oncotomskСибирский онкологический журналSiberian journal of oncology1814-48612312-3168Tomsk National Research Medical Сепtеr of the Russian Academy of Sciences10.21294/1814-4861-2019-18-5-45-53oncotomsk-1187Research ArticleЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯLABORATORY AND EXPERIMENTAL STUDIESИЗМЕНЕНИЕ ПРОФИЛЯ МЕТИЛИРОВАНИЯ ДНК В ТАМОКСИФЕН-РЕЗИСТЕНТНЫХ СУБЛИНИЯХ КЛЕТОК MCF-7CHANGES IN DNA METHYLATION PROFILE IN TAMOXIFEN-RESISTANT MCF-7 SUBLINEShttps://orcid.org/0000-0002-6015-6619АндрееваО. Е.AndreevaО. Е.

Андреева Ольга Евгеньевна, кандидат биологических наук, научный сотрудник

г. Москва, 115478, Каширское шоссе, 23

SPIN-код: 3607-9431

AuthorID (РИНЦ): 745734

Researcher ID (WOS): K-1304-2015

Author ID (Scopus): 37015258000

Olga E. Andreeva, PhD

24, Kashirskoye Shosse, Moscow, 115478

Researcher ID (WOS): K-1304-2015

Author ID (Scopus): 37015258000

tilberta@gmail.comСигинВ. О.SiginV. О.

Сигин Владимир Олегович, младший научный сотрудник

г. Москва, 115522, ул. Москворечье, 1

SPIN-код: 9156-3209

AuthorID (РИНЦ): 974871

Vladimir O. Sigin, Junior Researcher

1, Moskvorechye St., Moscow, 115522, 

1, Ostrovityanov St., Moscow, 117997

sigin.vladimir@gmail.comhttps://orcid.org/0000-0001-9283-902XСтрельниковВ. В.StrelnikovV. V.

Стрельников Владимир Викторович, доктор биологических наук, доцент, Медико-генетический научный центр, Российский национальный исследовательский медицинский университет им. Н.И. Пирогова Министерства здравоохранения Российской Федерации

г. Москва, 115522, ул. Москворечье, 1, 

г. Москва, 117997, ул. Островитянова, 1

SPIN-код: 9118-7267

AuthorID (РИНЦ): 97849

Researcher ID (WOS): D-1576-2012

Author ID (Scopus): 6603557133

Vladimir V. Strelnikov, PhD, DSc, Research Centre for Medical Genetics, Pirogov Russian National Research Medical University

1, Moskvorechye St., Moscow, 115522, 

1, Ostrovityanov St., Moscow, 117997

Researcher ID (WOS): D-1576-2012

Author ID (Scopus): 6603557133

vstrel@yandex.ruhttps://orcid.org/0000-0002-2177-6743ТанасА. С.TanasA. S.

Танас Александр Сергеевич, кандидат биологических наук, ведущий научный сотрудник, Медико-генетический научный центр, Российский национальный исследовательский медицинский университет им. Н.И. Пирогова Министерства здравоохранения Российской Федерации

г. Москва, 115522, ул. Москворечье, 1, 

г. Москва, 117997, ул. Островитянова, 1

SPIN-код: 2947-7067

AuthorID (РИНЦ): 619620

Researcher ID (WOS): D-2145-2012

Author ID (Scopus): 36006579000

Alexander S. Tanas, PhD, Senior Researcher, Research Centre for Medical Genetics, Pirogov Russian National Research Medical University

1, Moskvorechye St., Moscow, 115522, 

1, Ostrovityanov St., Moscow, 117997

Researcher ID (WOS): D-2145-2012

Author ID (Scopus): 36006579000

https://orcid.org/0000-0002-2974-9555ЩербаковА. М.ScherbakovA. M.

Щербаков Александр Михайлович, кандидат биологических наук, ведущий научный сотрудник

г. Москва, 115478, Каширское шоссе, 23

SPIN-код: 9526-0047

AuthorID (РИНЦ): 136087

Researcher ID (WOS): F-4914-2013

Author ID (Scopus): 7003636718

Alexander M. Scherbakov, PhD, Senior Researcher

24, Kashirskoye Shosse, Moscow, 115478

Researcher ID (WOS): F-4914-2013

Author ID (Scopus): 7003636718

alex.scherbakov@gmail.comhttps://orcid.org/0000-0002-5902-7633КрасильниковМ. А.KrasilnikovM. A.

