Научная статья на тему 'AUTOPHAGY ACTIVATION IN BREAST CANCER CELLS IN VITRO AFTER THE TREATMENT WITH PI3K/AKT/MTOR INHIBITORS'

AUTOPHAGY ACTIVATION IN BREAST CANCER CELLS IN VITRO AFTER THE TREATMENT WITH PI3K/AKT/MTOR INHIBITORS Текст научной статьи по специальности «Биотехнологии в медицине»

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Ключевые слова
BREAST CANCER / AUTOPHAGY / GLUCOCORTICOID / MTOR / RAPAMYCIN / WORTMANNIN / PHOSPHOINOSITIDE 3-KINASES / PROTEIN KINASE B

Аннотация научной статьи по биотехнологиям в медицине, автор научной работы — Grigoreva D. D., Zhidkova E. M., Lylova E. S., Enikeev A. D., Kirsanov K. I.

Introduction. Current chemotherapy of breast cancer has a wide range of disadvantages, in particular, the development of therapy-related infections and hormonal imbalance. Combination of main cytostatic with glucocorticoids allows to broaden its therapeutic interval and to decrease the total toxicity of the treatment. However, long-term treatment with glucocorticoids leads to the development of severe side effects via activation of multiple molecular mechanisms. Thus, glucocorticoids activate prosurvival mTOR-dependent autophagy. Therefore, the evaluation of PI3K (phosphoinositide 3-kinases) / Akt (protein kinase B) / mTOR (mammalian target of rapamycin) inhibitors as adjuvants for breast cancer therapy is important for optimization of treatment protocol.Aim. Analysis of the effects of PI3K/Akt/mTOR inhibitors, rapamycin, wortmannin and LY-294002 in combination with glucocorticoids in breast cancer cell lines of different subtypes.Materials and methods. We demonstrated the inhibition of PI3K/Akt/mTOR signaling and the autophagy induction after the treatment of breast cancer cells with rapamycin, wortmannin and LY-294002 by Western blotting analysis of Beclin-1, phospho-Beclin-1 (Ser93 and Ser30).Conclusion. PI3K/Akt/mTOR inhibitors in combination with Dexamethasone cooperatively inhibited mTOR signaling and activated autophagy in breast cancer cells in vitro.

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Похожие темы научных работ по биотехнологиям в медицине , автор научной работы — Grigoreva D. D., Zhidkova E. M., Lylova E. S., Enikeev A. D., Kirsanov K. I.

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Текст научной работы на тему «AUTOPHAGY ACTIVATION IN BREAST CANCER CELLS IN VITRO AFTER THE TREATMENT WITH PI3K/AKT/MTOR INHIBITORS»

DOI: 10.17650/2313-805x-2022-9-4-61-70

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Autophagy activation in breast cancer cells in vitro after the treatment with PI3K/AKT/mTOR inhibitors

Keywords: breast cancer, autophagy, glucocorticoid, mTOR, rapamycin, wortmannin, LY-294002, phosphoinositide 3-ki-nases, protein kinase B

For citation: Grigoreva D.D., Zhidkova E.M., Lylova E.S. et al. Autophagy activation in breast cancer cells in vitro after the treatment with PI3K/AKT/mTOR inhibitors. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2022;9(4):61-70. DOI: 10.17650/2313-805X-2022-9-4-61-70

Активация аутофагии в клетках рака молочной железы in vitro после воздействия ингибиторами PI3K/AKT/mTOR

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D.D. Grigoreva1, E.M. Zhidkova1, E.S. Lylova1, A.D. Enikeev1, K.I. Kirsanov1, 2, G.A. Belitsky1, o

M.G. Yakubovskaya1, E.A. Lesovaya1, 3 ^

'N.N. Blokhin National Medical Russian Research Center of Oncology, Ministry of Health of Russia; 24 Kashirskoye Shosse, Moscow 115522, Russia;

2Peoples'Friendship University of Russia; 6Miklukho-Maklaya St., Moscow 117198, Russia;

3I.P. Pavlov Ryazan State Medical University, Ministry of Health of Russia; 9 Vysokovol'tnaya St., Ryazan 390026, Russia

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Contacts: Diana Dmitrievna Grigoreva grigodidmit@gmail.com

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Introduction. Current chemotherapy of breast cancer has a wide range of disadvantages, in particular, the development >

of therapy-related infections and hormonal imbalance. Combination of main cytostatic with glucocorticoids allows o to broaden its therapeutic interval and to decrease the total toxicity of the treatment. However, long-term treatment

with glucocorticoids leads to the development of severe side effects via activation of multiple molecular mechanisms. ^

Thus, glucocorticoids activate prosurvival mTOR-dependent autophagy. Therefore, the evaluation of PI3K (phosphoino- ^

sitide 3-kinases) / Akt (protein kinase B) / mTOR (mammalian target of rapamycin) inhibitors as adjuvants for breast i_

cancer therapy is important for optimization of treatment protocol. ®

Aim. Analysis of the effects of PI3K/Akt/mTOR inhibitors, rapamycin, wortmannin and LY-294002 in combination with O

glucocorticoids in breast cancer cell lines of different subtypes. ^

Materials and methods. We demonstrated the inhibition of PI3K/Akt/mTOR signaling and the autophagy induction O

after the treatment of breast cancer cells with rapamycin, wortmannin and LY-294002 by Western blotting analysis >s

of Beclin-1, phospho-Beclin-1 (Ser93 and Ser30). x Conclusion. PI3K/Akt/mTOR inhibitors in combination with Dexamethasone cooperatively inhibited mTOR signaling and activated autophagy in breast cancer cells in vitro.

