TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFα and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

We conclude that oral sirolimus markedly delays the loss of renal function and retards cyst development in Han:SPRD rats with ADPKD. Our data also suggest that activation of the S6K signalling pathway plays an important role in the pathogenesis of PKD. Sirolimus could be a useful drug to retard progressive renal failure in patients with ADPKD.

Abstract-A central regulator of cell growth that has been implicated in responses to stress such as hypoxia is mTOR (mammalian Target Of Rapamycin). We have shown previously that mTOR is required for angiogenesis in vitro and endothelial cell proliferation in response to hypoxia. Here we have investigated mTOR-associated signaling components under hypoxia and their effects on cell proliferation in rat aortic endothelial cells (RAECs). Hypoxia (1% O 2 ) rapidly (Ͼ30 minutes) and in a concentration-dependent manner promoted rapamycin-sensitive and sustained phosphorylation of mTOR-Ser2448 followed by nuclear translocation in RAECs. Similarly, hypoxia induced phosphorylation of the mTORC2 substrate Akt-Ser473 (3 to 6 hours at 1% O 2 ) and a brief phosphorylation peak of the mTORC1 substrate S6 kinase-Thr389 (10 to 60 minutes). Phosphorylation of Akt was inhibited by mTOR knockdown and partially with rapamycin. mTOR knockdown, rapamycin, or Akt inhibition specifically and significantly inhibited proliferation of serum-starved RAECs under hypoxia (PϽ0.05; nՆ4). Similarly, hypoxia induced Akt-dependent and rapamycinsensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1␣ knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (PϽ0.001; nϭ7), and mTORC1 inhibition by raptor silencing, partially (PϽ0.05; nϭ7), inhibited hypoxia-induced RAEC proliferation. Thus, mTOR mediates an early response to hypoxia via mTORC1 followed by mTORC2, promoting endothelial proliferation mainly via Akt signaling. mTORC1 and especially mTORC2 might therefore play important roles in diseases associated with hypoxia and altered angiogenesis. (Circ Res. 2007;100:79-87.)

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The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway-addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system.

Proteins encoded by oncogenes represent suitable drug targets and several have been validated clinically. However, certain non-oncogenes, which are downstream effectors of oncogenes, may be similarly useful as potential targets of therapy. We have developed a high-throughput strategy using a shRNA genomic library and tumor cells addicted to the mTOR pathway to identify genes (oncogenes or non-oncogenes) which control the oncogenic phenotype. mTOR addicted cells were generated from IL-3 dependent cells following frame-shift mutagenesis and selection for IL-3 independence. mTOR addicted cells obtained from this selection carried frame-shift mutations in the PTEN gene. mTOR addiction was revealed by sensitivity to rapamycin and confirmed biochemically by examining phosphorylation of the mTOR targets S6K and PKB. Interestingly (and of importance to our shRNA-based screening strategy), addition of IL-3 rescued cells from mTOR addiction, as this growth factor antagonized the apoptotic effect of rapamycin. Using robotics (BioMek-NX) and a genomic retroviral shRNA library we evaluated 14’000 genes for a potential role in maintaining mTOR addicted growth. Our strategy was to screen cells in the presence and absence of IL-3 to identify shRNAs that antagonize growth of cells in the absence of IL-3. We identified around 300 genes required for mTOR addicted growth. This set of genes was enriched in highly significant manner for genes known to be involved in cancer, providing proof of concept for the strategy. Examples of identified genes include ras, raf, PKB and mTOR. Also, genes with no cancer link and genes with unknown function were identified. Use of Ingenuity software allowed to place many of the identified genes into functionally distinct groups or pathways. In addition numerous hits affecting metabolic functions, redox functions, and ROS generating systems were identified including the cytoplasmic NADPH complex and complex I-IV of the respiratory chain. Combining the genes et with an Affymetrix gene overexpression analysis in the same cells and analysing by Oncomine software, we identified a glioblastoma gene signature consisting of 13 genes (p-value 2.4E-5) containing 3 oncogenes and several signaling elements. Downregulation of several of these genes in human cancer lines induced apoptosis. Some of he genes identified represent novel potential drug targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2197.