Mixture of PI3K and RSK blockade overcomes resistance to PI3K inhibition in RSK overexpressing cells. The observations described above recommend that activation from the ERK RSK pathway serves as a mechanism to circumvent PI3K inhibitor sensitivity. As a result, we would reverse the resistance phenotype and also the molecular markers associated with resistance observed in RSK overexpressing cells. To test this hypothesis, we combined PI3K inhibitors together with the MEK inhib itor NVP MEK162 or the pan RSK precise inhib itor dihydropteridinone. In MCF7 cells, RSK3 or RSK4 expression decreased response to therapy with any of the PI3K inhibitors alone. Nevertheless, the mixture of PI3K inhibi tion with MEK162 or BI D1870 completely reversed the resistance of RSK expressing cells. BI D1870 has previously been demonstrated to inhibit the cell cycle regulators PLK1 and Aurora B, albeit at a lot greater con centrations than RSK inhibition.
To verify the specific efficacy of BI D1870, we treated AKT overexpressing cells with combined PI3K inhibitors selleck chemicals EGFR Inhibitors and RSK or MEK inhibitors. As expected, MCF7 cells overexpressing AKT1 have been refractory to combined PI3K and MEK RSK inhibition, confirming the precise efficacy of this com bination for cells with activation in the MEK ERK RSK pathway. We observed that rpS6 and eIF4B phos phorylation was entirely attenuated only when MCF7 RSK cells have been treated using the mixture of BEZ235 and BI D1870 or yet another MEK inhibitor, in agreement together with the effects on cell viability. Accordingly, we also observed an inhibition of RSK phosphoryla tion at Ser380, which serves as a marker of RSK activity, in MCF7 RSK4 cells upon treatment with AZD6244 or MEK162, verifying that MEK inhibition downregulates the function of overexpressed RSK.
In addition, combined inhibi tion of PI3K and RSK diminished rpS6 phosphorylation levels and proliferation compared selleck chemicals MLN8237 with either inhibitor alone in breast can cer cell lines with high levels of RSK. Given that RSK4 overexpression renders cells resistant to the proapoptotic effects of PI3K inhibitors, we hypothesized that combined inhibition of RSK and PI3K would improve apop tosis compared with either compound alone. Indeed, combined inhibition of PI3K and RSK substantially enhanced apoptosis to levels comparable to these in handle GFP overexpressing cells com pared with RSK4 overexpressing MCF7 cells and in breast cancer cell lines exhibiting elevated levels of RSK4. Similarly, targeted knockdown of RSK4 improved the sensitivity to PI3K inhibition in various RSK4 over expressing breast cancer cell lines, substantiating the function of RSK4 in mediating resistance to PI3K inhibition. Importantly, the degree of apop tosis was practically identical in RSK4 knockdown cells versus MEK inhibition when combined having a PI3K inhibitor.