferase reporter plasmid PGL 3 which contained either the wild type or mutant FMOD promoter sequence  which was injected into the mice every day for 7 days while the control group mice received PBS solution. At the time of animal sacrifice, tumors were excised and cells were lysed for western blot, Quantitative Real-time PCR, and immunofluorescence.Six to eight week old female BALB/c nude mice (nu/nu) were obtained from Guangdong Province Medical Animal Centre and maintained and monitored in a pathogenic free environment. The animal study was reviewed and approved by the Administrative Committee on Animal Research of the Graduate School at Shenzhen, Tsinghua University. 6 × 10Formalin-fixed, paraffin-embedded frozen sections (4–10 µm) of tissue obtained from breast carcinoma patients were selected for fluorescent immunohistochemical analysis. The tumor tissue was surgically excised and cut into small sections for further fixation with 4% paraformaldehyde for 2 h. The fixed tumor tissue samples were dehydrated in a series of 10%, 20%, and 30% sucrose in PBS solutions and the samples were then wrapped in Tissue-Tek O.C.T. compound  and frozen at −80 °C for 2 h. The frozen samples were then sectioned into 4–10 µm thick sections. After blocking with 3% BSA/0.2% Triton X-100 in PBS for 1 h, the sections were incubated with anti β-catenin (D10A8), and anti-FMOD antibodies  overnight at 4 °C. FITC conjugated goat anti-rabbit IgG antibody (1:50 Pierce) was used as a secondary antibody. To observe the nucleus, DAPI was used. Sections were observed using an Olympus confocal laser scanning microscope with imaging software  .t-test of unpaired data or two-way ANOVA . P values less than 0.05 indicates statistical significance.All experiments were repeated a minimum of 3 times and maximum of 8 times and the data are presented as means ± SD. The difference between data groups was evaluated for statistical significance using the Student Breast cancer cell migration was suggested to be inhibited by Aspirin , and to To understand the role of FMOD gene expression in breast cancer metastasis in relation to Aspirin and the Wnt/β-catenin signaling pathway, we measured the mRNA and protein levels of FMOD, in the presence and absence of interference of the Wnt/β-catenin pathway, by qPCR and Western blot. FMOD mRNA and protein levels were markedly reduced in MDA-MB-231 , or by oNext, the direct relationship between the FMOD protein and BCCMI was investigated. Activation of the Wnt/β-catenin signaling pathway by overexpressing Wnt-3a or FMOD in MDA-MB-231 cells enhanced breast cancer cell migration   for regulatory DNA binding elements and three binding sites that show a high degree of homology to the core consensus sequence of TCF4 binding (5AAAG-3/)  were ideSubsequently, we tested whether β-catenin, TCF4, and LEF1 directly regulate endogenous FMOD gene expression at the mRNA and protein levels. Lentivirus-mediated shRNA was used to knock-down β-catenin, TCF4, and LEF1 individually, and the specificity and efficiency of the RNAi was confirmed by qPCR and western blot. β-catenin mRNA  expression and cancer metastasis  expression regulation and its role in cancer progression, or about the action targets and mechanisms of the anticancer benefits of Aspirin. We find that FMOD is essential in promoting breast cancer metastasis, FMOD expression is regulated positively by the Wnt/β-catenin signaling pathway, wherein the β-catenin/TCF4/LEF1 complex binds to the FMOD promoter for its transcription, and Aspirin inhibits breast cancer metastasis by attenuating the Wnt/β-catenin pathway and suppressing FMOD expression, evidently Our findings identify a critical role of FMOD in cancer metastasis, reveal a mechanism regulating FMOD transcription and impacting tumor metastasis, unveil action targets and mechanism for the anticancer activity of Aspirin, and expand the understanding of the Wnt/β-catenin pathway and tumor metastasis, which are valuable for the development of cancer therapeutics.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All the animal studies were carried out according to the protocol after receiving approval from the Administrative Committee on Animal Research of the Graduate School at Shenzhen, Tsinghua University. Written informed consent was obtained from the owners for the participation of their animals in this study.LH and FK conceptualized and designed the research. FK conducted most of the the experiments and data analysis with the participation and assistance of XD, KL, YW, CS, HT, and HC. FK, NO-T, and LH drafted and revised the paper.This work was supported with funding awarded to LH from China MOST National Key Basic Research 973 Program (2005CCA03500), China NSFC  Guangdong Province NSF (05010197), Shenzhen Municipal Science & Technology Programs and the Program for Building Shenzhen and State Key Laboratories , and Shenzhen Development and Reform Commission Discipline Development Project [2017]1434.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.


