* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
[CANCER RESEARCH 63, 8451– 8460, December 1, 2003] Differential Effects of Rapamycin on Mammalian Target of Rapamycin Signaling Functions in Mammalian Cells Aimee L. Edinger,1 Corinne M. Linardic,2 Gary G. Chiang,3 Craig B. Thompson,1 and Robert T. Abraham3 1 Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania; 2Departments of Pediatrics and Pharmacology and Cancer Biology, Duke University, Durham, North Carolina; and 3Program in Signal Transduction Research, The Burnham Institute, La Jolla, California ABSTRACT Rapamycin and its analogues have shown promising anticancer activities in preclinical and clinical studies. However, the mechanism whereby rapamycin inhibits signaling through the mammalian target of rapamycin (mTOR) remains poorly understood. Here, we show that the FKBP12/ rapamycin complex is an essentially irreversible inhibitor of mTOR kinase activity in vitro. However, we observe no suppression of mTOR catalytic activity after immunoprecipitation from rapamycin-treated cells. These results suggest either that rapamycin acts as a reversible kinase inhibitor in intact cells or that the cellular effects of rapamycin are not mediated through global suppression in mTOR kinase activity. To better understand the cellular pharmacology of rapamycin, we compared the individual and combined effects of rapamycin and kinase-inactive mTOR expression on a panel of mTOR-dependent cellular responses. These studies identified glycolytic activity, amino acid transporter trafficking, and Akt kinase activity as novel, mTOR-modulated functions in mammalian cells. Whereas kinase-inactive mTOR did not enhance the decreases in cell size and glycolysis induced by rapamycin, expression of this mTOR mutant significantly enhanced the inhibitory effects of rapamycin on cell proliferation, 4EBP1 phosphorylation, and Akt activity. Unexpectedly, amino acid transporter trafficking was perturbed by kinase-inactive mTOR but not by rapamycin, indicating that this process is rapamycin insensitive. These results indicate that rapamycin exerts variable inhibitory actions on mTOR signaling functions and suggest that direct inhibitors of the mTOR kinase domain will display substantially broader anticancer activities than rapamycin. INTRODUCTION The TOR4 proteins were first identified during a screen for mutations that suppressed the growth-inhibitory effects of rapamycin in budding yeast (1– 4). TOR homologs were subsequently identified in flies and mammals and, like the yeast TORs, shown to be the relevant targets of rapamycin in these organisms (5–10). Based on its exquisite specificity for TOR as an intracellular target, rapamycin has been broadly used as a chemical probe to delineate TOR-dependent responses in eukaryotic cells. Indeed, our understanding of the functions of mTOR stems largely from observations made with rapamycintreated cells. Genetic analyses in budding yeast offered strong evidence to support the conclusion that rapamycin is actually a pro-drug that is converted to the proximate inhibitor of TOR via the formation of a complex with the immunophilin FKBP12 (11). Although it is generally assumed that FKBP12 is a requisite cofactor for rapamycin Received 6/26/03; revised 8/29/02; accepted 9/3/03. Grant support: Fellowship from the Helen Hay Whitney Foundation (A. L. E.), NIH grant CA76193 and grant from Johnson and Johnson (R. T. A.), NCI grant 5T32CA09307 (Research Training and Cancer Chemotherapy; C. M. L.), and in part by a NCI grant (C. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robert T. Abraham, Program in Signal Transduction Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037. Phone: (858) 646-3182; Fax: (858) 713-6274; E-mail: [email protected] 4 The abbreviations used are: TOR, target of rapamycin; FRB, FKBP12/rapamycin binding; TORC, TOR-containing complex; PI3k, phosphatidylinositol 3⬘-kinase; PI, propidium iodide; RIPA, radioimmunoprecipitation assay; GST, glutathione S-transferase; HEK, human embryonic kidney; Ci, microcurie; HA, hemagglutinin. activity in all eukaryotic cell types, this notion has not been tested rigorously in systems other than budding yeast. It is particularly noteworthy in this regard that studies of rapamycin action in the fission yeast led to the conclusion that the FKBP12/rapamycin complex was not responsible for the suppressive effect of this drug on sexual development in this organism (12). The latter results raise the possibility that the pharmacological actions of rapamycin in mammalian cells may not be entirely dependent on the formation of FKBP12/ rapamycin complexes. A related area of uncertainty surrounds the mechanism whereby this drug interferes with TOR signaling in eukaryotic cells. Several reports have documented that exposure of anti-mTOR immunoprecipitates to FKBP12/rapamycin leads to inhibition of mTOR kinase activity (13–15). However, it is noteworthy that the FRB domain of mTOR lies outside of the catalytic domain. Thus, although the binding of FKBP12/rapamycin to the FRB domain may affect the catalytic activity of mTOR through an allosteric mechanism, we cannot exclude the possibility that the drug perturbs the interactions of mTOR with critical regulatory proteins and/or its downstream target proteins. Given the interest in rapamycin as a potential anticancer agent, a detailed knowledge of the underlying pharmacology is crucial if we are to understand how this drug affects tumor growth in vivo, and it may facilitate the creation of second-generation inhibitors of mTOR as cancer therapeutics. A compelling body of genetic evidence indicates that TOR is a central regulator of cell growth in budding yeast and flies (reviewed in Refs. 16 and 17). In contrast, our understanding of mTOR function rests almost entirely on studies of rapamycin-treated mammalian cells. This approach to the study of mTOR has been highly informative; however, an important caveat is that in budding yeast, the phenotypic consequences of rapamycin exposure are substantially less severe than those induced by the depletion of both TOR proteins. TOR2 is an essential gene in this organism, whereas rapamycin exposure produces G1 arrest but not cell death (18). The segregation of TOR functions based on rapamycin sensitivity can now be explained by the existence of two different TORCs in yeast. TORC1 contains either TOR1 or TOR2 and is functionally suppressed by rapamycin. On the other hand, the second complex, TORC2, contains only TOR2 and is not susceptible to rapamycin (19). In contrast to yeast, mammalian cells express a single TOR protein, and the available evidence strongly supports the existence of a TORC1-like complex but not a TORC2like complex in these cells (19 –21). Nonetheless, the potential existence of multiple mTOR complexes in mammalian cells raises the possibility that the downstream events governed by mTOR may also show variable sensitivities to rapamycin. Interest in mTOR as an anticancer drug target has surged recently, based in part on reports that rapamycin and related compounds exert selective cytostatic/cytotoxic effects on PTEN ⫺/⫺ tumors in vivo (22, 23). The PTEN tumor suppressor protein is lost or mutated in many human cancers, particularly those that have progressed to an advanced stage (24, 25). Loss of PTEN leads to deregulated signaling through the PI3k pathway and, in turn, to the generation of a host of cell growth- and survival-promoting signals. A pivotal target for PI3k-derived second messengers is the proto-oncogene product Akt. 8451 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY MnCl2, and 1 mM DTT], and kinase reactions were initiated with 1 g of GST-p70S6k fragment (amino acids 332– 414), 10 M ATP, and 10 Ci of [␥-32P]ATP (6000 Ci/mmol; DuPont NEN). Reactions were incubated for 20 min at 30°C and terminated with SDS-PAGE sample buffer. For Akt kinase assays, HEK 293 cells were transfected with a HA-tagged wild-type Akt expression vector. After 48 h, the transfected cells were harvested in lysis buffer [50 mM Tris-Cl (pH 7.4), 100 mM NaCl, 50 mM ␤-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 10 mg/ml aprotinin, 1 mg/ml pepstatin A, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 20 M microcystin-LR, and 25 mM NaF]. Clarified lysates were immunoprecipitated with anti-HA antibody (12CA5). Immune complexes were collected on protein A-Sepharose (Sigma) prebound with rabbit antimouse antibodies (Pierce) and washed three times in lysis buffer and one time in Akt kinase buffer [25 mM Tris-HCl (pH 7.4), 5 mM ␤-glycerophosphate, 10% MgCl2, and 1 mM DTT]. Immunoprecipitates were resuspended in Akt kinase buffer, and kinase reactions were initiated with 1 g of GST-mTOR RD fragment (amino acids 2405–2517; Ref. 28), 10 M ATP, 10 Ci of [␥-32P]ATP (6000 Ci/mmol; DuPont NEN). Reactions were incubated for 20 min at 30°C and terminated with SDS-PAGE sample buffer. GST-FKBP12-Binding Assays. Five hundred g of ammonium sulfatefractionated rat brain extract (9) were incubated with 5 g of purified GSTFKBP12 (9) and either 10 M rapamycin or ethanol vehicle alone. GSTFKBP12 was collected by incubation with glutathione-Sepharose (Amersham Biosciences), and the resulting precipitates were washed once with precipitation buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 50 mM ␤-glycerophosphate, 10% glycerol (w/v), 1 mM EDTA, 10 g/ml aprotinin, 1 g/ml pepstatin A, 10 g/ml leupeptin, 2 mM phenylmethylsulphonyl fluoride, 20 M microcystin-LR, and 25 mM NaF] containing 0.02% Tween 20. The precipitates MATERIALS AND METHODS were washed three times as indicated in precipitation buffer supplied with Materials. Rapamycin was purchased from Calbiochem, and L-mimosine one of the following detergent mixtures: 0.02% Tween 20; 1% Tween 20; and BrdUrd were from Sigma. The anti-mTOR, anti-phospho-4EBP (37/45), 1% Triton X-100; 1% NP40; 0.5% 3-[(3-cholamidopropyl)dimethylammonio]anti-4EBP1, anti-phospho-p70S6k (421/424), anti-p70S6k, anti-phospho- 1-propanesulfonic acid; or modified RIPA buffer [50 mM Tris-HCl (pH 7.4), p44/42 MAP kinase, anti-p44/42 MAP kinase, anti-phospho-S6 (235/236), 150 mM NaCl, 1 mM EDTA, 1% NP40, 1% sodium deoxycholate, and 0.1% anti-S6, anti-phospho-Akt (Ser473), anti-Akt, and horseradish peroxidase- SDS]. For those samples indicated “high salt,” samples were washed an conjugated goat antirabbit antibodies were purchased from Cell Signaling additional time with high-salt buffer [100 mM Tris-HCl (pH 7.4) and 500 mM Technologies. The anti-phospho-Akt (Thr308) antibody was from Affinity LiCl]. Samples were resuspended in SDS-PAGE sample buffer, resolved by Bioreagents. The anti-4F2hc and the FITC-conjugated anti-BrdUrd antibodies SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immuwere obtained from BD PharMingen. The monoclonal AU-1 and 12CA5 noblotted with anti-mTOR antibodies. FL5.12 Cell Immunoblotting. Cells were washed with PBS and lysed in antibodies were from Babco, and the horseradish peroxidase-conjugated goat antimouse antibody was purchased from Promega. PI, Hoechst 33342, and RIPA buffer [150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Alexa488-conjugated secondary were from Molecular Probes. Tritiated glu- and 50 mM Tris-HCl (pH 8.0)] containing protease and phosphatase inhibitors cose was from Amersham Biosciences. The bicinchoninic acid protein assay [Complete (Roche) and Phosphatase Inhibitor Set 1 (Calbiochem)]. Fifty to 100 g clarified lysate/lane were loaded onto Tris-glycine SDS-PAGE gels kit (Pierce) was used to determine total protein in cell lysates. Cell Culture. HEK 293 cells were maintained in DMEM containing 10% (Invitrogen). Proteins were transferred to nitrocellulose, and membranes were FCS, antibiotics, and L-glutamine. All mTOR constructs were expressed in blocked with BLOTTO (5% nonfat dry milk and 0.1% Tween 20 in PBS) and HEK 293 cells in pcDNA3 (Invitrogen) and transfected using FuGene 6 incubated with the indicated antibodies before probing with enhanced chemi(Roche Molecular Biochemicals, Indianapolis, IN). Plasmids were linearized luminescence (Amersham Biosciences). Measurement of Cellular Growth and Proliferation. The growth rate of with SalI before transfection. Stable transfectants were selected with 1 mg/ml G418 (Invitrogen). Clonogenic assays were performed by seeding equal num- HEK 293 cells was determined with a CellTiter 96 Aqueous One Solution Cell bers of HEK 293 cells into 60-mm dishes. The medium was replaced every 3rd Proliferation kit (Promega). FL5.12 proliferation assays were performed by day until colonies were visible to the naked eye. Colonies were stained with plating 50,000 cells/ml in the presence or absence of 20 nM rapamycin and crystal violet (0.1% in 20% methanol). FL5.12 cells were maintained in RPMI measuring cell number at 24-h intervals with a Coulter Z2 particle analyzer. supplemented with 10% FCS, 8% WEHI-conditioned medium, 10 mM HEPES, For size analyses, live cells were incubated in medium containing 10 g/ml 55 M ␤-mercaptoethanol, antibiotics, and L-glutamine. All experiments were Hoechst 33342 (Molecular Probes) and 10 g/ml PI for 30 min at 37°C and conducted in medium containing 500 pg/ml recombinant IL-3 (BD PharMin- analyzed with a Becton Dickinson LSR flow cytometer. To confirm that gen). mTOR constructs were stably expressed in FL5.12 cells using the EF6 L-mimosine blocked DNA synthesis, cells were incubated for 1 h with 10 M BrdUrd, washed, fixed, and stained with anti-BrdUrd FITC as recommended vector (Invitrogen) and Blasticidin S (Invitrogen) selection. Immunoprecipitations and Kinase Assays. For mTOR immunoprecipi- by the manufacturer. Fluorescence Microscopy. FL5.12 cells were fixed for 10 min at room tations, cells were solubilized in lysis buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 50 mM ␤-glycerophosphate, 10% glycerol (w/v), 1% Tween 20, 1 temperature in 1% paraformaldehyde in PBS. Cells were permeabilized with wash buffer (2% FCS and 0.03% saponin in PBS) and then incubated sequenmM EDTA, 10 g/ml aprotinin, 1 g/ml pepstatin A, 10 g/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 20 M microcystin-LR, and 25 mM NaF]. tially with primary and secondary antibodies for 30 min at room temperature Clarified lysates were immunoprecipitated with anti-mTOR antibodies. Im- in PBS containing 10% FCS and 0.3% saponin. Cells were evaluated on a mune complexes were collected on protein A-Sepharose (Sigma) and washed Nikon E800 fluorescence microscope equipped with a CCD camera, and three times in lysis buffer, once in high-salt buffer [100 mM Tris-HCl (pH 7.4) images were analyzed using the Metamorph software package. Measurement of Glycolytic Rate. One million FL5.12 cells were resusand 500 mM LiCl], and once in mTOR kinase wash buffer [10 mM HEPES (pH 7.4), 50 mM NaCl, 50 mM ␤-glycerophosphate, and 10% glycerol]. Immuno- pended in 0.5 ml of RPMI 1640 that had been pre-equilibrated in a 37°C precipitates were resuspended in mTOR kinase assay buffer [10 mM HEPES incubator under 5% CO2. Ten Ci of 5-[3H]glucose were added to each well, (pH 7.4), 50 mM NaCl, 50 mM ␤-glycerophosphate, 10% glycerol, 10 mM and samples were incubated for 1 h at 37°C in a humidified incubator under 8452 Oncogenic versions of Akt promote cell growth and survival through a mTOR-dependent mechanism (26). Thus, the rapamycin sensitivity of PTEN-deficient tumors may stem from an acquired “addiction” to the PI3k-AKT signaling