Abstract
The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A2A adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (Treg) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4+ and CD8+ T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated Treg. Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4+ T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of Treg but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.
Similar content being viewed by others
Abbreviations
- CFDA-SE:
-
Carboxyfluorescein diacetate succinimidyl ester
- ENTPD1:
-
Ectonucleoside triphosphate diphosphohydrolase 1
- FITC:
-
Fluorescein isothiocyanate
- NK:
-
Natural killer (cells)
- OvCA:
-
Ovarian cancer
- PBMC:
-
Peripheral blood mononuclear cells
- shRNA:
-
Short hairpin RNA
- siRNA:
-
Short interfering RNA
References
Pectasides D, Pectasides E (2006) Maintenance or consolidation therapy in advanced ovarian cancer. Oncology 70(5):315–324. doi:10.1159/000097943
Sugiyama T, Konishi I (2008) Emerging drugs for ovarian cancer. Expert Opin Emerg Drugs 13(3):523–536. doi:10.1517/14728214.2010.502888
Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10(9):942–949. doi:10.1038/nm1093
Blay J, White TD, Hoskin DW (1997) The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res 57(13):2602–2605
Ohta A, Gorelik E, Prasad SJ, Ronchese F, Lukashev D, Wong MK, Huang X, Caldwell S, Liu K, Smith P, Chen JF, Jackson EK, Apasov S, Abrams S, Sitkovsky M (2006) A2A adenosine receptor protects tumors from antitumor T cells. Proc Natl Acad Sci USA 103(35):13132–13137. doi:10.1073/pnas.0605251103
Hasko G, Cronstein BN (2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol 25(1):33–39. doi:10.1016/j.it.2003.11.003
Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, Hopner S, Centonze D, Bernardi G, Dell’Acqua ML, Rossini PM, Battistini L, Rötzschke O, Falk K (2007) Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood 110(4):1225–1232. doi:10.1182/blood-2006-12-064527
Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, Kuchroo VK, Strom TB, Robson SC (2007) Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 204(6):1257–1265. doi:10.1084/jem.20062512
Resta R, Yamashita Y, Thompson LF (1998) Ecto-enzyme and signaling functions of lymphocyte CD73. Immunol Rev 161:95–109. doi:10.1111/j.1600-065X.1998.tb01574.x
Csoka B, Himer L, Selmeczy Z, Vizi ES, Pacher P, Ledent C, Deitch EA, Spolarics Z, Nemeth ZH, Hasko G (2008) Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function. FASEB J 22(10):3491–3499
Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, Park D, Woodson RI, Ostankovich M, Sharma P, Lysiak JJ, Harden TK, Leitinger N, Ravichandran KS (2009) Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461(7261):282–286
Aymeric L, Apetoh L, Ghiringhelli F, Tesniere A, Martins I, Kroemer G, Smyth MJ, Zitvogel L (2010) Tumor cell death and ATP release prime dendritic cells and efficient anticancer immunity. Cancer Res 70(3):855–858. doi:10.1158/0008-5472.CAN-09-3566
Künzli BM, Berberat PO, Giese T, Csizmadia E, Kaczmarek E, Baker C, Halaceli I, Büchler MW, Friess H, Robson SC (2007) Upregulation of CD39/NTPDases and P2 receptors in human pancreatic disease. Am J Physiol Gastrointest Liver Physiol 292(1):G223–G230. doi:10.1152/ajpgi.00259.2006
Dzhandzhugazyan KN, Kirkin AF, Thor Straten P, Zeuthen J (1998) Ecto-ATP diphosphohydrolase/CD39 is overexpressed in differentiated human melanomas. FEBS Lett 430(3):227–230
Shi XJ, Knowles AF (1994) Prevalence of the mercurial-sensitive EctoATPase in human small cell lung carcinoma: characterization and partial purification. Arch Biochem Biophys 315(1):177–184
Morrone FB, Oliveira DL, Gamermann P, Stella J, Wofchuk S, Wink MR, Meurer L, Edelweiss MI, Lenz G, Battastini AM (2006) In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model. BMC Cancer 6:226
Braganhol E, Morrone FB, Bernardi A, Huppes D, Meurer L, Edelweiss MI, Lenz G, Wink MR, Robson SC, Battastini AM (2009) Selective NTPDase2 expression modulates in vivo rat glioma growth. Cancer Sci 100(8):1434–1442
Zhou X, Zhi X, Zhou P, Chen S, Zhao F, Shao Z, Ou Z, Yin L (2007) Effects of ecto-5′-nucleotidase on human breast cancer cell growth in vitro and in vivo. Oncol Rep 17(6):1341–1346
Stagg J, Divisekera U, McLaughlin N, Sharkey J, Pommey S, Denoyer D, Dwyer KM, Smyth MJ (1547) Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis. Proc Natl Acad Sci USA 107(4):1547–1552. doi:10.1073/pnas.0908801107
Wang L, Zhou X, Zhou T, Ma D, Chen S, Zhi X, Yin L, Shao Z, Ou Z, Zhou P (2008) Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells. J Cancer Res Clin Oncol 134(3):365–372. doi:10.1007/s00432-007-0292-z
Jin D, Fan J, Wang L, Thompson LF, Liu A, Daniel BJ, Shin T, Curiel TJ, Zhang B (2010) CD73 on tumor cells impairs antitumor T-Cell responses: a novel mechanism of tumor-induced immune suppression. Cancer Res 70(6):2245–2255
Panjehpour M, Castro M, Klotz KN (2005) Human breast cancer cell line MDA-MB-231 expresses endogenous A2B adenosine receptors mediating a Ca2+ signal. Br J Pharmacol 145(2):211–218
