Translational Cancer Mechanisms and Therapy

Targeting anAutocrineRegulatory Loop inCancerStem-like Cells Impairs the Progression andChemotherapy Resistance of Bladder CancerKai-Jian Wang1, Chao Wang1, Li-He Dai1, Jun Yang1, Hai Huang1, Xiao-Jing Ma2,Zhe Zhou1, Ze-Yu Yang1,Wei-Dong Xu1, Mei-Mian Hua1, Xin Lu1, Shu-Xiong Zeng1,Hui-Qing Wang1, Zhen-Sheng Zhang1, Yan-Qiong Cheng1, Dan Liu1, Qin-Qin Tian1,Ying-Hao Sun1, and Chuan-Liang Xu1

Abstract

Purpose: Cancer stem-like cells (CSCs) contribute to blad-der cancer chemotherapy resistance and progression, but theassociated mechanisms have not been elucidated. This studydetermined whether blocking an autocrine signaling loop inCSCs improves the therapeutic effects of cis-platinum onbladder cancer.

Experimental Design: The expression of the epithelialmarker OV6 and other markers in human bladder cancerspecimens was examined by IHC. The CSC properties ofmagnetic-activated cell sorting (MACS)-isolated OV6þ andOV6� bladder cancer cells were examined.Molecularmechan-isms were assessed through RNA-Seq, cytokine antibodyarrays, co-immunoprecipitation (co-IP), chromatin immuno-precipitation (ChIP) and other assays. An orthotopic bladdercancer mouse model was established to evaluate the in vivoeffects of a YAP inhibitor (verteporfin) and a PDGFR inhibitor(CP-673451) on the cis-platinum resistance of OV6þ CSCsin bladder cancer.

Results: Upregulated OV6 expression positively associat-ed with disease progression and poor prognosis for bladdercancer patients. Compared with OV6� cells, OV6þ bladdercancer cells exhibited strong CSC characteristics, includingself-renewal, tumor initiation in NOD/SCID mice, andchemotherapy resistance. YAP, which maintains the stem-ness of OV6þ CSCs, triggered PDGFB transcription byrecruiting TEAD1. Autocrine PDGF-BB signaling through itsreceptor PDGFR stabilized YAP and facilitated YAP nucleartranslocation. Furthermore, blocking the YAP/TEAD1/PDGF-BB/PDGFR loop with verteporfin or CP-673451inhibited the cis-platinum resistance of OV6þ bladder cancerCSCs in an orthotopic bladder cancer model.

Conclusions: OV6 could be a helpful indicator of diseaseprogression and prognosis for patients with bladder cancer,and targeting the autocrine YAP/TEAD1/PDGF-BB/PDGFRloop might serve as a remedy for cis-platinum resistance inpatients with advanced bladder cancer.

IntroductionBladder cancer is one of the most frequently diagnosed and

lethal cancers worldwide, with an estimated 429,800 new casesand 165,100 deaths in 2012 (1). Although surgical resection is thepreferred treatment for patients with bladder cancer, most tumorsrecur andprogress tomuscle-invasive bladder cancers (MIBC) andmetastatic bladder cancer, which are prone to develop chemo-therapy resistance after treatment and are associated with poorprognosis (2, 3). Cancer stem-like cells (CSCs) are a small sub-

population of tumor cells that are functionally defined by theirstrong stem-like properties, including self-renewal, drug resis-tance, and tumor initiation capacity upon serial passage, and theheterogeneity, progression and chemotherapy resistance of manycancers, including bladder cancer, have been attributed to CSCs(4–6). Therefore, unveiling the molecular mechanisms by whichCSCs are regulated will likely facilitate the development of noveland efficacious therapies for advanced and chemoresistant blad-der cancer.

Bladder CSCs can be isolated using several markers, such asCD44, SOX2, CD133, and aldehyde dehydrogenase 1 A1(ALDH1A1; refs. 7, 8). In addition, many signaling pathwayshave been reported to regulate the self-renewal, chemotherapyresistance, and tumor initiation properties of bladder CSCs,including hedgehog signaling, the Wnt/b-catenin pathway, andthe KMT1A-GATA3-STAT3 and E2F1-EZH2-SUZ12 cascades(9–12). A recent study reveals that variants of ARID1A, GPRC5A,and MLL2 drive the self-renewal of bladder CSCs, as indicated bysingle-cell sequencing (13). However, the mechanisms underly-ing the maintenance of the stem-like properties of bladder CSCsremain insufficiently understood.

Hippo signaling was initially identified in Drosophila and hasbeen more recently examined for its regulatory role in mamma-lian cells; the Hippo pathway was found to play crucial roles in

1Department of Urology, Changhai Hospital, Second Military Medical University(Naval Medical University), Shanghai, China. 2Department of Microbiology andImmunology, Weill Cornell Medicine, New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

K.-J. Wang, C. Wang, and L.-H. Dai contributed equally to this article.

Corresponding Authors: Chuan-Liang Xu, Changhai Hospital, Second MilitaryMedical University, 168 Changhai Road, Shanghai 200438, China. Phone: 8602-1311-61716; Fax: 8602-1350-30006; E-mail: xuchuanliang@vip.126.com; andYing-Hao Sun, sunyhsmmu@126.com

doi: 10.1158/1078-0432.CCR-18-0586

�2018 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 25(3) February 1, 20191070

mediating carcinogenesis and self-renewal in stem cells and CSCs(14). The Hippo pathway effector Yes-associated protein (YAP) isnegatively regulated by the mammalian Ste20-like kinases 1/2(MST1/2) and large tumor suppressor 1/2 (LATS1/2), and thisnegative regulation results in retention of phosphorylated YAP inthe cytoplasm (15). Conversely, YAP that is not phosphorylatedenters the nucleus and functions as a transcription coactivator fortranscription factors of the TEA domain transcription factor(TEAD) family to induce the expression of downstream genesthat promote cell proliferation and differentiation (15). Recentstudies have demonstrated that YAP is required for CSC self-renewal and expansion in various cancer types, such as breastcancer, prostate cancer, glioblastoma, and lung cancer (16–18).However, the role of YAP in bladder CSCs and the relatedmechanisms have yet to be established.

The epithelialmarkerOV6serves as aputativemarker forhepaticoval cells, bile duct epithelial cells, and also a CSC marker inepithelium-derivedmalignant tumors; it is associated with patientprognosis as shown in many studies (19–24). In this study, wefound that highOV6 expression in specimens positively associatedwith disease progression and poor prognosis of patients withbladder cancer. Moreover, OV6þ bladder cancer cells harboredstrong stem-like properties, and the pattern of OV6 expression inbladder CSCs was very similar to those of CD44 and CD133. Inaddition, we demonstrated that YAP drives the self-renewal ofOV6þCSCs to facilitate the invasion,migrationanddrug resistanceof bladder cancer. Furthermore, YAP triggers PDGFB transcriptionvia TEAD1, and reciprocally, PDGF-BBbinds to its receptor PDGFRand stabilizes YAP by preventing its phosphorylation by LATS1/2,thus forming an autocrine-regulatory loop in OV6þ CSCs. Finally,anorthotopic bladder cancermousemodelwas employed to showthat blocking the autocrine regulatory loop in OV6þ CSCs using aYAPor PDGFR inhibitor overcame the resistance to cis-platinum inadvanced bladder cancer.

Materials and MethodsPatients and specimens

Two cohorts, cohort 1 (n ¼ 130, January 2009 to December2013) and cohort 2 (n¼ 95, January 2004 to December 2008), of

patients with bladder cancer from Changhai Hospital (Shanghai,China) were recruited in this study. The patients weremerged andrandomly divided by 1:1 ratio into a training (n ¼ 113) and avalidation set (n ¼ 112). In addition to the cohorts above,this study also included a patient with recurrent bladder cancer(n ¼ 1), patients with metastatic bladder cancer (n ¼ 6), andpatients with bladder cancer treated with cis-platinum (n ¼ 5).Histologic grade and tumor stage were assessed according to theAmerican Joint Committee on Cancer (2010) and the WorldHealth Organization classification (2004; ref. 25).

Clinical follow-up data were available for all patients. Themedian follow-up periodwas 59.17months (mean 50.3months)for cohort 1 and 122.1 months (mean 91.5months) for cohort 2.All patients were investigated according to a uniform method ofthe Department of Urology, Changhai Hospital (Shanghai, Chi-na) based on the guidelines (26, 27). Cystoscopy was applied forpatients treated with TURBT at the first 3months after surgery. Forpatients with low-risk tumors, cystoscopy was performed 1 yearafter surgery if thefirst cystoscopywasnegative, and then yearly for5 years. For patients with high-risk tumors, cystoscopy was doneevery 3 months for 2 years, then every 6 months for 2 years, andthen yearly. Patients with intermediate-risk tumors had an in-between follow-up scheme using cystoscopy, which is adaptedaccording to personal and subjective factors. For patients treatedwith radical cystectomy, Return visits were generally performedpostoperatively at least every 3 to 4 months for the first year,semiannually for the second year, and annually thereafter. Fol-low-up visits consisted of a physical examination, urine cytology,serum chemistry evaluation, chest radiography, ultrasound exam-ination (including liver, kidney and retroperitoneum), CT/MRI ofthe abdomen and pelvis.

The samples were obtained after written informed consentwas provided by the patients according to an establishedprotocol approved by the Ethics Committee of ChanghaiHospital (Shanghai, China). The study was conducted inaccordance with the International Ethical Guidelines forBiomedical Research Involving Human Subjects (CIOMS).

IHCIHC was performed as described previously (28), and the

following primary antibodies were used: mouse anti-OV6(MAB2020, R&D Systems), rabbit anti-YAP (ab52771), mouseanti-Vimentin (ab8978), and rabbit anti-PDGF receptor beta(ab32570; Abcam). The percentage of positive cells (% of PPs)and the staining intensity (SI value) were determined and mul-tiplied to obtain the immunoreactive score (IRS value; ref. 29),which ranged from aminimum score of 0 to a maximum score of12. Time-dependent receiver-operating characteristic (ROC) anal-ysis was performed to determine the cut-off value and AUC ofOV6 expression in predicting 5-year cancer-specific survival (30)using R software (4.3.3). Patients with bladder cancer weredivided into 2 groups: a low expression (IRS values of 0–3) groupand a high expression (IRS values of 4–12) group.