pathway, which increases the dependency of such tumors on mTOR signaling functions. Based on encouraging results in Phase I clinical cancer trials, three rapamycin analogues, CCI-779 (Wyeth-Ayerst), AP23573 (Ariad Pharmaceuticals), and RAD001 (Novartis), are in Phase II and III trials in patients with renal cancer and other tumors (27). These clinical studies have validated mTOR as an anticancer drug target and have fueled broad interest in the development of novel compounds that inhibit this protein kinase through mechanisms distinct from that of rapamycin. The objectives of the present study were to examine further the impact of rapamycin on mTOR signaling functions and to determine the individual and combined effects of rapamycin treatment and kinase-inactive mTOR expression on a battery of mTOR-dependent cellular responses. We identified three categories of mTOR-dependent responses that ranged from fully sensitive to rapamycin to largely resistant to this mTOR inhibitor. These findings have important implications both for the use of rapamycin as a probe to analyze mTOR-dependent signaling pathways in mammalian cells and for the future development of mTOR inhibitors as cancer chemotherapeutic agents. Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY 5% CO2. Reactions were terminated with 0.5 ml of 0.2 N HCl, and 100 l of the cell/HCl mixture were added to open PCR tubes, which were then placed upright in 4-ml scintillation vials containing 0.5 ml of H2O. The vials were capped, sealed with parafilm, and incubated for 2 days at room temperature. During the incubation, [3H]2O generated by glycolysis diffused from the PCR tube into the H2O in the scintillation vial through evaporation and condensation. The contents of the PCR tube were transferred to a new scintillation vial with 0.5 ml of H2O, the PCR tube was discarded, and scintillation fluid was added to both the original (diffused counts) and second (undiffused counts) vials. Vials were evaluated for tritium content with a Wallac Microbeta 1450. The fraction of [3H]2O that diffused in 2 days was determined with a control PCR tube containing 1 Ci of [3H]2O. The background diffusion ratio was determined with a cell-free control sample. To calculate the glycolytic rate, the sample diffusion ratio (diffused counts/undiffused counts) minus the background diffusion ratio was divided by the diffusion fraction from the [3H]2O control. This number was multiplied by 5500 (the nmols of glucose in 0.5 ml of RPMI) to obtain the nmol glucose consumed/million cells/h. RESULTS Cellular Treatment with Rapamycin Does Not Lead to Irreversible Inhibition of mTOR Kinase Activity. During early attempts to isolate mTOR by affinity purification over a FKBP12/ rapamycin column, we made the empirical observation that once formed, the ternary complex of FKBP12/rapamycin-mTOR was poorly reversible under nondenaturing conditions (13). To reexamine this finding in a more systematic fashion, we precipitated mTOR from rat brain extract with an immobilized GST-FKBP12 fusion protein loaded with rapamycin (Fig. 1A). As predicted, precipitation of mTOR by GST-FKBP12 was completely dependent on the presence of rapamycin. To evaluate the stability of this complex, parallel samples were washed with buffered solutions containing nonionic detergents (Tween 20, NP40, and Triton X-100), ionic detergents (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and RIPA buffer), or nonionic detergent plus high-salt concentrations (0.5 M LiCl). The GST-FKBP12/rapamycin-bound mTOR was not dislodged from the affinity matrix by any of the wash buffers used in this experiment. Based on these results, we reasoned that endogenous FKBP12/ rapamycin-mTOR complexes should easily survive cell lysis and precipitation in 1% Tween 20-containing, isotonic extraction buffer. Given that the interaction with FKBP12/rapamycin strongly inhibits mTOR kinase activity (13–15), we would expect that formation of the ternary complex in rapamycin-treated cells would lead to a clear reduction in the protein kinase activity present in anti-mTOR immunoprecipitates from these cells. To examine the impact of rapamycin treatment on mTOR kinase activity, HEK 293 cells were serum starved for 16 h and incubated for 30 min with 100 nM rapamycin or 10 M wortmannin. Cells were then stimulated for 10 min with serum, and mTOR activity was determined in immune complex kinase assays (Fig. 1B). Treatment of the cells with rapamycin had no effect on the mTOR kinase activity as measured with either GST-p70S6k (Fig. 1B) or PHAS-I/4E-BP1 (data not shown) as the substrate. Similar results were obtained after cells were exposed to rapamycin at concentrations up to 10 M (data not shown). In contrast, treatment of cells with wortmannin, which binds irreversibly to the mTOR kinase domain (29, 30), strongly inhibited mTOR-dependent GST-p70S6k phosphorylation. The HEK 293 cells were responsive to rapamycin, because endogenous p70S6k was extensively dephosphorylated (indicated by an increase in electrophoretic mobility) in the drug-treated cells (Fig. 1B). Identical results were obtained when the experiment was repeated with MCF-7 breast carcinoma cells as the test cell line (data not shown). Collectively, these observations suggest that treatment of intact cells with rapamycin does not lead to poorly reversible sup- Fig. 1. Effect of cellular exposure to rapamycin on mTOR kinase activity. A, poorly reversible binding of mTOR to the FKBP12/rapamycin complex. Rat brain extract was incubated for 2 h with GST-FKBP12 in the absence or presence of rapamycin. GSTFKBP12 was then captured with glutathione-Sepharose beads, and precipitates were washed with the indicated buffer solutions. Top panel, anti-mTOR Western blot of glutathione-Sepharose-bound GST-FKBP12 samples. Bottom panel, amido black-staining of the same protein blot. B, kinase activity of mTOR isolated from rapamycin-treated cells. Top panel, Lanes 1– 4, HEK 293 cells were maintained for 16 h in DMEM ⫹ 0.1% FBS. Cells were treated with 100 nM rapamycin (Rap) or 10 M wortmannin (Wort) as indicated for 30 min before stimulation with serum for an additional 10 min. Lanes 5 and 6, HEK 293 cells were grown for 16 h in DMEM ⫹ 10% FBS in the absence or presence of 20 nM rapamycin (Rapⴱ). Immune complex kinase assays were performed with anti-mTOR immunoprecipitates and a GST-p70S6k fragment (amino acids 332– 414) as substrate. Middle panel, anti-mTOR immunoprecipitates were immunoblotted with antimTOR antibodies. Bottom panel, whole-cell extracts were immunoblotted with antip70S6k antibodies. pression of mTOR kinase activity as would be expected if the FKBP12/rapamycin complex were the predominant effector of mTOR inhibition in this context (see “Discussion”). Generation of Stably Transfected HEK 293 Cells Expressing a Dominant Interfering mTOR Mutant. The biochemical observations described above raise the possibility that rapamycin exerts subtle and reversible effects on mTOR signaling functions as opposed to acting solely as a potent inhibitor of mTOR kinase activity in vivo. To define further the inhibitory effect of rapamycin on mTOR signaling functions, we transfected HEK 293 cells with a mTOR double mutant bearing a Ser20353 Ile (SI) substitution in the FKBP12-rapamycinbinding domain and an inactivating Asp23383 Ala (DA) substitution in the catalytic domain (28). This SIDA mTOR double mutant has a markedly reduced binding affinity for FKBP12-rapamycin and is catalytically inactive. Studies in yeast have shown that both the rapamycin-sensitive and -insensitive functions of the TOR proteins are contingent on the expression of an intact catalytic domain (18). By analogy to many other catalytically inactive protein