(332), re2. [DOI: 10.1126/scisignal.2005326] 7Science SignalingLópez-Rodríguez (1 July 2014) Jose Aramburu, M. Carmen Ortells, Sonia Tejedor, Maria Buxadé and Cristina

Transcriptional regulation of the stress response by mTOR`

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T R A N S C R I P T I O N

Transcriptional regulation of the stress responseby mTORJose Aramburu,1* M. Carmen Ortells,2 Sonia Tejedor,1 Maria Buxadé,1 Cristina López-Rodríguez1*

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The kinase mammalian target of rapamycin (mTOR) is a central regulator of cell growth and prolifer-ation that integrates inputs from growth factor receptors, nutrient availability, intracellular ATP (aden-osine 5′-triphosphate), and a variety of stressors. Since early works in the mid-1990s uncovered the role ofmTOR in stimulating protein translation, this kinase has emerged as a rather multifaceted regulator ofnumerous processes. Whereas mTOR is generally activated by growth- and proliferation-stimulatingsignals, its activity can be reduced and even suppressed when cells are exposed to a variety of stressconditions. However, cells can also adapt to stress while maintaining their growth capacity and mTORfunction. Despite knowledge accumulated on how stress represses mTOR, less is known about mTOR in-fluencing stress responses. In this review, we discuss the capability of mTOR, in particular mTOR com-plex 1 (mTORC1), to activate stress-responsive transcription factors, and we outline open questions forfuture investigation.

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mTOR Complexes in Mammalian Cells

The serine/threonine kinase target of rapamycin (TOR) belongs to thefamily of phosphatidylinositol 3-kinase (PI3K)–related kinases (PIKKs)and is a main activator of biosynthetic processes needed for cell growthin all eukaryotic organisms (1). TOR is the most frequently used acronymfor this kinase, but earlier works also used the name FRAP, for FK506-binding protein 12–rapamycin–associated protein. The acronym mTORrefers to “mammalian TOR,” although recently “mechanistic target ofrapamycin” is commonly used. The kinase mTOR functions in larger mul-tiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2(mTORC2). mTORC1 is defined by regulatory-associated protein of mTOR(Raptor) (2, 3), whereas mTORC2 contains the proteins rapamycin-insensitive companion of mTOR (Rictor) and stress-activated mitogen-activated protein kinase (MAPK)–interacting protein 1 (Sin1) (4, 5). Theseand additional mTOR-interacting proteins regulate its activity and speci-ficity toward various substrates (6). mTORC1 is activated by growth fac-tors, nutrients, and energy and promotes cell growth by enhancing thetranslation rate of diverse proteins by activating the ribosomal S6 subunitkinases (S6K) 1 and 2 (1, 7, 8) and inactivating the translation repressorprotein eukaryotic translation initiation factor 4E (eIF4E)–binding protein1 (4E-BP1) (9). mTORC1 also enhances protein synthesis indirectly byincreasing the activity of RNA polymerases I and III, which transcribegenes encoding ribosomal and transfer RNAs (10).

In addition, mTOR influences diverse transcription factors and the ex-pression of gene products involved in the control of metabolism, ribosomalbiogenesis, growth, and proliferation (11–16). mTORC2 was originallyshown to be essential for the function of the actin cytoskeleton (4, 17),but is now also known to regulate cell growth, differentiation, proliferation,and lipid homeostasis (18, 19). At least part of the growth-promoting ac-tivity of mTORC2 is mediated by activating and stabilizing the kinase Akt[also known as protein kinase B (PKB)], which in turn enhances the ac-tivity of mTORC1 (20, 21). A defining feature of mTOR is its inhibitionby rapamycin (22, 23), a compound originally isolated from the bacterium

1Department of Experimental and Health Sciences, Universitat Pompeu Fabra,Barcelona 08003, Spain. 2Centre for Genomic Regulation and UniversitatPompeu Fabra, Barcelona 08003, Spain.*Corresponding author. E-mail: jose.aramburu@upf.edu (J.A.); cristina.lopez-rodriguez@upf.edu (C.L.-R.)

Streptomyces hygroscopicus that binds to the intracellular chaperoneFK506-binding protein 1A, 12 kD (FKBP12). The rapamycin-FKBP12complex in turn binds with high affinity to the FKBP12-rapamycin bind-ing (FRB) domain in TOR. This domain is accessible to the rapamycin-FKBP12 complex in mTORC1, but not in mTORC2. Structural studiesshow that mTORC1 complexes are dimeric, with two molecules of mTORand Raptor per complex (24). This work, together with earlier biochem-ical analysis, shows that rapamycin-FKBP12 alters the conformation ofthe mTOR-Raptor complex, weakening their interaction and destabilizingthe mTORC1 dimer (2, 24). Notably, activation of mTORC1 by nutrientscauses a conformational change in the mTOR-Raptor complex that makesit more sensitive to the destabilizing effect of rapamycin (2). Rapamycin-FKBP12 rapidly suppresses the activity of mTORC1 toward many, al-though not all, of its substrates (25–27). However, when cells are incubatedwith rapamycin for long periods (hours to days), rapamycin-FKBP12 canbind to newly synthesized mTOR before it can assemble into new mTORC1and mTORC2 complexes, precluding the regeneration of their pools andinhibiting both mTORC1- and mTORC2-dependent functions (28).