The mechanisms underlying how cells subjected to genotoxic stress reestablish reduction-oxidation (redox) homeostasis to scavenge genotoxic stress-induced reactive oxygen species (ROS), which maintains the physiological function of cellular processes and cell survival, remain unclear. Herein, we report that, via a TCF-independent mechanism, genotoxic stress induces the enrichment of β-catenin in chromatin, where it forms a complex with ATM phosphorylated-JDP2 and PRMT5. This elicits histone H3R2me1/H3R2me2s-induced transcriptional activation by the recruitment of the WDR5/MLL methyltransferase complexes and concomitant H3K4 methylation at the promoters of multiple genes in GSH-metabolic cascade. Treatment with OICR-9429, a small-molecule antagonist of the WDR5-MLL interaction, inhibits the β-catenin/JDP2/PRMT5 complex-reestablished GSH metabolism, leading to a lethal increase in the already-elevated levels of ROS in the genotoxic-agent treated cancer cells. Therefore, our results unveil a plausible role for β-catenin in reestablishing redox homeostasis upon genotoxic stress and shed light on the mechanisms of inducible chemotherapy resistance in cancer. It is known that genotoxic stress induces high levels of ROS and deplete cellular glutathione stores. Here, Cao et al. uncover a β-catenin-dependent TCF/LEF-independent mechanism that promotes histone-mediated transcriptional activation of glutathione synthesis. Acting as signaling molecules, ROS are essential for the efficient and proper execution of a large number of cellular processes, such as regulation of intracellular signal transduction and gene expression patterns3. However, excessive or prolonged ROS generation results in considerable damage to cellular constituents, various diseased conditions, and the process of ageing5. On the other hand, intracellular thiols, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), play crucial roles in defense against oxidative stress and scavenging of ROS, resulted in maintaining biological redox homeostasis7. Hence, maintaining the redox homeostasis or reestablishing the redox balance in response to stress stimuli is fundamental for physiological cellular function and survival. As the most abundant endogenous low-molecular-weight redox molecule within mammalian cells, glutathione (GSH) plays pleiotropic roles in preventing damage induced by either external or intracellular stimuli. GSH either functions as an antioxidant to scavenge ROS directly or serves as an electron donor for other redox systems, such as glutaredoxin (Grx) and glutathione peroxidase (GPX), to scavenge peroxide-related products. Meanwhile, GSH could also represent a storage sink for cysteine, which displays cellular toxicity when present at high concentrations, and exert detoxification effects by conjugation with and exporting toxicants and xenobiotic compounds out of cells10. Thus, maintaining optimal intracellular GSH levels are crucial for cellular homeostasis and organismal fitness.Reactive oxygen species (ROS) are composed of free radicals with unpaired electron and non-radical oxygen species containing oxygen, such as superoxide  and thioredoxin/glutaredoxin (TRX/GRX), have been also demonstrated to contribute to GSH homeostasis15. For instance, γ-GGT could enhance cellular GSH synthesis by increasing the availability of component amino acids, and TRX/GRX could regulate cellular GSH homeostasis by reduction of oxidized forms, such as glutathione disulfide (GSSG) and glutathione mixed disulfide with protein thiols (GS-R), back to the reduced form of GSH15. By contrast, under conditions of intensive external insults, such as genotoxic stress, high levels of different types of intracellular ROS were