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An expanding role for mTOR in cancerDavid A. Guertin1 and David M. Sabatini1,2

1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02141, USA2Massachusetts Institute of Technology, Department of Biology, Cambridge, MA 02141, USA

Rapamycin, a valuable drug with diverse clinical appli-

cations, inhibits mTOR (mammalian target of rapamy-

cin), which is a protein kinase that controls cell growth

by regulating many cellular processes, including protein

synthesis and autophagy. The sensitivity of select tumor

cells to rapamycin has ignited considerable excitement

over its potential as an anti-cancer therapeutic. Recent

findings identified a rapamycin-insensitive function of

mTOR in regulating a cell-survival pathway that is

hyperactive in many cancers, particularly those with

elevated PtdIns3K signaling or harboring mutations in

the tumor suppressor PTEN (phosphatase and tensin

homolog deleted on chromosome 10). These new

findings suggest that targeting this function of mTOR

might have broader applications in cancer therapy. In

this article, we re-evaluate mTOR signaling, suggesting

a more central role for mTOR in cancers with defective

PtdIns3K–PTEN signaling and conceptually discuss

these implications in the context of drug discovery.

New horizons for mTOR signaling

Advances in our understanding of cancer have made clearthat even tumors of the same pathological classificationcan have markedly different signaling-pathway profilesand gene-expression patterns. Effective treatments willprobably require the use of molecular markers to decipherthese signatures on a patient-to-patient basis and thecustom design of appropriate treatment strategies. Bio-markers indicate that the mTOR (mammalian target ofrapamycin) growth pathway is hyperactive in certaincancers, suggesting mTOR as an attractive target forcancer therapy [1,2]. The general but unproven beliefis that activated mTOR provides certain tumor cellswith a growth advantage by promoting proteinsynthesis, which is the best-described physiologicalfunction of mTOR signaling. Recently, a new functionfor mTOR in regulating the AKT (also known asprotein kinase B or PKB) kinase was described [3].AKT, which is a crucial downstream effector in thephosphatidylinositol 3-kinase (PtdIns3K)–PTEN (phos-phatase and tensin homolog deleted on chromosome10) pathway, controls cell proliferation and survival,thus expanding our view of the role of mTOR in signaltransduction and the molecular pathology of cancer.Because hyperactive AKT is implicated in the etiologyof cancers with defective PtdIns3K–PTEN signaling,targeting this function of mTOR might have thera-peutic potential.

Corresponding author: Sabatini, D.M. (sabatini@wi.mit.edu).

www.sciencedirect.com 1471-4914/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved

Here, we discuss evidence suggesting that the inhi-bition of mTOR by rapamycin is a promising anti-cancerstrategy. The prevailing view is that mTOR lies down-stream of and is activated by AKT. This model predictsthat the aberrant activation of AKT upregulates mTORgrowth signaling, sensitizing transformed cells to rapa-mycin treatment. Although this model is still controver-sial, adding to the debate is new evidence implicatingmTOR as a direct AKT activator, placing mTOR on bothsides of the AKT signaling hub. These differentialfunctions of mTOR can be explained by the existence ofat least two distinct mTOR complexes containing uniqueinteracting proteins. We discuss each mTOR complex withrespect to pathway specificity and conclude with acomment on the value of mTOR inhibitors for thetreatment of cancer.

mTOR signaling in cancer

The best-described genetic deficiencies commonly found inhuman cancer and impinging upon mTOR signaling aremutations in the PTEN gene [4]. PTEN mutations areassociated with a spectrum of cancers, including prostate,breast, lung, bladder, melanoma, endometrial, thyroid,brain, renal carcinomas and others, making it one of themost frequently mutated tumor-suppressor genes(Table 1) [5–7]. PTEN, a lipid phosphatase, counteractsthe lipid-kinase activity of class I PtdIns3Ks (Figure 1).Following growth-factor stimulation, activated receptortyrosine kinases, or intermediaries such as the insulin-receptor substrates IRS-1 and IRS-2, recruit PtdIns3K tothe membrane. PtdIns3K phosphorylates phosphatidyl-inositol (4,5) bisphosphate [PtdIns(4,5)P2] at the mem-brane to generate PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 servesas a docking site for effector proteins with pleckstrin-homology (PH) domains, including the AKT and3-phosphoinositide-dependent protein kinase 1 (PDK1)protein kinases. In addition to PTEN deletions, amplifica-tions of both PtdIns3K and AKT occur in several cancers[4,8]. Transforming events not affecting the PtdIns3K,PTEN or AKT genes directly, such as the BCR–ABLtranslocation and HER-2/neu or epidermal growth-factorreceptor (EGFR) amplification, also activate PtdIns3Ksignaling [8]. Considering the prevalence of PTENmutations and aberrantly activated PtdIns3K–AKT sig-naling in cancer, identifying the associated downstreamsignaling events that are crucial for tumor progression isan area of intense investigation.

