Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin, Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

Abstract

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser473 while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser473 and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.

Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells

“…4C). Similarly, stimulation of human pancreatic cancer PANC-1 cells with neurotensin, a neuropeptide that acts through endogenously expressed GPCRs in these cells (50,51), promoted rapid YAP nuclear import and dephosphorylation at Ser 127 (Fig. 4D) (Fig.…”

“…Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin, and kept at 37°C in a humidified atmosphere containing 10% CO 2 and 90% air. HEK-293 cells and PANC-1 cells were maintained in culture in DMEM supplemented with 10% FBS, as described previously (51,75). Stock cultures were sub-cultured every 3-4 days.…”

Mechanism of Metformin-dependent Inhibition of Mammalian Target of Rapamycin (mTOR) and Ras Activity in Pancreatic Cancer

“…Recent studies report that diabetic cancer patients who take metformin exhibit improved outcomes as compared with patients taking other antidiabetic drugs (10), and this has spurred interest in possible clinical applications of metformin for cancer therapy. One of the hallmarks of metformin action is associated with inhibition of the mTOR signaling in both cancer and non-cancer tissues and cells (15)(16)(17)(18)(19)(20)(22)(23)(24)(25). For exam- A, RNA interference with siRNA against EGFR (siEGFR) decreased cell proliferation.…”

“…Metformin inhibits growth and induces apoptosis and other antineoplastic responses in multiple cancer cell lines, and this is accompanied by modulation of different pathways and genes. Several studies demonstrate that metformin activates AMP-activated protein kinase␣ (AMPK␣), which results in the inhibition of the mTOR 2 signaling pathway and downstream effects (15)(16)(17)(18)(19)(20)(22)(23)(24)(25), and this compliments one of the proposed mechanisms of action of metformin as an antidiabetic drug (29,30).…”

“…The potential clinical applications for metformin in cancer chemotherapy also stems from studies on the anticancer activities of this drug in cancer cells in culture and in in vivo models (1)(2)(3)(4)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Metformin inhibits growth and induces apoptosis and other antineoplastic responses in multiple cancer cell lines, and this is accompanied by modulation of different pathways and genes.…”