MDM4 actively restrains cytoplasmic mTORC1 by sensing nutrient availability
- Francesca Mancini1, 2Email author,
- Emanuela Teveroni1,
- Giusy Di Conza3, 4,
- Valentina Monteleone1,
- Ivan Arisi5,
- Marsha Pellegrino1,
- Marianna Buttarelli1,
- Luisa Pieroni6,
- Mara D’Onofrio5,
- Andrea Urbani6, 7,
- Alfredo Pontecorvi2,
- Massimiliano Mazzone3, 4 and
- Fabiola Moretti1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 13 September 2016
Accepted: 27 February 2017
Published: 7 March 2017
Many tumor-related factors have shown the ability to affect metabolic pathways by paving the way for cancer-specific metabolic features. Here, we investigate the regulation of mTORC1 by MDM4, a p53-inhibitor with oncogenic or anti-survival activities depending on cell growth conditions.
MDM4-mTOR relationship was analysed through experiments of overexpression or silencing of endogenous proteins in cell culture and using purified proteins in vitro. Data were further confirmed in vivo using a transgenic mouse model overexpressing MDM4. Additionally, the Cancer Genome Atlas (TCGA) database (N = 356) was adopted to analyze the correlation between MDM4 and mTOR levels and 3D cultures were used to analyse the p53-independent activity of MDM4.
Following nutrient deprivation, MDM4 impairs mTORC1 activity by binding and inhibiting the kinase mTOR, and contributing to maintain the cytosolic inactive pool of mTORC1. This function is independent of p53. Inhibition of mTORC1 by MDM4 results in reduced phosphorylation of the mTOR downstream target p70S6K1 both in vitro and in vivo in a MDM4-transgenic mouse. Consistently, MDM4 reduces cell size and proliferation, two features controlled by p70S6K1, and, importantly, inhibits mTORC1-mediated mammosphere formation. Noteworthy, MDM4 transcript levels are significantly reduced in breast tumors characterized by high mTOR levels.
Overall, these data identify MDM4 as a nutrient-sensor able to inhibit mTORC1 and highlight its metabolism-related tumor-suppressing function.
KeywordsMDM4 mTOR Nutrient deprivation Aminoacid p53
In the last years, many studies have reported the crosstalk between the pathways that control tumor development and cellular metabolism. MDM4 (also MDMX) is a crucial regulator of the tumor suppressor Tp53 [1, 2]. It cooperates with MDM2 by forming a MDM2/MDM4 heterodimer that efficiently reduces p53 levels and activity [3, 4]. As such, it possesses oncogenic features and accordingly its cancer promoting function has been reported [5, 6]. Conversely, under severe DNA damage, the two MDM proteins dissociate and MDM4 promotes p53-proapoptotic function by favouring the pro-apoptotic phosphorylation of p53 by the kinase HIPK2  and the mitochondrial activity of p53 [8, 9]. According to these last activities, its presence is correlated to the beneficial effects of chemotherapy in wild type p53 tumors [8–10]. Under mild cytostatic DNA damage, the protein is actively degraded and this allows p53 to execute its growth arrest response . Finally, MDM4 promotes chromosome and genome stability in long-term in vitro cultures, and suppresses tumorigenesis, independently of p53 . Thus, MDM4 appears to be sensitive to the cell growth conditions and its function to be consequently determined. To date, no direct activity has been reported for p53 and its MDM regulators towards mTORC1 function.
The kinase target of rapamycin (TOR) is one of the hubs that control cell physiology based on availability of nutrients, growth factors, and energy . Mammalian (recently, also mechanistic) TOR, mTOR, develops its kinase activity within two hetero complexes: mTORC1 and mTORC2 with mTORC1 integrating the signals from all previous factors. Mammalian TORC1 promotes cell growth and proliferation, a reason whereby its activity and/or levels are frequently increased in human tumors . Two main targets of mTORC1 are p70S6 kinase 1 (also S6K1) and eukaryotic initiation factor 4E-binding protein 1 (eIF4), both regulating mRNA translation initiation and progression, thus the rate of protein synthesis [12, 14]. The active form of mTORC1 resides at the lysosomes where it directly prevents autophagy and controls lysosome function . In response to nutrient deprivation, mTORC1 is released from activating partners and re-localizes from the lysosomal surface to the cytosolic compartment. The features underlying mTORC1 cytoplasmic localization are presently undefined.