induced via several mechanisms, which resulted in prominent depletion of cellular stores of the reduced form of GSH18. Therefore, restoring GSH levels in cells subjected to genotoxic stress is crucial to maintain physiological cellular function and survival. Interestingly, the genotoxic stress-mediated reduction of GSH level could speedily recovery back and even further elevated after a few hours18. However, the mechanisms underlying the genotoxic stress-treated cells reestablished GSH homeostasis remain unclear.Under physiological conditions, intracellular levels of GSH are mostly maintained by de novo synthesis from the precursor amino acids cysteine, glutamate, and glycine, which process is mediated by multiple components in the GSH-metabolic cascade, including the cystine–glutamate transporter SLC7A11 and the GSH rate-limiting enzymatic γ-glutamylcysteine ligase (GCL) and GSH synthetase (GSS)In this study, we report that genotoxic stress activates TCF-independent β-catenin signaling that contributes to reestablishing GSH metabolism and the rapid reduction in genotoxic stress-induced ROS. We demonstrate that β-catenin forms a complex with Jun Dimerization Protein 2 (JDP2) and arginine methyltransferase 5 (PRMT5) that elicit histone H3R2me1/H3R2me2s-induced transcriptional activation via a TCF-independent mechanism, by recruitment of WD repeat domain 5 (WDR5)/myeloid/lymphoid or mixed-lineage leukemia protein (MLL) methyltransferase complexes at the promoters of multiple genes in the GSH-metabolic cascade. Taken together, our results unveil a plausible role for β-catenin in reestablishing redox homeostasis upon genotoxic stress.21. Interestingly, we observed that in response to genotoxic stresses induced by camptothecin (CPT), irradiation (IR), or cisplatin (CDDP), 293FT, OVCAR3, MCF-7, and A549 cells exhibited rapid enrichment of β-catenin in their chromatin, in less than 15 min, but displayed decreased β-catenin/TCF4 complex formation and transcriptional activity , showed 57.4 million total reads and 55.6 million mapped reads, which β-catenin signals was associated with 20,521 peaks, including 6925 peaks in the promoter, 4265 peaks in intergenic, 6821 peaks in intron, 1112 peaks in the exon, 688 peaks in 5′ UTR, 125 peaks in 3′ UTR, 365 peaks in TTS in CPT-treated 293FT cells (PRJNA543097)  and RNA sequencing (RNA-seq) assays were conducted in CPT-treated 293FT cells. Analysis of pooled ChIP-seq data using two replicate data sets, correlated significantly with each other , LEF1, TCF3(TCF7L1), and TCF4 TCF7L2), had no effects on the expression of GSH-metabolic genes and the enrichment of β-catenin on the promoters of these GSH-metabolic genes in genotoxic agent-treated cells , relapse , FIGO stage , SLC7A11 expression  and GSH level , but was associated with shorter overall/relapse-free survival in patients with ovarian cancer treated with platinum-based therapy (both P < 0.05). Moreover, the positive correlation between JDP2 expression and genotoxic stress was further confirmed by gene set enrichment analysis (GSEA), in which JDP2 expression correlated strongly with gene signatures for cisplatin resistance , incorporated into dioleoylphosphatidylcholine (DOPC) nanoliposomes, dramatically enhanced the anti-tumor effect of Topotecan, resulting in a lower growth rate of tumor and GSH levels, but higher ROS levels and apoptosis-positive cells in the tumors , which functions as both a nucleophile and a reductant, plays roles in various cellular processes through regulation of the thiol-redox status. GSH could effectively scavenge ROS  via nonenzymatic reduction or eliminate hydroperoxides required enzymatic catalysis21. Typically, Wnt-ligand-dependent activation of β-catenin signaling results in the interaction of β-catenin with TCF/LEF factors and activation of TCF-dependent transcription. Interestingly, several studies have documented that H2O2 treatment-induced ROS-dependent signaling could shift β-catenin binding from TCF to forkhead box O proteins (FOXOs) and induce FOXO-mediated transcription, resulting in removal of H2O2 via upregulation of manganese superoxide dismutase and catalase and decreased β-catenin/TCF transcriptional activity23, which suggested that β-catenin signaling plays roles in the reduction of ROS via a TCF-independent mechanism. Meanwhile, it was also reported that genotoxic stress-induced