kinases, we predicted that SIDA mTOR would exert dominant inhibitory effects on mTOR signaling in transfected cells. The SI mutation was incorporated into the kinase-inactive mTOR construct to ensure that the potential dominant inhibitory activity of the kinase-inactive protein was not affected by rapamycin and that the kinase-inactive mTOR mutant was not simply acting as a sink for FKBP12-rapamycin complexes in drug-treated cells. Our underlying prediction was that 8453 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY cells grew at approximately the same rate as control cells. Cells expressing SIDA mTOR grew more slowly than control cells, consistent with the predicted dominant inhibitory effect of the kinaseinactive mTOR mutant. The growth rates of these cell lines were next measured in the presence of 20 nM rapamycin, a maximally effective drug concentration in this assay (Fig. 2C; data not shown). As expected, growth of the control cell lines was suppressed by rapamycin. In contrast, cells expressing SI mTOR were highly resistant to the drug, indicating that the growth-inhibitory effect of rapamycin was due to its effects on mTOR. In contrast, when SIDA mTOR-expressing cells were treated with rapamycin, cell growth was almost completely arrested. The slight increase in cell mass observed after 60 h in culture may reflect the degradation of rapamycin, which has a finite half-life (⬃10 h) in aqueous solution (31). Thus, the antiproliferative activity of rapamycin toward HEK 293 cells is dramatically enhanced in the presence of SIDA mTOR. Effect of Rapamycin Treatment and SIDA mTOR Expression on Clonogenic Activity under Nutrient-Replete and -Restricted Conditions. To examine further the overall contribution of mTOR to cell growth, clonogenic assays were performed. Rapamycin treatment decreased the size of colonies formed by vector control cells, whereas SI mTOR-expressing cells were relatively resistant to the antiproliferative effect of this drug (Fig. 3). In the absence of rapamycin, the SIDA mTOR-expressing cells displayed slightly lower plating efficiencies and a modest reduction in average colony size. However, when SIDA mTOR-expressing cells were treated with rapamycin, very few colonies emerged after 6 days in culture. These results indicate that the inhibitory effect of rapamycin on clonogenic activity was enhanced by SIDA mTOR. Clonogenic assays were also conducted in nutrient- and growth factor-deficient medium. Cells were seeded in complete medium and cultured overnight. The culture medium was then replaced with DMEM containing 2% FCS and 20% of the normal level of amino Fig. 2. mTOR stimulation of cell growth includes a rapamycin-insensitive component. A, HEK 293 lines stably expressing AU-1 tagged SI and SIDA mTOR or empty vector (VEC) were created and evaluated for transgene expression by Western blot. B, the growth rates of vector control, SI-, and SIDA-expressing HEK 293 cells were determined with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium tetrazolium assay. C, cellular growth rates were evaluated as in B, except that cells were incubated in the presence of 20 nM rapamycin. All measurements were performed in triplicate. Error bars represent SD; one representative of three similar experiments is shown. mTOR-dependent cellular responses that are only partially inhibited by rapamycin in normal cells should be suppressed further in drugtreated cells that express the dominant inhibitory SIDA mTOR protein. To confirm that the measured cellular responses were mTOR dependent, cells expressing the kinase active mTOR SI mutant were also generated. In mTOR SI-expressing cells, mTOR signaling should be largely refractory to rapamycin treatment. Clonal lines of SI or SIDA mTOR-expressing HEK 293 cells were derived (Fig. 2A), and their growth rates were compared with that of control cells expressing vector alone (Fig. 2B). The term “growth” is used here to refer to the accumulation of total cell mass due to both cell division and changes in single-cell volume. SI mTOR-expressing Fig. 3. The clonogenic growth-promoting activity of mTOR includes a rapamycininsensitive component. Equivalent numbers of HEK 293 cells expressing the indicated mTOR constructs were plated in 60-mm dishes. Twenty-four h later, the medium was replaced with complete DMEM or with DMEM containing 2% FCS and 20% of the normal level of amino acids. The indicated samples (RAP) received 20 nM (final concentration) rapamycin. After 6 days of growth, cells were stained with crystal violet and photographed. 8454 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY acids. Vector control and SI mTOR-expressing cells formed readily detectable colonies under conditions of nutrient-stress, although the average colony size was reduced (Fig. 3). In contrast, SIDA mTORexpressing cells failed to generate visible colonies under these conditions. Rapamycin treatment resulted in a slight decrease in the colony size of control cells under nutrient-limiting conditions, but the magnitude of this effect was quantitatively less than that seen in complete medium. These results demonstrate that expression of SIDA mTOR leads to greater sensitivity to growth factor/nutrient limitation than does treatment of HEK 293 cells with rapamycin. SIDA mTOR Fails to Enhance the Suppressive Effect of Rapamycin on Cell Size. We have used the term cellular growth to encompass both cellular proliferation and increases in individual cell mass. Because mTOR regulates mammalian cell size (20, 32), we wished to evaluate the sensitivity of cell size control to rapamycin. However, HEK 293 cells in monolayer culture are ill suited for these studies due to their intrinsic variation in cell size at various stages of confluence and to the rapid loss of cell viability after detachment from the plastic surface. Hematopoietic cells are more amenable to cell volume measurements, because these cells grow naturally in suspension and exhibit nearly spherical morphology. Consequently, we generated stable transfectants expressing empty vector, SI mTOR, or SIDA mTOR in the murine IL-3-dependent hematopoietic cell line, FL5.12 (Fig. 4A). To confirm that the regulation of cell growth by mTOR contained a rapamycin-insensitive component, we compared the proliferative rates of these FL5.12 cell lines in the presence or absence of rapamycin. Expression of SI mTOR had no effect on the proliferation of FL5.12 cells, whereas the expression of SIDA mTOR slowed cellular proliferation, confirming that the kinase-inactive mTOR mutant dominantly interferes with mitogenic signal transduction in FL5.12 cells (Fig. 4B). As expected, rapamycin treatment limited cellular proliferation in vector control FL5.12 cells, whereas cell lines expressing SI mTOR were resistant to treatment with the drug (Fig. 4C). Treatment of SIDA mTOR-expressing FL5.12 cells with rapamycin produced a more dramatic antiproliferative effect than was observed in control cells. Importantly, identical results were obtained when these experiments were repeated with a 10-fold higher concentration of rapamycin (data not shown), indicating that the standard drug concentration (20 nM) used in our studies caused the maximal suppression of mTOR function that can be obtained with rapamycin alone. These observations were consistent with the results obtained with HEK 293 cells, i.e., mTOR function is essential for FL5.12 cell proliferation, and the contribution of mTOR to this process is only partially suppressed by rapamycin. We next evaluated whether the regulation of cell size by mTOR included a rapamycin-insensitive component. Relative cell volumes were measured by forward light scatter on a flow cytometer. The confounding effects of