An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1

Abstract

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.

FIGURE 1.

Torin1 is a potent and selective mTOR inhibitor. A, Torin1 inhibits mTORC1 and mTORC2 in vitro. mTORC1 and mTORC2 were purified from HEK-293T stably expressing FLAG-Raptor and HeLa cells expressing FLAG-Protor-1, respectively. Following FLAG purification, each complex was subjected to in vitro kinase assays using S6K1 as a substrate for mTORC1 and Akt1 as a substrate for mTORC2. Assays were then analyzed by immunoblotting for the indicated proteins and phosphorylation states. B, Torin1 is an ATP-competitive inhibitor. The in vitro kinase activity of purified mTORC1 toward S6K1 was assayed in the presence of 20 nm Torin1 and increasing concentrations of ATP, as indicated. Assays were then analyzed by immunoblotting for the indicated proteins and phosphorylation states. C, Torin1 is a potent mTORC1 and mTORC2 inhibitor in cells. MEFs (p53^-/-) were treated with increasing concentrations of Torin1 or dual mTOR/PI3K inhibitors PI-103 and BEZ-235 for 1 h and then analyzed by immunoblotting for the indicated proteins and phosphorylation states. D, Torin1 has little effect on PI3K at concentrations where mTOR is completely inhibited. The experiment was performed as in C using mLST8-null MEFs and phosphorylation of Akt at Thr-308 was determined by immunoblotting. In mLST8-null MEFs, mTORC2 is inactive and Akt Ser-473 is constitutively dephosphorylated and so PDK1-mediated phosphorylation of Thr-308 only reflects PI3K activity. E, Torin1 is selective for mTOR over related kinases. IC₅₀ values for Torin1 were determined using in vitro kinase assays for mTOR (3 nm), hVps34 (3 μm), PI3K-α (1.8 μm), DNA-PK (1.0 μm), and ATM (0.6 μm). IC₅₀ values for PI-103 for mTOR (120 nm), PI3K-α (100 nm), DNA-PK (40 nm) were determined by the same assays. IC₅₀ values for PI-103 for hVps34 (10 μM) and ATM (1.0 μm) were determined previously (21). F, Torin1 is selective for mTOR over other PI3K isoforms. EC₅₀ values were determined for the indicated PI3K isoforms using the Invitrogen Adapta platform. The EC₅₀ for mTOR was determined using the cell-based LanthaScreen platform.

FIGURE 2.

mTOR inhibition prevents cell growth and proliferation through an mTORC2-independent mechanism. A, mTOR inhibition by Torin1 but not rapamycin prevents the proliferation of wild-type MEFs. MEF (p53^-/-) cells were grown in the presence of vehicle (blue), 50 nm rapamycin (orange), or 250 nm Torin1 (green) for 4 days. Cell proliferation was measured in triplicate at indicated time points using the CellTiterGlo viability assay. B, Torin1 causes a G₁/S cell cycle arrest in wild-type MEFs. MEF (p53^-/-) cells were treated with vehicle (DMSO), 50 nm rapamycin (rapa), or 250 nm Torin1 for 48 h. Cells were then harvested, stained with propidium iodide, and analyzed by flow cytometry. C, normalized cell size distributions for Torin1 and rapamycin-treated wild-type MEFs. MEF (p53^-/-) cells were treated with vehicle (blue, mean 17.81 μm), 50 nm rapamycin (orange, mean 17.58), or 250 nm Torin1 (green, mean 16.46 μm) for 24 h. Cell sizes were measured using a particle counter and are displayed as a histogram. D, experiment was performed as in A using Rictor^-/-, p53^-/- MEFs. E, experiment was performed as in B using Rictor^-/-, p53^-/- MEFs. F, experiment was performed as in C using Rictor^-/-, p53^-/- MEFs. Cells were treated with vehicle (blue, mean diameter 17.85 μm), 50 nm rapamycin (orange, mean diameter 17.33 μm), or 250 nm Torin1 (green, mean diameter 16.24 μm).