Cell cultureThe cell lines used in this study were obtained from the Cell

Bank of the Type Culture Collection of the Chinese Academy ofSciences (Shanghai, China) in June 2016. UMUC3 and J82 cellswere cultured in minimum essential medium (1871544, Gibco)supplemented with FBS (10%, 10099-141, Gibco). RT4 and T24cells were maintained in McCoy 5A medium (1849736, Gibco)

Translational Relevance

Bladder cancer progression and chemotherapy resistancehave been attributed to the existence of cancer stem-like cells(CSCs). Thus, unveiling the molecular mechanisms by whichCSCs are regulated will aid in the development of new ther-apies for patientswith bladder cancer.Our study demonstratesthat OV6 is a good indicator for disease progression and theprognosis of patients with bladder cancer and that the auto-crine signaling loop YAP/TEAD1/PDGF-BB/PDGFR sustainsthe self-renewal of OV6þ CSCs, which facilitates bladdercancer invasion and chemotherapy resistance. Furthermore,targetingOV6þCSCs by blocking this autocrine signaling loopusing a YAP or PDGFR inhibitor improves the efficacy of cis-platinum in a treatment model of orthotopic bladder cancer.In conclusion, YAP or PDGFR may be potential therapeutictargets for the control of OV6þ CSCs and the inhibition ofchemotherapy resistance in bladder cancer.

Targeting CSCs Impairs the Progression of Bladder Cancer

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modified with FBS (10%, 10099-141, Gibco). SW780 cells werecultured in Leibovitz L-15medium (1828421, Gibco) containing10% FBS (Gibco). All cell lines were supplemented with 1%penicillin/streptomycin (15140122, Gibco) and cultured at 37�Cin 5% CO2.

The cell lines in this study were authenticated by short tandemrepeat (STR) profiling and tested for Mycoplasma contaminationwith a Mycoplasma Detection Kit (B39032, Selleck Chemicals).The most recent tests were performed in December 2017. The celllines used in this study were within 40 passages.

Flow cytometry assay and magnetic cell sortingFlow cytometric assays were performedwith a Cyan ADP Sorter

(Beckman Coulter). Bladder cancer cell lines were magneticallylabeled with APC-conjugated-OV6 antibody (FAB2020A, R&DSystems), FITC-conjugated–CD44antibody(130-095-195,MiltenyiBiotec) or PE-conjugated-CD133 antibody (130-113-670, MiltenyiBiotec). The magnetic-activated cell sorting (MACS) assay wasperformed with a MiniMACS Cell Sorter (Miltenyi Biotec). Bladdercancer cell lines were magnetically labeled with OV6 antibody(MAB2020, R&DSystems), CD44 antibody (130-110-082,MiltenyiBiotec), or CD133 antibody (130-090-423, Miltenyi Biotec) andthen subsequently incubated with rat anti-mouse IgG1 microbe-ads and separated on a MACS MS column (Miltenyi Biotec). Allthe procedures were carried out according to the manufacturer'sinstructions.

Spheroid formation assayBriefly, after magnetic sorting, single-cell suspensions with

3,000 cells were seeded in 6-well ultra-low attachment cultureplates (Corning) and cultured in serum-free DMEM/F12 (Gibco)supplemented with B27 (1:50, 17504-044, Thermo Fisher Scien-tific), N2 (1:100, 17502048, Thermo Fisher Scientific), 20 ng/mLEGF (Gibco), 10 ng/mL bFGF (Gibco), and ITS (1:100, 13146,Sigma) for 5 days. The number of spheroids formed was deter-mined via microscopy, and representative images were obtained.

The spheroid formation assay was carried out in semisolidmedium. In brief, single-cell suspensions with 3,000 cells wereresuspended in 1:1 growth factor–reduced Matrigel (BD Bios-ciences, 356231)/serum-free DMEM/F12 (supplemented withB27,N2, EGF, bFGF, and ITS) in a total volumeof 200 mL. Sampleswere plated around the rims ofwells in a 12-well plate and allowedto solidify at 37�C for 10 minutes before 1 mL of serum-freeDMEM/F12 (supplemented with B27, N2, EGF, bFGF, and ITS)was added. Medium was replenished every 3 days. Ten days afterplating, spheres with a diameter over 50 mm were counted viamicroscopy, and representative images were obtained.

For the single-cell spheroid formation assay, bladder cancer cellswere deposited by FACS in wells from the ultra-low-adhesion96-well plates containing 200 mL stem cell medium (serum-freeDMEM/F12 supplemented with B27, N2, EGF, bFGF, and ITS) at aconcentrationof a single cell perwell,whichwas confirmedvisuallyby microscope. Wells containing either none or more than 1 cellwere excluded for further analysis. The single-cell wells werechecked daily and a further 100 mL of stem cell mediumwas addedafter 5 days. Ten days after plating, wells with sphere were countedunder a microscope, and representative images were obtained.

Cell proliferation, invasion, and migration assaysThe proliferation of bladder cancer cells under the indicated

conditions was evaluated using a CCK-8 kit (CK-04, Dojindo),

which was described in our previous study (31). The prolifer-ation rates are presented as a proportion of the control value,which was obtained from the treatment-free groups. Invasionand migration assays were carried out in transwell chambers(Millipore) with or without Matrigel (BD Biosciences) accord-ing to the manufacturer's instructions, which was explained inour previous study (22).

Assessment of apoptosisApoptotic cells were evaluated with ANXA5 and propidium

iodide (PI) staining (Invitrogen, A13201) according to the man-ufacturer's instructions and analyzed by flow cytometry with aCyan ADP Sorter (Beckman Coulter).

Gene knockdown and plasmid transfectionThe short hairpin RNA (shRNA) interference vector pLKO.1-

GFP, containing a U6 promoter upstream of the shRNA, and thelentivirus packaging vectors pVSVG-I and pCMV-GAG-POL wereobtained from Shanghai Integrated Biotech Solutions Co., Ltd.(Shanghai, China). TheUMUC3/J82 cell linewas transducedwiththe shRNA-expressing lentivirus (sh-YAP or sh-TEAD1) or controllentivirus. After 72 hours of transduction, the cells were observedand photographed under microscope. Stable UMUC3/J82 knock-down of YAP and TEAD1 were also generated using lentiviralconstructs. The shRNA sequences are presented in SupplementaryTable S18. Cell transfection with YAP and TEAD1 plasmid wascarried out using Lipofectamine 3000 reagent (L3000015, Invi-trogen) according to the manufacturer's protocol, and thesequence of the YAP and TEAD1 plasmid is shown in Supple-mentary Table S18.

Real-time PCRTotal RNA was extracted with RNAiso Plus (9109, Takara), and

cDNAs were synthesized using a PrimeScript One Step RT reagentKit (RR037B, Takara). Real-time PCR was performed with SYBRGreen Real-Time PCR Master Mix (QPK201, Toyobo) on an ABIPRISM 7300HT Sequence Detection System. The results werenormalized to the expression of b-actin. Fold change relative tothe mean value was determined by 2�DDCt. The primer sequencesare presented in Supplementary Table S18.

Western blotting and co-IP analysisThe Western blot analysis was performed as we reported

previously (24). Nuclear and cytoplasmic proteins from bladdercancer cells were extracted using an NE-PER Nuclear and Cyto-plasmic Extraction Kit (#78899, Thermo Fisher Scientific). Thefollowing primary antibodies were used: rabbit anti-b-tubulin(#2128) and rabbit anti-Histone H3 (#4499), rabbit anti-phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204; #4370), rabbitanti-phospho-MEK1/2 (Ser217/221; #9154) from Cell SignalingTechnology; rabbit anti-LATS1þLATS2 (ab70565), rabbit anti-LATS1þLATS2 (phospho-T1079þT1041; ab111344), rabbit anti-PDGF receptor beta (ab32570), rabbit anti-YAP (ab52771), andrabbit anti-YAP (phospho-S127; ab76252), rabbit anti-PDGFreceptor beta (phospho Y1021) (ab134048), rabbit anti-ERK1þ ERK2 (ab17942), and rabbit anti-MEK1þMEK2 (ab178876)from Abcam; and mouse anti-TEF-1 (610922) from BD Bio-sciences. The secondary antibodies were horse anti-mouse IgG-HRP–linked antibody (#7076S) or goat anti-rabbit IgG-HRP–linked antibody (#7074S) from Cell Signaling Technology.Co-immunoprecipitation (co-IP) experiments were performed

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according to previously published protocols (24). The antibodiesused are listed above.

Immunofluorescence analysisThe immunofluorescence analysis was performed as we

reported previously (24). The primary antibodies rabbit anti-YAP(ab52771, Abcam) and mouse anti-OV6 (MAB2020, R&D Sys-tems) were incubated with samples at 4�C overnight. Nuclei werestained with DAPI (E607303, Sangon). Fluorescence images wereobserved and collected under a Leica DM5000B fluorescencemicroscope (Leica).

Antibody–microarray experiment and ELISAsThe antibody–microarray experiment was performed as

reported previously (31). Cytokine profiles were examinedby Quantibody Human Inflammatory Array 3 (QAH-INF-G3,RayBiotech) containing 40 inflammation-associated cytokines.The PDGF-BB and ICAM-1 levels in cell culture medium weremeasured using ELISA Kits for PDGF-BB (DBB00, R&D Systems)and ICAM-1 (DCD540, R&D Systems) according to the manu-facturer's instructions.

Chromatin immunoprecipitation and luciferase reporter assayChromatin immunoprecipitation (ChIP) assays were per-

formed according to our previously published study (31). Primerscomplementary to the promoter region of PDGFB (forward: 50-TGGCAGAGCAGGTTCCCACATA; reverse: 50-TGCTGAGAC-CACCGTGCTGT-30) were used to detect PDGFB genomic DNA,and primers specific to the humanGAPDHpromoter were used asthe control (kit supplied). Enrichment of the targets was calcu-lated as follows: fold enrichment ¼ 2[Ct (PDGFB-ChIP) � Ct (IgG)].

The TEAD1-binding sites of the PDGFB promoter(NC_000022.11: �3000 to þ100 relative to the PDGFB tran-scription site) or its mutant sequence (�2732 to �2723,CTCATTCCAT) were cloned into a pGL3-basic luciferase reportervector (Promega). UMUC3 cells were cotransfected with 10 ngpTK-RL reporter control plasmid and 200 ng pGL3-basic-PDGFB-WT or pGL3-basic-PDGFB-Mut using Lipofectamine 3000reagents (L3000015, Invitrogen) according to the manufacturer'sprotocols. Cells were collected 48 hours after transfection, andPDGFB transcription activity was evaluated by measuring lumi-nescencewith aDual-Luciferase Assay Kit (E1910, Promega). Foldinduction was derived relative to normalized reporter activity.

