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Background: The ribosomal protein S6 kinase 1 (S6K1) is one of the main components of the mTOR/S6K signal transduction pathway, which controls cellular metabolism, autophagy, growth, and proliferation. Overexpression of S6K1 was detected in tumors of different origin including breast cancer, and correlated with the worse disease outcome. In addition, significant accumulation of S6K1 was found in the nuclei of breast carcinoma cells suggesting the implication of kinase nuclear substrates in tumor progression. However, this aspect of S6K1 functioning is still poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with the focus on cell migration. Methods: Multicellular spheroids of MCF-7 cells were generated using agarose-coated Petri dishes. Cell migration was induced by spheroids seeding onto adhesive growth surface and subsequent cultivation for 24 to 72 hours. The subcellular localization of S6K1 was studied in human normal breast and cancer tissue samples, 2D and 3D MCF-7 cell cultures using immunofluorescence analysis and confocal microscopy. Results: Analysis of histological sections of human breast tissue samples revealed predominantly nuclear localization of S6K1 in breast malignant cells and its mainly cytoplasmic localization in conditionally normal cells. In vitro studies of MCF-7 cells demonstrated that the subcellular localization of S6K1 depends on the cell density in the monolayer culture. S6K1 relocalization from the cytoplasm into the nucleus was detected in MCF-7 cells migrating from multicellular spheroids onto growth surface. Immunofluorescence analysis of S6K1 and immunocoprecipitation assay revealed the colocalization and interaction between S6K1 and transcription factor TBR2 (T-box brain protein 2) in MCF-7 cells. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.

Titel:	Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro [version 2; referees: 2 approved]
Autor/in / Beteiligte Person:	Kosach, Viktoriia ; Shkarina, Kateryna ; Kravchenko, Anastasiia ; Tereshchenko, Yuliia ; Kovalchuk, Evelina ; Skoroda, Larysa ; Krotevych, Mykhailo ; Khoruzhenko, Antonina
Link:	View record at F1000Research (Volltext)
Zeitschrift:	F1000Research, Jg. 7 (2018-12-17), S. 1332
Veröffentlichung:	London, UK: F1000 Research Limited, 2018
Medientyp:	academicJournal
ISSN:	2046-1402 (print)
DOI:	10.12688/f1000research.15447.2
Schlagwort:	Research Article Articles S6K1 subcellular localization mTOR/S6K1 signaling pathway breast cancer TBR2 Eomesodermin MCF-7 cell line
Sonstiges:	Nachgewiesen in: F1000Research Document Type: research-article Author Affiliations: 1 = Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, 03143, Ukraine ; 2 = Educational and Scientific Center , Taras Shevchenko National University of Kyiv, Kyiv, 03022, Ukraine ; 3 = National Cancer Institute, Kyiv, 03022, Ukraine Rights: Copyright: © 2018 Kosach V et al. Notes: Revised Amendments from Version 1 In version 2 of the manuscript we have modified the abstract and conclusions to more accurately represent the obtained data. Particularly, we have removed the statement about possible phosphorylation of transcription factor TBR2 by kinase S6K1, as it was predicted only by bioinformatic analysis. However, the existence of TBR2-S6K1 protein complex was shown by co-immunoprecipitation and colocalization assays for the first time, that is why we have left this finding in the abstract. Information about the supplier of antibodies, that were not represented in the main figures and results (against transcription factors ERG and CDX-2), has been moved to the legend of Dataset 4. We have removed the first introductory paragraph of the Results section, because of the feedback of both reviewers. The paragraph was descriptive, and no information about results of the research was lost. The style and language of the manuscript was greatly improved in version 2. Some accidental typographical errors were corrected in the new version. An additional affiliation has been listed for Kateryna Shkarina who, although was a Masters student at the Educational and Scientific Center, Taras Shevchenko National University of Kyiv at the time of version 1, carried out almost all the research at The Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine. ; [version 2; referees: 2 approved]

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Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro [version 2; referees: 2 approved]