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Д.Д. Григорьева1, Е.М. Жидкова1, Е.С. Лылова1, А.Д. Еникеев', К.И. Кирсанов'2, Г.А. Белицкий1, М.Г. Якубовская1,

Е.А. Лесовая1,3

ФГБУ«Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава России; Россия, 115522

Москва, Каширское шоссе, 24;

2ФГАОУ ВО «Российский университет дружбы народов»; Россия, 117198 Москва, ул. Миклухо-Маклая, 6;

3ФГБОУ ВО «Рязанский государственный медицинский университет им. И.П. Павлова» Минздрава России; Россия, 390026

Рязань, ул. Высоковольтная, 9

Контакты: Диана Дмитриевна Григорьева grigodidmit@gmail.com

Введение. Химиотерапия рака молочной железы имеет широкий спектр недостатков, в частности развитие сопутствующих инфекций и гормональных нарушений. Комбинация с синтетическими глюкокортикоидами позволяет расширить терапевтический интервал и снизить общую токсичность препаратов основной линии терапии. Однако длительное применение глюкокортикоидов способствует развитию ряда побочных эффектов, которые могут реали-зовываться за счет различных молекулярных механизмов. Так, глюкокортикоиды могут инициировать индукцию аутофагии, ведущую к выживанию опухолевых клеток. Запуск механизма аутофагии является mTOR-зависимым, в связи с чем актуальной является оценка возможности введения в качестве адъювантов в терапию рака молочной

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железы ингибиторов сигнального пути PI3K (фосфоинозитид-3-киназа)/А№ (протеинкиназа B)/mTOR (мишень рапамицина млекопитающих).

Цель работы - анализ действия ингибиторов PI3K/Akt/mTOR рапамицина, вортманнина и LY-294002 в комбинации с глюкокортикоидами на запуск аутофагии в клеточных линиях рака молочной железы различного гистогенеза. Материалы и методы. Методом Вестерн-блоттинга было показано, что рапамицин, вортманнин и LY-294002 инги-бируют активность сигнального пути PI3K/Akt/mTOR и индуцируют аутофагию в клетках рака молочной железы, о чем судили по повышению уровня ключевого белка макроаутофагии, Beclin-1, и его фосфорилированных форм phospho-Beclin-1 по остаткам серина Ser93 и Ser30.

Заключение. В ходе работы было показано, что ингибиторы сигнального пути PI3K/Akt/mTOR в комбинации с дек-саметазоном кооперативно подавляют сигнальный путь mTOR и активируют аутофагию в клетках РМЖ in vitro.

Ключевые слова: рак молочной железы, аутофагия, глюкокортикоид, мишень рапамицина млекопитающих, рапамицин, вортманнин, LY-294002, фосфоинозитид-3-киназа, протеинкиназа B

Для цитирования: Григорьева Д.Д., Жидкова Е.М., Лылова Е.С. и др. Активация аутофагии в клетках рака молочной железы in vitro после воздействия ингибиторами PI3K/AKT/mTOR. Успехи молекулярной онкологии 2022;9(4):61-70. DOI: 10.17650/2313-805X-2022-9-4-61-70. (На англ.).

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INTRODUCTION

Incidence of breast cancer (BC) in 2020 is about 2.26 million new cases. It is the first common cancer accounting for approximately 12 % of all cancer worldwide [1]. Breast cancer subtypes are characterized by high heterogeneity in histogenesis, genetic abnormalities, clinical progression of disease and prognosis. Molecular classification of BC is based on the presence/absence of the expression of estrogen and progesterone receptors (ER, PR) as well as epidermal growth factor 2 (HER2). Hormone-dependent BC, characterized by the ER, PR and HER2 expression, is well curable [2]. The treatment usually includes ER antagonists and selective modulators tamoxifen, raloxifen and some others. Long-term therapy course requires the combination of the main anti-cancer drug with glucocorticoids (GC) [3]. ER-negative BC subdivides to triple negative BC (TNBC) and HER2-positive BC. There BC subtypes are associated with poor prognosis compared to luminal BC. HER2 amplification and hyperexpression in BC allows to apply targeted anti-HER2 therapy with the high efficacy [4, 5]. Triple negative BC accounts for 15 % of all BC cases and is characterized by higher aggressiveness and the percentage of relapses as well as poor prognosis. Triple negative BC treatment is the combination of surgery, radio- and chemotherapy with platina derivatives, paclitaxel and doxo-rubicin.

Therefore, therapy of hormone-resistant BC consists of cytostatic drugs associated with high systemic toxicity and severe adverse effects. Also modern BC treatment is characterized by the fast development of drug resistance.

