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For a long time, the genocentric paradigm of the etiology and pathogenesis of tumor diseases dominated in the field of molecular oncology. According to this paradigm, cancer is a genetic disease caused by the accumulation of somatic mutations that lead to a functional imbalance between tumor suppressor and oncogenic signals. Today, it is convincingly established that naturally occurring processes of dedifferentiation/transdifferentiation closely related to the phenomenon of cellular plasticity, play an important role in the control of cancer progression [1]. The biological program underlying all these processes involves the reciprocal transitions from epithelial to mesenchymal identity termed EMT/MET, while the ability of cells to undergo EMT is currently referred as epithelial–mesenchymal plasticity (EMP). EMT is coopted by cancer cells to acquire of a more aggressive tumor phenotype tightly interrelated with conversion of noninvasive tumor cells into stem–like states, increase of therapy resistance and metastatic potential. In addition, cancer cells, to increase their migratory and invasive potential, can acquire amoeboid features (MAT), as a more effective cellular state within EMT spectra [2]. Molecular strategies that orchestrate EMP include context–dependent dynamic changes in extracellular environment; consequent modulation of receptor–mediated signaling and metabolic networks; fine control of epigenetic events; control of gene and miRNA expression patterns, translation and post–translational modifications, which are realized in changes in the phenotype of cells and their biological responses.The results of systemic studies obtained in the Department of Cell Signaling by using tumor cells of different tissue origins and in vitro and in vivo models demonstrated that the stable forced expression/down–regulation of SH3–containning adaptor protein Ruk/CIN85 is accompanied by activation/inhibition of signaling pathways that initiates global nuclear and metabolic reprogramming leading to EM–MA/AM–ME transitions depending on the optimal stoichiometry of Ruk/CIN85 in intracellular signaling complexes [3]. Taken together, the data obtained provide evidence to consider adaptor protein Ruk/CIN85 as a potent reprogramming factor that utilizes cellular plasticity to regulate the phenotype of tumor cells and may be useful for the development of new therapeutic approaches.

Aim. This study is aimed at investigation of the CIN85 effect on osteosarcoma (OS) cells collagen adhesion and invasion. Conclusions. Our results demonstrate that increased CIN85 expression in osteosarcoma cells is associated with elevated collagen adhesion and invasion and may thereby contribute to metastasis.

Fullerene C 60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C 60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C 60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C 60 was synthesized by covalent conjugation of C 60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C 60 from the medium. Light-emitting diode lamp (100 mW cm −2 , λ = 420–700 nm) was used for excitation of accumulated C 60 . A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C 60 and visible light. The cytotoxic effect of photoexcited C 60 was comparable with that induced by H 2 O 2 , as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C 60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H 2 O 2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C 60 . The cytotoxic effect of C 60 in combination with visible light irradiation was demonstrated also on leukemic L1210 cells both sensitive and resistant to cisplatin. It was shown that relative value of mitochondrial membrane potential measured with tetramethylrhodamine ethyl ester perchlorate (TMRE) probe was lower in resistant cells in comparison with sensitive cells and the drop of mitochondrial potential corresponded to further decrease of resistant cell viability after C 60 photoexcitation. The data obtained allow to suggest that C 60 -mediated photodynamic treatment is a candidate for restoration of drug-resistant leukemic cell sensitivity to induction of mitochondrial way of apoptosis.

Osteosarcoma (OSA) is the most common primary malignant bone tumor in children and adolescents, characterized by high metastatic potential and poor prognosis. The adaptor protein CIN85 is known to be upregulated in various cancers and is involved in cell motility and invasion. Aim. This study was purposed to investigate the role of CIN85 in the migra-tion of osteosarcoma cells. Methods. In vitro, scratch assay, xCELLigence, and Transwell assay were used to evaluate cell migration, while RNA-seq, qPCR, and Western blotting assessed gene expression. Results. Public datasets revealed elevated CIN85/SH3KBP1 expression in OSA tissues compared to normal bone, with even higher levels observed in metastases. Functional studies using CIN85-overexpressing and CIN85-silenced HOS and SAOS-2 osteosarcoma cells demonstrated that CIN85 promotes OSA cell migration and invasion in both 2D and 3D models. RNA-seq analysis identified differentially expressed genes and enriched pathways related to migration, extracellular matrix, adhesion, and cell signaling. CIN85-driven motility was shown to depend on the expression of COL3A1 and MMP2, Akt/mTOR signaling, and NOX activity. Conclusion. These findings support CIN85 as a potential biomarker and therapeutic target in osteosarcoma.

Aim. We previously demonstrated that mouse breast adenocarcinoma 4T1 cells overexpressing adaptor protein Ruk/CIN85 (4T1 RukUp) acquired a malignant phenotype associated with cancer stem cells features compared with parental 4T1 cells (4T1 Mock) [1]. VPA, a broad Class I histone deacetylases inhibitor, is widely used in cancer therapy [2]. The study aimed was to evaluate the effect of VPA on Warburg effect dependent on Ruk/CIN85 expression in 4T1 cells. Conclusions. The results obtained demonstrate that VPA restores the coupling between oxidative phosphorylation and glycolysis in 4T1 RukUp cells indicating a novel mechanism of its anti-cancer effects.

Aim. Conventional anticancer drugs have a number of known limitations but the development of new ones takes a long time, requires significant funding, and has a low success rate. Repurposing of pharmacologically active plant alkaloids Berberine (Ber) and Piperlongumine (Pl) and finding nanoplatforms for their delivery to cancer cells seems promising to be addressed. The ability of the C60 fullerene (C60) carbon nanostructure to be functionalized with therapeutic agents makes it a potential drug carrier. The aim of this study was to prove a non-covalent functionalization of C60 with Ber or PL and to check the effectiveness of these nanocomplexes against Lewis lung carcinoma (LLC) both in vitro and in vivo. Conclusions. The proposed nanocomplexes of alkaloids with C60 represent promising drugs for the treatment of metastatic lung cancer.

Aim. This study is focused on a comprehensive overview of mechanisms and processes involved in the acquisition of cancer cell plasticity in a manner dependent on the adapter protein Ruk/CIN85 (in rodents, Ruk — regulator of ubiquitous kinase; in human CIN85 — Cbl-interacting protein of 85 kDa, encoded by SH3KBP1 gene).. Methods. Gene expression was evaluated using RT2-PCR and Western blotting, cell proliferation and survival were analyzed using MTT and/or dye exclusion assays, motility was assessed by scratch test and Transwell assay, enzyme activities were measured using spectrophotometric assays. In vivo metastasis were studies using experimental metastasis model. Conclusion. This study discloses various aspects of cancer cells plasticity, such as EMT, stemness, metabolic changes, ECM components, and drug resistance in dependence on adaptor protein Ruk/CIN85 expression level.

Aim. To evaluate an impact of different S6K1 isoforms expression in MCF7 cells on initiation of Epithelial to Mesenchymal Transition (EMT). Methods. Immunocytochemical analysis, Real-Time PCR, Western blot. Results. We have demonstrated that unbalanced expression of p60, p70 and p85-S6K1 isoforms in MCF-7, namely downregulation of p70 and p85, and basal p60 expression, mediated by the CRISPR-Cas9 gene editing resulted in altered morphology and increased cell motility. Such changes were associated with the expression of genes whose products are involved in the regulation of cell motility, interaction with the extracellular matrix and loss of cellular adhesion demonstrating their increased potential for invasion and metastatic activity. qPCR analysis confirmed increased expression of a whole spectrum of genes associated with mesenchymal characteristics and loss of epithelial specific markers. Additionally, we observed a complete repression of the estrogen receptor 1 (ESR1) expression and downregulation of HER2/NEU. Conclusions. Our data demonstrate for the first time the implication of S6K1 isoforms in the regulation of EMT in MCF-7 cells by triggering for the concomitant onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type and has a strong negative impact on ESR1 expression. At the tumor level that can mean breast tumor transition to a more aggressive most probably triple negative molecular subtype.

Aim. To isolate and characterize extracellular vesicles (EVs) produced by mouse renal car-cinoma Renca cells with different expression levels of the adaptor protein Ruk/CIN85 under normoxia and hypoxia conditions. Methods. The density gradient centrifugation was used to isolate EVs from the conditioned medium of Renca cells cultured under normoxia and hy-poxia conditions. Further characterization of EVs was performed by using nanoparticle tracking analysis (NTA), electron microscopy and Western Blot analysis. Results. Significant differences in average particle size between EVs produced by sublines studied under experimental conditions were not found. At the same time, concentration of particles pro-duced by Ruk/CIN85 overexpressing cells turned out to be an order of magnitude higher in hypoxia in comparison to normoxia conditions. It was shown that under normoxia condi-tions the content of both Ruk/CIN85 and EVs’ markers Alix and CD81 was increased in vesicles produced by Renca cells with Ruk/CIN85 overexpression in comparison with those from control mock-transfected cells. Under hypoxia conditions, the content of studied pro-teins decreased by more than two orders of magnitude in EVs secreted by Renca cells with up-regulation of adaptor protein whereas the content of Ruk/CIN85 and CD81 increased in EVs from mock-transfected cells. Conclusions. It has been demonstrated that the adaptor protein Ruk/CIN85 is a novel component of EVs produced by tumor cells that may play a role in the control of EV composition under normoxia and hypoxia.

Aim. To study the effect of adaptor protein Ruk/CIN85 overexpression on the dynamics of migration and Matrigel invasion as well as transendothelial migration of murine 4T1 breast adenocarcinoma cells. Methods. Dynamics of 4T1 cells migration/invasion was monitored in real time using the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument equipped with a CIM-plate 16. Transendothelial migration (TEM) of 4T1 cells was performed through the layer of primary mouse lung endothelial cells seeded on gelatin-coated 24-well transwell inserts (8-μm pores).The two-tailed Student’s t-test for unequal variances was used for statistical analysis. Results. Ruk/CIN85-overexpression in 4T1 cells are indices a significantly increased motility, Matrigel invasiveness and migration through endothelial cells layer. Conclusions. The Ruk/CIN85 adaptor protein may play a potential role in the control of metastasis in vivo.