Starting from a shotgun proteomic comparative analysis of the untransformed breast cell line MCF10A, we have demonstrated that knocking down of MDM4 alters the function of the p70S6K signalling. Our results demonstrate that MDM4 contributes to maintain mTORC1 in its inactive state in the cytoplasm, thus providing MDM4 of the ability to sense metabolic stress and to control mTORC1-dependent oncogenic properties.
Cell cultures, transfections and treatments
HeLa, 293 T, HCT116, MDA-MB231 cells were maintained in DMEM/10% FBS (Life Technologies, USA), p53−/−Mdm4−/−MEFs, p53−/−Mdm2−/−MEFs, and p53−/−MEFs in DMEM high glucose/10% FBS (Cambrex). MCF10A cells in MEGM (Lonza, Switzerland). MDM4 and control (CTL) siRNA were by Invitrogen (Stealth RNAi), siRNA for S6K1 were from Ambion. siRNA and plasmids transfection were performed with RNAiMAX and Lipofectamine Plus respectively according to manufacturer’s instructions (Invitrogen). mTOR_1 shRNA was obtained from D. Sabatini through Addgene. Rapamycin (Sigma) was used 50nM unless specifically indicated. Torin2 was used 50nM. For amino acid starvation, cells were incubated for 3 h in amino acid free RPMI (US Biological) supplemented with 10% inactivated FBS, and stimulated with amino acid mixture for the indicated time. For serum and amino acid starvation, cells were incubated in EBSS (Invitrogen) for 50’, and stimulated with amino acids mixture or complete medium for the indicated time.
Shotgun proteomic analysis
MCF10A cells were transfected with stealth MDM4-specific (siMDM4-MCF10A) or stealth control RNA (siCTL-MCF10A), and after 48 h were lysed. The proteomic analysis was performed on proteins extracted from cytoplasmic cell lysate of MCF10 cells, through a label-free data-independent differential proteomic analysis by nUPLC-MSE. Details of the analysis are reported in .
Mammosphere forming assay
For mammosphere formation assay, cell culture dishes have been coated with pHEMA (poly(2-hydroxyethyl methacrylate) 10 mg/ml, dried and rinse with PBS. MDA-MB231 were interfered for siRNA control or siMDM4 for 16 h. Afterwards, cells were detached and seeded at 2000 cells/well in pHEMA coated 6 wells dishes for 72 h in DMEM/F12 supplemented with 2 mM Glutamine, 100U/ml Penicillin/streptomicin, 5%FBS, 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 10ug/ml Insulin.
Immunoprecipitation, western blot and cell fractionation
For immunoprecipitation (IP), cells were lysed in CHAPS lysis buffer (40 mM Hepes pH7.4, 120 mM NaCl, 2 mM EDTA, 0.3% CHAPS) containing mix of protease inhibitors (Boehringer), plus 5 mM NaF, 10 mM glycerophosphate and 1 mM Na3VO4. For IP lysates were pre-incubated with protein G-Agarose (Pierce) and then with the indicated antibody, under gentle rocking at 4 °C overnight. For Western blot (Wb) cells were lysed in RIPA buffer. Membranes were developed using the enhanced chemiluminescence (ECL Amersham) by chemiluminescence imaging system, Alliance 2.7 (UVITEC Cambridge) and quantified by the software Alliance V_1607. Primary antibodies used: MDM4 BL1258 (Bethyl laboratory), MDM4 C82 (Sigma), MDM4 8C6 (Millipore) p53 FL393 (Santa Cruz), α-tubulin DM1A (Sigma), actin C-40 (Sigma), mTOR (Santa Cruz), mTOR (Cell Signaling), anti-FLAG M2 affinity gel (Sigma), phosphoSer473-AKT (Cell Signaling), phosphor-Thr389-S6K (Cell Signaling), Akt (Cell Signaling), S6K1 (Santa Cruz), Raptor (Cell Signaling), Raptor (Santa Cruz).
Fractionation of lysates into heavy membrane and light membrane/cytosolic fractions was performed according to Menon et al. 2014.
