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1.  Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2 
PLoS Biology  2009;7(2):e1000038.
The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.
Author Summary
Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that (like all kinases) delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression.
Cells rely on the mammalian target of rapamycin kinase (mTOR) to sense growth factors. Inhibition of all forms of mTOR using newly developed inhibitors of its active site reveals new insights into the function of two mTOR-containing protein complexes and their potential as therapeutic targets.
doi:10.1371/journal.pbio.1000038
PMCID: PMC2637922  PMID: 19209957
2.  Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes 
BMC Cancer  2009;9:363.
Background
Human hemangiosarcoma (HSA) tends to have a poor prognosis; its tumorigenesis has not been elucidated, as there is a dearth of HSA clinical specimens and no experimental model for HSA. However, the incidence of spontaneous HSA is relatively high in canines; therefore, canine HSA has been useful in the study of human HSA. Recently, the production of angiogenic growth factors and their receptors in human and canine HSA has been reported. Moreover, the growth-factor environment of HSA is very similar to that of pathophysiological angiogenesis, which some homeobox genes regulate in the transcription of angiogenic molecules. In the present study, we established 6 xenograft canine HSA tumors and detected the expression of growth factors, their receptors, and angiogenic homeobox genes.
Methods
Six primary canine HSAs were xenografted to nude mice subcutaneously and serially transplanted. Subsequently, the expressions of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factors (bFGF), flt-1 and flk-1 (receptors of VEGF-A), FGFR-1, and angiogenic homeobox genes HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 were investigated in original and xenograft tumors by histopathology, immunostaining, and reverse transcription polymerase chain reaction (RT-PCR), using canine-specific primer sets.
Results
Histopathologically, xenograft tumors comprised a proliferation of neoplastic cells that were varied in shape, from spindle-shaped and polygonal to ovoid; some vascular-like structures and vascular clefts of channels were observed, similar to those in the original tumors. The expression of endothelial markers (CD31 and vWF) was detected in xenograft tumors by immunohistochemistry and RT-PCR. Moreover, the expression of VEGF-A, bFGF, flt-1, flk-1, FGFR-1, HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 was detected in xenograft tumors. Interestingly, expressions of bFGF tended to be higher in 3 of the xenograft HSA tumors than in the other tumors.
Conclusion
We established 6 xenograft canine HSA tumors in nude mice and found that the expressions of angiogenic growth factors and their receptors in xenograft HSAs were similar to those in spontaneous HSA. Furthermore, we detected the expression of angiogenic homeobox genes; therefore, xenograft models may be useful in analyzing malignant growth in HSA.
doi:10.1186/1471-2407-9-363
PMCID: PMC2768746  PMID: 19825192
3.  Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma 
The mammalian target of rapamycin (mTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.
PMCID: PMC3016100  PMID: 21228924
morphoproteomics; phospholipase D1; mTORC2; endometrial carcinoma
4.  Insulin Stimulates Adipogenesis through the Akt-TSC2-mTORC1 Pathway 
PLoS ONE  2009;4(7):e6189.
Background
The signaling pathways imposing hormonal control over adipocyte differentiation are poorly understood. While insulin and Akt signaling have been found previously to be essential for adipogenesis, the relative importance of their many downstream branches have not been defined. One direct substrate that is inhibited by Akt-mediated phosphorylation is the tuberous sclerosis complex 2 (TSC2) protein, which associates with TSC1 and acts as a critical negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Loss of function of the TSC1-TSC2 complex results in constitutive mTORC1 signaling and, through mTORC1-dependent feedback mechanisms and loss of mTORC2 activity, leads to a concomitant block of Akt signaling to its other downstream targets.
Methodology/Principal Findings
We find that, despite severe insulin resistance and the absence of Akt signaling, TSC2-deficient mouse embryo fibroblasts and 3T3-L1 pre-adipocytes display enhanced adipocyte differentiation that is dependent on the elevated mTORC1 activity in these cells. Activation of mTORC1 causes a robust increase in the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), which is the master transcriptional regulator of adipocyte differentiation. In examining the requirements for different Akt-mediated phosphorylation sites on TSC2, we find that only TSC2 mutants lacking all five previously identified Akt sites fully block insulin-stimulated mTORC1 signaling in reconstituted Tsc2 null cells, and this mutant also inhibits adipogenesis. Finally, renal angiomyolipomas from patients with tuberous sclerosis complex contain both adipose and smooth muscle-like components with activated mTORC1 signaling and elevated PPARγ expression.
