retinoic acid related orphan receptor c regulates ......molecular cell biology retinoic...

Molecular Cell Biology

Retinoic Acid–Related Orphan ReceptorC Regulates Proliferation, Glycolysis, andChemoresistance via the PD-L1/ITGB6/STAT3Signaling Axis in Bladder CancerDalong Cao1,2,3, Zihao Qi4, Yangyang Pang5, Haoran Li2,3,6, Huyang Xie7,Junlong Wu1,3, Yongqiang Huang1,3, Yao Zhu1,3, Yijun Shen1,3, Yiping Zhu1,3,Bo Dai1,3, Xin Hu2, Dingwei Ye1,2,3, and Ziliang Wang2,8

Abstract

Retinoic acid–related orphan receptor C (RORC) is amember of the nuclear orphan receptor family and performscritical regulatory functions in cell proliferation, metastasis,and chemoresistance in various types of malignant tumors.Here we showed that expression of RORC is lost in tumortissues of bladder cancer patients. Enhanced expression ofRORC suppressed cell proliferation and glucose metabolismand increased cisplatin-induced apoptosis in vitro and in vivo.RORC bound the promoter region of programmed deathligand-1 (PD-L1) and negatively regulated PD-L1 expression.PD-L1 directly interacted with integrin b6 (ITGB6) andactivated the ITGB6/FAK signaling pathway. RORC pre-vented the nuclear translocation of STAT3 via suppression

of the PD-L1/ITGB6 signaling pathway, which further inhib-ited bladder cell proliferation and glucose metabolism andincreased cisplatin-induced apoptosis. These findings revealthat RORC regulates bladder cancer cell proliferation, glu-cose metabolism, and chemoresistance by participating inthe PD-L1/ITGB6/STAT3 signaling axis. Moreover, this newunderstanding of PD-L1 signaling may guide the selection oftherapeutic targets to prevent tumor recurrence.

Significance: These findings suggest that RORC-mediatedregulation of a PD-L1/ITGB6/FAK/STAT3 signaling axis inbladder cancer provides several potential therapeutic targetsto prevent tumor progression.

IntroductionBladder cancer is one of the most common malignancies

worldwide and is also the most common cancer of the geni-tourinary system in China, which ranked the eighth among all

cancers and accounted for nearly 80.5 per 100,000 new casesand 32.9 per 100,000 deaths in 2015 (1, 2). Approximately70% of bladder cancer cases are non–muscle-invasive bladdercancer, treated by transurethral resection of bladder tumor andfollowed by intravesical treatment. However, up to two thirdsof patients will relapse or progress to muscle-invasive bladdercancer (MIBC; ref. 3). For MIBC, radical cystectomy is oftenperformed, followed by chemotherapy and radiotherapy, butonly 50% of patients will survive for 5 years (3, 4). Thus, thereis still a need for finding new methods for the treatment ofbladder cancer.

Nuclear receptors (NR) are a class of ligand-based transcriptionfactors that are widely distributed in organisms. There are variousmembers of NRs, which constitute a large family and can bedivided into three categories: steroid hormone receptors, nonste-roid hormone receptors, and nuclear orphan receptors. NRs,served as molecular switches, play key roles in diverse cellularprocesses, including cell proliferation, differentiation, andhomeostasis (5). NRs are involved in a multitude of pathologicprocesses, including carcinogenesis, and hence are supposed asnovel therapeutic targets (6, 7). Nuclear orphan receptors referredto those molecules that sequence similar to known receptorswithout an identified natural ligand.

Receptor retinoic acid–related orphan receptor C (RORC, alsonamedRORg) is one of the familymembers of the nuclear orphanreceptors and functions as aDNA-binding transcription factor (8).Recently, the regulatory role of RORC in tumorigenesis has beenestablished. In breast cancer, RORC expression is decreased in

1Department of Urology, Fudan University Shanghai Cancer Center, Shanghai,China. 2Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai,China. 3Department of Oncology, Shanghai Medical College, Fudan University,Shanghai, China. 4Huadong Hospital Affiliated to Fudan University, Shanghai,China. 5Department of Urology, Jiading District Central Hospital AffiliatedShanghai University of Medicine and Health Sciences, Shanghai, China. 6Depart-ment of Gynecologic Oncology, Fudan University Shanghai Cancer Center,Shanghai, China. 7Department of Urology, Affiliated Hospital of Nantong Uni-versity, Nantong, China. 8Department of Obstetrics and Gynecology, XinhuaHospital Affiliated to Shanghai Jiaotong University School Medicine, Shanghai,China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

D.L. Cao, Z.H. Qi, Y.Y. Pang, and H.R. Li contributed equally to this article.

Corresponding Authors: Ziliang Wang, Xinhua Hospital Affiliated to ShanghaiJiaotong University School Medicine, 1665 Kongjiang Road, Shanghai 200092,China. Phone: 86-21-34777310; E-mail: [email protected] [email protected]; and Dingwei Ye, Department of Urology, Fudan Univer-sity Shanghai Cancer Center, Department of Oncology, Shanghai MedicalCollege, Fudan University, 270 Dong'an Road, Shanghai 200032, China. E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-3842

�2019 American Association for Cancer Research.

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aggressive basal-like breast cancer and negatively associated withhistologic grade in several human cohorts (9). In melanoma, theexpression of RORC was lower in melanomas than in nevi anddecreased during tumor progression, with lowest levels found inprimary melanomas at stages III and IV and in melanoma metas-tases (10). RORC-deficient mice are relatively healthy and fertile.However, the expression and functionof RORC inhumanbladdercancer cells remain largely unexplored.

In this study, we identified that the expression of RORC wasdownregulated in tumors from bladder cancer patients, withsignificantly lower levels found in bladder cancer patients withadvanced tumor stage and resistant to chemotherapy. RORCfunctions as a key determinant of programmed death ligand-1(PD-L1) overexpression and aberrant signaling in bladder cancercells. Furthermore, RORC was determined to regulate cell prolif-eration, glucose metabolism, and chemoresistance via suppres-sing the PD-L1/ITGB6/STAT3 signaling axis in bladder cancer.Thus, our findings establish RORC as a previously unsuspectedkey player and a novel therapeutic target for bladder cancer.

