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Published OnlineFirst February 26, 2019; DOI: 10.1158/0008-5472.CAN-18-3842

Cancer Molecular Cell Biology Research

Retinoic Acid–Related Orphan Receptor C Regulates Proliferation, Glycolysis, and Chemoresistance via the PD-L1/ITGB6/STAT3 Signaling Axis in Bladder Cancer Dalong 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 a of the PD-L1/ITGB6 signaling pathway, which further inhib- member of the nuclear orphan receptor family and performs ited bladder cell proliferation and glucose metabolism and critical regulatory functions in cell proliferation, metastasis, increased cisplatin-induced apoptosis. These findings reveal and chemoresistance in various types of malignant tumors. that RORC regulates bladder cancer cell proliferation, glu- Here we showed that expression of RORC is lost in tumor cose metabolism, and chemoresistance by participating in tissues of bladder cancer patients. Enhanced expression of the PD-L1/ITGB6/STAT3 signaling axis. Moreover, this new RORC suppressed cell proliferation and glucose metabolism understanding of PD-L1 signaling may guide the selection of and increased cisplatin-induced apoptosis in vitro and in vivo. therapeutic targets to prevent tumor recurrence. RORC bound the promoter region of programmed death ligand-1 (PD-L1) and negatively regulated PD-L1 expression. Significance: These findings suggest that RORC-mediated PD-L1 directly interacted with integrin b6 (ITGB6) and regulation of a PD-L1/ITGB6/FAK/STAT3 signaling axis in activated the ITGB6/FAK signaling pathway. RORC pre- bladder cancer provides several potential therapeutic targets vented the nuclear translocation of STAT3 via suppression to prevent tumor progression.

Introduction cancers and accounted for nearly 80.5 per 100,000 new cases and 32.9 per 100,000 deaths in 2015 (1, 2). Approximately Bladder cancer is one of the most common malignancies 70% of bladder cancer cases are non–muscle-invasive bladder worldwide and is also the most common cancer of the geni- cancer, treated by transurethral resection of bladder tumor and tourinary system in China, which ranked the eighth among all followedbyintravesicaltreatment.However,uptotwothirds of patients will relapse or progress to muscle-invasive bladder 1Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, cancer (MIBC; ref. 3). For MIBC, radical cystectomy is often China. 2Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, performed, followed by chemotherapy and radiotherapy, but China. 3Department of Oncology, Shanghai Medical College, Fudan University, only 50% of patients will survive for 5 years (3, 4). Thus, there 4 Shanghai, China. Huadong Hospital Affiliated to Fudan University, Shanghai, is still a need for finding new methods for the treatment of 5 fi China. Department of Urology, Jiading District Central Hospital Af liated bladder cancer. Shanghai University of Medicine and Health Sciences, Shanghai, China. 6Depart- ment of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Nuclear receptors (NR) are a class of ligand-based transcription Shanghai, China. 7Department of Urology, Affiliated Hospital of Nantong Uni- factors that are widely distributed in organisms. There are various versity, Nantong, China. 8Department of Obstetrics and Gynecology, Xinhua members of NRs, which constitute a large family and can be Hospital Affiliated to Shanghai Jiaotong University School Medicine, Shanghai, divided into three categories: steroid hormone receptors, nonste- China. roid hormone receptors, and nuclear orphan receptors. NRs, Note: Supplementary data for this article are available at Cancer Research served as molecular switches, play key roles in diverse cellular Online (http://cancerres.aacrjournals.org/). processes, including cell proliferation, differentiation, and D.L. Cao, Z.H. Qi, Y.Y. Pang, and H.R. Li contributed equally to this article. homeostasis (5). NRs are involved in a multitude of pathologic Corresponding Authors: Ziliang Wang, Xinhua Hospital Affiliated to Shanghai processes, including carcinogenesis, and hence are supposed as Jiaotong University School Medicine, 1665 Kongjiang Road, Shanghai 200092, novel therapeutic targets (6, 7). Nuclear orphan receptors referred China. Phone: 86-21-34777310; E-mail: [email protected] or to those molecules that sequence similar to known receptors [email protected]; and Dingwei Ye, Department of Urology, Fudan Univer- without an identified natural ligand. sity Shanghai Cancer Center, Department of Oncology, Shanghai Medical Receptor retinoic acid–related orphan receptor C (RORC, also College, Fudan University, 270 Dong'an Road, Shanghai 200032, China. E-mail: named RORg) is one of the family members of the nuclear orphan [email protected] receptors and functions as a DNA-binding transcription factor (8). doi: 10.1158/0008-5472.CAN-18-3842 Recently, the regulatory role of RORC in tumorigenesis has been 2019 American Association for Cancer Research. established. In breast cancer, RORC expression is decreased in