Stress-Sensitive Checkpoints in mTOR Activity

The activity of mTOR is sensitive to complex signaling networks, and thenumber of proteins that at some point can influence mTOR function isprobably in the hundreds (29). From this perspective, it is conceivable thatdiverse sources of stress may affect mTOR complexes by acting on differentcomponents in these networks, and indeed, there are several well-characterizedregulators and signaling circuits that inhibit the activity of mTOR understress (30–33) (Fig. 1, A and B).

A substantial part of mTORC1 inhibitory inputs are channeled throughthe tuberous sclerosis (TSC) proteins TSC1 and TSC2 (34, 35). mTORC1activity is primarily regulated by its associated protein Ras homolog en-riched in brain (Rheb), a small guanosine triphosphatase (GTPase) that,in its guanosine triphosphate (GTP)–loaded state, activates mTORC1. Rhebenables the activation of mTORC1 in response to growth factors and,together with the Ragulator complex, to amino acids (36), a process thatoccurs at the lysosome surface (37–39). TSC2 acts as a GTPase-activatingprotein (GAP) and promotes GTP hydrolysis to convert Rheb-GTP to Rheb-GDP (guanosine diphosphate), thereby inactivating mTORC1 (40). TSC2exists in a heterodimeric complex together with TSC1, which has no GAP

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activity but is needed to stabilize TSC2 (41, 42). The GAP activity of theTSC1/TSC2 complex is assisted by a third component of TSC, Tre2-Bub2-cdc16 (TBC) 1 domain family, member 7 (TBC1D7) (43). The GAPactivity of TSC2 is adjusted through inhibitory and activating phospho-rylations. TSC2 phosphorylation by the growth factor–activated kinaseAkt/PKB disrupts its association with TSC1 and causes the dissociationof the TSC complex from the lysosome, enabling the regeneration of Rheb-GTP and the assembly of mTORC1 with the Ragulator complex and Rheb(39, 44). TSC can also be inactivated through phosphorylation of TSC2 bythe p90 ribosomal protein S6 kinase a-1 (RSK1) (45). On the other hand,TSC is activated by phosphorylations mediated by glycogen synthase kinase3b (GSK3b) (46) and adenosine 5′monophosphate (AMP)–activated kinase(AMPK) in response to energy stress and reactive oxygen species (ROS)(47, 48). AMPK is a major sensor of energy stress and is activated inresponse to the increase in the intracellular ratio of AMP and adenosine5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) (49–51). AnothermTORC1-suppressive mechanism under energy stress is the inhibitory

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phosphorylation of Rheb by the complexformed by p38b with the p38-regulated/activated protein kinase [PRAK, also knownas the MAPK-activated protein kinase 5(MK5)] (52). TSC2 activity can also be en-hanced by the DNA damage and hypoxia-inducible protein regulated in developmentand DNA damage responses (REDD1), alsoknown as DNA damage–inducible transcript4 (DDIT4) (30, 53), which facilitates the dis-sociation of TSC2 from the scaffold protein14-3-3 (54). TSC2 is also activated by theREDD1 homolog REDD2 (30, 53) and bysestrins 1 and 2 induced by the transcrip-tion factor p53 in response to DNA damage(55). The activity of mTORC1 can also bedecreased under energy stress conditionsthrough the inhibitory phosphorylation ofRaptor at Ser722/792 by AMPK (56) and theAMPK-related kinase MAP/microtubuleaffinity-regulating kinase 4 (MARK4) (57).

TSC2-activating and inhibitory circuitsare also regulated by changes in the abun-dance of their components. For instance, inresponse to DNA damage, the tumor sup-pressor and transcriptional regulator p53can enhance the expression of genes encod-ing mTORC1 repressors, including TSC2,AMPK, REDD1, sestrin 2, and the PI3K an-tagonist phosphatase and tensin homolog(PTEN) (58). REDD1 is also induced in ap53-independent manner in response to en-ergy stress (59) and by the hypoxia-induciblefactor (HIF) during hypoxia (60, 61), inwhich mitochondrial ATP synthesis is com-promised while production of mitochondrialROS is enhanced.

Activation of mTORC2 depends on PI3Ksignaling (26, 62) and would be expectedto be sensitive to perturbations of this path-way under stress. Notably, mTORC2 is par-tially repressed, although not fully inactivated,upon phosphorylation of its essential compo-

nent Rictor by the mTORC1-activated S6K1 and by negative feedbackthrough mTORC1 on insulin receptor signaling (63–65). Another intriguingaspect of mTORC2 is its positive regulation by TSC2, an effect that is in-dependent of the GAP activity of TSC1/2 and inhibition of mTORC1 (66).The finding that mTORC1 can attenuate mTORC2 activity does not mean,though, that mTORC2 is not functioning in cells with active mTORC1. Rather,both complexes are active in growing and proliferating cells (26, 67) andshould be viewed as continuously communicating and balancing each other(Fig. 1B).

Regulation of Stress Response Transcription Factorsby mTOR

Adaptive mechanisms to survive and maintain functionality under stressoften involve changes in gene expression patterns. Although a large num-ber of transcription regulators are directly or indirectly sensitive to pertur-bations of the intracellular milieu, which in itself could be used by cells to

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Fig. 1. mTOR sensitivity to stressors, and stress-responsive transcription factors stimulated by mTOR.