Materials and MethodsPatients and tissue samples

Tissue microarrays were fabricated using formalin-fixedparaffin-embedded tissues of carcinoma (n ¼ 155) and para-carcinoma (n¼115) frompatientswith bladder urothelial cancer,who underwent radical cystectomy at the Fudan UniversityShanghai Cancer Center (FUSCC) from 2008 to 2012. Clinicaland pathologic characteristics for all participants included in thisstudy were collected from the patients' electronic database atFUSCC and then evaluated (Supplementary Table S1). Briefly,themedian ageof the patientswas 62 years (range, 33–83years). Atotal of 86 (55.5%) patients were diagnosed with stage I–II while69 (44.5%) patients with stage III–IV, and 11 (7.1%) patientspossessed with low-grade urothelial cancer while 144 (92.9%)patients with high-grade urothelial cancer. For chemotherapeuticresponse, 35 (67.3%) patients were resistant to platinum-basedchemotherapy and 17 (32.7%) were sensitive to the same treat-ment. Progression-free survival (PFS) was calculated as the timefrom the date of surgery to the occurrence of progression, orrelapse. Overall survival (OS) was measured as the length of timefrom the initiation of surgery to death from any cause or until themost recent follow-up. PFS less than 6 months was defined asresistant to the last platinum-based chemotherapy, or else itwas defined as sensitivity to the last platinum-based chemother-apy. Searches involving human participants in this study wereapproved by the Ethics Committee of FUSCC. Written informedconsent was also approved and obtained from each participant,and each clinical investigation was conducted according to theprinciples expressed in the Declaration of Helsinki consent.

Cell lines and cultureThe human bladder cancer cell lines 5637 and UC3 were

obtained from the Cell Bank of the Chinese Academy of Science.Identities of cell lines were confirmed by DNA profiling(short tandem repeat, STR). These cell lines were conserved inour laboratory and subjected to routine cell line quality examina-tions (e.g., morphology, Mycoplasma) by HD Biosciences every3 months. All cells were maintained in RPMI-1640 (GibcoBRL) supplemented with 10% fetal bovine serum (Gibco, LifeTechnologies), 100 U/mL penicillin (Biowest), and 100 U/mL

streptomycin (Biowest) and incubated at 37�C in a humidifiedincubator with 5% CO2.

Whole-body 18F-FDG positron emission tomography/computed tomography

Briefly, 18F-FDG was automatically made by a cyclotron(Siemens CTI RDS Eclipse ST) using an Explora FDG4 module.Patients had been fasting for more than 6 hours. Scanning started1 hour after intravenous injection of the tracer (7.4 MBq/kg). Theimages were acquired on a Siemens biograph 16HR positronemission tomography/computed tomography (PET/CT) scannerwith a transaxial intrinsic spatial resolution of 4.1mm. CT scanningwas first initiated from the proximal thighs to the head, with120 kV, 80–250 mA, pitch 3.6, and rotation time of 0.5 seconds.Image interpretation was carried out on a multimodality computerplatform (Syngo; Siemens). Quantification of metabolic activitywas acquired using the standard uptake value (SUV) normalizedto body weight, and the SUVmax for each lesion was calculated.

Plasmid construction and viral infectionThe recombinant plasmid pENTER-RORC containing human

full cDNA sequence of RORC was purchased from Vigene Bios-ciences. Then the cDNA sequence of RORC was subcloned intolentivirus vector. Lentivirus was produced by cotransfecting 293Tcells with pRSV-Rev, pMD2.G, pMDLg/pRRE, and pCDH-puroexpression vectors. The virus was harvested after 48 hours byfiltering the virus-containing medium through 0.45-mm Steriflipfilter (Millipore). Human bladder cancer cell lines 5637 and UC3were infected by incubating cells with medium containing indi-cated virus and 1 ng/mL polybrene (Sigma) for 24 hours. Estab-lished stable cell lines expressing RORC were constructed asabove. By following the same protocol, control cell lines weregenerated using infection with viruses containing the emptyvector. In addition, the human full cDNA sequence of PD-L1 waspurchased from Vigene Biosciences.

Promoter sequences of PD-L1 (�3000–0 bp) were cloned fromgenomic DNA prepared from human bladder cancer samplesusing the Genomic DNA Extraction Kit (TIANGEN BIOTECHCO.LID). Then, these promoter sequences were subcloned intopGL3-basic vector and established the recombinant plasmid,pGL-PD-L1-promoter. The pGL-PD-L1-promoter recombinantplasmid was cotransfected with phRl-TK vector into 5637 andUC3 cells using Lipofectamine 2000 according to the manufac-turer's instructions. Potential transcript factor binding sites in thepromoter region of PD-L1 were predicted using GeneCards soft-ware (www.genecards.org) and the UCSC Genome Bioinformat-ics Site (http://genome.ucsc.edu/).

RNA-seq data analysisTotal RNA (1 mg) was isolated from 5637 cells and treated with

VAHTS mRNA Capture Beads (Vazyme) to enrich polyAþ RNAbefore constructing RNA libraries. RNA library preparation wasperformed by using VAHTS mRNA-seq v2 Library Prep Kit fromIllumina (Vazyme). Paired-end sequencing was performed withIllumina HiSeq 3000 at RiboBio Co., Ltd. For computationalanalysis of RNA-seq data, sequencing readswere aligned using thespliced read aligner HISAT2, which was supplied with the Ensem-ble Human Genome Assembly (Genome Reference ConsortiumGRCh38) as the reference genome. Gene-expression levels werecalculated by the fragments per kilobase of transcript per million

The RORC/PD-L1/ITGB6/STAT3 Signaling Axis in Bladder Cancer

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mapped reads (FPKM).Gene Set EnrichmentAnalysis (GSEA)wasused for gene functional annotation.

IHC assayThe 10 � 12 tissue microarray (TMA) was made by the

FUSCC Tissue Bank. IHC was performed on 7-mm-thick TMAsections using the antibody against RORC (ab207082, Abcam,1:100 dilution), PD-L1 (ab205921, Abcam, 1:100 dilution),ITGB6 (ab201638, Abcam, 1:100 dilution), STAT3 (#9139 CellSignaling Technology, 1:100 dilution), and phospho-STAT3(#9145, Cell Signaling Technology, 1:100 dilution). Each casehad two cores made from separate sources to preclude theheterogeneity of tumors. A known positive case sample wasincluded as a positive control, and the primary antibody wasreplaced with nonimmune mouse/rabbit serum for negativecontrol. The immunoreactive score (IRS) was multiplicity ofthe staining intensity and positive cancer percentage. Finally,the assessment of protein expression was defined as negative(�1þ) and positive (>2þ to �3þ).

Detection and quantification of the metabolic profileMetabolites were extracted and analyzed as previously

described (11). Briefly, metabolites were extracted from bladdercancer tissues and para-carcinoma tissues. Metabolite levels werenormalized to the total of all metabolites detected.

