1 Mutations in critical domains confer the human mTOR gene strong tumorigenicity Avaniyapuram Kannan Murugan, Ali Alzahrani, and Mingzhao Xing Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Baltimore, MD 21287 Running title: Tumorigenicity of mutated mTOR gene Address all correspondence and requests for reprints to: Michael Mingzhao Xing, MD., Ph.D. Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 1830, East Monument street, Suite 333, Baltimore, MD 21287, USA, Tel.: 1-410-955-3663;E-mail: [email protected]Key words: mTOR gene; mTOR mutation; proto-oncogene; tumorigenicity; PI3K/Akt pathway --------------------------------------------------------------------------------------------------------------------- Capsule Background: The tumorigenic potential of mTOR has not been established. Results: Mutations of mTOR gene confer it gain-of-function and induce cell transformation, anchorage independent growth, invasion and tumorigenesis. Conclusion: mTOR gene is tumorigenic upon mutation. Significance: The results demonstrate for the first time the tumorigenicity of mTOR, hence establishing its oncogenicity and important role in human tumorigenesis. Abstract Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which regulates cell growth, proliferation and survival. mTOR is frequently activated in human cancers and is a commonly sought anticancer therapeutic target. However, whether the human mTOR gene itself is a proto- oncogene possessing tumorigenicity has not been firmly established. To answer this question, we mutated evolutionarily conserved amino acids and generated eight mutants in the HEAT repeats (M938T), FAT (W1456R and G1479N), and kinase (P2273S, V2284M, V2291I, T2294I, and E2288K) domains of mTOR and studied their oncogenicity. On transient expression in HEK293T cells, these mTOR mutants displayed elevated protein kinase activities accompanied by activated mTOR/p70S6K signaling at varying levels, demonstrating the gain of function of the mTOR gene with these mutations. We selected P2273S and E2288K, the two most catalytically active mutants, to further examine their oncogenicity and tumorigenecity. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated the mTOR/p70S6K signaling, induced cell transformation and invasion, and, remarkably, caused rapid tumor formation and growth in athymic nude mice after subcutaneous inoculation of the transfected cells. This study confirms the oncogenic potential of mTOR suggested previously and demonstrates for the first time its tumorigenicity. Thus, beyond the pivotal position of mTOR to relay the oncogenic signals from the upstream phosphatidylinositol-3 kinase/Akt pathway in human cancer, mTOR is http://www.jbc.org/cgi/doi/10.1074/jbc.M112.399485 The latest version is at JBC Papers in Press. Published on January 15, 2013 as Manuscript M112.399485 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on July 3, 2020 http://www.jbc.org/ Downloaded from
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Mutations in critical domains confer the human mTOR gene strong tumorigenicity Avaniyapuram Kannan Murugan, Ali Alzahrani, and Mingzhao Xing Laboratory for Cellular and Molecular Thyroid Research, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Baltimore, MD 21287 Running title: Tumorigenicity of mutated mTOR gene Address all correspondence and requests for reprints to: Michael Mingzhao Xing, MD., Ph.D. Division of Endocrinology and Metabolism, The Johns Hopkins University School of Medicine, 1830, East Monument street, Suite 333, Baltimore, MD 21287, USA, Tel.: 1-410-955-3663;E-mail: [email protected] Key words: mTOR gene; mTOR mutation; proto-oncogene; tumorigenicity; PI3K/Akt pathway --------------------------------------------------------------------------------------------------------------------- Capsule Background: The tumorigenic potential of mTOR has not been established. Results: Mutations of mTOR gene confer it gain-of-function and induce cell transformation, anchorage independent growth, invasion and tumorigenesis. Conclusion: mTOR gene is tumorigenic upon mutation. Significance: The results demonstrate for the first time the tumorigenicity of mTOR, hence establishing its oncogenicity and important role in human tumorigenesis. Abstract Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which regulates cell growth, proliferation and survival. mTOR is frequently activated in human cancers and is a commonly sought anticancer therapeutic target. However, whether the human mTOR gene itself is a proto-oncogene possessing tumorigenicity has not been firmly established. To answer this question, we mutated evolutionarily conserved amino acids and generated eight mutants in the HEAT repeats (M938T), FAT (W1456R and G1479N),
and kinase (P2273S, V2284M, V2291I, T2294I, and E2288K) domains of mTOR and studied their oncogenicity. On transient expression in HEK293T cells, these mTOR mutants displayed elevated protein kinase activities accompanied by activated mTOR/p70S6K signaling at varying levels, demonstrating the gain of function of the mTOR gene with these mutations. We selected P2273S and E2288K, the two most catalytically active mutants, to further examine their oncogenicity and tumorigenecity. Stable expression of the two mTOR mutants in NIH3T3 cells strongly activated the mTOR/p70S6K signaling, induced cell transformation and invasion, and, remarkably, caused rapid tumor formation and growth in athymic nude mice after subcutaneous inoculation of the transfected cells. This study confirms the oncogenic potential of mTOR suggested previously and demonstrates for the first time its tumorigenicity. Thus, beyond the pivotal position of mTOR to relay the oncogenic signals from the upstream phosphatidylinositol-3 kinase/Akt pathway in human cancer, mTOR is
http://www.jbc.org/cgi/doi/10.1074/jbc.M112.399485The latest version is at JBC Papers in Press. Published on January 15, 2013 as Manuscript M112.399485
Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
potentially capable of playing a direct role in human tumorigenesis if mutated. These results also support further that mTOR is a major therapeutic target in human cancers. Introduction Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase involved in the regulation of a variety of cellular functions, including cell growth and proliferation (1-3). mTOR belongs to the family of phosphatidylinositol-3 kinase (PI3K)-related kinases and contains a long stretch of protein-protein interaction modules in the N-terminus. These modules include the HEAT (Huntington, elongation factor 3, protein phosphatase 2A, and TOR1) repeats and the FAT (FRAP, ATM and TRRAP) domain. The C-terminus of mTOR contains a protein kinase domain as well as a short FAT domain at the extreme C-terminus (FATC), which is critical for the kinase activity of mTOR.
In mediating the upstream signalings, particularly that of the PI3K/Akt pathway, mTOR exists in two molecule complexes, mammalian target of rapamycin complex 1 (mTORC1) and mammalian target of rapamycin complex 2 (mTORC2). Both mTORC1 and mTORC2 use mTOR as the catalytic subunit and a major distinction between the two is that the former contains the rapamycin-sensitive regulatory associated protein of mTOR (raptor) whereas the latter contains rapamycin-insensitive companion of mTOR (rictor) (3). In response to growth factors and nutrients, mTORC1 regulates cell growth and proliferation through the phosphorylation and regulation of downstream effector substrates, among which the most important and best characterized is ribosomal protein S6 kinase 1 (S6K1; also
known as p70S6K) (4). The function of mTORC2 is not well understood but is involved in the modulation of the PI3K/Akt pathway (5, 6, 3).