(A) mTOR integrates signaling from growth factor, nutrient, and energy sensors to stimulate growth-promoting processes and can be inhibited by diverse types of stress conditions. (B) A schematic viewof mTORC1 inhibition by the TSC1/2 complex in response to diverse stressors, by other negative reg-ulators (such as AMPK, PRAK, and MARK4) under energy stress, or through deactivation of the Ragu-lator complex under amino acid deprivation. (C) Stress-responsive transcription factors that can bestimulated by mTORC1.

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react to various stressors, certain transcription factors have critical rolesin stress responses. For example, HIF-1a, ROS-activated nuclear factor(erythroid-derived 2)–like 2 (NFE2L2 or Nrf2), hypertonicity-activatednuclear factor of activated T cells 5 (NFAT5), heat shock response factor(HSF1), DNA damage–activated p53, and the homeostasis regulatorforkhead box O (FOXO) proteins orchestrate stress-specific adaptation re-sponses and are sensitive to mTOR activity (Fig. 1C).

mTORC1 and HIF-1aThe hypoxia response factor HIF is a heterodimer formed by a constitutivesubunit, HIF-1b, and an inducible subunit, HIF-1a. HIF-1a is continuous-ly synthesized, but under normoxic conditions, it is rapidly degraded (68).HIF-1b can also dimerize with the HIF-1a homolog HIF-2a, which is alsoregulated through continuous synthesis and degradation. Although bothHIF-1a and HIF-2a can activate hypoxia-protective responses, they donot have entirely overlapping functions (69, 70). Under normoxic condi-tions, HIF-1a is inhibited by two oxygen-dependent, ROS-sensitive typesof enzymes: prolyl hydroxylases (PHDs) and factor inhibiting HIF-1 (FIH).Both proteins use molecular oxygen and a-ketoglutarate to hydroxylatekey residues in HIF-1a. PHD-mediated hydroxylation of Pro402 andPro564 in HIF-1a targets it for ubiquitylation by the von Hippel–Lindau(VHL) E3 ubiquitin ligase complex and subsequent proteasomal degrada-tion. FIH hydroxylates Asn803, a modification that inhibits HIF-1 transcrip-tional activity, although it does not cause its degradation (71, 72). Hypoxiacauses the activation of HIF-1–dependent responses by suppressing PHDand FIH. Decreasing oxygen concentration to 0.5 to 2% causes a rapidaccumulation of HIF-1a and expression of its target genes (73, 74). Amajor effect of activating HIF-1a is the switch in the mode of energy produc-tion in the mitochondria from respiration-dependent to glycolysis-dependentATP synthesis (75). This aspect is quite interesting because it means thatHIF-1a is an effective regulator of energy and metabolite resources. In-deed, it is now clear that some cell types, such as activated lymphocytesand tumor cells, also use HIF-1a to boost glucose uptake and glycolysisduring normoxia as well to respond to increased biosynthetic and ener-getic demands (76, 77).

mTORC1 can enhance HIF-1a activity in both normoxia and hypoxia.During normoxia, and despite the activity of PHDs, mTORC1 facilitatesthe accumulation of HIF-1a in normal and tumor cells by increasing itstranslation rate (76–80). mTORC1 can also enhance HIF-1a protein abun-dance during hypoxia and chemically induced hypoxia-like conditions(81, 82) and increase its transcriptional activity under hypoxia by facilitat-ing the interaction of HIF-1a with the transcriptional coactivator p300(83). mTORC1 activity can be inhibited during hypoxia; one mechanismby which this occurs is through HIF-1a–induced expression of the geneencoding mTORC1 inhibitor REDD1 (54, 60, 84), which suggests recip-rocal regulation and a balance between mTOR activity and the intensity ofthe hypoxia response.

mTORC1 and Nrf2The transcription factor Nrf2 responds to increases in ROS concentrationsand enhances the expression of genes encoding diverse proteins involvedin maintaining cellular redox balance, such as glutathione-synthesizing en-zymes, thioredoxin reductases, or peroxiredoxins (85). The abundanceof Nrf2 is controlled by a repressor, Kelch-like ECH-associated protein1 (Keap1), which facilitates the ubiquitylation and subsequent proteasome-mediated degradation of Nrf2 (86–88). Increased intracellular ROS inducesthe oxidation of various cysteines in Keap1, triggering a conformation-al change that causes it to dissociate from Nrf2, which then escapes theubiquitylation-degradation cycle and induces antioxidant response genes(85). Activation of Nrf2 in the absence of overt oxidative stress can also

have important effects, as seen in tumors bearing mutations in eitherKEAP1 or NFE2L2 that disrupt the repression of Nrf2 by Keap1 (85, 89).Enhanced Nrf2 activity in tumors increases survival not only to endogenousROS but also to chemotherapeutic drugs and confers stronger antiapoptoticdefenses and cell proliferation capability (90).