Western blotting assayAntibodies against P53, BCL-2, HK2, GLUT1, GLUT4, LDHB,

RORC, PDL-1, ITGB6, STAT3, FAK, and AKT1 were from Protein-tech. Antibodies against p-STAT3 (Tyr705), p-FAK (Tyr925), andp-AKT1 (Ser473) were from Cell Signaling Technology. All theprimary antibodies were used at 1:1,000 dilutions and secondaryantibodies at 1:5,000 dilutions. Three independent experimentswere done for final analyses. Western blotting assay was per-formed as previously described (12).

RT-qPCRFor tissue samples, total RNA was extracted with TRIzol

(Invitrogen) according to the manufacturer's instructions. RT-qPCR was performed as previously described (13). Primersequences were shown in Supplementary Table S2.

Immunofluorescence assayImmunofluorescence assay was performed as previously

described (13). Primary antibodies against RORC, PD-L1, andp-STAT3 were purchased from Abcam. DNA dye 40,6-diamidino-2-phenylindole (DAPI) was obtained from Molecular Probes.Secondary antibodies used were the cy3-conjugated donkeyanti-rabbit IgG (Jackson ImmunoResearch Laboratory). Allstained cells were examined and photographed with a Leica SP5confocal fluorescence microscope.

Luciferase reporter assayLuciferase reporter assaywas performed as previously described

(13). A human PD-L1 gene promoter region was inserted into apGL3 basic vector as pGL3- PD-L1-Promoter. One hundred nano-grams (ng) of constructed plasmid and 7 ng Renilla luciferasecontrol plasmid were transfected into cells expressing 5637/RORC cDNA and UC3/RORC cDNA in 6-well plates. Forty-eighthours later, luciferase activities were measured using the DualLuciferase Assay Kit (Promega). Renilla luciferase was used to

normalize reporter luciferase activities, which were then rescaledto vector control signals equal to unit 1.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assays were per-

formed using the Pierce Agarose ChIP Kit (Thermo, #27177).Briefly, 5637 or UC3 cells were crosslinked by 1% formaldehydefor 10 minutes at 37�C. The cross-linking reaction was quenchedby glycine, and cells were lysed in SDS buffer containing theprotease inhibitor cocktail. Cell lysates were sonicated to shearchromatin DNA into fragments with 200–1,000 base pairs in sizeand then subjected to immunoprecipitation with 4 mL IgG (CellSignaling Technology), 10 mL RORC (ab32 207082, Abcam), or 2mL Polymerase II (Imgenex) antibodies. After washing with aseries of low and high salt concentration washing buffers, immu-noprecipitated DNA fragments were de-crosslinked at 77�C inhigh salt condition, purified using the QIAquick PCR purificationkit (Qiagen), and then analyzed by qRT-PCR.

Using the GAPDH promoter primers (Kit) confirmed the effec-tiveness of conventional PCR chip results. The correct chip resultsshould be that only the input and RNA polII samples will havepositive results, which could be shown as a 300-bp band PCR, andthe other three groups (IgG and RORC) showed no band. In theopen reading frame (ORF) region of the human PD-L1 gene,which located within upstream 3,000 bp long of the target gene, apair of primers was designed by using Primer7.0 every 300 bp orso. The primer sets used to amplify the BCL-2 promoter withputative STAT3 binding sites were as follows: F: 50-CTTCATT-TATCCAGCAGCTT-30 and R: 50-GAGGGGACGATGAAGGAG-30.The primer sets used to amplify the P53 promoter with putativeSTAT3 binding sites were as follows: F: 50-GGGCCCGTGTT-GGTTCATC-30 and R: 50-CCGCGAGACTCCTGGCACAA-30.

Subcellular fractionationBy following themanufacturer's protocol, cytosolic andnuclear

fractionation of indicated cells were performed using the nuclearand cytoplasmic extraction Kit (Tiangen Biotech).

ImmunoprecipitationCells were collected and lysed in RIPA lysis buffer (Beyotime)

and protease inhibitor cocktail (Roche Diagnostics).Whole-celllysates (2 mg) were precleared with 30 mL protein G beads (LifeTechnologies), and then 2 mg isotype-matched IgG control orindicated antibodies was added incubating for 2 hours on arocking platform. Immunoprecipitates were collected by centri-fugation and then resolved by SDS-PAGE.

F€orster resonance energy transfer and fluorescence lifetimeimaging

For F€orster resonance energy transfer (FRET)-fluorescencelifetime imaging (FLIM) experiments, donor proteins (fused toGFP) were expressed from vectors pCMV3-C-GFPSpark, andacceptor proteins (fused to RFP) were expressed from vectorCMV3-C-OFPSpark. FRET-FLIM experiments were performed ona Leica TCS SMD FLCS confocal microscope excitation with WLL(white light laser) and emission collected by an SMD SPAD(single photon-sensitive avalanche photodiodes) detector. The5637 or UC3 cells transiently coexpressing donor and acceptor, asindicated in the figures, were visualized 36 hours after agroinfil-tration. Accumulation of the GFP- and RFP-tagged proteins wasestimated before measuring lifetime. The tunable WLL set at

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489 nm with a pulsed frequency of 40 MHz was used forexcitation, and emission was detected using SMD GFP/RFP FilterCube (with GFP: 500–550 nm). The fluorescence lifetime shownin thefigures corresponding to the averagefluorescence lifetime ofthe donor (t) was collected and analyzed by PicoQuant Sym-phoTime software. Lifetime is normally an amplitude-weightedmean value using the data from the single (GFP-fused donorprotein only orGFP-fused donor proteinwith free RFP acceptor orwith noninteracting RFP-fused acceptor protein) or biexponentialfit (GFP-fused donor protein interacting with RFP-fused acceptorprotein). Mean lifetimes are presented as means � SD based onmore than 10 cells from at least three independent experiments.FRET efficiency was calculated according to the formula E ¼ 1 �tDA/tD, where tDA is the average lifetime of the donor in thepresence of the acceptor and tD is the average lifetimeof thedonorin the absence of the acceptor.

Glycolysis analysisTheGlucoseUptakeColorimetric AssayKit (BioVision), Lactate

Colorimetric Assay Kit (BioVision), ATP Assay Kit (SIGMA ALOR-ICH), and Amplite Colorimetric NADPHAssay Kit (AAT BioquestInc.) were purchased to examine the glycolysis process in bladdercancer cells according to the manufacturers' protocol.

