Abstract
The PI3K/AKT/mTOR pathway, which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic ductal adenocarcinoma (PDAC) cells with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) BEZ235 blocked mTORC1/S6K activation (scored by S6 phosphorylation at Ser(240/244)), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr(37/46)), and mTORC2-mediated AKT phosphorylation at Ser(473), in a concentration-dependent manner. Strikingly, BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK overactivation coincided with complete inhibition of phosphorylation of AKT and 4E-BP1. ERK overactivation was induced by other PI3K/TOR-KIs, including PKI-587 and GDC-0980. The MEK inhibitors U126 or PD0325901 prevented ERK overactivation induced by PI3K/TOR-KIs. The combination of BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells indicate that PI3K/TOR-KIs act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated AKT phosphorylation on Ser(473), suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA markedly attenuated the enhancing effect of BEZ235 on ERK phosphorylation. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases
BEZ235 inhibited the phosphorylation of 4E-BP1 at Thr37/46 (Fig. 1A), sites that are sensitive to active-site mTOR inhibitors but not to rapamycin in PDAC cells [23]
Augmented PI3K/AKT activity in response to mTORC1/S6 kinase (S6K) inhibition by rapamycin and its analogs is well documented in a variety of cell types [18,19,20,21], including PDAC [23], overactivation of the MEK/ERK pathway by mTOR inhibitors has been less explored [18]
Summary
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human diseases. The estimated incidence of PDAC in the United States has increased to 44,000 new cases in 2012 and is the fourth leading cause of cancer mortality in both men and women [1]. The PI3K/AKT/mTOR pathway, a key module in the regulation of metabolism, migration, survival, autophagy, and growth [2], plays a pivotal role in the pancreas, mediating acinar-to-ductal metaplasia, and PDAC formation [3, 4] and is active in premalignant pancreatic lesions and pancreatic cancer tissues [4,5,6]. MTORC1, a complex including RAPTOR, phosphorylates and controls at least two regulators of protein synthesis, the 40S ribosomal protein subunit S6 kinase (S6K) and the translational repressor 4E-binding protein 1, referred as 4E-BP1. The PI3K/mTOR pathway functions downstream of RAS [8], which is mutated in 90% of PDACs, and plays a key role in insulin/IGF receptor signaling. Mutation of p53, as seen during the progression of 50% to 75% of PDAC, has been recognized to upregulate the insulin/IGFI/mTORC1 pathway [14]. Cross-talk between insulin/IGFI receptors and G protein-coupled
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