Abstract 5453: Dual mTOR/PI3K inhibitors induce ERK pathway overactivation in pancreatic cell lines: Rationale for combinatory therapy
Authors: Soares Heloisa P. , Ming Ming , Mellon Michelle , Sinnet-Smith James , Rozengurt Enrique 
Published: May 2015

Abstract
Pancreatic ductal adenocarcinoma (PDAC), which comprises 90% of all human pancreatic cancers, is a devastating disease, with overall 5-year survival rate of only 3-5%. As the current therapies offer very limited survival benefits, novel molecular therapeutic targets and strategies are urgently needed to treat this aggressive disease. The complementary activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1 and MEK/ERK signaling pathways in many cancer cells, including PDAC, potently stimulates their proliferation and survival. However, mTORC1 also mediates negative feedback loops that restrain upstream signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops by selective mTORC1 inhibitors, e.g. by rapamycin and its analogs, unleashes over-activation of the PI3K/Akt pathway that potentially opposes the anti-proliferative effects of mTOR inhibitors. This prompted the development of active-site mTORC1/2 kinase inhibitors (TOR-KIs) and dual PI3K and mTOR inhibitors (PI3K/TOR-KIs). It is not known whether PI3K/TOR-KIs induce any effect on the activity of the ERK pathway in PDAC cells. Consequently, we treated serum-starved cultures of PDAC cells (MiaPaca-2 and PANC-1) with increasing concentrations of the dual PI3K/TOR-KI NPV-BEZ235 followed by stimulation with insulin and neurotensin, a potent mitogenic combination for these cells. As expected, prior exposure to NPV-BEZ235 potently inhibited mTORC1 activation (scored by S6 phosphorylation at Ser-240/244) and mTORC2-mediated Akt phosphorylation at Ser-473, in a concentration-dependent manner. Strikingly, we also demonstrate, for the first time, that exposure to NPV-BEZ235 markedly enhanced the phosphorylation of ERK at Thr-202 and Tyr-204. Maximal ERK over-activation coincided with complete inhibition of Akt phosphorylation at Ser-473 (produced at 100-500 ηM NPV-BEZ235). ERK over-activation was also seen when PDAC cells were stimulated with 2% fetal bovine serum and when a different PI3K/TOR-KI (PKI-587) was used instead of NPV-BEZ235. Treatment with the MEK inhibitors U126 or PD0325901 (1-5 μM) prevented ERK over-activation induced by PI3K/TOR-KIs. Crucially, a combination of NPV-BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor added individually. Thus, the elucidation of novel feedbacks loops and crosstalk mechanisms can assist in developing rational and effective strategies for developing combination therapies in PDAC.
Citation Format: Heloisa P. Soares, Ming Ming, Michelle Mellon, James Sinnet-Smith, Enrique Rozengurt. Dual mTOR/PI3K inhibitors induce ERK pathway overactivation in pancreatic cell lines: Rationale for combinatory therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5453. doi:10.1158/1538-7445.AM2014-5453