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

aggressive basal-like breast cancer and negatively associated with streptomycin (Biowest) and incubated at 37C in a humidified histologic grade in several human cohorts (9). In melanoma, the incubator with 5% CO2. expression of RORC was lower in melanomas than in nevi and decreased during tumor progression, with lowest levels found in Whole-body 18F-FDG positron emission tomography/ primary melanomas at stages III and IV and in melanoma metas- computed tomography tases (10). RORC-deficient mice are relatively healthy and fertile. Briefly, 18F-FDG was automatically made by a cyclotron However, the expression and function of RORC in human bladder (Siemens CTI RDS Eclipse ST) using an Explora FDG4 module. cancer cells remain largely unexplored. Patients had been fasting for more than 6 hours. Scanning started In this study, we identified that the expression of RORC was 1 hour after intravenous injection of the tracer (7.4 MBq/kg). The downregulated in tumors from bladder cancer patients, with images were acquired on a Siemens biograph 16HR positron significantly lower levels found in bladder cancer patients with emission tomography/computed tomography (PET/CT) scanner advanced tumor stage and resistant to chemotherapy. RORC with a transaxial intrinsic spatial resolution of 4.1 mm. CT scanning functions as a key determinant of programmed death ligand-1 was first initiated from the proximal thighs to the head, with (PD-L1) overexpression and aberrant signaling in bladder cancer 120 kV, 80–250 mA, pitch 3.6, and rotation time of 0.5 seconds. cells. Furthermore, RORC was determined to regulate cell prolif- Image interpretation was carried out on a multimodality computer eration, glucose metabolism, and chemoresistance via suppres- platform (Syngo; Siemens). Quantification of metabolic activity sing the PD-L1/ITGB6/STAT3 signaling axis in bladder cancer. was acquired using the standard uptake value (SUV) normalized Thus, our findings establish RORC as a previously unsuspected to body weight, and the SUVmax for each lesion was calculated. key player and a novel therapeutic target for bladder cancer. Plasmid construction and viral infection Materials and Methods The recombinant plasmid pENTER-RORC containing human full cDNA sequence of RORC was purchased from Vigene Bios- Patients and tissue samples ciences. Then the cDNA sequence of RORC was subcloned into Tissue microarrays were fabricated using formalin-fixed lentivirus vector. Lentivirus was produced by cotransfecting 293T paraffin-embedded tissues of carcinoma (n ¼ 155) and para- cells with pRSV-Rev, pMD2.G, pMDLg/pRRE, and pCDH-puro carcinoma (n ¼ 115) from patients with bladder urothelial cancer, expression vectors. The virus was harvested after 48 hours by who underwent radical cystectomy at the Fudan University filtering the virus-containing medium through 0.45-mm Steriflip Shanghai Cancer Center (FUSCC) from 2008 to 2012. Clinical filter (Millipore). Human bladder cancer cell lines 5637 and UC3 and pathologic characteristics for all participants included in this were infected by incubating cells with medium containing indi- study were collected from the patients' electronic database at cated virus and 1 ng/mL polybrene (Sigma) for 24 hours. Estab- FUSCC and then evaluated (Supplementary Table S1). Briefly, lished stable cell lines expressing RORC were constructed as the median age of the patients was 62 years (range, 33–83 years). A above. By following the same protocol, control cell lines were total of 86 (55.5%) patients were diagnosed with stage I–II while generated using infection with viruses containing the empty 69 (44.5%) patients with stage III–IV, and 11 (7.1%) patients vector. In addition, the human full cDNA sequence of PD-L1 was possessed with low-grade urothelial cancer while 144 (92.9%) purchased from Vigene Biosciences. patients with high-grade urothelial cancer. For chemotherapeutic Promoter sequences of PD-L1 (3000–0 bp) were cloned from response, 35 (67.3%) patients were resistant to platinum-based genomic DNA prepared from human bladder cancer samples chemotherapy and 17 (32.7%) were sensitive to the same treat- using the Genomic DNA Extraction Kit (TIANGEN BIOTECH ment. Progression-free survival (PFS) was calculated as the time CO.LID). Then, these promoter sequences were subcloned into from the date of surgery to the occurrence of progression, or pGL3-basic vector and established the recombinant plasmid, relapse. Overall survival (OS) was measured as the length of time pGL-PD-L1-promoter. The pGL-PD-L1-promoter recombinant from the initiation of surgery to death from any cause or until the plasmid was cotransfected with phRl-TK vector into 5637 and most recent follow-up. PFS less than 6 months was defined as UC3 cells using Lipofectamine 2000 according to the manufac- resistant to the last platinum-based chemotherapy, or else it turer's instructions. Potential transcript factor binding sites in the was defined as sensitivity to the last platinum-based chemother- promoter region of PD-L1 were predicted using GeneCards soft- apy. Searches involving human participants in this study were ware (www.genecards.org) and the UCSC Genome Bioinformat- approved by the Ethics Committee of FUSCC. Written informed ics Site (http://genome.ucsc.edu/). consent was also approved and obtained from each participant, and each clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki consent. RNA-seq data analysis Total RNA (1 mg) was isolated from 5637 cells and treated with Cell lines and culture VAHTS mRNA Capture Beads (Vazyme) to enrich polyAþ RNA The human bladder cancer cell lines 5637 and UC3 were before constructing RNA libraries. RNA library preparation was obtained from the Cell Bank of the Chinese Academy of Science. performed by using VAHTS mRNA-seq v2 Library Prep Kit from Identities of cell lines were confirmed by DNA profiling Illumina (Vazyme). Paired-end sequencing was performed with (short tandem repeat, STR). These cell lines were conserved in Illumina HiSeq 3000 at RiboBio Co., Ltd. For computational our laboratory and subjected to routine cell line quality examina- analysis of RNA-seq data, sequencing reads were aligned using the tions (e.g., morphology, Mycoplasma) by HD Biosciences every spliced read aligner HISAT2, which was supplied with the Ensem- 3 months. All cells were maintained in RPMI-1640 (Gibco ble Human Genome Assembly (Genome Reference Consortium BRL) supplemented with 10% fetal bovine serum (Gibco, Life GRCh38) as the reference genome. Gene-expression levels were Technologies), 100 U/mL penicillin (Biowest), and 100 U/mL calculated by the fragments per kilobase of transcript per million

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mapped reads (FPKM). Gene Set Enrichment Analysis (GSEA) was normalize reporter luciferase activities, which were then rescaled used for gene functional annotation. to vector control signals equal to unit 1.

