Published OnlineFirst November 12, 2018; DOI: 10.1158/1078-0432.CCR-18-2297 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Targeting Purinergic Receptor P2Y2 Prevents the Growth of Pancreatic Ductal Adenocarcinoma by Inhibiting Cancer Cell Glycolysis Li-Peng Hu1, Xiao-Xin Zhang2, Shu-Heng Jiang2, Ling-Ye Tao3, Qing Li2, Li-Li Zhu1, Ming-Wei Yang3, Yan-Miao Huo3, Yong-Sheng Jiang3, Guang-Ang Tian2, Xiao-Yan Cao2, Yan-Li Zhang2, Qin Yang2, Xiao-Mei Yang2, Ya-Hui Wang2, Jun Li2, Gary Guishan Xiao4,5, Yong-Wei Sun3, and Zhi-Gang Zhang1,2 Abstract Purpose: Extensive research has reported that the tumor lular ATP in tumor microenvironment promoted PDAC microenvironment components play crucial roles in tumor growth and glycolysis. Further studies showed that the progression. Thus, blocking the supports of tumor microen- agonist-activated P2RY2 triggered PI3K/AKT–mTOR signal- vironment is a promising approach to prevent cancer progres- ing by crosstalk with PDGFR mediated by Yes1, resulting in sion. We aimed to determine whether blocking extracellular elevated expression of c-Myc and HIF1a, which subsequent- ATP–P2RY2 axis could be a potential therapeutic approach for ly enhanced cancer cell glycolysis. Genetic and pharmaco- PDAC treatment. logic inhibition of P2RY2 impaired tumor cell growth Experimental Design: Expression of P2RY2 was determined in subcutaneous and orthotopic xenograft model, as well in 264 human PDAC samples and correlated to patient sur- as delayed tumor progression in inflammation-driven vival. P2RY2 was inhibited in human PDAC cell lines by PDAC model. In addition, synergy was observed when antagonist and shRNA, respectively, and cell viability, clono- AR-C118925XX, the selective antagonist of P2RY2 receptor, genicity, and glycolysis were determined. RNA sequencing of and gemcitabine were combined, resulting in prolonged PDAC cell line was applied to reveal underlying molecular survival of xenografted PDAC mice. mechanisms. Multiple PDAC mouse models were used to Conclusions: These findings reveal the roles of the assess the effects of the P2RY2 inhibition on PDAC progression. P2RY2 in PDAC metabolic reprogramming, suggesting that Results: P2RY2 was upregulated and associated with poor P2RY2 might be a potential metabolic therapeutic target prognosis in PDAC. Activated P2RY2 by increased extracel- for PDAC. Introduction mately 8% (1). Despite much efforts have been invested, there is still no effective drug available for therapy of the disease, and Pancreatic ductal adenocarcinoma (PDAC) is one of the most the prognosis of PDAC has shown less improvement in decades lethal human cancers with a 5-year survival rate of approxi- (2, 3). It is thus an unmet need for understanding of the mechanism underlying regulation of PDAC to develop effective 1State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, School targets for therapy of PDAC. of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, P.R. China. Many studies have reported that the tumor microenviron- 2 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer ment, including the cellular and noncellular components of Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, the tumor niche, plays a critical role in tumor progression (4). Shanghai, P.R. China. 3Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China. 4School Cancer cells form intimate associations with stromal cells, of Pharmaceutical Science and Technology, Dalian University of Technology, resulting in an aberrant increase in growth factors (5), cyto- Dalian, P.R. China. 5Functional Genomics and Proteomics Laboratory, kines (6), chemokines (7), and metabolites (8) within the Osteoporosis Research Center, Creighton University Medical Center, Omaha, tumor microenvironment. The receptors and transporters Nebraska. activated by extracellular proteins or metabolites create the Note: Supplementary data for this article are available at Clinical Cancer crosstalk between cancer and stromal cells, which results in Research Online (http://clincancerres.aacrjournals.org/). metabolic reprograming in the microenvironment, leading L.-P. Hu, X.-X. Zhang, and S.