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

  • Sebastian F. M. Häusler
  • Itsaso Montalbán del Barrio
  • Jenny Strohschein
  • P. Anoop Chandran
  • Jörg B. Engel
  • Arnd Hönig
  • Monika Ossadnik
  • Evi Horn
  • Birgitt Fischer
  • Mathias Krockenberger
  • Stefan Heuer
  • Ahmed Adel Seida
  • Markus Junker
  • Hermann Kneitz
  • Doris Kloor
  • Karl-Norbert Klotz
  • Johannes Dietl
  • Jörg Wischhusen
Original article

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.

Keywords

Ovarian cancer Immune escape Adenosine CD39 CD73 

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

Notes

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.

References

  1. 1.
    Pectasides D, Pectasides E (2006) Maintenance or consolidation therapy in advanced ovarian cancer. Oncology 70(5):315–324. doi: 10.1159/000097943 PubMedCrossRefGoogle Scholar
  2. 2.
    Sugiyama T, Konishi I (2008) Emerging drugs for ovarian cancer. Expert Opin Emerg Drugs 13(3):523–536. doi: 10.1517/14728214.2010.502888 PubMedCrossRefGoogle Scholar
  3. 3.
    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 PubMedCrossRefGoogle Scholar
  4. 4.
    Blay J, White TD, Hoskin DW (1997) The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res 57(13):2602–2605PubMedGoogle Scholar
  5. 5.
    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 PubMedCrossRefGoogle Scholar
  6. 6.
    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 PubMedCrossRefGoogle Scholar
  7. 7.
    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 PubMedCrossRefGoogle Scholar
  8. 8.
    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 PubMedCrossRefGoogle Scholar
  9. 9.
    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 PubMedCrossRefGoogle Scholar
  10. 10.
    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):3491–3499PubMedCrossRefGoogle Scholar
  11. 11.
    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):282–286PubMedCrossRefGoogle Scholar
  12. 12.
    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 PubMedCrossRefGoogle Scholar
  13. 13.
    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 PubMedCrossRefGoogle Scholar
  14. 14.
    Dzhandzhugazyan KN, Kirkin AF, Thor Straten P, Zeuthen J (1998) Ecto-ATP diphosphohydrolase/CD39 is overexpressed in differentiated human melanomas. FEBS Lett 430(3):227–230PubMedCrossRefGoogle Scholar
  15. 15.
    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):177–184PubMedCrossRefGoogle Scholar
  16. 16.
    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 6:226PubMedCrossRefGoogle Scholar
  17. 17.
    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):1434–1442PubMedCrossRefGoogle Scholar
  18. 18.
    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):1341–1346PubMedGoogle Scholar
  19. 19.
    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 CrossRefGoogle Scholar
  20. 20.
    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 PubMedCrossRefGoogle Scholar
  21. 21.
    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):2245–2255Google Scholar
  22. 22.
    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):211–218PubMedCrossRefGoogle Scholar
  23. 23.
    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):7338–7348PubMedGoogle Scholar
  24. 24.
    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):49–52PubMedGoogle Scholar
  25. 25.
    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 YorkGoogle Scholar
  26. 26.
    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):1273–1285PubMedCrossRefGoogle Scholar
  27. 27.
    Neufeld HA, Towner RD, Pace J (1975) A rapid method for determining ATP by the firefly luciferin-luciferase system. Experientia 31(3):391–392PubMedCrossRefGoogle Scholar
  28. 28.
    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):51–56PubMedCrossRefGoogle Scholar
  29. 29.
    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 PubMedCrossRefGoogle Scholar
  30. 30.
    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):475–481PubMedGoogle Scholar
  31. 31.
    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):1203–1206PubMedGoogle Scholar
  32. 32.
    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):7–10PubMedCrossRefGoogle Scholar
  33. 33.
    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 PubMedCrossRefGoogle Scholar
  34. 34.
    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):527–535PubMedGoogle Scholar
  35. 35.
    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 PubMedGoogle Scholar
  36. 36.
    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 PubMedCrossRefGoogle Scholar
  37. 37.
    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 PubMedCrossRefGoogle Scholar
  38. 38.
    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):1493–1505PubMedCrossRefGoogle Scholar
  39. 39.
    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):7176–7186PubMedCrossRefGoogle Scholar
  40. 40.
    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 PubMedCrossRefGoogle Scholar
  41. 41.
    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 PubMedCrossRefGoogle Scholar
  42. 42.
    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):2311–2320PubMedCrossRefGoogle Scholar
  43. 43.
    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 PubMedGoogle Scholar
  44. 44.
    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 PubMedCrossRefGoogle Scholar
  45. 45.
    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 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Sebastian F. M. Häusler
    • 1
    • 4
  • Itsaso Montalbán del Barrio
    • 1
    • 4
  • Jenny Strohschein
    • 1
  • P. Anoop Chandran
    • 1
    • 3
  • Jörg B. Engel
    • 1
  • Arnd Hönig
    • 1
  • Monika Ossadnik
    • 1
    • 4
  • Evi Horn
    • 1
  • Birgitt Fischer
    • 1
  • Mathias Krockenberger
    • 1
  • Stefan Heuer
    • 1
  • Ahmed Adel Seida
    • 1
    • 4
  • Markus Junker
    • 1
    • 4
  • Hermann Kneitz
    • 2
  • Doris Kloor
    • 5
    • 6
  • Karl-Norbert Klotz
    • 7
  • Johannes Dietl
    • 1
  • Jörg Wischhusen
    • 1
    • 4
  1. 1.Department of Obstetrics and GynaecologyUniversity of Würzburg, School of MedicineWürzburgGermany
  2. 2.Department of DermatologyUniversity of Würzburg, School of MedicineWürzburgGermany
  3. 3.Graduate School for Life SciencesUniversity of Würzburg, School of MedicineWürzburgGermany
  4. 4.Interdisciplinary Centre for Clinical ResearchUniversity of Würzburg, School of MedicineWürzburgGermany
  5. 5.Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and ToxicologyEberhard-Karls-University Hospitals and ClinicsTübingenGermany
  6. 6.Interfaculty Centre of Pharmacogenomics and Pharmaceutical Research (ICePhA)University of TübingenTübingenGermany
  7. 7.Institute of Pharmacology and ToxicologyUniversity of WürzburgWürzburgGermany

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