Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2

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.

Figure 1. NPV-BEZ235 induces over-activation of ERK phosphorylation in PDAC cells

A: Cultures of MiaPaCa-2 cells were incubated in the absence or in the presence of NPV-BEZ235 (0,005–1 μM) for 2h. Then, the cells were stimulated for 30 min with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) and lysed with SDS–PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies illustrated in the figure. B: Quantification of phosphorylated ERK at Thr²⁰² and Tyr²⁰⁴, S6 at Ser ^240/244 and AKT at Ser⁴⁷³ was performed using Multi Gauge V3.0. in 3 independent experiments similar to Fig 1A. C: Cultures of MiaPaCa-2 cells were incubated in the absence or in the presence of 1μM of NPV-BEZ235 for 2h and then stimulated with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) for various times and lysed. Immunoblotting was performed as described in panel A. D: Cultures of BxPC-3 and AsPC-1 cells were treated with or without 1μM of NPV-BEZ235 (BEZ) for 2h and then stimulated with NT and Ins for 30 min and lysed. Immunoblotting was performed as in panel A. Image Editing: Irrelevant lanes were removed (indicated by a thin, vertical black line) from the acquired digital images and flanking lanes juxtaposed using Adobe Photoshop

Figure 2. MEK inhibitors suppresses ERK over-activation induced by NPV-BEZ235

A and B: Cultures of MiaPaCa-2 cells (A) and PANC-1 (B) cells were incubated for 2 h in the absence or presence of increasing doses of NPV-BEZ235 with or without U0126 at 1μM or 5μM. C and D: Cultures of MiaPaCa-2 cells (C) and PANC-1 (D) cells were incubated in the presence of increasing doses of NPV-BEZ235 with or without PD0325901 (PD) at 1μM or 5μM for 2 h. Then, for panels A–D, the cells were stimulated for 2 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with SDS–PAGE sample buffer. E and F: Cultures of MiaPaCa-2 (E) and PANC-1 (F) cells were incubated for 2 h in the absence or presence of increasing doses of NPV-BEZ235 with or without the addition of PD0325901 (PD) at 1μM and 5μM. Then, the cells were stimulated for 2 h with 2% fetal bovine serum (serum) and lysed with SDS–PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting with the antibodies that detect phosphorylated or total proteins, as described in each panel.

Figure 3. MEK inhibition blunts the enhancement of ERK activity induced by PKI-587 or GDC-0980

A and B: Cultures of MiaPaCa-2 cells (A) and PANC-1 (B) cells were incubated for 2 h in the absence or presence of increasing doses of PKI-587 with or without PD0325901 (PD) at 1μM or 5μM. Then, the cells were stimulated for 90 min with 5 nM neurotensin and 10 ng/ml insulin and lysed with SDS–PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting using the antibodies that detect phosphorylated or total proteins, as described in each panel. C: Cultures of MiaPaCa-2 cells were incubated in the absence or presence of 1μM of GDC-0980 for 2 h, and then the cells were stimulated for 15, 30, 60 or 120 min with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) and lysed with SDS–PAGE sample buffer. The extracts were then analyzed as in Panels A and B.

Figure 4. Dual PI3K/mTOR kinase inhibitors and PD0325901 inhibit the proliferation of PDAC cells