RNA-Seq and analysisRNA was isolated from OV6� and OV6þ UMUC3 cells using

TRIzol reagent (Invitrogen). The total RNA was purified with aQiagenRNeasyMini Kit (Qiagen), and then, the purifiedRNAwaschecked to determine the quantity. Single- and double-strandedcDNA was synthesized from mRNA samples. The double-strand-ed cDNA was then purified for end repair, dA tailing, adaptorligation, and DNA fragment enrichment. The libraries were quan-tified using Qubit (Invitrogen) according to the Qubit user guide.The constructed librarywas sequenced on an IlluminaHiseq 4000sequencer. The paired end raw reads were aligned using TopHatversion 1.2.0, which allowed 2 mismatches in the alignment. Thealigned reads were assembled into transcripts using cufflinksversion 2.0.0. The alignment quality and distribution of the readswere estimated using SAM tools. From the aligned reads, the geneand transcript expression was assessed using cufflinks version1.3.036. The differential transcripts were analyzed via cuffdiff.

Finally, GO and pathway functional analyses were performed forthe differentially expressed transcripts.

Animal experimentsAll experimental animal procedures were approved by the

Animal Care and Use Committee of the Second Military MedicalUniversity (Shanghai, China). UMUC3 cells were transfectedwiththe luciferase reporter gene. For the in vivo limiting dilution assay,sorted tumor cells were diluted at an appropriate cell dose andinjected into NOD/SCID mice (Shanghai Laboratory AnimalCenter, China); the number of tumors formed from each celldose injected was then scored. The frequency of CSCs was cal-culated using the ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html; The Walter and Eliza Hall Institute,Parkville, Victoria, Australia). In the animal experiments, humanrecombinant PDFG-BB (10 ng/mL), PDGF-BB–neutralizing anti-body (100 ng/mL), or CP-673451 (500 nmol/mL) was added tothe culture medium of sorted cells for 4 days before subcutane-ously injecting into the mice.

For the in vivo metastasis assay, 5 � 105 cells in 200 mL of PBSwere intravenously injected through the tail vein of 6-week-oldmale NOD/SCID mice. Mice were sacrificed and examined5 weeks after tumor cell injection. For mouse orthotopic bladdercancer, 6-week-old female NOD/SCID mice were used in thisstudy. Briefly, 5 � 105 UMUC3-Luc-OV6� cells or UMUC3-Luc-OV6þ cells in 50 mL of PBS were respectively installed into thebladder via a 24-gauge catheter and maintained for 3 hoursaccording to a previously described protocol (32). Tumor growthwas monitored weekly by live-animal bioluminescence opticalimaging using an IVIS Lumina II imaging system (PerkinElmer)after intraperitoneal injection of D-luciferin (150 mg/kg; GoldBiotech) in 100 mL of DPBS. Mice were sacrificed 4 weeks aftertumor implantation.

For the in vivo treatment model, orthotopic tumor-bearingmice, generated by installation of 1 � 106 UMUC3-Luc-OV6þ

cells or T24-Luc-OV6þ cells in 50 mL of PBS, were divided into 4groups on day 5 after tumor implantation. Cis-platinum (S1166,Selleck Chemicals), verteporfin (5305, Tocris Bioscience), andCP-673451 (HY-12050, MedChemExpress) were stocked inDMSO and prepared in PBS. Mice were treated by intraperitonealinjection of cis-platinum (3 mg/kg) alone or combined withverteporfin (100 mg/kg) or CP-673451 (30 mg/kg) every 2 days.Necropsies were performed after 6 weeks.

Statistical analysisNumerical data are expressed as the mean � SD. Statistical

differences between variables were analyzed with a 2-tailedStudent t test, x2 test, or Fisher exact test for categorical/binarymeasures or ANOVA for continuous measures. Correlation stud-ies were analyzed by Pearson. Survival curves were plotted usingthe Kaplan–Meier method and compared via log-rank analysis.Variables with P < 0.1 on the univariate analysis were included inmultivariate Cox proportional hazards analysis. Differences wereconsidered significant at P < 0.05. Time-dependent ROC analysiswas used to determine the cut-off value of censored data with"survival ROC" package, R software 3.4.4. Time-dependent areaunder the receiver-operating characteristic curve (AUC) was com-puted with the "time ROC" package. All the analyses were per-formed using the GraphPad Prism 5 (GraphPad Software, Inc.),SPSS 21.0 (IBMCorporation) software andR Project for StatisticalComputing (version 3.4.4).

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ResultsOV6 is associated with disease progression and prognosis ofpatients with bladder cancer

On the basis of the previous studies that OV6 closely associateswith progression and prognosis of patients with tumors (22–24),we also examinedwhether the epithelialmarkerOV6 is associatedwith the clinicopathologic characteristics and prognosis ofpatients with bladder cancer. First, we analyzed OV6 expressionin specimens via IHC. Thepositive and selective staining ofOV6 innormal bladder epithelial cells staining ofOV6 in normal bladderepithelial cells (Fig. 1A) suggested that it could serve as anepithelial marker. In addition, OV6 expression in bladder cancerspecimens was determined by immunoreactive scores (IRS; rang-ing from a minimum of 0 to a maximum of 12; the rules aredescribed in the "Materials andMethods"). Interestingly, theOV6expression was higher in the muscle-invasive bladder cancer(MIBC) than in the nonmuscle-invasive bladder cancer (NMIBC)samples (Fig. 1A). In addition, OV6 expression was higher in themuscular layer than in the mucosa of the same specimen frompatients with MIBC (Supplementary Fig. S1A). Of note, higherOV6 expression was observed in the invasive fronts of bladdercancer than in the core (Supplementary Fig. S1A). Metastaticlesions or postchemotherapy samples exhibited higher OV6expression than primary tumors or prechemotherapy samples(Fig. 1B and C). These data indicate that OV6 is positivelycorrelated with progression of bladder cancer.

Then, we determined whether OV6 expression in specimenswas associated with disease progression and the prognosis ofpatients with bladder cancer. First, the OV6 expression level inspecimens was assessed by IHC from patients with bladdercancer (Fig. 1D), and then, a time-dependent ROC curveanalysis was performed to determine the cutoff between lowand high OV6 expression in cohort 1; this analysis demon-strated that the best cut-off value was 4 (IRS score; Supple-mentary Fig. S1B). Thus, patients with bladder cancer fromcohort 1 were divided into 2 groups: a low OV6 expressiongroup (IRS values of 0–3) or a high OV6 expression (IRS valuesof 4–12) group, and OV6 expression in bladder cancer sampleswas significantly associated with multiple malignant clinico-pathologic features of bladder cancer tumors, such as tumorstage, tumor grade, lymph node status, and TNM stage (Sup-plementary Table S1). More importantly, Kaplan–Meier anal-ysis revealed that patients with higher OV6 expression exhib-ited markedly worse cancer-specific survival (CSS; P < 0.001),progression-free survival (PFS; P < 0.001), and overall survival(OS; P < 0.001) than their counterparts (Supplementary Fig.S1C–S1E). Furthermore, univariable and multivariable Coxregression analyses indicated that OV6 was an independentrisk factor for prognosis of patients with bladder cancer (Sup-plementary Table S2). In addition, another cohort (cohort 2;Supplementary Table S3), comprising 95 consecutive bladdercancer patients, was employed as a validation set using the cut-off value (IRS ¼ 4) derived from cohort 1. Kaplan–Meieranalysis revealed that patients with higher OV6 expressionexhibited markedly worse CSS (P ¼ 0.018), PFS (P ¼ 0.033),and OS (P < 0.001) than their counterparts (Supplementary Fig.S1F–S1H). Moreover, incorporating OV6 expression to TNMstage improved accuracy in predicting CSS of patients withbladder cancer as shown by time-dependent AUC analysis bothin cohort 1 and cohort 2 (Supplementary Fig. S1I and S1J).

Furthermore, patients with bladder cancer in cohort 1 weremerged with cohort 2, and all 225 patients with bladder cancerwere randomly split into a training set (n¼ 113) and a validationset (n ¼ 112) at 1:1 ratio. A time-dependent ROC curve analysiswas performed todetermine the cutoff between lowandhighOV6expression. Consistent with the best cutoff derived from cohort 1,the best cut-off value was also 4 (IRS score; Fig. 1E). Using thecutoff 4 for high and lowOV6 expression, highOV6 expression inbladder cancer sampleswas also significantly associatedwith hightumor stage, tumor grade, positive lymph node status, and highTNM stage in both the training set and the validation set (Sup-plementary Table S4). More importantly, Kaplan–Meier analysisrevealed that patients with higher OV6 expression exhibitedmarkedly worse CSS (P < 0.001, training set; P¼ 0.007, validationset), PFS (P<0.001, training set;P¼0.043, validation set), andOS(P < 0.001, training set; P ¼ 0.003, validation set) than theircounterparts (Fig. 1F–K). In addition, univariate andmultivariateCox regression analyses indicated that OV6 was an independentrisk factor for the CSS of patients with bladder cancer in both thetraining and validation sets (Supplementary Table S5). Moreover,improved accuracy in predicting CSS of patients with bladdercancer was also observed by combining OV6 expression with theTNM stage in both sets in time-dependent AUC analysis (Sup-plementary Fig.S1K and S1L). These results demonstrate that OV6could serve as a helpful indicator of disease progression andprognosis for patients with bladder cancer.

OV6þ cancer cells possess stem-like properties and facilitatebladder cancer's in situ tumorigenicity, invasion, migration,and metastasis

Given that OV6 is associated with disease progression andprognosis of patients with bladder cancer and serves as a CSCmaker for other tumors, we speculated that OV6 was a putativeCSCmarker for bladder cancer. First,wedetected thepercentage ofOV6 in bladder cancer cell lines and determined whether thepattern of OV6 expression in bladder cancer CSCs was similar toother CSC markers. The flow cytometry analysis demonstratedthat higher OV6 expression was observed in sphere-formingbladder cancer cells (which were reported to possess CSC prop-erties; ref. 33) than in adherent cells from various bladder cancercell lines (Fig. 2A). Second, immunofluorescence confirmedcolocalization of OV6 with other CSC markers, such as CD44 orCD133, in bladder cancer specimens or spheres from the bladdercancer cell line UMUC3 (Fig. 2B and C; Supplementary Fig. S2Aand S2B). Third, multimarker analyses indicated that OV6þ

UMUC3 or J82 cells also expressed CD44 or CD133 and that thepercentage of OV6þCD44þ or OV6þCD133þ cells was, respec-tively, increased in spheres (Supplementary Fig. S2C and S2D).Fourth, flow cytometry showed that OV6 was increased inCD133þ UMUC3 cells, which were sorted by magnetic-activatedcell sorting (MACS), and CD133 was also upregulated in OV6þ

UMUC3 cells (Supplementary Fig. S2E and S2F). These datasuggest that the pattern of OV6 expression in bladder cancer CSCsmay be similar to that of CD44 or CD133.

