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Cellular Oncology

, Volume 39, Issue 6, pp 583–589 | Cite as

Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report

  • Xavier TekpliEmail author
  • Vidar Skaug
  • Rita Bæra
  • David H. Phillips
  • Aage Haugen
  • Steen Mollerup
report

Abstract

Purpose

In the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells.

Methods

ERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n = 87) and adjacent histologically normal lung tissue samples from lung cancer patients (n = 184), primary normal bronchial epithelial-derived cell cultures (n = 11), immortalized bronchial epithelial-derived cell lines (n = 3) and NSCLC derived cell lines (n = 9).

Results

Using RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panel of cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts.

Conclusions

Taken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.

Keywords

Estrogen receptor Lung cancer Gene expression DNA methylation DNA adducts 

Notes

Acknowledgments

This study was supported by grants from the Norwegian Cancer Society. We are grateful to Mrs. Tove Andreassen and Mrs. Elin Einarsdottir Thorner for excellent technical assistance. The authors gratefully acknowledge collaboration of Dr. Lodve Stangeland, Haukeland University Hospital, Bergen, for recruiting the lung cancer patients and collecting the tissue specimens.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

13402_2016_295_MOESM1_ESM.pptx (226 kb)
Supplementary Figure 1 ER expression detailed for each individual cell line a-b: ERα and ERβ gene expression was analyzed in 11 primary cultured human lung cells (NHBE), 3 immortalized human bronchial epithelial cell lines (HBEC), and 9 human lung adenocarcinoma cell lines (HLAC). (PPTX 225 kb)
13402_2016_295_MOESM2_ESM.pptx (47 kb)
Supplementary Figure 2 Schematic representation of the location of the different assays designed to study (a) ERα and (b) ERβ promoter DNA methylation. (PPTX 47 kb)
13402_2016_295_MOESM3_ESM.pptx (75 kb)
Supplementary Figure 3 DNA methylation of ER promoters detailed for each individual cell line: DNA methylation levels were obtained by analyzing the mean methylation of all the CpG sites present in the different regions of ERα (a) or ERβ (b) promoters. We used 11 primary cultured human lung cells (NHBE), 3 immortalized human bronchial epithelial cell lines (HBEC), and 9 human lung adenocarcinoma cell lines (HLAC). (PPTX 75 kb)
13402_2016_295_MOESM4_ESM.pptx (73 kb)
Supplementary Figure 4 Effect of AZA on ERs expression detailed for each individual cell line: a-b: AZA effects on ER expression in human lung cultured cells. Cells were exposed or not for 5 days during proliferation phase to the demethylating agent AZA (0.5 μm). Expression of ER mRNA was normalized to β-actin mRNA levels. AZA effects on gene expression are presented as fold induction of gene expression induced by AZA. (PPTX 72 kb)
13402_2016_295_MOESM5_ESM.docx (14 kb)
Supplementary Table 1 (DOCX 14 kb)
13402_2016_295_MOESM6_ESM.docx (17 kb)
Supplementary Table 2 (DOCX 16 kb)

