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Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation

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Abstract

Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4+ regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4+ Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4+CD25+Foxp3+ T levels. Foxp3 expression in CD4+ Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4+ Tregs, we established a coculture system using NSCLC cell line and healthy CD4+ T cells and showed that SPC-A1 induced IL-10 and TGF-β1 secretion by affecting the function of CD4+ Tregs. The activity of DNA methyltransferases from CD4+ T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4+ T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4+ T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

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Abbreviations

AP-1:

Activator protein-1

DNMTs:

DNA methyltransferases

ELISA:

Enzyme-linked immunosorbent assay

Foxp3:

Forkhead box protein P3

IL:

Interleukin

NF-AT:

Nuclear factor of activated T cells

NSCLC:

Non-small-cell lung cancer

PBMCs:

Peripheral blood mononuclear cells

SAM:

S-adenosylmethionine

SCC:

Squamous cell carcinoma

TGF:

Transforming growth factor

Tregs:

Regulatory T cells

TSDR:

Treg-specific demethylated region

TSS:

Transcriptional start site

References

  1. He YQ, Bo Q, Yong W, Qiu ZX, Li YL, Li WM (2013) Foxp3 genetic variants and risk of non-small cell lung cancer in the Chinese Han population. Gene 531:422–425

    Article  CAS  PubMed  Google Scholar 

  2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM (2010) Estimates of worldwide burden of cancer in 2008: gLOBOCAN 2008. Int J Cancer 127:2893–2917

    Article  CAS  PubMed  Google Scholar 

  3. Wang F, Xu J, Zhu Q, Qin X, Cao Y, Lou J, Xu Y, Ke X, Li Q, Xie E, Zhang L, Sun R, Chen L, Fang B, Pan S (2013) Downregulation of IFNG in CD4+ T cells in lung cancer through hypermethylation: a possible mechanism of tumor-induced immunosuppression. PLoS ONE 8:e79064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Forde PM, Reiss KA, Zeidan AM, Brahmer JR (2013) What lies within: novel strategies in immunotherapy for non-small cell lung cancer. Oncologist 18:1203–1213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mockler MB, Conroy ML, Lysaght J (2014) Targeting T cell immunometabolism for Cancer Immunotherapy; understanding the impact of the tumor microenvironment. Front Oncol 4:107

    Article  PubMed  PubMed Central  Google Scholar 

  6. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chain (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164

    CAS  PubMed  Google Scholar 

  7. Nishikawa H, Sakaguchi S (2010) Regulatory T cells in tumor immunity. Int J Cancer 127:759–767

    CAS  PubMed  Google Scholar 

  8. Wang L, Liu R, Li W, Chen C, Katoh H, Chen GY, McNally B, Lin L, Zhou P, Zuo T, Cooney KA, Liu Y, Zheng P (2009) Somatic single hits inactivate the X-linked tumor suppressor FOXP3 in the prostate. Cancer Cell 16:336–346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Braga WM, da Silva BR, de Carvalho AC, Maekawa YH, Bortoluzzo AB, Rizzatti EG, Atanackovic D, Colleoni GW (2014) FOXP3 and CTLA4 overexpression in multiple myeloma bone marrow as a sign of accumulation of CD4(+) T regulatory cells. Cancer Immunol Immunother 63:1189–1197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. da Silva Martins M, Piccirillo CA (2012) Functional stability of Foxp3+ regulatory T cells. Trends Mol Med 18:454–462

    Article  Google Scholar 

  11. Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, Sakaguchi S (2004) Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 16:1643

    Article  CAS  PubMed  Google Scholar 

  12. Hall BM, Verma ND, Tran GT, Hodgkinson SJ (2011) Distinct regulatory CD4+T cell subsets; differences between naïve and antigen specific T regulatory cells. Curr Opin Immunol 23:641–647

    Article  CAS  PubMed  Google Scholar 

  13. Beyer M, Schultze JL (2006) Regulatory T cells in cancer. Blood 108:804

    Article  CAS  PubMed  Google Scholar 

  14. Landskron J, Helland Ø, Torgersen KM, Aandahl EM, Gjertsen BT, Bjørge L, Taskén K (2015) Activated regulatory and memory T-cells accumulate in malignant ascites from ovarian carcinoma patients. Cancer Immunol Immunother 64:337–347