IHC assay Chromatin immunoprecipitation assay The 10 12 tissue microarray (TMA) was made by the Chromatin immunoprecipitation (ChIP) assays were per- FUSCCTissueBank.IHCwasperformedon7-mm-thick TMA formed using the Pierce Agarose ChIP Kit (Thermo, #27177). sections using the antibody against RORC (ab207082, Abcam, Briefly, 5637 or UC3 cells were crosslinked by 1% formaldehyde 1:100 dilution), PD-L1 (ab205921, Abcam, 1:100 dilution), for 10 minutes at 37C. The cross-linking reaction was quenched ITGB6 (ab201638, Abcam, 1:100 dilution), STAT3 (#9139 Cell by glycine, and cells were lysed in SDS buffer containing the Signaling Technology, 1:100 dilution), and phospho-STAT3 protease inhibitor cocktail. Cell lysates were sonicated to shear (#9145, Cell Signaling Technology, 1:100 dilution). Each case chromatin DNA into fragments with 200–1,000 base pairs in size had two cores made from separate sources to preclude the and then subjected to immunoprecipitation with 4 mL IgG (Cell heterogeneity of tumors. A known positive case sample was Signaling Technology), 10 mL RORC (ab32 207082, Abcam), or 2 included as a positive control, and the primary antibody was mL Polymerase II (Imgenex) antibodies. After washing with a replaced with nonimmune mouse/rabbit serum for negative series of low and high salt concentration washing buffers, immu- control. The immunoreactive score (IRS) was multiplicity of noprecipitated DNA fragments were de-crosslinked at 77Cin the staining intensity and positive cancer percentage. Finally, high salt condition, purified using the QIAquick PCR purification the assessment of protein expression was defined as negative kit (Qiagen), and then analyzed by qRT-PCR. (1þ) and positive (>2þ to 3þ). Using the GAPDH promoter primers (Kit) confirmed the effec- tiveness of conventional PCR chip results. The correct chip results Detection and quantification of the metabolic profile should be that only the input and RNA polII samples will have Metabolites were extracted and analyzed as previously positive results, which could be shown as a 300-bp band PCR, and described (11). Briefly, metabolites were extracted from bladder the other three groups (IgG and RORC) showed no band. In the cancer tissues and para-carcinoma tissues. Metabolite levels were open reading frame (ORF) region of the human PD-L1 gene, normalized to the total of all metabolites detected. which located within upstream 3,000 bp long of the target gene, a pair of primers was designed by using Primer7.0 every 300 bp or Western blotting assay so. The primer sets used to amplify the BCL-2 promoter with Antibodies against P53, BCL-2, HK2, GLUT1, GLUT4, LDHB, putative STAT3 binding sites were as follows: F: 50-CTTCATT- RORC, PDL-1, ITGB6, STAT3, FAK, and AKT1 were from Protein- TATCCAGCAGCTT-30 and R: 50-GAGGGGACGATGAAGGAG-30. tech. Antibodies against p-STAT3 (Tyr705), p-FAK (Tyr925), and The primer sets used to amplify the P53 promoter with putative p-AKT1 (Ser473) were from Cell Signaling Technology. All the STAT3 binding sites were as follows: F: 50-GGGCCCGTGTT- primary antibodies were used at 1:1,000 dilutions and secondary GGTTCATC-30 and R: 50-CCGCGAGACTCCTGGCACAA-30. antibodies at 1:5,000 dilutions. Three independent experiments were done for final analyses. Western blotting assay was per- Subcellular fractionation formed as previously described (12). By following the manufacturer's protocol, cytosolic and nuclear fractionation of indicated cells were performed using the nuclear RT-qPCR and cytoplasmic extraction Kit (Tiangen Biotech). For tissue samples, total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions. RT- Immunoprecipitation qPCR was performed as previously described (13). Primer Cells were collected and lysed in RIPA lysis buffer (Beyotime) sequences were shown in Supplementary Table S2. and protease inhibitor cocktail (Roche Diagnostics).Whole-cell lysates (2 mg) were precleared with 30 mL protein G beads (Life Immunofluorescence assay Technologies), and then 2 mg isotype-matched IgG control or Immunofluorescence assay was performed as previously indicated antibodies was added incubating for 2 hours on a described (13). Primary antibodies against RORC, PD-L1, and rocking platform. Immunoprecipitates were collected by centri- p-STAT3 were purchased from Abcam. DNA dye 40,6-diamidino- fugation and then resolved by SDS-PAGE. 2-phenylindole (DAPI) was obtained from Molecular Probes. Secondary antibodies used were the cy3-conjugated donkey Forster€ resonance energy transfer and fluorescence lifetime anti-rabbit IgG (Jackson ImmunoResearch Laboratory). All imaging stained cells were examined and photographed with a Leica SP5 For Forster€ resonance energy transfer (FRET)-fluorescence confocal fluorescence microscope. lifetime imaging (FLIM) experiments, donor proteins (fused to GFP) were expressed from vectors pCMV3-C-GFPSpark, and Luciferase reporter assay acceptor proteins (fused to RFP) were expressed from vector Luciferase reporter assay was performed as previously described CMV3-C-OFPSpark. FRET-FLIM experiments were performed on (13). A human PD-L1 gene promoter region was inserted into a a Leica TCS SMD FLCS confocal microscope excitation with WLL pGL3 basic vector as pGL3- PD-L1-Promoter. One hundred nano- (white light laser) and emission collected by an SMD SPAD grams (ng) of constructed plasmid and 7 ng Renilla luciferase (single photon-sensitive avalanche photodiodes) detector. The control plasmid were transfected into cells expressing 5637/ 5637 or UC3 cells transiently coexpressing donor and acceptor, as RORC cDNA and UC3/RORC cDNA in 6-well plates. Forty-eight indicated in the figures, were visualized 36 hours after agroinfil- hours later, luciferase activities were measured using the Dual tration. Accumulation of the GFP- and RFP-tagged proteins was Luciferase Assay Kit (Promega). Renilla luciferase was used to estimated before measuring lifetime. The tunable WLL set at

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

489 nm with a pulsed frequency of 40 MHz was used for Cell apoptosis analysis excitation, and emission was detected using SMD GFP/RFP Filter To detect apoptosis, adherent cells were incubated with cis- Cube (with GFP: 500–550 nm). The fluorescence lifetime shown platin at different concentrations. After 48 hours, cells were in the figures corresponding to the average fluorescence lifetime of collected, washed twice with cold 1 PBS, and resuspended in the donor (t) was collected and analyzed by PicoQuant Sym- 200 mL binding buffer at a density of 1 107 cells/mL. Then, we phoTime software. Lifetime is normally an amplitude-weighted stained cells with 5 mL Annexin V and propidium iodide (BD mean value using the data from the single (GFP-fused donor Biosciences) using an apoptosis detection kit (BD Biosciences) protein only or GFP-fused donor protein with free RFP acceptor or and subjected to analysis by flow cytometry (Cytomics FC with noninteracting RFP-fused acceptor protein) or biexponential 500 MPL, Beckman Coulter). Early apoptosis was determined þ fit (GFP-fused donor protein interacting with RFP-fused acceptor based on the percentage of cells with Annexin V /PI , while late þ þ protein). Mean lifetimes are presented as means SD based on apoptosis was that of cells with Annexin V /PI . Experiments more than 10 cells from at least three independent experiments. were done at least three times for final analyses. FRET efficiency was calculated according to the formula E ¼ 1 tDA/tD, where tDA is the average lifetime of the donor in the Animal studies presence of the acceptor and tD is the average lifetime of the donor Animal experiments were approved by the Ethics Committee at fl in the absence of the acceptor. FUSCC. Brie y, female BALB/c nude (Shanghai Slac Laboratory Animal Co. Ltd, 4–6 weeks) were subcutaneously injected with 6 Glycolysis analysis RORC-overexpressing 5637 cells (5 10 suspended in 0.1 mL PBS 6 The Glucose Uptake Colorimetric Assay Kit (BioVision), Lactate for each mouse) and RORC-overexpressing UC3 cells (5 10 Colorimetric Assay Kit (BioVision), ATP Assay Kit (SIGMA ALOR- suspended in 0.1 mL PBS for each mouse). The mice were weighed fi ICH), and Amplite Colorimetric NADPH Assay Kit (AAT Bioquest every 3 days and sacri ced for detecting of the size, number, and Inc.) were purchased to examine the glycolysis process in bladder weight of celiac metastasis of different cell lines. For the subcuta- cancer cells according to the manufacturers' protocol. neous xenograft model, tumor growth with a digital caliper was measured every 3 days. Tumor volumes were calculated by Oxygen consumption rate and extracellular acidification rate the dimensional size of each tumor with the following formula: V ¼ L W 2 Cellular mitochondrial function was measured using the Sea- (volume) (length) (width) 0.72. Once reaching an 3 horse XF Cell Mito stress test Kit and the Bioscience XF96 Extra- average tumor volume of 100 mm , before being treated with cellular Flux Analyzer, according to the manufacturers' instruc- cisplatin, all the mice were subjected to perform PET/CT scan. The tions. Glycolytic capacity was determined using the Glycolysis glucose uptake of the tumor was evaluated by the SUV. Then, they Stress Test Kit as per the manufacturer's instructions. Briefly, 4 were intraperitoneally treated with cisplatin (5 mg/kg). Adminis- 104 cells were seeded onto 96-well plates and incubated overnight. tration of vehicle or agents and measurement of tumor volume fl After washing the cells with Seahorse buffer (DMEM with phenol were done every 3 days. Animals were also subjected to uores- fi red containing 25 mmol/L glucose, 2 mmol/L sodium pyruvate, cence imaging. Finally, mice were weighed and sacri ced, and and 2 mmol/L glutamine), 175 mL of Seahorse buffer plus 25 mL tumors were weighed and dissected. RT-PCR and IHC of xenograft each of 1 mmol/L oligomycin, 1 mmol/L FCCP, and 1 mmol/L tumor were done according to the protocol above. rotenone was automatically injected to measure the oxygen con- Statistical analysis sumption rate (OCR). Then, 25 mL each of 10 mmol/L glucose, The data in this study were calculated using GraphPad Prism 1 mmol/L oligomycin, and 100 mmol/L 2-deoxy-glucose were and reported as mean SD. Comparisons between controls and added to measure the extracellular acidification rate (ECAR). The treated groups were determined by paired t test or one-way OCR and ECAR values were calculated after normalization to the ANOVA, followed by Tukey multiple comparison tests. Clinico- cell number and were plotted as the mean SD. pathologic characteristics analysis was performed using SPSS 23.0 (SPSS Inc.). The relationship between RORC and PD-L1, ITGB6, Cell viability assay STAT3, and p-STAT3 was conducted using Spearman correlation Multiple cultures of bladder cancer cells were plated in 96-well coefficient. The association between RORC, PD-L1, ITGB6, STAT3, plates at a density of 1 103 cells/well supplemented with a and p-STAT3 expression and clinicopathologic characteristics 100 mL maintenance medium to evaluate the cell proliferation rate. was evaluated using the c2 test. The Kaplan–Meier method with Each day one set of cultures was collected and counted. The log-rank analysis were used to obtain estimates of PFS and OS. proliferation rate equaled the experimental OD value/the control Variables with a value of P < 0.05 in univariate analysis were OD value. Cell viability was also assessed by CCK-8. We plated included in the subsequent multivariate analysis on the basis of 8 103 cancer cells per well in 96-well plates. The next day, cells the Cox proportional hazards model. A probability less than 0.05 were treated with various concentrations of cisplatin. Cell viability was considered statistically significantly different. used to measure the number of viable cells was determined by measurement of absorbance at 470 nm by a Microplate Reader Acronyms and abbreviations (Synergy H4, Bio-Tek). All experiments were done in triplicate. The acronyms and abbreviations used in this article are shown in Supplementary Table S3. Colony formation assay Cells were seeded in six-well plates at a density of 500 per well. The cells were cultured with fresh medium and allowed to grow at Results least for 1 week. Colonies with more than 50 cells were counted RORC is downregulated in bladder urothelial cancer patients after being fixed with ice-cold methanol and stained with Crystal To evaluate whether RORC expression was associated with violet (Solarbio). clinicopathologic features of bladder cancer patients, we first