References

  1. 1.
    E. A. Musgrove, R. L. Sutherland, Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 9, 631–643 (2009)CrossRefPubMedGoogle Scholar
  2. 2.
    L. P. Stabile, S. Dacic, S. R. Land, D. E. Lenzner, R. Dhir, M. Acquafondata, R. J. Landreneau, J. R. Grandis, J. M. Siegfried, Combined analysis of estrogen receptor beta-1 and progesterone receptor expression identifies lung cancer patients with poor outcome. Clin Cancer Res 17, 154–164 (2011)Google Scholar
  3. 3.
    A. H. Wu, B. E. Henderson, M. C. Pike, M. C. Yu, Smoking and other risk factors for lung cancer in women. J Natl Cancer Inst 74, 747–751 (1985)PubMedGoogle Scholar
  4. 4.
    D. J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, R. M. Evans, The nuclear receptor superfamily: the second decade. Cell 83, 835–839 (1995)CrossRefPubMedGoogle Scholar
  5. 5.
    G. Early, Breast Cancer Trialists’ Collaborative, Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365(1687–1717) (2005)Google Scholar
  6. 6.
    V. Kirn, R. Shi, S. Heublein, J. Knabl, M. Guenthner-Biller, U. Andergassen, C. Fridrich, W. Malter, J. Harder, K. Friese, D. Mayr, U. Jeschke, Estrogen receptor promoter methylation predicts survival in low-grade ovarian carcinoma patients. J Cancer Res Clin Oncol 140, 1681–1687 (2014)CrossRefPubMedGoogle Scholar
  7. 7.
    S. Mishra, Q. Tai, X. Gu, J. Schmitz, A. Poullard, R. J. Fajardo, D. Mahalingam, X. Chen, X. Zhu, L. Z. Sun, Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer. Oncotarget 6, 44388–44402 (2015)PubMedPubMedCentralGoogle Scholar
  8. 8.
    R. Kennelly, D. O. Kavanagh, A. M. Hogan, D. C. Winter, Oestrogen and the colon: potential mechanisms for cancer prevention. Lancet Oncol 9, 385–391 (2008)CrossRefPubMedGoogle Scholar
  9. 9.
    D. C. Marquez-Garban, H. W. Chen, M. C. Fishbein, L. Goodglick, R. J. Pietras, Estrogen receptor signaling pathways in human non-small cell lung cancer. Steroids 72, 135–143 (2007)CrossRefPubMedGoogle Scholar
  10. 10.
    L. P. Stabile, A. L. Davis, C. T. Gubish, T. M. Hopkins, J. D. Luketich, N. Christie, S. Finkelstein, J. M. Siegfried, Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor alpha and beta and show biological responses to estrogen. Cancer Res 62, 2141–2150 (2002)PubMedGoogle Scholar
  11. 11.
    H. Kawai, A. Ishii, K. Washiya, T. Konno, H. Kon, C. Yamaya, I. Ono, Y. Minamiya, J. Ogawa, Estrogen receptor alpha and beta are prognostic factors in non-small cell lung cancer. Clin Cancer Res 11, 5084–5089 (2005)CrossRefPubMedGoogle Scholar
  12. 12.
    M. J. Fasco, G. J. Hurteau, S. D. Spivack, Gender-dependent expression of alpha and beta estrogen receptors in human nontumor and tumor lung tissue. Mol Cell Endocrinol 188, 125–140 (2002)CrossRefPubMedGoogle Scholar
  13. 13.
    S. Mollerup, K. Jorgensen, G. Berge, A. Haugen, Expression of estrogen receptors alpha and beta in human lung tissue and cell lines. Lung Cancer 37, 153–159 (2002)CrossRefPubMedGoogle Scholar
  14. 14.
    L. Di Nunno, L. G. Larsson, J. J. Rinehart, R. S. Beissner, Estrogen and progesterone receptors in non-small cell lung cancer in 248 consecutive patients who underwent surgical resection. Arch Pathol Lab Med 124, 1467–1470 (2000)PubMedGoogle Scholar
  15. 15.
    A. G. Schwartz, G. M. Prysak, V. Murphy, F. Lonardo, H. Pass, J. Schwartz, S. Brooks, Nuclear estrogen receptor beta in lung cancer: expression and survival differences by sex. Clin Cancer Res 11, 7280–7287 (2005)CrossRefPubMedGoogle Scholar
  16. 16.
    C. Thomas, J. A. Gustafsson, The different roles of ER subtypes in cancer biology and therapy. Nat Rev Cancer 11, 597–608 (2011)CrossRefPubMedGoogle Scholar
  17. 17.