In vitro kinase assay
Kinase assays were performed as previously described  with some modifications. Flag-mTOR immunoprecipitate was washed twice in CHAPS lysis buffer and twice in 25 mM HEPES (pH 7.4), 20 mM potassium chloride. Kinase assays were performed at 30 °C for 20 min in a final volume of 30 μl consisting of mTORC1 kinase buffer (25 mM HEPES [pH 7.4], 50 mM KCl, 10 mM MgCl2, 250 μM ATP) and inactive GST-S6K1 purified (by GST-Agarose gel, Sigma), from Hela cells transfected with GST-S6K1 plasmid and treated with EBSS and 20 μM LY294002 for 1 h. Reactions were stopped by the addition of sample buffer and boiling for 5 min. When used, 150 ng of GST-MDM4 was added to mTORC1 10 min before the addition of ATP to the kinase assay.
Cell viability and cell cycle analysis
Cell proliferation was determined by Cell Titer Blue colorimetric assay or Cell Live/Dead kit according to the manufacturer’s instructions (Promega and Invitrogen, respectively). Cell cycle profiles and forward scatter determination (FSC-H) were evaluated by fixing cells in cold 70% ethanol for 1 h on ice and staining DNA for 30 min at room temperature with 50 μg/mL propidium iodide (PI) in PBS containing 1 mg/mL RNase A. FSC-H evaluation was performed by previous gating of cells in G1 phase. FACScan flowcytometer (Becton Dickinson, USA) was used and data analysed by CellQuest Software (Becton-Dickinson).
Hela cells were fixed with 4% formaldehyde 5’ 37 °C, permeabilized with TritonX100 0.2% 15’ RT, and blocked with 0.25% BSA. Cells were stained with DAPI and primary antibodies: anti-MDM4 (1:100 Origene 4B5), and anti-mTOR (1:400 Cell Signaling). Cyanine (Cy3)-conjugated and Cyanine (Cy2)-conjugated secondary antibodies were used.
Mouse maintenance and treatment
Control (WT) and Mdm4 transgenic (TG) mice  were maintained and treated in accordance with the Guidelines on the protection of animals used for scientific purposes (European Directive 63/2010/EU and Italian Law DL116/1992 and DL 26/2014). Relative ethical approval has been obtained by Animal Welfare Body “Fondazione S. Lucia” (Protocol Number: 969/2015-PR). For in vivo assessment of mTOR activity, 13–15 week old male mice were fasted overnight and after 16 h intraperitoneally injected with leucine (120 mg/kg) or saline solution (control) in 0.2 ml volume. Ten minutes after injection, mice were sacrificed, tissues were snap frozen in liquid nitrogen and samples processed in RIPA lysis buffer for Western blot analysis.
The FH1t-UTG Mdm4 3' UTR-GFP lentiviral construct was obtained by Marine’s Lab by cloning shRNA sequence for MDM4-3'UTR (ACAGTCCTTCAGCTATTTCATTTCAAGAGAATGAAATAGCTGAAGGACTGTTTTTT) into the FH1tUTG vector, which constitutively expresses GFP . MCF10A, Hela and 293 T cells were infected with FH1t-UTG Mdm4 3' UTR-GFP lentivirus to generate TET-shMDM4 inducible cell line by doxycycline (DOX).
MDM4 inhibits p70S6K1 phosphorylation
Regulation of eIF4 and p70S6K signalling pathway: protein targets
Entrez Gene Name
Fold of induction
integrin, alpha 5
ribosomal protein S12
ribosomal protein SA
These data were observed also in p53+/+HCT116 and syngenic p53−/−HCT116 cells, pointing to a p53-independent effect of MDM4 on S6K1 (Fig. 1b).
Since S6K1 phosphorylation is mostly controlled by the mammalian target of rapamycin complex 1, mTORC1 , we analysed whether MDM4 activity is developed through inhibition of this complex. Human embryonic kidney 293 T cells, a mammalian cell line in which mTORC1 activity has been well characterized , were transfected with MDM4 siRNA and the levels of pS6K1 analysed in the presence of the mTORC1 inhibitor rapamycin (RAPA). In this cell line too, MDM4-KD increased the levels of pS6K1 but it was ineffective in the presence of RAPA (Fig. 1c). Basal S6K1 phosphorylation was inhibited by RAPA confirming the block of mTORC1 function. Similar results were obtained in MCF10A (Additional file 1: Fig. S1a) and in HeLa cells (Additional file 1: Fig. S1c). These data suggest that MDM4 inhibits mTORC1-mediated S6K1 phosphorylation. Given the inactivation of p53 both in 293 T and in HeLa cells, these data further support the p53-independent activity of MDM4.