We then isolated OV6þ and OV6� UMUC3 or J82 cells viaMACS to compare their stem-like properties (Supplementary Fig.S2G). First, higher expression levels of stem-associated genes wereobserved in OV6þ cells than in control OV6� cells via real-timePCR (Fig. 2D). Second, a spheroid assay in serum-free medium orin semisolid medium revealed that significantly more spheres

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Figure 1.

OV6 is associated with disease progression and the prognosis of patients with bladder cancer. A, Representative hematoxylin and eosin (H&E) staining andimmunohistochemistry (IHC) staining images and scores of OV6 expression in normal, nonmuscle-invasive bladder cancer (NMIBC), and muscle-invasive bladdercancer (MIBC) specimens are presented (scale bar¼ 20 mm; (M, muscle; T, tumor). The IHC scores for OV6 in the corresponding groups are presented (right). B and C,Representative H&E staining and IHC staining for OV6 in the following groups of tissues: primary bladder cancer (BCa) tissues versus metastasis tissues (Case1#) andprechemotherapy tissues versus postchemotherapy tissues (Case2#) from the same patient with bladder cancer (scale bar ¼ 20 mm). The IHC scores for OV6 in thecorresponding groups are presented (right). D, According to the expression level of OV6 (immunoreactive score; IRS was described in the section "Materials andMethods") in specimens, patients with bladder cancer were divided into 2 groups: an OV6low group (IRS values of 0–3) and an OV6high group (IRS values of 4–12).Representative H&E and IHC staining of OV6 in tissues from patients with bladder cancer are shown (scale bar ¼ 20 mm). E, A time-dependent receiver-operatingcharacteristics (ROCs) analysis was performed to determine the optimum cut-off value of the OV6 IRS score to predict 5-year cancer-specific survival in thetraining set (n ¼ 113). F–H, Kaplan–Meier curves for cancer-specific survival (F), progression-free survival (G) and overall survival (H) of bladder cancer patientswere analyzed according to OV6 expression (training set, n ¼ 113). I–K, Kaplan–Meier curves for cancer-specific survival (I), progression-free survival (J), and overallsurvival (K) of patients with bladder cancer were analyzed according to OV6 expression (validation set, n ¼ 112).

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Figure 2.

OV6þ cancer cells possessed stem-like properties and facilitated bladder cancer in situ tumorigenicity, invasion, migration, and metastasis. A, Flow cytometric analysiswas performed to detect the percentage of OV6 (APC) in spheres and adherent cells from bladder cancer (BCa) cell lines. Immunofluorescent staining of OV6 (red),CD44 (green) and their colocalization (yellow) in tissues from patients with bladder cancer (BCa; B) or in spheres formed by the bladder cancer UMUC3 cellline (C) was performed (scale bar ¼ 10 mm). D, The expression of stem-associated genes in OV6þ and OV6� cells sorted from UMUC3 or J82 cultures was detectedwith real-time PCR. E, Single-cell suspensions with 3,000 cells were seeded in 6-well ultra-low attachment culture plates and cultured in serum-free mediumsupplemented with other reagents for 5 days. The number of spheroids formed was determined via microscopy, and representative pictures are shown. Representativeimages and the numbers of OV6þ and OV6� cell-derived spheres from 3 serial passages were compared (scale bar ¼ 75 mm). F, A total of 5,000, 10,000, or20,000 OV6þ or OV6� UMUC3 or J82 cells were subcutaneously injected into 6-week-old, male, nonobese, diabetic, severe combined immunodeficient (NOD/SCID)mice (n ¼ 6/group). The tumor xenografts derived from OV6þ and OV6� cells and the tumor incidence in 2 generations are shown. G, UMUC3 or J82 cells weretreated with cis-platinum (10 mmol/L) for 3 days, and the percentage of OV6þ cells in the total population was analyzed by flow cytometry. H, OV6þ and OV6� cellssorted from UMUC3 or J82 cells were treated with cis-platinum for 3 days. The cell viability was measured through a CCK-8 assay, and the data are presented asthe fold change relative to the treatment-free groups. I,OV6þ and OV6� cells sorted from UMUC3 or J82 cells were treated with cis-platinum (10 mmol/L) for 3 days, andthe annexin V/PI staining percentage was analyzed via flow cytometry. All the data represent the means � SD (�, P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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were formed by OV6þ bladder cancer cells than by OV6� cells(Fig. 2E; Supplementary Fig. S2H). In addition, single-cellspheroid formation assay revealed that OV6þ bladder cancercells had significantly stronger capacity to generate spheroidsthan OV6� cells (Supplementary Fig. S2I), indicating thatOV6þ bladder cancer cells harbored strong self-renewal. Third,a tumor limited dilution assay to analyze OV6þ bladder cancercells stably labeled with luciferase that were subcutaneouslyinjected of into nonobese, diabetic, severe combined immu-nodeficient (NOD/SCID) mice was employed to determinewhether OV6þ bladder cancer cells were more tumorigenicthan control OV6� cells in vivo. As expected, OV6þ bladdercancer cells initiated tumors with markedly higher frequencythan OV6� cells in 2 serial generations. (Fig. 2F; Supple-mentary Fig. S2J and S2K; Supplementary Table S6). Further-more, we showed that OV6þCD44þ UMUC3 cells exhibitedhigher tumor initiation incidence and frequency thanOV6�CD44þ cells in NOD/SCID mice, but there was not astatistical difference in tumorigenicity between OV6þCD44þ

and OV6þCD44� cells (Supplementary Fig. S2L; Supplemen-tary Table S7), indicating that OV6 plays a stronger role thanCD44 in the tumor initiation capacity of CSCs. Moreover, giventhat CSCs were resistant to chemotherapeutic drugs, we alsoexamined whether OV6þ bladder cancer cells were more che-moresistant compared with OV6� cells. Because chemothera-peutic drugs can enrich CSCs in tumors, a flow cytometricanalysis was performed to detect the percentage of OV6þ cellsin bladder cancer cells after treatment with chemotherapeuticdrugs. As expected, cis-platinum enriched the ratio of OV6þ

cells in UMUC3 or J82 cell populations (Fig. 2G). A CellCounting Kit-8 (CCK-8) assay demonstrated that OV6þ CSCsexhibited higher cell survival during cis-platinum treatmentthan OV6� cells (Fig. 2H). In addition, annexin V/ propidiumiodide (PI) double staining revealed that cis-platinum treat-ment induced fewer apoptotic events in OV6þ CSCs (UMUC3,11.1% � 1.1%; J82, 12.89% � 2.1%) but considerably moreapoptotic events in OV6� cells (UMUC3, 47.5% � 5.1%; J82,85.4% � 9.45%; Fig. 2I). These results indicate that OV6þ

bladder cancer cells possess strong stem-like properties.Given that CSCs possess stronger tumorigenicity and facilitate

tumor metastasis (28), we assessed the in situ tumorigenicity andmetastatic abilities of OV6þ CSCs in bladder cancer. First, OV6þ

and OV6� cells obtained from UMUC3 cell populations stablylabeled with luciferase were perfused into murine bladdersthrough a urethral catheter, and the resulting bioluminescencewas detected using an in vivo imaging system to monitor tumorgrowth (Supplementary Fig. S2M). OV6þCSCs exhibited a higherrate of tumor formation in bladders than control OV6� cells(Supplementary Fig. S2M-O). Third, Matrigel invasion assays andtranswell migration assays respectively showed that OV6þ CSCsexhibited a higher rate of cell invasion and migration than OV6�

UMUC3 or J82 cells (Supplementary Fig. S2P and S2Q). Inaddition, OV6þ and OV6� UMUC3 cells were injected into thecaudal vein of mice, and the injection of OV6þ cells yieldedincreases in the incidence, number and size of lung metastasesthan OV6� cells (Supplementary Fig. S2R–S2T). Epithelial-to-mesenchymal transition (EMT) is an essential step in tumormetastasis (34). We thus assessed the expression of the EMTmarker Vimentin inOV6þUMUC3 cells-derivedmetastatic lesionby IHC. As expected, higher Vimentin expression was observed inspecimens from OV6þ cell–generated lung metastasis (Supple-

mentary Fig. S2R–S2T). These results demonstrate that OV6þ

CSCs can facilitate in situ tumorigenicity andmetastasis of bladdercancer cells.

YAP is required for maintenance of stem-like properties ofOV6þ CSCs in bladder cancer

To identify strategies for targeting OV6þ CSCs and herebyreversing bladder cancer progression and chemotherapy resis-tance, we next explored the mechanisms that promote the expan-sion and self-renewal of OV6þCSCs. First, OV6þCSCs andOV6�

UMUC3 cells were analyzed by RNA-Seq to search for the criticalgenes or pathways involved in maintaining the stem-like prop-erties of OV6þ CSCs. (Fig. 3A; Supplementary Fig. S3A–SD;Supplementary Table S8–S10). The results revealed that OV6þ

CSCs exhibited differential expression of Hippo pathway–relatedgenes, such as the higher expression of YAP (Fig. 3A). Real-timePCR confirmed that YAP mRNA was upregulated in OV6þ CSCsfrom UMUC3 and J82 cultures, and Western blotting also cor-roborated the increased YAP protein level in the nucleus of OV6þ

CSCs (Fig. 3B and C; Supplementary Fig. S3E), suggesting thatYAP was activated in OV6þ bladder cancer CSCs. In addition, theRNA-Seq results indicated that OV6þ CSCs presented elevatedexpression of YAP target genes (Fig. 3D), which were validated forconnective tissue growth factor (CTGF) and cysteine-rich angio-genic inducer 61 (CYR61; Fig. 3B). In addition, p-YAP phosphor-ylated by p-LATS1/2 is well known to be retained in the cyto-plasm, but only activated YAP (no phosphorylation) enters thenucleus to exert its biological function. Accordingly, Westernblotting demonstrated that reduced YAP and LATS1/2 phosphor-ylation was observed in the cytoplasm of OV6þ CSCs (Fig. 3C;Supplementary Fig. S3E). In addition, relative to YAP expressionin OV6� cells, Western blotting and immunofluorescence assaysboth revealed that YAPwasmainly located in the nucleus ofOV6þ

CSCs (Fig. 3C and E; Supplementary Fig. S3E and S3F). Takentogether, these data suggest that YAP signaling is activated inOV6þ bladder CSCs.