Oxygen consumption rate and extracellular acidification rateCellular mitochondrial function was measured using the Sea-

horse XF Cell Mito stress test Kit and the Bioscience XF96 Extra-cellular Flux Analyzer, according to the manufacturers' instruc-tions. Glycolytic capacity was determined using the GlycolysisStress Test Kit as per the manufacturer's instructions. Briefly, 4 �104 cellswere seededonto96-well plates and incubatedovernight.After washing the cells with Seahorse buffer (DMEMwith phenolred containing 25 mmol/L glucose, 2 mmol/L sodium pyruvate,and 2 mmol/L glutamine), 175 mL of Seahorse buffer plus 25 mLeach of 1 mmol/L oligomycin, 1 mmol/L FCCP, and 1 mmol/Lrotenone was automatically injected to measure the oxygen con-sumption rate (OCR). Then, 25 mL each of 10 mmol/L glucose,1 mmol/L oligomycin, and 100 mmol/L 2-deoxy-glucose wereadded to measure the extracellular acidification rate (ECAR). TheOCR and ECAR values were calculated after normalization to thecell number and were plotted as the mean � SD.

Cell viability assayMultiple cultures of bladder cancer cells were plated in 96-well

plates at a density of 1 � 103 cells/well supplemented with a100 mLmaintenancemedium to evaluate the cell proliferation rate.Each day one set of cultures was collected and counted. Theproliferation rate equaled the experimental OD value/the controlOD value. Cell viability was also assessed by CCK-8. We plated8 � 103 cancer cells per well in 96-well plates. The next day, cellswere treated with various concentrations of cisplatin. Cell viabilityused to measure the number of viable cells was determined bymeasurement of absorbance at 470 nm by a Microplate Reader(Synergy H4, Bio-Tek). All experiments were done in triplicate.

Colony formation assayCells were seeded in six-well plates at a density of 500 per well.

The cells were cultured with freshmedium and allowed to grow atleast for 1 week. Colonies with more than 50 cells were countedafter being fixed with ice-cold methanol and stained with Crystalviolet (Solarbio).

Cell apoptosis analysisTo detect apoptosis, adherent cells were incubated with cis-

platin at different concentrations. After 48 hours, cells werecollected, washed twice with cold 1 � PBS, and resuspended in200 mL binding buffer at a density of 1 � 107 cells/mL. Then, westained cells with 5 mL Annexin V and propidium iodide (BDBiosciences) using an apoptosis detection kit (BD Biosciences)and subjected to analysis by flow cytometry (Cytomics FC500 MPL, Beckman Coulter). Early apoptosis was determinedbased on the percentage of cells with Annexin Vþ/PI�, while lateapoptosis was that of cells with Annexin Vþ/PIþ. Experimentswere done at least three times for final analyses.

Animal studiesAnimal experiments were approved by the Ethics Committee at

FUSCC. Briefly, female BALB/c nude (Shanghai Slac LaboratoryAnimal Co. Ltd, 4–6 weeks) were subcutaneously injected withRORC-overexpressing5637cells (5�106 suspended in0.1mLPBSfor each mouse) and RORC-overexpressing UC3 cells (5 � 106suspended in 0.1mL PBS for eachmouse). Themice were weighedevery 3 days and sacrificed for detecting of the size, number, andweight of celiac metastasis of different cell lines. For the subcuta-neous xenograft model, tumor growth with a digital caliperwas measured every 3 days. Tumor volumes were calculated bythe dimensional size of each tumor with the following formula:V (volume) ¼ L (length) � W (width)2 � 0.72. Once reaching anaverage tumor volume of 100 mm3, before being treated withcisplatin, all the mice were subjected to perform PET/CT scan. Theglucose uptake of the tumor was evaluated by the SUV. Then, theywere intraperitoneally treated with cisplatin (5 mg/kg). Adminis-tration of vehicle or agents and measurement of tumor volumewere done every 3 days. Animals were also subjected to fluores-cence imaging. Finally, mice were weighed and sacrificed, andtumors were weighed and dissected. RT-PCR and IHC of xenografttumor were done according to the protocol above.

Statistical analysisThe data in this study were calculated using GraphPad Prism

and reported as mean � SD. Comparisons between controls andtreated groups were determined by paired t test or one-wayANOVA, followed by Tukey multiple comparison tests. Clinico-pathologic characteristics analysis was performed using SPSS 23.0(SPSS Inc.). The relationship between RORC and PD-L1, ITGB6,STAT3, and p-STAT3 was conducted using Spearman correlationcoefficient. The associationbetweenRORC,PD-L1, ITGB6, STAT3,and p-STAT3 expression and clinicopathologic characteristicswas evaluated using the c2 test. The Kaplan–Meier method withlog-rank analysis were used to obtain estimates of PFS and OS.Variables with a value of P < 0.05 in univariate analysis wereincluded in the subsequent multivariate analysis on the basis ofthe Cox proportional hazards model. A probability less than 0.05was considered statistically significantly different.

Acronyms and abbreviationsThe acronyms and abbreviations used in this article are shown

in Supplementary Table S3.

ResultsRORC is downregulated in bladder urothelial cancer patients

To evaluate whether RORC expression was associated withclinicopathologic features of bladder cancer patients, we first






performed immunostaining with antibodies against RORC intissue microarrays from 155 bladder cancer patients (FUSCCcohort) and found that RORC immunostaining was highlyexpressed in 51% (n ¼ 79) of patients and underexpressed inthe other 49% (n¼ 76) of patients (Fig. 1A and B).Meanwhile, weexamined expression profiles of RORC in 115 para-carcinomabladder tissue samples and observed that RORC expression levelsin para-carcinoma tissues were much higher (78/115, 67.8%)than those in tumor tissues (Fig. 1A and B).

In addition, we further analyzed RORC expression in theFUSCC cohort and detected that expression levels of RORC weresignificantly lower inpatientswhowere resistant to chemotherapythan those who remained sensitive to chemotherapy (P ¼0.035; Fig. 1C). Moreover, SUVs were obtained from the PET/CTscan data from 29 bladder urothelial carcinoma patients toevaluate the association of SUV values with RORC expression inbladder cancer tissues. And then we found that patients with lowRORC staining showed significantly higher SUVmax valuesof primary tumor than patients with high RORC staining (P <0.05; Fig. 1D and E). To examine in more detail the changes incellular glucose metabolism in bladder cancer, we performed aglobal metabolomic analysis with bladder cancer tissues andpara-carcinoma tissues. We observed that glycolysis and pen-tose phosphate pathway intermediates were increased in blad-der cancer tissues with low RORC expression, compared withthose in high RORC expression and para-carcinoma tissues(Fig. 1F and G). These results indicated that RORC was closelyrelated with bladder cancer progression, glucose metabolism,and chemoresistance.

Furthermore, the correlation of RORC staining with prognosisof bladder cancer patients was analyzed using Kaplan–Meieranalysis with the log-rank test, and then we found that patientswith low RORC expression levels had significantly shorter PFSand OS than patients whose tumors had high RORC expression(P¼ 0.0409 and 0.009, respectively; Supplementary Fig. S1A andS1B). Meanwhile, analyses stratifying by different clinicopatho-logic data indicated that patients with low RORC expressionpossessed poor PFS and OS compared with patients with highRORC expression, either in patients with stage I–II, stage III–IV, orpresence of chemotherapy resistance (all P < 0.05; SupplementaryFig. S1A and S1B). The multivariate analysis with the Cox pro-portional hazards model revealed that chemotherapeuticresponse, RORC expression, and TNM stage were independentprognostic factors for OS in bladder cancer (Supplementary TableS2). In conclusion, decreased expression of RORC was an inde-pendent predictive factor for OS in bladder cancer.

Modulation of RORC levels affects expression of genes involvedin glucose metabolism, apoptosis, and chemosensitivity

Due to relative lower background expression level of RORCin human bladder cancer cell lines 5637 and UC3, we thenselected these two cell lines for further experiments. RORC wasstably overexpressed in 5637 and UC3 cells (5637/RORCOE andUC3/RORC OE, respectively).

To explore the potential significance of RORC in bladdercancer, we used gene-chip assays to compare the expression ofRORC-related genes in 5637/RORC OE cells with their controlcells. We found that some important regulatory genes involved inglycolysis, oxidative phosphorylation, apoptosis and response tocisplatin were enriched in cells with high RORC expression(Fig. 2A and B). Results of the chip assay were validated by

qRT-PCR in 5637/RORCOE and UC3/RORCOE cells, comparedwith their control cells, respectively (Fig. 2C). All these signsindicated that RORC may play a critical role in bladder cancercell proliferation, apoptosis, and response to cisplatin throughinvolvement in glycolysis.

Cell proliferation and glycolysis are inhibited by RORC, whileconcomitantly inducing apoptosis in cultured bladder cancercells

To determine the role of RORC in regulating cell proliferation,we performed colony formation assays. Overexpression of RORCsuppressed cell growth and reduced the number and size of thecolonies in 5637 and UC3 cell lines compared with control cells(Supplementary Fig. S2A and S2B). We then performed glucoseuptake, lactate and ATP, NADPH uptake assays to test whetherRORC negatively regulates glucose metabolism in bladder cancercells. The results showed that glucose uptake, lactate and ATP,NADPH uptake, ECAR, and OCR were all dramatically decreasedin cells overexpressing RORC compared with controls (P < 0.05;Supplementary Fig. S2C–S2H).

We also found that overexpression of RORC resulted in moreapoptotic bladder cancer cells (Supplementary Fig. S2I–S2J).Western blotting results demonstrated that RORC overexpressionreduced expression levels of BCL-2, GLUT1, HK2, and LDHB butincreased proapoptotic protein P53 in 5637 and UC3 cell lines(Supplementary Fig. S2K). The above results indicated that RORCexert its anticancer effect by inhibiting cell proliferation andglucosemetabolism,while concomitantly inducing cell apoptosisin bladder cancer cells.

Bladder cancer cells in culture are sensitized to cisplatin by theactivation of RORC

To investigate the impact of RORC on the chemosensitivityof human bladder cancer cells to cisplatin, we treated 5637 andUC3 cells with cisplatin in a dose- and time-dependent manner.CCK-8 assays (Supplementary Fig. S3A), clone formation assay(Supplementary Fig. S3B and S3C), and flow cytometry analysis(Supplementary Fig. S3D and S3E) revealed that RORC over-expression increased the sensitivity of 5637 and UC3 cells'response to cisplatin.

Furthermore, in order to test whether overexpression ofRORC enhances the effect of cisplatin on glucose metabolism,RORC-overexpressing and control cancer cells were pretreatedwith cisplatin, and then subjected to glucose uptake, lactate,and ATP production assay. We found overexpression of RORCsynergized with cisplatin to inhibit cell glucose metabolism(Supplementary Fig. S3F–S3H). Results obtained from theimmunoblotting assay showed that overexpression of RORCled to the upregulation of P53 but downregulation of BCL-2,GLUT1, HK2, and LDHB by a dose-dependent manner in bothcell lines (Supplementary Fig. S3I).

Taken together, these results suggested that RORC-overexpres-sing cells had a higher rate of apoptosis than control cells inresponse to cisplatin treatment, and also that RORC overexpres-sion synergized with cisplatin to inhibit cell proliferation andglucose metabolism.

PD-L1 is a direct target of RORC in bladder cancerTo probe into the antitumor role of RORC in bladder cancer

cells, we analyzed the potential enrichment factors when over-expressing RORC and found high enrichment of RORC at the

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Figure 1.

RORC expression in bladder urothelial cancer patients. A, Representative images from human bladder carcinoma sections and para-carcinoma bladder tissuesamples with low or high RORC IHC staining (�4 and�400). B, RORC expression in human bladder carcinoma sections (n¼ 155, right) and para-carcinomabladder tissue samples (n¼ 115; left). C, Expression levels of RORC in patients resistant to chemotherapy or sensitive to chemotherapy. D, Representative18F-FDG-PET-CT images from bladder cancer patients with low (left) or high (right) RORC expression (all�200). E, SUVmax of bladder urothelial cancer patientswith low or high RORC expression (n¼ 29). F, The flow-process diagram of carbohydrate metabolism. G, Heat map comparing relative levels of intermediates inbladder cancer tissues with those in para-carcinoma tissues (n¼ 3 samples of each group). Blue, downregulation; red, upregulation.






promoter of PD-L1 using UCSC Genome Bioinformatics Site(http://genome.ucsc.edu/), which suggested that PD-L1 might bethe potential target of RORC in bladder cancer. To confirm thishypothesis, we first asked whether there is an inverse relation-ship between RORC and PD-L1 expression. We found PD-L1mRNA was downregulated when RORC was overexpressed inthe bladder cancer cells 5637 and UC3 (Fig. 3A). Immunoflu-orescence experiments in 5637 and UC3 cells also confirmedRORC negatively regulated the expression of PD-L1 in the cellmembrane (Fig. 3B).

Next, we constructed a PD-L1 promoter luciferase reporterplasmid and performed a luciferase reporter assay to confirmthe mechanistic link between RORC and PD-L1. First, we trans-fected PD-L1 promoter plasmids into 5637 and UC3 cell lineswith stably overexpressing RORC. Compared with the controlgroups, RORC significantly inhibited PD-L1 promoter activity(Fig. 3C). To confirm the exact region of RORC binding siteswithin the PD-L1 promoter, we performed ChIP in 5637 cell linesand found that there were two RORC binding regions, containingfour binding sites that existed at approximately1200 bp upstream

Figure 2.