The mTORC1-mediated phosphorylation of S6K1 is tightly regulated by nutrient availability and has been particularly well characterized by amino acids signalling [24, 25]. To further analyse the inhibitory function of MDM4 towards mTORC1, the activity of this last was blocked by cell starvation and then re-stimulated by amino acids (aa) addition. Indeed, cell treatment with Earle's Balanced Salt Solution (EBSS) depleted pS6K1 levels that were rescued by addition of amino acids (aa) mixture (Fig. 1d, Additional file 1: Fig. S1d). Under these conditions, MDM4-KD enhanced the increase of pS6K1 caused by aa addition, indicating that MDM4 antagonizes S6K1 phosphorylation by restraining mTORC1 activity (Fig. 1d, Additional file 1: Fig. S1d). Consistently, MDM4-KD was ineffective in the presence of RAPA (Fig. 1d). Similarly, amino acid deprivation restrained mTORC1 activity and the presence of MDM4 reduced the recovery of pS6K1 (Fig. 1e). Conversely, the over-expression of MDM4 strongly decreased the levels of pS6K1 induced by aa supplementation (Fig. 1f), overall indicating that MDM4 inhibits mTORC1 in response to aa depletion. To further confirm that MDM4 effect on pS6K1 are mediated through regulation of mTOR, the knockdown of mTOR prevented the upregulation of pS6K1 by siMDM4 (Fig. 1g). Similar effect were observed by pharmacological inhibition of mTOR with Torin2, a potent ATP-competitive inhibitor  although with less efficiency (Additional file 1: Fig. S1e).
To ascertain that MDM4 displays this activity in vivo too, we used a transgenic mouse model overexpressing Mdm4 (TG) . Since mTORC1 activity is strongly regulated in hepatocytes, we analysed pS6K1 levels in the liver of TG and age-matched control (WT) mice. Animals were fasted overnight and after 16 h injected intraperitoneally with the amino acid leucine (Leu), a specific activator of mTORC1 . In control WT mice, Leu increased phosphorylation of S6K1 compared to saline treated mice (Fig. 2b). Of note, such increase was almost abolished in Mdm4 TG mice (Fig. 2b), indicating that the overexpression of Mdm4 restrains mTORC1 activity in vivo too. Evaluation of mTOR levels in these samples did not show significant differences between WT and TG mice thus excluding an impact of Mdm4 on the total amount of the kinase.
MDM4 binds and inhibits mTOR
Since both proteins are mainly cytoplasmic [15, 29, 30], these data prompted us to investigate a possible interaction between MDM4 and mTOR, the kinase effector of the mTORC1 complex. Indeed, overexpressed MDM4 co-immunoprecipitated Flag-mTOR, indicating that the two proteins interact (Fig. 3c). Interestingly, the amount of co-immunoprecipitated mTOR was lower in presence of aa, supporting the inhibitory activity of MDM4 towards mTORC1 under nutrient deprivation (Fig. 3d). Analysis of endogenous proteins confirmed the interaction between MDM4 and mTOR during starvation whereas this was almost undetectable in presence of aa (Fig. 3e). To ascertain whether the binding between the two proteins mediates the MDM4 inhibitory activity, map of the binding of MDM4 to mTOR was performed by using different MDM4 deletion mutants (Fig. 3f) whose cytoplasmic localization was previously reported . The results revealed that the MDM4ΔBD, lacking the aminoacids 1–106 (consisting of the p53 binding domain) was unable to bind mTOR (Fig. 3f and g), indicating that the N-terminal domain of MDM4 is involved in the interaction. Of note, the MDM4ΔBD mutant did not decrease pS6K1 levels compared to the full-length MDM4 (Fig. 3h), indicating that the interaction between MDM4 and mTOR is required for the inhibition of this last. Overall, these data indicate that MDM4 binds mTOR during aa starvation and contributes to silence the kinase activity of the complex.