Nrf2 is sensitive to mTOR activity, although different studies show dif-ferent effects of mTOR on Nrf2. Rapamycin represses Nrf2-dependentantioxidant responses in renal carcinoma cells (91), suggesting a positiveregulation by mTORC1. Activation of Nrf2 by its release from Keap1 andinduction of cytoprotective target genes are also enhanced by mTORC1during selective autophagy (92). However, a study in HepG2 hepatocarci-noma cells showed that although rapamycin reduced basal abundance ofNrf2, it did not inhibit its accumulation in response to (R)-a-lipoic acid, adithiol redox-active compound that induces Nrf2-dependent gene expres-sion (93), which suggests that oxidative stress can increase Nrf2 indepen-dently from mTORC1. On the other hand, prolonged treatment of humanfibroblasts with rapamycin reduced Keap1 abundance and enhanced Nrf2accumulation (94). Therefore, mTORC1 can have both positive and neg-ative effects on Nrf2 in different cell types and experimental settings. Aswill be discussed further in the following sections, mTORC1 can also en-hance ROS production in epithelial stem cells and hematopoietic stemcells (HSCs) (95, 96), which suggests that the potential protective functionof mTORC1 may be offset by its ROS-promoting effects in some cell typesand conditions.

mTORC1 and HSF1HSF1 is a heat stress–activated transcription factor that is ubiquitouslyexpressed in mammalian cells and exists as a monomeric inactive formin unstressed cells (97). Heat, as well as heat-independent stressors, such asH2O2, low pH, and hypoosmotic and hyperosmotic stress, activates HSF1by inducing its trimerization, which enables it to bind DNA (97). HSF1induces the expression of genes encoding various heat shock proteins(HSPs), including HSP70, to chaperone other proteins at risk of aggrega-tion and misfolding (98). In turn, accumulating HSP70 induces negativefeedback on HSF1 by binding HSF1 and repressing its transcriptional ac-tivity (99, 100). HSF1 can also play substantial roles in addition to its stress-responsive function, as shown by the recent identification of numerous genesregulated by HSF1 that support oncogenic processes in tumor cells (101).

Short-term heat shock has been shown to activate the kinase S6K down-stream of mTORC1 (102, 103) and enhance the transcriptional activity ofHSF1 and its induction of HSPs in a human cancer cell line by phosphoryl-ating it in Ser326 (103). The later study showed that heat shock–enhancedS6K1 phosphorylation and activation of HSF1 were rapamycin-sensitive, sug-gesting the involvement of mTORC1. Moreover, a recent screening fortumor-suppressing compounds showed that translation inhibitors, includ-ing rapamycin and other inhibitors of the PI3K-mTOR pathway, preventedHSF1 from binding DNA in various cancer cell types (104), which pro-vides another mechanism by which mTOR activity may enhance HSF1function. However, a study in Saccharomyces cerevisiae showed thatsustained activation of yeast HSF1 inhibited TOR signaling (105). Al-though both studies were done in very different organisms, they suggestthat mTORC1-activated HSF1 could negatively feedback on mTORC1to modulate its activity.

mTORC1 and NFAT5NFAT5 belongs to the family of proteins with a Rel-like DNA binding do-main, which also comprises calcineurin-activated NFAT1, NFAT2, NFAT3,and NFAT4 and nuclear factor kB (NF-kB) (106, 107). One of the mostextensively characterized functions of NFAT5 is its ability to protect cellsfrom osmotic stress caused by excessive extracellular tonicity, which it

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does by inducing various chaperones, osmolite transporters, and en-zymes that enable cells to survive and adapt to prolonged osmotic stress(108, 109). Osmotic stress can inhibit the activity of mTORC1 and mTORC2complexes, although the degree of inhibition differs with stress intensityand duration. Exposure of diverse cell lines to intense, short-term osmoticstress (more than 600 mosmol/kg for up to 1 hour) causes a substantialdecrease in the phosphorylation of major mTORC1 and mTORC2 targets,such as S6K1 or Akt (110–112). Intriguingly, one work reported that a30-min pulse of hyperosmotic stress (800 mosmol/kg) enhanced the ac-tivity of S6K1 in fibroblasts, although whether this was due to increasedmTORC1 activity was not tested (113). Although the inhibitory effect ofosmostress on mTOR has been known for years (30, 31), the mechanismsinvolved have not been fully elucidated. The ability of AMPK to respondto diverse stressors, including osmotic stress (51), would make it a possi-ble candidate to inhibit mTORC1 under osmostress. However, recent stu-dies do not support a main role for AMPK in regulating mTORC1 in cellsexposed to hypertonicity (67, 114). Earlier studies showed that inhibitionof S6K1 and Akt in cells exposed to high, short-term osmostress was me-diated by a calyculin A–sensitive phosphatase (110, 111). In this regard, itwas recently shown that short-term osmotic stress activated the intrinsickinase activity of mTORC1 through the phosphorylation of Raptor byc-Jun N-terminal kinase (JNK), but still inhibited the phosphorylation ofS6K1 and 4E-BP1 in cells (114). Calyculin A abrogated the inhibition ofS6K1 and 4E-BP1 and enabled their robust phosphorylation by mTORC1under osmotic stress (114). These findings indicate that high-intensity os-motic stress can interfere with signaling pathways downstream of mTORC1without inhibiting its kinase activity. On the other hand, mammalian cellsexposed to milder hypertonic conditions (500 mosmol/kg) exhibited amoderate inhibition of mTOR signaling (67), but maintained the functionof mTOR complexes to sustain protein synthesis, cell growth, and cell pro-liferation under osmostress (67, 115).