Long-term treatment of BC includes GC. Their application allows to broaden the therapeutic range of main cy-tostatic drug, to diminish its side effects: nausea, vomits, inflammation [6—10]. Glucocorticoids also reveal antipro-liferative effects on the cancer cells of various subtypes [6—10]. Synthetic GC are usually used in the therapy of solid tumors including BC because of immunosuppressive, anti-inflammatory and anti-vomiting effects as well as an-ti-proliferative action on cancer cells [6—10]. However, chronic treatment with GC lead to the different metabolic

complications associated with the induction of the expression of a number GC-dependent genes: REDD1 [11, 12], FKBP51 [13], KLF5 [14], SGK1 [15], MKP-1 [16, 17], ROR1 [18], YAP [19] and others. Additionaly to direct regulation of gene expression by glucocorticoid receptor (GR) binding with GR-responsive elements in gene promotors and enhancers, GR could also regulate cell viability by the protein-protein interaction with key molecules of pro-proliferative and anti-apoptotic signaling pathways. Thus, GR suppresses the activity of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), AP-1, Wnt, mTORC (mammalian target of rapamycin) [20—22]. In our previous studies we demonstrated the efficacy of the combined application of GC and PI3K (phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR modulators with the ability to inhibit the expression of GC-dependent gene REDD1, to attenuate the viability of leukemia and lymphoma cells [7, 23, 24]. Moreover, we showed that side effects of GC are realized via multiple mechanisms including pro-survival autophagy activation [25].

Autophagy is the cell process of catabolism of cytoplas-mic macromolecules and organelles. Autophagy is divided to macroautophagy associated with the autophagosome assemble and degradation of organelles and genetic material, microautophagy, which is realized via endosome and lysosome formation, and shaperon-related autophagy associated with the activation of heat shock proteins [26]. Macroautophagy induction promotes the shifts in the expression of oncogenes and tumor suppressor genes, the elimination of damaged organelles and the decrease of chromosomal instability [26, 27]. At the same time mac-roautophagy leads to cell death in tumors associated with the resistance to apoptosis induction [26—32]. Macroauto-phagy is activated in cells in conditions of hypoxia, stress and nutrient deficiency [26], and allows cells to resist the metabolic stress and the loss of sensitivity to treatment [28]. Autophagy activation is regulated by PI3K /Akt /mTOR signaling [29, 30] via the activation of Beclin-1, key component of PI3K III complex [31, 32]. It was demonstrated that Beclin-1 expression in BC cell line MCF-7 is lower

compared to the normal cells [33]. Along with this observation, Beclin-1 stimulation leads to the induction of au-tophagy, inhibition of proliferation in vitro and suppression of malignant transformation in xenograft model in vivo [33]. Mice with the loss of heterozygosity of BECN1 gene demonstrated higher frequency of spontanic tumor development [14, 34, 35]. Low BECN1 expression in HER2-po-sitive BC is associated with HER2 amplification and poor prognosis [36].

Role of autophagy in the pathogenesis of BC is complicated due to difficulties in separation of microautophagy from macroautophagy in different tumors and in evaluation of the contribution of both processes in cell death and survival. Glucocorticoid-dependent autophagy via increase in REDD1 (regulated in development and DNA damage response 1) and FKBP51 expression was demonstrated in non-transformed cells of epidermis [12] and muscle [37]. It is known that GC-dependent kinase SGK1 (serum and glucocorticoid-inducible kinase 1) affects the activation of autophagy via PI3K/Akt/mTOR signaling [15]. Up-regulation of SGK1 is detected in many tumors including BC and is associated with metastasis and chemoresistance [15, 38, 39]. As GC-induced autophagy belongs to prosurvival autophagy type, we propose that GC induce microauto-phagy associated with the development of the drug resistance. Thus, GC activate the prosurvival autophagy in glio-ma and blood cancer cells [25, 40—42]. At the same time, the treatment of cancer cells with the combination of GC with PI3K inhibitors 3-methyladenine and chloroquine leads to the apoptosis induction [40].

Role of the autophagy in GC-induced resistance to chemotherapeutics remains unclear. Phosphoinositide 3-kinases inhibitors 3-methyladenine and hydroxychloro-quine restore the sensitivity of lapatinib-resistance HER2-positive BC to lapatinib in vitro [43], PI3K inhibitor LY-294002 decrease the ER-related resistance of ovarian cancer to paclitaxel [44], and rapamycin (Rapa) restore the

sensitivity of the cancer cells of various subtypes to chemo-therapeutics [45—47]. Based of these data we assume that targeted regulation of autophagy by PI3K/Akt/mTOR inhibitors is promising for the optimization of GC-based combined BC therapy (fig. 1) [48, 49].

Therefore, targeted regulation of autophagy could be the option to restore the sensitivity of cancer cells to chemo-therapeutics. The application of PI3K/Akt/mTOR inhibitors in combined anti-cancer therapy is promising for autophagy induction [48, 49].

The aim of the study — the present study is devoted to evaluation of autophagy activation by PI3K/Akt/mTOR modulators rapamycin (Rapa), wortmannin (WM) and LY-294002 (LY) individually and in combination with Dexa-methasone (Dex) in BC cells.

MATERIALS AND METHODS

Cell cultures. Breast cancer cells were cultured in DMEM (MCF-7 and MDA-MB-231 cell lines) or RPMI-1640 (HCC-1954 cell line) with 10 % fetal embryonic serum, penicillin (50 ME / ml) and streptomycin (50 ME / ml) ("Paneco", Russia) at 37 °C and 5 % C02.

Cell treatment. Cells were pretreated with solvent, Rapa, WM, LY (10 nM, "LC Labs", USA) for 4 h and then were treated with Dex (10 mM, "KRKA", Czech Republic) for 24 h as described [39].

