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DNA methylation in the upstream CpG island of the GPER locus and its relationship with GPER expression in colon cancer cell lines

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Abstract

The G-protein coupled estrogen receptor (GPER), a proposed tumor suppressor, relays short-term non-genomic responses in target cells and tissues. It frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. Liu and co-workers recently reported loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. We hypothesized that GPER expression is inversely correlated with methylation in the upstream CpG island (upCpGi) in the GPER locus. Methylation in the upCpGi was analysed by bisulfite sequencing and correlated with GPER expression in a panel of colon cancer cell lines. Eight downstream CpGs of the upCpGi was differentially methylated across the cell lines. Methylation in this differentially methylated region (DMR) correlated inversely with GPER expression. Two cell lines, namely SW620 and COLO-320DM, were compared in terms of their viability in response to varying concentrations of G1, a GPER specific agonist. SW-620 cells, which had the least methylated DMR and the highest level of GPER expression, showed significant loss of viability with 1 µM G1. COLO-320DM, which had the most methylated DMR and the lowest level of GPER expression, did not show a significant response to 1 µM G1. At 5 µM G1, SW620 cells showed a greater reduction in viability than COLO-320DM cells. DNA methylation in the DMR is inversely correlated with GPER expression. DNA methylation-dependent silencing of GPER may be, at least in part, the underlying reason behind the loss of estrogen’s oncoprotective effect via GPER in the colon.

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References

  1. Bray F, Ferlay J, Soerjomataram I et al (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394–424. https://doi.org/10.3322/caac.21492

    Article  PubMed  Google Scholar 

  2. Simon MS, Chlebowski RT, Wactawski-Wende J et al (2012) Estrogen plus progestin and colorectal cancer incidence and mortality. J Clin Oncol 30:3983–3990. https://doi.org/10.1200/JCO.2012.42.7732

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lin KJ, Cheung WY, Lai JY-C, Giovannucci EL (2012) The effect of estrogen vs. combined estrogen-progestogen therapy on the risk of colorectal cancer. Int J cancer 130:419–430. https://doi.org/10.1002/ijc.26026

    Article  CAS  PubMed  Google Scholar 

  4. Rossouw JE, Anderson GL, Prentice RL et al (2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333. https://doi.org/10.1001/jama.288.3.321

    Article  CAS  PubMed  Google Scholar 

  5. Quaresma M, Coleman MP, Rachet B (2015) 40-Year trends in an index of survival for all cancers combined and survival adjusted for age and sex for each cancer in England and Wales, 1971–2011: a population-based study. Lancet 385:1206–1218. https://doi.org/10.1016/S0140-6736(14)61396-9

    Article  PubMed  Google Scholar 

  6. Nilsson S, Mäkelä S, Treuter E et al (2001) Mechanisms of estrogen action. Physiol Rev 81:1535–1565. https://doi.org/10.1152/physrev.2001.81.4.1535

    Article  CAS  PubMed  Google Scholar 

  7. Hammes SR, Levin ER (2007) Extranuclear steroid receptors: nature and actions. Endocrinol Rev 28:726–741. https://doi.org/10.1210/er.2007-0022

    Article  CAS  Google Scholar 

  8. Hammes SR, Levin ER (2011) Minireview: recent advances in extranuclear steroid receptor actions. Endocrinology 152:4489–4495. https://doi.org/10.1210/en.2011-1470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lange CA, Gioeli D, Hammes SR, Marker PC (2007) Integration of rapid signaling events with steroid hormone receptor action in breast and prostate cancer. Annu Rev Physiol 69:171–199. https://doi.org/10.1146/annurev.physiol.69.031905.160319

    Article  CAS  PubMed  Google Scholar 

  10. Losel RM, Falkenstein E, Feuring M et al (2003) Nongenomic steroid action: controversies, questions, and answers. Physiol Rev 83:965–1016. https://doi.org/10.1152/physrev.00003.2003

    Article  PubMed  Google Scholar 

  11. Chaudhri RA, Schwartz N, Elbaradie K et al (2014) Role of ERα36 in membrane-associated signaling by estrogen. Steroids 81:74–80. https://doi.org/10.1016/j.steroids.2013.10.020

    Article  CAS  PubMed  Google Scholar 

  12. Sołtysik K, Czekaj P (2015) ERα36–another piece of the estrogen puzzle. Eur J Cell Biol 94:611–625. https://doi.org/10.1016/j.ejcb.2015.10.001

    Article  CAS  PubMed  Google Scholar 

  13. Xu S, Yu S, Dong D, Lee LTO (2019) G protein-coupled estrogen receptor: a potential therapeutic target in cancer. Front Endocrinol (Lausanne) 10:725. https://doi.org/10.3389/fendo.2019.00725