Aberrant activation of mTOR signaling, particularly mTORC1, occurs commonly in human cancers (2, 7). Thus, mTOR has become an attractive therapeutic target in the development of cancer treatments in recent years (8), illustrating well the importance of mTOR/p70S6K signaling in human cancers and its clinical relevance. However, there is little genetic evidence to support this as mutations of the mTOR gene have been rarely found in human cancers. Activation of the mTOR in cancer has been known to be mainly a result of genetic-driven deregulation of upstream signaling, particularly that of the PI3K/Akt and MAP kinase pathways (9, 10, 3). Several previous studies attempted to characterize activating mutations in the mTOR gene. For example, expression of a deletion mutant ∆TOR (amino acids 2430-2450 in the kinase domain) in the HEK293 cells displayed a 3.5-10 fold increase in kinase activity, accompanied by increased phosphorylation of p70S6K (11). Expression of this ∆TOR deletion mutant in p53-/- MEF cells increased colony formation (12). Caffeine- or rapamycin-resistant mutations were identified in budding yeast TOR1 and introduction of these mutations in human mTOR resulted in elevated kinase activities (13). Artificial fission yeast screen with methyl-nitro-nitrosoguanidine mutagen identified a large number of activating mutations in the FAT and kinase domains which exhibited Rheb independence and hyperactive Tor2 phenotypes. Expression of these mutants in mammalian cells conferred constitutive activation of mTOR/p70S6K signaling (14). Three hyperactive Tor2 mutants have
been isolated from the yeast (S. cerevisiae), construction of one of these mutants in human mTOR was made (FLAG-mTORSL1) and its expression in HeLa cells demonstrated its increased kinase activity; when combined with the FRB domain mutant I2017T (FLAG-mTORSL1+IT), it further increased the kinase activity, showing an addictive effect on the mTOR kinase activity, but the hyperactive mTOR mutant did not seem to significantly affect the formations of foci (15). Some of the mTOR mutations (A8S, S2215Y, P2476L and R2505P) selected from human cancer genome database have been introduced into wt-mTOR and expressed in HEK293T cells. The cells expressing S2215Y and R2505P mutants showed increased mTOR kinase activities and activation of mTOR/p70S6K signaling. These mutants retained the phosphorylation of mTORC1 substrates even under nutrient starved conditions. Increased number of foci has been observed in Rat 1 cells when the mTOR mutant (S2215Y) co-expressed with mutant K-ras but, no much significant difference was observed when the mutant alone compared to wt mTOR (16).
These previous studies demonstrated the impact of mutations on the function of mTOR and in vitro oncogenicity in some cases. However, the oncogenicity of the human mTOR gene has not been firmly established. This is particularly the case given the lack of in vivo data on the tumorigenicity of the mTOR gene, since in vitro documentation of oncogenicity is only preliminary. In the present study, we introduced mutations into the evolutionarily conserved amino acid residues in key functional domains of the human mTOR and examined their impact on the biological activities and tumorigenecity of mTOR.
Experimental procedures Multiple amino acid sequence alignment and identification of conserved amino acid residues in mTOR Original amino acid sequences of mTOR of various species were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/protein/), including H_sapiens (NP_004949.1), C_lupus familiaris (XP_535407.2), B_taurus (XP_001788280.1), M_musculus (NP_064393.1), R_norvegicus (NP_063971.1), G_gallus (XP_417614.2), D_rerio (NP_001070679.2), D_melanogaster (NP_524891.1), and A_gambiae (XP_317619.4). These amino acid sequences were compared using a multiple sequence alignment program (http://pir.georgetown.edu/pirwww/search/multialn.shtml).
Based on the functional importance of the domains of the mTOR, weightage was given to each domain of mTOR as 12.5% for HEAT repeats, 25% for FAT domain and 62.5% for kinase domain. On this basis, 12, 25 and 63 highly conserved amino acid residues were identified from the HEAT repeats, FAT, and kinase domain, respectively. We then narrowed down the selection randomly to 1 of 12 from the HEAT repeats (M938), 2 of 25 from the FAT domain (W1456 and G1479), and 5 of 63 from the kinase domain (P2273, V2284, V2291, V2294 and E2288). These amino acid residues of mTOR were changed by site-directed in vitro mutagenesis as described below. The choices for amino acid change were made based on speculated possible impact on the function of the mTOR kinase as suggested by the biochemical properties of amino acids or in analogy with known mutations of other oncogenes. This is explained in details in Supplementary Table 1.
Expression vector and site-directed mutagenesis A mammalian expression vector (pCMV6) containing the human wild-type mTOR cDNA with c-myc tag at the C-terminal (Catalog No. RC220457) was obtained from OriGene Technologies, Inc. (Rockville, MD). This expression vector carrying the wild-type mTOR cDNA was used to generate 8 mTOR mutants M938T, W1456R, G1479N, P2273S, V2284M, V2291I, T2294I, and E2288K with a Quick Change XL II Site-Directed mutagenesis kit (Stratagene, Lajolla CA) according to the instructions of the manufacturer. The primers were designed using a template specific mutagenic primer design program. The primer sequences are as follows: 1) M938T: sense, mTOR-T2813C 5’-TAGTGAAATGCTGGTCAACACGGGAAACTTGCCTC-3’; antisense, mTOR-T2813C 5’-GAGGCAAGTTTCCCGTGTTGACCAGCATTTCACTA-3’. 2) W1456R: sense, mTOR-T4366C 5’-GAGAAACTGCACGAGCGGGAGGATGCCCTTG -3’; antisense, mTOR- T4366C 5’-CAAGGGCATCCTCCCGCTCGTGCAGTTTCTC-3’. 3) G1479N: sense, mTOR-G4435+6A 5’-CAGAGCTGATGCTGAACCGCATGCGCTGCC-3’; antisense, mTOR-G4435+6A 5’- GGCAGCGCATGCGGTTCAGCATCAGCTCTG-3’. 4) P2273S: sense, mTOR-C6817T 5’-CATCATGTTGCGGATGGCTTCGGACTATGACC-3’; antisense, mTOR-C6817T 5’-GGTCATAGTCCGAAGCCATCCGCAACATGATG-3’. 5) V2284M: sense, mTOR-G6850A 5’- TGACTCTGATGCAGAAGATGGAGGTGTTTGAGCAT-3’; antisense, mTOR-
G6850A 5’- ATGCTCAAACACCTCCATCTTCTGCATCAGAGTCA-3’. 6) V2291I: sense, mTOR-G6871A 5’-AGGTGTTTGAGCATGCCATCAATAATACAGCTGGG-3’; antisense, mTOR-G6871A 5’-CCCAGCTGTATTATTGATGGCATGCTCAAACACCT-3’. 7) T2294I: sense, mTOR-C6881T 5’- GTTTGAGCATGCCGTCAATAATATAGCTGGGGACGA-3’; antisense, mTOR-C6881T 5’-TCGTCCCCAGCTATATTATTGACGGCATGCTCAAAC-3’. 8) E2288K: sense, mTOR-G6862A 5’-ATGCAGAAGGTGGAGGTGTTTAAGCATGCCGTC-3’; antisense, mTOR-G6862A 5’-GACGGCATGCTTAAACACCTCCACCTTCTGCAT-3’. The mutations were confirmed in the vectors by sequencing with the primer SEQ-1(2821) _F 5’-AGTGAACATTGGCATGATAGAC-3’, SEQ-2(4291)_F 5’- GAGTGTTAGAATATGCCATG-3’, and SEQ-3(6671)_F 5’-TTCCTTCTAAAAGGCCATGAAG-3’. Plasmid DNAs for the transfection experiments were purified using a mini prep kit (Cat # K2100-11, Invitrogen, Carlsbad CA). Cell culture, transient transfection and cell lysate preparation HEK293T cells obtained from American Type Culture Collection (ATCC) were grown at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were transiently transfected with an empty vector, wild-type or each of the mutant mTOR expression vectors using the Lipofectamine 2000 transfection reagent per manufacturer’s instructions (Invitrogen Life Technologies, CA). Medium was
changed 24 h after transfection. After 48 hr of transfection, cells were washed twice with ice-cold Tris-buffered saline (TBS) and lysed on ice using a freshly prepared ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% tween-20 (w/v), 1mM EDTA, 20 mM microcystein-LR, 25mM NaF, and a complete, EDTA-free protein inhibitor cocktail (Roche Applied Science, Mannheim Germany) at 20 µl per mL of lysis buffer. Cell lysates were collected in a 1.5 mL microfuge tube and disrupted by ultra-sonication for 15 sec on ice and further incubated on ice for 30 min. Cell lysates were then centrifuged at 20,000g for 10 min at 4 °C. The supernatants were collected and protein concentrations were measured using a DC protein assay kit (Bio-Rad Laboratories, Hercules CA). This cell lysate was used for Western blotting and immunoprecipitation. Immunoprecipitation For vector, wild-type and mutant lysates, the protein concentration was adjusted to 1 µg/µl using lysis buffer and 500 µg total lysate protein from each sample was used for immunoprecipitation. The 500-µg lysate was pre-cleared by adding 15µl of protein-G-agarose beads (Roche Applied Science, Mannheim Germany), followed by an incubation for 15 min at 4ºC. The protein-G-agarose beads were pelleted by centrifugation at 4,000 rpm in a microcentrifuge for 5 min at 4ºC. The pre-cleared lysate was transferred to a clean tube, added with 5 µg of anti-c-myc antibody, and incubated on a rotator for 1 hr at 4ºC. The lysates were then added with 50 µl protein-G-agarose and further incubated on the rotator for 90 min at 4ºC. The protein-G-agarose beads were pelleted by centrifugation in the microcentrifuge at 4,000 rpm for 5 min at 4ºC. The resulting
supernatant was carefully removed and discarded. The pelleted beads were washed four times with 500 µl of lysis buffer and one time with kinase assay buffer supplied with the K-LISA mTOR (Recombinant) Activity kit (Cat # CBA104, Calbiochem-EMD Chemical Inc., Philadelphia, PA). Half of the immunoprecipitate was used for mTOR kinase assay and the other half was resolved in a SDS/PAGE and subjected to Western blotting analyses. Protein kinase assay of mTOR The kinase activity of mTOR was assayed as described previously (17-19), using a commercially available K-LISA mTOR (Recombinant) Activity kit (Cat # CBA104, Calbiochem-EMD Chemical Inc., Philadelphia, PA) according to the manufacturer’s instructions. Briefly, immunoprecipitates obtained above were resuspended in 50 µl of 2X kinase buffer and gently mixed with 50µl of mTOR substrate, followed by an incubation at 30ºC for 30 min. The mTOR substrate used in this assay was a p70S6K-GST fusion protein, which is phosphorylated at threonine 389 of p70S6K by active mTOR in the presence of ATP. After the reaction, the reaction mixture was incubated in a glutathione-coated 96-well plate to facilitate the binding of GST-fused p70S6K to the plate. The phosphorylated substrate was detected using anti-p70S6K-pT389 antibody, followed by detection with HRP-antibody conjugate and TMB substrate. Sensitivity was increased by the addition of ELISA stop solution and relative activities were determined by reading at the absorbance wave length of 450 nm. Western blotting Cells were washed once with phosphate-buffered saline (PBS) and lyzed in RIPA
lysis Buffer (Cat. No. sc-24948, Santa Cruz Biotechnology, Santa Cruz CA). Western blotting was performed using 45 μg of cell lysates resolved on SDS/PAGE and transferred to a PVDF membrane (Millipore Co., Bedford, MA). The membrane was blocked with 5% skim-milk/PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temperature and the membrane was then sliced based on the molecular weights. Membranes were incubated overnight at 4ºC with primary antibodies, including anti-Myc (Cat # sc-40, Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p70S6K (Cat # 9234, Cell Signaling Technology, Danvers, MA), anti-mTOR (Cat # 2972, Cell Signaling Technology, Danvers MA), anti-phospho-4EBP1 (Cat # 2855, Cell Signaling Technology, Danvers MA), anti-phospho-Akt (Cat # sc-7985, Santa Cruz Biotechnology), anti-phospho-S6 Ribosomal protein (Cat # 4858 & 2215, Cell Signaling Technology, Danvers MA) or anti-β-actin (Cat # sc-1616R, Santa Cruz Biotechnology) antibody. The membranes used for phospho-4EBP1, phospho-p70S6K, phospho-Akt and phospho-S6 Ribosomal protein were stripped off for total 4EBP1 (Cat # sc-81149, Santa Cruz Biotechnology), p70S6K (Cat # sc-8418, Santa Cruz Biotechnology), total Akt (Cat # sc-5298, Santa Cruz Biotechnology) and total S6 Ribosomal protein (Cat # 2217, Cell Signaling Technology, Danvers MA), respectively. After washing four times with PBST, blots were incubated with respective HRP conjugated anti-rabbit or anti-mouse secondary antibodies (Cat # sc-2004 and # c-2005, Santa Cruz Biotechnology) for 1 h at room temperature. After washing with PBST, protein bands on the membrane were detected with enhancement chemiluminescence (ECL) reaction
reagents (Amersham Biosciences, Piscataway, NJ) and exposure to X-ray films. Cell culture, transfection and focus formation assay of NIH3T3 cells NIH3T3 cells (ATCC) were cultured in DMEM supplemented with fetal calf serum (FCS). Focus formation assay was done as described previously (20). Briefly, NIH3T3 cells were transfected with equal amount of empty vector or vectors expressing the wild-type mTOR or mutant mTOR P2273S or E2288K in a 6-well plate (Costar® Corning, NY) using the Lipofectamine 2000 transfection reagent as instructed by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). After 48 hrs of transfection, cells were washed once with PBS, tripsinized briefly, and transferred to a T-75 plate containing DMEM supplemented with 10% FCS and 800µg/mL G418. Medium was changed every 3-4 days. After 3 weeks, morphologically transformed, multi-layered foci were counted and photographed (Nikon Eclipse Ti-U, Tokyo, Japan) Cell focus cloning and stable cell line establishment For the vector, wild-type mTOR, mutant mTOR P2273S, and mutant mTOR E2288K, each single multi-layered foci formed from the afore-mentioned focus formation assay was cloned using cloning cylinders (Cat # TR-1004, Millipore, Billerica MA). Cells from a single colony were cultured, expanded and maintained in DMEM containing 10% FCS and 800µg/ml G418. All the clones were subjected to genomic DNA isolation, PCR amplification, sequencing and Western blotting for confirmation of integration and expression of the transfected plasmid constructs. Briefly, a portion of each
stably transfected NIH3T3 cell clone was lysed and genomic DNA was isolated by standard phenol-chloroform extraction, using MaXtract high-density gel tubes (Qiagen) as described previously (21). Integration of the plasmid and presence of the mutation in the mTOR ORF and myc-tag were confirmed by PCR amplification of the genomic DNA using a forward primer binding in the mTOR cDNA and the reverse primer binding in the vector back bone of pCMV6. The amplified PCR products were directly sequenced using a BigDye terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems) and an ABI PRISM 3730 genetic analyzer (Applied Biosystems). All the single clones were also analyzed by Western blotting as described above for over-expression of mTOR and activation of the mTOR/p70S6K signaling pathway. The verified clones were used in further experiments for functional studies. Determination of morphological transformation Morphological transformation of NIH3T3 cells expressing mTOR mutants was determined as described previously (22). Briefly, NIH3T3 cells stably transfected with the empty vector, wild-type mTOR, and the indicated mTOR mutants were plated in 60-mm culture plates (Corning, NY) at a density of 3 X 105 cells. After cultivation of the cells for one day in regular medium (DMEM+10% FCS+G418 800µg/ml), cell morphology was examined under a microscope (Nikon Eclipse Ti-U, Tokyo, Japan). Soft-agar colony formation assay Soft-agar colony formation assay was performed as previously described (20). Briefly, NIH3T3 cells stably expressing the indicated type of mTOR were seeded at 1.0 X 104 cells/well on 6-well plates
(Costar® Corning, NY) in 0.3% agar (Cat # 214010, BD Biosciences) over a bottom layer of 0.6% agar. After 4 weeks, the colonies of > 0.1 mm were counted and photographed (Zeiss Axiovert 200M, CarlZeiss, Germany). Invasion assay Cell invasion assay was performed as described previously (20). Briefly, the assay was performed using matrigel invasion chambers consisting of BD FalconTM cell culture inserts containing a polyethylene terephthalate (PET) membrane with 8 µm pores coated with matrigel matrix (BD BioCoatTM MatrigelTM Invasion Chamber, BD Biosciences, Bedford, MA). NIH3T3 cells stably transfected with the empty vector, the wild-type mTOR or the indicated mutant mTOR were completely serum-starved for 8 h and then collected and re-suspended with 5 X 104 cells in 500 µL of serum-free DMEM supplemented with 0.1% BSA. Culture inserts were placed in the wells of a BD Falcon 24-well multi-well companion plate and 750 µL of DMEM containing 1% serum was added to the lower compartment of each well. Cell suspensions were added to each culture inserts. After a 22-h incubation at 37ºC with 5% CO2, the non-invading cells on top of the matrigel were removed using cotton swab and invaded cells on the lower side of the membrane were fixed with 70% ethanol and stained with Coomassie Brilliant Blue (CBB). Invading cells were counted and photographed under a microscope with 10X magnification (Nikon Eclipse Ti-U, Tokyo, Japan) Xenograft tumorigenicity assay Four-week-old female nude mice (Hsd: Athymic Nude-Foxn1nuMICE) were purchased (Harlon, Frederick MD) and maintained under standard conditions and
in vivo experiments were performed per our institution’s guidelines for the use of laboratory animals. NIH3T3 cells stably expressing the empty vector, wild-type mTOR or the indicated mTOR mutants were washed once with PBS, briefly trypsinized , pelleted, and re-suspended in serum-free medium (DMEM). An aliquot of 150 µl medium containing 2.0 X 107 cells were injected subcutaneously into the right flank of nude mice using 23-gauge needles. Each group consisted of 5 animals. Tumor formation was monitored. After 2 weeks, tumors were assessed and photographed. Unless otherwise indicated, data are presented as representative of at least three independently performed experiments. Results Identification and selection of evolutionarily highly conserved amino acid residues in mTOR for site-directed in vitro mutagenesis. The goal of this study was to test and definitely confirm the oncogenic potential of the human mTOR gene and its candidacy for proto-oncogene by introducing mutations into mTOR gene and testing their impact on the functions of mTOR. To this end, we first performed amino acid sequence alignment analyses of mTOR from various species as described in the Materials and Methods and illustrated in Fig. 1 and identified the evolutionarily most conserved amino acid residues. As shown in Fig. 1A, by giving weightage to the importance of the functional domains of the mTOR, we identified and selected 8 amino acid residues— M938, W1456, G1479, P2273, V2284, V2291, V2294 and E2288 for site-directed in vitro mutagenesis. As illustrated in Fig. 1B, among the 8 amino acid residues, one was from the HEAT repeats (M938), two from the FAT domain
(W1456 and G1479), and five from the kinase domain (P2273, V2284, V2291, V2294 and E2288). These amino acid residues were randomly mutated to M938T, W1456R, G1479N, P2273S, V2284M, V2291I, T2294I and E2288K for further analysis of their impact on the function of mTOR and its oncogenicity. We have cross-checked somatic mutation (Catalogue of Somatic Mutations in Cancer - COSMIC) and single nucleotide polymorphism (SNP) data bases (http://www.ncbi.nlm.nih.gov/projects/SNP/ and http://useast.ensembl.org/Homo_sapiens/Gene/Variation_Gene/) and assessed whether these mutations are already identified in human cancers. To date, none of these mutations has been enlisted in these databases. mTOR mutations conferred gain of function with enhanced protein kinase activities To determine the functional consequences of mTOR mutations, we tested and compared the in vitro protein kinase activities of immunoprecipated mTOR mutants from transiently expressed in HEK293T cells. As shown in Fig. 2A, the 8 mTOR mutants, including M938T, W1456R, G1479N, P2273S, V2284M, V2291I, T2294I and E2288K, showed significantly increased protein kinase activities on the substrate p70S6K-GST fused protein in comparison with the wild-type mTOR, ranging from 4-11 folds. As expected, the empty vector transfection alone did not show activities. As shown in Fig. 2B, expression of mTOR protein both in immunoprecipitates and in whole cell lysates was confirmed by Western blotting for the wild-type mTOR and all the 8 mTOR mutants. As expected, the empty vector did not express the mTOR protein. Thus, all these introduced mTOR