FIGURE 3.

Torin1 inhibits mTORC1-dependent processes that are resistant to rapamycin. A, Torin1 but not rapamycin (Rapa) causes LC3 to relocalize from the cytoplasm to autophagosomes. Wild-type (p53^-/-) or Rictor-null (p53^-/-) MEFs were transiently transfected with GFP-LC3 and treated with vehicle (Veh) (DMSO), 50 nm rapamycin, or 250 nm Torin1 for 3 h before being fixed and processed. Cells were also stained with Hoechst to visualize nuclei and imaged at ×63. B, amino acid starvation and Torin1, but not rapamycin, cause LC3 degradation. Wild-type (p53^-/-) and Rictor-null (p53^-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin, 250 nm Torin1 or grown in amino acid (AA)-free conditions for 0, 1, 3, or 6 h. Cells were lysed at the indicated time points and analyzed by immunoblotting. Induction of autophagy causes the degradation of the native LC3B (LC3B-I) protein and the transient accumulation of the faster running lipidated version (LC3B-II). C, Torin1 suppresses global protein synthesis through a rapamycin-resistant and mTORC2-independent process. Wild-type (p53^-/-) and Rictor-null (p53^-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin (Rap), 250 nm Torin1, or 10 μg/ml cycloheximide (Chx) for 2.5 h and then pulsed with ³⁵S-labeled methionine and cysteine for 30 min. The amount of ³⁵S incorporation was determined by scintillation counting. Measurements were made in triplicate, and error bars indicate standard deviation.

FIGURE 4.

mTORC1 regulation of 4E-BP1 phosphorylation and binding to eIF-4E reveals rapamycin-resistant functions. A, phosphorylation of 4E-BP1 at Thr-37/46 and Ser-65 is dependent on mTORC1 but resistant to rapamycin. Wild-type (p53^-/-) and Rictor-null (p53^-/-) MEFs were treated with the indicated concentrations of Torin1 or rapamycin for 1 h and then lysed. Cell lysates were analyzed by immunoblot using antibodies specific for the indicated proteins or phosphorylation states. B, Torin1 increases the amount of 4E-BP1 bound to eIF-4E to a degree that far exceeds the effects of rapamycin. Wild-type (p53^-/-) and Rictor-null (p53^-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin, or 250 nm Torin1 for 1 h before lysis. eIF-4E was purified from lysates using 7-methyl-GTP-Sepharose and analyzed by immunoblotting for the indicated proteins. C, phosphorylation of Thr-36/47 on 4E-BP1 requires Raptor but not Rictor. MEFs (p53^-/-) were infected with lentivirus expressing either control, Raptor-specific, or Rictor-specific shRNAs. Cells were grown for 4 days and then treated with vehicle (DMSO), 50 nm rapamycin, or 250 nm Torin1 for 1 h. Cell lysates were then analyzed by immunoblot using antibodies specific for the indicated proteins or phosphorylation states. D, prolonged mTOR inhibition alters the expression of key cell cycle regulators. Wild-type (p53^-/-) and Rictor-null (p53^-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin, or 250 nm Torin1 for 48 h. Cell lysates were then analyzed by immunoblotting using antibodies specific for the indicated proteins. E, Torin1 prevents phosphorylation of rapamycin-resistant sites in human cancer cell lines. MCF7, HCT116, HeLa, and HEK-293T cell lines were treated with vehicle (Veh), rapamycin (Rap) (50 or 250 nm), or Torin1 (50 or 250 nm) for 1 h and then analyzed by immunoblotting for the indicated proteins and phosphorylation states. F, Torin1 increases the amount of 4E-BP1 bound to eIF-4E in human cancer cell lines. MCF7 and HCT116 cells were treated with vehicle (DMSO), 50 nm rapamycin, 50 nm Torin1, or 250 nm Torin1 for 48 h before lysis. eIF-4E was purified from lysates using 7-methyl-GTP-Sepharose and analyzed by immunoblotting for the indicated proteins.