poly-ADP-ribosylated PARP1 and upregulated Ku70, which competed for the interaction of TCF4 with β-catenin and reduced β-catenin/TCF transcriptional activity31. However, the biological role of β-catenin signaling in response to genotoxic stress remains unclear. In the present study, we observed that, unlike H2O2 treatment, genotoxic stresses did not increase the β-catenin/FOXOs interaction, suggesting that the effect of β-catenin signaling on the reduction of genotoxic stress-induced ROS might be through other mechanisms. We further demonstrated that genotoxic stresses induced the formation of β-catenin/JDP2/PRMT5 complex, which facilitated the restoration of GSH homeostasis via transcriptional upregulation of multiple genes in the GSH-metabolic cascade, resulting in elimination of genotoxic stress-induced ROS. Therefore, our results revealed a mechanism by which β-catenin signaling maintains redox homeostasis in genotoxic stress-treated cells. However, it is worth to note that expression level of four TCF/LEF factors individually silenced in this study was still detectable. It would be better to knockout all four TCF/LEF factors simultaneously via CRISPR-Cas9 system to further rule out the possibility of functional redundancy of these TCF/LEF factors on GSH regulation.The β-catenin signaling pathway plays vital roles in regulating embryonic development, stem cell maintenance, tissue homeostasis, and the progression and development of cancer via TCF-dependent and -independent mechanisms33. Further studies demonstrated that JDP2-mediated transcriptional repression acts via distinct mechanisms, including decreasing histone acetylation through associating with histone deacetylases (HDACs), inhibiting p300-mediated acetylation by directly interacting with the core histones, or by promoting supercoiling into circular DNA in the presence of core histones26. Meanwhile, JDP2 also acts as a transcriptional activator, such as contributing to the promotion of progesterone receptor- or sRANKL (receptor activator of nuclear factor kappa B ligand)-mediated transcriptional activation35. However, the precise mechanism of JDP2-mediated transcriptional activation remains unknown. In this study, we validated that genotoxic stress-triggered JDP2 formed a complex with β-catenin and PRMT5, which inhibited the binding of JDP2 to histones and HDACs, thereby acting as transcriptional co-activator complex that upregulated multiple GSH-metabolic genes. Therefore, our results unveiled a mechanism by which JDP2-mediated transcriptional activation. Contrastingly, JDP2 was reported to play a role in the antioxidant response through an association with the NF-E2-related factor 2 (NRF2)/ MAF BZIP transcription factor K (MAFK) complex36. However, the mechanism by which JDP2-reduced ROS production has not been clarified. In this study, we demonstrated that JDP2 was enriched at gene promoters and transcriptionally upregulated multiple GSH-metabolic genes upon genotoxic stress, resulting in restoration of GSH metabolism and a reduction in genotoxic stress-induced ROS, consequently leading to resistance of cancer to genotoxic chemotherapeutics. Thus, our results also uncovered a plausible mechanism for the JDP2-mediated maintenance of ROS homeostasis and chemoresistance.Human JDP2, originally identified as an AP-1 repressor, is involved in the transcriptional repression of TRE-dependent and CRE-dependent genes via heterodimerization with c-Jun or ATF-238. For instance, ROS-induced cysteine S-glutathionylation promoted TAZ stability that was critical for ROS-mediated transactivation of TAZ, and oxidative stress-induced S-glutathionylation of mitochondrial thymidine kinase 2 (TK2) have significant impacts on TK2 degradation and mitochondrial DNA precursor synthesis40. Herein, we demonstrated that β-catenin formed a complex with ATM phosphorylated-JDP2 and PRMT5 that associated with the promoters of multiple genes in the GSH-metabolic cascade, resulted in reestablishing GSH homeostasis upon genotoxic stress. However, we found that genotoxic stress did not induce either S-glutathionylational modification or stabilization of either JDP2, or β-catenin, or ATM, or PRMT5 , such as S-glutathionylation of sulfur-containing amino acids, controls a wide range of intracellular protein activities and is involved in the response of oxidative stress41. Marullo