mutations were gain of function in nature and conferred the mTOR increased protein kinase activities to various extents. mTOR mutants activated the mTOR/p70S6K and Akt signaling pathway in HEK293T cells Given the elevated kinase activities of the mTOR mutants on the substrate p70S6K-GST fused protein, we next examined the impact of these mTOR mutants on the endogenous downstream mTOR/ p70S6K signaling activities in HEK293T cells. As shown in Fig. 3A, this signaling, as reflected by the elevated phosphorylation level at threonine 389 of the immediate downstream substrate p70S6K in HEK293T cells was significantly enhanced by transfection with various mTOR mutants in comparison with the wild-type mTOR. As illustrated in Fig. 3B, quantification of the phosphorylation bands of p70S6K with normalization for total protein of p70S6K more clearly shows the dramatic increase in the mTOR/ p70S6K signaling activities, up to several hundreds folds in some cases compared with the wild-type mTOR. This dramatic increase in the mTOR/ p70S6K signaling was particularly prominent with mutants W1456R, P2273S and E2288K, consistent with the finding that these mutants displayed the highest proteins kinase activities on the p70S6K-GST fused protein in the in vitro assay on immunoprecipated mTOR proteins (Fig. 2). To test whether the mTOR mutants affected the mTORC2 function, we performed Western blotting to examine the phosphorylation of Akt at S473, a direct substrate of mTORC2, in transiently transfected HEK293T cells. As shown in Fig 3A and 3C, these mTOR mutants caused Akt phosphorylation to various extents. Among the 8 mutants, W1456R, P2273S, and E2288K displayed the highest impact on both the mTOR/p70S6K and Akt signaling. We next examined whether the mTOR mutants confer activation of 4EBP1 and the downstream effector of p70S6K, rpS6. As illustrated in Supplementary Fig 2, these mutants minimally enhanced phosphorylation of 4EBP1 compared to wild-type mTOR. We found a robust
phosphorylation of rpS6 at the basal state and could not see further increase in this phosphorylation by mTOR mutants in HEK293T cells (Supplementary Fig 2). Expression of mTOR mutants promoted focus formation of NIH3T3 cells We took the next step to explore the oncogenic potential of mTOR gene by selecting two kinase domain mutants of mTOR, P2273S and E2288K, which exhibited the highest gained activities (Fig. 2 and 3), to test their impact on focus formation of NIH3T3 cells—loss of contact inhibition of cell growth—a cell phenotypic reflection of oncogenic property of oncogenes. We stably transfected NIH3T3 cells with corresponding vectors as indicated in Fig. 4. After a 3-week culture, cells transfected with P2273S and E2288K readily formed multi-layered foci whereas, in contrast, cells transfected with the wild-type mTOR or empty vector did not (Fig. 4A and Supplementary Fig 1). As shown in Fig. 4B, mutational analysis of the transfected NIH3T3 cell foci showed the presence of corresponding introduced mTOR mutations. Fig. 4C quantitatively illustrates the increased focus formation of cells with mTOR mutants in comparison with the wild-type mTOR or empty vector. As a positive control, we also examined the impact of oncoprotein Hras G12V on focus formation of NIH3T3 cells and, as expected, observed similar results as with mTOR mutants (Fig. 4A and 4C). These findings suggest that the mTOR mutants are likely oncogenic. Expression of mTOR mutants caused transformation of NIH3T3 cells The cell focus-forming ability of mTOR mutants demonstrated above suggested that the mTOR mutant-expressing cells might have been transformed. To further test
this, we also examined the cell morphology that could have altered as a reflection of transformation. Indeed, as shown in Fig. 5A, we found that NIH3T3 cells stably expressing the vector or the wild-type mTOR resembled the parental cells in morphology, but cells stably expressing mTOR mutants P2273S and E2288K exhibited striking morphological changes with more aggressive appearance, including the loss of complete adherence, cell thickening and twisting, spindle-shaping, and stacking growth of cells on one and another. These are all characteristic features of cell transformation. As shown in Fig. 5B and 5C, Western blotting of lysates from these cells confirmed the increased expression of mTOR in the cells transfected with the wild-type mTOR or mTOR mutants P2273S and E2288K. Over-activated mTOR/p70S6K and Akt signaling, as reflected by the dramatically increased threonine 389 phosphorylation of p70S6K and serine 473 phosphorylation of Akt, respectively, were also seen in cells expressing the mutants, but not in cells expressing the empty vector or the wild-type mTOR (Fig. 5B, 5D and E). We also examined whether these mutants confers phosphorylation of 4EBP1 and the downstream effector of p70S6K, rpS6. As illustrated in Supplemental Fig 3, we found strong activation of 4EBP1 as reflected by the phosphorylation of 4EBP1 at threonine 37/46. We also found robust rpS6 phosphorylation at S235/236 and S240/244. These results corroborate that, unlike HEK293T cells, mTOR mutants robustly activate the classical mTOR/p70S6K/rpS6 and 4EBP1signaling in NIH3T3 cells that lead to protein synthesis, transformation and oncogenecity. To further examine the cell transforming ability of mTOR mutants
P2273S and E2288K, we also performed anchorage-independent soft agar colony formation assay. As shown in Fig. 5F and 5G, NIH3T3 cells stably expressing the two mTOR mutants formed significantly more colonies with extremely large size on soft agar in striking contrast with the empty vector or wild-type mTOR-expressing cells. These results thus demonstrate the strong cell transforming ability of mTOR mutants, further supporting their oncogenicity. Expression of mTOR mutants promoted invasion of NIH3T3 cells To drive cancer cell invasion is characteristic of many oncogenes. A previous study demonstrated an important role of activated p70S6K in cell-motility (23). We were therefore interested in finding out whether mTOR mutants could promote cell invasion. As shown in Fig. 6A, NIH3T3 cells stably expressing mTOR mutants P2273S or E2288K exhibited a much more avid invasion on Matrigel matrix-coated membrane than the empty vector or wild-type mTOR-expressing cells. As shown in Fig. 6B, the number of invading cells was significantly higher with the mTOR mutants than with the vector and wild-type mTOR, again suggesting that these mTOR mutants are oncoproteins. Expression of mTOR mutants induced tumor formation in nude mice To firmly establish the oncogenicity of strongly activated mTOR, we next tested tumorigenicity of selected mTOR mutants, P2273S and E2288K, in nude mice. Shown in Fig. 7A are large tumors that developed at two weeks after subcutaneous inoculation into athymic nude mice of NIH3T3 cells stably expressing the two mTOR mutants. All the five animals in each of the two mutant groups developed
tumors. Tumors in some animals displayed necrotic changes. In contrast, no significant tumor developed at two weeks with cells stably transfected with empty vector or the wild-type mTOR, although a small tumor-like structure developed in some animals with wild-type mTOR at 4-5 weeks. Thus, this in vivo result on tumorigenicity of mTOR mutants definitely demonstrated the oncogenicity of genetically activated mTOR. Fig. 7B schematically illustrates the pivotal position of mTOR in this tumorigenicity.