RNA-sequencing after upregulating RORC. A, GSEA analysis was performed using 5637 cells with RORC overexpression and the corresponding controls. Thesignature was defined by genes with significant expression changes. B, Heat map shows that the RORC-altered genes are involved in glycolysis, apoptosis,response to cisplatin, and oxidative phosphorylation. Gene expression profiling was performed with RNA-sequencing. C, qRT-PCR analysis of indicated genes in5637/RORC OE and UC3/RORCOE and control cells. Data are shown as mean� SD. Significance was calculated using the Student t test. �, P < 0.01.

Cao et al.




http://genome.ucsc.edu/http://cancerres.aacrjournals.org/

of the ORF of PD-L1 (Fig. 3D). Moreover, to further determinebinding sites of RORC in the promoter of PD-L1, we individuallymutated each of these binding sites and repeated the luciferaseassay (Fig. 3E). Data showed thatmutation of the first binding sitealone abrogated luciferase activity (Fig. 3F).

PD-L1 rescues the antitumor effect of RORC on bladder cancercells

To validate that PD-L1 is a critical target gene of RORC, weperformed the rescue experiment by overexpressing PD-L1 andRORC simultaneously and observed the impact on cell

Figure 3.

PD-L1 was regulated directly by RORC in bladder cancer cells. A, The expression of PD-L1 was detected by the qRT-PCR assay in 5637 and UC3 cellsoverexpressing RORC and control cells. B, Representative images showing RORC inhibited the expression of PD-L1 in cytoplasm (red), with immunofluorescencestaining (�1,000). Blue dye DAPI indicates the nucleus. C, Luciferase reporter assay in 5637 and UC3 cell lines with RORC enhancement. D, PCR results of ChIPanalysis showed that RORC bound to the PD-L1 gene promoter region. E, Themap of RORC binding sits in the promoter region of PD-L1. F, Luciferase reporterassay was used for the detection of RORC-mutant sites in the promoter region of PD-L1. � , P < 0.05; �� , P < 0.01.






proliferation, apoptosis, and glycolysis. In both 5637 andUC3 cell lines, PD-L1 overexpression partially reversed thechange of cell proliferation caused by the RORC overexpression(Supplementary Fig. S4A and S4B). In addition, PD-L1 over-expression significantly reverted RORC overexpression–mediatedcell apoptosis, glucose consumption, and lactate production(Supplementary Fig. S4C–S4F), as well as the RORC overexpres-sion–mediated ATP and NADPH production (SupplementaryFig. S4G and S4H). Similar results were also observed in Westernblotting. More specifically, PD-L1 and RORC overexpressiondecreased expression of the proapoptotic protein P53, butincreased glycolysis-related proteins GLUT1 and LDHB, as wellas upregulated the antiapoptotic protein BCL-2 (SupplementaryFig. S4I). Taken together, these findings demonstrate that RORCmay block tumor progression by suppressing PD-L1 expression.

Tumor suppression properties of RORC are the result ofbinding, activation, and regulation of the PDL1/ITGB6/STAT3signaling pathway

To further elucidate the molecular mechanism underlying theantitumor effect of RORC, we analyzed the potential enrichment

pathways when overexpressing RORC. We found higher enrich-ment of the integrin pathway (Fig. 4A) from RNA-sequencingdata. Next, we found that overexpression of RORC decreased theexpression level of the members of the integrin family anddownstream FAK/AKT1 signaling pathway in 5637 and UC3 cells(Fig. 4B), and introduction of PD-L1 cDNA rescued expressionlevels of ITGB6, p-FAK (Tyr397), p-AKT1 (Ser473), and p-STAT3(Tyr705; Fig. 4G). Our previous studies had validated the directbinding of PD-L1 and ITGB4 (14); we here found PD-L1 mightbind to ITGB6 detected by coimmunoprecipitation assay(Fig. 4C). Results of immunofluorescence assay (Fig. 4D) andFRET-FLIM (Fig. 4E and F) further demonstrated the direct inter-action between PD-L1 and ITGB6. To further demonstrate thatITGB6 is a critical target gene of RORC and PD-L1, we performedthe rescue experiment by overexpressing PD-L1 and RORC simul-taneously and observed the impact on protein expression levels ofthe integrin signaling pathway.

Interestingly, we also found that RORC overexpression signif-icantly inhibited the expression levels of STAT-family membersfrom RNA-sequencing data (Fig. 5A). The most obvious changewas observed in STAT3 (Fig. 5A and B). Immunofluorescence

Figure 4.

Tumor-suppressed properties of RORCmay be achieved by the PD-L1/ITGB6 signaling pathway.A, GSEA analysis showed high enrichment of the integrinpathway from RNA-sequencing data. B, RORC significantly changed the expression of proteins in the integrin signaling pathway. C, Coimmunoprecipitationassay demonstrated the interaction between PD-L1 and ITGB6.D, Immunofluorescence assay detected the colocation of PD-L1 and ITGB6 in 5637 and UC3 cells.E and F, Interaction between PD-L1 and ITGB6 confirmed by FRET-FLIM upon transient coexpression. FE, FRET efficiency. Asterisks indicate a statisticallysignificant difference (�� , P < 0.01) according to a Student t test. G, Representative images of the integrin pathway–related protein expression detected byWestern blotting in a rescue experiment. � , P < 0.05; �� , P < 0.01.

Cao et al.





assay also confirmed that RORC overexpression reduced theexpression of p-STAT3 (Tyr705) in the nucleus (Fig. 5C). Further-more,we foundRORC reduced the accumulation of protein in thenucleus but had no impact on cytoplasmic levels of STAT3 andp-STAT3 (Tyr705; Fig. 5D). Then, ChIP assay was performed todetermine whether RORC weakened the binding of STAT3 to thepromoter of STAT3-mediated target genes, such asBCL-2 and P53.As shown in Fig. 5E, we found that overexpression of RORCattenuated the binding of STAT3 to the promoter of BCL-2 andenhanced the binding of STAT3 to the promoter of P53, which

demonstrated that RORC might exert its inhibitory function oncell proliferation and glucose metabolism by blocking the bind-ing of STAT3 to the promoter of STAT3-mediated genes throughthe PD-L1/ITGB6/FAK/AKT1 signal pathway.