AKT belongs to the cAMP-dependent, cGMP-dependentprotein kinase C (AGC) family of protein kinases thatincludes ribosomalS6kinases (S6K1andS6K2), serum-and

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Table 1. mTOR signaling in disease

Diseasea Linked genetic mutation and clinical

pathologyb

Predicted functional link to mTOR

signalingc

Refs

Tumor-prone syndromes

TSC (tuberous sclerosis

complex)

TSC1 or TSC2; harmatomas in multiple

organs

TSC1 and 2 negatively regulate Rheb [60,61]

LAM

(lymphangioleiomyomatosis)

TSC2; abnormal proliferation of smooth-

muscle-like cells in the lung

TSC1 and 2 negatively regulate Rheb [100]

Cowden’s disease PTEN; harmatomatous tumor syndrome Might promote AKT-dependent

inhibition of TSC2 and mTOR

phosphorylation

[62]

Proteus syndrome PTEN; harmatomatous tumor syndrome Might promote AKT-dependent

inhibition of TSC2 and mTOR

phosphorylation

[62]

Lhermitte–Duclos disease PTEN; harmatomatous tumor syndrome Might promote AKT-dependent

inhibition of TSC2 and mTOR

phosphorylation

[62,101]

PJS (Peutz–Jeghers syndrome) STK11/LKB1; gastrointestinal

harmatoma tumor syndrome

STK11 activates AMPK, a positive

regulator TSC2

[52–54]

HCM (familial hypertrophic

cardiomyopathy)

AMPK; myocardial hypertrophy AMPK promotes TSC2 function [102]

Cancer

Prostate PTEN PTEN loss promotes AKT activation [103,104]

Breast PTEN; PtdIns3K, AKT or Her2/neu

amplification or hyperactivation

PTEN loss or gene amplifications

promote AKT activation

[4,104–106]

Lung PTEN; HER amplification PTEN loss or gene amplifications

promote AKT activation

[4,107]

Bladder PTEN Promotes AKT activation [108]

Melanoma PTEN Promotes AKT activation [4]

Renal-cell carcinoma PTEN Promotes AKT activation [4]

Ovarian PTEN; PtdIns3K, AKT or Her2/neu

amplification or hyperactivation

PTEN loss or gene amplifications

promote AKT activation

[4,105,106,109]

Endometrial PTEN Promotes AKT activation [4]

Thyroid PTEN; PtdIns3K, AKT or Her2/neu

amplification or hyperactivation

PTEN loss or gene amplifications

promote AKT activation

[4]

Brain (glioblastoma) PTEN Promotes AKT activation [4,104,110]

CML (chronic myeloid leukemia) BCR–ABL translocation Promotes AKT activation [8]aList is not comprehensive, but rather attempts to indicate diseases commonly linked to genes involved in mTOR signaling.bListed only are the linked mutations that might affect mTOR signaling.cBest prediction based on existing data.

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glucocorticoid protein kinase (SGK) and protein kinase C(PKC) [9]. Two essential phosphorylation sites character-ize AGC kinases: one in the activation loop and one in aC-terminal hydrophobic motif. At the membrane, phos-phorylation on Thr308 in the activation loop and Ser473 inthe hydrophobic motif activates AKT. In mice andhumans, three AKT isoforms exist (Akt1 or PKBa, Akt2or PKBb and Akt3 or PKBg, respectively, for the mouseand human forms), which are the products of distinctgenes. Mouse knockout models indicate that, in spite oftheir structural similarity, all three have distinct roles ingrowth and metabolism, whereas overexpression studiesimplicate Akt1 in cell and organ size control [10–17].Because distinct roles for each AKT isoform are onlybeginning to be understood, we will refer to all threecollectively as AKT.

Normally, active AKT regulates cell survival, cellproliferation and metabolism (particularly glucose metab-olism) by phosphorylating proteins such as BAD, FOXO,NF-kB, p21Cip1, p27Kip1, glycogen synthase kinase(GSK)3b and others [4,9,18]. AKT might also promotecell growth by phosphorylating targets such as TSC2(a negative regulator of mTOR) andmTOR itself, althoughthe physiological significance of these interactions is stillunder investigation [19]. AKT activation probably pro-motes cellular transformation and resistance to apoptosis

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by collectively promoting growth, proliferation and survi-val while inhibiting apoptotic pathways. Elevated AKTsignaling might additionally cause cells to adopt a moreglycolytic metabolism [8]. Such a glycolytic shift could helpto sustain cellular metabolism in the harsh nutrient-limiting environment of a tumor and discourage apoptosisby promoting mitochondrial membrane potential[8,20,21]. Thus, inhibitors of AKT signaling are sought-after drugs and current strategies target both the kinaseand PH-domain functions of AKT directly, in addition toits upstream activators [5,22,23].

