210 © 2020 N. Ya. Hotsuliak et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio- polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577+576 Sensitivity of MCF-7 cells with differential expression of S6K1 isoforms to the regulatory impact of fbroblasts N. Ya. Hotsuliak 1 , A.O. Kravchenko 1,2 , V. V. Kosach 1 , I. O. Tykhonkova 1 , A. I. Khoruzhenko 1 1 Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143 2 Educational and Scientifc Center "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 a.i.khoruzhenko@imbg.org.ua Aim. To investigate the involvement of mTOR/S6K1 cell signaling network, with focus on S6K, in response of tumor cells to regulatory impact of fbroblasts. Methods. Cell culture, including co-cultivation of fbroblasts and tumor cells, immunofuorescence analysis, Western blot analysis, assessment of cell migration by scratch test, and transformation of multicellular spheroid in monolayer cell colony. Results. The present work showed the positive effect of stromal cells on the phosphorylation level of the components of mTOR/S6K1 signaling cascade: p85S6K1, p70S6K1 and mTOR in human breast adenocarcinoma MCF-7 cells. To determine, which of the S6K1 isoforms, p85S6K1, p70S6K1 or p60S6K1, is the most sensitive to the extracellular environment, the stable MCF-7 cell lines with edited expression of S6K1 isoforms were used. It was found that selective expression of p60S6K1 led to the changes in morpho- logical features of tumor cells under both two- and three-dimensional culture conditions. These cells also exhibited high levels of focal adhesion kinase (FAK) phosphorylation and large protein content of Zo-1, CD29, CD44 compatible with their high migration potential in the scratch test. Besides, the cells, selectively expressing p60S6K1, were resistant to fbroblast- producing factors and rapamycin. It was also demonstrated that fbroblasts increased tumor cell motility in scratch test and spheroid outspreading assay under co-cultivation conditions in paracrine manner, whereas the direct contact of tumor spheroids with the fbroblast mon- olayer signifcantly reduced the velocity of spheroid outspreading. Conclusions. The data obtained indicate that not only the differential expression of S6K1 isoforms in MCF-7 cells but also their ratio are important signaling parameters determining cell survival and response to microenvironment factors. Keywords: S6K1, migration, tumor microenvironment. N. Ya. Hotsuliak, A.O. Kravchenko, V. V. Kosach Genomics, Transcriptomics and Proteomics ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2020. Vol. 36. N 3. P 210–228 doi: http://dx.doi.org/10.7124/bc.000A2E