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performed immunostaining with antibodies against RORC in qRT-PCR in 5637/RORC OE and UC3/RORC OE cells, compared tissue microarrays from 155 bladder cancer patients (FUSCC with their control cells, respectively (Fig. 2C). All these signs cohort) and found that RORC immunostaining was highly indicated that RORC may play a critical role in bladder cancer expressed in 51% (n ¼ 79) of patients and underexpressed in cell proliferation, apoptosis, and response to cisplatin through the other 49% (n ¼ 76) of patients (Fig. 1A and B). Meanwhile, we involvement in glycolysis. examined expression profiles of RORC in 115 para-carcinoma bladder tissue samples and observed that RORC expression levels Cell proliferation and glycolysis are inhibited by RORC, while in para-carcinoma tissues were much higher (78/115, 67.8%) concomitantly inducing apoptosis in cultured bladder cancer than those in tumor tissues (Fig. 1A and B). cells In addition, we further analyzed RORC expression in the To determine the role of RORC in regulating cell proliferation, FUSCC cohort and detected that expression levels of RORC were we performed colony formation assays. Overexpression of RORC significantly lower in patients who were resistant to chemotherapy suppressed cell growth and reduced the number and size of the than those who remained sensitive to chemotherapy (P ¼ colonies in 5637 and UC3 cell lines compared with control cells 0.035; Fig. 1C). Moreover, SUVs were obtained from the PET/CT (Supplementary Fig. S2A and S2B). We then performed glucose scan data from 29 bladder urothelial carcinoma patients to uptake, lactate and ATP, NADPH uptake assays to test whether evaluate the association of SUV values with RORC expression in RORC negatively regulates glucose metabolism in bladder cancer bladder cancer tissues. And then we found that patients with low cells. The results showed that glucose uptake, lactate and ATP, RORC staining showed significantly higher SUVmax values NADPH uptake, ECAR, and OCR were all dramatically decreased of primary tumor than patients with high RORC staining (P < in cells overexpressing RORC compared with controls (P < 0.05; 0.05; Fig. 1D and E). To examine in more detail the changes in Supplementary Fig. S2C–S2H). cellular glucose metabolism in bladder cancer, we performed a We also found that overexpression of RORC resulted in more global metabolomic analysis with bladder cancer tissues and apoptotic bladder cancer cells (Supplementary Fig. S2I–S2J). para-carcinoma tissues. We observed that glycolysis and pen- Western blotting results demonstrated that RORC overexpression tose phosphate pathway intermediates were increased in blad- reduced expression levels of BCL-2, GLUT1, HK2, and LDHB but der cancer tissues with low RORC expression, compared with increased proapoptotic protein P53 in 5637 and UC3 cell lines those in high RORC expression and para-carcinoma tissues (Supplementary Fig. S2K). The above results indicated that RORC (Fig. 1F and G). These results indicated that RORC was closely exert its anticancer effect by inhibiting cell proliferation and related with bladder cancer progression, glucose metabolism, glucose metabolism, while concomitantly inducing cell apoptosis and chemoresistance. in bladder cancer cells. Furthermore, the correlation of RORC staining with prognosis of bladder cancer patients was analyzed using Kaplan–Meier Bladder cancer cells in culture are sensitized to cisplatin by the analysis with the log-rank test, and then we found that patients activation of RORC with low RORC expression levels had significantly shorter PFS To investigate the impact of RORC on the chemosensitivity and OS than patients whose tumors had high RORC expression of human bladder cancer cells to cisplatin, we treated 5637 and (P ¼ 0.0409 and 0.009, respectively; Supplementary Fig. S1A and UC3 cells with cisplatin in a dose- and time-dependent manner. S1B). Meanwhile, analyses stratifying by different clinicopatho- CCK-8 assays (Supplementary Fig. S3A), clone formation assay logic data indicated that patients with low RORC expression (Supplementary Fig. S3B and S3C), and flow cytometry analysis possessed poor PFS and OS compared with patients with high (Supplementary Fig. S3D and S3E) revealed that RORC over- RORC expression, either in patients with stage I–II, stage III–IV, or expression increased the sensitivity of 5637 and UC3 cells' presence of chemotherapy resistance (all P < 0.05; Supplementary response to cisplatin. Fig. S1A and S1B). The multivariate analysis with the Cox pro- Furthermore, in order to test whether overexpression of portional hazards model revealed that chemotherapeutic RORC enhances the effect of cisplatin on glucose metabolism, response, RORC expression, and TNM stage were independent RORC-overexpressing and control cancer cells were pretreated prognostic factors for OS in bladder cancer (Supplementary Table with cisplatin, and then subjected to glucose uptake, lactate, S2). In conclusion, decreased expression of RORC was an inde- and ATP production assay. We found overexpression of RORC pendent predictive factor for OS in bladder cancer. synergized with cisplatin to inhibit cell glucose metabolism (Supplementary Fig. S3F–S3H). Results obtained from the Modulation of RORC levels affects expression of genes involved immunoblotting assay showed that overexpression of RORC in glucose metabolism, apoptosis, and chemosensitivity led to the upregulation of P53 but downregulation of BCL-2, Due to relative lower background expression level of RORC GLUT1, HK2, and LDHB by a dose-dependent manner in both in human bladder cancer cell lines 5637 and UC3, we then cell lines (Supplementary Fig. S3I). selected these two cell lines for further experiments. RORC was Taken together, these results suggested that RORC-overexpres- stably overexpressed in 5637 and UC3 cells (5637/RORC OE and sing cells had a higher rate of apoptosis than control cells in UC3/RORC OE, respectively). response to cisplatin treatment, and also that RORC overexpres- To explore the potential significance of RORC in bladder sion synergized with cisplatin to inhibit cell proliferation and cancer, we used gene-chip assays to compare the expression of glucose metabolism. RORC-related genes in 5637/RORC OE cells with their control cells. We found that some important regulatory genes involved in PD-L1 is a direct target of RORC in bladder cancer glycolysis, oxidative phosphorylation, apoptosis and response to To probe into the antitumor role of RORC in bladder cancer cisplatin were enriched in cells with high RORC expression cells, we analyzed the potential enrichment factors when over- (Fig. 2A and B). Results of the chip assay were validated by expressing RORC and found high enrichment of RORC at the

