Ribosomal protein S6 kinase 1 (S6K1) is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1) participating primarily in the regulationA number of downstream effects of mTORC1, including protein biosynthesis, cell growth, proliferation and survival [4,5] are mediated via ribosomal protein S6 kinase 1 (S6K1), a well-studied mTORC1 substrate.The S6K1 gene (RPS6KB1) was shown to encode two well-known S6K1 isoforms, p85-S6K1 and p70-S6K1, that differ only by the presence of the Nterminal 23 a.a. extension in p85-S6K1 due to the use of alternative (the first and second ATG) translational start sites [6]. Recently, it has been discovered that the splicing factor SF2/ASF promotes the expression of the oncogenic and the only known S6K1 splice variant termed p31-S6K1 or S6K1-isoform 2 that is truncated from the C-terminus [7]. A mechanism underlying oncogenic properties of p31-S6K1 is unclear but it seems to be kinase-independent, since the kinase domain of the given isoform is severely truncated. e x p e r i m e n T a l w o r K S e x p e r i m e n T a l w o r K S

To generate HEK-293 cells with disrupted expression of S6K1 isoforms: p85, p70 and p60. Methods. CRISPR/Cas9 gene editing, Western blotting, immunofluorescent analysis, RT-PCR analysis, MTT assay, scratch assay. Results. Several clones of HEK-293 cells with a complete loss of p85/p70/p60-S6K1 protein expression were generated. The effects of p85/ p70/p60-S6K1 knockout on Akt/mTORC1/S6K1 signaling and cell proliferation and migration were assessed. Conclusions. The generated cell lines can be used to study a role played by S6K1 in cell physiology and to gain more detailed insight into cellular functions of the S6K1 isoforms. The HEK-293 cells exhibit downregulation of Akt phosphorylation on Ser473 and subsequent attenuation of cell growth rate, as well as inhibition of cell motility. K e y w o r d s: mTOR/S6K1 signaling, CRISPR/Cas9, S6K1 isoforms. К л ю ч е в ы е с л о в а: mTOR/S6K1 сигналинг, CRISPR/Cas9, изоформы S6K1.

To generate and characterize MCF-7 cell lines with altered expression of p85, p70 and p60 S6K1 isoforms: p85-/p70-/p60-MCF-7 and p85-/p70-/p60+MCF-7. Methods. CRISPR/ Cas9 gene editing, western blot analysis, immunofluorescence analysis, scratch assay. Results. Modified MCF-7 cells with knocked down expression of p85, p70, p60 or only p85 and p70 S6K1 isoforms were generated. Selective inhibition of only p85 and p70 isoforms in p85-/p70-/p60+MCF-7 cells was accompanied by actin cytoskeleton rearrangements, appearance of fibroblast-like cell morphology and significantly increased cell locomotor activity. Downregulation of all three S6K1 isoforms in p85-/p70-/p60 -MCF-7 cells inhibited cell migration with no changes in the cell morphology. Alterations in had a different impact on the ribosomal protein S6 phosphorylation and Akt signaling. Conclusion. Analysis of the modified MCF-7 cell lines revealed different impact of expression of S6K1 isoforms on MCF7 cell locomotor activity and the S6K1-and Akt-dependent signaling. Our data suggest that p60-S6K1 could be involved in regulation of the cell migration. The generated cells can be used for further analysis of functional activity of the S6K1 isoforms. K e y w o r d s: mTOR/S6K1 signaling, MCF-7, S6K1, CRISPR/Cas9, breast cancer К л ю ч е в ы е с л о в а: mTOR/S6K1 сигналинг, MCF-7, S6K1, CRISPR/Cas9, рак молочной железы.

Aim.To generate the p85-S6K1 knockout MCF-7 breast cancer cell line and to evaluate the effect of p85-S6K1 on cell growth, migration and survival under stress conditions. Methods. CRISPR/Cas9 genome editing, Western blotting, immunofluorescence staining, MTT assay, in vitro scratch assay. Results. We generated two clones of the p85-S6K1 knockout MCF-7 cell line and tested their survival upon hydrogen peroxide treatment as well as the proliferation and migration rates. The generated cell clones display an impaired ability to survive under oxidative stress, exhibit inhibition of cell growth, cell motility and downregulation of rpS6 phosphorylation on Ser235/236/240/244 under cell starvation compared to the control cells. Conclusions. The p85-S6K1 isoform could be involved in modulation of cancer cell behaviour promoting cell growth, migration and survival. The obtained clones can be further used to study the participation of different S6K1 isoforms in the control of cell function. K e y w o r d s: mTOR/S6K1 signaling, CRISPR/Cas9, p85-S6K1 isoform.

Migration capacity is an important feature of tumor cells. There are several approaches to analyze the dynamics of cancer cell migration in vitro. The model of initiation of cell migration from 3D multicellular spheroids onto growth surface is one of the closest to the in vivo conditions. Aim. Optimization of the model to study tumor cell mobility for several days. Methods. 2D and 3D MCF-7 cell culture, immunofluorescence analysis and image analysis using the Fiji computer software. Results. Unification of spheroid size allowed avoiding a significant data deviation. The obtained spheroids spread completely for three days. The highest migration ratio was observed on the second day. The proliferation level was similar during each day of the three-day experiment; it did not exceed 3 %. The validity of the model was tested after migration inhibition by a mTOR signaling inhibitor rapamycin. Additionally, this model was successfully applied to immunofluorescence study of p85S6K1 subcellular localization in moving MCF-7 cells. Conclusions. Double filtration of multicellular spheroids allowed unification of their size; this promotes an adequate interpretation of the migration assay. This model allows to study tumor cell migration dynamics and can be further used for development of anticancer drugs.