{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Citation":"https:\/\/open.library.ubc.ca\/terms#identifierCitation","Contributor":"http:\/\/purl.org\/dc\/terms\/contributor","CopyrightHolder":"https:\/\/open.library.ubc.ca\/terms#rightsCopyright","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","PeerReviewStatus":"https:\/\/open.library.ubc.ca\/terms#peerReviewStatus","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","PublisherDOI":"https:\/\/open.library.ubc.ca\/terms#publisherDOI","Rights":"http:\/\/purl.org\/dc\/terms\/rights","RightsURI":"https:\/\/open.library.ubc.ca\/terms#rightsURI","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Subject":"http:\/\/purl.org\/dc\/terms\/subject","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Other UBC","@language":"en"},{"@value":"Non UBC","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Citation":[{"@value":"Journal of Experimental & Clinical Cancer Research. 2021 Mar 01;40(1):88","@language":"en"}],"Contributor":[{"@value":"Vancouver Coastal Health Authority. Research Institute","@language":"en"}],"CopyrightHolder":[{"@value":"The Author(s)","@language":"en"}],"Creator":[{"@value":"Su, Wenjie","@language":"en"},{"@value":"Zhu, Shikai","@language":"en"},{"@value":"Chen, Kai","@language":"en"},{"@value":"Yang, Hongji","@language":"en"},{"@value":"Tian, Mingwu","@language":"en"},{"@value":"Fu, Qiang","@language":"en"},{"@value":"Shi, Ganggang","@language":"en"},{"@value":"Feng, Shijian","@language":"en"},{"@value":"Ren, Dianyun","@language":"en"},{"@value":"Jin, Xin","@language":"en"},{"@value":"Yang, Chong","@language":"en"}],"DateAvailable":[{"@value":"2021-03-03T18:14:01Z","@language":"en"}],"DateIssued":[{"@value":"2021-03-01","@language":"en"}],"Description":[{"@value":"Background:\r\n                WD repeat domain 3 (WDR3) is involved in a variety of cellular processes including gene regulation, cell cycle progression, signal transduction and apoptosis. However, the biological role of WDR3 in pancreatic cancer and the associated mechanism remains unclear. We seek to explore the immune-independent functions and relevant mechanism for WDR3 in pancreatic cancer.\r\n              \r\n              \r\n                Methods:\r\n                The GEPIA web tool was searched, and IHC assays were conducted to determine the mRNA and protein expression levels of WDR3 in pancreatic cancer patients. MTS, colony formation, and transwell assays were conducted to determine the biological role of WDR3 in human cancer. Western blot analysis, RT-qPCR, and immunohistochemistry were used to detect the expression of specific genes. An immunoprecipitation assay was used to explore protein-protein interactions.\r\n              \r\n              \r\n                Results:\r\n                Our study proved that overexpressed WDR3 was correlated with poor survival in pancreatic cancer and that WDR3 silencing significantly inhibited the proliferation, invasion, and tumor growth of pancreatic cancer. Furthermore, WDR3 activated the Hippo signaling pathway by inducing yes association protein 1 (YAP1) expression, and the combination of WDR3 silencing and administration of the YAP1 inhibitor TED-347 had a synergistic inhibitory effect on the progression of pancreatic cancer. Finally, the upregulation of YAP1 expression induced by WDR3 was dependent on an interaction with GATA binding protein 4 (GATA4), the transcription factor of YAP1, which interaction induced the nuclear translocation of GATA4 in pancreatic cancer cells.\r\n              \r\n              \r\n                Conclusions:\r\n                We identified a novel mechanism by which WDR3 plays a critical role in promoting pancreatic cancer progression by activating the Hippo signaling pathway through the interaction with GATA4. Therefore, WDR3 is potentially a therapeutic target for pancreatic cancer treatment.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/77432?expand=metadata","@language":"en"}],"FullText":[{"@value":"RESEARCH Open AccessOverexpressed WDR3 induces theactivation of Hippo pathway by interactingwith GATA4 in pancreatic cancerWenjie Su1, Shikai Zhu2,3, Kai Chen2,3, Hongji Yang2,3, Mingwu Tian2,3, Qiang Fu2,3,4, Ganggang Shi5, Shijian Feng5,Dianyun Ren6, Xin Jin6 and Chong Yang2,3*AbstractBackground: WD repeat domain 3 (WDR3) is involved in a variety of cellular processes including gene regulation,cell cycle progression, signal transduction and apoptosis. However, the biological role of WDR3 in pancreatic cancerand the associated mechanism remains unclear. We seek to explore the immune-independent functions andrelevant mechanism for WDR3 in pancreatic cancer.Methods: The GEPIA web tool was searched, and IHC assays were conducted to determine the mRNA and proteinexpression levels of WDR3 in pancreatic cancer patients. MTS, colony formation, and transwell assays were conductedto determine the biological role of WDR3 in human cancer. Western blot analysis, RT-qPCR, and immunohistochemistrywere used to detect the expression of specific genes. An immunoprecipitation assay was used to explore protein-protein interactions.Results: Our study proved that overexpressed WDR3 was correlated with poor survival in pancreatic cancer and thatWDR3 silencing significantly inhibited the proliferation, invasion, and tumor growth of pancreatic cancer. Furthermore,WDR3 activated the Hippo signaling pathway by inducing yes association protein 1 (YAP1) expression, and thecombination of WDR3 silencing and administration of the YAP1 inhibitor TED-347 had a synergistic inhibitory effect onthe progression of pancreatic cancer. Finally, the upregulation of YAP1 expression induced by WDR3 was dependenton an interaction with GATA binding protein 4 (GATA4), the transcription factor of YAP1, which interaction induced thenuclear translocation of GATA4 in pancreatic cancer cells.Conclusions: We identified a novel mechanism by which WDR3 plays a critical role in promoting pancreatic cancerprogression by activating the Hippo signaling pathway through the interaction with GATA4. Therefore, WDR3 ispotentially a therapeutic target for pancreatic cancer treatment.Keywords: Pancreatic Cancer, WDR3, GATA4, YAP1, Hippo signaling pathway\u00a9 The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http:\/\/creativecommons.org\/licenses\/by\/4.0\/.The Creative Commons Public Domain Dedication waiver (http:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.