et al. reported that CDDP treatment upregulated ROS levels in cancer cells were dependent on the mitochondria instead of nuclear DNA damage signaling in DU145 and DU145ρ° cells 42. Furthermore, hydroxyl radical scavenger was found to be essential for ameliorating the nephrotoxicity following CDDP chemotherapy via protecting mitochondria and preventing oxidative stress43. Consistently, exposing cells to either γ-radiation or α-particles significantly enhanced cellular ROS levels, such as superoxide anions and hydrogen peroxide, via inducing a reversible mitochondrial permeability transition that attributable to normal cell metabolism45. Therefore, genotoxic stress provoked by inhibitor of DNA topoisomerase, or chemotherapeutic drug (CDDP), or γ-radiation could induce multiple types of ROS. On the other hand, several studies have provided evidence that depletion of GSH could potentiate apoptosis provoked by CPT, or CDDP, or ionizing radiation. These results suggest that cytotoxicity provoked by these reagents were linked to intracellular GSH alteration, which was related with chemoresistance and radioresistance and cancer progression48. Thus, exploring the mechanism underlying the reprogramming of GSH metabolism in genotoxic stress may aid the identification of therapeutic targets for cancer and avoid the side effects of chemoradiotherapy. According to our finding that genotoxic stress-triggered β-catenin/JDP2/PRMT5 complex formation resulted in WDR5/MLL methyltransferase complex-mediated restoration of GSH homeostasis, we demonstrated that combined treatment with OICR-9429, an antagonist of the WDR5-MLL interaction, could inhibit the GSH-metabolism reestablishing and lead to a lethal increase in the already-elevated levels of ROS in genotoxic chemotherapeutics-treated cancer cells. Therefore, understanding the precise mechanism underlying genotoxic stress-induced reestablishing redox homeostasis would be not only benefit the treatment of a large group of cancer patients, but also would increase our knowledge of the biological basis of cancer progression.Genotoxicity-based chemotherapy and radiotherapy, which induce severe DNA damage, ROS production, and apoptosis of cancer cells, remain the standard first-line treatments for most human cancers. For instance, Sen et al. have previously reported that CPT-induced ROS elevation, especially the level of superoxide radical and hydroxyl radical, promoted apoptotic cell death via dysfunction of cellular respiration and mitochondrial hyperpolarizationThe OVCAR3 ovarian cancer cell line, A549 lung cancer cell line, MCF7 breast cancer cell line, and human embryonic kidney 293FT cell line were obtained from American Type Culture Collection  and were grown in the DMEM medium  supplemented with 10% fetal bovine serum . All the cell lines have been tested for mycoplasma contamination, and were authenticated by short tandem repeat (STR) fingerprinting at Medicine Lab of Forensic Medicine Department of Sun Yat-Sen University (China).The ORFs of JDP2 , β-catenin, PRMT5 and truncated-JDP2 and -β-catenin fragments were cloned into pCDNA3 or pSin-EF2-vector. ShRNAs targeting JDP2 were cloned into the pSuper Retro viral vector. The TCF/LEF1 reporter plasmids contain wild-type  or mutated . Transfection of siRNAs or plasmids was performed using the Lipofectamine 3000 reagent  according to the manufacturer’s instruction. All siRNA oligonucleotides are listed as Supplementary Data GAPDH to control the variability in expression levels and calculated as 2C−[(tCof gene) – (tof GAPDH)], where Ct represents the threshold cycle for each transcript. All primers are listed as Supplementary Data The total RNA was extracted from indicated cell using the Trizol (Life Technologies) reagent according to the manufacturer’s instruction. Real-time reverse transcription–polymerase chain reaction (PCR) primers and probes were designed with the assistance of the Primer Express v 2.0 software . Expression data were normalized to the geometric mean of housekeeping gene Camptothecin (CPT), Cisplatin (CDDP), Topotecan, ATM inhibitor (KU55933), and WDR5 inhibitor (OICR-9429) PRMT5 inhibitor (GSK591) were purchased from MedChemexpress . Human recombinant His-JDP2 protein was purchased from Abcam .IB was performed according to a standard protocol with the following antibody: anti-β-catenin , anti-α-catenin , and