An earlier study in yeast had already shown that TOR promoted sur-vival under salt stress (116). In mammalian cells, mTORC1 stimulated theexpression of several osmoprotective gene products under moderate osmo-stress and enhanced histone acetylation in their promoters and the re-cruitment of NFAT5 and RNA polymerase II (67). mTORC1 was found toinfluence the expression of a subset of NFAT5-regulated genes as well asseveral NFAT5-independent ones, suggesting that mTOR influences othermechanisms or factors in the osmotic stress response (67). One such NFAT5-dependent osmotic stress–responsive gene encodes REDD1, the abun-dance of which is increased at both the mRNA and protein levels by mTOR.REDD1 can inhibit mTORC1 activity in various stress contexts (54, 61),but intriguingly, its induction by osmotic stress does not appear to inhibitmTORC1 (67). Hypertonic stress can enhance mitochondrial ROS pro-duction (117, 118), which can inhibit mTORC1 (119). Because REDD1attenuates the generation of mitochondrial ROS (120), it is possible thatthe mTORC1 inhibitory function of REDD1 under osmotic stress couldbe compensated by a protective antioxidant effect.

mTORC1 and p53The tumor suppressor transcription factor p53 is well known for its respon-siveness to DNA damage and its ability to prevent cellular transformationby inducing gene products that control the cell cycle, DNA repair, senes-cence, and apoptosis (121–123). Among other targets, p53 induces the ex-pression of various genes encoding proteins capable of inhibiting mTORactivity directly or indirectly [including PTEN, AMPK, TSC2, REDD1,sestrins, and TIGAR (TP53-induced glycolysis and apoptosis regulator)(58)], thus slowing down cell growth and attenuating biosynthetic pro-cesses that could favor the expansion of altered cells. Through AMPK-mediated phosphorylation of p53 at Ser15, p53 can also repress growth

and proliferation under energy stress and in the absence of DNA damage(124). Besides these stress-activated functions, p53 can influence mTORactivity by modulating glucose utilization and energy production byinhibiting various steps in glycolysis and enhancing mitochondrial res-piration (125) and by providing antioxidant defenses through the in-duction of proteins that are involved in ROS neutralization, such assestrins (126, 127).

As found for other stress-responsive transcription factors, p53 is alsosensitive to mTORC1 activity. mTORC1 can increase p53 activity by en-hancing its translation rate (128, 129) or by activating S6K1 that then se-questers and neutralizes the E3 ubiquitin protein ligase mouse doubleminute 2 (MDM2), a p53 repressor (130). It is interesting that increasedactivation of mTOR in TSC- or REDD1-deficient cells increases their sen-sitivity to stressors that activate p53; thus, in a sense, mTORC1 can beviewed as an amplifier of the proapoptotic function of p53 under stress.A similar stress sensitization mechanism in TSC1 mutant cells functionsthrough the enhanced mTORC1-dependent translation of alternative read-ing frame (ARF), a cell cycle repressor and p53 activator (131). In addition,mTORC1 can enhance p53-dependent induction of replicative senescencein cells subjected to moderate DNA damage and also in nonstressed cells(132, 133). The stimulatory effect of mTORC1 on p53 could be one reasonwhy TSC-deficient cells that maintain p53 functionality mostly give rise tobenign tumors (128, 134).

mTOR complexes and FOXOTranscription factors of the FOXO family are important regulators ofcellular homeostasis that play protective roles under diverse types of stressbut can also enhance cell death mechanisms (135). In different contexts,FOXO transcription factors can induce the expression of genes encodingproteins that arrest the cell cycle, activate apoptosis, enhance antioxidantresponses, promote autophagy, or modulate receptor signaling, includingsignaling in mTOR-mediated pathways (135).

Stress conditions that stimulate FOXO activity are generally inhibitoryfor mTOR complexes. Several kinases that activate FOXO, such as JNKin response to ROS (136), AMPK under energy stress (137, 138), and MK5(or PRAK) in response to DNA damage (139), can either inhibit mTORC1directly or inhibit upstream signaling to mTORC1 and mTORC2. FOXO canalso oppose mTORC1 by inducing its negative regulator sestrin 3 (140). Con-versely, by activating Akt (141, 142) or serum- and glucocorticoid-regulatedkinase 1 (SGK1) (143, 144), mTORC2 represses FOXO activity. In glio-blastoma tumors, mTORC2 also opposes FOXO by inhibiting its deacet-ylation by Sirt1 (145). Intriguingly, FOXO promotes the expression of thegene encoding PI3K and enhances PI3K signaling in certain tumor cells(146) as well as the expression of the gene encoding the mTORC2 com-ponent Rictor (140), which suggests that FOXO can induce its own neg-ative feedback through the expression of several repressors.

However, although these observations reveal some degree of antago-nism between FOXO and both mTOR complexes, there are also specificcontexts in which FOXO can benefit from the ability of mTORC1 to at-tenuate mTORC2. In T lymphocytes, excess mTORC1 activation throughdeletion of TSC1 leads to reduced mTORC2 function along with a sub-stantial reduction in mTORC2 and Akt-dependent repressive phosphorylationof FOXO1 and FOXO3 in response to T cell receptor (TCR) activation (147).TSC1-deficient T lymphocytes also exhibit a stronger induction of apoptosisupon TCR activation and greater expression of several FOXO-regulatedgenes encoding proteins that promote apoptosis and cell cycle arrest, suchas Bcl-2–like protein 11 (BCL2L11) and cyclin-dependent kinase inhibi-tor 1A (CDKN1A, also known as p21Cip1) (147). Additionally, rapamycintreatment in human colon cancer cell lines inactivates FOXO concomitant-ly with increased Akt activity (148).