mTOR–raptor, a regulator of growth

Because hyperactive AKT signaling is associated withelevated mTOR signaling in some cancers, the mTORinhibitor rapamycin could have promise as an anti-cancertherapeutic [1,2,8,24,25]. mTOR is a highly conservedprotein belonging to the PtdIns3K-related kinase family(PIKK family) of serine/threonine protein kinases thatincludes ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-depen-dent protein kinase (DNA-PKcs). In cells, mTOR exists inat least two distinct complexes: a rapamycin-sensitivecomplex defined by its interaction with the accessoryprotein raptor (regulatory-associated protein of mTOR)and a rapamycin-insensitive complex defined by its

mTORRaptor GβL

mTOR

Rictor GβL

RhebPTEN

TSC1 TSC2

AktRapamycin

S6K

PDK1

PDK1

PI3K

IRS

Growthfactors

(amino acids and glucose)

SurvivalProliferation

P P

P P

P

P

PIP3

IR

InsulinIGF-1

AMPKP

RTK

??

4E-BPP

eIF4E

Transcription Autophagy

?

?

GTP FKBP12

Growth

Nutrients

Proteinsynthesis

Hypoxia or stress

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Figure 1. mTOR signaling in cell growth, proliferation and survival. Growth factors, including insulin and IGFs, bind to their receptors [denoted as the insulin receptor (IR) or

receptor tyrosine kinase (RTK) for non-insulin-like growth factors]. This triggers PtdIns3K (PI3K) membrane recruitment and activation directly, or through IRS proteins for

insulin or insulin-like growth factors. At the cell membrane, PtdIns3K phosphorylates PtdIns(4,5)P2 to generate PtdIns(3,4,5)P3 (PIP3). The tumor suppressor PTEN, a lipid

phosphatase, counteracts PtdIns3K lipid-kinase activity. PIP3 serves as a membrane-docking site for the PH-domain-containing proteins AKT and PDK1. Active PDK1

phosphorylates the activation loop of S6K1 and AKT, two AGC family kinases that are key effectors of cell growth and cell-survival pathways, respectively. Growth-factor

signaling, along with nutrient availability (particularly amino acids and glucose), regulates cell growth by controlling the activity of the mTOR–raptor complex. Most available

evidence suggests the TSC complex (TSC1 and TSC2) integrates these diverse inputs along with inputs from energy status, stress response and hypoxia pathways to mTOR–

raptor through a small GTPase called Rheb. Energy status is communicated to TSC2 through direct phosphorylation by AMPK, whereas the mediators of hypoxia, stress and

nutrient availability are unknown. It remains possible that nutrients and growth factors, by unknown mechanisms, could also signal to mTOR independently of the TSC

complex. mTOR–raptor controls cellular growth in part by phosphorylating the hydrophobic motif of S6K1 and activating the kinase. mTOR–raptor also phosphorylates and

inhibits 4E-BPs, which themselves inhibit the eIF4E-dependent translation of capped mRNAs. mTOR–raptor has less defined but important roles in regulating transcription

and autophagy. Rapamycin (when bound to FKBP12) specifically inhibits the nutrient-sensitive mTOR–raptor complex. Compelling evidence suggests that a negative-

feedback loop enables the nutrient-sensitive mTOR–raptor pathway, through S6K1, to desensitize insulin signaling. S6K1 mediates the feedback by phosphorylating and

inactivating IRS1. Recent findings indicate that mTOR, when bound to rictor instead of raptor, activates AKT by phosphorylating its hydrophobic motif in a manner sensitive

to growth factors. AKT is a crucial regulator of cell proliferation and survival pathways and is hyperactivated in many cancers. mTOR is also phosphorylated on a C-terminal

site (not shown), but which mTOR complex is phosphorylated and why is unknown and thus we have excluded that information from the figure. Evidence also suggests that

AKT phosphorylates and inhibits TSC2, thus activating the mTOR–raptor growth pathway. Other studies have questioned the significance of this interaction, so although it is

an attractive model to explain why some tumors with hyperactive AKT signaling are sensitive to rapamycin, debate on its integrity is ongoing. The main pathways are

depicted with black arrows, whereas feedback loops are in red. The physiological relevance of these feedbacks for modulating normal mTOR signaling is still under

investigation but, regardless, it appears that considerable cross-talk exists. We have indicated selected direct phosphorylation sites with a ‘P’, but only for sites discussed in

the main text. Broken lines and question marks indicate poorly defined molecular connections.

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interaction with rictor (rapamycin-insensitive companionof mTOR) [26–30]. Both complexes additionally containthe small WD-repeat protein Gb-like (GbL) and possiblyother unidentified proteins [28,31].