anti-PARP1  antibodies were purchased from BD company . Anti-JDP2 , anti-PRMT5 , anti-SLC7A11 ab37185,1:200), anti-GCLM , anti-TCF4 , anti-p-SQ/TQ ATM/ATP , anti-H3 , anti-H4 , anti-ATM , anti-ATF3 , anti-HDAC3, anti-FOXO3 , anti-TCF1 , anti-LEF1 , anti-cleaved PARP1  and anti-GSH  antibodies were purchased from Abcam . Anti-c-Jun , anti-cleaved Caspase 3 , anti-His , anti-TCF3 , anti-SMARCA4 , and anti-HNRNPA2B1  antibodies were purchased from cell signaling technology . Anti-WDR5 antibody  was purchased from Millipore . Anti-Flag  and anti-HA  antibodies were purchased from Sigma-Aldrich . Membranes were stripped and re-probed with an anti-α-tubulin antibody , or anti-GAPDH antibody , or anti-β-actin antibody  as a protein loading control. Uncropped images of Immunoblotting were provided in the Supplementary Fig. 5,1:200, IF staining was carried out on tumor cell chamber slide cultures (Thermo Fisher Scientific), and followed by the antibody: β-catenin  and anti-JDP2  antibodies. The secondary antibody was goat anti-rabbit IgG (H + L) conjugated with Alexa Fluor 568 (Thermo Fisher Scientific). Then cells were mounted with Antifade Mountant with DAPI (Thermo Fisher Scientific). The images were captured using the AxioVision Rel.4.6 computerized image analysis system .IHC analysis was performed to determine altered protein expression in 146 paraffin-embedded ovarian cancer tissues with Rabbit anti-JDP2  and anti-SLC7A11  antibodies overnight at 4 °C. The degree of immunostaining of formalin-fixed, paraffin-embedded sections were reviewed and scored separately by two independent pathologists uninformed of the histopathological features and patient data of the samples. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The scores given by the two independent pathologists were combined into a mean score for further comparative evaluation. Tumor cell proportions were scored as follows: 0, no positive tumor cells; 1, < 10% positive tumor cells; 2, 10–35% positive tumor cells; 3, 35–75% positive tumor cells; 4, > 75% positive tumor cells. Staining intensity was graded according to the following standard: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); 4, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated protein expression in benign esophageal epithelia and malignant lesions by determining the SI, with possible scores of 0, 2, 3, 4, 6, 8, 9, 12, and 16. Samples with a SI ≥ 8 were determined as high expression and samples with a SI < 8 were determined as low expression. Cutoff values were determined on the basis of a measure of heterogeneity using the log-rank test with respect to overall survival.2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and protease inhibitor mixture), and incubated for 5 min on ice. The cell pellet was collected by low-speed centrifugation . After washed once in buffer A, the cell pellet was lysed in 200 µl of buffer B  for 10 min on ice. Insoluble chromatin was collected by centrifugation , washed once in buffer B, and resuspended in SDS sample buffer.Briefly, cells were harvested and resuspended in 200 µl of buffer A , was treated with DNAase (stemcell Technologies) for 1.0 h and then incubated with anti-β-catenin antibody  overnight at 4 °C. After then the supernatants were incubated with 20 μl of protein G-agarose beads overnight at 4 °C. The agarose beads were then washed six times with wash buffer . The eluates were pooled and concentrated in a 10-kDa MW cutoff filter unit  to a volume of 30 μl. The eluates were denaturation by addition of 10 μl 4 × sample buffer and were subjected to mass spectrometry (MS) analysis.The chromatin fraction, prepared from CPT -treated β-catenin-transduced 293FT cells  underneath the skin of female NOD-SCID IL-2rγ−/− (NSG) mice (4–8 weeks old). Tumor growth was monitored by measurements of tumor diameters once weekly, and the tumor volume was calculated as /2. Recipient mice bearing ~0.2 cm3 size of tumors were intraperitoneally treated with vehicle (control), CDDP (5 mg/kg), Topotecan (10 mg/kg), or OICR-9429 (3 mg/kg) alone, or combined OICR-9429 (3 mg/kg) and Topotecan (10 mg/kg), or combined OICR-9429 (3 mg/kg) and CDDP (5 mg/kg body weight), three times per week for up to 6 weeks. In the intraperitoneal tumor model, OVCAR3 luciferase expressing cells (5 × 106) were injected intraperitoneally into female nu/nu nude mice. Recipient