MDM4 affects cell size and proliferation
The data presented in this work highlight a p53-independent link between MDM4 and mTOR, with MDM4 acting as inhibitor of mTORC1 kinase activity.
MDM4 acts at two different levels: 1) by anchoring the cytoplasmic inactive form of mTORC1, 2) by inhibiting the kinase function of the mTORC1. The activity of mTORC1 is regulated by different pathways that alter the composition and/or the post-translational modifications of the complex. Overall, these pathways affect the localization of mTORC1 that sways between its lysosomal active and its cytoplasmic inactive site . This movement is accompanied by its association with activating complexes, represented by an activated heterodimer of Rag GTPases in presence of amino acids [31, 40, 41]. Most of the studies have therefore investigated how the cell senses the amino acids and activates mTORC1 . Conversely, it is not known whether an active mechanism maintains mTOR in its inhibited state. By demonstrating that the lack of MDM4 increases the presence of mTORC1 at the lysosomes and the phosphorylation of its target p70S6K1, our data provide the proof of concept of an active mechanism able to control cytoplasmic inactive mTORC1. Furthermore, the ability of MDM4 to inhibit the kinase function of mTORC1 in vitro indicates the existence of an active inhibition of the mTOR enzymatic activity.
The binding of MDM4 to mTOR is stimulated by nutrient depletion suggesting a mechanism whereby MDM4 senses this condition. However, manipulation of MDM4 both in vitro and in vivo is able to alter mTORC1 localization and/or activity in normal growth conditions too, suggesting that the intracellular balance between the two proteins is determinant for the control of the growth-promoting function of mTORC1. The inverse correlation between MDM4 and mTOR observed in human breast cancer specimens is in agreement with this hypothesis.
Interestingly, the same MDM4 region is involved in p53 and mTOR binding, i.e. the N-terminus. Although no data have been reported about competitive activities between these two hubs of cell growth, the reduced correlation between MDM4 and mTOR observed in tumor harbouring wt-p53 compared to those with mutant p53 might indeed suggest an exclusive mode of MDM4 function. Furthermore, these data well reconcile with the reported anti-oncogenic properties of Mdm4 in absence of p53 .
The metabolism of tumor cells is emerging as an important field in which distinct metabolic pathways provide tumor cells of advantageous activities for their growth. In these last years, many studies have reported the frequent crosstalk between the pathways that control tumor development and cellular metabolism; accordingly, different oncogenes have demonstrated their ability to enhance and/or promote alternative ways of obtaining necessary nutrients thus establishing the hallmarks of cancer metabolism .
The data presented in this work add another member to this community, MDM4, endowed of p53-independent growth suppressive properties. This MDM4 function is in agreement with its pro-apoptotic activity under DNA damage and support a model of MDM4 with anti-oncogenic activities in stress conditions . Furthermore, these data together with the previous report of MDM4 functioning as a bridge for phosphorylation of p53 , contribute to define MDM4 as a cytoplasmic scaffold ready to sense different stimuli – i.e. DNA damage, cell starvation – and to accordingly regulate cell growth by recruiting different partners.
Overall, these data demonstrate a new p53-independent function of MDM4 in inhibiting mTOR. They highlight an additional way of de-regulation of mTORC1 activity in human tumors and include MDM4 among the proteins affecting both cell metabolism and tumorigenesis.
Earle’s Balanced Salt Solution
Eukaryotic initiation factore 4E-binding protein 1
Homeodomain Interacting Protein Kinase 2
Mouse double minute 2 homolog
Mouse double minute 4, human homolog of p53-binding protein
Mouse embryo fibroblast
Mammalian target of rapamycin complex 1
Ribosomal protein S6 kinase beta-1
Target of rapamycin
We thank Dr. G. Lozano for the Mdm4 transgenic mice, and Dr. JC. Marine (Leuven) for the doxycycline-inducible shMDM4 constructs and for p53−/−Mdm4−/−MEFs.
This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) (IG-12767) to F.M.; FIRB RBAP1153LS_007 to A.P. F. Mancini was recipient of a fellowship from FIRB RBAP1153LS_007.
Availability of data and materials
Additional data are available as Supplementary information.