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Negative effects of mTOR during stress responses

Although mTORC1 can be beneficial for the stress response, augmentedmTORC1 activity can be detrimental. For instance, TSC2-deficient cellscannot shut down mTORC1 when their DNA is damaged and die fasterthan wild-type cells because mTORC1 enhances the accumulation of p53(128). Likewise, TSC2-deficient cells poorly survive glucose depriva-tion because they cannot cope with the energy drain caused by mTORC1-

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driven protein synthesis (149). Studies inflies show that enhancing TORC1 activityby overexpressing Rheb increases theirsensitivity to oxidative stress (150), and otherstudies in flies and mice reveal that excessTORC1 activity worsens neuronal deteri-oration caused by huntingtin aggregates,whereas inhibition of TORC1 activates anautophagic response that clears protein ag-gregates and improves neuron survival(151, 152). These findings indicate that ex-cessive mTORC1 activity could overloadmechanisms of protein quality control, asconfirmed in a recent article showing thatTSC2-deficient fibroblasts exhibit de-creased mRNA translation fidelity (153).

Increased activation of mTORC1 by thesuppression of TSC1 or overexpression ofRheb also impairs hematopoiesis and self-renewal of HSCs (96, 154). This repressiveeffect correlates with increased mitochon-drial biogenesis and increased amounts ofROS, and the neutralization of ROS re-stores HSC function in Tsc1-deficient mice(96). mTORC1 also increases ROS in nor-mal oral keratinocytes and epithelial stemcells by decreasing the abundance of mito-chondrial manganese superoxide dismutase(MnSOD) (95). These effects seem at oddswith the ability of mTORC1 to activate anti-oxidant defenses through Nrf2 describedin other cells (92), suggesting that the bal-ance between the ROS-protective and ROS-promoting activities of mTORC1 may differsubstantially in different cell types and mi-croenvironments.

If reducing mTORC1 activity can helpcells to resist certain stresses, is it then par-adoxical that mTORC1 can also stimulateprotective stress responses? Both possibil-ities can be reconciled by proposing that par-tial suppression of mTORC1 may protectstressed cells by reducing the rate of bio-synthesis and the cellular energy demandwhile enabling sufficient mTORC1 activi-ty to support prosurvival stress responses(Fig. 2, A and B). As described above, ex-amples where the sustained activation ofmTORC1 reduces resistance to stress includeits roles in increasing mitochondrial ROSproduction; enhancing p53’s antiproliferative,proapoptotic, and senescence-promoting

functions; exacerbating ATP depletion in nutrient-restricted cells; or disrupt-ing proteostasis through excessive protein translation (95, 128, 149, 153). Acommon theme in these effects may be the persistence of energy-drainingmTORC1-driven biosynthetic processes and the overloading of mechanismsfor eliminating endogenous noxious and waste products (Fig. 2C). On theother hand, the stimulation of stress resistance responses by mTORC1 isobserved in cells where mTORC1 is not constitutively augmented, underconditions that can either maintain or transiently enhance its activity [such

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mTORC1

Protection from stress Increased sensitivity to stress

Fig. 2. Potential scenario compatible with mTOR being partially inhibited under stress but still capable of

enhancing adaptive stress responses. (A) Moderate stress intensity can cause a partial suppression ofmTOR activity and initiate stress responses with induction of stress-adaptation genes. Even if mTORactivity fell below the threshold required to sustain rapid proliferation or growth rate, cells could stillhave sufficient mTOR to enhance protective responses and facilitate a faster adaptation to stress. Phar-macological mTOR inhibitors such as rapamycin or Torin1 can prevent mTOR from enhancing stress re-sponses, as shown for HIF-1a, HSF1, Nrf2, or NFAT5. High-intensity stress can suppress mTOR so that itcannot further stimulate some stress responses. In this model, lack of an enhanced stress responsemediated by mTOR may not be necessarily detrimental for cells, and they could survive stress—despiteinducing a less robust response—by decreasing high-energy expending and stress-sensitizing bio-synthetic processes. (B) In cells with normally regulated mTORC1, partial suppression of mTORC1 bystress could attenuate processes that can negatively affect stress resistance. Remaining mTORC1 activitywould contribute to enhance transcriptionally regulated stress protection programs. (C) By contrast, in cellsunable to suppress mTOR activity, such as TSCmutants or cells overexpressing Rheb, the combination ofstress with the inability to attenuate energy expenditure and the overloading of repair or disposal of dam-aged components would worsen sensitivity to stress. Even if some stress adaptation responses could bestimulated by mTORC1, they would not be sufficient to counteract stress. In addition, overactive mTORC1can enhance the activation of p53 in response to various stressors, driving cells to senescence or death.