Conclusions/Significance
This study demonstrates that activation of mTORC1 signaling is a critical step in adipocyte differentiation and identifies TSC2 as a primary target of Akt driving this process. Therefore, the TSC1-TSC2 complex regulates the differentiation of mesenchymal cell lineages, at least in part, through its control of mTORC1 activity and PPARγ expression.
doi:10.1371/journal.pone.0006189
PMCID: PMC2703782  PMID: 19593385
5.  Aberrant hyperactivation of Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in sporadic chordomas 
Statement of Translational Relevance
Inhibitors of mammalian target of rapamycin complex 1 (mTORC1), such as rapamycin and its analogues, are currently being tested in clinical trial for TSC as well as many human cancers, which display hyperactivated mTORC1 signaling. mTORC1 has emerged as a critical integrator of signals from growth factor, nutrient, oxygen, and energy to regulate cell growth and proliferation. This study demonstrates for the first time that mTORC1 signaling is aberrantly hyperactivated in primary chordoma tumors/cell lines and PTEN deficiency may be frequently associated with sporadic chordomas. Furthermore, we show that the mTOR inhibitor rapamycin suppresses mTORC1 signaling and proliferation of chordoma-derived cell line. Therefore, this study not only reveals pathogenic mechanisms of chordomas, but also provides a rationale for initiating clinical trials of Akt/mTORC1 inhibition in patients with sporadic chordomas.
Purpose
Chordomas are rare, malignant bone neoplasms in which the pathogenic mechanisms remain unknown. Interestingly, Tuberous Sclerosis Complex (TSC) is the only syndrome where the incidence of chordomas has been described. We previously reported the pathogenic role of the TSC genes in TSC-associated chordomas. In this study, we investigated whether aberrant TSC/mTORC1 signaling pathway is associated with sporadic chordomas.
Experimental Design
We assessed the status of mTORC1 signaling in primary tumors/cell lines of sacral chordomas and further examined upstream of mTORC1 signaling, including PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor. We also tested the efficacy of the mTOR inhibitor rapamycin on signaling and growth of chordoma cell lines.
Results
Sporadic sacral chordoma tumors and cell lines examined commonly displayed hyperactivated Akt and mTORC1 signaling. Strikingly, expression of PTEN, a negative regulator of mTORC1 signaling, was not detected or significantly reduced in chordoma-derived cell lines and primary tumors. Furthermore, rapamycin inhibited mTORC1 activation and suppressed proliferation of chordoma-derived cell line.
Conclusions
Our results suggest that loss of PTEN as well as other genetic alterations which result in constitutive activation of Akt/mTORC1 signaling may contribute to the development of sporadic chordomas. More importantly, a combination of Akt and mTORC1 inhibition may provide clinical benefits to chordoma patients.
doi:10.1158/1078-0432.CCR-08-2364
PMCID: PMC2701205  PMID: 19276265
chordomas; tuberous sclerosis complex; mTOR; PTEN; Akt
6.  TORC-specific phosphorylation of mTOR: phospho-Ser2481 is a marker for intact mTORC2 
Cancer research  2009;69(5):1821-1827.
The mammalian target of rapamycin (mTOR) Ser/Thr kinase is the catalytic component of two evolutionarily conserved signaling complexes. mTOR signaling complex 1 (mTORC1) is a key regulator of growth factor and nutrient signaling. S6 kinase (S6K) is the best characterized downstream effector of mTORC1. mTOR signaling complex 2 (mTORC2) has a role in regulating the actin cytoskeleton and activating Akt through S473 phosphorylation. Herein we demonstrate that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2 and that intact complexes are required for these mTORC-specific mTOR phosphorylations. Specifically, we find that mTORC1 contains mTOR phosphorylated predominantly on S2448 whereas mTORC2 contains mTOR phosphorylated predominantly on S2481. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, we find that mTORC2 formation is in fact rapamycin-sensitive in several cancer cell lines in which it had been previously reported that mTORC2 assembly and function were rapamycin-insensitive. Thus phospho-S2481 on mTOR serves as a biomarker for intact mTORC2 and its sensitivity to rapamycin.