Western blotting. Western blot analysis was performed as following: after the incubation cells were washed with PBS (phosphate buffered saline), then were lysed in RIPA (radioimmunoprecipitation assay) buffer with protease and phosphatase inhibitors ("Sigma-Aldrich", USA). Protein concentration was evaluated as described in [50]. Proteins were resolved in 10 % PAGE (polyacrylamide gel electrophoresis) in Tris-glycin buffer with 1 % SDS (sodium dodecyl sulfate) and transferred on PVDF (polyvi-nylidene fluoride) membrane (pore diameter 0,22 um). Membranes were blocked with 5 % non-fat milk in TBS

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Fig. 1. Regulation of autophagy (adapted from [25, 26, 29]). PI3K — phosphoinositide 3-kinases; mTOR — mammalian target of rapamycin complex; Akt — protein kinase B; ULK — uncoordinated 51-like kinase

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Fig. 2. The effects of PI3K (phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin complex) inhibitors on the level of p-4E-BP1 andpS6 in breast cancer cells individually and in the combination with Dexamethasone (Dex). The Beclin-1 level was evaluated by Western blotting with the specific antibodies. Densitometry results were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The treatment effects were compared by one-way ANOVA: a — statistically significant difference from the control; b — statistically significant difference from the samples treated with Dex (p <0.05). Rapa — rapamycin; WM — wortmannin; LY — LY-294002;pS6 — phospho-S6ribosomalprotein

(TBS) and incubated with primary antibody overnight at 4 °C. The following antibodies ("Cell Signaling Technology", USA) were used: p-Beclin-1 (Ser30), #54101, p-Be-clin-1 (Ser93), #14717, Beclin-1, #4122, pS6 (phospho-S6 ribosomal protein), #5364, p-4E-BP1, #2855. Then membranes were incubated with anti-rabbit /anti-mouse IgG secondary antibodies ("Abcam", UK). To verify equal protein loading and adequate transfer, the membranes were probed with anti-glyceraldehyde-3-phosphatehehydro-henase (GAPDH, ab181602, "Abcam", UK). Protein bands were visualized by Clarity™ Western ECL Substrate ("Bio-Rad", USA) on ImageQuant™ LAS 4000 ("General Electric", USA). Quantitative analysis were performed by ImageJ software.

Antiproliferative activity. Cell were cultured in 24-well plates (25 000 cell/well) and treated as described above. Antiproliferative effects were evaluated by trypan blue staining using cell counter ("Bio-Rad", USA).

Induction of apoptosis. Cells were cultured in 24-well plates (50 000 cells/well) and treated as described above. For PI (propidium iodide) staining cells were resuspended in 70 % ethanol, fixed for 2 h at -20 °C, placed in PBS

containing 5 ^L PI, 0,1 % sodium citrate and 0,3 % Tri-ton-X100 and incubated for 30 min at room temperature. Analysis by FACScan flow cytometer (Becton Dickinson) was carried out to discriminate between live and apoptotic cells.

Statistical analysis. Mean and standard deviation values were calculated using Microsoft Excel software. The treatment effects in each experiment were compared by one-way ANOVA or t-test.

RESULTS

Effects of combined application of Dexamethasone and PI3K/Akt/mTOR modulators on mTOR activity. Effects of Rapa, WM, LY and Dex on mTOR inhibition on BC cells were evaluated by the phosphorylation level of key down-stream targets of mTOR: 4E-BP1 (eukaryotic initiation factor 4E (eIF4E) binding protein-1 (Thr37 /46), p-4E-BP1) and S6 (phospho-S6 Ribosomal Protein (Ser240/244), pS6) using Western blotting. It has to be mentioned that MCF-7 and HCC-1954 expressed the mutant PIK3CA leading to hyperactivation of PI3K/Akt mTOR signaling.

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Fig. 3. The effects of PI3K (phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR (phosphoinositide 3-kinases) inhibitors on the expression of Beclin-1 protein in breast cancer cells individually and in the combination with Dexamethasone (Dex). The Beclin-1 level was evaluated by Western blotting with the specific antibodies. Densitometry results were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The treatment effects were compared by one-way ANOVA: a — statistically significant difference from the control; b — statistically significant difference from the samples treated with Dex (p <0.05). Rapa — rapamycin; WM — wortmannin; LY — LY-294002

Dexamethasone increased the level of p-4E-BP1 (phospho eukaryotic initiation factor 4E (eIF4E) binding protein-1) in TNBC cells MDA-MB-231. PI3K/Akt/mTOR inhibitors did not affect GC-induced phosphorylation of 4E-BP1 (fig. 2). Rapamycin suppressed the phosphorylation of ribosomal protein S6 in all three BC cell lines individually and in combination with Dex, and inhibited the phosphorylation of 4E-BP1 in MCF-7 and HCC-1954 cells. Wortmannin and LY in combination with Dex decreased the level of p-4E-BP1 and pS6 in HCC-1954 cells (fig. 2). The level of mTOR activity suppression varied between different BC subtypes.

Effect of Dexamethasone and PI3k/Akt/mTOR on the activation of autophagy. Beclin-1 is the main regulator of autophagy in cells [31, 51], and its cleavage in stress conditions induced the shift in cell metabolism to apoptosis activation [31].

Incubation of BC cells with all studied molecules individually and in combination did not lead to Beclin-1 cleavage as well as they did not affect Beclin-1 expression in MDA-MB-231 cells (fig. 3). At the same time combination of WM and LY with Dex induced the increase

in Beclin-1 protein level in MCF-7 and HCC-1954 cells. Rapamycin stimulated the expression of Beclin-1 in combination with Dex in HCC-1954 cell line.