We subsequently examined whether YAP was required formaintaining the stem-like properties of OV6þ CSCs. After thesuccessful knockdown of YAP in bladder cancer cells was con-firmed by Western blot analysis (Supplementary Fig. S3G), lowerexpression of stem-associated genes and fewer sphereswere foundin YAP-silenced OV6þ CSCs than in control OV6þ cells (Fig. 3Fand G; Supplementary Fig. S3H and S3I). Furthermore, com-pared with control OV6þ CSCs, YAP-silenced OV6þ CSCsshowed decreased tumor initiation in 2 serial generations, andenhanced apoptosis and decreased cell survival were observedin OV6þ CSCs under cis-platinum treatment (Fig. 3H and I;Supplementary Fig. S3J–S3L). In addition, to test whether upre-gulation of YAP could rescue these inhibitory effects of YAPknockdown on the stem-like properties of OV6þ CSCs, we reex-pressed either wild-type (WT) or mutant (MT) YAP in YAP-knockdown OV6þ CSCs from UMUC3 and J82 cells. Westernblotting demonstrated that bladder cancer cells transfected withMT-YAP exhibited a better overexpression effect (SupplementaryFig. S3M). Subsequently, exogenous expression of MT-YAPenhanced the expression of stem-associated genes and thesphere-forming capacity of YAP shRNA-transfected-OV6þ CSCs(Supplementary Fig. S3N and S3O). In addition, YAP overexpres-sion increased the expression of stem-associated genes, the num-ber of spheres, and the tumorigenicity and chemoresistance ofOV6þ CSCs in bladder cancer (Fig. 3J–M; Supplementary

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Fig. S3P–S3T; Supplementary Table S11). Thus, YAP is crucial formaintaining the stem-like properties of OV6þ CSCs.

In addition, IHC analyses of specimens from patients withbladder cancer were performed (Supplementary Fig. S3U), andthe results revealed a positive correlation betweenOV6 expressionand nuclear YAP expression (Supplementary Table S12; P <0.001). Furthermore, all patients were classified into 4 groupsaccording to OV6 and nuclear YAP expression in specimens(Supplementary Table S13), and concomitantly elevated expres-sion of OV6 and nuclear YAP in bladder cancer specimenswas found to be associated with the poorest CSS (P < 0.001),PFS (P < 0.001), and OS (P < 0.001; Fig. 3N–P).

PDGF-BB/PDGFR–mediated signaling sustains persistentactivation of YAP in OV6þ CSCs

Given that autocrine signaling maintains the stem-like prop-erties of CSCs (35), we investigated the crucial inflammatoryfactors in mediating OV6þ bladder cancer CSCs using a RayBioHuman Cytokine Antibody Array (Fig. 4A–C; Supplementary Fig.S4A-C; Supplementary Table S14). As expected, the level of manycytokines was significantly upregulated in the conditioned medi-um (CM) from OV6þ CSCs from UMUC3 or J82 cell culturesrelative to those in CM from OV6� cells (Fig. 4A and B; Supple-mentary Fig. S4A-C; Supplementary Table S14). We then com-pared the significantly differentially expressed cytokines using aVennplot and found that PDGF-BB and ICAM-1were consistentlyincreased in both cell types (Fig. 4C; Supplementary Table S14).On the basis of validation through ELISA analysis, the secretion ofPDGF-BB but not ICAM-1was significantly higher in the CM fromOV6þ bladder CSCs than in the CM from OV6� cells (Fig. 4D),which prompted us to investigate whether PDGF-BB was respon-sible for maintaining the stemness of OV6þ CSCs. First, humanrecombinant PDGF-BB protein was added to OV6þ CSC cultures,which resulted in higher expression of stem-related genes andhigher sphere formation than that detected in na€�ve OV6þ CSCcultures (Fig. 4E and F; Supplementary Fig. S4D and S4E). Second,PDGF-BB–treatedOV6þCSCspresented ahigher tumor incidencethan control OV6þ cells (Fig. 4G). Conversely, the addition of aneutralizing antibody against PDGF-BB to the CM of OV6þ CSCssuppressed the stem-like properties (Fig. 4E–G; SupplementaryFig. S4D and S4E). In addition, PDGF-BB facilitated the stem-likeproperties ofOV6� bladder cancer cells (Supplementary Fig. S4F–S4H; Supplementary Table S15). Thus, PDGF-BB is required forthe stem-like features of OV6þ bladder CSCs.

Given that PDGF-BB triggers the intrinsic signaling of tumorcells through its receptor, PDGFR (36), we also found thatrecombinant PDGF-BB protein activated the phosphorylation ofPDGFR and its downstream kinases (MEK and ERK; ref. 37) inOV6þ CSCs (Supplementary Fig. S4I). We then determinedwhether the PDGFR inhibitor CP-673451 (38) suppressed thestem-like characteristics of OV6þ CSCs. As expected, CP-673451inhibited the stem-like characteristics of OV6þ CSCs, includingstem-associated gene expression, self-renewal, and tumorigenicity(Fig. 4E–G; Supplementary Fig. S4D and S4E). Thus, autocrinePDGF-BB/PDGFR signaling is required for maintenance of thestem-like characteristics of OV6þ CSCs in bladder cancer.

Because YAP was required for the stem-like properties of OV6þ

CSCs, we next investigated whether autocrine PDGF-BB/PDGFR–mediated YAP activity. First, recombinant PDGF-BB proteininhibited YAP phosphorylation in the cytoplasmwhile increasingYAP expression in the nucleus ofOV6þCSCs, whereas CP-673451had the opposite effects (Fig. 4H; Supplementary Fig. S4K).Second, YAP knockdown alleviated the promoting role ofPDGF-BB in YAP stabilization and the stem-like characteristicsof OV6þ CSCs, including stem-associated gene expression, self-renewal, and tumorigenicity (Fig. 4I-K; Supplementary Fig. S4Land S4M). Therefore, PDGF-BB/PDGFR facilitated OV6þ CSCsthrough stabilizing YAP and promoting YAP entry into thenucleus.

Moreover, we investigated how PDGF-BB/PDGFR mediatedYAP activity inOV6þCSCs. First, we tested whether PDGFR coulddirectly interactwithYAP to forma complex andprevent LATS1/2-dependent YAP phosphorylation. A co-IP analysis demonstratedthat endogenous PDGFR directly interacted with YAP in OV6þ

UMUC3 CSCs (Fig. 4L). Second, PDGF-BB enhanced the inter-action between PDGFR and YAP, while CP-673451 abated theeffects in OV6þ CSCs from UMUC3 or J82 cultures (Fig. 4M). Inthe samemanner, PDGF-BB decreased the interaction between p-LATS1/2 and YAP, while CP-673451 abrogated the promotingeffects of PDGF-BB (Fig. 4M). Third, PDGF-BB decreased thephosphorylation of YAP and LATS1/2 in the cytoplasm of OV6þ

CSCs (Fig. 4H). In addition, PDGF-BB–treated OV6þ CSC–derived xenografts presented activated PDGFR and YAP, whichwere inhibited in CP-673451-treated OV6þ CSC–derived xeno-grafts (Supplementary Fig. S4N-Q). Therefore, PDGFR activatedby PDGF-BB can directly interact with YAP, which preventsp-LAST1/2 interaction with YAP and inhibits YAP phosphoryla-tion in OV6þ CSCs.

Figure 3.YAP is required for maintenance of the stem-like properties of OV6þ CSCs in bladder cancer. A, RNA-Seq was applied, and a heatmap depicting thesignificantly expressed genes in OV6þ and OV6� UMUC3 cells from 3 independent experiments is shown. B, The mRNA levels of YAP, CTGF, and CYR61 in OV6þ

and OV6� cells from UMUC3 or J82 cultures were detected by real-time-PCR. C,Western blot analysis of p-LATS1/2, LATS1/2, p-YAP, and YAP in cytoplasmic (Cyt)and nuclear (Nuc) fractions of OV6þ and OV6� cells from UMUC3 cultures. b-Tubulin and Histone H3 served as internal controls for the cytoplasmic andnuclear fractions, respectively. D, A heatmap depicting the significantly differentially expressed LATS1, LATS2, YAP, and target genes of YAP in OV6þ and OV6�

UMUC3 cells from 3 independent experiments is shown. E, Immunofluorescent staining of OV6 and YAP in OV6þ and OV6� cells from UMUC3 cultures was analyzed(scale bar¼ 10mm).F,Expression of stem-associatedgenes inOV6þUMUC3 cells transfectedwithYAP shRNA (#1 and#2) or control shRNA.G,Thenumbers ofOV6þ

UMUC3 cells transfected with YAP shRNA (#1 or #2) or control shRNA-derived spheres were compared among serial passages. H, OV6þ UMUC3 cells withYAP shRNA#1 or control shRNA were subcutaneously injected into NOD/SCID mice (n ¼ 6/group), and images of xenografts derived from OV6þ UMUC3 cellswith different treatments are presented. I,OV6þ UMUC3 cells transfected with YAP shRNA (#1 and #2) or control shRNAwere treated with cis-platinum (10 mmol/L)for 3 days. The annexin V/PI staining percentage was analyzed by flow cytometry. J, Expression of stem-associated genes in OV6þ UMUC3 cells transfectedwithout or with YAP plasmid, examined via real-time PCR. K, The number spheres derived from OV6þ UMUC3 cells treated without or with YAP plasmid wascompared among serial passages. L,OV6þUMUC3cellswithout orwithYAPplasmidwere subcutaneously injected intoNOD/SCIDmice (n¼6/group), and imagesofxenografts are presented. M, OV6þ UMUC3 cells without or with YAP plasmid were treated with cis-platinum (10 mmol/L) for 3 days. The annexin V/PIstaining percentage was analyzed via flow cytometry. N–P, Kaplan–Meier curves for CSS, PFS, and OS of patients with bladder cancer were analyzed accordingto OV6 and YAP expression (cohort1, n ¼ 130; � , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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YAP/TEAD1/PDGF-BB/PDGFR forms an autocrine regulatoryloop in OV6þ CSCs