doi:10.1158/0008-5472.CAN-08-3014
PMCID: PMC2652681  PMID: 19244117
Kinase; phosphorylation; biomarker; rapamycin; cell growth
7.  Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D 
Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.
doi:10.1186/1478-811X-11-3
PMCID: PMC3560233  PMID: 23311350
PDGF; PI3K; mTOR; Rictor; Raptor; Akt; PLC; PKC; PLD; S6
8.  Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR) 
Biochemical Journal  2009;421(Pt 1):29-42.
mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.
doi:10.1042/BJ20090489
PMCID: PMC2708931  PMID: 19402821
Akt/protein kinase B (PKB); cancer; kinase inhibitor; phosphoinositide 3-kinase (PI3K); p70 ribosomal S6 kinase (S6K); serum and glucocorticoid protein kinase (SGK); AGC family, protein kinase A/protein kinase G/protein kinase C family; AMPK, AMP-activated protein kinase; DTT, dithiothreitol; eIF4E, eukaryotic initiation factor 4E; 4E-BP1, eIF4E-binding protein 1; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HEK-293 cell, human embryonic kidney 293 cell; IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NDRG1, N-Myc downstream-regulated gene-1; PDK, 3-phosphoinositide-dependent protein kinase; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRAS40, proline-rich Akt substrate of 40 kDa; RSK, ribosomal S6 kinase; S6K, p70 ribosomal S6 kinase; SGK, serum and glucocorticoid protein kinase; SPHK, sphingosine kinase
9.  Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells 
PLoS ONE  2013;8(7):e69104.
Aims
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.
Methods
We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.
Results
In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.
Conclusion
Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.
doi:10.1371/journal.pone.0069104
PMCID: PMC3712950  PMID: 23874880
10.  Alcohol-Induced Modulation of Rictor and mTORC2 Activity in C2C12 Myoblasts 
Background
The mTOR kinase controls cell growth, proliferation and metabolism through two distinct multi-protein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2 and mTORC1.
Methods
C2C12 myoblasts were incubated with EtOH for 18-24 h. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by 35S-methionine/cysteine incorporation into cellular protein.
Results
EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, PRR5 and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, PRAS40, mSin1, Deptor and GβL. Activated PP2A was associated with decreased Akt and eEF2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the two mTOR complexes following EtOH treatments in myoblasts.
Conclusions
EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the IRS-I/PI3K signaling pathway to regulate protein synthesis.
doi:10.1111/j.1530-0277.2011.01480.x
PMCID: PMC3503252  PMID: 21438886
Ethanol; rictor; shRNA; protein synthesis
11.  Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells 
Respiratory Research  2014;15(1):26.
Background
mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.
Methods
Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.
Results
IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.
Conclusions
In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.
doi:10.1186/1465-9921-15-26
PMCID: PMC3941688  PMID: 24571487
mTOR; mTORC1; mTORC2; E-Cadherin repressor complexes; Raptor; Rictor; EMT
12.  Morphoproteomic Profiling of the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Desmoplastic Small Round Cell Tumor (EWS/WT1), Ewing’s Sarcoma (EWS/FLI1) and Wilms’ Tumor(WT1) 
PLoS ONE  2013;8(7):e68985.
Background
Desmoplastic small round cell tumor (DSRCT) is a rare sarcoma in adolescents and young adults. The hallmark of this disease is a EWS-WT1 translocation resulting from apposition of the Ewing’s sarcoma (EWS) gene with the Wilms’ tumor (WT1) gene. We performed morphoproteomic profiling of DSRCT (EWS-WT1), Ewing’s sarcoma (EWS-FLI1) and Wilms’ tumor (WT1) to better understand the signaling pathways for selecting future targeted therapies.
Methodology
This pilot study assessed patients with DSRCT, Wilms’ tumor and Ewing’s sarcoma. Morphoproteomics and immunohistochemical probes were applied to detect: p-mTOR (Ser2448); p-Akt (Ser473); p-ERK1/2 (Thr202/Tyr204); p-STAT3 (Tyr 705); and cell cycle-related analytes along with their negative controls.