RORC blocks the progression of bladder cancer and sensitizescancer cells' response to cisplatin in vivo

We next tested antitumor effects of RORC in vivo. To observesubcutaneous tumor formation, we injected 5637 and UC3 cellseither overexpressing RORC or harboring empty vector into the

Figure 5.

STAT3 is identified as a key target of the RORC/PDL-1/ITGB6 signaling pathway. A, GSEA showed high enrichment of the STAT3 pathway from RNA-sequencing data. B, The expression of STAT-family genes was performed by qRT-PCR assay in 5637 and control cells. C, Representative images showingRORC inhibited the expression of STAT3 in the cell nucleus (green), with immunofluorescence staining (�1,000). Blue dye DAPI indicates the nucleus. D,Enhancement of RORC significantly changed the distribution and expression of STAT3. E, ChIP results of the binding of STAT3 to the promoter of P53 andBCL-2. �� , P < 0.01; �� , P < 0.001.






Figure 6.

RORC blocked the progression of bladder cancer and sensitized cancer cells to cisplatin-induced apoptosis in vivo. A, Representative image of nudemice bearing tumors formed by 5637/RORC OE and UC3/RORC OE and their control cells. B, The average tumor volume of nude mice bearingtumors formed by 5637/RORC OE and UC3/RORC OE and their control cells. C, The average tumor weight of nude mice bearing tumors formed by5637/RORC OE and UC3/RORC OE and their control cells. D, Representative image of PET-CT was used for the detection of glucose uptake in 5637/RORC OE and UC3/RORC OE xenografts and their controls before the administration of cisplatin. E, Average SUVmax values of nude mice bearingtumors before the administration of cisplatin. F, Representative image of nude mice bearing tumors formed by 5637/RORC OE and UC3/RORC OEand their control cells after cisplatin treatment. G, The average tumor volume of nude mice bearing tumors formed by 5637/RORC OE and UC3/RORC OE and their control cells after cisplatin treatment. H, The average tumor weight of nude mice bearing tumors formed by 5637/RORC OE andUC3/RORC OE and their control cells after cisplatin treatment. Error bars, 95% CIs. � , P < 0.05; �� , P < 0.01.

Cao et al.





flanks of nude mice. As is shown in Fig. 6A–C, overexpression ofRORC slowed the speed of tumor growth and reduced overalltumor weight in vivo. In addition, PET-CT analysis showed thatoverexpression of RORC significantly suppressed the glucoseuptake of xenograft cells in vivo and resulted in a lower SUVmaxvalue (Fig. 6D and E). IHC assay with xenograft tissues showedthat RORC inhibited the expression of PD-L1, ITGB6, andp-STAT3 (Tyr705; Supplementary Fig. S5A). Subsequently, weextracted mRNA from transplanted murine tumors and per-formed RT-qPCR to further validate the role of RORC in glycolysisand apoptosis. In accordance with earlier results obtained in celllines, RORC could decrease the expression of proteins involved inglycolysis and antiapoptosis, upregulate proapoptosis proteins(Supplementary Fig. S5B and S5C).

To validate RORC-sensitized bladder cancer cells response tocisplatin in vivo, when the tumor volume reached 100 mm3, micewere treated with cisplatin on alternate days. The results showedthat RORCoverexpression coordinates with cisplatin treatment tofurther reduce tumor volume and weight, relative to cells harbor-ing an empty vector (Fig. 6F–H).

Expression of PDL1, ITGB6, STAT3, and p-STAT3 is associatedwith poor survival in bladder urothelial cancer patients

To determine the clinical significance of PDL1, ITGB6, STAT3,and p-STAT3 in bladder cancer, we assessed their expression in abladder tissue microarray (n ¼ 155). PDL1 was expressed at highlevels in 68.4% (106/155) of bladder cancer tissues, 85 (54.8%)patients showed "high" expression of ITGB6, 98 (63.2%)patients showed "high" expression of STAT3, and 103 (66.5%)patients showed "high" expression of p-STAT3 (Fig. 7A and B;Supplementary Table S2). In addition, high expression of PDL1(log-rank,P¼0.0281), ITGB6 (log-rank,P¼0.0325), STAT3 (log-rank, P ¼ 0.0193), and p-STAT3 (log-rank, P ¼ 0.0314) was allcorrelated with poor OS (Supplementary Fig. S6A). Simulta-neously, high expression of PDL1, ITGB6, STAT3, and p-STAT3was also associated with poor PFS (P ¼ 0.0348, 0.0078, 0.0366,and 0.0308, respectively, Supplementary Fig. S6B). The correla-tion of PDL1, ITGB6, STAT3, and p-STAT3 with clinicopathologiccharacteristics in bladder cancer patients was shown in Supple-mentary Table S4. Moreover, consistent with our previous data,expression levels of PDL1, ITGB6, STAT3, and p-STAT3 in patientswith low RORC expression were significantly higher than those inpatients with high RORC expression (Fig. 7C). Finally, we iden-tified the RORC/ PDL1/ITGB6/STAT3 signaling axis in bladdertumorigenesis (Fig. 7D).

DiscussionIn this study, we illustrated the regulatory function of RORC in

the regulation of bladder cancer carcinogenesis. Here, we identi-fied RORC as an important tumor suppressor gene in bladdercancer and the role of the RORC/PD-L1/ITGB6/STAT3 signalingaxis in bladder tumorigenesis, which served as a potential futuremolecular marker for the prognosis of bladder cancer.

NRs, binding with their corresponding ligands and coregula-tors, regulate the coordinated expressionof genes and thenplay animportant role in the growth and development of the body,metabolism, cell differentiation, and many physiologic processesin vivo (5). Dysfunction ofNRswill lead to a range of diseases suchas cancer (15), infertility (16), obesity (17), and diabetes (18).NRs are promising drug design targets and can bind small mole-

cules that have been modified by drug design to regulate relateddiseases such as cancer and diabetes. Therefore, the search forligands and signaling pathways of orphan receptors has become apromising research area. Our results determined the anticancereffect of RORC in bladder cancer and found that loss of RORCwasprevalent in bladder cancer tissues. RORC also represented a verylowhazard ratio frommultiple Cox regression analysis, indicatingRORC expression is associated with improved survival outcomes.That is consistent with previous observations. Furthermore, theloss of RORC alters multiple cellular processes accounting forcancer progression. A literature review showed that RORC inhib-ited FasL and cytokine gene expression to regulate apoptosis (19).RORCalso attenuates TGFb/EMT signaling, thereby regulating themetastasis process. More specifically, key genes in this pathway,for example, TGFb and SMAD3, were direct targets of RORC (20).Besides, RORC was also involved in DNA-repair gene expres-sion (9) and angiogenesis (21). However, the expression andfunction of RORC in glucose metabolism remain largely unex-plored. Our data showed that RORC strongly reduced the glucosemetabolism of bladder cancer. These findings reveal a novelmechanism underlying the suppressive effect of RORC on cellproliferation in cancer cells.

