CHANGES IN DNA METHYLATION PROFILE IN TAMOXIFEN-RESISTANT MCF-7 SUBLINES

Abstract Introduction. We have previously shown the feasibility of hormonal resistance horizontal distribution from cell to cell, with the joint cultivation of sensitive and resistant cells and/or through exosomes secreted by resistant cells. What is the mechanism of such resistance distribution, and how do cells with secondary resistance reproduce the characteristics of donor resistant cells? To answer these questions, we analyzed the overall level of DNA methylation in MCF-7 estrogen-dependent breast cancer cells and estrogen-independent sublinia.

The purpose of the study was to analyze DNA methylation profiles for the development of hormonal resistance by breast cancer cells and for resistant phenotype further accession.

Methods. DNA methylation was evaluated by the RRBS (Reduced Representation Bisulfite Sequencing) method in MCF-7 breast cancer cells and their resistant sublines.

Results. 19 CpG dinucleotides, differentially and generally unidirectionally methylated in cells with primary and secondary resistance to tamoxifen, were detected. Differential changes in methylation were found for DNA regions that regulated the expression of six protein-coding genes: PRKCZ, TRAPPC9, AS IC2, C2CD4A, ZNF787, CRTAC 1. Bioinformatics analysis showed that two of these six genes, PRKCZ (protein kinase C Zeta) and TRAPPC9 (Trafficking Protein Particle Complex Subunit 9) were directly involved in the regulation of NF-κB activity.

Conclusion. The data obtained indicate the existence of common DNA patterns, the methylation of which varies in the same direction in cells with primary and secondary resistance. The involvement of two of the identified genes in the regulation of NF-κB may indicate the inclusion of the latter in the formation of a resistant phenotype of tumor cells, even under conditions of horizontal transfer of resistance.

1. Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D.M., Pineros M., Znaor A., Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019 Apr 15; 144(8): 1941–1953. doi: 10.1002/ijc.31937.

2. Meric-Bernstam F. Heterogenic loss of BRCA in breast cancer: the «two-hit» hypothesis takes a hit. Ann Surg Oncol. 2007. 14(9): 2428–2429. doi: 10.1245/s10434-007-9379-7.

3. Di Cosimo S., Baselga J. Management of breast cancer with targeted agents: importance of heterogeneity. [corrected]. Nat Rev Clin Oncol. 2010 Mar; 7(3): 139–47. doi: 10.1038/nrclinonc.2009.234.

4. Sachs N., de Ligt J., Kopper O., Gogola E., Bounova G., Weeber F., Balgobind A.V., Wind K., Gracanin A., Begthel H., Korving J., van Boxtel R., Duarte A.A., Lelieveld D., van Hoeck A., Ernst R.F., Blokzijl F., Nijman I.J., Hoogstraat M., van de Ven M., Egan D.A., Zinzalla V., Moll J., Boj S.F., Voest E.E., Wessels L., van Diest P.J., Rottenberg S., Vries R.G.J., Cuppen E., Clevers H. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell. 2018 Jan 11; 172(1–2): 373–386.e10. doi: 10.1016/j.cell.2017.11.010.

5. Keller R.R., Gunther E.J. Evolution of Relapse-Proficient Subclones Constrained by Collateral Sensitivity to Oncogene Overdose in Wnt-Driven Mammary Cancer. Cell Rep. 2019 Jan 22; 26(4): 893–905. e4. doi: 10.1016/j.celrep.2018.12.096.

6. Condorelli R., Spring L., O’Shaughnessy J., Lacroix L., Bailleux C., Scott V., Dubois J., Nagy R.J., Lanman R.B., Iafrate A.J., Andre F., Bardia A. Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer. Ann Oncol. 2018 Mar 1; 29(3): 640–645. doi: 10.1093/annonc/mdx784.

7. Feinberg A.P., Ohlsson R., Henikoff S. The epigenetic progenitor origin of human cancer. Nature reviews. Genetics. 2006. 7(1): 21–33. doi: 10.1038/nrg1748.

8. Klinge C.M. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res. 2001. 29(14): 2905–2919. doi: 10.1093/nar/29.14.2905.

9. Safe S., Kim K. Non-classical genomic estrogen receptor (ER)/ specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol. 2008 Nov; 41(5): 263–75. doi: 10.1677/JME-08-0103.

10. Schiff R., Reddy P., Ahotupa M., Coronado-Heinsohn E., Grim M., Hilsenbeck S.G., Lawrence R., Deneke S., Herrera R., Chamness G.C., Fuqua S.A., Brown P.H., Osborne C.K. Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo. J Natl Cancer Inst. 2000 Dec 6; 92(23): 1926–34. doi: 10.1093/jnci/92.23.1926.

