According to the National Cancer Registry, malignant tumors of the mammary gland make up the largest proportion in the structure of the cancer incidence of female population in Ukraine. Therefore, the study of molecular mechanisms involved in the regulation of breast cancer cell proliferation and migration is necessary to understand the processes of metastasis and to improve anticancer therapy.
The study of genetic disorders in breast adenocarcinomas revealed amplification of the RPS6K1 gene located in the 17q22-24 chromosomal region. The amplification of this gene results in an increased expression of S6K1 protein kinase, which is an important link in the PI3K/mTOR/S6K signaling pathway. Moreover, the alterations of the nuclear-cytoplasmic distribution of protein kinase S6K1 toward accumulation in the nuclei may potentially indicate resistance to anti-tumor therapy and a worse prognosis for patients. However, the causes and patterns of S6K1 intracellular localization change in breast malignant neoplasms are currently unknown. Therefore, the study of the features of S6K1 nuclear-cytoplasmic re-arrangement and the reasons leading to it is an urgent task for researchers.
Such studies are complicated by the fact that S6K1 has a number of isoforms formed as a result of an alternative translation of the mRNA and alternative splicing. In human cells there are several S6K1 isoforms: p85S6K1, p70S6K1, p60S6K1 and p31S6K1. So, there is a need to create cellular and animal models which provide selective expression of individual S6K1 isoforms, to study their functions and intracellular localization.
Therefore, the presented work is devoted to the study of the role of subcellular localization of mTOR and S6K1 kinases in the processes of division and migration of human adenocarcinoma cells in vitro, and the study of the participation of p60S6K1 in regulation of these processes.
An analysis of S6K1 and mTOR kinases distribution in mitotic cells revealed an increase of S6K1 content in MCF-7 mammary adenocarcinoma cells in course of division. Moreover, for the first time it was described the co-localization of the phosphorylated mTOR kinase form (phospho-mTOR S 2481) with condensed chromosomes at metaphase stage of MCF-7 cell mitosis.
The nuclear-cytoplasmic distribution of S6K1 was studied in invasive adenocarcinoma cells and conditionally normal human breast mammary tissue. A prominent reaction against S6K1 was detected in the nuclei of malignant cells. At the same time, in conditionally normal tissue S6K1 was predominantly located in the cytoplasm.
The immunofluorescence analysis of three-dimensional spheroid MCF-7 cell cultures, allowed to establish that S6K1 kinase is localized in the cytoplasm. However, in a monolayer culture with low cell density, a bright nuclear reaction was observed against the studied protein kinase. The resulting discrepancy has prompted us to assume that the nuclear-cytoplasmic arrangement of S6K1 may depend on cell density in a culture.
So, immunofluorescence analysis of S6K1 intracellular localization was applied to MCF-7 cells cultured at different density. It was found that at low density 10,000 cells/well, the nuclear reaction against S6K1 was observed in all cells. At a moderate cell density 50,000 cells/well, coloration appeared in the cytoplasm of cells along with the nuclear reaction. When cultivating 100,000 cells/well, which corresponded to 100% confluent monolayer, the nuclear reaction almost completely disappeared and only cytoplasmic one was observed.
The redistribution of S6K1 was also detected in the migrating MCF-7 cells in vitro. To find out the role of nuclear translocation of the studied kinase, the analysis for possible S6K1 target proteins in the moving cells was performed. Co-immunoprecipitation and double immunofluorescence assays revealed the existence of the protein-protein complex of S6K1 and the transcription factor TBR2, which is involved in the regulation of cell migration. Possible sites of TBR2 phosphorylation by S6K1 kinase were identified using bioinformatics analysis.
Application of CRISPR/cas9 genome editing system allowed us to obtain MCF-7 cell lines with selective expression of S6K1 isoforms, namely: with suppressed expression of all isoforms (MCF-7 p85-/p70-/p60-) and selective expression of p60S6K1 (MCF-7 p85-/p70-/p60+).
The study of p60S6K1 expression effect on the proliferation activity of MCF-7 showed that MCF-7 p85-/p70-/p60+ cells had a 1.5-fold higher mitotic index in comparison to the wild type cells. Besides, morphological analysis of the generated cell lines showed that MCF-7 p85-/p70-/p60+ cells lost a typical epithelial morphology. Moreover, it was found that migration rate of MCF-7 p85-/p70-/p60+ cell line increased more than twice as compared to wild-type cells. The obtained results suggest that the p60S6K1 isoform can possess oncogenic potential, and also may be involved in epithelial-mesenchymal cell transition.
