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.

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.

The ribosomal protein S6 kinase 1 (S6K1) is one of the main Background: 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, which was associated with a 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 poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with focus on cell migration.Multicellular spheroids of MCF-7 cells were generated using Methods: agarose-coated Petri dishes. Cell migration was initiated by spheroids seeding onto growth surface and subsequent cultivation for 24 and 72 hours. S6K1 subcellular localization was studied in human breast cancer and normal tissue, 2D and 3D MCF-7 cell culture using immunofluorescence analysis and confocal microscopy.Analysis of histological sections of human breast cancer and Results: normal tissue revealed predominantly nuclear localization of S6K1 in breast malignant cells and mainly cytoplasmic one in conditionally normal cells. In studies of MCF-7 cells showed that the subcellular localization of S6K1 vitro 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. Bioinformatical analysis revealed existence of several phosphorylation sites in TBR2 for S6K1 suggesting that TBR2 can be a target for phosphorylation and regulation by S6K1. 2018, 7:1332 Last updated: 17 MAY 2019 that TBR2 can be a target for phosphorylation and regulation by S6K1.Subcellular localization of S6K1 depends on the density and Conclusions: locomotor activity of the MCF-7 cells.

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