Феномен подавления ДНК-метилтрансферазы 3А при формировании резистентного фенотипа в клетках рака молочной железы

Введение. Переключение сигнальных путей и активация параллельных сигнальных каскадов относятся к ключевым факторам, определяющим развитие резистентности опухолевых клеток, и изучение механизмов подобной реаранжировки является одной из актуальных задач современной онкологии.

Материалы и методы. Настоящая работа выполнена на ERα-положительных  (ERα – эстрогеновый рецептор α) клетках рака молочной железы MCF-7 и полученных из них сублиниях, устойчивых к ингибитору mTOR рапамицину или антиэстрогену тамоксифену, а также на ERα-отрицательных клетках рака молочной железы MDA-MB-231. Используемые методы включают тест МТТ, транзиторную трансфекцию, иммуноблоттинг, полимеразную цепную реакцию в реальном времени и анализ метилирования с помощью бисульфитного пиросеквенирования.

Результаты. Мы показали, что резистентность клеток рака молочной железы к таргетным и гормональным препаратам связана с подавлением ДНК-метилтрансферазы 3А (DNMT3A) и соответствующими изменениями в метилировании ДНК. Нокдаун DNMT3A приводит к частичной резистентности к обоим препаратам, что демонстрирует ключевую роль подавления DNMT3A в развитии резистентности опухолевых клеток.

Заключение. В целом, полученные результаты свидетельствуют о возможном механизме формирования устойчивости опухолевых клеток к таргетным/гормональным препаратам, основанном на дерегуляции экспрессии DNMT, и демонстрируют прямую связь между подавлением DNMT3A и развитием резистентности.

1. Mansoori B., Mohammadi A., Davudian S. et al. The Different mechanisms of cancer drug resistance: a brief review. Adv Pharm Bull 2017;7(3):339–48. DOI: 10.15171/apb.2017.041

2. Aleksakhina S.N., Kashyap A., Imyanitov E.N. Mechanisms of acquired tumor drug resistance. Biochim Biophys Acta Rev Cancer 2019;1872(2):188310. DOI: 10.1016/j.bbcan.2019.188310

3. Cree I.A., Charlton P. Molecular chess? Hallmarks of anti-cancer drug resistance. BMC Cancer 2017;17(1):10. DOI: 10.1186/s12885-016-2999-1

4. Konjevic G., Mirjacic Martinovic K., Vuletic A. et al. Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients. Clin Exp Metastasis 2007;24(1):1–11. DOI: https://doi.org/10.1007/s10585-006-9043-9

5. Scherbakov A.M., Krasil’nikov M.A., Kushlinskii N.E. Molecular mechanisms of hormone resistance of breast cancer. Bull Exp Biol Med 2013;155(3):384–95. DOI: https://doi.org/10.1007/s10517-013-2160-y

6. Clarke R., Tyson J.J., Dixon J.M. Endocrine resistance in breast cancer – an overview and update. Mol Cell Endocrinol 2015;418 Pt. 3(03):220–34. DOI: https://doi.org/10.1016/j.mce.2015.09.035

7. Araki K., Miyoshi Y. Mechanism of resistance to endocrine therapy in breast cancer: the important role of PI3K/Akt/mTOR in estrogen receptor-positive, HER2-negative breast cancer. Breast Cancer 2018;25(4)392–401. DOI: https://doi.org/10.1007/s12282-017-0812-x

8. Citi V., Del Re M., Martelli A. et al. Phosphorylation of AKT and ERK1/2 and mutations of PIK3CA and PTEN are predictive of breast cancer cell sensitivity to everolimus in vitro. Cancer Chem Pharmacol 2018;81(4):745–54. DOI: https://doi.org/10.1007/s00280-018-3543-6

9. Xie W., Sun H., Li X. et al. Ovarian cancer: epigenetics, drug resistance, and progression. Cancer Cell Int 2021;21(1):1–16. DOI: https://doi.org/10.1186/s12935-021-02136-y

10. Hazra A., Bose P., Sunita P. et al. Molecular epigenetic dynamics in breast carcinogenesis. Arch Pharm Res 2021;44(8):741–63. DOI: https://doi.org/10.1007/s12272-021-01348-0

11. Andreeva O.E., Shchegolev Y.Y., Scherbakov A.M. et al. The phenomenon of the cross-resistance of breast cancer to target and hormonal drugs: the role of epigenetic reconstruction. Med Sci Forum 2023;20(1):5. DOI: https://doi.org/10.3390/IECC2023-14220

12. Vinokurova S. Epigenetics of virus-induced tumors: perspectives for therapeutic targeting. Curr Pharm Des 2017;23(32):4842–61. DOI: https://doi.org/10.2174/1381612823666170822100627

13. Ariazi E.A., Taylor J.C., Black M.A. et al. A new role for ERalpha: silencing via DNA methylation of basal, stem cell, and emt genes. Mol Cancer Res 2017;15(2):152–64. DOI: https://doi.org/10.1158/1541-7786.MCR-16-0283

14. Lin X., Li J., Yin G. et al. 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;15(6):R119. DOI: https://doi.org/10.1186/bcr3588

15. Jin B., Robertson K.D. DNA methyltransferases, DNA damage repair, and cancer. Adv Exp Med Biol 2013;754:3–29. DOI: https://doi.org/10.1007/978-1-4419-9967-2_1

16. Moore L.D., Le T., Fan G. DNA methylation and its basic function. Neuropsychopharmacology 2013;38(1):23–38. DOI: https://doi.org/10.1038/npp.2012.112

17. Jahangiri R., Mosaffa F., Emami Razavi A. et al. Altered DNA methyltransferases promoter methylation and mRNA expression are associated with tamoxifen response in breast tumors. J Cell Physiol 2018;233(9):7305–19. DOI: https://doi.org/10.1002/jcp.26562