All the above in vitro and in vivo experiments using stable cell transfectants used cell foci, including cells transfected with the wild-type mTOR that formed very few foci. But, oncogenicity was seen only with mutant transfectants, not the wild-type mTOR. Formation of small tumors was seen with wild-type mTOR after three weeks of cell inoculation in animal studies (data not shown) in contrast to the rapid formation of large tumors with mTOR mutants. The results are presented as representatives, representing at least two experiments.
Discussion
As a ubiquitously expressed serine/threonine protein kinase and a key signaling molecule downstream the PI3K/Akt pathway, mTOR is widely known to play a critical role in controlling human cancer cell growth and proliferation and is being actively tested as a major therapeutic target for cancers both preclinically and clinically (8, 9). The current known mechanism for persistent activation of mTOR in human cancers is the epigenetic and genetic alterations of the upstream signaling molecules, such as the methylation of PTEN (24), mutations of Ras, PIK3CA, and PTEN (9, 25, 8,), and genetic copy gain of receptor tyrosine kinase genes and other genes in the
PI3K/Akt and MAP kinase pathways (26, 10). Mutation of the mTOR gene itself has been only occasionally found in some cancers (27-29). The tumorigenic potential of the human mTOR gene has not been established.
In the present study, by introducing mutations into evolutionarily conserved amino acid residues in major functional domains of the human mTOR, we were able to generate mTOR mutants that possessed dramatically increased protein kinase activities with strong oncogenicity in vitro and tumorigenicity in vivo. We thus for the first time definitively demonstrate that the human mTOR gene is a proto-oncogene that possesses strong tumorigenicity when genetically activated. The present results also provide further support that mTOR lies in a pivotal position to relay the oncogenic signals from constitutively activated upstream signaling cascades in cancer cells, assuring further that mTOR is a key cancer therapeutic target as widely tested.
It has been suggested that mutations affecting preferentially residues that are highly conserved in evolution are likely of critical functional importance (30). Indeed, the majority of cancer-associated oncogenic mutations identified so far are highly conserved evolutionarily (31, 32). Our strategy of identifying and selecting the highly conserved amino acids through the multiple-amino acid sequence alignment approach to create functional mutations in major function domains of mTOR proved to be successful in studying the oncogenic potential of the mTOR gene. The amino acid residues that we mutated in mTOR in the present study are evolutionarily highly conserved over the divergence of different species. Like mTOR, these amino acid residues are also highly conserved within other members of the PIKK family (33). These regions are
less evolved because they are located in the catalytically important regions. To date, the three-dimensional structure of mTOR has not been available. Recently, a predicted structure for the C-terminal region of the mTOR including the FAT, kinase and FATC domains was proposed based on the amino acid sequence alignment of the known crystal structure of the PIKK family member PI3KCγ (33). The region consisting of amino acids 2180-2381 of mTOR is considered to be a similar catalytic loop to that of PI3KCγ. All the kinase domain mutations analyzed in the present study (P2273S, V2284M, E2288K, V2291I and T2294I) are located in this catalytic loop and in the predicted helix equivalent to kα5 of PI3KCγ. They are also located adjacent to the region kα3, which is one of the three kinase active sites [region I(Kα3), II(kα9) and III(kα11)] of mTOR where activating mutations of fission yeast TOR2 have been found to be clustered (33, 34). A conformational change associated with mutations may be responsible for the elevated protein kinase activity of mTOR observed in the present study.
The mTOR mutants generated in the present study displayed varying elevated protein kinase activities on the downstream substrate p70S6K either in cell-free enzymatic assays or in cell systems to different extents. Some of the mTOR mutants generated from mutations in the kinase domain, such as P2273S and E2288K, exhibited the highest enzymatic activities and the highest capability of activating the p70S6K signaling. Mutant W1456R from a FAT domain mutation also exhibited remarkable activation whereas mutant M938T from a mutation in the HEAT domain exhibited only a moderate activation. These results are interestingly consistent with a previous observation that mutagen induced random
mutagenesis in fission yeasts resulted in the accumulation of activating point mutations mostly in the kinase and FAT domains of TOR2 (14). These results are not surprising as the kinse and FAT domains, particularly the former, are the most important to the kinase activities of mTOR.
It is striking to see the several hundreds-fold activation of the p70S6K signaling in cells expressing some of the mTOR mutants, suggesting a high oncogenic power of these mutants. Although these mutants enhanced the phosphorylation of 4EBP1 (Thr37/46) in HEK293T cells, the effect was small (Supplemental Fig 2). However, stable expression of mutants P2273S and E2288K in NIH3T3 cells strongly phosphorylated both 4EBP1 at Thr37/46 and p70S6K (Supplemental Fig 3). Thus, these mTOR mutants seem to signal mainly through p70S6K in some cells, but can activate both p70S6K and 4EBP1 in other cells. The p70S6K preferentiality of the mTOR mutants in HEK293T cells is consistent with a previous report that p70S6K is more than 10 fold preferred than 4EBP1 as a substrate by mTOR in these cells (19). A recent study demonstrated that in urothelial cells (RT112 and T24), ShRNA-mediated silencing of mTOR resulted in dephosphorylation of p70S6K1 but only had a minor effect on 4EBP1 (35). This provides further evidence that p70S6K is a much more preferred substrate than 4EBP1 for mTOR in certain cells. Moreover, we found a robust full phosphorylation of rpS6 at the basal state which was not changed by mTOR mutants in HEK293T cells, although stable expression of mutants P2273S and E2288K in NIH3T3 cells dramatically phosphorylated rpS6 at both serine 235/236 and serine 240/244. These results suggest that mTOR might not signal
to rpS6 in HEK293T cells, unlike in NIH3T3 cells. It has been suggested in recent years that the classical mTORC1/p70S6K1/rpS6 signaling may not be valid in all cell systems and an alternative signaling that leads to the phosphorylation of rpS6 independently of mTOR/p70S6K in some cell systems also exists (36). Mutants P2273S and E2288K conferred mTOR remarkable oncogenicity, as demonstrated by their strong power to cause and promote NIH3T3 cell transformation as reflected by the unique cell morphological changes, contact-inhibition-free focus formation, and anchorage-independent cell growth. Moreover, the mTOR mutants also conferred cells high invasiveness—another common feature of classical oncogenes. Most remarkably, driven by mTOR mutants, NIH3T3cells can develop large tumors in nude mice, which were necrotic in some cases, demonstrating strong tumorigenicity of these mTOR mutants. Thus, these in vitro and in vivo data all support that these mTOR mutants meet the classical criteria for oncoproteins. mTOR is thus another classical example that proto-oncogenes are usually genes that play a critical role in signaling pathways, gene transcription, protein synthesis, and metabolism pathways (37).