Upon entering cells, rapamycin binds to its intracellu-lar receptor, FKBP12, and the complex binds to andspecifically inhibits mTOR [19,32]. This small lipophilicmacrolide antibiotic is already a clinically valuable drugused as an immunosuppressant and in preventing rest-enosis after balloon angioplasty [25]. The inhibition ofmTOR with rapamycin suppresses the enhanced growthphenotype observed in certain mouse cells that are null for

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PTEN or expressing constitutively activated AKT [33,34].Similar effects occur in transformed chicken embryofibroblasts, multiple myeloma cells and other transformedcells [35,36]. Although the reason why rapamycin isparticularly effective against some transformed cells andnot others remains unknown, such studies provide therationale for exploringmTOR inhibition as a treatment forcancers with PTEN inactivation.

Why might rapamycin be an effective drug againsttumor cells with PTEN loss of function? One hypoth-esis is that the loss of PTEN activates a rapamycin-sensitive mTOR growth pathway, sensitizing such

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transformed cells to the drug. Evaluating this hypoth-esis requires a brief discussion of current thought as tohow the mTOR growth pathway is regulated.

The rapamycin-sensitive mTOR–raptor complex con-trols cellular growth (the accumulation of cellular mass)by regulating protein synthesis and probably also bysuppressing autophagy. In doing so, mTOR–raptor coordi-nates growth-promoting signals from nutrient and energyavailability, particularly from amino acids, and relaysthem to downstream targets [19,32]. One target is thetranslational regulator S6K1, which mTOR–raptor phos-phorylates in coordination with the PDK1 protein kinase(Figure 1). Through its interaction with S6K1, raptordirects mTOR to phosphorylate Thr389 in the hydrophobicmotif of S6K1 in a rapamycin-sensitive manner, whereasthe PDK1 kinase phosphorylates Thr229 in the activationloop [37]. A second S6 kinase (S6K2), which is O80%identical to S6K1, is also phosphorylated in a rapamycin-sensitive manner [38,39]. Considerably less is knownabout S6K2, but genetic models suggest S6K1 and S6K2have redundant and distinct functions [38,40–42].Although more attention should be given to S6K2, wewill not discuss it further because S6K1 appears to bemore crucial for growth control. mTOR–raptor can alsophosphorylate and inhibit the eIF4E-binding proteins(4E-BPs), which are a family of negative regulators ofeukaryotic translation-initiation factor 4E (eIF4E)-depen-dent translation [19]. Studies implicate both S6K1 andeIF4E in cellular transformation [43–45].

The normal function of S6K1 and 4E-BP1 in proteinsynthesis has been extensively described elsewhere[19,32,37]. In brief, S6K1 is believed to drive translationin part by phosphorylating the ribosomal protein S6, butthe function of this event is still being studied. Thus,conclusive evidence as to the role of S6K1 (and S6K2) ingrowth remains elusive. Two recent reports furthersuggest that S6K1 can phosphorylate mTOR on aC-terminal site previously thought to be anAKT-phosphorylation site, but the functional significanceof this event is also unknown [19,46,47]. 4E-BP1 inhibitseIF4E which, in complex with other translationalregulators, binds to the 5 0 m7GpppN cap of nuclear-transcribed mRNA. This association recruits the cap-dependent translational activator eIF4G and the 40Sribosomal subunit. Hypophosphorylated 4E-BP1 competi-tively inhibits the interaction between eIF4G and eIF4Eby tightly binding to the same domain. mTOR–raptorrelieves 4E-BP1 inhibition by promoting its phosphoryl-ation and dissociation. Other less-characterized roles forthe rapamycin-sensitive mTOR complex in ribosomebiogenesis, transcription, cytoskeletal rearrangementsand autophagy have also been described [19,32,37].

The TSC connection

The tuberous sclerosis complex (TSC) is a heterodimer oftwo proteins, hamartin (TSC1) and tuberin (TSC2), whichis predicted to integrate signals derived from nutrientavailability, cellular energy status and hypoxia into acommon growth regulatory signal to the mTOR–raptorcomplex (Figure 1) [19]. The uncharacterized nutrient-derived growth signal feeds to mTOR–raptor through

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TSC2, a GAP (GTPase-activating protein)-domain-con-taining protein that negatively regulates a smallGTPase called Rheb [19]. Adding credence to thismodel are recent biochemical data showing thatGTP-bound Rheb can bind to and activate mTOR inan amino-acid-sensitive manner [48–50].

By phosphorylating TSC2, hyperactive AKT mightpromote mTOR–raptor-dependent cell growth (Figure 1).Evidence indicates that AKT-dependent phosphorylationof TSC2 inactivates the TSC complex, leading to mTOR–raptor activation and the hyperphosphorylation of S6K1and 4E-BP1 [19]. By activating mTOR–raptor, tumorsmight gain a growth advantage, suggesting one expla-nation as to why rapamycin is an effective cytostatic agentagainst cancer cells with hyperactive AKT signaling.Importantly, the relationship between PTEN inactivation,which presumably hyperactivates AKT, and rapamycinsensitivity is not overly compelling and predictingrapamycin sensitivity based on PTEN status alone is notparticularly effective.

