Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro

Abstract

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, 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. Methods: Multicellular spheroids of MCF-7 cells were generated using 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. Results: Analysis of histological sections of human breast cancer and normal tissue revealed predominantly nuclear localization of S6K1 in breast malignant cells and mainly cytoplasmic one in conditionally normal cells. In vitro studies of MCF-7 cells showed 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. 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. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.