* Correspondence: yangchong@med.uestc.edu.cn2Clinical Immunology Translational Medicine Key Laboratory of SichuanProvince & Organ Transplantation Center, Sichuan Provincial People\u2019sHospital, University of Electronic Science and Technology of China, Chengdu611731, Sichuan, China3Chinese Academy of Sciences Sichuan Translational Medicine ResearchHospital, Chengdu 610072, Sichuan, ChinaFull list of author information is available at the end of the articleSu et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 https:\/\/doi.org\/10.1186\/s13046-021-01879-wBackgroundPancreatic ductal adenocarcinoma (PDAC) is a highlymalignant disease with a 5-year survival rate of only 10%for all stages combined [1]. According to GLOBOCAN2020 estimates, PDAC is the seventh leading cause ofcancer-related mortality worldwide and accounting forabout 495,773 new cases and 466,003 deaths [2]. Surgicalresection remains the only treatment that offers a cura-tive potential, whereas most cases lost the chance be-cause of those presenting with advanced stages at thetime of diagnosis [3]. Currently, molecular targeted ther-apy showed great potential for improving the survivalrates of PDAC patients [4]. Then, it is significant for de-veloping new therapeutic strategies and potential thera-peutic targets for pancreatic cancer treatment.WD repeat domain 3 (WDR3), also known as DIP2 orUTP12, belongs to the WD-repeat family and is a com-ponent of the 80 S complex of the small subunit proces-some, which is implicated in the 40 S ribosome synthesispathway [5]. It has been reported that WDR3 is involvedin a variety of cellular processes including genome sta-bility, cell proliferation, signal transduction, and apop-tosis [6\u20138]. McMahon et al proved that suppression ofWDR3 reduced breast carcinoma cell proliferation andfocus formation [7]. Also, Akdi et al indicated WDR3gene expression is associated with thyroid cancer risk inspecial populations [9], and WDR3 can modulate gen-ome stability in thyroid cancer patients [10]. These stud-ies revealed WDR3 confer growth and proliferativeadvantages of some malignant cancer, whereas the bio-logical role of WDR3 in pancreatic cancer and the rele-vant mechanism remain unclear.Studies proved the Hippo signaling pathway plays acritical role in modulating cell proliferation and has beendemonstrated to contribute to the progression of malig-nant cancers, including pancreatic cancer [11\u201314]. Thecore components of the Hippo signaling pathway, in-cluding yes association protein 1 (YAP1), promote themigration, invasion, and malignancy of cancer cells [15],and inhibiting YAP1 expression suppresses pancreaticcancer progression by disrupting tumor-stroma inter-action [16, 17]. YAP1 usually enters the nucleus and in-teracts with other transcription factors, including TEAdomain (TEAD) family members, to regulate down-stream gene targets [18\u201320]. Study of connective tissuegrowth factor (CTGF) and cysteine rich angiogenic in-ducer 61 (CYR61), the major downstream gene targetsregulated by YAP1, has provided new insights into thephysiological\/pathological functions of Hippo pathwayeffectors [12, 21]. CYR61 and CTGF had been reportedto act as factors stimulating aggressiveness in a variety ofcancers. CYR61 expression was exorbitantly higher incancer cells and significantly triggered the aggressivephenotype in PADC [22]. CTGF was a fibrosis-relatedgene related to pancreatic cancer progression by protect-ing pancreatic cancer cells from hypoxia-mediated apop-tosis, and tumor cell-derived CTGF was vital forpancreatic cancer growth [23]. Therefore, exploringmethods to inhibit YAP1 expression is essential for im-proving pancreatic cancer therapy.In our study, we proved that overexpressed WDR3was correlated with poor survival in pancreatic cancerpatients. Furthermore, WDR3 silencing could signifi-cantly decrease the proliferative and invasive abilities ofpancreatic cancer cells by inducing YAP1 inhibition,which was found to rely on the interaction betweenWDR3 and GATA4. Taken together, our resultsemphasize the importance of WDR3 as a therapeutictarget in pancreatic cancer.Materials and methodsCell cultureThe PANC-1, MIA PaCa-2, and BxPC-3 cell lines werepurchased from the Type Culture Collection Cell Bankof the Chinese Academy of Sciences (Shanghai, China).PANC-1 and MIA PaCa-2 cells were cultured in Dulbec-co\u2019s Modified Eagle\u2019s Medium (DMEM) (#30030,Thermo Fisher Scientific) supplemented with 10% fetalbovine serum (FBS) (#10099141, Thermo Fisher Scien-tific) and 1% penicillin\/streptomycin at 37 \u00b0C in a 5%CO2 incubator. BxPC-3 cells were cultured in RPMI-1640 medium (#88365, Thermo Fisher Scientific) sup-plemented with10% FBS and 1% penicillin\/streptomycinat 37 \u00b0C in a 5% CO2 incubator.Antibodies and chemicalsAn anti-WDR3 antibody (#ab176817, working dilution1:1000) was purchased from Abcam. An anti-GAPDHantibody (#10494\u20131-AP, working dilution: 1:3000), anti-GATA4 antibody (#19530\u20131-AP, working dilution: 1:1000), and anti-YAP1 antibody (#13584\u20131-AP, workingdilution: 1:1000) were acquired from Proteintech. TED-347 (HY-125269, working concentration: 10 \u03bcM) wasprocured from MedChemExpress (USA).Immunoprecipitation and western blot analysisWhole cell lysates were obtained with RIPA lysis buffer(Cell Signaling Technology, Danvers, MA) containing1% protease and phosphatase inhibitors (Sigma-Aldrich)on ice. The resulting cell lysates were centrifuged at 12,000 rpm for 15min at 4 \u00b0C to remove undissolved im-purities and collect the supernatants. The protein con-centration was quantified using a BCA assay (#P0012S,Beyotime). Protein extracts (500 \u03bcg) were incubated withappropriate primary antibody beads overnight for an im-munoprecipitation assay or directly evaluated for west-ern blot analysis. The precipitated immune complexeswere subjected to SDS-PAGE, transferred to 0.45-\u03bcmSu et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 2 of 15polyvinylidene difluoride (PVDF) membranes, and thenimmunoblotted with specific primary antibodies. Thesignal intensities of bands were quantified using ImageJsoftware.Liquid chromatography-tandem mass spectrometry\/massspectrometry (LC-MS\/MS) analysisTo identify potential WDR3-binding proteins, 293 T cellstransduced with pcDNA3-WDR3 were collected for as-says. WDR3 