    S. A. Belinsky, S. S. Snow, K. J. Nikula, G. L. Finch, C. S. Tellez, W. A. Palmisano, Aberrant CpG island methylation of the p16(INK4a) and estrogen receptor genes in rat lung tumors induced by particulate carcinogens. Carcinogenesis 23, 335–339 (2002)CrossRefPubMedGoogle Scholar
  18. 18.
    M. Esteller, D. N. A. Aberrant, Methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicology 45, 629–656 (2005)Google Scholar
  19. 19.
    J. P. Issa, B. A. Zehnbauer, C. I. Civin, M. I. Collector, S. J. Sharkis, N. E. Davidson, S. H. Kaufmann, S. B. Baylin, The estrogen receptor CpG island is methylated in most hematopoietic neoplasms. Cancer Res 56, 973–977 (1996)PubMedGoogle Scholar
  20. 20.
    J. F. Lechner, A. Haugen, I. A. McClendon, E. W. Pettis, Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18, 633–642 (1982)CrossRefPubMedGoogle Scholar
  21. 21.
    R. D. Ramirez, S. Sheridan, L. Girard, M. Sato, Y. Kim, J. Pollack, M. Peyton, Y. Zou, J. M. Kurie, J. M. Dimaio, S. Milchgrub, A. L. Smith, R. F. Souza, L. Gilbey, X. Zhang, K. Gandia, M. B. Vaughan, W. E. Wright, A. F. Gazdar, J. W. Shay, J. D. Minna, Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64, 9027–9034 (2004)CrossRefPubMedGoogle Scholar
  22. 22.
    H. K. Oie, E. K. Russell, D. N. Carney, A. F. Gazdar, Cell culture methods for the establishment of the NCI series of lung cancer cell lines. J Cell Biochem Suppl 24, 24–31 (1996)CrossRefPubMedGoogle Scholar
  23. 23.
    S. E. Olivo-Marston, L. E. Mechanic, S. Mollerup, E. D. Bowman, A. T. Remaley, M. R. Forman, V. Skaug, Y. L. Zheng, A. Haugen, C. C. Harris, Serum estrogen and tumor-positive estrogen receptor-alpha are strong prognostic classifiers of non-small-cell lung cancer survival in both men and women. Carcinogenesis 31 (1778–1786)Google Scholar
  24. 24.
    X. Tekpli, S. Zienolddiny, V. Skaug, L. Stangeland, A. Haugen, S. Mollerup, DNA methylation of the CYP1A1 enhancer is associated with smoking-induced genetic alterations in human lung. Int J Cancer 131, 1509–1516 (2012)CrossRefPubMedGoogle Scholar
  25. 25.
    Z. Birsu Cincin, M. Unlu, B. Kiran, E. Sinem Bireller, Y. Baran, B. Cakmakoglu, Anti-proliferative, apoptotic and signal transduction effects of hesperidin in non-small cell lung cancer cells. Cell Oncol 38(195–204) (2015)Google Scholar
  26. 26.
    A. Koren, E. Sodja, M. Rijavec, M. Jez, V. Kovac, P. Korosec, T. Cufer, Prognostic value of cytokeratin-7 mRNA expression in peripheral whole blood of advanced lung adenocarcinoma patients. Cell Oncol 38(387–395) (2015)Google Scholar
  27. 27.
    C. Zeng, W. Fan, X. Zhang, RRM1 expression is associated with the outcome of gemcitabine-based treatment of non-small cell lung cancer patients--a short report. Cell. Oncol 38(319–325) (2015)Google Scholar
  28. 28.
    D. Nojima, L. C. Li, A. Dharia, G. Perinchery, L. Ribeiro-Filho, T. S. Yen, R. Dahiya, CpG hypermethylation of the promoter region inactivates the estrogen receptor-beta gene in patients with prostate carcinoma. Cancer 92, 2076–2083 (2001)CrossRefPubMedGoogle Scholar
  29. 29.
    C. Zhao, E. W. Lam, A. Sunters, E. Enmark, M. T. De Bella, R. C. Coombes, J. A. Gustafsson, K. Dahlman-Wright, Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation. Oncogene 22, 7600–7606 (2003)CrossRefPubMedGoogle Scholar
  30. 30.
    F. Suzuki, J. Akahira, I. Miura, T. Suzuki, K. Ito, S. Hayashi, H. Sasano, N. Yaegashi, Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5′-untranslated region in human epithelial ovarian carcinoma. Cancer Sci 99, 2365–2372 (2008)CrossRefPubMedGoogle Scholar
  31. 31.
    X. Zhang, Y. K. Leung, S. M. Ho, AP-2 regulates the transcription of estrogen receptor (ER)-beta by acting through a methylation hotspot of the 0 N promoter in prostate cancer cells. Oncogene 26, 7346–7354 (2007)CrossRefPubMedGoogle Scholar
  32. 32.