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
F. Mancini, performed experimental design, most experiments, acquisition of data and analysis, and wrote the manuscript.designed E.T. performed the lentiviral, the shmTOR and siS6K1 experiments. G.D.C. performed the mammosphere experiments. V. M. performed the experiments with the animals. M.P. and M.B. maintained and analysed the TG mice. M.P. contributed to the FACS analyses. I.A. and M.D’O. contributed to analyse Atlas data, L.P. and A.U. performed the proteomic and bioinformatics analysis. A. P. contributed analytical and scientific tools. M.M. contributed to scientific and critical evaluation of data. F. Moretti conducted scientific direction, analysed and discussed the data and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study involves animals that were maintained and treated in accordance with the Guidelines on the protection of animals used for scientific purposes (European Directive 63/2010/EU and Italian Law DL116/1992 and DL 26/2014). Relative ethical approval has been obtained by Animal Welfare Body “Fondazione S. Lucia” (Protocol Number: 969/2015-PR).
This study does not involve human participants. Not applicable.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Lenos K, Jochemsen AG. Functions of MDMX in the modulation of the p53-response. J Biomed Biotechnol. 2011;2011:876173.View ArticlePubMedPubMed CentralGoogle Scholar
- Marine JC, Francoz S, Maetens M, Wahl G, Toledo F, Lozano G. Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ. 2006;13:927–34.View ArticlePubMedGoogle Scholar
- Pant V, Xiong S, Iwakuma T, Quintas-Cardama A, Lozano G. Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability. Proc Natl Acad Sci U S A. 2011;108:11995–2000.View ArticlePubMedPubMed CentralGoogle Scholar
- Shadfan M, Lopez-Pajares V, Yuan ZM. MDM2 and MDMX: alone and together in regulation of p53. Transl Cancer Res. 2012;1:88–9.PubMedPubMed CentralGoogle Scholar
- Xiong S, Pant V, Suh YA, Van Pelt CS, Wang Y, Valentin-Vega YA, Post SM, Lozano G. Spontaneous tumorigenesis in mice overexpressing the p53-negative regulator Mdm4. Cancer Res. 2010;70:7148–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Lenos K, de Lange J, Teunisse AF, Lodder K, Verlaan-De Vries M, Wiercinska E, van der Burg MJ, Szuhai K, Jochemsen AG. Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts. Mol Cancer. 2011;10:111.View ArticlePubMedPubMed CentralGoogle Scholar
- Mancini F, Pieroni L, Monteleone V, Luca R, Fici L, Luca E, Urbani A, Xiong S, Soddu S, Masetti R, et al. MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response. Oncogene. 2016;35:228–40.View ArticlePubMedGoogle Scholar
- Mancini F, Di Conza G, Pellegrino M, Rinaldo C, Prodosmo A, Giglio S, D’Agnano I, Florenzano F, Felicioni L, Buttitta F, et al. MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway. EMBO J. 2009;28:1926–39.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu Y, Regunath K, Jacq X, Prives C. Cisplatin causes cell death via TAB1 regulation of p53/MDM2/MDMX circuitry. Genes Dev. 2013;27:1739–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen SH, Forrester W, Lahav G. Schedule-dependent interaction between anticancer treatments. Science. 2016;351:1204–8.View ArticlePubMedGoogle Scholar
- Matijasevic Z, Steinman HA, Hoover K, Jones SN. MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice. Mol Cell Biol. 2008;28:1265–73.View ArticlePubMedGoogle Scholar
- Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Menon S, Manning BD. Common corruption of the mTOR signaling network in human tumors. Oncogene. 2008;27 Suppl 2:S43–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Bahrami BF, Ataie-Kachoie P, Pourgholami MH, Morris DL. p70 Ribosomal protein S6 kinase (Rps6kb1): an update. J Clin Pathol. 2014;67:1019–25.View ArticleGoogle Scholar
- Betz C, Hall MN. Where is mTOR and what is it doing there? J Cell Biol. 2013;203:563–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, Carr SA, Sabatini DM. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell. 2007;25:903–15.View ArticlePubMedGoogle Scholar