Interestingly, in addition to its direct impact on tumor growthof RORC, we found that overexpression of RORC conferredsensitivity to cisplatin and increased cisplatin-induced apoptosisthrough the mitochondrial apoptotic pathway. Chemotherapy isthe effective adjuvant treatment for bladder cancer. Platinum isstill the most commonly used drug in chemotherapeutic regi-mens. Notwithstanding, platinum resistance remains a seriousproblem for bladder cancer patients and is one of the burningissues of our times. Recent studies have concluded that cisplatincan exert an inhibitory effect on glycolysis in cancer cells.Currently, combined treatment modalities that target glycolyticpathways hold promise for the treatment of chemoresistantcancer cells (22). Increasing evidence shows that inhibition ofglycolysis enhances drug-induced apoptosis in ovarian cancer,lung cancer, and leukemia (23–25). Because RORC is a keyregulator in glucose metabolism, targeting RORC may play animportant role in restoring cisplatin sensitivity. In our research,we found that RORC-overexpressing cells had a higher rate ofapoptosis than control cells in response to cisplatin treatment.RORC overexpression synergized with cisplatin to inhibit cellglucose metabolism and regulated the cancer cell, which mayaccount for the cisplatin sensitivity mediated by RORC.

A special feature of nuclear receptors is to act as importantinitiators of gene transcription. Moreover, nuclear receptors canrecruit a variety of transcription factors and coregulators to targetpromoters. In our study, we discovered that RORC interacteddirectly with the PD-L1 promoter and regulated the expression ofPD-L1 mRNA. PD-L1, also known as CD274 and B7-H1, wasoriginally identified in a murine T-cell hybridoma and a hemato-poietic progenitor cell line. High expression of PD-L1 in tumorcells was associated with poor prognosis in multiple cancer types,including papillary thyroid carcinoma (26), breast cancer (27),esophageal squamous cell carcinoma (28), hepatocellular carci-noma (29), and other solid tumors. Mechanistically, PD-L1expression is regulated by oncogenic transcription factors, suchas c-MYC and hypoxia-inducible factor (HIF; refs. 30, 31). Ourresults first reported that RORC directly bound to the promoterregionofPD-L1 andpromoted its downstream signaling pathway,reinforcing the importance of RORC in PD-L1–mediated






Figure 7.

IHC staining of PD-L1, ITGB6, STAT3, and p-STAT3 in bladder urothelial cancer patients. A, Representative images of biopsies containing low and highexpression of PD-L1, ITGB6, STAT3, and p-STAT3 (�4 and �400). B, PD-L1, ITGB6, STAT3, and p-STAT3 expression in human bladder carcinomasections (n ¼ 155; right) and para-carcinoma bladder tissue samples (n ¼ 115; left). C, Correlation of RORC expression with PD-L1, ITGB6, STAT3, andp-STAT3. D, Schematic model showing the role of the RORC/PDL-1/ITGB6/STAT3 signaling axis in the regulation of cell proliferation, apoptosis,chemosensitivity, and glycolysis.

Cao et al.





tumorigenesis. Furthermore, increasing PD-L1 expression in blad-der cancer cells reversed the inhibition of cell progression inducedby RORC overexpression.

Tumor-intrinsic functions of PD-L1 and their interplays withother oncogenic pathways have gained much attention. Highexpression of PD-L1 interacted with mTOR signaling to promotecell growth and autophagy in ovarian cancer cells and melano-ma (32), regulated BTK signaling to reverse the immune meta-bolic dysfunctions of monocytes in chronic lymphocytic leuke-mia (33), bind to ITGB4 to trigger AKT/GSK3b and SNAI1/SIRT3signaling in cervical cancer (14). The above studies demonstratedthat PD-L1 plays a critical role in tumor progression and metab-olism. This is the first study to find that ITGB6 is a previouslyunrecognized target of PD-L1 in cancer cells. Subsequently, ITGB6activated FAK/AKT/STAT3 signaling. Thus, this novel PD-L1/ITGB6 signaling axis critically contributed to the Warburg effectin bladder cancer cells and, as a result, to the development andprogression of bladder cancer.

In summary, our results demonstrate that the expression levelof RORC is negatively correlated with the prognosis of humanbladder cancer patients. Enhanced RORC suppressed cell growthandmetabolic reprogrammingby inhibiting the promoter activityof PD-L1, and further inactivated PD-L1/ITGB6/FAK/AKT/STAT3signaling. Thus, RORC may present as a new biomarker offavorable prognosis in bladder cancer and as a potential noveltarget for the treatment of bladder cancer patients.

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

Authors' ContributionsConception and design: D. Cao, Z. Qi, D. Ye, Z. WangDevelopment of methodology: D. Cao, Z. Qi, D. YeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Cao, H. Li, H. Xie, J. Wu, Y. Huang, Y. Zhu,Y. Shen, Y. Zhu, B. Dai, D. YeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.Cao, Z.Qi, H. Xie, J.Wu, Y.Huang, Y. Zhu, Y. Shen,Y. Zhu, B. Dai, D. YeWriting, review, and/or revision of the manuscript: D. Cao, H. Xie, J. Wu,Y. Huang, Y. Zhu, Y. Shen, Y. Zhu, B. Dai, D. Ye, Z. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):D. Cao, Y. Pang, H. Xie, J. Wu, Y. Huang, X. Hu,D. Ye, Z. WangStudy supervision: D. Ye, Z. Wang

AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China (No. 81502235 to Z. Wang; No. 81302213 to D. Cao; Nos.81872099 and 81672544 to D. Ye), by the Natural Science Foundationof Science and Technology Commission Shanghai Municipality (No.18ZR1407700 to D. Cao), and by the Youth Foundation of ShanghaiMunicipal Commission of Health and Family Planning (No. 20174Y0102to D. Cao).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedDecember 10, 2018; revised February 1, 2019; accepted February 21,2019; published first February 26, 2019.

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2019;79:2604-2618. Published OnlineFirst February 26, 2019.Cancer Res Dalong Cao, Zihao Qi, Yangyang Pang, et al. Signaling Axis in Bladder CancerGlycolysis, and Chemoresistance via the PD-L1/ITGB6/STAT3

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retinoic acid related orphan receptor c regulates ......molecular cell biology retinoic...

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