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

Figure 1. RORC expression in bladder urothelial cancer patients. A, Representative images from human bladder carcinoma sections and para-carcinoma bladder tissue samples with low or high RORC IHC staining (4and400). B, RORC expression in human bladder carcinoma sections (n ¼ 155, right) and para-carcinoma bladder tissue samples (n ¼ 115; left). C, Expression levels of RORC in patients resistant to chemotherapy or sensitive to chemotherapy. D, Representative 18F-FDG-PET-CT images from bladder cancer patients with low (left) or high (right) RORC expression (all 200). E, SUVmax of bladder urothelial cancer patients with low or high RORC expression (n ¼ 29). F, The ﬂow-process diagram of carbohydrate metabolism. G, Heat map comparing relative levels of intermediates in bladder cancer tissues with those in para-carcinoma tissues (n ¼ 3 samples of each group). Blue, downregulation; red, upregulation.

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Figure 2. RNA-sequencing after upregulating RORC. A, GSEA analysis was performed using 5637 cells with RORC overexpression and the corresponding controls. The signature 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 in 5637/RORC OE and UC3/RORC OE and control cells. Data are shown as mean SD. Significance was calculated using the Student t test. , P < 0.01.

promoter of PD-L1 using UCSC Genome Bioinformatics Site Next, we constructed a PD-L1 promoter luciferase reporter (http://genome.ucsc.edu/), which suggested that PD-L1 might be plasmid and performed a luciferase reporter assay to confirm the potential target of RORC in bladder cancer. To confirm this the mechanistic link between RORC and PD-L1. First, we trans- hypothesis, we first asked whether there is an inverse relation- fected PD-L1 promoter plasmids into 5637 and UC3 cell lines ship between RORC and PD-L1 expression. We found PD-L1 with stably overexpressing RORC. Compared with the control mRNA was downregulated when RORC was overexpressed in groups, RORC significantly inhibited PD-L1 promoter activity the bladder cancer cells 5637 and UC3 (Fig. 3A). Immunoflu- (Fig. 3C). To confirm the exact region of RORC binding sites orescence experiments in 5637 and UC3 cells also confirmed within the PD-L1 promoter, we performed ChIP in 5637 cell lines RORC negatively regulated the expression of PD-L1 in the cell and found that there were two RORC binding regions, containing membrane (Fig. 3B). four binding sites that existed at approximately1200 bp upstream

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

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 cells overexpressing RORC and control cells. B, Representative images showing RORC inhibited the expression of PD-L1 in cytoplasm (red), with immunoﬂuorescence staining (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 ChIP analysis showed that RORC bound to the PD-L1 gene promoter region. E, The map of RORC binding sits in the promoter region of PD-L1. F, Luciferase reporter assay was used for the detection of RORC-mutant sites in the promoter region of PD-L1. , P < 0.05; , P < 0.01.

of the ORF of PD-L1 (Fig. 3D). Moreover, to further determine PD-L1 rescues the antitumor effect of RORC on bladder cancer binding sites of RORC in the promoter of PD-L1, we individually cells mutated each of these binding sites and repeated the luciferase To validate that PD-L1 is a critical target gene of RORC, we assay (Fig. 3E). Data showed that mutation of the ﬁrst binding site performed the rescue experiment by overexpressing PD-L1 and alone abrogated luciferase activity (Fig. 3F). RORC simultaneously and observed the impact on cell

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Figure 4. Tumor-suppressed properties of RORC may be achieved by the PD-L1/ITGB6 signaling pathway. A, GSEA analysis showed high enrichment of the integrin pathway from RNA-sequencing data. B, RORC significantly changed the expression of proteins in the integrin signaling pathway. C, Coimmunoprecipitation assay 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 statistically significant difference (, P < 0.01) according to a Student t test. G, Representative images of the integrin pathway–related protein expression detected by Western blotting in a rescue experiment. , P < 0.05; , P < 0.01.

proliferation, apoptosis, and glycolysis. In both 5637 and pathways when overexpressing RORC. We found higher enrich- UC3 cell lines, PD-L1 overexpression partially reversed the ment of the integrin pathway (Fig. 4A) from RNA-sequencing change of cell proliferation caused by the RORC overexpression data. Next, we found that overexpression of RORC decreased the (Supplementary Fig. S4A and S4B). In addition, PD-L1 overexpression level of the members of the integrin family and expression significantly reverted RORC overexpression–mediated downstream FAK/AKT1 signaling pathway in 5637 and UC3 cells cell apoptosis, glucose consumption, and lactate production (Fig. 4B), and introduction of PD-L1 cDNA rescued expression (Supplementary Fig. S4C–S4F), as well as the RORC overexpres- levels of ITGB6, p-FAK (Tyr397), p-AKT1 (Ser473), and p-STAT3 sion–mediated ATP and NADPH production (Supplementary (Tyr705; Fig. 4G). Our previous studies had validated the direct Fig. S4G and S4H). Similar results were also observed in Western binding of PD-L1 and ITGB4 (14); we here found PD-L1 might blotting. More specifically, PD-L1 and RORC overexpression bind to ITGB6 detected by coimmunoprecipitation assay decreased expression of the proapoptotic protein P53, but (Fig. 4C). Results of immunofluorescence assay (Fig. 4D) and increased glycolysis-related proteins GLUT1 and LDHB, as well FRET-FLIM (Fig. 4E and F) further demonstrated the direct inter- as upregulated the antiapoptotic protein BCL-2 (Supplementary action between PD-L1 and ITGB6. To further demonstrate that Fig. S4I). Taken together, these findings demonstrate that RORC ITGB6 is a critical target gene of RORC and PD-L1, we performed may block tumor progression by suppressing PD-L1 expression. the rescue experiment by overexpressing PD-L1 and RORC simultaneously and observed the impact on protein expression levels of Tumor suppression properties of RORC are the result of the integrin signaling pathway. binding, activation, and regulation of the PDL1/ITGB6/STAT3 Interestingly, we also found that RORC overexpression signif- signaling pathway icantly inhibited the expression levels of STAT-family members To further elucidate the molecular mechanism underlying the from RNA-sequencing data (Fig. 5A). The most obvious change antitumor effect of RORC, we analyzed the potential enrichment was observed in STAT3 (Fig. 5A and B). Immunofluorescence