was pulled down by IP using an anti-WDR3antibody and protein A +G agarose (#P2012, Beyotime) at4 \u00b0C. LC-MS\/MS analysis was performed using a ThermoUltimate3000 liquid phase combined with Q Exactive Plushigh-resolution mass spectrometry at Shanghai AppliedProtein Technology. The data were retrieved with max-quant (v1.6.6) software, and the database retrieval algo-rithm was Andromeda. The database used in the searchwas the human proteome reference database of UniProt.The results were screened with a 1% FDR at the proteinand peptide levels.RNA-seqA total amount of 1 \u03bcg of RNA per sample was used asthe input material for RNA sample preparations. Se-quencing libraries were generated using the NEBNext\u00aeUltraTM RNA Library Prep Kit for Illumina\u00ae (NEB,USA) following the manufacturer\u2019s recommendations,and index codes were added to attribute sequences toeach sample. Clustering of the index-coded samples wasperformed on the cBot Cluster Generation System usingthe TruSeq PE Cluster Kit v3-cBot-HS (Illumina) ac-cording to the manufacturer\u2019s instructions. After clustergeneration, the library preparations were sequenced onan Illumina Novaseq platform, and 150-bp paired-endreads were generated. FeatureCounts v1.5.0-p3 was usedto count the read numbers mapped to each gene. Differ-ential expression analysis of two conditions\/groups (twobiological replicates per condition) was performed usingthe DESeq2 R package (1.16.1). We used the cluster Pro-filer R package to test the statistical enrichment of differ-entially expressed genes in KEGG pathways.Quantitative RT-PCR assayTotal RNA was extracted with RNAiso Plus (#15596026,Invitrogen). The PrimeScript RT Reagent Kit (#RR047A,TAKARA, Japan) was used for reverse transcription. Real-time PCR (RT-PCR) was conducted with a TB Green\u2122Fast qPCR Mix kit (#RR430A, TAKARA, Japan). The2-\u0394Ct method was used to quantify fold changes withnormalization to GAPDH. Detailed information on theprimer sequences is shown in Table S1.RNA interferenceSh-Control and gene-specific shRNAs were procured fromSigma-Aldrich, and si-Control and gene-specific siRNAswere provided by RiboBio. Pancreatic cancer cells weretransfected with siRNA using Lipofectamine 2000(#11668019, Thermo Fisher Scientific) in accordance withthe manufacturer\u2019s instructions for 24 h, and then the Li-pofectamine 2000-containing medium was replaced withfresh DMEM containing 10% FBS. 293 T cells were trans-fected with shRNA plasmids and packaging plasmids(pVSV-G and pEXQV) in Lipofectamine 2000 accordingto the manufacturer\u2019s instructions for 24 h, and the Lipo-fectamine 2000-containing medium was replaced withfresh DMEM containing 10% FBS and 1mM sodiumpyruvate. At 48 h post transfection, the virus culturemedium was collected and added to pancreatic cancercells for 24 h of culture, after which the infected cells wereselected with 1 \u03bcg\/ml puromycin. The shRNA and siRNAsequences are shown in Table S2.Chromatin immunoprecipitation (ChIP) and ChIP-qPCRChIP was performed with the Chromatin Extraction Kit(#ab117152, Abcam) and ChIP Kit Magnetic-One Step(#ab156907, Abcam) according to the manufacturer\u2019s in-structions. Purified DNA was analyzed using RT-PCRwith a TB Green\u2122 Fast qPCR Mix kit (#RR430A, TAKARA, Japan) following the manufacturer\u2019s protocols. TheChIP-qPCR primers are shown in Table S3.Nuclear and cytoplasmic extracts preparationCells were collected and the cell pellet was resuspended in1mL of Buffer A (10mM HEPES-KOH, pH 7.9, 1.5 mMMgCl2, 10mM KCl, 0.1% NP-40) to lyse the cells on icefor 10min. Samples were spined down at 6500 rpm 4 \u00b0Cfor 3 min to pellet the nuclei. Nuclei pellet was washedwith Buffer A and spined down at 3500 rpm for 5min at4 \u00b0C. The cell pellet was lysed by IP buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 1% Triton X-100, 1% sodiumdeoxycholate, and 1% protease inhibitor cocktails) on icefor more than 30min. Protein concentration was deter-mined by BCA protein quantification assay.Colony formation assayFor colony formation assays, 500 pancreatic cancer cellstransfected with sh-Control or sh-WDR3s were seededin a six-well plate and cultured for approximately 10\u201312days. Then, the colonies were fixed in methanol for 30mins and stained with a 4 g\/l crystal violet solution for30 mins. The colonies were photographed, and the num-ber of colonies was counted. All assays were performedin triplicate.Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 3 of 15MTS assayFor MTS assays, transfected pancreatic cancer cells wereseeded in 96-well plates with 2500 cells per well. After72 h of culture, [3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium](MTS reagent) (Abcam, #ab197010, USA) was added toeach well for three hours of culture according to themanufacturer\u2019s instructions. The absorbance in each wellwas measured with a microplate reader at 490 nm. Eachexperiment included five replicates and was performedin triplicate.Cell invasion assayCell invasion assays were performed using transwellchambers (8-\u03bcm pore size; Millipore) with a Matrigel (BDBiosciences, CA, USA) matrix. In brief, 600 \u03bcl of completemedium supplemented with 30% FBS was added to thebottom chamber, and 105 transfected pancreatic cancercells were suspended in 200 \u03bcL of serum-free medium andadded to the upper chamber. After culturing for 12\u201324 h,the cells on the top surface of the membrane were mech-anically removed using a cotton swab. The cells on thebottom surface of the membrane were fixed in methanolfor 30mins and stained with a 4 g\/l crystal violet solutionfor 30mins. The invaded cells were counted under amicroscope, with five fields per well evaluated. Each ex-periment was performed in triplicate.Bioinformatic miningGene correlation analyses between the mRNA expres-sion levels of WDR3 and YAP1 were carried out withthe GEPIA database (http:\/\/gepia.cancer-pku.cn\/) for allgiven sets of GTEx and TCGA expression data. TheEukaryotic Promoter Database (https:\/\/epd.epfl.ch\/\/index.php) was used to determine the potential bindingsites of GATA4 in the promoter of the YAP1 gene.PDAC xenografts in nude miceAnimal experiments were approved by the Ethical Com-mittee on Animal Experiments of the Sichuan ProvincialPeople\u2019s Hospital in Chengdu, China. PANC-1 cells (3 \u00d7106) infected with sh-Control or sh-WDR3 #1 were sub-cutaneously