Results from several previous functional studies on genetic manipulation of the human mTOR and yeast TOR genes support the conclusion of the present study on mTOR as a proto-oncogene. For example, a small deletion of amino acids 2430-2450 in the kinase domain close to the carboxyl terminus of the mTOR could moderately increase the kinase activity of mTOR and cause colony formation of p53-/- MEF cells (11, 12). Introduction of mTOR mutations S2215Y and R2505P from the human cancer genome database into the wild-type mTOR could increase its
kinase activity and enhance the mTOR/p70S6K signaling when expressed in HEK293T cells (16). Introduction of yeast TOR mutations into the human mTOR also increased the activities of the latter (13, 15). Interestingly, artificial fission yeast screen with methyl-nitro-nitrosoguanidine mutagen identified a large number of activating TOR mutations in the FAT and kinase domains and expression of these mutants in mammalian cells conferred constitutive activation of the mTOR/p70S6K signaling (14). However, unlike the present study, none of the above studies tested the tumorigenicity of the mTOR gene.
The strong oncogenicity and tumorigenicity of some of the human mTOR mutants demonstrated in the present study, alone with the previous studies discussed above, including the high induciblility of TOR mutations in yeast, suggest that oncogenic mTOR mutations likely exist in certain human cancers. Further, it will be reasonable to extensively search for oncogenic mTOR mutations in human cancers as recently pursued for renal cancer which interestingly revealed intra-tumor heterogeneity of an occasional mTOR mutation (38). Genetic alterations in the PI3K/Akt pathway confer certain cancers a poorer prognosis (39-41) and cancer cell sensitivity to the inhibitors of the PI3K/Akt/mTOR pathway (42-45). It is thus expected that discovery of oncogenic mTOR mutations in human cancers may facilitate molecular-based prognostication and therapeutic targeting of such cancers. Acknowledgements This work was supported by NIH grant R01CA134225 (to MX). Conflict of Interest The authors declare no competing financial interests.
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Legends to Figures
Fig. 1. Identification and selection of evolutionarily conserved wild-type amino acid residues in mTOR for the generation of mTOR mutants. A. Amino acid sequence alignment of the mTOR proteins from 9 different species. The eight selected amino acid residues M938 in the HEAT repeats, W1456 and G1479 in the FAT domain, and P2273, V2284, E2288, V2291 and T2294 in the kinase domain are evolutionarily highly conserved among these different species. The selection criteria are as detailed in the Materials and Methods. B. Schematic diagram of the major functional domains of the mTOR protein. Shown are the mutations generated by site-directed mutagenesis of the 8 amino acids in Fig. 1A and the domains of mTOR carrying them. The numbers indicate amino acid or codon positions, with the initiation codon (methionine) of the protein defined as number 1. Fig. 2. mTOR mutants show increased protein kinase activities. A. In vitro assay of protein kinase activities of mTOR mutants. HEK293T cells were transiently transfected with myc-tagged vector, wild-type mTOR (Wt), and each of the 8 mTOR mutants as indicated. Cell lysates were immunoprecipitated with the anti-c-myc antibody and immunoprecipitates were assayed for protein kinase activity of mTOR as described in the Materials and Methods. B. Expression of mTOR mutants in the HEK293T cells corresponding to the assays in Fig. 2A. HEK293T cells transiently transfected with the indicated vector constructs as described in Fig. 2A and cell lysate proteins were subjected to immunoprecipitation followed by the corresponding protein kinase assays in Fig. 2A. A part of these immunoprecipitates and whole cell lysate from the above indicated transfectants were visualized by SDS-PAGE and Western blotting analyses and for the indicated proteins using appropriate antibodies as described in the Materials and Methods. Successful immunoprecipitation of myc-tagged wild-type mTOR and each of the mTOR mutants is shown in the top row of Fig. 2B. Successful expression of the wild-type and each of mTOR mutants was reconfirmed by analyzing the whole cell lysate shown in the subsequent row. β-actin is used for quality control of the loading proteins.