Krockenberger M, Dombrowski Y, Weidler C, Ossadnik M, Honig A, Häusler S, Voigt H, Becker JC, Leng L, Steinle A, Weller M, Bucala R, Dietl J, Wischhusen J (2008) Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by down-regulating NKG2D. J Immunol 180(11):7338–7348
Aversa GG, Suranyi MG, Waugh JA, Bishop AG, Hall BM (1988) Detection of a late lymphocyte activation marker by A1, a new monoclonal antibody. Transplant Proc 20(1):49–52
Dörken B, Möller P, Pezzuto A, Schwartz-Albiez R, Moldenhauer G (1989) Part I: B-cell antigens. In: Knapp W, Dörken B, Gilks WR et al (eds) Leukocyte typing IV: white cell differentiation antigens. Oxford University Press, New York
Buira SP, Albasanz JL, Dentesano G, Moreno J, Martin M, Ferrer I, Barrachina M (2010) DNA methylation regulates adenosine A(2A) receptor cell surface expression levels. J Neurochem 112(5):1273–1285
Neufeld HA, Towner RD, Pace J (1975) A rapid method for determining ATP by the firefly luciferin-luciferase system. Experientia 31(3):391–392
Häusler SF, Ossadnik M, Horn E, Heuer S, Dietl J, Wischhusen J (2010) A cell-based luciferase-dependent assay for the quantitative determination of free extracellular adenosine with paracrine signaling activity. J Immunol Methods 361(1–2):51–56
Dyer BW, Ferrer FA, Klinedinst DK, Rodriguez R (2000) A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal Biochem 282(1):158–161. doi:10.1006/abio.2000.4605
Crack BE, Pollard CE, Beukers MW, Roberts SM, Hunt SF, Ingall AH, McKechnie KC, Ijzerman AP, Leff P (1995) Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br J Pharmacol 114(2):475–481
Krug F, Parikh I, Illiano G, Cuatrecasas P (1973) α,β-methylene-adenosine 5′-triphosphate. A competitive inhibitor of adenylate cyclase in fat and liver cell membranes. J Biol Chem 248(4):1203–1206
Ongini E, Dionisotti S, Gessi S, Irenius E, Fredholm BB (1999) Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 359(1):7–10
Brown CE, Wright CL, Naranjo A, Vishwanath RP, Chang WC, Olivares S, Wagner JR, Bruins L, Raubitschek A, Cooper LJ, Jensen MC (2005) Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing. J Immunol Methods 297(1–2):39–52. doi:10.1016/j.jim.2004.11.021
Hoskin DW, Mader JS, Furlong SJ, Conrad DM, Blay J (2008) Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (review). Int J Oncol 32(3):527–535
Shi J, Wan Y, Di W (2008) Effect of hypoxia and re-oxygenation on cell invasion and adhesion in human ovarian carcinoma cells. Oncol Rep 20(4):803–807. doi:10.3892/or_00000077
Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, Makrigiannakis A, Gray H, Schlienger K, Liebman MN, Rubin SC, Coukos G (2003) Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 348(3):203–213. doi:10.1056/NEJMoa020177
Havre PA, Abe M, Urasaki Y, Ohnuma K, Morimoto C, Dang NH (2008) The role of CD26/dipeptidyl peptidase IV in cancer. Front Biosci 13:1634–1645. doi:10.2741/2787
Eltzschig HK, Abdulla P, Hoffman E, Hamilton KE, Daniels D, Schönfeld C, Löffler M, Reyes G, Duszenko M, Karhausen J, Robinson A, Westerman KA, Coe IR, Colgan SP (2005) HIF-1-dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia. J Exp Med 202(11):1493–1505
Mandapathil M, Hilldorfer B, Szczepanski MJ, Czystowska M, Szajnik M, Ren J, Lang S, Jackson EK, Gorelik E, Whiteside TL (2010) Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells. J Biol Chem 285(10):7176–7186
Mandapathil M, Szczepanski MJ, Szajnik M, Ren J, Lenzner DE, Jackson EK, Gorelik E, Lang S, Johnson JT, Whiteside TL (2009) Increased ectonucleotidase expression and activity in regulatory T cells of patients with head and neck cancer. Clin Cancer Res 15(20):6348–6357. doi:1078-0432.CCR-09-1143
Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K, Robson SC, Colgan SP (2003) Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J Exp Med 198(5):783–796. doi:10.1084/jem.20030891
Enjyoji K, Kotani K, Thukral C, Blumel B, Sun X, Wu Y, Imai M, Friedman D, Csizmadia E, Bleibel W, Kahn BB, Robson SC (2008) Deletion of CD39/ENTPD1 results in hepatic insulin resistance. Diabetes 57(9):2311–2320
Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, Colgan SP (2002) Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 110(7):993–1002. doi:10.1172/JCI15337
Tanganelli S, Sandager Nielsen K, Ferraro L, Antonelli T, Kehr J, Franco R, Ferre S, Agnati LF, Fuxe K, Scheel-Krüger J (2004) Striatal plasticity at the network level. Focus on adenosine A2A and D2 interactions in models of Parkinson’s disease. Parkinsonism Relat Disord 10(5):273–280. doi:10.1016/j.parkreldis.2004.02.015
Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF (2001) A novel technique for the fluorometric assessment of T lymphocyte antigen specific lysis. J Immunol Methods 249(1–2):99–110. doi:10.1016/S0022-1759(00)00329-X
Acknowledgments
We wish to thank Drs. George G. Holz and Oleg Chepurny (Upstate Medical University, Syracuse, NY) for providing the RIP1-CRE-luc reporter gene construct used for adenosine measurement.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Häusler, S.F.M., Montalbán del Barrio, I., Strohschein, J. et al. Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity. Cancer Immunol Immunother 60, 1405–1418 (2011). https://doi.org/10.1007/s00262-011-1040-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00262-011-1040-4