Synergistic interactions of the Hsp90 inhibitor 17AAG and the PI3K inhibitor LY294002 in non-small cell lung cancer (NSCLC) cell lines involve disruption of Akt and c-raf signalling

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Background: 17-allylamino-demethoxygeldanamycin (17AAG) inhibits the chape-roning action of heat-shock protein 90 (Hsp90) and promotes the proteasomal degradation of multiple oncogenic signalling molecules such as EGFR and Akt. 17AAG has shown promising growth inhibition in a variety of cancer cell lines and xenografts and is currently in clinical development. Non-small cell lung cancer (NSCLC) cells rely on multiple genetic abnormalities for their survival. Thus, on the basis of combinatorial attack of multiple targets by Hsp90 inhibitors the effects of 17AAG on human NSCLC cell lines were investigated. Methods and Results: Exposure of H1792, H292, H596, H1299, A549, H460 and H157 cells to 17AAG resulted in dose and time dependent inhibition of proliferation exhibiting IC50 (inhibitory concentration) values for cell viability at 72 h that ranged from 1.07 ± 0.28 to 7.87 ± 1.27 uM (Mean ± SE) as measured by MTT assays. In A549 and H157 cells flowcytometry revealed 17AAG-induced G1 cell cycle arrest and subsequent apoptosis and immunoblot analysis showed a decline in c-raf and Akt expression levels. Since Akt is a central effector of NSCLC cell proliferation and survival we reasoned that dual targeting of Akt signalling by exposure of A549 and H157 cells to 17AAG plus LY294002, a PI3K inhibitor, would enhance the 17AAG-induced effects. Indeed, isobologram analysis and calculation of the combination indices (CI) at the IC50 level demonstrated highly synergistic interactions. The combination enhanced 17AAG-induced cell cycle and proapoptotic effects as evidenced by both flowcytometry and immunoblot analysis of relevant proteins and led to a further decline in Akt and c-raf signalling. Inducible expression of a constitutively active gag-Akt construct introduced in H1299 cells attenuated but did not completely abolish the observed synergism. Moreover, dowregulation of c-raf signalling by combined treatment with 17AAG plus U0126, a MEK inhibitor produced synergistic antiproliferative effects in A549 and H157 cell lines. Enhancement of 17AAG-induced cell cycle effects was shown in both cell lines, whereas potentiation of proapoptotic effects was only evident in A549 cells that exhibited a decline in Akt activation along with the downregulation in c-raf signalling. Conclusions: Taken together the above findings demonstrate that the efficacy of 17AAG against lung cancer cell lines can be potentiated by the combination with either a PI3K or a MEK inhibitor and that the synergism between 17AAG and LY294002 is mediated through concurrent interference with at least Akt and c-raf signalling. The information obtained from the present study could have direct applications in the management of NSCLC (supported by NCI P50 CA97007).