PI3K/Akt/mTOR inhibitors combined with Dex induced the phosphorylation of Beclin-1 by Ser93 residue in cells with PIK3CA hyperexpression. Thus, we demonstrated the 1.9 ± 0.5-fold increase in p-Beclin-1 level after incubation of MCF-7 cells with Rapa + Dex, 3.0 ± 0.7-fold increase with WM+Dex, and 2.5 ± 0.2-fold increase with LY + Dex. Weaker effects were showed in HCC-1954 cells: the average increase in p-Beclin-1 (Ser93) level was 1.5-fold (fig. 4).

Dexamethasone and PI3K/Akt/mTOR inhibitors induced the phosphorylation of Beclin-1 by Ser30 in HCC-1954 cells after individual treatment. We demonstrated the 1.5-fold increase in p-Beclin-1 (Ser30) level in MCF-7 and MDA-MB-231 cell after the treatment with Rapa. The similar effect was described in these cell lines after the treatment with WM+Dex (fig. 5).

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Cytotoxic effects of Dexamethasone and PI3K/Akt/ mTOR inhibitors in breast cancer cells. Dex did not reveal significant cytotoxic effects in BC cells in vitro [7] but

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Fig. 4. The effects of PI3K (phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR (phosphoinositide 3-kinases) inhibitors on the Phospho-Beclin-1 (Ser93) level in breast cancer cells individually and in the combination with Dexamethasone (Dex). The phosho-Beclin-1 level was evaluated by Western blotting with the specific antibodies. Densitometry results were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The treatment effects were compared by one-way ANOVA: a — statistically significant difference from the control; b — statistically significant difference from the samples treated with Dex (p <0.05). Rapa — rapamycin; WM — wortmannin; LY — LY-294002

induces the growth arrest in G1 phase. Rapamycin inhibited the proliferation of HCC-1954 and MDA-MB-231 cells by 50 and 30 %, respectively (fig. 6, a) as well as induced growth arrest in G1 phase in all studied cells (fig. 6, b). Combination of PI3K/Akt/mTOR inhibitors with Dex decreased the proliferative activity by 20-30 % in MCF-7 cell line.

DISCUSSION

It is well-known that partial mTOR inhibition leads to the development of chemoresistance in cancer cells [52, 53]. The suppression of S6 and 4E-BP1 is associated with antiproliferative effects of Rapa [54-56]. In vitro Rapa inhibited the phosphorylation of S6 h 4E-BP1 individually and in combination with Dex in BC cells. LY decrease the level of phosphorylated mTOR targets in MCF-7 cells. At the same time, LY and WM inhibited mTOR activity when used in combination with GC. The data obtained demonstrated partial mTOR inhibition and showed higher sensitivity of S6 to partial inhibitors of PI3K/Akt/mTOR signaling [54]. We demonstrated for the first time the potency of combined application of PI3K/Akt/mTOR modulators

and GC as these compounds did not reveal antagonistic mode of action.

Also we observed higher cytotoxic effects of Dex, Rapa, WM and LY when applied in combination rather than individually in BC cells in vitro (fig. 6). Taken together with the absence of Beclin-1 cleavage, these results demonstrated the autophagy contribution to growth arrest in G1 phase (fig. 3).

Phosphorylation of Beclin-1 by Ser30 and Ser93 residues is associated with the activation of autophagy [57]. Our results showed the autophagy induction in BC cells by PI3K/Akt/mTOR inhibitors as well as cooperative effects of Dex and PI3K/Akt/mTOR inhibitors on autophagy activation in BC cells with PI3K excessive activation (MCF-7 and HCC-1954).

Dex ability to activate autophagy but not BC cell death in vitro demonstrated the induction of prosurvival micro-autophagy leading to the development of chemoresistance of cancer cells. The combination of GC with PI3K/Akt/mTOR inhibitors allows to activate GC-de-pendent macroautophagy related to PI3K/Akt/ mTOR suppression.

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Fig. 5. The effects of PI3K (phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR (phosphoinositide 3-kinases) inhibitors on the Phospho-Beclin-1 (Ser30) level in breast cancer cells individually and in the combination with Dexamethasone (Dex). The phosho-Beclin-1 level was evaluated by Western blotting with the specific antibodies. Densitometry results were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The treatment effects were compared by one-way ANOVA: a — statistically significant difference from the control; b — statistically significant difference from the samples treated with Dex (p <0.05). Rapa — rapamycin; WM — wortmannin; LY — LY-294002

CONCLUSION

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We demonstrated that the combination of PI3K/Akt/ mTOR with Dex cooperatively suppressed mTOR signaling

and activated autophagy in BC cells in vitro. Overall, our data provide the rationale for novel GC and PI3K/Akt/mTOR-based therapy for BC and further investigation of this approach.

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Fig. 6. Antiproliferative effect of PI3K(phosphoinositide 3-kinases)/Akt (protein kinase B)/mTOR (phosphoinositide 3-kinases) inhibitors individually and in combination with Dexamethasone (Dex) in breast cancer cells: a — cells were cultured with solvent, Dex, PI3K/Akt/m TOR inhibitors and their combinations. Cells were counted after the 24 h of treatment. Number of the viable cells is presented as percentage to solvent-treated control. a — statistically significant difference from the control (p <0.05); b — cell cycle phases were detected by flow cytometry with PI staining. The treatment effects were compared by t-test. Rapa — rapamycin; WM — wortmannin; LY — LY-294002

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REFERENCES/ЛИТЕРАТУРА

1. Rezanejad Gatabi Z., Mirhoseini M., Khajeali N. et al.

The Accuracy of electrical impedance tomography for breast cancer detection: a systematic review and meta-analysis. Breast J 2022;2022:8565490. DOI: 10.1155/2022/8565490

2. Onitilo A.A., Engel J.M., Greenlee R.T. et al. Breast cancer subtypes based on ER/PR and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res 2009; 7(1—2):4—13. DOI: 10.3121/cmr.2009.825

3. Vaidya J.S., Baldassarre G., Thorat M.A. et al. Role of glucocorticoids in breast cancer. Curr Pharm Des 2010;16(32):3593-600. DOI: 10.2174/138161210793797906

4. Slamon D.J., Leyland-Jones B., Shak S. et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344(11): 783-92. DOI: 10.1056/NEJM200103153441101

5. Piccart-Gebhart M.J., Procter M., Leyland-Jones B. et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005;353(16):1659-72. DOI: 10.1056/ NEJMoa052306

6. Cidlowski J.A. Glucocorticoids and their actions in cells. Retina 2009;29(Suppl. 6):21-3. DOI: 10.1097/IAE. 0b013e3181ad2636

7. Жидкова Е.М., Кузин К.А., Тилова Л.Р. и др. Сравнительный анализ биологических эффектов селективного агониста глю-кокортикоидного рецептора CpdA на клеточные линии рака молочной железы различных молекулярных подтипов. Сибирский онкологический журнал 2017;16(6):41-6.

DOI: 10.21294/1814-4861-2017-16-6-41-46 Zhidkova E.M., Kuzin K.A., Tilova L.R. et al. Comparative analysis of biological effects of selective activator of the glucocorticoid receptor CpdA on different subtypes of breast cancer cell lines.

Sibirskij onkologicheskij zhurnal = Siberian Journal of Oncology. 2017;16(6):41-6. (In Russ.). DOI: 10.21294/1814-4861-2017-16-641-46

8. Conzen S.D. Recent advances in understanding glucocorticoid receptor function in cancer. Clin Adv Hematol Oncol 2017;15(5):338-40.

9. Kach J., Conzen S.D., Szmulewitz R.Z. Targeting the glucocorticoid receptor in breast and prostate cancers. Sci Transl Med 2015;7(305):19. DOI: 10.1126/scitranslmed.aac7531

10. Vandewalle J., Luypaert A., De Bosscher K. et al. Therapeutic mechanisms of glucocorticoids. Trends Endocrinol Metab 2018;29(1):42-54. DOI: 10.1016/j.tem.2017.10.010

11. Britto F.A., Cortade F., Belloum Y. et al. Glucocorticoid-dependent REDD1 expression reduces muscle metabolism to enable adaptation under energetic stress. BMC Biol 2018;16(1):65.

DOI: 10.1186/s12915-018-0525-4

12. Baida G., Bhalla P., Kirsanov K. et al. REDD1 functions at the crossroads between the therapeutic and adverse effects of topical glucocorticoids. EMBO Mol Med 2015;7(1):42-58.

DOI: 10.15252/emmm.201404601

13. Baida G., Bhalla P., Yemelyanov A. et al. Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy. Oncotarget 2018;9(78):34772-83. DOI: 10.18632/ oncotarget.26194

14. Li Z., Chen B., Wu Y. et al. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors. BMC Cancer 2010;10:98. DOI: 10.1186/1471-2407-10-98

15. Zhu R., Yang G., Cao Z. et al. The prospect of serum and glucocorticoid-inducible kinase 1 (SGK1) in cancer therapy:

a rising star. Ther Adv Med Oncol 2020;12:1758835920940946. DOI: 10.1177/1758835920940946

16. Wu W., Chaudhuri S., Brickley D.R. et al. Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells. Cancer Res 2004;64(5):1757-64. DOI: 10.1158/0008-5472.can-03-2546

17. Melhem A., Yamada S.D., Fleming G.F. et al. Administration of glucocorticoids to ovarian cancer patients is associated with expression of the anti-apoptotic genes SGK1 and MKP1/DUSP1 in ovarian tissues. Clin Cancer Res 2009;15(9):3196-204.

DOI: 10.1158/1078-0432.CCR-08-2131

18. Karvonen H., Arjama M., Kaleva L. et al. Glucocorticoids induce differentiation and chemoresistance in ovarian cancer by promoting ROR1-mediated stemness. Cell Death Dis 2020;11(9):790.

DOI: 10.1038/s41419-020-03009-4

19. Sorrentino G., Ruggeri N., Zannini A. et al. Glucocorticoid receptor signalling activates YAP in breast cancer. Nat Commun 2017;8:14073. DOI: 10.1038/ncomms14073

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

20. Ohnaka K. Wnt signaling and glucocorticoid-induced osteoporosis. Clin Calcium 2006;16(11):1812-6. DOI: CliCa061118121816

21. Polman J.A., Hunter R.G., Speksnijder N. et al. Glucocorticoids modulate the mTOR pathway in the hippocampus: differential effects depending on stress history. Endocrinology 2012;153(9):4317-27. DOI: 10.1210/en.2012-1255

22. Hirose I., Kanda A., Noda K. et al. Glucocorticoid receptor inhibits Muller glial galectin-1 expression via DUSP1-dependent and -independent deactivation of AP-1 signalling. J Cell Mol Med 2019;23(10):6785-96. DOI: 10.1111/jcmm.14559

23. Lesovaya E.A., Savinkova A.V., Morozova O.V. et al. A novel approach to safer glucocorticoid receptor-targeted anti-lymphoma therapy via REDD1 (regulated in development and DNA damage 1) inhibition. Mol Cancer Ther 2020;19(9):1898-908.

