Advances in Molecular OncologyAdvances in Molecular OncologyУспехи молекулярной онкологии2313-805X2413-3787Publishing House ABV Press24110.17650/2313-805X-2019-6-4-47-57RESEARCH ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИVemurafenib resistant melanoma cells acquire mesenchymal stem cell-like propertiesРезистентные к вемурафениб у клетки меланомы приобретают свойства мезенхимальных стволовых клетокhttps://orcid.org/0000-0001-9342-5523VartanianA. A.ВартанянА. А.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

Амалия Арташевна Вартанян

115478 Москва, Каширское шоссе, 24

zhivotov57@mail.ruhttps://orcid.org/0000-0001-8897-0172BurovaO. S.БуроваО. С.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

VishnyakovaKh. S.ВишняковаХ. С.
Russian Federation

32 Vavilova St., Moscow 119991

119991 Москва, ул. Вавилова, 32

SamoylenkoI. V.СамойленкоИ. В.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0003-1349-2879MisyurinV. A.МисюринВ. А.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

EgorovE. E.ЕгоровЕ. Е.
Russian Federation

32 Vavilova St., Moscow 119991

119991 Москва, ул. Вавилова, 32

https://orcid.org/0000-0001-6295-3497RyabayaO. O.РябаяО. О.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

https://orcid.org/0000-0002-6688-8423BaryshnikovaM. A.БарышниковаМ. А.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of RussiaФГБУ «Национальный медицинский исследовательский центр онкологии им. Н. Н. Блохина» Минздрава РоссииV.A. Engelhardt Institute of Molecular Biology, Russian Academy of SciencesФГБУН «Институт молекулярной биологии им. В. А. Энгельгардта РАН»151220196447571512201915122019Copyright ©; 2019, Vartanian A.A., Burova O.S., Vishnyakova K.S., Samoylenko I.V., Misyurin V.A., Egorov E.E., Ryabaya O.O., Baryshnikova M.A.Copyright ©; 2019, Вартанян А.А., Бурова О.С., Вишнякова Х.С., Самойленко И.В., Мисюрин В.А., Егоров Е.Е., Рябая О.О., Барышникова М.А.2019Vartanian A.A., Burova O.S., Vishnyakova K.S., Samoylenko I.V., Misyurin V.A., Egorov E.E., Ryabaya O.O., Baryshnikova M.A.Вартанян А.А., Бурова О.С., Вишнякова Х.С., Самойленко И.В., Мисюрин В.А., Егоров Е.Е., Рябая О.О., Барышникова М.А.https://creativecommons.org/licenses/by/4.0https://umo.abvpress.ru/jour/article/view/241

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months.

Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.

Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.

Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells.

Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

Введение. Мутация в онкогене BRAF приводит к конститутивной активации BRAF-киназы и обеспечивает независимость пролиферации клеток меланомы от присутствия лиганда. Ингибиторы BRAF-киназы заметно увеличивают общую выживаемость больных метастатической меланомой, однако у половины больных через 6—8 мес приема препарата наступает резистентность.

Цель исследования — получение культуры клеток меланомы из опухолей больных, которые перестали отвечать на терапию вемурафенибом, а также изучение механизмов, вовлеченных в возникновение этой резистентности.

Материалы и методы. Были использованы двухмерное культивирование опухолевых клеток, включая получение культуры клеток из опухолевого материала больных, иммуноцитохимический анализ, иммунофлуоресцентный анализ, полимеразная цепная реакция в реальном времени, модели дифференцировки клеток.

Результаты. Из опухолевого материала больных меланомой кожи с мутацией BRAFV600E, резистентной к вемурафенибу, были получены 2 клеточные линии меланомы — Mel Ki и Mel F1702. Клетки имели типичную для меланом веретеноподобную форму. Значения IC50 для Mel Ki и Mel F1702 составляли 4,7 и 6,3 мкМ вемурафениба соответственно. Экспрессия раково-тестикулярных антигенов практически отсутствовала в обеих линиях клеток. Иммунофенотипический профиль клеток выявил высокую экспрессию маркеров мезенхимальных стволовых клеток, таких как CD90, CD105 и CD44. Обе клеточные линии меланомы обладали способностью дифференцироваться in vitro в остеобластподобные и адипоцитподобные клетки.

Заключение. Полученные результаты указывают на фенотипическую транзицию резистентных к вемурафенибу клеток меланомы в мезенхимальноподобные стволовые клетки.

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