The downregulation of mTOR–raptor activity alsooccurs through the TSC complex. In times of energy crisis,AMP-activated kinase (AMPK) promotes TSC inhibitionof mTOR–raptor by phosphorylating unique sites on TSC2[51,52]. Because AMPK responds to increasing levels ofAMP, it links growth to the cellular ATP:AMP ratio [52].By phosphorylating AMPK, the LKB1 kinase (also calledSTK11) contributes to AMPK activation and, accordingly,LKB1-null mouse-embryo fibroblasts exhibit elevatedmTOR–raptor signaling [53–56]. By a mechanism thatdiffers from AMPK, hypoxia promotes the TSC-dependentinhibition of mTOR through the hypoxia-inducible factor(HIF)-1-inducible genes REDD1 (DDIT4) and REDD2(DDIT4L) [57,58]. HIF-1a is a transcription factor thatalso promotes angiogenesis in response to low oxygen [59].Moreover, HIF-1a expression is sensitive to rapamycin[60,61], thus a complex interaction between mTOR andoxygen sensing exists.

The clinical value of inhibiting the mTOR–raptorpathway extends beyond cancer (Table 1). Mutations inthe TSC1 or TSC2 genes result in the hyperphosphoryla-tion of mTOR–raptor effectors and are linked to theharmatomatous syndrome tuberous sclerosis [62,63].Related hamartoma syndromes such as lymphangioleio-myomatosis (LAM), which is linked to tuberous sclerosis,Peutz–Jeghers syndrome (PJS), which is caused bymutations in LKB1, and Cowden’s disease, Lhermitte–Duclos disease and Bannayan–Riley–Ruvalcaba syn-drome, which are linked to PTEN inactivation, might allbe ameliorated by the inhibition of mTOR–raptor [62–64].Mutations in AMPK are associated with familial cardiachypertrophy, which also appears to be manifestedthorough mTOR–raptor [62]. Clinical trials with rapamy-cin are underway in patients with some of these diseases.

Feedback inhibition of IRS1

The hamartoma syndromes described above rarely pro-gress to metastatic cancer, raising the obvious question ofwhy not? Perhaps part of the answer lies in the recentconfirmation of a negative-feedback loop to the PtdIns3Kpathway. Compelling evidence shows that S6K1

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phosphorylates and inhibits the insulin-receptor sub-strate IRS1, an important mediator of insulin-receptor-dependent activation of PtdIns3K (Figure 1) [65–68].Chronic insulin stimulation leads to the phosphorylationand degradation of IRS1 protein in a rapamycin-sensitivemanner. Interestingly, the deletion of S6K1 hypersensi-tizes mice to insulin, protecting them from age- and diet-induced obesity [41]. These data predict that S6K1activation promotes growth, but by desensitizing insulin-receptor signaling through IRS1, S6K1 indirectly deacti-vates PtdIns3K–AKT signaling. Whether this negativefeedback exclusively targets IRS1 remains to be seen. Infact, AKT phosphorylation in response to platelet-derivedgrowth factor (PDGF) is reduced in some TSC-null cells asa result of reduced expression of the PDGF receptor, whichcould be analogous to IRS1 downregulation [69].

Biologically, this feedback might be important tocontrol the insulin-dependent influx of nutrients to cells,balancing intake with expenditure. Pathologically, TSC-deficient cells, which have elevatedmTOR–raptor activity,fail to activate AKT following insulin stimulation [65].Amino acids, which also activate mTOR–raptor activity,can similarly attenuate PtdIns3K signaling followinginsulin stimulation [70,71]. Thus, activating mTOR–raptor could inhibit the AKT-dependent pathways thatcontribute to cellular transformation, whichmight explainthe benign nature of tuberous sclerosis. Importantly,rapamycin treatment of TSC2-null cells enables thestimulation of AKT phosphorylation in response to insulinand insulin growth factor (IGF)-1; thus, inhibiting themTOR–raptor pathway with rapamycin in some tumorcells could promote AKT-dependent survival pathways[66,67]. Unlike a TSC-null deficiency, PTEN-null cancersmight exhibit cumulative tumorigenic signals that over-ride any potential negative feedback to IRS1; alterna-tively, PtdIns3K could be activated independently of IRS1.

mTOR–rictor, a missing link to AKT activation

To this point, we have focused on targeting the mTOR–raptor growth pathway in cancers characterized byhyperactive AKT. However, AKT has key regulatoryroles in cell-cycle control, apoptosis and metabolism, inaddition to cell growth. In fact, the physiological signifi-cance of AKT-dependent phosphorylation of TSC2 hasbeen challenged in Drosophila in a recent report showingthat a nonphosphorylatable version of TSC2 completelyrescues the lethality of TSC2-null flies [72]. OtherAKT-dependent processes are likely to be crucial fortumor progression. Therefore, inhibitors of AKT or itsupstream activators might have broader applications thanmTOR–raptor inhibitors.

mTOR–rictor: the elusive PDK2?

