Ming

Dual PI3K/mTOR inhibitors induce rapid over-activation of the MEK/ERK pathway in human pancreatic cancer cells through suppression of mTORC2

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 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, 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 Ser473, 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. Mol Cancer Ther; 14(4); 1014–23. ©2015 AACR.

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling

We examined whether class IIa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1 (PKD1) in IEC-18 intestinal epithelial cells. Our results show that class IIa HDAC4, HDAC5, and HDAC7 are prominently expressed in these cells. Stimulation with ANG II, a potent mitogen for IEC-18 cells, induced a striking increase in phosphorylation of HDAC4 at Ser(246) and Ser(632), HDAC5 at Ser(259) and Ser(498), and HDAC7 at Ser(155). Treatment with the PKD family inhibitors kb NB 142-70 and CRT0066101 or small interfering RNA-mediated knockdown of PKD1 prevented ANG II-induced phosphorylation of HDAC4, HDAC5, and HDAC7. A variety of PKD1 activators in IEC-18 cells, including vasopressin, lysophosphatidic acid, and phorbol esters, also induced HDAC4, HDAC5, and HDAC7 phosphorylation. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5 with Ser(259) and Ser(498) mutated to Ala was localized to the nucleus in unstimulated and stimulated cells. Treatment of IEC-18 cells with specific inhibitors of class IIa HDACs, including MC1568 and TMP269, prevented cell cycle progression, DNA synthesis, and proliferation induced in response to G protein-coupled receptor/PKD1 activation. The PKD1-class IIa HDAC axis also functions in intestinal epithelial cells in vivo, since an increase in phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of crypt cells from PKD1 transgenic mice compared with matched nontransgenic littermates. Collectively, our results reveal a PKD1-class IIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling.

Abstract 670: Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Significant attention has been given to molecular target therapies in pancreatic cancer due to the limited survival benefits provided by standard chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway which is aberrantly stimulated in pancreatic ductal adenocarcinoma (PDAC) has emerged as a target for therapy. Inhibitors of PI3K and/or mTOR (PI3K/TOR-KIs) are been developed to target this pathway. 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 a panel of pancreatic ductal adenocarcinoma (PDAC) cells including PANC-1, MiaPaCa-2, AsPC-1 and BxPC-3 with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) NPV-BEZ23 induces a novel negative feedback loop leading to increased activity of MEK/ERK pathway in PDAC cells. Exposure of the above mentioned PDAC cells to NPV-BEZ235 potently blocked mTORC1 activation 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, NPV-BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK over-activation coincided with complete inhibition of phosphorylation of Akt and 4E-BP1. Other PI3K/TOR-KIs, including PKI-587 and GDC-0980 also induced ERK over-activation. The MEK inhibitors PD0325901 or Trametinib prevented ERK over-activation induced by PI3K/TOR-KIs at least acutely. The combination of NPV-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 using a PIP3 fluorescent reporter indicated that NPV-BEZ235, PKI-587 and GDC-0980 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 Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. 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. The therapeutic effectiveness of PI3K/TOR-KIs inhibitors in PDAC and possibly other malignancies can be diminished by activation of MEK/ERK that opposes their anti-proliferative effects and leads to drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Heloisa P. Soares, Ming Ming, Michelle Mellon, Steven H. Young, Liang Han, James Sinnet-Smith, Enrique Rozengurt. Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 670. doi:10.1158/1538-7445.AM2015-670

Abstract A109: Dual mTOR/PI3K inhibitors induce compensatory MEK/ERK activation in human pancreatic cancer cells: A mechanism contributing to drug resistance

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.

Tu1915 Suppression of the mTOR/PI3K Pathway Promotes ERK Pathway Activation in Human Pancreatic Cancer Cells

Background: The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway plays a pivotal role in the proliferation and survival of pancreatic ductal adenocarcinoma (PDAC) cells and is aberrantly activated in pancreatic cancer tissues. In addition to growth-promoting signaling, mTORC1/S6K 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 overactivation of the PI3K/Akt pathway that potentially oppose 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). Recently, we reported that TOR-KIs induce an unexpected increase in the activity of the ERK pathway in PDAC cells through a PI3K-independent pathway. Here, we examined whether PI3K/TOR-KIs also induce ERK pathway over-activation in PDAC cells. Results: To determine the effect of dual PI3K/TOR inhibition on ERK activation, we treated serum-starved cultures of PDAC cells (MiaPaca-2 and PANC-1) with increasing concentrations of the dual PI3K/TOR-KI NPVBEZ235 for 2 h followed by stimulation with insulin and neurotensin, a potent mitogenic combination for these cells. As expected, prior exposure to NPV-BEZ235 potently blocked 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 increase in 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 100500 ηM NPV-BEZ235). ERK over-activation was also seen when PDAC cells were stimulated with 2% fetal bovine serum instead of insulin and neurotensin 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. 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 NPV-BEZ235, PD0325901 or a combination of NPV-BEZ235 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.