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as hypoxia, xenophagy, and short-term heat shock (79, 82, 83, 92, 103)]or reduce it [such as hyperosmotic stress (67)]. These observations maysuggest that sustained, increased activity of mTORC1 sensitizes cells toseveral types of stress, whereas moderate, regulated activity of mTORC1would favor stress adaptation responses (Fig. 2, B and C). Here, it mustbe noted that stress conditions under which mTORC1 can stimulate tran-scriptional responses [such as hypoxia (82, 83), hyperosmotic stress (67),or DNA damage (130)] can also inhibit mTORC1 (60, 67, 112, 134). Thissuggests that the extent of mTORC1 activity under increasing stress inten-sity will be important for determining its capacity to enhance protectiveresponses. These examples suggest that careful analysis of mTOR func-tion under moderate stress conditions could uncover other mTOR-sensitivetranscriptionally regulated processes. For instance, severe nutrient depriva-tion or intense endoplasmic reticulum (ER) stress can suppress mTOR, buta milder intensity of these stressors could allow enough mTOR activity tosupport relevant cell functions. In this regard, mTORC1 antagonizes the in-duction of the ER stress–responsive factors activating transcription fac-tor 4 (ATF4), ATF6, and CCAAT/enhancer binding protein homologousprotein (CHOP) (155), and emerging evidence suggests that a bidirectionalcrosstalk exists between mTOR complexes and the unfolded protein re-sponse (156).

Toward Future Directions: A Place for mTOR Complexes in theMap of Stress Responses?

As discussed above, mTORC1 can stimulate a number of stress-activatedtranscriptional responses, and mTORC2 can also enhance certain stress re-sponses, as shown in yeast and mammalian tumors. In S. cerevisiae, TORC2enhances cell survival to low amounts of DNA damage by stimulating theactivity of the SGK1-related kinases Ypk1 and Ypk2 (157), and suppressionof TORC2 in Schizosaccharomyces pombe impairs survival under hydrogen

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peroxide stress, salt stress, and DNA dam-age (158–160). Furthermore, in a positivefeedforward loop with Akt and the atypicalRIO kinases RIOK1 and RIOK2, mTORC2increases survival of mouse glioblastomacells to the DNA-damaging drugs doxoru-bicin and temozolomide (161).

Intensity and duration could be importantvariables in the effect of stress on mTOR ac-tivity and its capacity to enhance protectiveresponses. A given quantity of stress is likelyto have a different impact on cellular func-tions and elicit different responses depend-ing on a cell’s state of biosynthesis andgrowth; for example, cells undergoing activegrowth and intense metabolic activity maybenefit from the ability of mTOR to enhancestress survival responses. In this regard, it isinteresting that different types of tumorsoften exhibit augmented mTOR signaling(162) together with enhanced activity of somestress response factors like HIF-1 (163–165),Nrf2 (92, 166), or HSF (101, 103, 104). Also,mTOR hypomorphic mice have a lower in-cidence of spontaneous malignant tumors(167). Although this does not imply thatstronger mTOR signaling necessarily en-hances anti-stress capabilities in tumors, thecoexistence of elevated activities of mTOR

and various stress-responsive transcription factors in different tumorscan provide an interesting scenario to study how mTOR signaling maycontribute to cellular resilience—or fragility—under stress. These studiesmight also reveal an opportunistic weakness in tumors that are over-relianton sustained mTOR activity to survive stresses.

Elucidating how mTOR complexes influence the balance between re-sistance and enhanced sensitivity to stress might also aid understandingtheir role in aging. Reduction of mTOR activity by pharmacological orgenetic means increases life span in yeast, worms, flies, and mice, an ob-servation that opens exciting challenges regarding the possibility of slow-ing aging by manipulating mTOR (6, 168–171). The precise mechanismsunderlying the longevity phenotype in mice with reduced mTOR activityare still being elucidated, but it is already apparent that some processesbenefit more than others from lower mTOR activity (168). Mice withlow mTOR expression throughout their entire life, and therefore reducedactivity of both mTORC1 and mTORC2, show improved neuromotor andcognitive performance and a lower incidence of malignant tumors at oldage (167). Decreasing mTOR activity in old mice with rapamycin also im-proves self-renewal capacity of HSCs (172), which agrees with the findingthat overactive mTORC1 impairs HSC renewal (96, 154). However,complete lack of mTORC1 impairs HSC regeneration (173). It is also im-portant to note that mTOR-null organisms are not viable (174, 175), andthat decreasing global mTOR activity below a certain threshold can se-verely weaken immune defenses against infections (167), an observation thatunderscores extensive evidence of a fundamental role of mTOR complexesin immune responses (176–180).

Prolonging longevity in an organism requires a good coordination be-tween extending the life span of terminally differentiated cells, maintainingtheir proper function in tissues, and ensuring the replicative and differen-tiation potential of progenitor cells. These processes need not be equallysensitive to changes in mTOR activity. Because cells in any organism will

mTOR

mTOR

Cytoplasm

Nucleus

Inducible mTOR mutant(Rapamycin/Torin1-insensitive)

Inducible release from repressor

mTOR

Endogenous mTOR

RapamycinTorin1

Inducible promoter

mTOR

R

R

Fig. 3. Schematic outline of approaches to address mTOR function in stress responses. Potentially,

mTOR could be reversibly and quantifiably manipulated in cells engineered to express inducible re-combinant mTOR mutants resistant to either rapamycin or Torin1. Endogenous mTOR complexescould be inhibited with rapamycin or Torin1, and resistant mutants (represented with a red dot) couldbe either expressed from an inducible promoter or activated by release from a repressor (for instance,estrogen receptor) in a time-controlled manner. These approaches could use wild-type or TSC1/2-deficientcells, and one could design ectopic mTOR mutants that are capable of interacting only with Raptor orRictor, to selectively reactivate mTORC1 or mTORC2 functions.