mice-bearing tumors when luminescence signal reached > 2 × 107 p/sec/cm2/sr were treatment with Topotecan (10 mg/kg) for up to 6 weeks. Tumor burden was measured weekly in mice injected with d-luciferin using the IVIS Spectrum In Vivo Imager.All of the animal procedures were complied with all relevant ethical regulations for animal testing and research, and the ethical approval was approved by the Sun Yat-sen University Animal Care Committee. Two models were examined in this study. In the subcutaneous patient-derived xenografts (PDX) tumor model, a fragment of freshly tumor isolated clinical ovarian cancer patient tissues were placed subcutaneously , or stained with TUNEL  and DAPI (Thermo Fisher Scientific) according to manufacturer’s protocols. The images were captured using the AxioVision Rel.4.6 computerized image analysis system .7 cells. The cross-linked protein/DNA complexes were collected by magnetic pull down, and then were eluted from beads by elution buffer. After reverse cross-link of protein/DNA complexes to free DNA, PCR was performed using specific primers. All ChIP primers are listed as Supplementary Data The entire procedure was performed with the chromatin immunoprecipitation (ChIPs) assay kit  according to the manufacturer’s instructions. Briefly, indicated cells were grown to 70–80% confluence on 100-mm culture dish, and were fixed with 1% formaldehyde to cross-link proteins to DNA. The cell lysates were sonicated to shear DNA into small uniform fragments. Equal aliquots of chromatin supernatants were then immunoprecipitated overnight at 4 °C using anti-JDP2 (ab40916), or anti-β-catenin (BD 610154), anti-PRMT5 NBP2-19935), anti-TCF4 (ab76151), anti-WDR5 (07-706), anti-H3 (ab1791), anti-H3R2me1(ab15584), anti-H3R2me2s (ab194684), anti-H3R8me2s (ab130740), anti-H2AR3me2s (ab22397), anti-H4R3me2s (ab5823), anti-H3K4me3 (ab12209), anti-RNAP (ab76123), anti-TAF3 antibodies, or an anti-Anti-IgG antibody  respectively, with protein G magnetic beads. Two micrograms of each antibody was used for ChIP per 105, anti-Tg for 5 min. The cell pellet was washed with PBS (pH 7.4) 2–3 times and then transferred to a 1.5 -mL microcentrifuge tube and centrifuged at 500 × g for 2–3 min. The cell pellet was resuspended in 200 μl ice-cold CER I and vortexed the tube vigorously and incubated on ice for 10 min. The cells were added 11 μl ice-cold CER II to the tube, vortexed for 5 s and centrifuged for 5 min at 16,000 × g. The supernatant (cytoplasmic extract) was immediately transfer to a clean pre-chilled tube. The pelleted nuclei were resuspended in 100 μl ice-cold NER, which contains nuclei and vortexed for seconds every 10 min, for a total of 40 min. The supernatant (nuclear extract) fraction was then centrifuged at ~16,000 × g for 10 min, and then immediately transferred the to a clean pre-chilled tube. Store extracts at −80 °C until use.Nuclear fraction was performed by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) according to the manufacturer’s instructions. The indicated cells were treated with 0.25% trypsin-EDTA, and subsequently harvested at centrifuge at 500 × Electrophoretic mobility shift assay was performed by using the LightShift Chemiluminescent EMSA kit (Thermo Fisher) according to the manufacturer’s instruction. To evaluate the effects of genotoxic stress on JDP2 binding to DNA in the nucleus, nuclear extracts from indicated cells were used in an EMSA. The biotin-labeled oligonucleotide probe was synthesized (Thermo Fisher), and unlabeled oligonucleotide with an identical was synthesized as a control. OCT-1 probe was used as a loading control. The sequences of probes are listed in Supplementary Data The GSH-Glo™ Glutathione Assay Kit  and the cysteine Detection Assay Kit  were used to determine the reduced GSH content and cysteine in indicated cells, and the luciferase signal was determined by Spectra Mas M5 (Molecular Devices). Experiments were performed with biological replicates.3 cells were cultured in a 96-well plate, and then cell permeable oxidative fluorescent dye 2’,7’ dichlorodihydrofluorescein diacetate (H2DCFDA) was added to wells and incubated for 1 h at 37 °C. ROS levels were quantified by measuring fluorescence intensity at excitation and emission wavelength of 490 and 525 nm, respectively using Spectro fluorimeter.Intracellular ROS levels were examined using Fluorometric