Fig. 3. Activation of the mTOR/p70S6K and Akt signaling pathways by mTOR mutants in HEK293T cells. A. Western blotting analysis of the HEK293T cells transfected with myc-tagged vector, wild-type mTOR and each of mTOR mutants. Activation is reflected by increased phosphorylation of p70S6K (P-p70S6K) and Akt (P-Akt). HEK293T cells were transiently transfected with c-myc-tagged vector, wild-type mTOR (Wt), and each of mTOR mutants as indicated. Cell lysates were subjected to Western blotting analyses for the indicated proteins using appropriate antibodies as described in the Materials and Methods. Shown, from top to bottom, are the expression of empty vector, wild-type mTOR (Wt), and eight mTOR mutants; phosphorylation levels of p70S6K (T389); total p70S6K; phosphorylation levels of Akt (S473); total Akt; and β-actin for quality controls of loading proteins. B and C. Quantitative presentation of the phosphorylation levels of p70S6K and Akt, respectively. Phosphorylation levels of p70S6K and Akt corresponding to the transfection conditions in Fig. 3A as indicated were normalized by dividing the intensities of P-p70S6K by the total p70S6K and P-Akt by the total Akt in Fig. 3A. Results represent mean ± S.D. of three independent experiments. Fig. 4. Focus-formation of NIH3T3 cells promoted by mTOR mutants. A. Cell focus-forming activities of mTOR mutants. Shown are images of adherent growth of NIH3T3 cells transfected with myc-tagged vector, wild-type mTOR, and each of the mTOR mutants indicated. Cells were cultured in regular medium with 10% FCS under standard conditions. Images of cell foci were photographed with 10X magnification after appropriate culture of cells as described in the Materials and Methods. Transfection of cells with Hras G12V as a positive control induced cell focus formation. B. Sequencing electropherogram of the mTOR gene. Each multilayered foci was cloned and cultured and genomic DNA was isolated, PCR amplified and sequenced as described in the Materials and Methods. As expected, sequencing electropherogram of the empty vector shows no amplification or a junk, two wild-types show no mutation in the corresponding positions of the mutants analyzed, and mutants show the expected introduced mutations for P2273S and E2288K. C. Number of cell foci formed with the indicated transfections. The number of transfectd foci was counted 21 days after cell transfection. Results represent mean ± S.D. of three independent experiments. Fig. 5. Morphologic transformation and anchorage-independent growth of cells transfected with mTOR mutants. A. Morphologic transformation of NIH3T3 cells stably expressing mTOR mutants. Cells were plated at low density, cultured, maintained and photographed as detailed in the Materials and Methods. Shown are representative images of morphology of NIH3T3 cells stably expressing empty vector, wild-type mTOR and the indicated mTOR mutants. B. Corresponding expression of the mTOR mutants and their activation of the mTOR/p70S6K and Akt signaling in NIH3T3 cells. NIH3T3 cells corresponding to Fig 5A stably transfected with the empty vector, wild-type mTOR, and the indicated mTOR mutants were subjected to lysis and Western blotting analyses as presented in Fig. 3A. Thus, stable transfection and expression of mTOR mutants in NIH3T3 cells also activated the mTOR/p70S6K and Akt signaling, consistent with the similar observations in transiently transfected HEK293T cells (Fig 3). C. Relative expression of the mTOR protein in stably transfected NIH3T3 cells. Densitometry was
performed to measure the density of the mTOR and β-actin bands in Fig. 5B. The relative mTOR levels were obtained by dividing the mTOR band density by the corresponding β-actin band density. D and E. Relative phosphorylation of the p70S6K and Akt protein in stably transfected NIH3T3 cells. Densitometry was performed to measure the densities of the phospho-p70S6K, p70S6K, phospho-Akt and Akt bands in Fig. 5B. The relative phospho-p70S6K and phospho-Akt levels were obtained by dividing the phospho-p70S6K and phospho-Akt band densities by the corresponding p70S6K and Akt band density, respectively. F. Anchorage-independent cell growth of mTOR mutants on soft agar. NIH3T3 cells stably transfected with the empty vector, wild-type mTOR, and the indicated mTOR mutants as confirmed in Fig. 5B were seeded in soft agar. Colonies formed 4 weeks later and were photographed with 40X magnification. G. Analyses of the number of colonies. The number of cell colonies corresponding to Fig. 5C that were > 0.1 mm in diameter was counted. Results represent mean ± S.D. of three independent experiments. Fig. 6. Cell invasion promoted by mTOR mutants. A. In vitro invasion assay of NIH3T3 cells with various transfections. Cells were stably transfected with the empty vector, wild-type mTOR, and the indicated mTOR mutants, followed by cell invasion assay performed as described in the Materials and Methods. Shown are the cells that invaded on the matrigel matrix-coated polyethylene terephthalate membrane after removal of the non-invasive cells. B. Number of invading cells with the indicated transfections. Results of each column represent the mean ± S.D. of the numbers of invasive cells from three independent experiments. Fig. 7. In vivo tumorigenicity of mTOR mutants in nude mice and mTOR mutant-promoted mTOR/p70S6K signaling. A. in vivo tumorigenic assay of NIH3T3 cells with various transfections. NIH3T3 cells stably transfected with the empty vector, wild-type mTOR, and the mTOR mutants P2273S or E2288K were inoculated subcutaneously into the athymic nude mice as described in Materials and Methods and subsequently observed for tumor formation. Photographs of the tumors and animals were taken two weeks after cell inoculation. Tumor necrosis was seen in some cases. Each group consisted of five mice. A representative mouse is shown for each group and the number shown in the bracket for each group represents the number of animals that formed tumor out of five mice. B. Schematic illustration of mutant mTOR signaling to promote tumorigenesis. mTOR normally regulates translation initiation and cell proliferation by integrating inputs from upstream signaling pathways, particularly that from the PI3K/Akt pathway activated by growth factors and receptor tyrosine kinases through the indicated signaling cascade. Upon mutation, mTOR becomes strongly activated and continuously phosphorylates and activates its substrates p70S6K and Akt to promote protein synthesis, cell proliferation and tumorigenesis.
H.sapiens………..929-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFI--978 C.lupus………….929-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFI--978 B.taurus………....929-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFI--978 M.musculus……..929-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFI--978 R.norvegicus……929-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSHHHTMVVQAITFI--978 G.gallus…………861-YSTSEMLVNMGNLPLDEFYPAVSMVALMRIFRDQSLSQHHTMVVQAITFI--910 D.rerio…………..912-YSTSEMLVNMGNLPLDEFYPAVA IVTLMRILRDPSLSNHHTMVVQAVTFI--961 D.melanogaster....904-I STAELLVNMGN –ALDEYYPAVA I AALMRILRDPTLSTRHTSVVQAVTFI--952 A.gambiae………913-LSTSEML I NMST - QLDEYYPAVV I STLMKILRDPTLSNHHLSVVQAITFT--961
H.sapiens………1453-LHEWEDALVAYDKKMDTNKD-DPELMLGRMRCLEALGEWGQLHQQCCE-1499 C.lupus………...1453-LHEWEDALVAYDKKMDTNKD-DPELMLGRMRCLEALGEWGQLHQQCCE-1499 B.taurus……….1453-LHEWEDALVAYDKKMDTNKD-DPELMLGRMRCLEALGEWGQLHQQCCE-1499 M.musculus…...1453-LHEWEDALVAYDKKMDTNKE-DPELMLGRMRCLEALGEWGQLHQQCCE-1499 R.norvegicus......1453-LHEWEDALVAYDKKMDTNKD-DPELMLGRMRCLEALGEWGQLHQQCCE-1499 G.gallus……......1385-LHEWEDALVAYDKKMDTNKD-DPELMLGRMRCLEALGEWGQLHQQCCE-1431 D.rerio………....1436-LHEWEDALVAYDKKIDMNK D-DPEL I LGRMRCLEALGEWGQLHQQCCE-1482 D.melanogaster..1420-LHNWDEALEHYERNLKTD S S-DLEARLGHMRCLEALGDW SEL SNVTKH-1466 A.gambiae…......1430-LHS WEQARSLYSEKLK SNP N -DLESRLGEMRCLEALG EW SAL NAVTTQ-1476