Given that PDGF-BB/PDGFR upregulated and activated YAP inOV6þ bladder CSCs, we next examined whether YAP couldreciprocally promote PDGFB expression and secretion. As shownin Fig. 5A, higher PDGFB expression was observed in OV6þ thanin OV6� CSCs from UMUC3 or J82 cells. However, YAP knock-down decreased the PDGFB levels in OV6þ CSCs (Fig. 5A), whileYAP overexpression caused the opposite effects (Fig. 5B). Second,the concentration of PDGF-BB inCM fromOV6þCSCswas higherthan that in CM from OV6� cells (Fig. 5C). Silencing of YAPdecreased the PDGF-BB concentration in CM from OV6þ CSCs(Fig. 5C), whereas YAP overexpression increased the PDGF-BBconcentration (Fig. 5D). Third, we reexpressed YAP in YAP-knockdown OV6þ CSCs, which reversed the inhibitory effects ofYAP knockdown, especially the decreases in PDGFB expressionand PDGF-BB concentration (Supplementary Fig. S5A and S5B).The data indicate that YAP promotes PDGFB expression in OV6þ

bladder CSCs.Then,we examined themechanisms underlying YAP regulation

of PDGFB inOV6þ bladder CSCs. YAP triggers the transcription ofdownstream genes by recruiting TEAD in tumors (39), whichprompted us to determine whether YAP upregulated PDGFBtranscription via TEAD1 in OV6þ CSCs. We observed an interac-tion between YAP and TEAD1 in OV6þ UMUC3 or J82 CSCs,which was further enhanced by TEAD1 overexpression or recom-binant human PDGF-BB treatment (Fig. 5E). Verteporfin, aninhibitor that blocks the interaction between YAP and TEAD(40), decreased PDGFB expression and PDGF-BB secretion byOV6þ CSCs (Fig. 5A and C). Third, TEAD1-knockdown attenu-ated the YAP-induced increase in PDGFB mRNA expression andPDGF-BB concentration in CM of OV6þ CSCs (Fig. 5B and D;Supplementary Fig. S5C). Furthermore, TEAD1 overexpressionincreased the levels of stem-associated genes and the number ofspheres formed by OV6þ CSCs (Fig. 5F-G; Supplementary Fig.S5D-F), and TEAD1 knockdown alleviated the promoting effectsof YAP or PDGF-BB on the stem-like properties of OV6þ CSCsfrom UMUC3 or J82 cells (Fig. 5F-G; Supplementary Fig. S5E-F).Therefore, YAP facilitates PDGFB expression in a TEAD1-depen-dent manner, contributing to the stem-like characteristics ofOV6þ CSCs.

In addition, online JASPAR software (http://jaspar.genereg.net)was employed to predict putative transcription factor–bindingsites of TEAD1 on the PDGFB promoter (Supplementary Fig.S5G). As expected, TEAD1 bound to the PDGFB promoter inOV6þ CSCs, as demonstrated through a chromatin immunopre-cipitation (ChIP) assay (Fig. 5H). In addition, though TEAD1 orYAP overexpression or PDGF-BB treatment enhanced PDGFBtranscriptional activity in OV6þ UMUC3 CSCs, mutated variantsof the TEAD1-binding sites on PDGFB abolished the effects,which was validated by luciferase assays (Fig. 5I). Thus, PDGF-BB/PDGFR–induced YAP upregulation reciprocally promotes thePDGFB transcription through TEAD1 in OV6þ bladder CSCs.

Blocking the YAP/TEAD1/PDGF-BB/PDGFR autocrineregulatory loop impairs chemotherapy resistanceofOV6þCSCs

As the autocrine regulatory loop YAP/TEAD1/PDGF-BB/PDGFR is required inOV6þCSCs,we further investigatedwhetherblocking the loop using verteporfin or CP-673451 could improvethe therapeutic effect of cis-platinum in an orthotopic bladdercancer mouse model. OV6þ CSCs from UMUC3 or T24 culturesstably expressing a luciferase reporter were perfused into murinebladders through a urethral catheter, and tumor growth wasmonitored using an in vivo imaging system (Fig. 6A; Supplemen-tary Fig. S6A). After the perfusion, mice were treated with cis-platinum or cis-platinum (Cis) combined with verteporfin (VP),or with CP-673451 (CP). As shown in Fig. 6A and B and Sup-plementary Fig. S6A andS6B, therewas no significant difference intumor growth between OV6þ CSC–derived xenografts treatedwith cis-platinum and na€�ve xenografts, indicating that OV6þ

CSCs were resistant to cis-platinum in vivo, which is consistentwith the in vitro experimental results. However, cis-platinumcombinedwith verteporfinorCP-673451 inhibited tumor growthand reduced the expression levels of OV6 and YAP in tissues fromOV6þ CSC–derived xenografts (Fig. 6A–D; Supplementary Fig.S6A–S6D). These results indicate that blocking the YAP/TEAD1/PDGF-BB/PDGFR autocrine regulatory loop in OV6þ CSCs withverteporfin or CP-673451 can reduce cis-platinum resistance inbladder cancer.

Furthermore, we examined the clinical significance of OV6 andPDGFR in patients with bladder cancer. We found a positivecorrelation between OV6 and PDGFR expression in bladder

Figure 4.PDGF-BB/PDGFR sustains persistent activation of YAP in OV6þ CSCs. A and B, Cytokine profiles were analyzed using a RayBio Human Cytokine AntibodyArray. Heatmaps of significantly differentially expressed cytokines in conditioned medium (CM) from OV6þ and OV6� J82 (A) or UMUC3 (B) cells. C, A Venn plot ofsignificantly upregulated cytokines in CMof OV6þ cells (relative to CM of OV6� cells) fromUMUC3 or J82 cultures is shown.D,An ELISAwas performed to detect thePDGF-BB or ICAM-1 concentration (pg/ml) in CM from OV6þ and OV6� bladder cancer (BCa) cells. E, Expression of stem-associated genes in OV6þ UMUC3 cellswithout or with recombinant PDGF-BB (10 ng/mL), a neutralizing antibody for PDGF-BB (100 ng/mL) or the PDGFR inhibitor CP-673451 (500 nmol/mL) for4 days. F, The numbers of spheres derived from OV6þ UMUC3 cells without or with recombinant PDGF-BB (10 ng/mL), a neutralizing antibody for PDGF-BB(100 ng/mL), or CP-673451 (500 nmol/mL) for 4 days were compared among serial passages. G, OV6þ UMUC3 cells without or with recombinantPDGF-BB (10 ng/mL), a neutralizing antibody for PDGF-BB (100 ng/mL) or CP-673451 (500 nmol/mL) for 4 dayswere subcutaneously injected into NOD/SCIDmice(n ¼ 6/group). Images of xenografts derived from OV6þ UMUC3 cells with different treatments in 2 generations are presented. H, Western blot analysisof p-LATS1/2, LATS1/2, p-YAP, and YAP in cytoplasmic (Cyt) and nuclear (Nuc) fractions of OV6þ UMUC3 or J82 cells without or with recombinant PDGF-BB(10 ng/mL) or CP-673451 (500 nmol/mL) treatment for 4 days. b-Tubulin and Histone H3 served as internal controls for the cytoplasmic and nuclear fractions,respectively. I, Expression of stem-associated genes in OV6þ UMUC3 cells without or with recombinant PDGF-BB (10 ng/mL, 4 days) or with PDGF-BBplus YAP knockdown (shYAP1# and shYAP2#). J, The numbers of spheres derived fromOV6þ UMUC3 cells without or with recombinant PDGF-BB (10 ng/mL, 4 days)or with PDGF-BB plus YAP knockdown (shYAP1# and shYAP2#) were compared among serial passages. K, OV6þ UMUC3 cells without or with recombinantPDGF-BB (10 ng/mL, 4 days) or with PDGF-BB plus YAP knockdown (shYAP1#) were subcutaneously injected into NOD/SCID mice (n ¼ 6/group). Images ofxenografts derived from OV6þ UMUC3 cells with different treatments in 2 generations were presented. L, A Western blot analysis was performed to detectcoimmunoprecipitation (co-IP) of endogenous PDGFR with YAP from OV6þ UMUC3 cells. IgG served as the IP control. M, Western blot analysis of co-IP ofendogenous PDGFR or p-LATS1/2 with YAP from OV6þ UMUC3 or J82 cells without or with recombinant PDGF-BB (10 ng/mL) or CP-673451 (500 nmol/mL)for 4 days (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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Figure 5.

YAP/TEAD1/PDGF-BB/PDGFR forms an autocrine regulatory loop in OV6þ CSCs. A, mRNA expression of PDGFB in OV6� and OV6þ UMUC3 or J82 cellstreated without or with YAP shRNA#1, shRNA#2 or the YAP inhibitor verteporfin (500 nmol/mL, 3 days). B, PDGFB mRNA expression was analyzed in control OV6þ

cells and YAP-overexpressing OV6þ cells without or with TEAD1 shRNA#1 or TEAD1 shRNA#3. C, An ELISA was performed to detect the PDGF-BB concentration(pg/mL) in CM from OV6� and OV6þ UMUC3 or J82 cells treated without or with YAP shRNA#1, YAP shRNA#2, or verteporfin (500 nmol/mL, 3 days). D, AnELISA was performed to detect the PDGF-BB concentration (pg/mL) in CM from control OV6þ cells and YAP-overexpressing OV6þ cells without or withTEAD1 shRNA#1 or TEAD1 shRNA#3.E,Western blot analysis of the co-IP of endogenousYAPwith TEAD1 fromOV6þUMUC3or J82 cells treatedwithout orwith TEAD1overexpression or recombinant PDGF-BB treatment (10 ng/mL, 4 days). F, Expression of stem-associated genes in control OV6þ cells, TEAD1-overexpressingOV6þ cells or YAP-overexpressing OV6þ cells without or with TEAD1 shRNA#3. G, The numbers of spheres derived from control OV6þ cells, TEAD1-overexpressingOV6þ cells or YAP-overexpressing OV6þ cells without or with TEAD1 shRNA#3 were compared among serial passages. H, ChIP-PCR analysis confirmed thebinding of TEAD1 to the PDGFBpromoter inOV6� andOV6þbladder cancer cells. I, TEAD1-binding sites inOV6þUMUC3 cellswere blocked using reporter constructsharboring mutant TEAD1 variants. Luciferase assays were performed to detect PDGFB transcription activity in OV6þ UMUC3 cells without or with TEAD1overexpression, YAP overexpression and PDGF-BB (10 ng/mL, 4 days) treatment (� , P < 0.05; ��, P < 0.01; and ��� , P < 0.001).

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Figure 6.

