Combined effects if inhibition of mTOR and EGFR signaling on apoptosis and kinase pathways in non-small cell lung cancer cell lines

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Based on recent reports that inhibition of the PI3 kinase/Akt pathway mediates the anitumor effects of epidermal growth factor receptor (EGFR) inhibitors and their limited clinical therapeutic success, in this study we studied molecular mechanisms that might underlie the anti-tumor effects of combinations of two agents that specifically target EGFR and mTOR signaling, the EGFR tyrosine kinase inhibitor AG1478 and rapamycin (Rap). We used a panel of non-small cell lung cancer cell lines (A549, H460, H157, H1155), selected on the basis of AKT expression and we studied treatment effects on cell growth, apoptosis and expression levels and /or activities of key intermediate signaling components as assessed by immunoblotting. Cell growth in the presence of serum was inhibited at relatively high concentrations of Rap (20-50nM) and AG1478 (5nM), correlating with increase in annexin V staining. A gradual increase in PARP cleavage with increased time and concentration exposure of cells from 24-48 hours and 10 to 50 nM respectively was seen associated with a gradual decrease in expression of AKT (Ser473) at 24 and 48 hours. Similar effects were seen with AG1478 using a concentration of 5μM and increase in time exposure from 1 to 3 hours. However in H157, H1155 and H460 cell lines despite dose and time –dependent apoptotic effect of AG1478 alone, AKT (Ser473) remained unchanged, while rapamycin induced apoptotic effect at 24 hours without effects on AKT (Ser473). Strikingly the levels of p70 ribosome 6S protein kinase (S6k) (Thr389) were markedly decreased with rapamycin alone and eliminated with the combination of AG1478 and rapamycin (synchronous exposure), correlating also with at least additive effects in growth inhibition at 24 and 48 hours for the 4 cell lines investigated. These results suggest that constitutive activation of pathways downstream Akt such as the S6K could underlie resistance of NSCLC cells to EGFR inhibitors alone and that combinations that inhibit multiple such growth-promoter signal transduction pathways might be novel strategies to treat these tumors. Supported by the ASCO CDA to V.P.