To investigate the involvement of mTOR/S6K1 cell signaling network, with focus on S6K, in response of tumor cells to regulatory impact of fibroblasts. Methods. Cell culture, including co-cultivation of fibroblasts and tumor cells, immunofluorescence 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 morphological 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 fibroblastproducing factors and rapamycin. It was also demonstrated that fibroblasts 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 fibroblast monolayer significantly 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.
To determine the paracrine effect of cultured fibroblasts on HeLa cell motility and mTOR/ S6K1 phosphorylation status in vitro. Background. High cancer cell motility is a feature of the malignant tumor. The mTOR/S6K1 signaling network is one of the key links in regulation of cell migration. The cancer cell motility is also dependent on the tumor microenvironment but the effect of stroma on cancer cell migration is insufficiently studied. Methods. Cell culture, Western blot analysis, scratch test, statistical analysis. Results. Application of the media conditioned by a highly confluent monolayer culture of primer human dermal fibroblasts or NIH 3T3 fibroblasts leads to a significant increase of the mTOR and S6K1 phosphorylation in HeLa cells. These conditioned media also have an inhibitory effect on the motility of tumor cells similar to that of mTOR/S6K1 signaling inhibitor rapamycin. Moreover, the combination of rapamycin and fibroblast-conditioned medium does not additionally change the cancer cell motility in comparison to rapamycin or fibroblast conditioned medium alone. Conclusion. The tumor microenvironment can significantly modulate the behavior of cancer cells and the efficiency of anticancer drugs. This should be taken into consideration when developing anticancer drugs.
Kinase mTOR is one of the main links in signal transduction from variety of growth factors/hormones into the cell. Earlier it was shown its overactivation in numerous of cancers. mTOR inhibitors are regarded as antitumor drugs.Immuunofluorescent analysis was applied to detect subcellular localization of mTOR in MCF‐7 cells and human breast tumor. The effect of rapamycin on cultured cells was tested by MTT‐test, migration, adhesion/spreading assay, zymography, actin detection with falloidin, confocal microscopy.Cell behavior under the condition of inhibited mTOR activity was analyzed in vitro. It was revealed decrease of MMP‐9 activity, cell adhesion (up to 40%), cell spreading, migration in “wound healing” model (up to 80%). It was shown the apparent change in actin cytoskeleton organization in paranuclear space.Detection of mTOR localization with anti N‐terminal antibodies revealed a stained network in cells. Confocal microscopy analysis shown a strong colocalization of mTOR and intermediated filaments in MCF‐7 and Hela as well at histological sections of normal and malignant breast tissue.The effect of mTOR activity inhibition on cell behavior can be explained by its relation with cytoskeleton.
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