18. He D., Wang X., Zhang Y. et al. DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of The Cancer Genome Atlas. Chin Med J (Engl) 2019;132(2): 161–70. DOI: https://doi.org/10.1097/CM9.0000000000000054

19. Chen B.F., Chan W.Y. The de novo DNA methyltransferase DNMT3A in development and cancer. Epigenetics 2014;9(5):669–77. DOI: https://doi.org/10.4161/epi.28324

20. Shchegolev Y.Y., Sorokin D.V., Scherbakov A.M. et al. Exosomes are involved in the intercellular transfer of rapamycin resistance in the breast cancer cells. Bioimpacts 2023;13(4):313–21. DOI: https://doi.org/10.34172/bi.2023.27490

21. Scherbakov A.M., Sorokin D.V., Tatarskiy V.V.Jr. et al. The phenomenon of acquired resistance to metformin in breast cancer cells: the interaction of growth pathways and estrogen receptor signaling. IUBMB life 2016;68(4):281–92. DOI: https://doi.org/10.1002/iub.1481

22. Semina S.E., Scherbakov A.M., Kovalev S.V. et al. Horizontal transfer of tamoxifen resistance in MCF-7 cell derivates: proteome study. Cancer Invest 2017;35(8):506–18. DOI: https://doi.org/10.1080/07357907.2017.1368081

23. Iselt M., Holtei W., Hilgard P. The tetrazolium dye assay for rapid in vitro assessment of cytotoxicity. Arzneimittel-Forschung 1989;39(7):747–9.

24. Scherbakov A.M., Vorontsova S.K., Khamidullina A.I. et al. Novel pentacyclic derivatives and benzylidenes of the progesterone series cause anti-estrogenic and antiproliferative effects and induce apoptosis in breast cancer cells. Invest New Drugs 2023;41(1): 142–52. DOI: https://doi.org/10.1007/s10637-023-01332-z

25. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods (San Diego, Calif) 2001;25(4):402–8. DOI: https://doi.org/10.1006/meth.2001.1262

26. Scherbakov A.M., Komkov A.V., Komendantova A.S. et al. Steroidal pyrimidines and dihydrotriazines as novel classes of anticancer agents against hormone-dependent breast cancer cells. Front Pharmacol 2017;8:979. DOI: https://doi.org/10.3389/fphar.2017.00979

27. Scherbakov A.M., Lobanova Y.S., Shatskaya V.A. et al. Activation of mitogenic pathways and sensitization to estrogen-induced apoptosis: two independent characteristics of tamoxifen-resistant breast cancer cells? Breast Cancer Res Treat 2006;100(1):1–11. DOI: https://doi.org/10.1007/s10549-005-9075-x

28. Mruk D.D., Cheng C.Y. Enhanced chemiluminescence (ECL) for routine immunoblotting: an inexpensive alternative to commercially available kits. Spermatogenesis 2011;1(2):121–2. DOI: https://doi.org/10.4161/spmg.1.2.16606

29. Taylor S.C., Berkelman T., Yadav G. et al. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol 2013;55(3):217–26. DOI: https://doi.org/10.1007/s12033-013-9672-6 PMID: 23709336

30. Lander E.S., Linton L.M., Birren B. et al. Initial sequencing and analysis of the human genome. Nature 2001;409(6822):860–921. DOI: 10.1038/35057062

31. Yang A.S., Estecio M.R., Doshi K. et al. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004;32(3):e38. DOI: https://doi.org/10.1093/nar/gnh032

32. Dong C., Wu J., Chen Y. et al. Activation of PI3K/AKT/mTOR pathway causes drug resistance in breast cancer. Front Pharmacol 2021;12:628690. DOI: https://doi.org/10.3389/fphar.2021

33. Liu R., Chen Y., Liu G. et al. PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell Death Dis 2020;11(9):797. DOI: https://doi.org/10.1038/s41419-020-02998-6

34. Scherbakov A.M., Basharina A.A., Sorokin D.V. et al. Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance. Cancer Drug Resist 2023;6(1):103–15. DOI: https://doi.org/10.20517/cdr.2022.96

35. Meng D., Li Z., Ma X. et al. MicroRNA-1280 modulates cell growth and invasion of thyroid carcinoma through targeting estrogen receptor alpha. Cell Mol Biol (Noisy-le-grand) 2016;62 (3):1–6.

36. Xu C.G., Yang M.F., Ren Y.Q. et al. Exosomes mediated transfer of lncRNA UCA1 results in increased tamoxifen resistance in breast cancer cells. Eur Rev Med Pharmacol Sci 2016;20(20):4362–8.

37. Romero-Garcia S., Prado-Garcia H., Carlos-Reyes A. Role of DNA methylation in the resistance to therapy in solid tumors. Front Oncol 2020;10:1152. DOI: https://doi.org/10.3389/fonc.2020.01152

38. Nayak S.R., Harrington E., Boone D. et al. A role for histone H2B variants in endocrine-resistant breast cancer. Horm Cancer 2015;6(5–6):214–24. DOI: https://doi.org/10.1007/s12672-015-0230-5

39. Hamadneh L., Al-Lakkis L., Alhusban A.A. et al. Changes in Lactate production, lactate dehydrogenase genes expression and DNA methylation in response to tamoxifen resistance development in MCF-7 cell line. Genes (Basel) 2021;12(5). DOI: https://doi.org/10.3390/genes12050777

40. Андреева О.Е., Сигин В.О., Стрельников В.В. и др. Изменение профиля метилирования ДНК в тамоксифен-резистентных сублиниях клеток MCF-7. Сибирский онкологический журнал 2019;18(5):45–53. DOI: https://doi.org/10.21294/1814-4861-2019-18-5-45-53