injected into the left flank of BALB\/c-numice (4\u20135 weeks old, male) purchased from Vital River.Tumor sizes were assessed with a digital Vernier caliperevery three days. Tumors were harvested 3 weeks afterinjection, and tumor weights were measured.Orthotopic syngeneic model of pancreatic cancer toC57BL\/6 miceWe used 8-week-old wild-type C57BL\/6 mice in the ex-periments. For orthotopic implantation, mice were anes-thetized with pentobarbital sodium, and hair wasremoved from their abdomens. We incised each mouselongitudinally along the abdomen to expose the pan-creas, injected 20 \u03bcL of the cell suspension into the pan-creas, and closed the incision with sutures. Eachexperimental group consisted of five mice. All mice wereweighed and checked for signs of distress regularly. Ab-dominal palpation was used to monitor tumor size. Tu-mors were harvested 3 weeks after injection, and tumorweights were measured.Statistical analysisAll data are expressed as the mean \u00b1 standard deviation(SD) of three independent experiments. Comparisonsbetween two groups were performed using Student\u2019s t-test, and two-way ANOVA or one-way ANOVA to-gether with the Bonferroni post hoc test was used formultigroup analysis. A P value less than 0.05 was consid-ered significant. GraphPad Prism 6 software (GraphPadSoftware, Inc.) was used for statistical analysis.ResultsOverexpressed WDR3 was correlated with poor survival inpancreatic cancer patientsTo determine the expression level of WDR3 in malig-nant cancers, the GEPIA database was searched, and theresults showed that WDR3 was significantly overex-pressed in several malignant cancers, including pancre-atic cancer (Fig. 1a). Moreover, survival rate analysisresults indicated that the elevated expression level ofWDR3 was correlated with poor disease-free survival(DFS) and overall survival (OS) in pancreatic cancer, ra-ther than other malignant cancers (Fig. 1b-c). Therefore,we speculated that WDR3 plays an important biologicalrole in the progression of pancreatic cancer. Further-more, IHC analysis verified the overexpression of WDR3in pancreatic cancer patients (Fig. 1d-e). Finally, com-pared with normal pancreatic cells, pancreatic cancercells expressed high levels of WDR3 (Fig. 1f-g). There-fore, we concluded that WDR3 was significantly overex-pressed and positively correlated with poor survival inpancreatic cancer.Silenced WDR3 inhibited the proliferation and invasion ofpancreatic cancer cells in vitro and in vivoTo explore the biological role of WDR3 in pancreaticcancer, pancreatic cancer cell lines with WDR3 silencingwere established (Fig. 2a-b). Colony formation and MTSassays showed that WDR3 inhibition significantly inhib-ited the proliferation of pancreatic cancer cells (Fig. 2c-d), while a transwell invasion assay proved that WDR3inhibition decreased the invasive ability of pancreaticcancer cells (Fig. 2e). To investigate the biological role ofWDR3 in pancreatic cancer in vivo, PANC-1 cells withnormal WDR3 expression or silenced WDR3 expressionwere subcutaneously injected into the left flank of nudeSu et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 4 of 15Fig. 1 (See legend on next page.)Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 5 of 15mice under the same conditions for a xenograft assay.As Fig. 2f-h shows, the tumors formed by WDR3-silenced PANC-1 cells were smaller and lighter thanthose formed by WDR3 normal-expressing cells. Takentogether, our results indicated that silenced WDR3 sig-nificantly inhibited the proliferation and invasion abilityof pancreatic cancer cells in vitro and in vivo.Overexpressed WDR3 promoted the proliferation andinvasion of pancreatic cancer cells in vitro and in vivoTo further explore the biological role of overexpressedWDR3 in pancreatic cancer, pancreatic cancer cell lineswith WDR3 overexpression were established (Fig. 3a-b).Colony formation and MTS assays showed that WDR3overexpression significantly promoted the proliferationof pancreatic cancer cells (Fig. 3c-d), while a transwellinvasion assay proved that WDR3 overexpression in-creased the invasive ability of pancreatic cancer cells(Fig. 3e). To investigate the biological role of overex-pressed WDR3 in pancreatic cancer in vivo, PANC-1cells with normal WDR3 expression or overexpressedWDR3 expression were subcutaneously injected into theleft flank of nude mice under the same conditions for axenograft assay. As Fig. 3f-h shows, the tumors formedby WDR3-overexpressed PANC-1 cells were larger andheavier than those formed by WDR3 normal-expressingcells. Taken together, our results indicated that overex-pressed WDR3 significantly promoted the proliferationand invasion ability of pancreatic cancer cells in vitroand in vivo.Overexpressed WDR3 induced the activation of the hippopathway in pancreatic cancerWe have proved that overexpressed WDR3 could increasethe proliferation and invasion abilities of pancreatic cancercells and was correlated with poor survival in pancreaticcancer patients. However, the specific regulatory mechan-ism underlying these effects is still unclear. By silencingWDR3 expression in PANC-1 cells and performing anRNA-seq assay, we identified 490 differentially expressedgenes (DEGs), including 248 upregulated DEGs and 242downregulated DEGs (Fig. 4a-b). Additionally, KEGG sig-naling pathway analysis showed that the Hippo signalingpathway was significantly inhibited after WDR3 silencing(Fig. 4c), which indicated that WDR3 could regulate theactivation of the Hippo signaling pathway. Consistently,PCR and western blot analyses showed that both themRNA and protein levels of YAP1, the main effector ofthe Hippo signaling pathway, were significantly downregu-lated in WDR3-silenced pancreatic cancer cells (Fig. 4d-e),while both the mRNA and protein levels of YAP1 weresignificantly upregulated in WDR3-overexpressing pancre-atic cancer cells (Fig. 4f-g). Furthermore, the mRNA ex-pression of CTGF and CYR61, the major downstreamregulatory genes in the Hippo signaling pathway, was alsopositively correlated with the expression level of WDR3 inpancreatic cancer cells (Fig. 4h-i). Furthermore, by overex-pressing YAP1 in WDR3 silenced PANC-1 cells, we foundthat overexpressed YAP1 reversed the inhibition of theproliferation and invasion ability in pancreatic cancer cellsinduced by WDR3 silencing (Supplementary Fig. 1). Theseresults indicated that WDR3 promoted the proliferationand invasion ability of pancreatic cancer cells by transcrip-tionally upregulating YAP1 expression and activating theHippo signaling pathway in pancreatic cancer cells.WDR3 protein expression positively correlated with YAP1levels in cancer patient specimensWe demonstrated that WDR3 could regulate YAP1 ex-pression in pancreatic cancer cells, but the clinical rela-tionship between WDR3 and YAP1 in human pancreaticcancer specimens remains unclear. By conducting IHCanalysis of a cohort of PDAC patients (n = 31), we founda positive correlation between the protein expressionlevels of WDR3 and YAP1 in PDAC specimens (Spear-man correlation r = 0.5916, P < 0.001) (SupplementaryFig. 