Principal Findings
In DSRCT the PI3K/Akt/mTOR pathway is constitutively activated by p-Akt (Ser 473) expression in the nuclear compartment of the tumor cells and p-mTOR phosphorylated on Ser 2448, suggesting mTORC2 (rictor+mTOR) as the dominant form. Ewing’s sarcoma had upregulated p-Akt and p-mTOR, predominantly mTORC2. In Wilm’s tumor, the mTOR pathway is also activated with most tumor cells moderately expressing p-mTOR (Ser 2448) in plasmalemmal and cytoplasmic compartments. This coincides with the constitutive activation of one of the downstream effectors of the mTORC1 signaling pathway, namely p-p70S6K (Thr 389). There was constitutive activation of the Ras/Raf/ERK pathway p-ERK 1/2 (Thr202/Tyr204) expression in the Wilms tumor and metastatic Ewing’s sarcoma, but not in the DSRCT.
Conclusion
Morphoproteomic tumor analyses revealed constitutive activation of the mTOR pathway as evidenced by: (a) expression of phosphorylated (p)-mTOR, p-p70S6K; (b) mTORC 2 in EWS and DSRCT; (c) ERK signaling was seen in the advanced setting indicating these as resistance pathways to IGF1R related therapies. This is the first morphoproteomic study of such pathways in these rare malignancies and may have potential therapeutic implications. Further study using morphoproteomic assessments of these tumors are warranted.
doi:10.1371/journal.pone.0068985
PMCID: PMC3726753  PMID: 23922674
13.  Combination of mTOR and EGFR Kinase Inhibitors Blocks mTORC1 and mTORC2 Kinase Activity and Suppresses the Progression of Colorectal Carcinoma 
PLoS ONE  2013;8(8):e73175.
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKTS473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFRT1068). The parallel increase of AKTS473 and EGFRT1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.
doi:10.1371/journal.pone.0073175
PMCID: PMC3750018  PMID: 23991179
14.  mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling▿  
Molecular and Cellular Biology  2009;30(4):908-921.
The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.
doi:10.1128/MCB.00601-09
PMCID: PMC2815569  PMID: 19995915
15.  A complex interplay between Akt, TSC2, and the two mTOR complexes 
Biochemical Society transactions  2009;37(Pt 1):217-222.
Akt/PKB both regulates and is regulated by the TSC1-TSC2 complex. Downstream of phosphoinositide 3-kinase (PI3K), Akt directly phosphorylates TSC2 on multiple sites. While the molecular mechanism is not well understood, these phosphorylation events relieve the inhibitory effects of the TSC1-TSC2 complex on Rheb and mTOR complex 1 (mTORC1), thereby activating mTORC1 in response to growth factors. Through negative feedback mechanisms, mTORC1 activity inhibits growth factor stimulation of PI3K. This is particularly evident in cells and tumors lacking the TSC1-TSC2 complex, where Akt signalling is severely attenuated due, at least in part, to constitutive activation of mTORC1. An additional level of complexity in the relationship between Akt and the TSC1-TSC2 complex has recently been uncovered. The growth factor-stimulated kinase activity of mTOR complex 2 (mTORC2 or the mTOR-Rictor complex), which normally enhances Akt signalling by phosphorylating its hydrophobic motif (Ser473), was found to be defective in cells lacking the TSC1-TSC2 complex. This effect on mTORC2 can be separated from the inhibitory effects of the TSC1-TSC2 complex on Rheb and mTORC1. This review discusses our current understanding of the increasingly complex functional interactions between Akt, the TSC1-TSC2 complex and mTOR, which are fundamentally important players in a large variety of human diseases.
doi:10.1042/BST0370217
PMCID: PMC2778026  PMID: 19143635
16.  Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome 
First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo.
