Взаимодействие аутофагии и эпителиально-мезенхимального перехода в развитии опухолевой прогрессии

Autophagy and epithelial-to-mesenchymal transition (EMT) are the main biological processes involved in tumor progression, and are closely linked. On the one hand, activation of autophagy provides energy and essential nutrients for EMT during the metastases spreading, which is required for tumor cells survival in adverse environmental conditions. On the other hand, autophagy, acting as a tumor suppressor, tends to inhibit metastasis by selectively suppressing the transcription factors of EMT in the early stages. Therefore, inhibition of EMT by inhibitors or inducers of autophagy may be a new strategy for antitumor therapy. Thus, the aim of this review is to highlight current knowledge about the crosstalk between autophagy and EMT processes in tumor progression and to summarize data supporting the necessity of parallel regulation of two processes through signaling pathways.Аутофагия и эпителиально-мезенхимальный переход (ЭМП) являются основными биологическими процессами, участвующими в опухолевой прогрессии, и тесно взаимосвязаны между собой. С одной стороны, активация аутофагии обеспечивает энергию и основные питательные вещества для ЭМП во время распространения метастазов, что помогает клеткам выживать в неблагоприятных условиях окружающей среды. С другой стороны, аутофагия, выступающая в качестве функции, подавляющей опухолевый рост, склонна препятствовать метастазированию путем избирательного подавления основных транскрипционных факторов ЭМП на ранних стадиях. Следовательно, воздействие на ЭМП ингибиторами или активаторами аутофагии может быть стратегией, которая позволит предположить новые мишени для противоопухолевой терапии. Цель данного обзора – освещение современных знаний о перекрестном взаимодействии процессов аутофагии и ЭМП в развитии опухолевой прогрессии и суммирование данных, поддерживающих параллельное регулирование этих двух процессов через общие пути сигнализации

Резистентные к вемурафениб у клетки меланомы приобретают свойства мезенхимальных стволовых клеток

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

Роль ингибирования аутофагии в изменении цитотоксичности темозоломида на клеточных линиях меланомы

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.Введение. Несмотря на современные успехи в терапии метастатической меланомы кожи, эта нозология остается крайне резистентной к существующим препаратам. Современные данные свидетельствуют о том, что опухоли могут преодолевать гибель посредством аутофагии – процесса, при котором опухолевые клетки переваривают свои собственные белки и клеточные компоненты при недостатке энергии и дефиците питательных веществ.Цель работы – исследование влияния ингибиторов аутофагии, таких как хлорокин (CQ) и LY-294.002 (LY) на цитотоксичность темозоломида (TMZ) в клеточных линиях меланомы человека. Материалы и методы. Работа проведена на клеточных линиях меланомы Mel Z, Mel IL и Mel MTP, полученных от пациентов, проходивших лечение в РОНЦ им. Н. Н. Блохина. Оценку антипролиферативной активности TMZ в комбинации с ингибиторами аутофагии исследовали с помощью МТТ-теста и метода колониеобразования. Мы оценили изменение клеточного цикла, активацию апоптоза и изменение экспрессии основных маркеров аутофагии при комбинированной терапии.Результаты. CQ и LY усиливали цитотоксичность TMZ и снижали число жизнеспособных колоний во всех изученных линиях, при этом оба ингибитора увеличивали накопление популяции клеток в стадии G0 / G1 в линиях Mel Z, Mel IL, но не в Mel MTP. Показано также, что CQ и LY синергично активировали апоптоз во всех исследованных линиях. Анализ экспрессии матричной РНК ключевых генов аутофагии свидетельствовал о вовлечении данного процесса в цитотоксичность.Заключение. Инактивация аутофагии на разных этапах данного процесса позволяет преодолевать резистентность к TMZ и может быть рассмотрена как перспективная мишень для терапии меланомы

РОЛЬ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА И АУТОФАГИИ В ПРОТИВООПУХОЛЕВОМ ОТВЕТЕ КЛЕТОЧНЫХ ЛИНИЙ МЕЛАНОМЫ НА ТАРГЕТНОЕ ИНГИБИРОВАНИЕ MEK и mTOR КИНАЗ

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma.Введение. Возникновение резистентности и дальнейшая опухолевая прогрессия остаются актуальной проблемой в лечении меланомы кожи. Процесс аутофагии и эпителиально-мезенхимальный переход (ЭМП) тесно связаны между собой и играют ключевую роль в опухолевой прогрессии. Таргетное коингибирование МЕК и mTOR киназ является потенциальной мишенью для терапии меланомы, нацеленной на блокирование ЭМП.Цель работы – изучение влияния ко-ингибирования МЕК и mTOR киназ на выживаемость, возможность формирования 3D-сфероидов и миграционные способности клеточных линий меланомы, а также взаимосвязь этих изменений с маркерами ЭМП и аутофагии.Материал и методы. Работа проведена на клеточных линиях меланомы Mel Z и Mel MTP, полученных от пациентов, проходивших лечение в НМИЦ онкологии им. Н.Н. Блохина. Оценку антипролиферативной активности биниметиниба и/или рапамицина исследовали МТТ-тестом. 3D-сфероиды получали на основе RGDпептидов, миграционную способность и инвазивность оценивали к камере Бойдена и базальном матриксе. Изменения экспрессии маркеров аутофагии и ЭМП исследованы иммуноцитохимически или иммуноблоттингом.Результаты. Рапамицин усиливал цитотоксичность биниметиниба как в 2D-, так и в 3D-культурах клеточных линий меланомы. При этом биниметиниб и рапамицин снижали инвазию, но не миграцию клеток меланомы in vitro. Эффективность комбинации связана со снижением маркеров ЭМП N-кадхерина и β-катенина и аутофагии в клетках меланомы – Беклин 1, р62/SQST M1 и LC3BII .Заключение. Инактивация аутофагии и ЭМП позволяет преодолевать резистентность к существующей терапии и может быть рассмотрена как перспективная мишень для терапии меланомы

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The interplay of autophagy and epithelial-to-mesenchymal transition in cancer progression

Autophagy and epithelial-to-mesenchymal transition (EMT) are the main biological processes involved in tumor progression, and are closely linked. On the one hand, activation of autophagy provides energy and essential nutrients for EMT during the metastases spreading, which is required for tumor cells survival in adverse environmental conditions. On the other hand, autophagy, acting as a tumor suppressor, tends to inhibit metastasis by selectively suppressing the transcription factors of EMT in the early stages. Therefore, inhibition of EMT by inhibitors or inducers of autophagy may be a new strategy for antitumor therapy. Thus, the aim of this review is to highlight current knowledge about the crosstalk between autophagy and EMT processes in tumor progression and to summarize data supporting the necessity of parallel regulation of two processes through signaling pathways

The role of autophagy inhibition in the enhanced cytotoxicity of temozolomide on melanoma cell lines

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment

THE ROLE OF EPITHELIAL-TO-MESENCHYMAL TRANSITION AND AUTOPHAGY IN ANTITUMORAL RESPONSE OF MELANOMA CELL LINES TO TARGET INHIBITION OF MEK AND mTOR KINASES

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma

Vemurafenib resistant melanoma cells acquire mesenchymal stem cell-like properties

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