cell cycle position on cell size were eliminated by gating on viable (PI-negative), G1-phase cells. To avoid artifacts due to sample processing, cells were maintained in complete medium during the staining and analysis procedures. As expected based on previous reports (20, 32), rapamycin treatment decreased the size of control cells, whereas the volume of SI mTOR-expressing cells was unaffected by the drug (Fig. 5). Interestingly, both SIDA mTORexpressing clones displayed a constitutive reduction in cell size resembling that provoked by rapamycin treatment in the control cells. Exposure of SIDA mTOR-expressing cells to rapamycin caused no additional decrease in cell size. Modulation of Amino Acid Transporter Trafficking by mTOR Includes a Rapamycin-Insensitive Component. Previous studies demonstrated that mTOR activity is required for activated forms of Akt to maintain cell surface expression of the 4F2hc amino acid Fig. 4. Growth rates of FL5.12 cells in the presence of rapamycin and kinase-inactive mTOR. A, FL5.12 cells stably expressing the indicated constructs were generated and screened for transgene expression by Western blot. B, the effect of kinase-inactive mTOR expression on cell growth rates was determined by plating 5 ⫻ 104 cells/ml and measuring cell concentration with a Coulter counter at the indicated time points. All measurements were performed in triplicate. C, growth rates were evaluated as in B, except that 20 nM rapamycin was added to the culture medium. Error bars represent SD; one of three similar experiments is shown. transporter in the absence of growth factors (26). In these earlier studies, however, rapamycin treatment did not alter 4F2hc localization in the presence of growth factors; therefore, it remained unclear whether mTOR regulated transporter trafficking under normal cell culture conditions. We hypothesized that control of amino acid transporter trafficking by mTOR might be partially rapamycin insensitive in mammalian cells. If so, expression of SIDA mTOR, either alone or in combination with rapamycin, might alter amino acid transporter localization in growth factor-stimulated cells. In control FL5.12 cells, a surface-staining pattern for 4F2hc was observed, with no staining of the intracellular compartment (Fig. 6). Consistent with our previous 8455 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY Fig. 5. Cell size regulation by mTOR is fully sensitive to rapamycin. FL5.12 cells expressing the indicated mTOR constructs were cultured for 24 h in the presence or absence of 20 nM rapamycin (RAP). Cell size was determined by forward light scatter (FSC) analysis with a flow cytometer. Error bars represent SD; one of four similar experiments is shown. transcripts are more strongly affected than others. In the present study, we examined the individual and combined effects of rapamycin and SIDA mTOR on the phosphorylation states of p70S6k and 4EBP1. In response to mitogenic stimuli, p70S6k undergoes multisite phosphorylation by several upstream kinases, including mTOR (14, 34, 35). Both rapamycin treatment and SIDA mTOR expression decreased the phosphorylation of p70S6k, as indicated by increased mobility in SDS-PAGE (Fig. 7). However, the protein mobility shift assay lacked the resolution needed to determine whether the combined effects of rapamycin and kinase-inactive mTOR were additive. Consequently, we examined the phosphorylation of a known p70S6k target, the ribosomal protein S6. Consistent with the observed effect on p70S6k phosphorylation, both rapamycin exposure and SIDA mTOR expression inhibited S6 phosphorylation. An unexpected finding was that S6 phosphorylation was also suppressed in the SI mTORexpressing clones. This alteration might reflect a compensatory adjustment made to the chronic elevation of mTOR signaling in the SI mTOR-expressing cells (see “Discussion”). Nonetheless, the observation that rapamycin alone provoked complete S6 dephosphorylation in the vector control FL5.12 line argues that this mTOR-dependent response is fully sensitive to the drug. Earlier studies demonstrated that rapamycin treatment leads to the dephosphorylation of five Ser and Thr residues in 4EBP1 (reviewed in Ref. 33). Treatment of control cells with rapamycin produced the expected reduction in 4EBP1 phosphorylation (Fig. 7). The effect of rapamycin on 4EBP1 phosphorylation was reversed by expression of rapamycin-resistant SI mTOR. Surprisingly, cells expressing SIDA mTOR exhibited greater 4EBP1 dephosphorylation than did control cells treated with rapamycin. Furthermore, the effects of rapamycin treatment and the expression of kinase-inactive mTOR on 4EBP1 phosphorylation were additive. These results indicate that the regulation of 4EBP1 phosphorylation by rapamycin includes both rapamycin-sensitive and -insensitive components. Chronic Inhibition of mTOR Suppresses Akt Kinase Activity. Earlier studies (23, 28, 36, 37) demonstrated that acute exposure to rapamycin does not inhibit the activity of AKT, a PI3k-regulated kinase that may reside upstream of mTOR (28, 38). However, the impact of longer-term exposure to rapamycin on Akt activity has not Fig. 6. Amino acid transporter trafficking is affected by kinase-inactive mTOR expression but not by rapamycin treatment. Vector control (VEC) or SIDA mTOR-expressing cells were maintained in the presence or absence of 20 nM rapamycin for 24 h before fixation and staining for 4F2hc expression. Scale bars denote 10 m; insets are enlarged to better demonstrate the intracellular staining pattern. studies (26), little change in this staining pattern was observed after a 24-h treatment with rapamycin. However, when SIDA mTORexpressing cells were examined for 4F2hc localization, many small 4F2hc-positive intracellular vesicles were apparent. This pattern of 4F2hc staining was not altered by rapamycin treatment. The effect of SIDA mTOR on amino acid transporter localization was attributable to the mutated catalytic domain, because expression of SI mTOR had no effect on 4F2hc localization (data not shown). These results suggest that the trafficking of 4F2hc is regulated by mTOR but is largely insensitive to rapamycin. Phosphorylation of 4EBP1 Is Partially Rapamycin Insensitive. In mammalian cells, mTOR regulates translation by promoting the phosphorylation of p70S6k and 4EBP1. Rapamycin treatment causes rapid decreases in the phosphorylation of these proteins, which results in the inactivation of p70S6k and increased binding of 4EBP1 to the translation initiation factor, eIF-4E (33). The net outcome of these events is a decrease in mRNA translation rates, although certain Fig. 7. Effects of rapamycin treatment and kinase-inactive mTOR expression on phosphorylation of p70S6k and 4EBP1. FL5.12 cells expressing the indicated mTOR constructs were cultured for 24 h in the presence or absence of 20 nM rapamycin (RAP). Detergent extracts were prepared and evaluated by Western blotting with the indicated antibodies. 