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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 showing RORC 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 and BCL-2. , P < 0.01; , P < 0.001.

assay also conﬁrmed that RORC overexpression reduced the demonstrated that RORC might exert its inhibitory function on expression of p-STAT3 (Tyr705) in the nucleus (Fig. 5C). Further- cell proliferation and glucose metabolism by blocking the bind- more, we found RORC reduced the accumulation of protein in the ing of STAT3 to the promoter of STAT3-mediated genes through nucleus but had no impact on cytoplasmic levels of STAT3 and the PD-L1/ITGB6/FAK/AKT1 signal pathway. p-STAT3 (Tyr705; Fig. 5D). Then, ChIP assay was performed to determine whether RORC weakened the binding of STAT3 to the RORC blocks the progression of bladder cancer and sensitizes promoter of STAT3-mediated target genes, such as BCL-2 and P53. cancer cells' response to cisplatin in vivo As shown in Fig. 5E, we found that overexpression of RORC We next tested antitumor effects of RORC in vivo. To observe attenuated the binding of STAT3 to the promoter of BCL-2 and subcutaneous tumor formation, we injected 5637 and UC3 cells enhanced the binding of STAT3 to the promoter of P53, which either overexpressing RORC or harboring empty vector into the

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Figure 6. RORC blocked the progression of bladder cancer and sensitized cancer cells to cisplatin-induced apoptosis in vivo. A, Representative image of nude mice bearing tumors formed by 5637/RORC OE and UC3/RORC OE and their control cells. B, Theaveragetumorvolumeofnudemicebearing tumors formed by 5637/RORC OE and UC3/RORC OE and their control cells. C, The average tumor weight of nude mice bearing tumors formed by 5637/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 bearing tumors before the administration of cisplatin. F, Representative image of nude mice bearing tumors formed by 5637/RORC OE and UC3/RORC OE and 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 and UC3/RORC OE and their control cells after cisplatin treatment. Error bars, 95% CIs. , P < 0.05; , P < 0.01.

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flanks of nude mice. As is shown in Fig. 6A–C, overexpression of cules that have been modified by drug design to regulate related RORC slowed the speed of tumor growth and reduced overall diseases such as cancer and diabetes. Therefore, the search for tumor weight in vivo. In addition, PET-CT analysis showed that ligands and signaling pathways of orphan receptors has become a overexpression of RORC significantly suppressed the glucose promising research area. Our results determined the anticancer uptake of xenograft cells in vivo and resulted in a lower SUVmax effect of RORC in bladder cancer and found that loss of RORC was value (Fig. 6D and E). IHC assay with xenograft tissues showed prevalent in bladder cancer tissues. RORC also represented a very that RORC inhibited the expression of PD-L1, ITGB6, and low hazard ratio from multiple Cox regression analysis, indicating p-STAT3 (Tyr705; Supplementary Fig. S5A). Subsequently, we RORC expression is associated with improved survival outcomes. extracted mRNA from transplanted murine tumors and per- That is consistent with previous observations. Furthermore, the formed RT-qPCR to further validate the role of RORC in glycolysis loss of RORC alters multiple cellular processes accounting for and apoptosis. In accordance with earlier results obtained in cell cancer progression. A literature review showed that RORC inhib- lines, RORC could decrease the expression of proteins involved in ited FasL and cytokine gene expression to regulate apoptosis (19). glycolysis and antiapoptosis, upregulate proapoptosis proteins RORC also attenuates TGFb/EMT signaling, thereby regulating the (Supplementary Fig. S5B and S5C). metastasis process. More specifically, key genes in this pathway, To validate RORC-sensitized bladder cancer cells response to for example, TGFb and SMAD3, were direct targets of RORC (20). cisplatin in vivo, when the tumor volume reached 100 mm3, mice Besides, RORC was also involved in DNA-repair gene expres- were treated with cisplatin on alternate days. The results showed sion (9) and angiogenesis (21). However, the expression and that RORC overexpression coordinates with cisplatin treatment to function of RORC in glucose metabolism remain largely unex- further reduce tumor volume and weight, relative to cells harbor- plored. Our data showed that RORC strongly reduced the glucose ing an empty vector (Fig. 6F–H). metabolism of bladder cancer. These findings reveal a novel mechanism underlying the suppressive effect of RORC on cell Expression of PDL1, ITGB6, STAT3, and p-STAT3 is associated proliferation in cancer cells. with poor survival in bladder urothelial cancer patients Interestingly, in addition to its direct impact on tumor growth To determine the clinical significance of PDL1, ITGB6, STAT3, of RORC, we found that overexpression of RORC conferred and p-STAT3 in bladder cancer, we assessed their expression in a sensitivity to cisplatin and increased cisplatin-induced apoptosis bladder tissue microarray (n ¼ 155). PDL1 was expressed at high through the mitochondrial apoptotic pathway. Chemotherapy is levels in 68.4% (106/155) of bladder cancer tissues, 85 (54.8%) the effective adjuvant treatment for bladder cancer. Platinum is patients showed "high" expression of ITGB6, 98 (63.2%) still the most commonly used drug in chemotherapeutic regi- patients showed "high" expression of STAT3, and 103 (66.5%) mens. Notwithstanding, platinum resistance remains a serious patients showed "high" expression of p-STAT3 (Fig. 7A and B; problem for bladder cancer patients and is one of the burning Supplementary Table S2). In addition, high expression of PDL1 issues of our times. Recent studies have concluded that cisplatin (log-rank, P ¼ 0.0281), ITGB6 (log-rank, P ¼ 0.0325), STAT3 (log- can exert an inhibitory effect on glycolysis in cancer cells. rank, P ¼ 0.0193), and p-STAT3 (log-rank, P ¼ 0.0314) was all Currently, combined treatment modalities that target glycolytic correlated with poor OS (Supplementary Fig. S6A). Simulta- pathways hold promise for the treatment of chemoresistant neously, high expression of PDL1, ITGB6, STAT3, and p-STAT3 cancer cells (22). Increasing evidence shows that inhibition of was also associated with poor PFS (P ¼ 0.0348, 0.0078, 0.0366, glycolysis enhances drug-induced apoptosis in ovarian cancer, and 0.0308, respectively, Supplementary Fig. S6B). The correla- lung cancer, and leukemia (23–25). Because RORC is a key tion of PDL1, ITGB6, STAT3, and p-STAT3 with clinicopathologic regulator in glucose metabolism, targeting RORC may play an characteristics in bladder cancer patients was shown in Supple- important role in restoring cisplatin sensitivity. In our research, mentary Table S4. Moreover, consistent with our previous data, we found that RORC-overexpressing cells had a higher rate of expression levels of PDL1, ITGB6, STAT3, and p-STAT3 in patients apoptosis than control cells in response to cisplatin treatment. with low RORC expression were significantly higher than those in RORC overexpression synergized with cisplatin to inhibit cell patients with high RORC expression (Fig. 7C). Finally, we iden- glucose metabolism and regulated the cancer cell, which may tified the RORC/ PDL1/ITGB6/STAT3 signaling axis in bladder account for the cisplatin sensitivity mediated by RORC. tumorigenesis (Fig. 7D). A special feature of nuclear receptors is to act as important initiators of gene transcription. Moreover, nuclear receptors can recruit a variety of transcription factors and coregulators to target Discussion promoters. In our study, we discovered that RORC interacted In this study, we illustrated the regulatory function of RORC in directly with the PD-L1 promoter and regulated the expression of the regulation of bladder cancer carcinogenesis. Here, we identi- PD-L1 mRNA. PD-L1, also known as CD274 and B7-H1, was fied RORC as an important tumor suppressor gene in bladder originally identified in a murine T-cell hybridoma and a hemato- cancer and the role of the RORC/PD-L1/ITGB6/STAT3 signaling poietic progenitor cell line. High expression of PD-L1 in tumor axis in bladder tumorigenesis, which served as a potential future cells was associated with poor prognosis in multiple cancer types, molecular marker for the prognosis of bladder cancer. including papillary thyroid carcinoma (26), breast cancer (27), NRs, binding with their corresponding ligands and coregula- esophageal squamous cell carcinoma (28), hepatocellular carci- tors, regulate the coordinated expression of genes and then play an noma (29), and other solid tumors. Mechanistically, PD-L1 important role in the growth and development of the body, expression is regulated by oncogenic transcription factors, such metabolism, cell differentiation, and many physiologic processes as c-MYC and hypoxia-inducible factor (HIF; refs. 30, 31). Our in vivo (5). Dysfunction of NRs will lead to a range of diseases such results first reported that RORC directly bound to the promoter as cancer (15), infertility (16), obesity (17), and diabetes (18). region of PD-L1 and promoted its downstream signaling pathway, NRs are promising drug design targets and can bind small mole- reinforcing the importance of RORC in PD-L1–mediated