Cellular growth is a fundamental process that requires constant adaptations to changing environmental conditions, like growth factor and nutrient availability, energy levels and more. Over the years, the insulin receptor/target of rapamycin pathway (InR/TOR) emerged as a key signaling system for the control of metazoan cell growth. Genetic screens carried out in the fruit fly Drosophila melanogaster identified key InR/TOR pathway components and their relationships. Phenotypes such as altered cell growth are likely to emerge from perturbed dynamic networks containing InR/TOR pathway components, which stably or transiently interact with other cellular proteins to form complexes and networks thereof. Systematic studies on the topology and dynamics of protein interaction networks become therefore highly relevant to gain systems level understanding of deregulated cell growth. Despite much progress in genetic analysis only few systematic protein interaction studies have been reported for Drosophila, which in most cases lack quantitative information representing the dynamic nature of such networks. Here, we present the first quantitative affinity purification mass spectrometry (AP–MS/MS) analysis on the evolutionary conserved InR/TOR signaling network in Drosophila. Systematic RNAi-based functional analysis of identified network components revealed key components linked to the regulation of the central effector kinase dTOR. This includes also dTTT, a novel dTOR-containing complex required for the control of dTORC1 and dTORC2 in vivo.
For systematic AP–MS analysis, we generated Drosophila Kc167 cell lines inducibly expressing affinity-tagged bait proteins previously linked to InR/TOR signaling. Bait expressing Kc167 cell lines were harvested before and after insulin stimulation for subsequent affinity purification. Following LC–MS/MS analysis and probabilistic data filtering using SAINT (Choi et al, 2010), we generated a quantitative network model from 97 high confidence protein–protein interactions and 58 network components (Figure 2). The presented network displayed a high degree of orthologous interactions conserved also in human cells and identified a number of novel molecular interactions with InR/TOR signaling components for future hypothesis driven analysis.
To measure insulin-induced changes within the InR/TOR interaction proteome, we applied a recently introduced label-free quantitative MS approach (Rinner et al, 2007). The obtained quantitative data suggest that 22% of all interactions in the network are regulated by insulin. Major changes could be observed within the membrane proximal InR/chico/PI3K signaling complexes, and also in 14-3-3 protein containing signaling complexes and dTORC1, a complex that contains besides dTOR all major orthologous proteins found also in human mTORC1 including the two dTORC1 substrates d4E-BP (Thor) and S6 Kinase (S6K). Insulin triggered both, dissociation and association of dTORC1 proteins. Among the proteins that showed enhanced binding to dTORC1 upon insulin stimulation we found Unkempt, a RING-finger protein with a proposed role in ubiquitin-mediated protein degradation (Lores et al, 2010). Besides dTORC1 our systematic AP–MS analysis also revealed the presence of dTORC2, the second major TOR complex in Drosophila. dTORC2 contains the Drosophila orthologous of human mTORC2 proteins, but in contrast to dTORC1 was not affected upon insulin stimulation. Interestingly, we also found a specific set of proteins that were not linked to the canonical TOR complexes TORC1 and TORC2 in dTOR purifications. These include LqfR (liquid facets related), Pontin, Reptin, Spaghetti and the gene product of CG16908. We found the same set of proteins when we used CG16908 as a bait, suggesting complex formation among the identified proteins. None of the dTORC1/2 components besides dTOR was identified in CG16908 purifications, indicating that these proteins form dTOR complexes distinct from dTORC1 and dTORC2. Based on known interaction information from other species and data obtained from this study we refer to this complex as dTTT (Drosophila TOR, TELO2, TTI1) (Horejsi et al, 2010; [18]Hurov et al, 2010; [20]Kaizuka et al, 2010). A directed quantitative MS analysis of dTOR complex components suggests that dTORC1 is the most abundant dTOR complex we identified in Kc167 cells.
We next studied the potential roles of the identified network components for controlling the activity of the dInR/TOR pathway using systematic RNAi depletion and quantitative western blotting to measure the changes in abundance of phosphorylated substrates of dTORC1 (Thor/d4E-BP, dS6K) and dTORC2 (dPKB) in RNAi-treated cells (Figure 5). Overall, we could identify 16 proteins (out of 58) whose depletion caused an at least 50% increase or decrease in the levels of phosphorylated d4E-BP, S6K and/or PKB compared with control GFP RNAi. Besides established pathway components, we found several novel regulators within the dInR/TOR interaction network. For example, RNAi against the novel insulin-regulated dTORC1 component Unkempt resulted in enhanced phosphorylation of the dTORC1 substrate d4E-BP, which suggests a negative role for Unkempt on dTORC1 activity. In contrast, depletion of CG16908 and LqfR caused hypo-phosphorylation of all dTOR substrates similar to dTOR itself, suggesting a positive role for the dTTT complex on dTOR activity. Subsequently, we tested whether dTTT components also plays a role in dTOR-mediated cell growth in vivo. Depletion of both dTTT components, CG16908 and LqfR, in the Drosophila eye resulted in a substantial decrease in eye size. Likewise, FLP-FRT-mediated mitotic recombination resulted in CG16908 and LqfR mutant clones with a similar reduced growth phenotype as observed in dTOR mutant clones. Hence, the combined biochemical and genetic analysis revealed dTTT as a dTOR-containing complex required for the activity of both dTORC1 and dTORC2 and thus plays a critical role in controlling cell growth.