8456 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY been investigated. Therefore, we evaluated whether a more prolonged (24-h) drug treatment affected Akt phosphorylation at the regulatory Thr308 and Ser473 residues. When control HEK 293 cells were treated with rapamycin, AKT phosphorylation was clearly decreased, with no change in total Akt levels (Fig. 8A, top panel). The dephosphorylation of Akt induced by long-term rapamycin treatment was blocked by SI mTOR expression. Similar decreases in Akt phosphorylation were observed in SIDA mTOR-expressing cells. Moreover, when rapamycin treatment was combined with SIDA mTOR expression, Akt phosphorylation was compromised further. Finally, we demonstrated that chronic rapamycin exposure had no effect on the phosphorylation of the p44 and p42 MAP kinase isoforms in HEK 293 cells, indicating that the drug did not globally suppress the activities of cytoplasmic protein kinases under these treatment conditions (Fig. 8A, bottom panel). Similar results were obtained in parallel experiments with FL5.12 cells (data not shown), indicating that chronic rapamycin treatment affects Akt phosphorylation in multiple cell types. To address the possibility that the observed inhibition of Akt phosphorylation was a secondary consequence of the antiproliferative activity of rapamycin, we treated HEK 293 cells for 24 h with 500 M L-mimosine and examined the phosphorylation status of Akt as described above. Consistent with the production of an early S-phase cell cycle arrest, L-mimosine treatment completely abrogated BrdUrd incorporation (data not shown). Despite the induction of a complete cell cycle arrest, L-mimosine treatment had no effect on Akt phosphorylation, whereas rapamycin-treated cells displayed the expected decreases in Akt phosphorylation at Ser308 and Thr473 (Fig. 8B). Immune complex kinase assays were performed to confirm that chronic rapamycin treatment actually suppressed Akt kinase activity. HEK 293 cells expressing empty vector, SI mTOR, or SIDA mTOR were transfected with HA-Akt and then treated with 20 nM rapamycin for 24 h. Rapamycin treatment alone caused a 50% reduction in Akt kinase activity present in anti-HA immunoprecipitates from control cells (Fig. 8C). As expected, Akt activity was not affected by rapamycin in the SI mTOR-expressing cells. In contrast, SIDA mTORexpressing cells displayed a constitutive decrease in Akt activity similar to rapamycin-treated control cells. Rapamycin treatment of the SIDA mTOR-expressing clone produced an even greater reduction in Akt kinase activity, to a level less than 25% of that observed in untreated controls. These results indicate that long-term inhibition of mTOR function leads to suppression of Akt kinase activity and that this mTOR-dependent outcome is partially suppressed by rapamycin. The Regulation of Glycolysis by mTOR Is Rapamycin Sensitive. Using DNA microarrays, others have shown that treatment of yeast and mammalian cells with rapamycin decreases the level of mRNA transcripts encoding glycolytic enzymes and increases the abundance of mRNAs coding for tricarboxylic acid cycle enzymes (39, 40). These findings hinted that the TOR proteins serve as rapamycin-sensitive stimulators of glycolytic activity in nutrient-replete Fig. 8. mTOR regulates Akt kinase activity by a mechanism that is partially inhibited by rapamycin. A, HEK 293 cells expressing the indicated mTOR constructs were incubated for 24 h in the presence or absence of 20 nM rapamycin (RAP). The cells were lysed, and detergent extracts were immunoblotted with the indicated antibodies. B, cellular extracts were analyzed as in A after a 24-h incubation with 500 M L-mimosine or 20 nM rapamycin as indicated. C, HEK 293-derived stable transfectants (pCDNA3, mTOR SI 5, and mTOR SIDA 7) were transiently transfected with an HA-tagged Akt expression plasmid. Twenty-four h post-transfection, the indicated samples were treated for 24 h with 20 nM rapamycin. Clarified lysates were immunoprecipitated with anti-HA antibodies (12CA5) and subjected to immune complex kinase assays. Top panel, immune complex kinase assays were performed with anti-HA immunoprecipitates and GST-mTOR RD (amino acids 2405–2517) as substrate. Middle panel, the same membrane was immunoblotted with anti-HA antibodies. Bottom panel, phosphorimager analysis of GST-mTOR RD phosphorylation. Substrate phosphorylation by HA-Akt in each sample was normalized to that obtained with the untreated HEK 293 vector-only cell line. 8457 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY Fig. 9. mTOR regulates glycolytic rate through a mechanism that is fully sensitive to rapamycin. FL5.12 cells expressing the indicated mTOR constructs were incubated in the presence or absence of 20 nM rapamycin (RAP), and glycolytic rates were determined by measuring the conversion of [3H]2O from [3H]glucose. Error bars represent SE; a representative experiment of three similar trials is shown. cells. In light of these reports, we examined whether mTOR regulates glycolytic activity in FL5.12 cells cultured in the presence of IL-3. As shown in Fig. 9, treatment with rapamycin decreased the rate of glycolysis in control cells but not in cells expressing rapamycinresistant SI mTOR. Interestingly, expression of SIDA mTOR decreased the rate of glycolysis to a level similar to that observed in rapamycin-treated control cells. The addition of rapamycin to SIDA mTOR-expressing cells produced no additional decrease in the glycolytic rate. These findings indicate that mTOR is a positive regulator of glycolysis in mammalian cells and that this activity of mTOR is fully suppressed by rapamycin. DISCUSSION Rapamycin has been used extensively to identify the contributions of mTOR to various signaling pathways and cellular responses. An implicit assumption in these experiments is that exposure to maximally effective concentrations of rapamycin translates into a “chemical knockout” of mTOR function in drug-treated cells. However, in this report, we demonstrate that rapamycin exerts differential inhibitory effects on mTOR-dependent responses in both epithelial and hematopoietic cells. Certain mTOR-regulated processes, such as glycolytic activity and cell size control, were inhibited to similar extents by either rapamycin treatment or SIDA mTOR expression. Other mTOR-dependent outcomes, such as 4EBP1 phosphorylation, Akt activity, and cellular growth, were only partially suppressed by rapamycin. Finally, amino acid transporter trafficking was disrupted by SIDA mTOR expression but was not altered by rapamycin treatment, indicating that some mTOR-dependent functions are rapamycin insensitive. These results strongly suggest that chemical genetic experiments involving rapamycin as the probe will not uncover the full range of mTOR-dependent responses in mammalian cells. The complex pharmacology of rapamycin was underscored by the unexpected finding that mTOR kinase activity was not reduced by treatment of intact cells with rapamycin. These results were not easily reconciled with the observation that the FKBP12/rapamycin complex, the presumed effector of intracellular mTOR inhibition, is a poorly reversible and highly effective inhibitor of mTOR kinase activity in vitro (see Fig. 1A; Refs. 13–15). The present findings suggest that if rapamycin functions primarily as a inhibitor of mTOR kinase activity in intact cells, it does so in a reversible fashion. An alternative, but nonexclusive possibility is that the drug interferes with the recognition of upstream regulatory signals by mTOR, and/or with the phosphorylation of downstream targets for this protein kinase. Moreover, certain functions of mTOR may be sensitive to rapamycin alone, whereas others may be inhibited by rapamycin only when the drug is complexed to FKBP12. If reversible interaction of rapamycin with the FRB domain has differential effects on signal transmission through mTOR, then a search for novel ligands for the FRB domain of mTOR could yield drugs with a different spectrum of immunosuppressive and anticancer activities than those exhibited by rapamycin. The presence of two TORCs (TORC1 and TORC2) provides a rational explanation for the existence of rapamycin-sensitive and -insensitive TOR functions in budding yeast (19). Although mammalian cells express a rapamycin-sensitive, TORC1-like (raptor-containing) complex (19 –21), the expression of additional mTOR complexes remains speculative. Our finding that mTOR carries out activities (e.g., amino acid transporter localization) that are relatively resistant to rapamycin hints that two (or more) mTOR-containing complexes may be present in mammalian cells. Alternatively, as discussed above, rapamycin binding to a single mTOR-containing complex could result in differential inhibition of the various efferent signaling outputs emanating from this complex. Clearly, additional studies of mTOR and its partner proteins in mammalian cells will be needed to distinguish between these alternative models. The finding that rapamycin exerts variable effects on mTOR functions also has important implications for cancer chemotherapy with rapamycin and other inhibitors of the mTOR signaling pathway now under preclinical and clinical development. In our analysis of mTORdependent outcomes, the suppressive effect of SIDA mTOR expression on endogenous mTOR function was typically comparable with or greater than that of rapamycin. Kinase-inactive proteins exert dominant inhibitory activities through sequestration of associated regulatory proteins and/or substrates of the endogenous protein kinase. Direct inhibitors of the mTOR kinase domain should result in effects similar to those induced by forced expression of SIDA mTOR. In fact, mTOR kinase inhibitors may be even more efficacious inhibitors of mTOR function, because the dominant-negative protein can be stably expressed only to a level compatible with continued cell growth. Our results suggest that small molecule inhibitors of the mTOR kinase domain will exert considerably broader effects on mTOR function than does rapamycin. Whether such drugs will show selectivity toward tumor cells remains an open question. Our results also indicate that mTOR regulates amino acid transporter trafficking in mammalian cells. Rapamycin has been shown previously to alter amino acid transporter localization in FL5.12 cells (26). However, this drug effect was only apparent when cells expressing an activated Akt mutant were deprived of growth factors. In the present report, disruption of mTOR function by SIDA mTOR expression altered transporter localization in the presence of growth factors. This observation correlated with the finding that SIDA mTOR expression was more effective than rapamycin as a suppressor of the clonogenic activity of HEK 293 cells cultured under growth factor/ nutrient-limited conditions. Interestingly, TOR-dependent amino acid transporter trafficking in yeast is sensitive to rapamycin in optimal growth medium (41, 42). Our findings suggest that multiple upstream signals converge on mTOR to regulate amino acid transporter expression and that some do so in a rapamycin-sensitive fashion (e.g., the Akt-dependent signal), whereas others are less affected by drug treatment. We also observed that mTOR regulates the glycolytic rate in mammalian cells. Although DNA microarray studies suggested that TOR controls glucose metabolism in eukaryotic cells (39, 40), direct measurements of glycolytic rates in rapamycin-treated cells had not been performed before this study. Because cancer cells characteristi- 8458 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY cally display abnormally elevated glycolytic activity (43), it is plausible that the antitumor effects of rapamycin reflect, in part, the suppression of glycolysis and the consequent depletion of energy supplies needed for tumor growth. A surprising observation was that chronic repression of mTOR signaling caused a significant reduction in Akt phosphorylation and catalytic activity in the host cells. Previous findings indicated that short-term (ⱕ2 h) rapamycin treatment had no effect on Akt kinase activity (23, 28, 36, 37). We found that a minimum rapamycin exposure time of 8 h was required to induce significant dephosphorylation of Akt. These results suggest that mTOR function contributes to sustained Akt activation in growth factor-stimulated cells. In contrast, chronic elevations of dTOR/p70S6k activity lead to decreased Akt activity in Drosophila larva (44, 45). Although the reason for this apparent discrepancy is unknown, it is possible that long-term mTOR suppression inhibits Akt via an indirect mechanism that targets a component(s) of Akt-containing protein complexes in mammalian cells (46). Regardless, the observation that long-term rapamycin exposure interferes with Akt function may be highly relevant to the anticancer activities of rapamycin-like compounds in human patients. As predicted, expression of the catalytically active SI mTOR mutant uniformly rescued rapamycin-sensitive mTOR functions in HEK 293 cells. An unexpected observation was that SI mTOR expression caused a consistent reduction in the level of phosphorylated S6 in these cells. These results may be linked to the recent observation that lethality associated with deletion of Tsc1 in Drosophila was rescued by manipulations that lowered p70S6k activity (44). Stable expression of SI mTOR in mammalian cells may resemble disruption of the Tsc1 gene in the fly in that both alterations would lead to chronically elevated TOR activity (47–51), which would need to be countered by a reduction in p7056K activity to maintain normal cell growth. In conclusion, the present findings indicate that rapamycin exerts surprisingly variable effects on mTOR-dependent signaling in mammalian cells. Our results additionally suggest that a direct inhibitor of the mTOR kinase domain will display a profile of pharmacological activities that only partially mimics those associated with rapamycin treatment. Clinical experience with rapamycin indicates that this drug possesses a high therapeutic index. As with many conventional anticancer agents, nonspecific toxicity to proliferating tissues may limit the clinical application of an agent that globally blocks signaling through mTOR. Nonetheless, our findings indicate that further development of mTOR inhibitors is clearly warranted and could yield a new class of anticancer drugs that selectively target anabolic metabolism and energy production in developing tumors. ACKNOWLEDGMENTS We thank the members of the Thompson and Abraham labs for helpful discussions and for technical advice. REFERENCES 1. Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R., and Hall, M. N. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell., 5: 105–118, 1994. 2. Heitman, J., Movva, N. R., and Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science (Wash. DC), 253: 905–909, 1991. 3. Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. Target of rapamycin in yeast. TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell, 73: 585–596, 1993. 4. Cafferkey, R., Young, P. R., McLaughlin, M. M., Bergsma, D. J., Koltin, Y., Sathe, G. M., Faucette, L., Eng, W. K., Johnson, R. K., and Livi, G. P. Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol. Cell. Biol., 13: 6012– 6023, 1993. 5. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C., and Neufeld, T. P. Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev., 14: 2712–2724, 2000. 6. Oldham, S., Montagne, J., Radimerski, T., Thomas, G., and Hafen, E. Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev., 14: 2689 –2694, 2000. 7. Chiu, M. I., Katz, H., and Berlin, V. RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex, Proc. Natl. Acad. Sci. USA, 91: 12574 –12578, 1994. 8. Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature (Lond.), 369: 756 –758, 1994. 9. Sabers, C. J., Martin, M. M., Brunn, G. J., Williams, J. M., Dumont, F. J., Wiederrecht, G., and Abraham, R. T. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem., 270: 815– 822, 1995. 10. Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell, 78: 35– 43, 1994. 11. Abraham, R. T., and Wiederrecht, G. J. Immunopharmacology of rapamycin. Annu. Rev. Immunol., 14: 483–510, 1996. 