Красильников Михаил Александрович, доктор биологических наук, профессор

г. Москва, 115478, Каширское шоссе, 23

SPIN-код: 3881-0919

AuthorID (РИНЦ): 80070

Researcher ID (WOS): R-6938-2019

Author ID (Scopus): 7005790120

Mikhail A. Krasilnikov, PhD, DSc, Professor

24, Kashirskoye Shosse, Moscow, 115478

Researcher ID (WOS): R-6938-2019

Author ID (Scopus): 7005790120

ФГБУ «Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Министерства здравоохранения Российской ФедерацииРоссияN.N. Blokhin National Medical Research Center of OncologyRussian FederationФГБНУ «Медико-генетический научный центр»РоссияResearch Centre for Medical GeneticsRussian FederationФГБНУ «Медико-генетический научный центр»; Российский национальный исследовательский медицинский университет им. Н.И. Пирогова Министерства здравоохранения Российской ФедерацииРоссияResearch Centre for Medical Genetics; Pirogov Russian National Research Medical UniversityRussian Federation2019311020191854553Copyright © Андреева О.Е., Сигин В.О., Стрельников В.В., Танас А.С., Щербаков А.М., Красильников М.А., 20192019Андреева О.Е., Сигин В.О., Стрельников В.В., Танас А.С., Щербаков А.М., Красильников М.А.Andreeva О.Е., Sigin V.О., Strelnikov V.V., Tanas A.S., Scherbakov A.M., Krasilnikov M.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.siboncoj.ru/jour/article/view/1187Введение

Введение. Ранее мы показали возможность горизонтального распространения гормональной резистентности от клетки к клетке при совместном культивировании чувствительных и резистентных клеток и/или через экзосомы, секретируемые резистентными клетками. Каков механизм подобного распространения резистентности, и в какой мере клетки со вторичной резистентностью воспроизводят характеристики донорских резистентных клеток – для ответа на эти вопросы был проведен анализ общего уровня метилирования ДНК в клетках эстрогензависимого рака молочной железы MCF-7 и эстрогеннезависимых сублиниях.

Цель исследования – изучение профиля метилирования ДНК при развитии гормональной резистентности и его значения в закреплении резистентного фенотипа клеток рака молочной железы.

Методы

Методы. Метилирование ДНК исследовали методом RRBS (Reduced Representation Bisulfite Sequencing) в клетках рака молочной железы MCF-7 и резистентных сублиниях.

Результаты

Результаты. Выявлено 19 динуклеотидов CpG, дифференциально и в целом однонаправленно метилированных в клетках c первичной и вторичной резистентностью к тамоксифену. Дифференциальное изменение метилирования было обнаружено для участков ДНК, регулирующих экспрессию шести белок-кодирующих генов: PRKCZ, TRAPPC9, AS IC2, C2CD4a, ZNF787, CRTAC 1. Проведенный биоинформатический анализ показал, что два из этих шести генов, PRKCZ (protein kinase С Zeta) и TRAPPC9 (Trafficking Protein Particle Complex Subunit 9), напрямую вовлечены в регуляцию активности NF-κB.

Заключение

Заключение. Полученные данные свидетельствуют о существовании общих паттернов ДНК, метилирование которых изменяется в одном направлении в клетках с первичной и вторичной резистентностью. Участие двух из идентифицированных генов в регуляции NF-κB может свидетельствовать о включении последнего в формирование резистентного фенотипа опухолевых клеток, в том числе в условиях горизонтального переноса резистентности.

Abstract Introduction

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

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

Results

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

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.

рак молочной железытамоксифенэкзосомыгормональная резистентностьметилированиеbreast cancertamoxifenexosomeshormonal resistancemethylationРабота финансировалась из средств гранта Российского научного фонда № 17-75-10212 (Анализ метилирования ДНК) и гранта Российского фонда фундаментальных исследований № 18-29-09016\18 (Получение и исследование активности препаратов экзосом)The study was supported by Russian Scientific Foundation No. 17-75-10212 (the analysis of DNA methylation) and by RFBR (Russian Foundation for Basic Research) No. 18-29-09016\18 (isolation and study of exosomes)ReferencesFerlay 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.Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D.M., Pineros M., Znaor A., Bray F. 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The authors declare that there are no conflicts of interest present.