Blocking the autocrine regulatory loop using verteporfin or CP-673451 impedes chemotherapy resistance of OV6þ CSCs. A, OV6þ cells with stable luciferaseexpressionwere perfused intomurine bladders using a urethral catheter, and then, themice were intraperitoneally injected with cis-platinum (3mg/kg; Cis) alone orcis-platinum combined with verteporfin (100 mg/kg; VP) or CP-673451 (30 mg/kg; CP) every 2 days. The tumor growth was monitored using an in vivo imagingsystem. Bioluminescence images and tumor images of orthotopic xenografts derived fromOV6þ UMUC3 cells with different treatments are presented. H&E and IHCstaining of OV6 and YAP in orthotopic tumors from mice in different groups was performed (scale bar ¼ 500 mm, 100 mm, or 20 mm). B, At the third weekpostperfusion, photon fluxwas examined in the different groups ofmice. The results are presented as the fold increase in tumor growth over time until the thirdweekpostinjection. C and D, IHC staining scores for OV6 (C) and YAP (D) in orthotopic tumors from mice in different groups are presented. E, Schematic diagram of theunderlying mechanisms described in our study and the clinical significance of our findings (� , P < 0.05; �� , P < 0.01; and ��� , P < 0.001).

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cancer specimens (Supplementary Fig. S6E; SupplementaryTable S16). On the basis of intratumor OV6 and PDGFR expres-sion, patients with bladder cancer were classified into 4 groups(Supplementary Table S17), and concomitantly elevated OV6and PDGFR expression in patients with bladder cancer resultedin the poorest CSS (P<0.001), PFS (P<0.001), andOS (P<0.001;Supplementary Fig. S6F–S6H).

DiscussionApproximately 75%of newly diagnosed patients are diagnosed

with NMIBC, and 25% are diagnosed with MIBC or metastaticdisease (41). Despite receiving radical cystectomy and pelviclymph node dissection for MIBC, more than half of patients willeventually develop tumors at distant sites (42). Bladder cancerrelapse and treatment failure in most patients have been attrib-uted to the existence of CSCs, which survive many commonlyemployed therapeutics, including chemotherapy. Althoughmanystudies have focused on bladder CSCs, the specific markers andunderlying mechanisms of chemotherapy resistance have notbeen elucidated. However, our study identified a new subset ofCSCs with OV6 expression that is closely associated with theprogression and prognosis of patients with bladder cancer. Fur-thermore, the results showed that targeting OV6þ CSCs inhibitedchemotherapy resistance and improved the therapeutic effects ofcis-platinum on MIBC (Fig. 6E). These data suggest that OV6 canalso serve as a potential therapeutic target. However, becauseOV6itself is a poorly understood molecular entity, the practicality ofusing it to mark CSCs awaits further delineation of its constituentcomplexity.

YAP is a transcription cofactor suppressed by the Hippopathway, and this cofactor exerts its protumorigenic functionand drives CSC self-renewal in various malignant tumors. Inbladder cancer, YAP has been reported to be an independentbiomarker for poor prognosis of patients and to promote cellgrowth and migration (43, 44). A recent study indicated thatYAP activation is a pharmacological target for enhancing theantitumor effects of DNA-damaging modalities in chemother-apy (45). However, the role and mechanism of YAP in bladderCSCs have not been clearly elucidated. In this study, thedifferentially expressed genes regulating bladder CSCs werescreened through transcriptome sequencing, and YAP wasfound to be a crucial factor. In addition, we found that YAPis necessary for maintenance of the stem-like properties ofOV6þ bladder CSCs and that the combined expression of YAPand OV6 predicts the prognosis of patients.

Many studies have demonstrated that autocrine signaling loopscontinuously activate intratumor pathways and regulate tumorgrowth and CSCs (10, 46, 47). To elucidate the mechanismsunderlying the sustained activation of YAP and its role in theself-renewal ofOV6þCSCs, we also identified a positive autocrineloop, namely, the YAP/TEAD1/PDGF-BB/PDGFR loop. PDGF-BBhas been reported to mediate mesenchymal stem cells and che-motherapy-resistant cancer cells (48, 49), but the biologicalfunction and mechanisms of PDGF-BB in CSCs are not fullyunderstood. In addition, although YAP is upregulated by stimu-lation with PDGF-BB in vascular smooth muscle cells (50), themechanisms underlying PDGF-BB regulation of YAP have notbeen elucidated. In our study, we found that PDGF-BB facilitatesthe stem-like characteristics of OV6þ CSCs by enhancing YAPstability in the cytoplasm and promoting increased YAP entry into

the nucleus. A recent study revealed that a PDGFR-Src familykinase (SFK) cascade regulates YAP activation via tyrosine phos-phorylation in cholangiocarcinoma (51). However, whetherPDGFR directly mediates YAP stabilization in tumors has notbeen reported. Our study demonstrated that PDGFR interactswith YAP to prevent YAP phosphorylation by LATS1/2, whichfacilitates YAP activation in OV6þ CSCs.

Given that OV6 is associated with bladder cancer progres-sion and the prognosis of patients with bladder cancer and theautocrine YAP/TEAD1/PDGF-BB/PDGFR signaling loop isrequired in OV6þ CSCs, we established a treatment model oforthotopic bladder cancer to examine whether blocking theautocrine loop inhibited the resistance of MIBC and augment-ed the therapeutic effect of cis-platinum. As expected, PDGFRor YAP suppression alleviated the chemotherapy resistance ofOV6þ CSCs to cis-platinum and achieved a favorable thera-peutic effect in vivo. We do, however, recognize that the YAP/TEAD1 inhibitor verteporfin used in our study has beenreported to exhibit YAP-independent antiproliferative andcytotoxic effects in endometrial cancer cells (52). On the basisof the results of this study, our future studies will furtherelucidate the interaction between CSCs and the microenviron-ment, which will aid in the discovery of more effective ther-apeutic targets for drug resistance and recurrence in patientswith bladder cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.-J. Wang, C. Wang, Y.-H. Sun, C.-L. XuDevelopment of methodology: C. Wang, S. Zeng, C.-L. XuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.-J. Wang, L.-H. Dai, J. Yang, Q.-Q. Tian, C.-L. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.-J. Wang, C. Wang, L.-H. Dai, J. Yang, H. Huang,X. Ma, X. Lu, S. Zeng, C.-L. XuWriting, review, and/or revision of the manuscript: C. Wang, X. Ma, S. Zeng,C.-L. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Wang, J. Yang, Z. Zhou, Z.-Y. Yang, W. Xu,M.-M. Hua, H.-Q. Wang, Y.-Q. Cheng, D. Liu, Y.-H. SunStudy supervision: Z. Zhang, Y.-H. Sun, C.-L. Xu

AcknowledgmentsThis work was supported by Innovation Program of Shanghai Municipal

Education Commission (no. 2017-01-07-00-07-E00014); the National NaturalScience Foundation of China (no. 81772720, 81773154, 81572509, and81301861); Shanghai Natural Science Foundation of China (no.13ZR1450700); Special Fund for Major Projects of Zhangjiang National Inno-vation Demonstration Zone; the National New Drug Innovation Program(2017ZX09304030); Shanghai Clinical Medical Center For Urinary SystemDiseases (2017ZZ01005); Shanghai Key Laboratory of Cell Engineering(14DZ2272300). Medical Discipline Construction Project of the Pudong NewDistrict (PWYgf2018-03). We also thank Dr. Zi-Wei Wang and Yu-xin Tan(Department of Urology, Changhai Hospital, Second Military Medical Univer-sity, Shanghai, China), Jian Lu, and Qi Chen (Department of Health Statistics,Second Military Medical University, Shanghai, China) for their contributionsin this study.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked adver-tisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 19, 2018; revised June 15, 2018; accepted November 1,2018; published first November 5, 2018.

Wang et al.

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References1. Torre Lindsey A, Freddie B, Siegel Rebecca L, Jacques F, Joannie L-T,

Ahmedin J. Global cancer statistics, 2012. CA Cancer J Clin2015;65:87–108.

2. Knowles Margaret A, Hurst Carolyn D. Molecular biology of bladdercancer: new insights into pathogenesis and clinical diversity. Nat RevCancer 2015;15:25–41.

3. Zhang H, Prado K, Zhang KX, Peek EM, Lee J, Wang X, et al. Biasedexpression of the FOXP3 3 Isoform in aggressive bladder cancer mediatesdifferentiation and cisplatin chemotherapy resistance. Clin Cancer Res2016;22:5349–61.

4. Park Nicole I, Paul G, Kinjal D, McAdam Rochelle F, Ellen L, ConnorMadlen O, et al. ASCL1 reorganizes chromatin to direct neuronal fate andsuppress tumorigenicity of glioblastoma stem cells. Cell Stem Cell2017;21:209–24.

5. Manuel G-HJ, Antonio L-C, Verdugo-Sivianes Eva M, Marco P, Amancio C.The Cargo Protein MAP17 (PDZK1IP1) regulates the cancer stem cell poolactivating the notch pathway by abducting numb. Clin Cancer Res2017;23:3871–83.

6. Park S-J, Shim JW, Park HS, Eum D-Y, Park M-T, Mi Yi J, et al. MacroH2A1downregulation enhances the stem-like properties of bladder cancer cellsby transactivation of Lin28B. Oncogene 2016;35:1292–301.

7. Chan KS, Espinosa I, Chao M, Wong D, Ailles L, Diehn M, et al. Identi-fication, molecular characterization, clinical prognosis, and therapeutictargeting of human bladder tumor-initiating cells. Proc Natl Acad Sci U S A2009;106:14016–21.

8. Fengyu Z,Weiqing Q, Haojie Z, Yu L, MingqingW, Yingyin Z, et al. SOX2 isa marker for stem-like tumor cells in bladder cancer. Stem Cell Rep2017;9:429–37.

9. Li C, Du Y, Yang Z, He L, Wang Y, Hao L, et al. GALNT1-Mediatedglycosylation and activation of sonic hedgehog signaling maintains theself-renewal and tumor-initiating capacity of bladder cancer stem cells.Cancer Res 2016;76:1273–83.

10. Deyong J, Li L, Sulaiman A, David A, Xuguang L, Jonathan L, et al. Anautocrine inflammatory forward-feedback loop after chemotherapy with-drawal facilitates the repopulation of drug-resistant breast cancer cells. CellDeath Dis 2017;8:e2932.

11. Zhao Y, Luyun H, Kaisu L, Yun Z, Aihua D, Yong L, et al. The KMT1A-GATA3-STAT3 circuit is a novel self-renewal signaling of human bladdercancer stem cells. Clin Cancer Res 2017;23:6673–85.

12. Lee SR, Roh YG, Kim SK, Lee JS, Seol SY, Lee HH, et al. Activation ofEZH2 and SUZ12 regulated by E2F1 predicts the disease progressionand aggressive characteristics of bladder cancer. Clin Cancer Res2015;21:5391–403.