DOI: 10.1158/1535-7163.MCT-19-1111

24. Григорьева Д.Д., Жидкова Е.М., Лылова Е.С. и др. Ингибиро-вание глюкокортикоидиндуцированной экспрессии REDD1 рапамицином в клетках рака молочной железы. Успехи молекулярной онкологии 2022;9(1):42-47. DOI: 10.17650/2313-805X-2022-9-1-42-47

Grigorieva D.D., Zhidkova E.M., Lylova E.S. et al. Inhibition of glucocorticoid-induced REDD1 expression by rapamycin

in breast cancer cells. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2022;9(1): 42-7. (In Russ.). DOI: 10.17650/2313-805X-2022-9-1-42-47

25. Molitoris J.K., McColl K.S., Swerdlow S. et al. Glucocorticoid elevation of dexamethasone-induced gene 2 (Dig2/RTP801/ REDD1) protein mediates autophagy in lymphocytes. J Biol Chem 2011;286(34):30181-9. DOI: 10.1074/jbc.M111.245423

26. Parzych K.R., Klionsky D.J. An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal 2014;20(3):460-73. DOI: 10.1089/ars.2013.5371

27. Yun C.W., Lee S.H. The roles of autophagy in cancer. Int J Mol Sci 2018;19:11. DOI: 10.3390/ijms19113466

28. Bhat P., Kriel J., Shubha Priya B. et al. Modulating autophagy in cancer therapy: advancements and challenges for cancer cell death sensitization. Biochem Pharmacol 2018;147:170-82. DOI: 10.1016/j.bcp.2017.11.021

29. Wang Y., Zhang H. Regulation of autophagy by mTOR signaling pathway. Adv Exp Med Biol 2019;1206:67-83. DOI: 10.1007/978-981-15-0602-4_3

30. Romero M.A., Bayraktar Ekmekcigil O., Bagca B.G. et al. Role of autophagy in breast cancer development and progression: opposite sides of the same coin. Adv Exp Med Biol 2019;1152:65— 73. DOI: 10.1007/978-3-030-20301-6_5

31. Kang R., Zeh H.J., Lotze M.T. et al. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 2011;18(4):571-80. DOI: 10.1038/cdd.2010.191

32. Vega-Rubin-de-Celis S. The role of Beclin 1-dependent autophagy in cancer. Biology (Basel) 2019;9(1). DOI: 10.3390/biology9010004

33. Liang X.H., Jackson S., Seaman M. et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999;402(6762):672-6. DOI: 10.1038/45257

34. Qu X., Yu J., Bhagat G. et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.

J Clin Invest 2003;112(12):1809-20. DOI: 10.1172/JCI20039

35. Valente G., Morani F., Nicotra G. et al. Expression and clinical significance of the autophagy proteins BECLIn 1 and LC3

in ovarian cancer. Biomed Res Int 2014;2014:462658. DOI: 10.1155/2014/462658

36. Tang H., Sebti S., Titone R. et al. Decreased BECN1 mRNA expression in human breast cancer is associated with estrogen receptor-negative subtypes and poor prognosis. EBioMedicine 2015;2(3):255-63. DOI: 10.1016/j.ebiom.2015.01.008

37. Gao J., Cheng T.S., Qin A. et al. Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes. Oncotarget 2016;7(19):26966-78. DOI: 10.18632/ oncotarget.9034

38. Loffing J., Flores S.Y., Staub O. Sgk kinases and their role in epithelial transport. Annu Rev Physiol 2006;68:461-90. DOI: 10.1146/annurev.physiol.68.040104.131654

39. Hall B.A., Kim T.Y., Skor M.N. et al. Serum and glucocorticoid-regulated kinase 1 (SGK1) activation in breast cancer: requirement for mTORC1 activity associates with ER-alpha expression. Breast Cancer Res Treat 2012;135(2):469-79. DOI: 10.1007/s10549-012-2161-y

40. Jiang L., Xu L., Xie J. et al. Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells. Cancer Biol Ther 2015;16(3):466-76. DOI: 10.1080/15384047.2015.1016658

41. Surjit M., Ganti K.P., Mukherji A. et al. Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor. Cell 2011;145(2):224-41. DOI: 10.1016/ j.cell.2011.03.027

42. Komakech A., Im J.H., Gwak H.S. et al. Dexamethasone interferes with autophagy and affects cell survival in irradiated malignant glioma cells. J Korean Neurosurg Soc 2020;63(5):566-78.

DOI: 10.3340/jkns.2019.0187

43. Chen S., Rehman S.K., Zhang W. et al. Autophagy is a therapeutic target in anticancer drug resistance. Biochim Biophys Acta 2010;1806(2):220-9. DOI: 10.1016/j.bbcan.2010.07.003

44. Mabuchi S., Ohmichi M., Kimura A. et al. Estrogen inhibits paclitaxel-induced apoptosis via the phosphorylation of apoptosis

СЧ СЧ

о

СЧ

>-

(J

о

—I

о и z о

ОС <

о ж

to

< >

а

<

о m

а.