    Article  Google Scholar 

  14. Kennelly R, Kavanagh DO, Hogan AM, Winter DC (2008) Oestrogen and the colon: potential mechanisms for cancer prevention. Lancet Oncol 9:385–391. https://doi.org/10.1016/S1470-2045(08)70100-1

    Article  CAS  PubMed  Google Scholar 

  15. Papaxoinis K, Triantafyllou K, Sasco AJ et al (2010) Subsite-specific differences of estrogen receptor beta expression in the normal colonic epithelium: implications for carcinogenesis and colorectal cancer epidemiology. Eur J Gastroenterol Hepatol 22:614–619. https://doi.org/10.1097/MEG.0b013e328335ef50

    Article  CAS  PubMed  Google Scholar 

  16. Konstantinopoulos PA, Kominea A, Vandoros G et al (2003) Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour’s dedifferentiation. Eur J Cancer 39:1251–1258. https://doi.org/10.1016/s0959-8049(03)00239-9

    Article  CAS  PubMed  Google Scholar 

  17. Jassam N, Bell SM, Speirs V, Quirke P (2005) Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes’ staging. Oncol Rep 14:17–21

    CAS  PubMed  Google Scholar 

  18. Elbanna HG, Ebrahim MA, Abbas AM et al (2012) Potential value of estrogen receptor beta expression in colorectal carcinoma: interaction with apoptotic index. J Gastrointest Cancer 43:56–62. https://doi.org/10.1007/s12029-010-9214-4

    Article  CAS  PubMed  Google Scholar 

  19. Rudolph A, Toth C, Hoffmeister M et al (2012) Expression of oestrogen receptor β and prognosis of colorectal cancer. Br J Cancer 107:831–839. https://doi.org/10.1038/bjc.2012.323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Saleiro D, Murillo G, Benya RV et al (2012) Estrogen receptor-β protects against colitis-associated neoplasia in mice. Int J cancer 131:2553–2561. https://doi.org/10.1002/ijc.27578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Williams C, DiLeo A, Niv Y, Gustafsson J-Å (2016) Estrogen receptor beta as target for colorectal cancer prevention. Cancer Lett 372:48–56. https://doi.org/10.1016/j.canlet.2015.12.009

    Article  CAS  PubMed  Google Scholar 

  22. Prossnitz ER, Barton M (2009) Signaling, physiological functions and clinical relevance of the G protein-coupled estrogen receptor GPER. Prostaglandins Other Lipid Mediat 89:89–97. https://doi.org/10.1016/j.prostaglandins.2009.05.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Maggiolini M, Picard D (2010) The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol 204:105–114. https://doi.org/10.1677/JOE-09-0242

    Article  CAS  PubMed  Google Scholar 

  24. Smith HO, Leslie KK, Singh M et al (2007) GPR30: a novel indicator of poor survival for endometrial carcinoma. Am J Obstet Gynecol 196:386.e1-386.e11. https://doi.org/10.1016/j.ajog.2007.01.004

    Article  CAS  Google Scholar 

  25. Smith HO, Arias-Pulido H, Kuo DY et al (2009) GPR30 predicts poor survival for ovarian cancer. Gynecol Oncol 114:465–471. https://doi.org/10.1016/j.ygyno.2009.05.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Prossnitz ER, Barton M (2014) Estrogen biology: new insights into GPER function and clinical opportunities. Mol Cell Endocrinol 389:71–83. https://doi.org/10.1016/j.mce.2014.02.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gaudet HM, Cheng SB, Christensen EM, Filardo EJ (2015) The G-protein coupled estrogen receptor, GPER: the inside and inside-out story. Mol Cell Endocrinol 418 Pt 3:207–219. https://doi.org/10.1016/j.mce.2015.07.016

    Article  CAS  PubMed  Google Scholar 

  28. Barton M (2016) Not lost in translation: Emerging clinical importance of the G protein-coupled estrogen receptor GPER. Steroids 111:37–45. https://doi.org/10.1016/j.steroids.2016.02.016

    Article  CAS  PubMed  Google Scholar 

  29. Jacenik D, Beswick EJ, Krajewska WM, Prossnitz ER (2019) G protein-coupled estrogen receptor in colon function, immune regulation and carcinogenesis. World J Gastroenterol 25:4092–4104. https://doi.org/10.3748/wjg.v25.i30.4092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu Q, Chen Z, Jiang G et al (2017) Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer. Mol Cancer 16:87. https://doi.org/10.1186/s12943-017-0654-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bustos V, Nolan ÁM, Nijhuis A et al (2017) GPER mediates differential effects of estrogen on colon cancer cell proliferation and migration under normoxic and hypoxic conditions. Oncotarget 8:84258–84275. https://doi.org/10.18632/oncotarget.20653