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endure different stressors to varying degrees throughout their life, it is pos-sible that cellular longevity may be influenced by the ability of mTOR com-plexes to regulate sensitivity and resistance to stress. Reduced mTORC1activity could attenuate cellular stress under aging-promoting conditions(such as DNA damage, perturbed proteostasis, or ROS) while simulta-neously being sufficient to enhance specific stress adaptation responses.However, this is but one among various possibilities and will need to beexperimentally tested.

Several conceptual and experimental questions arise regarding the rele-vance of mTOR-mediated regulation of stress responses and the mechan-isms involved. To know whether mTOR activity is predictive of a better orworse outcome under stress, it will be important to determine three things:(i) how cells quantitate stress and balance this information against mTORstimulatory inputs; (ii) which mTOR functions are more and less sensitive todefined stress conditions of varying intensity and duration; and (iii) howimportant it is for the cell to enhance specific response mechanisms in anmTOR-dependent manner to survive stress.

Identifying genes and proteins that are sensitive to varying degrees ofstimulation or inhibition of mTOR activity in different stress scenarios anddifferent cell types is technically feasible with current transcriptomic, ge-nomic, metabolomics, and proteomic tools (11, 15, 67, 181–183). It is alsopossible to identify and quantitate changes in posttranslational modifica-tions and the abundance of individual proteins associated with stress re-sponses that are regulated by mTOR (184). However, dissecting howmTOR’s contribution to stress responses may affect a cell’s behavior dur-ing and after stress has subsided poses several challenges. ManipulatingmTOR-sensitive stress-responsive genes or proteins could provide cluesabout how changes in their expression or activity would influence cellularbehavior under stress, but this could be difficult if the number of genesand proteins involved were relatively large, thus complicating conventionalapproaches that silence or overexpress candidate genes. However, this taskcould be simplified if mTOR-sensitive stress response networks could bedelineated, and their key nodes identified. Characterization of functionalnodes and links in these networks would enable the manipulation ofkey elements to dissect the contribution of specific pathways in mTOR-regulated stress responses. Also, devising tools that allowed reversible andquantitative modulation of mTOR activity at different stages along thestress response could be useful to understand how mTOR may influencecellular functions during and after stress. Alternating repression and acti-vation of mTOR in a time-controlled manner could be accomplished bycombining current chemical inhibitors, such as rapamycin or Torin1 (26),with inducible mTOR mutants engineered to be resistant to those inhibi-tors (Fig. 3). Rapamycin-resistant mTOR mutants already exist (185, 186),and the available structure of Torin1-bound mTOR (187) should facilitatethe design of Torin1-resistant mutants.

Concluding Remarks

The activity of mTOR complexes is sensitive to diverse forms of stress, whichin general means that cells under stress often suppress mTOR signaling.At least in the case of mTORC1, attenuating its activity can improve cell sur-vival under certain stress conditions, whereas abnormally increased mTORC1function during stress can be detrimental for the cell. On the other hand, bothmTORC1 and mTORC2 can also enhance cellular responses that supportadaptation and survival to stress, and stress-activated transcriptional regulatorssuch as HIF-1a, HSF1, NFAT5, p53, and FOXO are stimulated by mTORC1.Different findings suggest that the communication between mTOR and stressresponses may play an important role in cellular functions, tumor biology, andperhaps long-term fitness of the organism. Aside from identifying relevantmTOR targets in stress response pathways and the regulatory mechanisms

involved, it will be important to elucidate how these are regulated by the bal-ance between mTORC1 and mTORC2 activities. It will also be necessary todissect the role of mTOR complexes in stress separately from their otherfunctions, and to determine how the contribution of mTOR to stress responsesmay affect longer-term outcomes in cell survival, proliferative capacity, main-tenance of specialized functions, differentiation potential, or longevity.

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Acknowledgments: We thank the members of the J.A. and C.L.-R. groups for continuedand inspiring discussions. We also thank the editors and reviewers for insight and helpfulcomments. Our apologies go to those authors whose work was not cited because of ouroversight. Funding: J.A. and C.L.-R. are supported by grants from the Spanish Ministry ofEconomy and Competitiveness (SAF2011-24268 to J.A., SAF2012-36535 to C.L.-R.), byFundació la Marató TV3 (122530), and by Generalitat de Catalunya (2009 SGR601, 2014SGR1153). S.T. is supported by a predoctoral fellowship BES-2013-062670. Competing in-terests: The authors declare that they have no competing interests.

Submitted 31 March 2014Accepted 30 May 2014Final Publication 1 July 201410.1126/scisignal.2005326Citation: J. Aramburu,M.C.Ortells, S. Tejedor,M. Buxadé,C. López-Rodríguez, Transcriptionalregulation of the stress response by mTOR. Sci. Signal. 7, re2 (2014).

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