11. Zhou Y., Eppenberger-Castori S., Eppenberger U., Benz C.C. The NFkappaB pathway and endocrine-resistant breast cancer. Endocr Relat Cancer. 2005 Jul; 12 Suppl 1: S37–46. doi: 10.1677/erc.1.00977.

12. Ji Z., He L., Regev A., Struhl K. Inflammatory regulatory network mediated by the joint action of NF-kB, STAT3, and AP-1 factors is involved in many human cancers. Proc Natl Acad Sci USA. 2019 May 7; 116(19): 9453–9462. doi: 10.1073/pnas.1821068116.

13. Scherbakov A.M., Andreeva O.E., Shatskaya V.A., Krasil’nikov M.A. The relationships between snail1 and estrogen receptor signaling in breast cancer cells. J Cell Biochem. 2012 Jun; 113(6): 2147–55. doi: 10.1002/jcb.24087.

14. Stone A., Zotenko E., Locke W.J., Korbie D., Millar E.K., Pidsley R., Stirzaker C., Graham P., Trau M., Musgrove E.A., Nicholson R.I., Gee J.M., Clark S.J. DNA methylation of oestrogen-regulated enhancers defines endocrine sensitivity in breast cancer. Nat Commun. 2015 Jul 14; 6: 7758. doi: 10.1038/ncomms8758.

15. Trimarchi M.P., Mouangsavanh M., Huang T.H. Cancer epigenetics: a perspective on the role of DNA methylation in acquired endocrine resistance. Chin J Cancer. 2011 Nov; 30(11): 749–56. doi: 10.5732/cjc.011.10128.

16. Semina S.E., Scherbakov A.M., Vnukova A.A., Bagrov D.V., Evtushenko E.G., Safronova V.M., Golovina D.A., Lyubchenko L.N., Gudkova M.V., Krasil’nikov M.A. Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs. Molecules. 2018; 23(4). pii: E829. doi: 10.3390/molecules23040829.

17. Tanas A.S., Borisova M.E., Kuznetsova E.B., Rudenko V.V., Karandasheva K.O., Nemtsova M.V., Izhevskaya V.L., Simonova O.A., Larin S.S., Zaletaev D.V., Strelnikov V.V. Rapid and affordable genome-wide bisulfite DNA sequencing by XmaI-reduced representation bisulfite sequencing. Epigenomics. 2017; 9(6): 833–847. doi: 10.2217/epi-2017-0031.

18. Tanas A.S., Sigin V.O., Kalinkin A.I., Litviakov N.V., Slonimskaya E.M., Ibragimova M.K., Ignatova E.O., Simonova O.A., Kuznetsova E.B., Kekeeva T.V., Larin S.S., Poddubskaya E.V., Trotsenko I.D., Rudenko V.V., Karandasheva K.O., Petrova K.D., Tsyganov M.M., Deryusheva I.V., Kazantseva P.V., Doroshenko A.V., Tarabanovskaya N.A., Chesnokova G.G., Sekacheva M.I., Nemtsova M.V., Izhevskaya V.L., Kutsev S.I., Zaletaev D.V., Strelnikov V.V. Genome-wide methylotyping resolves breast cancer epigenetic heterogeneity and suggests novel therapeutic perspectives. Epigenomics. 2019 May; 11(6): 605–617. doi: 10.2217/epi-2018-0213.

19. Krueger F., Andrews S.R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011. 27(11): 1571–1572. doi: 10.1093/bioinformatics/btr167.

20. Semina S.E., Scherbakov A.M., Kovalev S.V., Shevchenko V.E., Krasil’nikov M.A. Horizontal Transfer of Tamoxifen Resistance in MCF-7 Cell Derivates: Proteome Study. Cancer Invest. 2017 Sep 14; 35(8): 506–518. doi: 10.1080/07357907.2017.1368081.

21. Orr B.O., Gorczyca D., Younger M.A., Jan L.Y., Jan Y.N., Davis G.W. Composition and Control of a Deg/ENaC Channel during Presynaptic Homeostatic Plasticity. Cell Rep. 2017 Aug 22; 20(8): 1855–1866. doi: 10.1016/j.celrep.2017.07.074.

22. Walz W. pH shifts evoked by neuronal stimulation in slices of rat hippocampus. Canad J Physiol Pharmacol. 1989; 67(6): 577–581. doi: 10.1139/y89-092.

23. Zhou Z.H., Song J.W., Li W., Liu X., Cao L., Wan L.M., Tan Y.X., Ji S.P., Liang Y.M., Gong F. The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis. J Exp Clin Cancer Res. 2017 Sep 19; 36(1): 130. doi: 10.1186/s13046-017-0599-9.

