Abstract A109: Dual mTOR/PI3K inhibitors induce compensatory MEK/ERK activation in human pancreatic cancer cells: A mechanism contributing to drug resistance
Authors: Soares Heloisa P. , Ming Ming , Sinnett-Smith James , Mellon Michelle , Rozengurt Enrique 
Published: October 2015

Abstract
Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway is aberrantly activated in premalignant pancreatic lesions and in pancreatic ductal adenocarcinoma (PDAC) tissues. The pathway functions downstream of RAS (mutated in 90% of PDAC) and plays a key role in tyrosine kinase receptor signaling. Consequently, mTORC1 and the upstream components of the cascade are attractive therapeutic targets in PDAC. However, mTORC1 also mediates potent negative feedback loops that restrain upstream signaling via insulin/IGF receptor and other tyrosine kinase receptors, as shown with first and second generation of mTOR inhibitors. Dual PI3K and mTOR inhibitors (PI3K/TOR-KIs) have been developed to block completely mTORC1 and mTORC2 and prevent upstream pathway activation. Surprisingly, we report here that dual PI3K/TOR-KIs induce robust MEK/ERK over-activation in PDAC cells via a PI3K-independent mechanism.
Results: Treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with NPV-BEZ235 (0.005-1µM), a clinically relevant dual PI3K/TOR-KI, potently blocked mTORC1/S6K activation (scored by S6 phosphorylation at Ser240/244), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr37/46) and mTORC2-mediated Akt phosphorylation at Ser473, in a concentration-dependent manner. Strikingly, exposure to NPV-BEZ235 markedly enhanced the increase in the phosphorylation of ERK at Thr202 and Tyr204. Maximal ERK over-activation (~3 fold) coincided with complete inhibition of phosphorylation of Akt and 4E-BP1 (0.1-0.5 µM NPV-BEZ235). ERK over-activation was demonstrated when PDAC cells were stimulated with serum growth factors or insulin and neurotensin as well as when different PI3K/TOR-KIs (PKI-587, GDC-0980) were used instead of NPV-BEZ235. In order to prove that dual PI3K/TOR-KIs over-activate ERK through a novel PI3K-independent pathway in PDAC cells, we determined the effect of NEV-BEZ235, PKI-587 and GDC-0980 on PI3K-generated accumulation of PIP3 in the plasma membrane of single MiaPaCa-2 cells, using distribution of Akt-PH-GFP to monitor PIP3. Stimulation with insulin induced striking translocation of the PIP3 sensor to the plasma membrane, indicative of robust PI3K activation. Exposure to NEV-BEZ235, PKI-587 or GDC-0980 completely prevented the translocation of Akt-PH-GFP to the plasma membrane, providing conclusive evidence that dual PI3K/mTOR inhibitors induce rapid MEK/ERK activation in PDAC cells with suppressed PI3K activity. Additionally, we showed that treatment with the MEK inhibitors U126 or PD0325901 (1-5 µM) prevented ERK over-activation induced by PI3K/TOR-KIs. PD0325901 released feedback inhibition of RAS/RAF, as revealed by over-phosphorylation of MEK in MiaPaCa-2 or PANC-1 cells. Interestingly, NVP-BEZ235 induced further enhancement of MEK phosphorylation in PDAC cells treated with PD-0325901, implying that dual PI3K/mTOR inhibitor enhanced Raf/MEK in cells without ERK-mediated negative feedbacks loops. Finally, in order to examine whether the over-activation of the ERK pathway counterbalances the growth-suppressive effect of PI3K/TOR-KIs, PDAC cells were treated with PI3K/TOR-KIs (NPV-BEZ235 or PKI-587 or GDC-0980) and PD0325901 or a combination of either PI3K/TOR-KIs and PD0325901. The combination of these drugs caused a more pronounced inhibition of cell growth than that produced by each inhibitor added individually.
Conclusion: Collectively, our results highlight the importance of discovering novel signaling crosstalk to anticipate mechanisms of tumor resistance to new drugs. The capability of predicting drug resistance can assist in developing rational and effective strategies for developing combination therapies in PDAC.
Citation Format: Heloisa P. Soares, Ming Ming, James Sinnett-Smith, Michelle Mellon, Enrique Rozengurt. Dual mTOR/PI3K inhibitors induce compensatory MEK/ERK activation in human pancreatic cancer cells: A mechanism contributing to drug resistance. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A109.