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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 high expression of PD-L1, ITGB6, STAT3, and p-STAT3 (4and400). B, PD-L1, ITGB6, STAT3, and p-STAT3 expression in human bladder carcinoma sections (n ¼ 155; right) and para-carcinoma bladder tissue samples (n ¼ 115; left). C, Correlation of RORC expression with PD-L1, ITGB6, STAT3, and p-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.

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tumorigenesis. Furthermore, increasing PD-L1 expression in blad- Authors' Contributions der cancer cells reversed the inhibition of cell progression induced Conception and design: D. Cao, Z. Qi, D. Ye, Z. Wang by RORC overexpression. Development of methodology: D. Cao, Z. Qi, D. Ye Tumor-intrinsic functions of PD-L1 and their interplays with Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Cao, H. Li, H. Xie, J. Wu, Y. Huang, Y. Zhu, other oncogenic pathways have gained much attention. High Y. Shen, Y. Zhu, B. Dai, D. Ye expression of PD-L1 interacted with mTOR signaling to promote Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cell growth and autophagy in ovarian cancer cells and melano- computational analysis): D. Cao, Z. Qi, H. Xie, J. Wu, Y. Huang, Y. Zhu, Y. Shen, ma (32), regulated BTK signaling to reverse the immune meta- Y. Zhu, B. Dai, D. Ye bolic dysfunctions of monocytes in chronic lymphocytic leuke- Writing, review, and/or revision of the manuscript: D. Cao, H. Xie, J. Wu, mia (33), bind to ITGB4 to trigger AKT/GSK3b and SNAI1/SIRT3 Y. Huang, Y. Zhu, Y. Shen, Y. Zhu, B. Dai, D. Ye, Z. Wang Administrative, technical, or material support (i.e., reporting or organizing signaling in cervical cancer (14). The above studies demonstrated data, constructing databases): D. Cao, Y. Pang, H. Xie, J. Wu, Y. Huang, X. Hu, that PD-L1 plays a critical role in tumor progression and metab- D. Ye, Z. Wang olism. This is the ﬁrst study to ﬁnd that ITGB6 is a previously Study supervision: D. Ye, Z. Wang unrecognized target of PD-L1 in cancer cells. Subsequently, ITGB6 activated FAK/AKT/STAT3 signaling. Thus, this novel PD-L1/ Acknowledgments ITGB6 signaling axis critically contributed to the Warburg effect This work was supported by the National Natural Science Foundation of in bladder cancer cells and, as a result, to the development and China (No. 81502235 to Z. Wang; No. 81302213 to D. Cao; Nos. progression of bladder cancer. 81872099 and 81672544 to D. Ye), by the Natural Science Foundation In summary, our results demonstrate that the expression level of Science and Technology Commission Shanghai Municipality (No. of RORC is negatively correlated with the prognosis of human 18ZR1407700 to D. Cao), and by the Youth Foundation of Shanghai bladder cancer patients. Enhanced RORC suppressed cell growth Municipal Commission of Health and Family Planning (No. 20174Y0102 and metabolic reprogramming by inhibiting the promoter activity to D. Cao). of PD-L1, and further inactivated PD-L1/ITGB6/FAK/AKT/STAT3 signaling. Thus, RORC may present as a new biomarker of The costs of publication of this article were defrayed in part by the payment of advertisement favorable prognosis in bladder cancer and as a potential novel page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. target for the treatment of bladder cancer patients.

Disclosure of Potential Conflicts of Interest Received December 10, 2018; revised February 1, 2019; accepted February 21, No potential conflicts of interest were disclosed. 2019; published first February 26, 2019.

References 1. Pang C, Guan Y, Li H, Chen W, Zhu G. Urologic cancer in China. Jpn J Clin beta4/FAK/SOX2/HIF-1alpha signaling pathway in gastric cancer. Oncol 2016;46:497–501. Oncogene 2018;37:744–55. 2. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics 14. Wang S, Li J, Xie J, Liu F, Duan Y, Wu Y, et al. Programmed death ligand 1 in China, 2015. CA Cancer J Clin 2016;66:115–32. promotes lymph node metastasis and glucose metabolism in cervical 3. Kukreja JB, Shah JB. Advances in surgical management of muscle invasive cancer by activating integrin beta4/SNAI1/SIRT3 signaling pathway. bladder cancer. Indian J Urol 2017;33:106–10. Oncogene 2018;37:4164–80. 4. Browne BM, Vanni AJ. Management of urethral stricture and bladder neck 15. Papi A, Guarnieri T, Storci G, Santini D, Ceccarelli C, Taffurelli M, et al. contracture following primary and salvage treatment of prostate cancer. Nuclear receptors agonists exert opposing effects on the inﬂammation Curr Urol Rep 2017;18:76. dependent survival of breast cancer stem cells. Cell Death Differ 2012;19: 5. Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. 1208–19. Mol Endocrinol 2005;19:1429–38. 16. Han SJ, O'Malley BW. The dynamics of nuclear receptors and nuclear 6. Robinson-Rechavi M, Escriva Garcia H, Laudet V. The nuclear receptor receptor coregulators in the pathogenesis of endometriosis. Hum Reprod superfamily. J Cell Sci 2003;116:585–6. Update 2014;20:467–84. 7. Lee JS, Kim KI, Baek SH. Nuclear receptors and coregulators in inﬂamma- 17. Swanson HI, Wada T, Xie W, Renga B, Zampella A, Distrutti E, et al. Role of tion and cancer. Cancer Lett 2008;267:189–96. nuclear receptors in lipid dysfunction and obesity-related diseases. 8. Benoit G, Cooney A, Giguere V, Ingraham H, Lazar M, Muscat G, et al. Drug Metab Dispos 2013;41:1–11. International Union of Pharmacology. LXVI. Orphan nuclear receptors. 18. Solt LA, Banerjee S, Campbell S, Kamenecka TM, Burris TP. ROR inverse Pharmacol Rev 2006;58:798–836. agonist suppresses insulitis and prevents hyperglycemia in a mouse model 9. Oh TG, Bailey P, Dray E, Smith AG, Goode J, Eriksson N, et al. PRMT2 and of type 1 diabetes. Endocrinology 2015;156:869–81. RORgamma expression are associated with breast cancer survival out- 19. Sun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, comes. Mol Endocrinol 2014;28:1166–85. et al. Requirement for RORgamma in thymocyte survival and lymphoid 10. Brozyna AA, Jozwicki W, Skobowiat C, Jetten A, Slominski AT. RORalpha organ development. Science 2000;288:2369–73. and RORgamma expression inversely correlates with human melanoma 20. Oh TG, Wang SM, Acharya BR, Goode JM, Graham JD, Clarke CL, et al. The progression. Oncotarget 2016;7:63261–82. Nuclear Receptor, RORgamma, Regulates Pathways Necessary for Breast 11. Finley LW, Carracedo A, Lee J, Souza A, Egia A, Zhang J, et al. SIRT3 opposes Cancer Metastasis. EBioMedicine 2016;6:59–72. reprogramming of cancer cell metabolism through HIF1alpha destabili- 21. Wang RX, Liu H, Xu L, Zhang H, Zhou RX. Melatonin downregulates zation. Cancer Cell 2011;19:416–28. nuclear receptor RZR/RORgamma expression causing growth-inhibitory 12. Li H, Hu S, Pang Y, Li M, Chen L, Liu F, et al. Bufalin inhibits glycolysis-induced and anti-angiogenesis activity in human gastric cancer cells in vitro and cell growth and proliferation through the suppression of Integrin beta2/FAK in vivo. Oncol Lett 2016;12:897–903. signaling pathway in ovarian cancer. Am J Cancer Res 2018;8:1288–96. 22. Ai Z, Lu Y, Qiu S, Fan Z. Overcoming cisplatin resistance of ovarian cancer 13. Gan L, Meng J, Xu M, Liu M, Qi Y, Tan C, et al. Extracellular matrix protein 1 cells by targeting HIF-1-regulated cancer metabolism. Cancer Lett 2016; promotes cell metastasis and glucose metabolism by inducing integrin 373:36–44.

www.aacrjournals.org Cancer Res; 79(10) May 15, 2019 2617

Cao et al.

23. Hulleman E, Kazemier KM, Holleman A, VanderWeele DJ, Rudin CM, cinoma related to IL-6 and T-cell immunosuppression. Oncotarget 2016;7: Broekhuis MJ, et al. Inhibition of glycolysis modulates prednisolone 7913–24. resistance in acute lymphoblastic leukemia cells. Blood 2009;113: 29. Gao Q, Wang XY, Qiu SJ, Yamato I, Sho M, Nakajima Y, et al. Over- 2014–21. expression of PD-L1 significantly associates with tumor aggressiveness and 24. Loar P, Wahl H, Kshirsagar M, Gossner G, Griffith K, Liu JR. Inhibition of postoperative recurrence in human hepatocellular carcinoma. Clin Cancer glycolysis enhances cisplatin-induced apoptosis in ovarian cancer cells. Res 2009;15:971–9. Am J Obstet Gynecol 2010;202:371e371–378. 30. Casey SC, Tong L, Li Y, Do R, Walz S, Fitzgerald KN, et al. MYC regulates the 25. Guo WH, Zhang Y, Chen T, Wang Y, Xue J, Zhang Y, et al. Efficacy of RNAi antitumor immune response through CD47 and PD-L1. Science 2016;352: targeting of pyruvate kinase M2 combined with cisplatin in a lung cancer 227–31. model. J Cancer Res Clin 2011;137:65–72. 31. Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, et al. 26. Chowdhury S, Veyhl J, Jessa F, Polyakova O, Alenzi A, MacMillan C, et al. PD-L1 is a novel direct target of HIF-1alpha, and its blockade under Programmed death-ligand 1 overexpression is a prognostic marker for hypoxia enhanced MDSC-mediated T cell activation. J Exp Med 2014; aggressive papillary thyroid cancer and its variants. Oncotarget 2016;7: 211:781–90. 32318–28. 32. Clark CA, Gupta HB, Sareddy G, Pandeswara S, Lao S, Yuan B, et al. Tumor- 27. Qin T, Zeng YD, Qin G, Xu F, Lu JB, Fang WF, et al. High PD-L1 expression intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy was associated with poor prognosis in 870 Chinese patients with breast in ovarian cancer and melanoma. Cancer Res 2016;76:6964–74. cancer. Oncotarget 2015;6:33972–81. 33. Qorraj M, Bruns H, Bottcher M, Weigand L, Saul D, Mackensen A, et al. The 28. Chen MF, Chen PT, Chen WC, Lu MS, Lin PY, Lee KD. The role of PD-L1 in PD-1/PD-L1 axis contributes to immune metabolic dysfunctions of mono- the radiation response and prognosis for esophageal squamous cell car- cytes in chronic lymphocytic leukemia. Leukemia 2017;31:470–8.

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Retinoic Acid−Related Orphan Receptor C Regulates Proliferation, Glycolysis, and Chemoresistance via the PD-L1/ITGB6/STAT3 Signaling Axis in Bladder Cancer

Dalong Cao, Zihao Qi, Yangyang Pang, et al.

Cancer Res 2019;79:2604-2618. Published OnlineFirst February 26, 2019.

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