Combination of the mammalian target of rapamycin (mTOR) inhibitor everolimus (E) with the insulin like growth factor-1-receptor (IGF-1-R) inhibitor NVP-AEW-541: A mechanistic based anti-tumor strategy

Abstract

3112 Background- Everolimus inhibits mTOR and has anti-tumor activity in vitro and in vivo. Interestingly, E induces up-stream phosphorylated Akt (p-Akt), which could in turn mediate resistance to E. To explore the mechanisms of E-induced p-Akt, we considered that mTOR negatively regulates insulin signaling, and thus p-Akt levels, via degradation of the insulin receptor substrate1 (IRS-1) in differentiated adypocites. Methods- Starved human prostate cancer cells DU-145 were stimulated with IGF-1 after a brief exposure (2 hours) or concomitantly with E. Small interfering RNAs for S6K (Dharmacon) were transfected using LipofectAMINE 2000, cells were treated 48 hours later with NVP-AEW-541. Cell growth inhibition was analysed after 72 hour - exposure to E and NVP-AEW-541 alone or in combination. Results- Treatment with IGF-1 resulted in a strong induction and posterior degradation of IRS-1. Similarly, p-Akt was rapidly induced and then gradually decreased. Conversely, pretreatment with E resulted in a persistent induction of IRS-1 and p-Akt. When E was added after an initial 2 hour IGF-1 stimulation, IGF-1 induced degradation of IRS-1 was inhibited for the rest of 6 hour - exposure to both IGF-1 and E, as if the cells had only been treated with IGF-1 for 2 instead of 8 hours. Experiments with siRNA demonstrated that a 70% knock down of the down-stream effector of mTOR S6K mimics the effect of E increasing the levels of p-Akt in parallel with those of IRS-1 and reducing those of p-S6. These results suggest that the inhibition of S6K by E results in a sustained activation of IGF-1-R signaling which in turn results in the activation of Akt. Accordingly, we demonstrated that NVP-AEW-541, at the dose able to inhibit the IGF-1-R pathway, prevents the activation of Akt induced by either siRNA-S6K or E; and that combined optimal and sub-optimal doses of NVP-AEW-541 and E significantly inhibited cell proliferation more than each drug alone (p<0.001). Conclusion - The IGF-1-R pathway is involved in the activation of Akt by E and dual mTOR and IGF-1-R targeting appears to be a rational and promising anti-tumor strategy. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis