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Not all substrates are treated equally: Implications for mTOR, rapamycin-resistance, and cancer therapy

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 5.01). 03/2009; 8(4):567-72. DOI: 10.4161/cc.8.4.7659
Source: PubMed

ABSTRACT The mTORC1 signaling pathway is a critical regulator of cell growth and is hyper activated in many different cancers. Rapamycin, an allosteric inhibitor of mTORC1, has been approved for treatment against renal cell carcinomas and is being evaluated for other cancers. Mechanistically, mTORC1 controls cell growth in part through its two well-characterized substrates S6K1 and 4E-BP1. In this review, we discuss the implications of a recent finding that showed differential inhibition of S6K1 and 4E-BP1 by rapamycin, leading to cell-type-specific repression of cap-dependent translation. We discuss potential mechanisms for this effect, and propose that mTOR-specific kinase inhibitors, instead of rapamycin, should be considered for mTOR-targeted cancer therapy.

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Available from: John Blenis, Mar 19, 2014
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    • "In the present study, we intended to inhibit 4E-BP1 phosphorylation during maturation of bovine oocytes, and we revealed its effects on meiotic progression. Rapamycin, however, was reported to block 4E-BP1 phosphorylation inefficiently (Choo and Blenis, 2009; Dowling et al., 2010; Livingstone and Bidinosti, 2012). Thus, we have used a member of a new class of mTor inhibitors, the so-called active-site inhibitor Torin2, for the first time in oocytes. "
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    ABSTRACT: The mammalian target of rapamycin (mTor), a Ser/Thr protein kinase, is implicated in the phosphorylation-triggered inactivation of translation repressors, the so called eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Previous observations in porcine and bovine oocytes revealed an increasing phosphorylation of 4E-BP1 during meiotic maturation. The factor is hypophosphorylated in the germinal vesicle (GV) stage and highest phosphorylated in the metaphase II (M II). In the present approach we intended to block 4E-BP1 phosphorylation specifically to impair initiation of translation and elucidate effects on resumption of meiosis. Torin2, which acts as an active-site mTor inhibitor, reduces 4E-BP1 phosphorylation without any effect on eIF4E and arrests up to 60% of the oocytes in the M I stage. Effects of Torin2 treatment, analyzed by site-specific substrate phosphorylation, were also observed at protein kinase B (Akt, PKB) and cyclin dependent kinases (CDKs). However, if at all, only minor side effects were found at protein kinase A, C (PKA, PKC), ATM/ATR (Ataxia telangiectasia mutated/AT and Rad3-related protein) and the mitogen activated protein kinases (MAPK) ERK1, 2. The inhibition of 4E-BP1 phosphorylation by Torin2 is reversible when cultivating oocytes for additional 24 h in Torin2-free medium. However, even so, oocytes persist in the M I stage. This may indicate the necessity of spatiotemporally regulated translation during meiosis, which cannot be restored later. In conclusion, Torin2 enables an effective and specific inhibition of 4E-BP1 phosphorylation and such an approach may be valuable to investigate maturation specific protein synthesis in more detail. Mol. Reprod. Dev. © 2014 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 04/2014; 81:363-375. DOI:10.1002/mrd.22305 · 2.68 Impact Factor
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    • "This suppresses most, if not all, mTOR activities. In contrast, rapamycin allosterically affects mTOR-substrate interactions and selectively blocks some mTOR actions, but not others in a cell-type specific manner (Edinger et al., 2003; Choo et al., 2008; Thoreen et al., 2009). Relevant examples include rapamycin-resistant mTOR-dependent phophorylation of Akt ((Jacinto et al., 2004; Sarbassov et al., 2004) and see Fig. 4) and capdependent mRNA translation (Choo et al., 2008; Choo and Blenis, 2009). "
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    ABSTRACT: We report that rapamycin, an allosteric inhibitor of certain but not all actions of the key cellular kinase mammalian target of rapamycin (mTOR), protects neurons from death in both cellular and animal toxin models of Parkinson's disease (PD). This protective action appears to be attributable to blocked translation of RTP801/REDD1/Ddit4, a protein that is induced in cell and animal models of PD and in affected neurons of PD patients and that causes neuron death by leading to dephosphorylation of the survival kinase Akt. In support of this mechanism, in PD models, rapamycin spares phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity. The capacity of rapamycin to provide neuroprotection in PD models appears to arise from its selective suppression of some but not all actions of mTOR, as indicated by the contrasting finding that Torin1, a full catalytic mTOR inhibitor, is not protective and induces Akt dephosphorylation and neuron death.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2010; 30(3):1166-75. DOI:10.1523/JNEUROSCI.3944-09.2010 · 6.75 Impact Factor
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    ABSTRACT: Abstract The serine/threonine kinase mTOR plays a critical role in the regulation of cellular proliferation, growth, differentiation, autophagy, and cytoskeletal organization. Two mTOR containing complexes exist in mammalian cells: mTORC1 and mTORC2. mTORC1 mediates its effects through the control of mRNA translation initiation via phosphorylation of its two major downstream targets: the 4E-BPs and S6Ks. In order to study the individual roles of S6Ks and 4E-BPs downstream of mTORC1, we utilized new active-site mTOR inhibitors and cells deficient for either 4E-BPs or S6Ks. Surprisingly, we found that 4E-BPs and S6Ks play distinct roles in mediating the activity of mTORC1, with the 4E-BPs mediating cell proliferation and the S6Ks regulating cell growth. The effects of active-site mTOR inhibitors on cell proliferation, cell cycle progression, and protein synthesis were highly attenuated in cells lacking expression of 4E-BPs (4E-BP DKO), while these processes were significantly inhibited in wild-type cells. However, the growth of 4E-BP DKO cells was equally sensitive to active site mTOR inhibitors, as compared to their wild-type counterparts. In contrast, the growth of S6K1/2 null cells was refractory to the effects of active site mTOR inhibitors, whereas these cells were fully sensitive to the effects of the inhibitors on proliferation and cell cycle progression. These data support a model whereby the mTORC1 proliferative and growth signals diverge in mammalian cells to provide a system that allows for increased flexibility in the maintenance of full body homeostasis. mTOR is often over-activated in human cancers and as a result, has emerged as a target for anti-cancer therapies. The anti-diabetic drug metformin was recently identified as a potential anti-cancer agent. To elucidate its mechanism of action, we studied its effects on breast cancer cell proliferation, mTOR signalling and mRNA translation. In breast cancer cells, metformin treatment led to a 30% decrease in global protein synthesis and caused a dose-dependent specific decrease in cap-dependent translation. The decrease in translation caused by metformin was associated with AMPK activation, mTOR inhibition and a decrease in the phosphorylation of its substrates: 4E-BP1 and S6K. Cells lacking the tumour suppressors LKB1 and TSC2 were unaffected by metformin, highlighting the importance of these proteins in the mechanism of action of metformin in the inhibition of mTOR signalling and protein synthesis in cancer cells. RésumémTOR est une sérine/thréonine kinase qui joue un rôle primordial dans lecontrôle de plusieurs fonctions cellulaires, incluant la prolifération, la croissance, ladifférentiation, l'autophagie et la réorganisation du cytosquelette. Les cellulesmammifères possèdent deux complexes protéiques contenant mTOR, soit mTORC1 andmTORC2. mTORC1 exerce son action en contrôlant l'initiation de la traduction desARN messagers via la phosphorylation de ses principaux effecteurs : les 4E-BP et lesS6K. Dans le but de mieux comprendre les fonctions spécifiques des 4E-BP et des S6Ken aval de la signalisation cellulaire initiée par mTORC1, nous avons utilisé de nouveauxinhibiteurs spécifiques au site actif de mTOR, ainsi que des cellules n'exprimant pas les4E-BP ou les S6K. Nous avons découvert que les 4E-BP et les S6K jouent des rôlesdistinct en aval de mTORC1, les 4E-BP étant principalement impliquées dans laprolifération cellulaire, alors que les S6K jouent un rôle dans le contrôle de la croissancecellulaire. Les effets des inhibiteurs spécifiques au site actif de mTOR sur la proliférationcellulaire, la progression à travers le cycle cellulaire et la synthèse protéique sontfortement atténués dans les cellules nulles pour les 4E-BP, alors qu'ils sont inhibés demanière importante dans les cellules de type sauvage. Cependant, la croissance descellules 4E-BP nulles, en présence des inhibiteurs spécifiques au site actif de mTOR, estla même que celle observée pour les cellules de type sauvage. À l'opposé, la croissancedes cellules S6K nulles n'est pas affectée par ces inhibiteurs, tandis que les inhibiteursconservent leurs effets négatifs sur la prolifération et la progression au travers du cyclecellulaire dans ces mêmes cellules. Ces résultats sont en accord avec un modèle danslequel la signalisation en aval de mTORC1, pour promouvoir la prolifération et lacroissance cellulaire, utilise des cibles distinctes, permettant ainsi une flexibilité accruedans le maintien de l'homéostasie.mTOR est fréquemment hyperactivée dans les tumeurs humaines etconséquemment est d'un grand intérêt comme cible anti-cancer potentielle. De plus, il arécemment été démontré que la metformin, un agent anti-diabétique, possède une activitéanti-tumorale. Afin d'étudier ce nouveau mécanisme d'action de la metformin, nousavons utilisé des cellules du cancer du sein. Dans ces cellules, le traitement à lametformin résulte en une inhibition d'environ 30% de la synthèse protéique. Un effet dedose dépendance dans l'inhibition de la traduction cap-dépendante est aussi observéaprès traitement avec la metformin. L'inhibition de la traduction est accompagnée parune activation de AMPK, par une inhibition de mTOR et par une diminution de laphosphorylation de ses substrats : mTOR et S6K. Cependant, des cellules nulles pourAMPK ou TSC2 ne sont pas affectées par la metformin, démontrant l'importance de cesprotéines dans l'inhibition de l'activité de mTOR par la metformin dans les cellulescancéreuses.
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