- Herold MJ, van den Brandt J, Seibler J, Reichardt HM. Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc Natl Acad Sci U S A. 2008;105:18507–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Nir T, Dor Y, Zisman P, Meyuhas O. Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 2005;19:2199–211.View ArticlePubMedPubMed CentralGoogle Scholar
- Fingar DC, Salama S, Tsou C, Harlow E, Blenis J. Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 2002;16:1472–87.View ArticlePubMedPubMed CentralGoogle Scholar
- Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J. mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol. 2004;24:200–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Pearson RB, Dennis PB, Han JW, Williamson NA, Kozma SC, Wettenhall RE, Thomas G. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J. 1995;14:5279–87.PubMedPubMed CentralGoogle Scholar
- Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998;95:1432–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002;110:163–75.View ArticlePubMedGoogle Scholar
- Bar-Peled L, Sabatini DM. Regulation of mTORC1 by amino acids. Trends Cell Biol. 2014;24:400–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Goberdhan DC, Wilson C, Harris AL. Amino Acid Sensing by mTORC1: Intracellular Transporters Mark the Spot. Cell Metab. 2016;23:580–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Q, Xu C, Kirubakaran S, Zhang X, Hur W, Liu Y, Kwiatkowski NP, Wang J, Westover KD, Gao P, et al. Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR. Cancer Res. 2013;73:2574–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Migliorini D, Lazzerini Denchi E, Danovi D, Jochemsen A, Capillo M, Gobbi A, Helin K, Pelicci PG, Marine JC. Mdm4 (Mdmx) regulates p53-induced growth arrest and neuronal cell death during early embryonic mouse development. Mol Cell Biol. 2002;22:5527–38.View ArticlePubMedPubMed CentralGoogle Scholar
- Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, Sabatini DM. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science. 2016;351:43–8.View ArticlePubMedGoogle Scholar
- Migliorini D, Danovi D, Colombo E, Carbone R, Pelicci PG, Marine JC. Hdmx recruitment into the nucleus by Hdm2 is essential for its ability to regulate p53 stability and transactivation. J Biol Chem. 2002;277:7318–23.View ArticlePubMedGoogle Scholar
- Li C, Chen L, Chen J. DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms. Mol Cell Biol. 2002;22:7562–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010;141:290–303.View ArticlePubMedPubMed CentralGoogle Scholar
- Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, Cantley LC, Manning BD. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell. 2014;156:771–85.View ArticlePubMedPubMed CentralGoogle Scholar
- Cargnello M, Tcherkezian J, Roux PP. The expanding role of mTOR in cancer cell growth and proliferation. Mutagenesis. 2015;30:169–76.View ArticlePubMedGoogle Scholar
- Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007;12:9–22.View ArticlePubMedGoogle Scholar
- Network CGA. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61–70.View ArticleGoogle Scholar
- Abdel-Fatah TM, Powe DG, Agboola J, Adamowicz-Brice M, Blamey RW, Lopez-Garcia MA, Green AR, Reis-Filho JS, Ellis IO. The biological, clinical and prognostic implications of p53 transcriptional pathways in breast cancers. J Pathol. 2010;220:419–34.PubMedGoogle Scholar
- Wazir U, Newbold RF, Jiang WG, Sharma AK, Mokbel K. Prognostic and therapeutic implications of mTORC1 and Rictor expression in human breast cancer. Oncol Rep. 2013;29:1969–74.PubMedGoogle Scholar
- Welcome - IARC TP53 Database [http://p53.iarc.fr/]
- Demetriades C, Doumpas N, Teleman AA. Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell. 2014;156:786–99.View ArticlePubMedPubMed CentralGoogle Scholar
- Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008;320:1496–501.View ArticlePubMedPubMed CentralGoogle Scholar
- Sancak Y, Sabatini DM. Rag proteins regulate amino-acid-induced mTORC1 signalling. Biochem Soc Trans. 2009;37:289–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Pavlova NN, Thompson CB. The Emerging Hallmarks of Cancer Metabolism. Cell Metab. 2016;23:27–47.View ArticlePubMedPubMed CentralGoogle Scholar
- Mancini F, Di Conza G, Monti O, Macchiarulo A, Pellicciari R, Pontecorvi A, Moretti F. Puzzling over MDM4-p53 network. Int J Biochem Cell Biol. 2010;42:1080–3.View ArticlePubMedGoogle Scholar