Taken together, these results illustrate how a systematic quantitative AP–MS approach when combined with systematic functional analysis in Drosophila can reveal novel insights into the dynamic organization of regulatory networks for cell growth control in metazoans.
Using quantitative mass spectrometry, this study reports how insulin affects the modularity of the interaction proteome of the Drosophila InR/TOR pathway, an evolutionary conserved signaling system for the control of metazoan cell growth. Systematic functional analysis linked a significant number of identified network components to the control of dTOR activity and revealed dTTT, a dTOR complex required for in vivo cell growth control by dTORC1 and dTORC2.
Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.
doi:10.1038/msb.2011.79
PMCID: PMC3261712  PMID: 22068330
cell growth; InR/TOR pathway; interaction proteome; quantitative mass spectrometry; signaling
17.  Dual phosphorylation of Sin1 at T86 and T398 negatively regulates mTORC2 complex integrity and activity 
Protein & Cell  2014;5(3):171-177.
Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.
doi:10.1007/s13238-014-0021-8
PMCID: PMC3967077  PMID: 24481632
18.  Dual phosphorylation of Sin1 at T86 and T398 negatively regulates mTORC2 complex integrity and activity 
Protein & Cell  2014;5(3):171-177.
Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.
doi:10.1007/s13238-014-0021-8
PMCID: PMC3967077  PMID: 24481632
19.  Signaling events downstream of mTOR complex 2 are attenuated in cells and tumors deficient for the tuberous sclerosis complex tumor suppressors 
Cancer research  2009;69(15):6107-6114.
Mutations in TSC1 and TSC2 tumor suppressor genes give rise to the neoplastic disorders tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM). Their gene products form a complex that is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and cell growth. We recently found that the TSC1-TSC2 complex promotes the activity of mTOR complex 2 (mTORC2), an upstream activator of Akt, and this occurs independent of its inhibitory effects on mTORC1. Loss of mTORC2 activity in cells lacking the TSC1-TSC2 complex, coupled with mTORC1-mediated feedback mechanisms, leads to strong attenuation of the growth factor-stimulated phosphorylation of Akt on S473. In this study, we demonstrate that both PI3K-dependent and independent mTORC2 substrates are affected by loss of the TSC1-TSC2 complex in cell culture models and kidney tumors from both Tsc2+/− mice (i.e., adenoma) and TSC patients (i.e., angiomyolipoma). These mTORC2 targets are all members of the AGC kinase family and include Akt, protein kinase C (PKCα), and serum and glucocorticoid-induced protein kinase (SGK1). We also demonstrate that the TSC1-TSC2 complex can directly stimulate the in vitro kinase activity of mTORC2. The interaction between these two complexes is mediated primarily through regions on TSC2 and a core component of mTORC2 called Rictor. Hence, loss of the TSC tumor suppressors results in elevated mTORC1 signaling and attenuated mTORC2 signaling. These findings suggest that the TSC1-TSC2 complex plays opposing roles in tumor progression, both blocking and promoting specific oncogenic pathways through its effects on mTORC1 inhibition and mTORC2 activation, respectively.
doi:10.1158/0008-5472.CAN-09-0975
PMCID: PMC2735013  PMID: 19602587
20.  TARGETED INHIBITION OF mTOR SIGNALING INHIBITS TUMORIGENESIS OF COLORECTAL CANCER 
PURPOSE
The mammalian target of rapamycin (mTOR) kinase acts downstream of PI3K/Akt to regulate cellular growth, metabolism and cytoskeleton. Since approximately 60% of sporadic colorectal cancers (CRCs) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs.