    Article  PubMed  PubMed Central  Google Scholar 

  32. Weißenborn C, Ignatov T, Poehlmann A et al (2014) GPER functions as a tumor suppressor in MCF-7 and SK-BR-3 breast cancer cells. J Cancer Res Clin Oncol 140:663–671. https://doi.org/10.1007/s00432-014-1598-2

    Article  CAS  PubMed  Google Scholar 

  33. Weissenborn C, Ignatov T, Nass N et al (2017) GPER promoter methylation controls GPER expression in breast cancer patients. Cancer Invest 35:100–107. https://doi.org/10.1080/07357907.2016.1271886

    Article  CAS  PubMed  Google Scholar 

  34. Manjegowda MC, Gupta PS, Limaye AM (2017) Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells. Gene 614:65–73. https://doi.org/10.1016/j.gene.2017.03.006

    Article  CAS  PubMed  Google Scholar 

  35. Manjegowda MC, Gupta PS, Limaye AM (2016) Validation data of a rabbit antiserum and affinity purified polyclonal antibody against the N-terminus of human GPR30. Data Br 7:1015–1020. https://doi.org/10.1016/j.dib.2016.03.054

    Article  Google Scholar 

  36. LOWRY OH, ROSEBROUGH NJ, RANDALL FARRAL RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  Google Scholar 

  37. Manjegowda MC, Deb G, Limaye AM (2014) Epigallocatechin gallate induces the steady state mRNA levels of pS2 and PR genes in MCF-7 breast cancer cell. Indian J Exp Biol 52:312–316

    CAS  PubMed  Google Scholar 

  38. John Mary DJS, Sikarwar G, Kumar A, Limaye AM (2020) Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of cystatin A in breast cancer cells. Mol Cell Endocrinol 504:110701. https://doi.org/10.1016/j.mce.2020.110701

    Article  CAS  PubMed  Google Scholar 

  39. Santolla MF, Lappano R, De Marco P et al (2012) G protein-coupled estrogen receptor mediates the up-regulation of fatty acid synthase induced by 17β-estradiol in cancer cells and cancer-associated fibroblasts. J Biol Chem 287:43234–43245. https://doi.org/10.1074/jbc.M112.417303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gilligan LC, Gondal A, Tang V et al (2017) Estrone sulfate transport and steroid sulfatase activity in colorectal cancer: implications for hormone replacement therapy. Front Pharmacol 8:103. https://doi.org/10.3389/fphar.2017.00103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gilligan LC, Rahman HP, Hewitt A-M et al (2017) Estrogen activation by steroid sulfatase increases colorectal cancer proliferation via GPER. J Clin Endocrinol Metab 102:4435–4447. https://doi.org/10.1210/jc.2016-3716

    Article  PubMed  PubMed Central  Google Scholar 

  42. Weißenborn C, Ignatov T, Ochel H-J et al (2014) GPER functions as a tumor suppressor in triple-negative breast cancer cells. J Cancer Res Clin Oncol 140:713–723. https://doi.org/10.1007/s00432-014-1620-8

    Article  CAS  PubMed  Google Scholar 

  43. Lv Q-Y, Xie B-Y, Yang B-Y et al (2017) Increased TET1 expression in inflammatory microenvironment of hyperinsulinemia enhances the response of endometrial cancer to estrogen by epigenetic modulation of GPER. J Cancer 8:894–902. https://doi.org/10.7150/jca.17064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The work was supported by financial assistance from the Indian Council of Medical Research, Govt. of India (Sanction letter No. 2019 − 0687/CMB/ADHOC-BMS, dated 13.11.2019). Infrastructural support from the Department of Biosciences and Bioengineering at IIT Guwahati is acknowledged. SG was a student of the M.Tech program supported by the Department of Biotechnology, Govt. of India, which also provided her fellowship.

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  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Uttariya Pal, Sujasha Ghosh & Anil Mukund Limaye

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  1. Uttariya Pal
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UP and AML designed the experiments. UP and SG performed the experiments. UP and AML analysed the results. UP, SG, and AML wrote the manuscript.

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Correspondence to Anil Mukund Limaye.

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Pal, U., Ghosh, S. & Limaye, A.M. DNA methylation in the upstream CpG island of the GPER locus and its relationship with GPER expression in colon cancer cell lines. Mol Biol Rep 47, 7547–7555 (2020). https://doi.org/10.1007/s11033-020-05817-5

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