A, Single-cell suspensions of MiaPaCa-2 cells were plated at a density of 10⁵ cells per dish. After 24 h, the cultures were shifted to media containing fetal bovine serum (FBS) with 100n NPV-BEZ235 (BEZ), 100 nM PD0325901 (PD) or combination of both drugs as indicated. After 72 h, cell numbers were determined from 6 plates per condition. Results are presented as means ± SEM. B, Single-cell suspensions of PANC-1 cells were plated at a density of 10⁵ cells per dish. After 24 h, the cultures were shifted to media containing FBS with 100 nM NPV-BEZ235 (BEZ), 500 nM PD0325901 (PD) or combination of both drugs as indicated. After 72 h, cell numbers were determined from 6 plates per condition. Results are presented as means ± SEM. C: Single-cell suspensions of MiaPaCa-2 cells were plated at a density of 10⁵ cells per dish. After 24 h, the cultures were shifted to media containing FBS with 100 nM PKI-587 (PKI), 100 nM of GDC-0980 (GDC), 100 nM PD0325901 (PD) and combinations of either PKI-587 or GDC-0980 with PD0325901, as indicated. After 72 h, cell numbers were determined from 6 plates per condition. Results are presented as means ± SEM. D: Cell colony formation was performed as described in the methods section. MiaPaCa-2 cells were incubated for 8 days with 5 nM of NPV-BEZ235 (BEZ), 5 nM PD0325901 (PD) or with a combination of both drugs. E: The bars represent the number of colonies (means ± SEM; n=3 dishes per condition). * T-test p values comparing the indicated 2 groups were < 0.001.

Figure 5. NPV-BEZ235 enhances ERK pathway activation through a pathway that does not require PI3K, EGFR, HER2, insulin receptor or IGF-1R

A: MiaPaCa-2 cells were transiently transfected with a plasmid encoding a fusion protein between GFP and the PH domain of AKT (AKT-PH-GFP). After 24h, the cultures were incubated in DMEM without or with NVP-BEZ235 (BEZ), PKI-587 (PKI) or GDC-0980 (GDC) each at 1μM for 1h prior to stimulation with 5 nM neurotensin and 10 ng/ml insulin. The intracellular distribution of AKT-PH-GFP was monitored under a fluorescence microscope. The selected cells displayed in the figures were representative of 90% of the population of GFP-positive cells. B: Graphic represents quantification from panel A (ratio of membrane/cytoplasm fluorescence). C, D and E: Cultures of MiaPaCa-2 cells were incubated for 2 h in the absence or presence of 1 μM NPV-BEZ235 (BEZ) with or without 1 μM of AG-1478 (AG, panel C), 1 μM of Lapatinib (Lp, panel D) or 1 μM of OSI-906 (OSI, panel E). Then, the cells were stimulated with 5 nM neurotensin and 10 ng/ml insulin and lysed with SDS–PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting with the antibodies that detect phosphorylated or total S6, AKT and ERK proteins, as indicated in each panel.

Figure 6. NPV-BEZ235 enhances ERK activation through a Rictor(mTORC2)-dependent but AKT- independent pathway

A: MiaPaCa-2 cells were transfected with siRNA targeting Rictor or non-targeted siRNA. After 3 days, cells were incubated with 1μM NPV-BEZ235 (BEZ) for 2h. Then, the cells were stimulated for 60 min with 5 nM neurotensin and 10 ng/ml insulin and lysed with SDS–PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. B: Representation of quantification of levels of Rictor protein after transfection with non-targeted siRNA or siRNA targeting Rictor. C: Fold increase of phosphorylated ERK. Quantification of phosphorylated ERK at Thr²⁰² and Tyr²⁰⁴ from 3 independent experiments was performed using Multi Gauge V3.0. D: MiaPaCa-2 cells were incubated for 2 h in the absence or presence of increasing doses of NVP-BEZ235 (BEZ) and then stimulated for 90 min with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) and lysed with SDS–PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting using the antibodies that detect phosphorylated Raf-1 (at Ser²⁵⁹) and ERK or total glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. E MiaPaCa-2 cells were incubated for 2 h in the absence or presence of 1μM NVP-BEZ235 (BEZ) and either MK2206 (MK) at 1μM or 5μM or GDC-0068 (GDC) at 1μM or 5μM, as indicated. The cultures were then stimulated for 60 min with 5 nM neurotensin (NT) and 10 ng/ml insulin (Ins) and lysed with SDS–PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting using the antibodies that detect phosphorylated AKT and ERK or total GAPDH as a loading control. Note that MK2206 (allosteric inhibitor) inhibited AKT phosphorylation whereas GDC0068 (active-site inhibitor) increased AKT phosphorylation, presumably by stabilizing a conformation that prevents AKT dephosphorylation.