2A-C). Consistently, the GEPIA searching resultshowed a positive correlation between WDR3 and YAP1mRNA expression levels in pancreatic cancer patientspecimens (Supplementary Fig. 2D). Therefore, all theseresults suggested a positive correlation between the ex-pression levels of WDR3 and YAP1 in pancreatic cancerspecimens.The WDR3 knockdown enhanced the anti-pancreaticcancer effect of YAP1 inhibitionTED-347 is a selective inhibitor of YAP1 at the proteinlevel that functions by inhibiting the binding of TEAD4(See figure on previous page.)Fig. 1 Overexpression of WDR3 is correlated with an unfavorable prognosis in pancreatic cancer. a The GEPIA database was searched for WDR3mRNA expression in several malignant cancers. Colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), liver hepatocellular carcinoma(LIHC), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD). *, P < 0.05. b-c The GEPIA web tool was searched for disease-free survival (b) and overall survival (c) data for several malignant cancer patients with a high or low WDR3 expression level. P values are shownin the Fig. d. IHC images of WDR3 staining in TMA tissue sections are shown. The scale bars are shown in the Fig. e. Dot plots show the IHCscores of WDR3 expression for TMA tissue sections (normal pancreatic specimens: n = 25, PAAD TMA specimens: n = 31, P < 0.001). Statisticalanalyses were performed with D\u2019Agostino & Pearson omnibus normality test. f and g Western blot analysis evaluated the expression of WDR3 innormal pancreatic ductal epithelial cells and pancreatic cancer cells (f). The protein expression levels of WDR3 were quantified with ImageJsoftware (g). GAPDH served as an internal referenceSu et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 6 of 15to full-length Yap1 in a dose- and time-dependent man-ner [24]. Since WDR3 silencing could inhibit YAP1 ex-pression at the mRNA and protein levels, wehypothesized that The WDR3 knockdown enhanced theanti-pancreatic cancer effect of YAP1 inhibition. RT-PCR analysis showed an enhanced effect for WDR3 si-lencing and TED-347 treatment, resulting in decreasedlevels of the downstream target genes of YAP1, CTGFFig. 2 Silencing WDR3 suppresses the aggressive behavior of pancreatic cancer cells in vitro and pancreatic tumor growth in vivo. a and b RT-PCR (a) and western blot analyses (b) of WDR3 expression in PANC-1, MIA PaCa-2, and BxPC-3 cells infected with sh-Control or sh-WDR3s. GAPDHserved as an internal reference. Data are presented as the mean \u00b1 SD of three independent experiments. Each sh-WDR3 group was comparedwith sh-Control group. Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019s tests. **, P < 0.01; ***,P < 0.001. C-E. PANC-1, MIA PaCa-2, and BxPC-3 cells were infected with sh-Control or sh-WDR3 #1. The cells were harvested for colony formation(c), MTS (d), and Transwell invasion assays (e) after 48 h of culture. Each bar represents the mean \u00b1 SD of three independent experiments. **, P <0.01; ***, P < 0.001. F-H. PANC-1 cells infected with sh-Control or sh-WDR3 #1 were subcutaneously injected into nude mice. The tumors wereharvested and photographed (f) on day 21. Data for tumor volume (g) and tumor mass (h) are shown as the mean \u00b1 SD (n = 5). Each sh-WDR3group was compared with sh-Control group. Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019stests. ***, P < 0.001Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 7 of 15and CYR61 (Fig. 5a). Besides, MTS and colony forma-tion assays indicated the synergistic effect of WDR3silencing and TED-347 treatment, demonstrating inhib-ition of the proliferative ability of pancreatic cancer cellsin vitro (Fig. 5b-c). Furthermore, by subcutaneouslyinjecting normal WDR3-expressing or WDR3-silencedPANC-1 cells into the left flank of nude mice under thesame conditions for a xenograft assay and treating themice with or without TED-347, we found that WDR3 si-lencing and TED-347 treatment could both slow tumorgrowth, and the combined treatment group showed fur-ther inhibition of tumor growth (Fig. 5d-f). Consistently,the orthotopic syngeneic model of pancreatic cancer inC57BL\/6 mice verified that the combined treatment ofWDR3 silencing and TED-347 treatment further inhib-ited of tumor growth of pancreatic cancer (Supplemen-tary Fig. 3). In brief, all these data showed that WDR3knockdown enhanced the anti-pancreatic cancer effectof YAP1 inhibition both in vitro and in vivo.The regulation of YAP1 induced by WDR3 was dependenton GATA4 in pancreatic cancer cellsAs mentioned above, WDR3 silencing could inactivate theHippo signaling pathway by decreasing YAP1 expression inpancreatic cancer cells. However, the specific mechanismremained unclear. We performed immunoprecipitation andFig. 3 Overexpressed WDR3 promoted the aggressive behavior of pancreatic cancer cells in vitro and pancreatic tumor growth in vivo. a and b RT-PCR (a) and western blot analyses (b) of WDR3 expression in PANC-1, MIA PaCa-2, and BxPC-3 cells infected with pcDNA3.1 or WDR3 plasmid. GAPDHserved as an internal reference. Data are presented as the mean \u00b1 SD of three independent experiments. WDR3 group was compared with pcDNA3.1group. Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019s tests. ***, P < 0.001. C-E. PANC-1, MIAPaCa-2, and BxPC-3 cells were infected with pcDNA3.1 or WDR3 plasmid. The cells were harvested for colony formation (c), MTS (d), and Transwellinvasion assays (e) after 48 h of culture. Each bar represents the mean \u00b1 SD of three independent experiments. **, P < 0.01; ***, P < 0.001. F-H. PANC-1cells infected with pcDNA3.1 or WDR3 plasmid were subcutaneously injected into nude mice. The tumors were harvested and photographed (f) onday 21. Data for tumor volume (g) and tumor mass (h) are shown as the mean \u00b1 SD (n = 5). WDR3 group was compared with pcDNA3.1 group.Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019s tests. ***, P < 0.001Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 8 of 15LC-MS\/MS assays to identify WDR3-associated proteins,and GATA4 was identified as a potential WDR3-interactingprotein (Fig. 6a-b and Supplementary Fig. 4), which wasverified by western blot analysis of immunoprecipitated sam-ples (Fig. 6c-d). Then, we hypothesized that the regulation ofYAP1 by WDR3 is dependent on the interaction withFig. 4 WDR3 transcriptionally increases YAP1 expression in pancreatic cancer cells. a and b. Volcano plot (a) and heatmap (b) showing the differentially expressedgenes in PANC-1 cells infected with si-Control or si-WDR3. The blue points represent the downregulated genes (n=248), while the red points represent theupregulated genes (n=242). c Heatmap showing a subset of WDR3 knockdown-regulated genes with a p-value < 0.1 participating in the Hippo signalingpathway in PANC-1 cells. si-WDR3 group was compared with si-Control group. Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019smultiple comparison\u2019s tests. d and e RT-PCR analysis (d) and western blot analysis (e) to detect the mRNA and protein expression levels of YAP1 in pancreaticcancer cells infected with sh-Control or sh-WDR3s. GAPDH served as an internal reference. Data are shown as the mean\u00b1SD (n=3). Statistical analyses wereperformed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019s tests. **, P<0.01; ***, P<0.001. F-G. RT-PCR analysis (f) and western blot analysis (g)to detect the mRNA and protein expression levels of YAP1 in pancreatic cancer cells infected with pcDNA3.1 or WDR3. GAPDH served as an internal reference.Data are shown as the mean\u00b1SD (n=3). Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiple comparison\u2019s tests. ***, P<0.001. h RT-PCR analysis to detect the mRNA expression levels of CTGF and CYR61 in pancreatic cancer cells infected with sh-Control or sh-WDR3s. GAPDH servedas an internal reference. Data are shown as the mean\u00b1SD (n=3). Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiplecomparison\u2019s tests. ***, P<0.001. i RT-PCR analysis to detect the mRNA expression levels of CTGF and CYR61 in pancreatic cancer cells infected with pcDNA3.1 orWDR3. GAPDH served as an internal reference. Data are shown as the mean\u00b1 SD (n=3). Statistical analyses were performed with one-way ANOVA followed byTukey\u2019s multiple comparison\u2019s tests. ***, P<0.001Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 9 of 15GATA4. GATA4 knockdown reversed not only the inhib-ition of YAP1 induced by WDR3 silencing (Fig. 6e) but alsothe upregulation of YAP1 expression induced by WDR3overexpression (Fig. 6f). Consistently, GATA4 knockdownalso reversed the inhibition of proliferation and invasion abil-ity of pancreatic cancer cells induced by WDR3 silencing(Supplementary Fig. 5). Furthermore, WDR3 silencingcouldn\u2019t regulate the total expression level of GATA4,whereas could increase the intranuclear expression level ofGATA4 (Fig. 6g). Therefore, the regulation of YAP1 byWDR3 was found to be dependent on GATA4 in pancreaticcancer cells.GATA4, acting as a transcription factor, transcriptionallyupregulated YAP1 expression in pancreatic cancer cellsAs a transcription factor, GATA4 can regulate the ex-pression of numerous tumor-related genes in pancreaticcancer [25]. Since the regulation of YAP1 by WDR3 wasfound to be dependent on GATA4 (Fig. 6), wespeculated that GATA4 can transcriptionally regulateYAP1 expression as a transcription factor. Consistently,RT-PCR and western blot assays indicated that GATA4knockdown could significantly inhibit YAP1 expressionat both the mRNA and protein levels (Fig. 7a-b), whileGATA4 overexpression could significantly induce YAP1expression at both the mRNA and protein levels (Fig.7c-d). More importantly, ChIP-qPCR analysis identifiedthat GATA4 could bind to the promoter of the YAP1gene (Fig. 7e-f), which could be inhibited by WDR3 si-lencing (Fig. 7g) and induced by WDR3 overexpression(Fig. 7h). In conclusion, we proved that GATA4, actingas a transcription factor, could transcriptionally upregu-late YAP1 expression in pancreatic cancer cells.DiscussionIn our study, we first proved that WDR3 was overex-pressed and positively correlated with poor survival inpancreatic cancer (Fig. 1). Akdi et al reported thatFig. 5 WDR3 knockdown enhanced the anti-pancreatic cancer effect of YAP1 inhibition. a RT-PCR analysis was used to detect the mRNA expressionlevels of CTGF and CYR61 in pancreatic cancer cells infected with sh-Control or sh-WDR3 #1 and treated with or without TED-347. GAPDH served as aninternal reference. Data are shown as the mean \u00b1 SD (n = 3). Statistical analyses were performed with one-way ANOVA followed by Tukey\u2019s multiplecomparison\u2019s tests. ***, P < 0.001. b-c. PANC-1 cells infected with sh-Control or sh-WDR3 #1 and treated with or without TED-347 were harvested forMTS (b) and colony formation assays (c). Data are shown as the mean \u00b1 SD (n = 3). Statistical analyses were performed with one-way ANOVA followedby Tukey\u2019s multiple comparison\u2019s tests. ***, P < 0.001. d-f. PANC-1 cells infected with sh-Control or sh-WDR3 #1 were subcutaneously injected intonude mice. The mice were treated with TED-347 3 times on days 1, 4, and 7 at a dose of 20mg\/kg. The tumors were harvested and photographed(d) on day 21. Data for tumor volume (e) and tumor mass (f) are shown as the mean \u00b1 SD (n = 5). Statistical analyses were performed with two-wayANOVA followed by Sidak\u2019s multiple comparison\u2019s tests. ***, P < 0.001Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 10 of 15WDR3 is overexpressed and a risk factor in thyroid can-cer [9]. Additionally, several groups have also reportedthe biological role of WDR3 in modulating genomestability [10], increasing cancer predisposition [26], pro-moting proliferation and arresting cancer cells in the G1phase of the cell cycle [7]. Consistently, our results alsoFig. 