Although phospho rpS6 was maintained in RSK1, RSK3, and RSK4 overex pressing cells, phospho eIF4B was only detectable in RSK3 and RSK4 overexpressing cells following PI3K inhibition. These benefits are in line with our proliferation studies suggesting that, even though RSK1, RSK3 and RSK4 reduce the sensitivity of cells to PI3K inhibitors, only RSK3 and RSK4 overexpressing cells exhibit a sturdy resistance phenotype. Two classes of protein kinases are identified to phosphorylate rpS6 directly. The kinases mainly responsible for rpS6 phosphoryla tion will be the mTOR regulated S6 kinases, which are very sensitive to PI3K mTOR inhibition. The second class is definitely the RSK family of kinases, which are regulated by ERK signaling and are activated following mitogenic stimulation.
Based on our observation that retention of rpS6 and eIF4B phosphorylation correlates with resistance to PI3K pathway inhibitors, we hypothesized that cell lines with larger levels of activated ERK and or RSK signaling may well keep larger levels of phosphorylated S6235 236 upon PI3K block ade and as a result be comparatively insensitive to PI3K inhibition. To investi gate this possibility, we surveyed 27 breast selleck chemicals cancer cell lines by immu noblotting and queried Oncomine to determine breast cancer cell lines with high levels of RSK4. Notably, the 2 breast cancer cell lines exhibiting higher levels of RSK4 in Oncomine, HCC1143 and HCC38, also demonstrated resistance to the PI3K inhibitor GSK 1059615. As anticipated, when subjected to remedy with PI3K inhibitors, cell lines with higher levels of RSK4 activity exhibited a reduce in sensi tivity compared with the sensitive cell line MCF7. Fur thermore, each AU565 and MDA MB 231, but not MCF7, retained rpS6 and eIF4B phosphorylation when treated with various PI3K pathway inhibitors.
These benefits recommend that, when selleckchem rpS6 and eIF4B phosphorylation is principally regulated by the PI3K AKT mTOR axis, inside the context of RSK overexpression or activation by upstream elements, RSKs can sustain rpS6 and eIF4B phosphorylation for the duration of PI3K pathway downregulation. In eukaryotic cells, initiation of protein translation is definitely the major price limiting step in protein synthesis. Current research have suggested that phosphorylation of Ser235 236 in rpS6 and eIF4B Ser422 is essential for cap dependent translation of mRNA. To figure out the effects of RSK4 overexpression on translation, we monitored new protein synthesis rates in vivo by labeling cells with S35 methionine. Certainly, we observed that RSK4 overexpressing cells had greater levels of total protein synthesis in each regular and PI3K inhibitor treated circumstances compared with control cells. Collectively, our information recommend that RSK overexpression prevents response to PI3K inhibition by means of upkeep of pro tein translation and by way of the inhibition of apoptosis.