    Article  CAS  PubMed  Google Scholar 

  15. Khaghanzadeh N, Samiei A, Ramezani M, Mojtahedi Z, Hosseinzadeh M, Ghaderi A (2014) Umbelliprenin induced production of IFN-γ and TNF-α, and reduced IL-10, IL-4, Foxp3 and TGF-β in a mouse model of lung cancer. Immunopharmacol Immunotoxicol 36:25–32

    Article  CAS  PubMed  Google Scholar 

  16. Sakurai T, Kudo M (2011) Signaling pathways governing tumor angiogenesis. Oncology 81(Suppl 1):24–29

    Article  CAS  PubMed  Google Scholar 

  17. Eusebio M, Kuna P, Kraszula L, Kupczyk M, Pietruczuk M (2014) Allergy-related changes in levels of CD8+CD25+FoxP3 (bright) Treg cells and FoxP3 mRNA expression in peripheral blood: the role of IL-10 or TGF-beta. J Biol Regul Homeost Agents 28:461–470

    CAS  PubMed  Google Scholar 

  18. Mason CM, Porretta E, Zhang P, Nelson S (2007) CD4+CD25+ transforming growth factor-beta-producing T cells are present in the lung in murine tuberculosis and may regulate the host inflammatory response. Clin Exp Immunol 148:537–545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Khattri R, Cox T, Yasayko SA, Ramsdell F (2003) An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 4:337–342

    Article  CAS  PubMed  Google Scholar 

  20. Buck-Koehntop BA, Defossez PA (2013) On how mammalian transcription factors recognize methylated DNA. Epigenetics 8:131–137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pan S, Zhang L, Gao L, Gu B, Wang F, Xu J, Shu Y, Yang D, Chen Z (2009) The property of methylated APC gene promotor and its influence on lung cancer cell line. Biomed Pharmacother 63:463–468

    Article  CAS  PubMed  Google Scholar 

  22. Hattori N, Ushijima T (2014) Compendium of aberrant DNA methylation and histone modifications in cancer. Biochem Biophys Res Commun 455:3–9

    Article  CAS  PubMed  Google Scholar 

  23. Subramaniam D, Thombre R, Dhar A, Anant S (2014) DNA methyltransferases: a novel target for prevention and therapy. Front Oncol 4:80

    Article  PubMed  PubMed Central  Google Scholar 

  24. Kar S, Deb M, Sengupta D, Shilpi A, Parbin S, Torrisani J, Pradhan S, Patra S (2012) An insight into the various regulatory mechanisms modulating human DNA methyltransferase 1 stability and function. Epigenetics 7:994–1007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Robertson KD (2001) DNA methylation, methyltransferases, and cancer. Oncogene 20:3139–3155

    Article  CAS  PubMed  Google Scholar 

  26. Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257

    Article  CAS  PubMed  Google Scholar 

  27. Kangaspeska S, Stride B, Métivier R, Polycarpou-Schwarz M, Ibberson D, Carmouche RP, Benes V, Gannon F, Reid G (2008) Transient cyclical methylation of promoter DNA. Nature 452:112–115

    Article  CAS  PubMed  Google Scholar 

  28. Mazzio EA, Soliman KF (2012) Basic concepts of epigenetics: impact of environmental signals on gene expression. Epigenetics 7:119–130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Reik W (2007) Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447:425–432

    Article  CAS  PubMed  Google Scholar 

  30. Janson PC, Winerdal ME, Marits P, Thörn M, Ohlsson R, Winqvist O (2008) FOXP3 promoter demethylation reveals the committed Treg population in humans. PLoS ONE 3:e1612

    Article  PubMed  PubMed Central  Google Scholar 

  31. Lal G, Bromberg JS (2009) Epigenetic mechanisms of regulation of Foxp3 expression. Blood 114:3727–3735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang F, Chi J, Peng G, Zhou F, Wang J, Li L, Feng D, Xie F, Gu B, Qin J, Chen Y, Yao K (2014) Development of virus-specific CD4+ and CD8+ regulatory T cells induced by human Herpesvirus 6 infection. J Virol 88:1011–1024

    Article  PubMed  PubMed Central  Google Scholar 

  33. La Rocca C, Carbone F, Longobardi S, Matarese G (2014) The immunology of pregnancy: regulatory T cells control maternal immune tolerance toward the fetus. Immunol Lett 162:41–48