24. Hu W.H., Pendergast J.S., Mo X.M., Brambilla R., Bracchi-Ricard V., Li F., Walters W.M., Blits B., He L., Schaal S.M., Bethea J.R. NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation. J Biol Chem. 2005; 280(32): 29233–29241. 10.1074/jbc.M501670200.

25. Zhang Y., Liu S., Wang H., Yang W., Li F., Yang F., Yu D., Ramsey F.V., Tuszyski G.P., Hu W. Elevated NIBP/TRAPPC9 mediates tumorigenesis of cancer cells through NFkappaB signaling. Oncotarget. 2015. 6(8): 6160–6178. doi: 10.18632/oncotarget.3349.

26. Qin M., Zhang J., Xu C., Peng P., Tan L., Liu S., Huang J. Knockdown of NIK and IKKbeta-Binding Protein (NIBP) Reduces Colorectal Cancer Metastasis through Down-Regulation of the Canonical NF-kappaBeta Signaling Pathway and Suppression of MAPK Signaling Mediated through ERK and JNK. PLoS One. 2017 Jan 26; 12(1): e0170595. doi: 10.1371/journal.pone.0170595.

27. Fu Z.H., Liu S.Q., Qin M.B., Huang J.A., Xu C.Y., Wu W.H., Zhu L.Y., Qin N., Lai M.Y. NIK and IKKbetabinding protein contributes to gastric cancer chemoresistance by promoting epithelialmesenchymal transition through the NFkappaB signaling pathway. Oncol Rep. 2018 Jun; 39(6): 2721–2730. doi: 10.3892/or.2018.6348.

28. Ariazi E.A., Taylor J.C., Black M.A., Nicolas E., Slifker M.J., Azzam D.J., Boyd J. A New Role for ERalpha: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes. Mol Cancer Res. 2017 Feb; 15(2): 152–164. doi: 10.1158/1541-7786.MCR-16-0283.

29. Lin X., Li J., Yin G., Zhao Q., Elias D., Lykkesfeldt A.E., Stenvang J., Brunner N., Wang J., Yang H., Bolund L., Ditzel H.J. Integrative analyses of gene expression and DNA methylation profiles in breast cancer cell line models of tamoxifen-resistance indicate a potential role of cells with stem-like properties. Breast Cancer Res. 2013 Dec 19; 15(6): R119. doi: 10.1186/bcr3588.

30. Ding L., Ni J., Yang F., Huang L., Deng H., Wu Y., Ding X., Tang J. Promising therapeutic role of miR-27b in tumor. Tumour Biol. 2017 Mar; 39(3): 1010428317691657. doi: 10.1177/1010428317691657.

31. Zhuang L., Qu H., Cong J., Dai H., Liu X. MiR-181c affects estrogen-dependent endometrial carcinoma cell growth by targeting PTEN. Endocr J. 2019 Jun 28; 66(6): 523–533. doi: 10.1507/endocrj.EJ18-0538.

32. Chanyshev M.D., Razumova Y.V., Ovchinnikov V.Y., Gulyaeva L.F. MiR-21 regulates the ACAT1 gene in MCF-7 cells. Life Sci. 2018 Sep 15; 209: 173–178. doi: 10.1016/j.lfs.2018.08.010.

33. Sachdeva M., Wu H., Ru P., Hwang L., Trieu V., Mo Y.Y. MicroRNA101-mediated Akt activation and estrogen-independent growth. Oncogene. 2011 Feb 17; 30(7): 822–31. doi: 10.1038/onc.2010.463.

34. Miller P.C., Clarke J., Koru-Sengul T., Brinkman J., El-Ashry D. A novel MAPK-microRNA signature is predictive of hormone-therapy resistance and poor outcome in ER-positive breast cancer. Clin Cancer Res. 2015 Jan 15; 21(2): 373–85. doi: 10.1158/1078-0432.CCR-14-2053.

35. Egeland N.G., Lunde S., Jonsdottir K., Lende T.H., Cronin-Fenton D., Gilje B., Janssen E.A., Soiland H. The Role of MicroRNAs as Predictors of Response to Tamoxifen Treatment in Breast Cancer Patients. Int J Mol Sci. 2015 Oct 14; 16(10): 24243–75. doi: 10.3390/ijms161024243.

36. Li X., Wu Y., Liu A., Tang X. MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3. Biochem Biophys Res Commun. 2016 Sep 2; 477(4): 768–773. doi: 10.1016/j.bbrc.2016.06.133.

37. Hysolli E., Tanaka Y., Su J., Kim K.Y., Zhong T., Janknecht R., Zhou X.L., Geng L., Qiu C., Pan X., Jung Y.W., Cheng J., Lu J., Zhong M., Weissman S.M., Park I.H. Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family. Stem Cell Reports. 2016 Jul 12; 7(1): 43–54. doi: 10.1016/j.stemcr.2016.05.014.