AKT activation requires the phosphorylation of Thr308 inthe activation loop and Ser473 in the hydrophobic motif[9]. Similar to S6K1, PDK1 phosphorylates AKT in itsactivation loop. The identity of the Ser473 kinase (oftenreferred to as PDK2) is controversial. In a recent report, itwas suggested that the mTOR–rictor complex fulfills therole of PDK2 in vitro and in vivo; importantly, this role isconserved in cultured Drosophila cells [3].

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Rictor was identified both in yeast (AVO3) andmammals as a novel mTOR-interacting protein defininga second, raptor-independent mTOR complex [28–30]. Asits name implies, the mTOR–rictor complex, unlike itsmTOR–raptor sibling, cannot bind to FKBP12–rapamycinand is insensitive to acute rapamycin treatment [28,29].This discovery provided solid evidence that rapamycintreatment does not represent a complete inhibition ofmTOR function, which had been speculated. The firstreports on the mTOR–rictor complex suggest that one ofits cellular functions is to regulate the cytoskeleton[29,30]. PKCa could mediate this function, because itsphosphorylation state is sensitive to RNA-interference(RNAi)-mediated silencing of mTOR and rictor and, inyeast, AVO3p regulates the actin cytoskeleton throughPKC1 [28,29]. PKC, interestingly, is an AGC-familykinase and its mTOR–rictor-dependent phosphorylationsite (Ser657) shares homology with the hydrophobic motifsites of S6K1 and AKT [73].

By using cells particularly sensitive to RNAi (such ascultured Drosophila cells) and lentiviral short-hairpinexpression systems in mammalian cells, it has beenpossible to achieve robust RNAi-mediated silencing ofmTOR-pathway components. A clue that mTOR has a rolein AKT phosphorylation was the observation thatsilencing raptor, but not mTOR or rictor, activates AKTon Ser473 (Ser505 in Drosophila), presumably by releas-ing the negative-feedback inhibition to the insulin–IGFpathway described above [3]. This suggested that mTORhas a positive role in regulating AKT phosphorylation thatis not shared with raptor but possibly is with rictor. Therealization that the Ser473 site in AKT is similar to thehydrophobic motif site in S6K1 and PKC prompted thediscovery that in both Drosophila and mammalian cells,the phosphorylation of AKT Ser473 decreases followingRNAi-mediated silencing of mTOR and rictor, but notraptor [3]. Biochemical experiments indicate that mTOR–rictor, but not mTOR–raptor, phosphorylates AKT Ser473in vitro and that full mTOR–rictor activity requiresgrowth factors, confirming the suspicion that mTOR,when bound to rictor, is a Ser473 kinase [3].

Still unknown is themechanism by whichmTOR–rictoris activated. Although mTOR–rictor activity is sensitive togrowth factors, the fact that constitutively activePtdIns3K alone triggers AKT Ser473 phosphorylation[4,8] suggests that if mTOR–rictor is the sole Ser473kinase, its activation lies downstream of PtdIns3K. Noknown component of either mTOR complex contains a PHdomain or other known lipid-binding domain. Thus,membrane recruitment following lipid phosphorylationis not an obvious possibility. Furthermore, a lack ofunambiguous cellular localization data for mTOR and itsinteracting proteins obscures attempts to identify sub-cellular compartments that might serve as mTORsignaling centers. Understanding how mTOR–rictoractivity is modulated and what signals promote theinteraction between mTOR–rictor and AKT is para-mount to understanding its role in cancer. It would beinteresting to determine whether elevated mTOR–rictor activity or overexpression is a hallmark of anyparticular cancer cell line.

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Considering the importance of AKT signaling indisease, attempts to identify the crucial Ser473 kinasehave been hotly pursued. Many other potential candidateshave been identified, including PDK1, integrin-linkedkinase (ILK), AKT itself and DNA-PKcs, but in each caseeither in vivo or in vitro data or conservation could not beconfirmed [74–77]. Because the mTOR–rictor complexfulfills all three criteria, we suggest that it is a Ser473kinase. One implication of this proposal, consideringcurrent models, is that mTOR–rictor lies upstream andcould indirectly regulate mTOR–raptor through the AKT–TSC2–Rheb axis. As previously mentioned, the debateconcerning the relevance of this interaction is ongoing.However, in the context of a transformed cell, hyperactiveAKT could become promiscuous and phosphorylate off-target proteins. If this were the case for TSC2, then byhijacking the mTOR–raptor pathway in transformed cells,the mTOR–rictor sibling (through AKT) would exert itsdominance in the pathogenesis of some cancers. Never-theless, if the role of mTOR–rictor in AKT activationwithstands future experimental challenges, then mTOR–rictor will attract considerable attention as a novel drugtarget. Determining whether mTOR–rictor is the crucialSer473 kinase in tumors characterized by hyperactiveAKT will be an essential step to this end.