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о ж.

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>-

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о ж

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signal-regulating kinase 1 in human ovarian cancer cell lines. Endocrinology 2004;145(1):49-58. DOI: 10.1210/en.2003-0792

45. Stephan S., Datta K., Wang E. et al. Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer. Clin Cancer Res 2004;10(20):6993-7000. DOI: 10.1158/1078-0432.CCR-04-0808

46. Abrams S.L., Steelman L.S., Shelton J.G. et al. Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy. Cell Cycle 2010;9(9):1839-46. DOI: 10.4161/cc.9.9.11544

47. Rexer B.N., Engelman J.A., Arteaga C.L. Overcoming resistance to tyrosine kinase inhibitors: lessons learned from cancer cells treated with EGFR antagonists. Cell Cycle 2009;8(1):18-22. DOI: 10.4161/cc.8.1.7324

48. Holloway R.W., Marignani P.A. Targeting mTOR and glycolysis

in HER2-positive breast cancer. Cancers (Basel) 2021;13(12):2922. DOI: 10.3390/cancers13122922

49. Mery B., Poulard C., Le Romancer M. et al. Targeting AKT in ERpositive HER2-negative metastatic breast cancer: from molecular promises to real life pitfalls? Int J Mol Sci 2021;22(24):13512. DOI: 10.3390/ijms222413512

50. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. DOI: 10.1006/ abio.1976.9999

51. Menon M.B., Dhamija S. Beclin 1 phosphorylation — at the center of autophagy regulation. Front Cell Dev Biol 2018;6:137.

DOI: 10.3389/fcell.2018.00137

52. Ducker G.S., Atreya C.E., Simko J.P. et al. Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors. Oncogene 2014;33(12):1590-600. DOI: 10.1038/onc.2013.92

53. Gremke N., Polo P., Dort A. et al. mTOR-mediated cancer drug resistance suppresses autophagy and generates a druggable metabolic vulnerability. Nat Commun 2020;11(1):4684. DOI: 10.1038/s41467-020-18504-7

54. Yellen P., Saqcena M., Salloum D. et al. High-dose rapamycin induces apoptosis in human cancer cells

by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1. Cell Cycle 2011;10(22):3948-56. DOI: 10.4161/ cc.10.22.18124

55. Easton J.B., Houghton P.J. Therapeutic potential of target

of rapamycin inhibitors. Expert Opin Ther Targets 2004;8(6):551-64. DOI: 10.1517/14728222.8.6.551

56. Dowling R.J., Topisirovic I., Alain T. et al. mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs. Science 2010;328(5982):1172-6. DOI: 10.1126/science.1187532

57. Qian X., Li X., Cai Q. et al. Phosphoglycerate kinase 1 phospho-rylates beclin1 to induce autophagy. Mol Cell 2017;65(5):917-31. DOI: 10.1016/j.molcel.2017.01.027

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Authors' contribution

D.D. Grigoreva: Western blot analysis, statistical analysis, article writing;

E.M. Zhidkova: cell culturing, preparation of the figures, article writing; E.S. Lylova: statistical analysis, article writing;

A.D. Enikeev: statistical analysis, preparation of the figures; K.I. Kirsanov: article writing; G.A. Belitsky: article edition;

M.G. Yakubovskaya: analysis of the literature, article edition; E.A. Lesovaya: design of the study, article edition. Вклад авторов

Д.Д. Григорьева: Вестерн-блот-анализ, статистический анализ, написание текста статьи;

Е.М. Жидкова: культивирование клеток, подготовка иллюстративного материала, написание текста статьи;

Е.С. Лилова: статистический анализ, написание текста статьи;

А.Д. Еникеев: статистический анализ, подготовка иллюстративного материала;

К.И. Кирсанов: написание текста статьи;

Г.А. Белицкий: редактирование;

М.Г. Якубовская: анализ литературы, редактирование;

Е.А. Лесовая: разработка дизайна исследования, редактирование.

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ORCID авторов / ORCID of authors

D.D. Grigoreva / Д.Д. Григорьева: https://orcid.org/0000-0003-2675-089X

E.M. Zhidkova / Е.М. Жидкова: https://orcid.org/0000-0003-3318-9391 E.S. Lylova / Е.С. Лылова: https://orcid.org/0000-0001-6388-1624 A.D. Enikeev / А.Д. Еникеев: https://orcid.org/0000-0002-7628-8616 K.I. Kirsanov / К.И. Кирсанов: https://orcid.org/0000-0002-8599-6833

M.G. Yakubovskaya / М.Г. Якубовская: https://orcid.org/0000-0002-9710-8178 E.A. Lesovaya / Е.А. Лесовая: https://orcid.org/0000-0002-1967-9637

Conflict of interest. The authors declare no conflict of interest.

Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов.

Funding. This work is supported by the Russian Science Foundation (grant No 17-75-20124).

Финансирование. Исследование выполнено при поддержке Российского научного фонда (грант № 17-75-20124).

Compliance with principles ofbioethics. The study protocol was approved by the biomedical ethics committee of N.N. Blokhin National Medical Russian Research Center of Oncology, Ministry of Health of Russia.

Соблюдение правил биоэтики. Протокол исследования одобрен локальным комитетом Научно-исследовательский институт онкологии ФГБУ «Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава России.

Article submitted: 22.05.2022. Accepted for publication: 17.11.2022. Статья поступила: 22.05.2022. Принята к публикации: 17.11.2022.

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