EXPERIMENTAL DESIGN
HCT116, KM20, Caco-2 and SW480 human CRC cells were utilized to determine the effects of pharmacological (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression and subcutaneous growth in vivo
RESULTS
We show that the mTOR complex proteins, mTOR, Raptor and Rictor, are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of some CRC cell lines (rapamycin-sensitive), while other cell lines were resistant to its effects (rapamycin-resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, while only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycin-sensitive and -resistant CRCs in vivo when implanted as tumor xenografts.
CONCLUSIONS
Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC.
doi:10.1158/1078-0432.CCR-09-1249
PMCID: PMC2898570  PMID: 19934294
mTOR; Raptor; Rictor; Akt; rapamycin; colorectal cancer
21.  The mTOR Pathway Controls Cell Proliferation by Regulating the FoxO3a Transcription Factor via SGK1 Kinase 
PLoS ONE  2014;9(2):e88891.
The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.
doi:10.1371/journal.pone.0088891
PMCID: PMC3928304  PMID: 24558442
22.  Rictor Phosphorylation on the THR-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2 
Molecular cancer research : MCR  2010;8(6):896-906.
In animal cells growth factors coordinate cell proliferation and survival by regulating the PI3K/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K dependent signaling kinase complex defined as mTORC2 functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to inhibition of PI3K, mTOR, or expression of ILK. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt indicating that the rictor Thr-1135 phosphorylation is not critical in regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells suggesting that this modification might play a role in regulation not only mTORC2 but also the mTORC2-independent function of rictor.
doi:10.1158/1541-7786.MCR-09-0409
PMCID: PMC2896829  PMID: 20501647
23.  The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention 
Background
Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs in vitro.
Results
We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell line-dependent.
Conclusion
This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.
doi:10.1186/1746-6148-8-73
PMCID: PMC3515332  PMID: 22647622
Canine; Cancer; PI3; AKT; MTOR; Therapeutic; Target
24.  Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin▿  
Molecular and Cellular Biology  2008;29(6):1411-1420.
mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin—with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.
doi:10.1128/MCB.00782-08
PMCID: PMC2648237  PMID: 19114562
25.  Constitutive activation with overexpression of the mTORC2-phospholipase D1 pathway in uterine leiomyosarcoma and STUMP: morphoproteomic analysis with therapeutic implications 
The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for therapy and drug development. Biologically, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR complex 1 (mTORC1) which is predominantly cytoplasmic in subcellular localization and mTOR complex 2 (mTORC2) which is both cytoplasmic and nuclear. mTORC1 is sensitive to the selective inhibitor rapamycin. By contrast, mTORC2 is relatively resistant to rapamycin. Moreover, its putative downstream effector, Akt phosphorylated on serine 473 represents a signal transduction pathway for tumor survival. Phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as an activator of mTOR signaling, including the direct phosphorylative activation of p70S6K atthreonine 389. The latter promotes cell cycle progression. In this study, we investigated the activation status and subcellular localization of mTOR and the relative expression of PLD1, as well as their downstream effectors in a spectrum of uterine smooth muscle tumors using normal myometria as controls. The results show significant activation with overexpression of phosphorylated mTORC2 complex in uterine leiomyosarcoma (ULMS) and smooth muscle tumors of uncertain malignant potential (STUMP) as evidenced by nuclear localization of p-mTOR (Ser 2448) in ULMS>STUMP>uterine leiomyoma and normal myometria (p<0.05) and with overexpression of PLD1(p<0.05). Cor-relatively, there are overexpressions of nuclear p-Akt (Ser 473) and nuclear p-p70S6K (Thr 389) in ULMS and STUMP (p<0.05). The activation with overexpression of components of the mTORC2-PLD1 pathway in ULMS and to a lesser degree in STUMP provides insight into their tumorigenic mechanisms. Thus the development of therapies designed to target mTORC2 and PLD1 activity may be beneficial in treating ULMS.
PMCID: PMC3037199  PMID: 21326806
Morphoproteomics; mTORC2; phospholipase D1; uterine leiomyosarcoma; STUMP

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