6 The regulation of YAP1 induced by WDR3 was dependent on GATA4 in pancreatic cancer cells. A-B. LC-MS\/MS identified an interactionbetween WDR3 and GATA4 (a) by detecting two peptides of GATA4 (b). c-d Coimmunoprecipitation showed the interaction between WDR3 andGATA4. e-f Western blot analysis showed the protein expression levels of specific genes. GAPDH served as an internal reference. g Western blotanalysis to show the GATA4 expression in the nucleus and cytoplasm of pancreatic cancer cells infected with sh-Control or sh-WDR3Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 11 of 15indicated that overexpressed WDR3 increased the prolif-eration and invasion abilities of pancreatic cancer cells(Fig. 2). However, the biological mechanism of WDR3overexpression in pancreatic cancer still needs furtherstudy.GATA binding protein 4 (GATA4), a protein in theGATA family of zinc-finger transcription factors, canrecognize the GATA motif, which is present in the pro-moter of many tumor-related genes. GATA4 regulatesthe expression of genes involved in multipleFig. 7 GATA4, acting as a transcription factor, transcriptionally upregulated YAP1 expression in pancreatic cancer cells. a-b RT-PCR analysis (a) andwestern blot analysis (b) were used to detect the mRNA and protein expression levels of YAP1 in pancreatic cancer cells infected with sh-Control orsh-GATA4s. GAPDH served as an internal reference. Data are shown as the mean \u00b1 SD (n = 3). Statistical analyses were performed with two-wayANOVA followed by Sidak\u2019s multiple comparison\u2019s tests. ***, P < 0.001. C-D. RT-PCR analysis (c) and western blot analysis (d) were used to detect themRNA and protein expression levels of YAP1 in pancreatic cancer cells infected with pcDNA3.1 or GATA4.GAPDH served as an internal reference. Dataare shown as the mean \u00b1 SD (n = 3). Statistical analyses were performed with two-way ANOVA followed by Sidak\u2019s multiple comparison\u2019s tests. ***, P <0.001. e The Eukaryotic Promoter Database was searched to evaluate potential YAP1 promoter binding by GATA4 (\u2212 649 bp, \u2212 467 bp, and 66 bp), andthe ChIP primer sequences (Table S3) were designed for the gene locus from \u2212 700 bp to \u2212 400 bp. f GATA4 ChIP-qPCR of YAP1 in PANC-1, MIA PaCa-2, and BxPC-3 cells were performed. All data are shown as the mean \u00b1 SD of three replicates. Statistical analyses were performed with two-way ANOVAfollowed by Sidak\u2019s multiple comparison\u2019s tests. ***, P < 0.001. G-H. GATA4 ChIP-qPCR of YAP1 in PANC-1, MIA PaCa-2, and BxPC-3 cells with WDR3silenced (g) or overexpressed (h). was performed. All data are shown as the mean \u00b1 SD of three replicates. Statistical analyses were performed withtwo-way ANOVA followed by Sidak\u2019s multiple comparison\u2019s tests. ***, P < 0.001Su et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 12 of 15pathological\/physiological processes, including embryo-genesis, myocardial differentiation and function, andnormal testicular development. It has been reportedGATA4 expression is associated with increased tumorsize, metastasis, and a poor prognosis [27]. GATA4mRNA expression is upregulated in pancreatic cancercell lines and tissues [25], and downregulation ofGATA4 expression increases drug sensitivity in cancercells [28]. GATA4 can decrease P53 protein expressionby transcriptionally activating the expression of MDM2[29], the primary negative regulatory factor of the P53protein that induces p53 ubiquitination and degradation[30]. GATA4 is also highly expressed in most hepato-blastomas and correlates with a mesenchymal, migratoryphenotype in hepatoblastoma cells by regulating the ex-pression of ADD3, AHNAK, and IGFBP1 [31]. Similarly,our results indicated that GATA4 could function as atranscription factor to induce YAP1 expression and acti-vate the Hippo signaling pathway, which resulted in pan-creatic cancer progression. GATA4 knockdown reversedthe inhibition of YAP1 and proliferation and invasion ofabilities of pancreatic cancer cells induced by WDR3 si-lencing and reversed the upregulation of YAP1 expres-sion induced by WDR3 overexpression (Fig. 6). Takentogether, our results provide new insights into the spe-cific mechanism by which GATA4 regulates the progres-sion of pancreatic cancer.The Hippo signaling pathway was first discovered in stud-ies of Drosophila melanogaster [32]. Hippo signaling governsnormal organ development and tissue regeneration underphysiological conditions [33]. Hippo signaling is an evolu-tionarily conserved network that plays a key role in regulat-ing cell proliferation, organ growth, and regeneration [34].YAP1 is the key downstream regulator in the Hippo pathwaythat exhibits upregulated expression in pancreatic cancer[35\u201337]. Aberrant transcriptional activity of YAP1 has crucialroles in pancreatic tumor cell biology, including roles ingrowth, epithelial-mesenchymal transition (EMT), microen-vironmental signaling transduction, and drug resistance [34].Then, inhibition of YAP1 expression is essential for pancre-atic cancer targeted therapy. Studies have shown that YAP1expression is induced by KRAS activation [35], aerobic gly-colysis [38], GNAS [39], and the cancer upregulated gene(CUG) 2 exhibiting upregulated expression in lung cancerwhich could increase the expression of YAP1 [40]. Interest-ingly, our results identified a protein interaction betweenWDR3 and GATA4 that led to the regulation of GATA4 nu-clear translocation and YAP1 expression in pancreatic can-cer. Silencing WDR3 significantly decreased the expressionlevels of YAP1 and the downstream target genes CTGF andCYR61 in pancreatic cancer. All these results emphasized theclinical significance of WDR3-targeted therapy.TED-347 is a potent, irreversible, covalent, allostericinhibitor of the YAP-TEAD protein-protein interaction[24]. TED-347 forms a covalent complex with TEAD4that inhibits TEAD4 binding to YAP1, blocks YAP1transcriptional activity, and suppresses the expression ofdownstream target genes, including CTGF and CYR61.Combined with the inhibition of YAP1 transcription in-duced by WDR3 knockdown, TED-347 treatmentFig. 8 Overexpressed WDR3 induces the activation of Hippo pathway by interacting with GATA4 in pancreatic cancer. The overexpressed WDR3promoted the nuclear translocation of GATA4 and increased the binding of GATA4 to the promoter of YAP1 by interacting with GATA4 in pancreaticcancer cells. As a transcription factor, the binding of GATA4 to the promoter of YAP1 induced the expression of YAP1. The upregulation of YAP1, themain effector of the Hippo signaling pathway, resulted in the activation of the Hippo pathway signaling and the overexpression of downstreameffector genes, CTGF and CYR61. Finally, the cell proliferation and invasion were promotedSu et al. Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 13 of 15further enhanced the ability of WDR3 silencing to in-hibit pancreatic cancer progression.ConclusionsOur study proved that overexpressed WDR3 was corre-lated with poor survival in pancreatic cancer and WDR3silencing significantly inhibited the proliferation, inva-sion, and tumor growth of pancreatic cancer. Further-more, WDR3 induced YAP1 expression by interactingwith GATA4 and inducing the nuclear translocation ofGATA4, the transcription factor of YAP1, in pancreaticcancer cells. Finally, the combination of WDR3 silencingand administration of the YAP1 inhibitor TED-347 hada synergistic inhibitory effect on the progression of pan-creatic cancer (Fig. 8). Therefore, WDR3 is potentially atherapeutic target for pancreatic cancer treatment.Supplementary InformationThe online version contains supplementary material available at https:\/\/doi.org\/10.1186\/s13046-021-01879-w.Additional file 1: Fig. S1. YAP1 overexpression reversed the inhibitionin the aggressive behavior of pancreatic cancer cells induced by WDR3silencing in vitro. Fig. S2. WDR3 expression was positively correlatedwith YAP1 expression in pancreatic cancer patient specimens. Fig. S3.WDR3 silencing enhanced the anti-pancreatic cancer effect of TED-347treatment in the orthotopic syngeneic model of pancreatic cancer. Fig.S4. LC-MS\/MS assay to identify the base peak of IgG IP and WDR3 IPsamples. Fig. S5. GATA4 silencing reversed the induction in the aggres-sive behavior of pancreatic cancer cells induced by WDR3 overexpressionin vitro. Table S1. The primer sequences for RT-qPCR. Table S2. TheshRNA sequences. Table S3. The primer sequences for ChIP-qPCR.AbbreviationsPDAC: Pancreatic ductal adenocarcinoma; WDR3: WD repeat domain 3;YAP1: Yes association protein 1; GATA4: GATA binding protein 4; TEAD: TEAdomain; CTGF: Connective tissue growth factor; CYR61: Cysteine richangiogenic inducer 61; KEGG: Kyoto Encyclopedia of Genes and Genomes;TCGA: The Cancer Genome Atlas; COAD: Colon adenocarcinoma;ESCA: Esophageal carcinoma; LIHC: Liver hepatocellular carcinoma;PAAD: Pancreatic adenocarcinoma; STAD: Stomach adenocarcinoma;DFS: Disease-free survival; OS: Overall survivalAcknowledgmentsThe author thanks AJE for editing grammar, spelling, and other commonerrors.Authors\u2019 contributionsConception and design: SK Zhu, C Yang, HJ Yang. Development ofmethodology: WJ Su, K Chen, Q Fu, MW Tian, C Yang. Acquisition of data(provided animals, provided facilities, etc.): X Jin, DY Ren, C Yang. Analysisand interpretation of data (statistical analysis, biostatistics, computationalanalysis, etc.): Q Fu, GG Shi, SJ Feng. Writing, review, and\/or revision of themanuscript: WJ Su, SK Zhu, HJ Yang, C Yang. Administrative, technical, ormaterial support (i.e., reporting or organizing data, constructing databases):GG Shi, SJ Feng, X Jin. Study supervision: SK Zhu, HJ Yang, C Yang. Theauthor(s) read and approved the final manuscript.FundingThis work was supported by The Programs of Sichuan Provincial HealthCommittee (No. 19PJ116 and No. 19PJ135).Availability of data and materialsThe datasets during and\/or analyzed during the current study available fromthe corresponding author on reasonable request. Gene correlation analysesbetween the mRNA expression levels of WDR3 and YAP1 were carried outwith the GEPIA database (http:\/\/gepia.cancer-pku.cn\/) for all given sets ofGTEx and TCGA expression data. The Eukaryotic Promoter Database (https:\/\/epd.epfl.ch\/\/index.php) was used to determine the potential binding sites ofGATA4 in the promoter of the YAP1 gene.Ethics approval and consent to participateAll experiments were conducted in accordance with the ethical standards ofthe ethics committee of Sichuan Provincial People\u2019s Hospital.Consent for publicationNot applicable.Competing interestsThe authors have declared that no competing interest exists.Author details1Department of Anesthesiology, Sichuan Provincial People\u2019s Hospital,University of Electronic Science and Technology of China, Chengdu 611731,Sichuan, China. 2Clinical Immunology Translational Medicine Key Laboratoryof Sichuan Province & Organ Transplantation Center, Sichuan ProvincialPeople\u2019s Hospital, University of Electronic Science and Technology of China,Chengdu 611731, Sichuan, China. 3Chinese Academy of Sciences SichuanTranslational Medicine Research Hospital, Chengdu 610072, Sichuan, China.4Transplant Center, Massachusetts General Hospital, Harvard Medical School,Boston, MA 02148, USA. 5Jack Bell Research Centre, University of BritishColumbia, Vancouver, BC V6H3Z6, Canada. 6Department of PancreaticSurgery, Union Hospital, Tongji Medical College, Huazhong University ofScience and Technology, Wuhan 430022, Hubei, China.Received: 5 November 2020 Accepted: 14 February 2021References1. 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Journal of Experimental & Clinical Cancer Research           (2021) 40:88 Page 15 of 15","@language":"en"}],"Genre":[{"@value":"Article","@language":"en"}],"IsShownAt":[{"@value":"10.14288\/1.0396030","@language":"en"}],"Language":[{"@value":"eng","@language":"en"}],"PeerReviewStatus":[{"@value":"Reviewed","@language":"en"}],"Provider":[{"@value":"Vancouver : University of British Columbia Library","@language":"en"}],"Publisher":[{"@value":"BioMed Central","@language":"en"}],"PublisherDOI":[{"@value":"10.1186\/s13046-021-01879-w","@language":"en"}],"Rights":[{"@value":"Attribution 4.0 International (CC BY 4.0)","@language":"en"}],"RightsURI":[{"@value":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/","@language":"en"}],"ScholarlyLevel":[{"@value":"Faculty","@language":"en"}],"Subject":[{"@value":"Pancreatic Cancer","@language":"en"},{"@value":"WDR3","@language":"en"},{"@value":"GATA4","@language":"en"},{"@value":"YAP1","@language":"en"},{"@value":"Hippo signaling pathway","@language":"en"}],"Title":[{"@value":"Overexpressed WDR3 induces the activation of Hippo pathway by interacting with GATA4 in pancreatic cancer","@language":"en"}],"Type":[{"@value":"Text","@language":"en"}],"URI":[{"@value":"http:\/\/hdl.handle.net\/2429\/77432","@language":"en"}],"SortDate":[{"@value":"2021-03-01 AD","@language":"en"}],"@id":"doi:10.14288\/1.0396030"}