With hindsight, there are clues in the literature aboutthe important role of rictor and mTOR in AKT activation.For example, a loss-of-function allele of pianissimo, theDictostylium orthologue of rictor [78], causes chemotaxisand cell-aggregation defects that are remarkably similarto those caused by loss of the Dictostylium orthologue ofAKT [79]. In addition, the orthologues of mTOR in bothDrosophila and Caenorhabditis elegans regulate thelifespan of the organism [80–82], a well-characterizedrole of AKT and its downstream effectors. mTOR waspreviously not considered an AKT kinase because Ser473phosphorylation is not sensitive to acute rapamycintreatment. But, because the rapamycin–FKBP12 complextightly binds to mTOR in the absence of associatedproteins, it remains possible that chronic exposure torapamycin might compromise the ability of newly syn-thesized mTOR protein to associate with rictor. Consistentwith this, long-term rapamycin treatment decreases AKTphosphorylation in at least one cell type [83]. This ideaprovides an alternative hypothesis for why rapamycin isparticularly effective at inhibiting the proliferation ofcertain PTEN-deficient cancer cells. Such a scenariowould necessitate a review of current rapamycin treat-ment regimens.

Considering the structural similarity between S6Ksand AKTs, particularly within the hydrophobic motif, itmakes sense in retrospect that mTOR is an AKT Ser473kinase. Indirectly, this finding strengthens the argumentthat mTOR directly phosphorylates S6K1 rather than anintermediate. Challenging this assumption was the factthat deleting a C-terminal domain of S6K1 rendered thephosphorylation of its hydrophobic motif insensitive torapamycin [84–86]. Interestingly, deleting this C-terminaldomain from S6K1 makes its hydrophobic motif siteaccessible to phosphorylation by the rapamycin-insensi-tive mTOR–rictor complex [86]. In fact, the S6K1

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C-terminal truncation mutant closely resembles thestructure of AKT which, outside of its PH domain, issimilar to S6K1 except that it lacks the C-terminaldomain. Although the mechanism of inhibition imposedon mTOR–rictor by this inhibitory domain is unclear, thisobservation does explain how the S6K1 C-terminaltruncation mutant can be rapamycin insensitive ifmTOR–raptor is the physiological S6K1 hydrophobic-motif kinase. The ability of mTOR to phosphorylatesimilar motifs in different kinases provides an excel-lent example of how one kinase, through specificinteractions with accessory proteins, can function indiverse cellular processes. It will be interesting todetermine if mTOR phosphorylates the hydrophobicmotifs of other AGC-family kinases and whetherdifferent accessory proteins are required.

Prospects for drug discovery

Three rapamycin analogs, CCI-779 (Wyeth: http://www.wyeth.com/), RAD001 (Novartis: http://www.novartis.com/)and AP23573 (Ariad Pharmaceuticals Inc: http://www.ariad.com/) are in clinical trials for the treatment of cancer[1,24,25]. Early reports indicate that the drugs showpromise in several tumor types, such as renal-cellcarcinoma, breast carcinomas and non-small-cell lungcarcinomas, and have tolerable side effects, including skinreactions, mucositis and myelosuppression [24,25]. Pre-liminary evidence suggested that rapamycin was particu-larly effective against tumors with PTEN inactivation, butan important point from the clinical trials is thatbiomarkers are needed to predict tumor sensitivity tothe drug. One study of breast cancer cell lines indicatesthat AKT phosphorylation and S6K1 overexpression,rather than PTEN status or S6K1 phosphorylation, arethe best predictors of rapamycin sensitivity [87]. More-over, in many instances, the disease remains progressivefollowing rapamycin treatment, even in PTEN-deficienttumors. In fact, one of the most favorable responses torapamycin was observed in mantle-cell lymphoma,which is characterized by cyclin-D1 overexpression[24]. The development and validation of biomarkersis crucial for determining which patients will benefitfrom rapamycin treatment.