-H. Jiang contributed equally to this article. to the reduced immune response and the accelerated growth Corresponding Authors: Zhi-Gang Zhang, Shanghai Cancer Institute, Ren Ji of cancer cell, and eventually apoptosis escaped. Therefore, Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Jiao Tong abolishing the extracellular supports from the tumor micro- University, 800 Dongchuan Road, Shanghai 200240, P.R. China. Phone: 8621- environment may be a promising approach for effective ther- 3420-6763; Fax: 8621-3420-6022; E-mail: [email protected]; Yong-Wei Sun, apy of cancer. [email protected]; and Gary Guishan Xiao, School of Pharmaceutical Science G-protein–coupled receptors (GPCR), the largest transmem- and Technology, Dalian University of Technology. E-mail: [email protected] brane receptor family in humans, are critical responders of doi: 10.1158/1078-0432.CCR-18-2297 extracellular stimulation and modulators of intracellular sig- Ó2018 American Association for Cancer Research. naling pathways (9). It is well established that GPCRs are 1318 Clin Cancer Res; 25(4) February 15, 2019 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 12, 2018; DOI: 10.1158/1078-0432.CCR-18-2297 Targeting P2RY2 Signaling Prevents the Growth of PDAC for the measurement of glucose and lactate concentration. Total Translational Relevance protein was extracted from the cell pellets and quantified Tumor microenvironment is widely reported to be by Bradford (Thermo Fisher Scientific, catalog no. 23227). involved in cancer progression. Abolishing the supports Glucose uptake was measured using Amplex Red Glucose/ from tumor microenvironment components would be an Glucose Oxidase Assay Kit (Invitrogen, catalog no. A22189). effective way to prevent cancer progression. Our study Glucose consumption was calculated by the net content of the revealed that increased extracellular ATP in pancreatic ductal original glucose concentration deduced from the measured adenocarcinoma (PDAC) microenvironment promotes can- glucose concentration in the medium. Lactate production was cer progression by activating Purinergic Receptor P2Y2 measured by using the Lactate Assay Kit (BioVision, catalog (P2RY2). Thus, activated P2RY2 resulted in activation of no. ABIN411683). Total proteins were used for normalization PI3K–mTOR pathway and elevation of cancer glycolysis by of the results obtained above. These experiments were per- crosstalk with PDGFRb, as intermediated by Src family formed in triplicate manner and repeated twice. kinase, Yes1. Blocking P2RY2 by a selective inhibitor AR-C 118925XX profoundly suppressed tumor progression, pro- Extracellular ATP measurement longed orthotopic PDAC mice survival, and exhibited The ATP levels were determined using a bioluminescent synergistic effect with gemcitabine. Together, P2RY2 might ATP Assay Kit (Beyotime, catalog no. S0027) according to the be a promising target for PDAC therapy. manufacturer's instructions. Tumor interstitial fluids were col- lected as reported previously (14). Briefly, tissues were sup- ported with triple-layered 10-mm nylon mesh in the tube and centrifuged at 50 Â g for 5 minutes to remove surface liquids of tissues, followed by centrifugation at 400 Â g for another crucial mediators in the communication between cancer cells 10 minutes to collect interstitial fluids. The ectonucleotidase and other components in the microenvironment (10–12). In inhibitor ARL 67156 trisodium salts were added to the tumor addition, GPCRs are critical pharmaceutical acceptable targets interstitial fluid throughout the procedure. Luminescence was for the treatment of diseases (13), which may also presumably measured using a luminometer (M1000 PRO, TECAN). The hold true for PDAC. standard ATP samples were used for preparation of the cali- Here, we aimed to identify novel therapeutic targets for bration curve. Results were normalized by total protein from PDAC. We found that P2RY2 expression was elevated in PDAC each sample. All experiments were performed in triplicate and and its high expression correlated with poor survival in patients repeated twice. with PDAC. Further studies revealed that activated P2RY2 promoted cancer progression by enhanced glycolysis. Genetic Human PDAC tissue array analysis or pharmacologic inhibition of P2RY2 significantly suppressed The study was conducted in accordance with International PDAC cell growth both in vitro and in vivo. Collectively, our Ethical Guidelines for Biomedical Research Involving Human results indicate that targeting P2RY2 may provide a new oppor- Subjects (CIOMS). The study was approved by the Research tunity for PDAC therapy. Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University (Shanghai, P.R. China). Written Materials and Methods informed consent was provided to all the patients before Seahorse analyses enrollment. The patient cohort of human pancreatic tissue The assays for extracellular acidification rate (ECAR) and array containing 264 PDAC specimens and corresponding oxygen consumption rate (OCR) in the cultured cells were per- noncancerous tissues were also obtained from Ren Ji Hospital formed with