    Article  PubMed  Google Scholar 

  34. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL (2002) CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502–507

    Article  CAS  PubMed  Google Scholar 

  35. Larmonier N, Marron M, Zeng Y, Cantrell J, Romanoski A, Sepassi M, Thompson S, Chen X, Andreansky S, Katsanis E (2007) Tumor-derived CD4+CD25+ regulatory T cell suppression of dendritic cell function involves TGF-beta and IL-10. Cancer Immunol Immunother 56:48–59

    Article  CAS  PubMed  Google Scholar 

  36. Erfani N, Mehrabadi SM, Ghayumi MA, Haghshenas MR, Mojtahedi Z, Ghaderi A, Amani D (2012) Increase of regulatory T cells in metastatic stage and CTLA-4 over expression in lymphocytes of patients with non-small cell lung cancer (NSCLC). Lung Cancer 77:306–311

    Article  PubMed  Google Scholar 

  37. Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6:295–307

    Article  CAS  PubMed  Google Scholar 

  38. Ju S, Qiu H, Zhou X, Zhu B, Lv X, Huang X, Li J, Zhang Y, Liu L, Ge Y, Johnson DE, Ju S, Shu Y (2009) CD13+CD4+CD25hi regulatory T cells exhibit higher suppressive function and increase with tumor stage in non-small cell lung cancer patients. Cell Cycle 8:2578–2585

    Article  CAS  PubMed  Google Scholar 

  39. Schneider T, Kimpfler S, Warth A, Schnabel PA, Dienemann H, Schadendorf D, Hoffmann H, Umansky V (2011) Foxp3+ regulatory T cells and natural killer cells distinctly infiltrate primary tumors and draining lymph nodes in pulmonary adenocarcinoma. J Thorac Oncol 6:432–438

    Article  PubMed  Google Scholar 

  40. Yang G, Li H, Yao Y, Xu F, Bao Z, Zhou J (2015) Treg/Th17 imbalance in malignant pleural effusion partially predicts poor prognosis. Oncol Rep 33:478–484

    CAS  PubMed  Google Scholar 

  41. Karimi S, Chattopadhyay S, Chakraborty NG (2015) Manipulation of regulatory T cells and antigen-specific cytotoxic T lymphocyte-based tumourimmunotherapy. Immunology 144:186–196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Shen X, Du J, Guan W, Zhao Y (2014) The balance of intestinal Foxp3+ regulatory T cells and Th17 cells and its biological significance. Expert Rev Clin Immunol 10:353–362

    Article  CAS  PubMed  Google Scholar 

  43. Yang R, Qu C, Zhou Y, Konkel JE, Shi S, Liu Y, Chen C, Liu S, Liu D, Chen Y, Zandi E, Chen W, Zhou Y, Shi S (2015) Hydrogen sulfide promotes Tet1- and Tet2-mediated Foxp3 demethylation to drive regulatory T cell differentiation and maintain immune homeostasis. Immunity 43:251–263

    Article  CAS  PubMed  Google Scholar 

  44. Anderson MR, Enose-Akahata Y, Massoud R, Ngouth N, Tanaka Y, Oh U, Jacobson S (2014) Epigenetic modification of the FoxP3 TSDR in HAM/TSP decreases the functional suppression of Tregs. J Neuroimmune Pharmacol 9:522–532

    Article  PubMed  PubMed Central  Google Scholar 

  45. Li E, Zhang Y (2014) DNA methylation in mammals. Cold Spring Harb Perspect Biol 6:a019133

    Article  PubMed  Google Scholar 

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Acknowledgments

We are grateful to the technical support from National Key Clinical Department of Laboratory Medicine of Jiangsu Province Hospital. This work was supported by National Natural Science Foundation of China (Nos. 81272324, 81371894) and Key Laboratory for Medicine of Jiangsu Province of China (No. XK201114), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Correspondence to Fang Wang or Shiyang Pan.

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The authors declared no financial or commercial conflict of interest.

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Xing Ke and Shuping Zhang have contributed equally to this work.

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Ke, X., Zhang, S., Xu, J. et al. Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation. Cancer Immunol Immunother 65, 587–599 (2016). https://doi.org/10.1007/s00262-016-1825-6

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