13. Zhao Y, Chong L, Zusen F, Hongjie L, Xiaolong Z, Zhiming C, et al. Single-cell sequencing reveals variants in ARID1A, GPRC5A and MLL2 drivingself-renewal of human bladder cancer stem cells. Eur Urol 2017;71:8–12.

14. Francesca Z, Michelangelo C, Stefano P. YAP/TAZ at the roots of cancer.Cancer Cell 2016;29:783–803.

15. Meng Z, Moroishi T, Guan KL. Mechanisms of Hippo pathway regulation.Genes Dev 2016;30:1–17.

16. Escoll M, Gargini R, Cuadrado A, Anton IM, Wandosell F. Mutant p53oncogenic functions in cancer stem cells are regulated byWIP throughYAP/TAZ. Oncogene 2017;36:3515–27.

17. Noto A, De Vitis C, Pisanu ME, Roscilli G, Ricci G, Catizone A, et al.Stearoyl-CoA-desaturase 1 regulates lung cancer stemness via stabilizationand nuclear localization of YAP/TAZ. Oncogene 2017;36:4573–84.

18. Guo Y, Cui J, Ji Z, Cheng C, Zhang K, Zhang C, et al. miR-302/367/LATS2/YAP pathway is essential for prostate tumor-propagating cells and pro-motes the development of castration resistance. Oncogene 2017;36:6336–47.

19. Crosby HA, Hubscher SG, Joplin RE, Kelly DA, Strain AJ. Immunolocaliza-tion ofOV-6, a putative progenitor cellmarker in human fetal and diseasedpediatric liver. Hepatology 1998;28:980–5.

20. Van Den Heuvel M. Expression of anti-OV6 antibody and anti-N-CAMantibody along the biliary line of normal and diseased human livers.Hepatology 2001;33:1387–93.

21. Wang C, Yang W, Yan HX, Luo T, Zhang J, Tang L, et al. Hepatitis B virus X(HBx) induces tumorigenicity of hepatic progenitor cells in 3,5-diethox-ycarbonyl-1,4-dihydrocollidine-treated HBx transgenic mice. Hepatology2012;55:108–20.

22. Chao W, Fei-hu Y, Jia-jun Z, Hai H, Qin-shu C, Wei D, et al. OV6 þ cancerstem cells drive esophageal squamous cell carcinoma progression throughATG7-dependent b-catenin stabilization. Cancer Lett 2017;391:100–13.

23. Chao W, Ming-da W, Peng C, Hai H, Wei D, Wei-wei Z, et al. Hepatitis Bvirus X protein promotes the stem-like properties of OV6þ cancer cells inhepatocellular carcinoma. Cell Death Dis 2017;8: e2560.

24. YangW,WangC, Lin Y, LiuQ, Yu LX, Tang L, et al.OV6(þ) tumor-initiatingcells contribute to tumor progression and invasion in human hepatocel-lular carcinoma. J Hepatol 2012;57:613–20.

25. Cheng L, Montironi R, Davidson DD, Lopez-Beltran A. Staging andreporting of urothelial carcinoma of the urinary bladder. Mod Pathol2009;22: S70–95.

26. Babjuk M, Bohle A, Burger M, Capoun O, Cohen D, Comperat EM, et al.EAU guidelines on non-muscle-invasive urothelial carcinoma of the blad-der: update 2016. Eur Urol 2017;71:447–61.

27. Alfred Witjes J, Lebret T, Comperat EM, Cowan NC, De Santis M, BruinsHM, et al. Updated 2016 EAU guidelines on muscle-invasive and meta-static bladder cancer. Eur Urol 2017;71:462–75.

28. Wang C, Fu SY, Wang MD, Yu WB, Cui QS, Wang HR, et al. Zinc fingerprotein X-linked promotes expansion of EpCAM(þ) cancer stem-like cellsin hepatocellular carcinoma. Mol Oncol 2017;11:455–69.

29. Kemal TM, Benjamin E, Alexander B, Ellen L, ChristianeW, Kai S, et al. The140-kD Isoform of CD56 (NCAM1) directs the molecular pathogenesis ofischemic cardiomyopathy. Am J Pathol 2013;182:1205–18.

30. Zheng Y, Katsaros D, Shan SJC, de la Longrais IR, Porpiglia M, Scorilas A,et al. A multiparametric panel for ovarian cancer diagnosis, prognosis, andresponse to chemotherapy. Clin Cancer Res 2007;13:6984–92.

31. Wang C, Peng G, Huang H, Liu F, Kong DP, Dong KQ, et al. Blocking thefeedback loop between neuroendocrine differentiation and macrophagesimproves the therapeutic effects of enzalutamide (MDV3100) on prostatecancer. Clin Cancer Res 2018;24:708–23.

32. Watanabe T, Shinohara N, Sazawa A, Harabayashi T, Ogiso Y, Koyanagi T,et al. An improved intravesicalmodel usinghumanbladder cancer cell linesto optimize gene and other therapies. Cancer Gene Ther 2000;7:1575–80.

33. Yongfang W, You Z, Feng T, Shoujie C, Xia X, Ying Y, et al. N-mycdownstream regulated gene 1(NDRG1) promotes the stem-like propertiesof lung cancer cells through stabilized c-Myc. Cancer Lett 2017;401:53–62.

34. Hur K, Toiyama Y, Takahashi M, Balaguer F, Nagasaka T, Koike J, et al.MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT)in human colorectal cancer metastasis. Gut 2013;62:1315–26.

35. AliceN, Gupta Vineet K, Patricia D,Nikita S, VikasD,NipunM, et al. NFkB-Mediated invasiveness in CD133þ Pancreatic TICs is regulated by auto-crine and paracrine activation of IL1 signaling. Mol Cancer Res2018;16:162–72.

36. Bauman JE, Eaton KD,Martins RG. Antagonism of platelet-derived growthfactor receptor in non small cell lung cancer: rationale and investigations.Clin Cancer Res 2007;13:4632s–6s.

37. Hitoshi Y, Yuko N, Masaharu T, Toshifumi M, Yoshinori T. Taurinesuppresses platelet-derived growth factor (PDGF) BB-induced PDGF-breceptor phosphorylation by protein tyrosine phosphatase-mediateddephosphorylation in vascular smooth muscle cells. Biochim BiophysActa 2005;1745:350–60.

38. Konotop G, Bausch E, Nagai T, Turchinovich A, Becker N, Benner A, et al.Pharmacological inhibition of centrosome clustering by slingshot-medi-ated cofilin activation and actin cortex destabilization. Cancer Res2016;76:6690–700.

39. Katschnig AM, Kauer MO, Schwentner R, Tomazou EM, Mutz CN, LinderM, et al. EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulationinhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.Oncogene 2017;36:5995–6005.

40. Liu-Chittenden Y, Huang B, Shim JS, Chen Q, Lee SJ, Anders RA, et al.Genetic and pharmacological disruption of the TEAD-YAP complex sup-presses the oncogenic activity of YAP. Genes Dev 2012;26:1300–5.

41. Zlotta Alexandre R, Thierry R, Cynthia K, Sultan A, Sandrine R, Anne L, et al.Select screening in a specific high-risk population of patients suggests astage migration toward detection of non–muscle-invasive bladder cancer.Eur Urol 2011;59:1026–31.

42. Karin B-D, Emil C, Iver N, Michael K, Ann T, Søren H, et al. Monitoringtreatment response and metastatic relapse in advanced bladder cancer byliquid biopsy analysis. Eur Urol 2018;73:535–40.

Targeting CSCs Impairs the Progression of Bladder Cancer

www.aacrjournals.org Clin Cancer Res; 25(3) February 1, 2019 1085

43. Liu JY, Li YH, Lin HX, Liao YJ, Mai SJ, Liu ZW, et al. Overexpression ofYAP 1 contributes to progressive features and poor prognosis ofhuman urothelial carcinoma of the bladder. Bmc Cancer 2013;13:349.

44. Liang D, Fan L, Wanjun W, Weiren H, Zhiming C. Transcriptional cofactorMask2 is required for YAP-induced cell growth and migration in bladdercancer cell. J Cancer 2016;7:2132–8.

45. Ciamporcero E, ShenH, Ramakrishnan S, Yu Ku S, Chintala S, Shen L, et al.YAP activation protects urothelial cell carcinoma from treatment-inducedDNA damage. Oncogene 2016;35:1541–53.

46. Jaya G, Jin-Mo K, Chandra RS, Hyunyoung J, Ying W, Suhrid B, et al. Dualinhibition of NOX2 and receptor tyrosine kinase by BJ-1301 enhancesanticancer therapy efficacy via suppression of autocrine-stimulatory factorsin lung cancer. Mol Cancer Ther 2017;16:2144–56.

47. Deyong J, Li L, Sulaiman A, David A, Xuguang L, Jonathan L, et al. Anautocrine inflammatory forward-feedback loop after chemotherapy with-drawal facilitates the repopulation of drug-resistant breast cancer cells. CellDeath Dis 2017;8:e2932.

48. Hung Ben P, HuttonDaphne L, Kozielski Kristen L, Bishop Corey J, Bilal N,Green Jordan J, et al. Platelet-derived growth factor BB enhances osteo-genesis of adipose-derived but not bone marrow-derived mesenchymalstromal/stem cells. Stem Cells 2015;33:2773–84.

49. Lau CK, Yang ZF, Ho DW, Ng MN, Yeoh GCT, Poon RTP, et al. An Akt/Hypoxia-Inducible Factor-1 /Platelet-Derived Growth Factor-BB autocrineloop mediates hypoxia-induced chemoresistance in liver cancer cells andtumorigenic hepatic progenitor cells. Clin Cancer Res 2009;15:3462–71.

50. Changqing X, YanhongG, Tianqing Z, Jifeng Z,MaPeter X, ChenY. Eugene.Yap1 protein regulates vascular smooth muscle cell phenotypic switch byinteraction with myocardin. J Biol Chem 2012;287:14598–605.

51. Smoot RL, Werneburg NW, Sugihara T, Hernandez MC, Yang L, Mehner C,et al. Platelet-derived growth factor regulates YAP transcriptional activityvia Src family kinase dependent tyrosine phosphorylation. J Cell Biochem2018;119:824–36.

52. Dasari VR, Mazack V, Feng W, Nash J, Carey DJ, Gogoi R. Verteporfinexhibits YAP-independent anti-proliferative and cytotoxic effects in endo-metrial cancer cells. Oncotarget 2017;8:28628–40.

Clin Cancer Res; 25(3) February 1, 2019 Clinical Cancer Research1086

Wang et al.