The cells were washed twice with DMEM containing 5 mM glucose and incubated in serum-free medium for 4 h and then treated as explained in individual experiments. Akt was used to verify equivalent gel loading.(TIF) pone.0057289.s001.tif (236K) GUID:?18391E09-0857-44D1-9B2B-FC54C626A8C5 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 bad opinions loops that attenuate signaling via insulin/IGF receptor and additional 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 malignancy 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 possess any stimulatory effect on ERK activation. The results imply that 1st 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 concern 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 providers JNJ7777120 potently inhibited the mTORC1/S6K axis. Intro The mammalian target of rapamycin (mTOR) is definitely a highly evolutionarily conserved protein kinase that takes on a key part in the integration of growth element, nutrient and energy status of the cells [1]. mTOR functions like a catalytic subunit in two unique multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1, characterized by the regulatory subunit Raptor, settings at least two regulators of protein synthesis, the 40S ribosomal protein subunit S6 kinase (S6K) and the eukaryotic translation initiation element 4E (eIF4E)-binding protein 1, referred as 4E-BP1 [1], [2]. The heterodimer of the tumor suppressor TSC2 (tuberin) and TSC1 (hamartin) represses mTORC1 signaling by acting as the GTPase-activator protein for the small G protein Rheb (Ras homolog enriched in mind), a potent activator of mTORC1 signaling in its GTP-bound state [3], [4]. Phosphorylation of TSC2 by Akt and/or ERK/p90RSK suppresses its GTPase activating activity towards Rheb, leading to mTORC1 activation [5]. mTORC1 is definitely acutely and allosterically inhibited by rapamycin through binding to FKBP12. mTORC2, characterized by Rictor, is not inhibited by short-term treatment with this agent and phosphorylates several AGC protein kinases, including Akt at Ser473 [6], [7]. The mTORC1 pathway plays a key part in insulin/IGF receptor signaling [8], [9] and is aberrantly activated in many cancers, including pancreatic ductal adenocarcinoma (PDAC), probably one of the most lethal human being diseases. Accordingly, PDAC cells communicate insulin and IGF-1 receptors and over-express IRS-1 and IRS-2 [10]C[12] and PDAC (but not normal) tissue display triggered (phosphorylated) IGF-1R [13]. Gene variations in the IGF-1 signaling system have been connected to worse survival in individuals with PDAC [14]. Inactivation of p53, as seen during the progression of 50C70% of PDAC, up-regulates the insulin/IGF-1/mTORC1 pathway [15]. Crosstalk between insulin/IGF-1 receptors and G protein-coupled receptor (GPCR) signaling systems potently stimulate mTORC1, DNA synthesis and cell proliferation inside a panel of PDAC cells [16]C[20]. JNJ7777120 mTORC1 signaling takes on a pivotal part in the proliferation and survival of PDAC cells [21] and is triggered in pancreatic malignancy cells [20], [22]C[24]. As a result, mTORC1 has emerged as a stylish therapeutic target in PDAC and additional common malignancies. In addition to growth-promoting signaling, mTORC1/S6K also mediates bad opinions loops that restrain signaling through insulin/IGF receptor and additional tyrosine kinase receptors via phosphorylation and transcriptional repression of IRS-1 [25]C[30] and phosphorylation of Grb10 [31], [32]. As a result, suppression of mTORC1 activity by rapamycin prevents inhibitory IRS-1 phosphorylations and degradation, therefore augmenting PI3K/Akt activation in several malignancy cell types [30], [33]C[35]. These studies imply that the potential anti-cancer activity of rapamycin (or analogs) can be counterbalanced by launch of opinions inhibition of PI3K/Akt activation [25], [30], [33]C[35]. Furthermore, rapamycin incompletely inhibits 4E-BP-1 phosphorylation [36]C[40]. Accordingly, the medical antitumor activity of rapamycin and its analogs (rapalogs) has been rather limited in many types of malignancy [41], [42], including PDAC [43], [44]. In an effort to target the mTOR pathway more effectively, novel inhibitors of mTOR that take action in the catalytic active site (active-site mTOR inhibitors) have been recognized, including PP242 [37], Torin [45], KU63794 [38] and its analogue AZD8055 [46]. These compounds inhibit 4E-BP-1 phosphorylation at rapamycin-resistant sites (e.g. Thr37/46) and block Akt phosphorylation at.The precise consequence of suppression of negative feedback loops mediated from the mTORC1/S6K axis in response to metformin remains poorly defined and, in particular, it is not known whether rapamycin, active-site mTOR inhibitors and metformin lead to over-activation of similar upstream pathways in PDAC cells. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic malignancy cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin, a combination of insulin and the GPCR agonist neurotensin or serum. in many malignancy cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates bad opinions loops that attenuate signaling via insulin/IGF receptor and additional 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 malignancy 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 possess any stimulatory effect on ERK activation. The results imply that 1st 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 concern in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating JNJ7777120 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. Therefore, 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 providers potently inhibited the mTORC1/S6K axis. Intro The mammalian target of rapamycin (mTOR) is definitely a highly evolutionarily conserved protein kinase that takes on a key part in the integration of growth element, nutrient and energy status of the cells [1]. mTOR functions like a catalytic subunit in two unique multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1, characterized by the regulatory subunit Raptor, settings at least two regulators of protein synthesis, the 40S ribosomal protein subunit S6 kinase (S6K) and the eukaryotic translation initiation element 4E (eIF4E)-binding protein 1, referred as 4E-BP1 [1], [2]. The heterodimer of the tumor suppressor TSC2 (tuberin) and TSC1 (hamartin) represses mTORC1 signaling by acting as the GTPase-activator protein for the small G protein Rheb (Ras homolog enriched in mind), a potent activator of mTORC1 signaling in its GTP-bound state [3], [4]. Phosphorylation of TSC2 by Akt and/or ERK/p90RSK suppresses its GTPase activating activity towards Rheb, leading to mTORC1 activation [5]. mTORC1 is definitely acutely and allosterically inhibited by rapamycin through binding to FKBP12. mTORC2, characterized by Rictor, is not inhibited by short-term treatment with this agent and phosphorylates several AGC protein kinases, including Akt at Ser473 [6], [7]. The mTORC1 pathway plays a key part in insulin/IGF receptor signaling [8], [9] and is aberrantly activated in many cancers, including pancreatic ductal adenocarcinoma (PDAC), probably one of the most lethal human being diseases. Accordingly, PDAC cells communicate insulin and IGF-1 receptors and over-express IRS-1 and IRS-2 [10]C[12] and PDAC (but not normal) tissue display triggered (phosphorylated) IGF-1R [13]. Gene variations in the IGF-1 signaling system have been connected to worse survival in individuals with PDAC [14]. Inactivation of p53, as seen during the progression of 50C70% of PDAC, up-regulates the insulin/IGF-1/mTORC1 pathway [15]. Crosstalk between insulin/IGF-1 receptors and G protein-coupled receptor (GPCR) signaling systems potently stimulate mTORC1, DNA synthesis and cell proliferation inside a panel of PDAC cells [16]C[20]. mTORC1 signaling takes on a pivotal part in the proliferation and survival of PDAC cells [21] and is triggered in pancreatic malignancy cells [20], [22]C[24]. As a result, mTORC1 has emerged as a stylish therapeutic target in PDAC and additional common malignancies. In addition to growth-promoting signaling, mTORC1/S6K also mediates bad opinions loops that restrain signaling through insulin/IGF receptor and additional tyrosine kinase receptors via phosphorylation and transcriptional repression of IRS-1 [25]C[30] and phosphorylation of Grb10 [31], [32]. As a result, suppression of mTORC1 activity by rapamycin prevents inhibitory IRS-1 Mouse monoclonal to SHH phosphorylations and degradation, thereby augmenting.