Intracellular ROS Kit . Briefly, 1 × 10DNA damage was measured by immunofluorescence with an anti-8-hydroxydeoxyguanosine (8OHdG) monoclonal antibody (Abcam), following the manufacturer’s instructions. The secondary antibody was goat anti-mouse IgG2α conjugated with Alexa Fluor 488. Then cells were mounted with Vectashield Mounting Medium with DAPI (Thermo Fisher Scientific). The images were captured using the AxioVision Rel.4.6 computerized image analysis system .51, with FDR ≤ 0.05. A peak was assigned to the transcriptional start site (TSS) of a RefSeq gene when falling into the surrounding 4 kb ( ± 2 kb). Promoters were defined as 6 kb regions ( ± 3 kb) surrounding the TSS. The promoter peaks analysis, KEGG Pathway, and GO analysis were implemented using KOBAS (version 3.0). P-value was calculated by Student’s t test.ChIP-seq assay was performed using Kit according to the manufacturer’s instructions. Briefly, chromatin was isolated from β-catenin-transduced 293FT cells treated with CPT . DNA library was labeled for high-throughput sequencing using the Illumina ChIP-seq Library kit following the manufacturer’s protocol. The reads were first mapped the human genome sequence (hg19) by using bowtie2 (version 2.2.9). The parameters were set as default, SAMtools (version 0.1.19) were then used to convert files to bam format, sort. Peaks were called using MACS (version 2.1.1)P-value was calculated by Student’s t test.β-catenin-silenced or control 293FT cells were treated with CPT , and the total RNA was extracted and purified using the Trizol (Life Technologies) reagent according to the manufacturer’s instruction. RNA quantitation and quality control were performed by Bioanalyzer 2100 (Agilent Technologies). Construction of stranded RNA-seq libraries for high-throughput sequencing were done on Illumina HiSeq X Ten following the manufacturer’s protocol. RNA-seq reads were mapped to reference genome of Illumina Ensembl genome GRCh37 using HISAT2 version 2.2.9. Mapped reads were summarized for each gene using htseq-count version 0.11.2. Differential expression analysis was implemented using DEGseq version 1.36.1. This study, which complied with all relevant ethical regulations for work with human participants, was conducted on a total of 146 paraffin-embedded ovarian cancer samples and 30 freshly collected ovarian cancer tissues. The clinical information regarding the samples is summarized in Supplementary Tables Ten thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of luciferase reporter plasmids or the control-luciferase plasmid, plus 5 ng of pRL-TK renilla plasmid , were transfected into indicated cells using the Lipofectamine 3000 reagent  according to the manufacturer’s recommendation. Luciferase and renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit  according to a protocol provided by the manufacturer. Three independent experiments were performed, and the data are presented as mean ± SD.3) were seeded in six-well culture plates and cultured with fresh median with CPT (10 μM). After incubation for an additional 10 days, the colonies were stained with 1% crystal violet for 30 s after fixation with 10% formaldehyde for 5 min. Colonies of at least 50 cells were quantified by Analysis software (Olympus Biosystems).The indicated cells , according to the manufacturer’s instruction. Indicated treated cells were washed with PBS and then with the Annexin-V binding solution. Subsequently, the cells were added 150 μl of an Annexin-V antibody in binding buffer and incubated for 15 min, and followed by addition of 1.5 μl of PI at 1 mg/ml for a further incubation 5 min. The apoptotic cells were analyzed on a flow cytometer .t test. Bivariate correlations between study variables were calculated by Spearman’s rank correlation coefficients. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The significance of various variables for survival was analyzed by univariate and multivariate Cox regression analyses. Statistical analyses were performed using the SPSS 11.0 statistical software package. Data represent mean ± SD. P-values of 0.05 or less were considered statistically significant.Statistical tests for data analysis included Fisher’s exact test, log-rank test, Chi-square test and Student’s two-tailed Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Description of Supplementary dataReporting SummarySource Data