    N. D. Freedman, M. F. Leitzmann, A. R. Hollenbeck, A. Schatzkin, C. C. Abnet, Cigarette smoking and subsequent risk of lung cancer in men and women: analysis of a prospective cohort study. Lancet Oncol 9, 649–656 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    D. H. Phillips, A. Hewer, C. N. Martin, R. C. Garner, M. M. King, Correlation of DNA adduct levels in human lung with cigarette smoking. Nature 336, 790–792 (1988)CrossRefPubMedGoogle Scholar
  34. 34.
    M. G. Raso, C. Behrens, M. H. Herynk, S. Liu, L. Prudkin, N. C. Ozburn, D. M. Woods, X. Tang, R. J. Mehran, C. Moran, J. J. Lee, I. I. Wistuba, Immunohistochemical expression of estrogen and progesterone receptors identifies a subset of NSCLCs and correlates with EGFR mutation. Clin Cancer Res 15, 5359–5368 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    A. Kerr, 2nd, J. F. Eliason, J. L. Wittliff, Steroid receptor and growth factor receptor expression in human nonsmall cell lung cancers using cells procured by laser-capture microdissection. Adv Exp Med Biol 617, 377–384 (2008)Google Scholar
  36. 36.
    J. A. Baron, Beneficial effects of nicotine and cigarette smoking: the real, the possible and the spurious. Br Med Bull 52, 58–73 (1996)CrossRefPubMedGoogle Scholar
  37. 37.
    X. Z. Zhao, Y. Liu, L. J. Zhou, Z. Q. Wang, Z. H. Wu, X. Y. Yang, Role of estrogen in lung cancer based on the estrogen receptor-epithelial mesenchymal transduction signaling pathways. Onco Targets Ther 8, 2849–2863 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    S. A. Belinsky, Unmasking the lung cancer epigenome. Annu Rev Physiol 77, 453–474 (2015)CrossRefPubMedGoogle Scholar
  39. 39.
    S. J. Ellem, G. P. Risbridger, Treating prostate cancer: a rationale for targeting local oestrogens. Nat Rev Cancer 7, 621–627 (2007)CrossRefPubMedGoogle Scholar
  40. 40.
    A. M. Traynor, J. H. Schiller, L. P. Stabile, J. M. Kolesar, J. C. Eickhoff, S. Dacic, T. Hoang, S. Dubey, S. M. Marcotte, J. M. Siegfried, Pilot study of gefitinib and fulvestrant in the treatment of post-menopausal women with advanced non-small cell lung cancer. Lung Cancer 64, 51–59 (2009)CrossRefPubMedGoogle Scholar
  41. 41.
    H. Niikawa, T. Suzuki, Y. Miki, S. Suzuki, S. Nagasaki, J. Akahira, S. Honma, D. B. Evans, S. Hayashi, T. Kondo, H. Sasano, Intratumoral estrogens and estrogen receptors in human non-small cell lung carcinoma. Clin Cancer Res 14, 4417–4426 (2008)CrossRefPubMedGoogle Scholar
  42. 42.
    G. Maillot, M. Lacroix-Triki, S. Pierredon, L. Gratadou, S. Schmidt, V. Benes, H. Roche, F. Dalenc, D. Auboeuf, S. Millevoi, S. Vagner, Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth. Cancer Res 69, 8332–8340 (2009)CrossRefPubMedGoogle Scholar
  43. 43.
    X. Yu, X. Zhang, I. B. Dhakal, M. Beggs, S. Kadlubar, D. Luo, Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells. BMC Cancer 12, 29 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    E. Prodromaki, A. Korpetinou, E. Giannopoulou, E. Vlotinou, M. Chatziathanasiadou, N. I. Papachristou, C. D. scopa, H. Papadaki, H. P. Kalofonos, D. J. Papachristou, Expression of the microRNA regulators Drosha, Dicer and Ago2 in non-small cell lung carcinomas. Cell Oncol 38, 307–317 (2015)Google Scholar

Copyright information

© International Society for Cellular Oncology 2016

Authors and Affiliations

  1. 1.Section for Toxicology and Biological Working Environment, Department of Biological and Chemical Working EnvironmentNational Institute of Occupational HealthOsloNorway
  2. 2.Department of Genetics, Institute for Cancer ResearchOslo University Hospital - The Norwegian Radium HospitalOsloNorway
  3. 3.Analytical and Environmental Sciences, MRC-PHE Centre for Environment and HealthKing’s College LondonLondonUK

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