12. Weisman, R., and Choder, M. The fission yeast TOR homolog, tor1⫹, is required for the response to starvation and other stresses via a conserved serine. J. Biol. Chem., 276: 7027–7032, 2001. 13. Brunn, G. J., Hudson, C. C., Sekulic, A., Williams, J. M., Hosoi, H., Houghton, P. J., Lawrence, J. C., Jr., and Abraham, R. T. Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science (Wash. DC), 277: 99 –101, 1997. 14. Burnett, P. E., Barrow, R. K., Cohen, N. A., Snyder, S. H., and Sabatini, D. M. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA, 95: 1432–1437, 1998. 15. Mothe-Satney, I., Brunn, G. J., McMahon, L. P., Capaldo, C. T., Abraham, R. T., and Lawrence, J. C., Jr. Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies. J. Biol. Chem., 275: 33836 –33843, 2000. 16. Miron, M., and Sonenberg, N. Regulation of translation via TOR signaling: insights from Drosophila melanogaster. J. Nutr., 131: 2988S–2993S, 2001. 17. Schmelzle, T., and Hall, M. N. TOR, a central controller of cell growth. Cell, 103: 253–262, 2000. 18. Zheng, X. F., Florentino, D., Chen, J., Crabtree, G. R., and Schreiber, S. L. TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell, 82: 121–130, 1995. 19. Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J. L., Bonenfant, D., Oppliger, W., Jenoe, P., and Hall, M. N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell, 10: 457– 468, 2002. 20. Kim, D. H., Sarbassov, D. D., Ali, S. M., King, J. E., Latek, R. R., ErdjumentBromage, H., Tempst, P., and Sabatini, D. M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell, 110: 163–175, 2002. 21. Hara, K., Maruki, Y., Long, X., Yoshino, K., Oshiro, N., Hidayat, S., Tokunaga, C., Avruch, J., and Yonezawa, K. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell, 110: 177–189, 2002. 22. Podsypanina, K., Lee, R. T., Politis, C., Hennessy, I., Crane, A., Puc, J., Neshat, M., Wang, H., Yang, L., Gibbons, J., Frost, P., Dreisbach, V., Blenis, J., Gaciong, Z., Fisher, P., Sawyers, C., Hedrick-Ellenson, L., and Parsons, R. An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten⫹/⫺ mice. Proc. Natl. Acad. Sci. USA, 98: 10320 –10325, 2001. 23. Neshat, M. S., Mellinghoff, I. K., Tran, C., Stiles, B., Thomas, G., Petersen, R., Frost, P., Gibbons, J. J., Wu, H., and Sawyers, C. L. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA, 98: 10314 –10319, 2001. 24. Cantley, L. C., and Neel, B. G. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA, 96: 4240 – 4245, 1999. 25. Mills, G. B., Lu, Y., and Kohn, E. C. Linking molecular therapeutics to molecular diagnostics: inhibition of the FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks PTEN mutant cells in vitro and in vivo. Proc. Natl. Acad. Sci. USA, 98: 10031–10033, 2001. 26. Edinger, A. L., and Thompson, C. B. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol. Biol. Cell, 13: 2276 –2288, 2002. 27. Hidalgo, M., and Rowinsky, E. K. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene, 19: 6680 – 6686, 2000. 28. Sekulic, A., Hudson, C. C., Homme, J. L., Yin, P., Otterness, D. M., Karnitz, L. M., and Abraham, R. T. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res., 60: 3504 –3513, 2000. 29. Walker, E. H., Pacold, M. E., Perisic, O., Stephens, L., Hawkins, P. T., Wymann, M. P., and Williams, R. L. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol. Cell, 6: 909 –919, 2000. 30. Brunn, G. J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J. C., Jr., and Abraham, R. T. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J., 15: 5256 –5267, 1996. 31. Hosoi, H., Dilling, M. B., Shikata, T., Liu, L. N., Shu, L., Ashmun, R. A., Germain, G. S., Abraham, R. T., and Houghton, P. J. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res., 59: 886 – 894, 1999. 8459 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. mTOR POSSESSES RAPAMYCIN-RESISTANT ACTIVITY 32. Fingar, D. C., Salama, S., Tsou, C., Harlow, E., and Blenis, J. Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev., 16: 1472–1487, 2002. 33. Gingras, A. C., Raught, B., and Sonenberg, N. Regulation of translation initiation by FRAP/mTOR. Genes Dev., 15: 807– 826, 2001. 34. Isotani, S., Hara, K., Tokunaga, C., Inoue, H., Avruch, J., and Yonezawa, K. Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase ␣ in vitro. J. Biol. Chem., 274: 34493–34498, 1999. 35. Dufner, A., and Thomas, G. Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res., 253: 100 –109, 1999. 36. Dennis, P. B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S. C., and Thomas, G. Mammalian TOR: a homeostatic ATP sensor. Science (Wash. DC), 294: 1102–1105, 2001. 37. Desai, B. N., Myers, B. R., and Schreiber, S. L. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc. Natl. Acad. Sci. USA, 99: 4319 – 4324, 2002. 38. Nave, B. T., Ouwens, M., Withers, D. J., Alessi, D. R., and Shepherd, P. R. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem. J., 344: 427– 431, 1999. 39. Hardwick, J. S., Kuruvilla, F. G., Tong, J. K., Shamji, A. F., and Schreiber, S. L. Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. Proc. Natl. Acad. Sci. USA, 96: 14866 –14870, 1999. 40. Peng, T., Golub, T. R., and Sabatini, D. M. The immunosuppressant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation. Mol. Cell. Biol., 22: 5575–5584, 2002. 41. Beck, T., Schmidt, A., and Hall, M. N. Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J. Cell Biol., 146: 1227–1238, 1999. 42. Schmidt, A., Beck, T., Koller, A., Kunz, J., and Hall, M. N. The TOR nutrient signaling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J., 17: 6924 – 6931, 1998. 43. Warburg, O. On the origin of cancer cells. Science (Wash. DC), 123: 309 –314, 1956. 44. Radimerski, T., Montagne, J., Hemmings-Mieszczak, M., and Thomas, G. Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling. Genes Dev., 16: 2627–2632, 2002. 45. Jaeschke, A., Hartkamp, J., Saitoh, M., Roworth, W., Nobukuni, T., Hodges, A., Sampson, J., Thomas, G., and Lamb, R. Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J. Cell Biol., 159: 217–224, 2002. 46. Basso, A. D., Solit, D. B., Chiosis, G., Giri, B., Tsichlis, P., and Rosen, N. Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol. Chem., 277: 39858 –39866, 2002. 47. Potter, C. J., Pedraza, L. G., and Xu, T. Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell. Biol., 4: 658 – 665, 2002. 48. Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J., and Cantley, L. C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell, 10: 151–162, 2002. 49. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol., 4: 648 – 657, 2002. 50. McManus, E. J., and Alessi, D. R. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nat. Cell Biol., 4: E214 –E216, 2002. 51. Gao, X., Zhang, Y., Arrazola, P., Hino, O., Kobayashi, T., Yeung, R. S., Ru, B., and Pan, D. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat. Cell Biol., 4: 699 –704, 2002. 8460 Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research. Differential Effects of Rapamycin on Mammalian Target of Rapamycin Signaling Functions in Mammalian Cells Aimee L. Edinger, Corinne M. Linardic, Gary G. Chiang, et al. Cancer Res 2003;63:8451-8460. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/63/23/8451 This article cites 50 articles, 32 of which you can access for free at: http://cancerres.aacrjournals.org/content/63/23/8451.full.html#ref-list-1 This article has been cited by 38 HighWire-hosted articles. Access the articles at: /content/63/23/8451.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected] To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected] Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 2003 American Association for Cancer Research.