Interestingly, rapamycin triggers apoptosis in somecell-culture systems, suggesting a role for the mTOR–raptor complex in cell survival [88–92]. Under serum-freeconditions in rhabdomyosarcoma cells lacking p53, rapa-mycin has been suggested to trigger apoptosis byactivating a stress response involving the apoptosis-signal-regulating kinase 1 (ASK1) and c-JUN [93].However, studies implicating rapamycin-dependent acti-vation of apoptosis in general rely on long-term rapamycintreatment, which might, in some cells, compromise AKTsignaling by disrupting the mTOR–rictor complex, assuggested above. Renal tumors bearing TSC2 mutationsare also sensitive to rapamycin-induced apoptosis [90].Perhaps these tumors, by upregulating mTOR–raptorsignaling in the absence of TSC2 function, activate thenegative-feedback loop to IRS1, preventing the fullactivation of AKT-dependent survival mechanisms andrendering the cells more susceptible to apoptosis. Other

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data indicate that in B-cell lymphoma, eIF4E overexpres-sion can promote cell survival in a rapamycin-insensitivemanner, probably because it lies downstream of mTOR–raptor [94,95]. In summary, mTOR–raptor signalingcontributes to a cell-survival mechanism in someinstances, but this is through apparently diverse meansthat are not necessarily sensitive to rapamycin treatment.

Clearly an mTOR–rictor inhibitor might be beneficialfor treating tumors with elevated AKT phosphorylation.Theoretically, such an inhibitor might downregulate thegrowth, proliferation and survival effects that are associ-ated with AKT activation and could be used in combi-nation with other chemotherapeutic agents or rapamycin.If mTOR–rictor is a crucial activator of AKT-dependentsurvival processes, such a drug might promote apoptosisin tumor cells that have adapted to AKT-dependentregulatory mechanisms. The essential element will be tofind an mTOR–rictor-specific inhibitor with a promisingtherapeutic window and acceptable toxicity. However, thelack of mTOR–rictor structural information might hindersuch efforts. Could a hyperactivated mTOR–raptor path-way, by TSC2 inhibition or another activator, drive cells todeplete energy stores? And, in combination with acompromised AKT-survival pathway, perhaps with anmTOR–rictor inhibitor, might apoptosis be potentlyactivated? At present, we can only speculate, but withthe newly discovered multifunctional roles for mTOR,open debate on new drug discovery and therapeuticapproaches should be stimulated.

Re-evaluating mTOR signaling

Considering these new findings, we feel the role of mTORin growth and disease warrants re-evaluation. Growth isdefined as an increase in size or mass. With respect tocellular growth, mTOR has been referred to as a ‘masterregulator’ because nutrient sensing through mTOR is anancient mechanism for determining individual cell mass.Extending the ‘master regulator’ role of mTOR to organand organism growth had been complicated by the factthat most organs additionally rely on a complex balancebetween cell proliferation and cell death to determine finalorgan size. The discovery that mTOR is a regulator of AKTimplies a direct role for mTOR in these biologicalprocesses. Mouse knockout models of mTOR exhibitearly embryonic lethality with cell growth and prolifer-ation defects, which supports this theory [96–98]. More-over, although not a major focus of this article, metabolismand aging, which are tightly linked to AKT-dependentprocesses, are probably more wired to mTOR signalingthan previously thought [18,99]. Many mutations thataffect insulin–IGF-1 signaling extend lifespan, as do somemutations that decrease TOR activity in C. elegans andDrosophila [18,99]. Perhaps the extended life span of TORmutants reflects in part the role of TOR in AKTphosphorylation. Considering that mTOR regulates cellproliferation and cell-survival pathways, in addition togrowth, we suggest a more global role for mTOR incontrolling organ and organism growth.

Rapamycin treatment for cancers with loss of PTENfunction is a potential clinical strategy. However, thetherapeutic potential of rapamycin has been considered

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mainly because the loss of PTEN is associated withhyperactive AKT signaling and existing evidence suggeststhat AKTregulates the TSC–mTOR–Raptor–S6K1 growthpathway. Because mTOR–rictor was recently shown todirectly regulate AKT in a manner that was insensitive toacute rapamycin treatment, mTOR probably has a morecentral role in cancers with hyperactive PtdIns3K–AKTsignaling. Designing new drugs that specifically inhibitmTOR–rictor might have great therapeutic value.

Concluding remarks

During the last few years, our understanding of mTORsignaling in many organisms has greatly advanced thanksto excellent reports on many fronts. Although thesediscoveries have led to considerable interest and evalu-ation of mTOR signaling, exemplified by the many reviewswritten on the subject, we eagerly anticipate learning themany secrets that this molecule still holds. In this article,we have addressed the prevailing views on the role ofmTOR in cancer and the hype surrounding rapamycintreatment as a promising anti-cancer strategy. However,new evidence suggests that mTOR might have a morecentral rapamycin-insensitive role in the pathology ofsome cancers. This discovery begins a new chapter in themTOR odyssey, importantly opening a new door in thesearch for promising anti-cancer drug targets.

Acknowledgements

We apologize to those authors whose primary work we did not referencedirectly in the text. We thank Deanna Stevens and Anne Carpenter forcomments on the manuscript. This work was supported by a grant fromthe NIH (RO1 A147389) to D.M.S. D.A.G. is supported by a grant from theDamon Runyon Cancer Research Foundation.

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