Oncogene (2013) 32, 3923–3932 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW The evolution of the TOR pathway and its role in cancer

EM Beauchamp1 and LC Platanias1,2

The target of rapamycin (TOR) pathway is highly conserved among eukaryotes and has evolved to couple nutrient sensing to cellular growth. TOR is found in two distinct signaling complexes in cells, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). These complexes are differentially regulated and act as effectors for the generation of signals that drive diverse cellular processes such as growth, proliferation, protein synthesis, rearrangement of the cytoskeleton, autophagy, metabolism and survival. Mammalian TOR (mTOR) is very important for development in embryos, while in adult organisms it is linked to aging and lifespan effects. In humans, the mTOR pathway is implicated in the tumorigenesis of multiple cancer types and its deregulation is associated with familial cancer syndromes. Because of its high biological relevance, different therapeutic strategies have been developed to target this signaling cascade, resulting in the emergence of unique pharmacological inhibitors that are either already approved for use in clinical oncology or currently under preclinical or clinical development. Multimodal treatment strategies that simultaneously target multiple nodes of the pathway and/or negative feedback regulatory loops may ultimately provide the best therapeutic advantage in targeting this pathway for the treatment of malignancies.

Oncogene (2013) 32, 3923–3932; doi:10.1038/onc.2012.567; published online 17 December 2012 Keywords: mTOR; cancer; leukemia

INTRODUCTION (PRAS40), DEP domain-containing mTOR-interacting protein (DEP- The target of rapamycin (TOR) pathway is only found in TOR), and mammalian lethal with SEC13 protein 8 (mLST8), with eukaryotes and is conserved in multiple species from yeast to RAPTOR and PRAS40 being the unique components, while the rest 15 mammals.1 It was originally discovered by the recognition of are shared with mTORC2. The major substrates for mTORC1 are mutations that conferred resistance to the growth inhibitory the p70 S6 kinase (S6K) and the translational repressor eIF4E- 16–18 effects of rapamycin in the yeast, Saccharomyces cerevisiae.2 The binding protein 1 (4E-BP1). mTORC1 activity is required for mammalian TOR (mTOR) was identified later, when multiple phosphorylation and subsequent activation of S6K, which then groups cloned the mTOR gene in mouse, human and rat.3–7 TOR is phosphorylates or binds proteins such as eukaryotic elongation present in at least two unique complexes with other proteins, TOR factor 2 kinase (eEF2K), S6K Aly/REF-like target (SKAR), ribosomal complex 1 (TORC1) and TOR complex 2 (TORC2).8,9 These protein S6 (rpS6) and eukaryotic initiation factor 4B (eIF4B), 19–21 complexes are differentially regulated by distinct upstream ultimately promoting translation initiation and elongation. signals, and their activation and functional engagement results Recently, a new substrate for S6K has been discovered, glioma- in control of distinct downstream effector pathways. Nutrients, associated oncogene homolog 1 (GLI1), which links mTORC1 to 22 growth factors, cellular energy and stress pathways regulate Hedgehog signaling. Thus, several distinct substrates are TORC1, while TORC2 appears to be primarily regulated by growth phosphorylated by S6K, resulting in activation of diverse factors.8,10,11 Recently, it was also shown that interferons (IFNs), downstream signals, reflecting a multifaceted role for S6K as a which are cytokines with growth inhibitory activities, could also major branch of the mTOR pathway. mTORC1 also phosphorylates activate mTORC112 and mTORC2.13 Other work has demonstrated and deactivates 4E-BP1, which in its unphosphorylated form that mTORC2 is inhibited by ER stress through phosphorylation of inhibits mRNA translation initiation by binding to the eukaryotic 23–25 rapamycin-insensitive companion of mTOR (RICTOR) by glycogen initiation factor 4E (eIF4E). Upon phosphorylation by mTORC1, synthase kinase 3b (GSK-3b).14 These complexes have distinct 4E-BP1 dissociates from eIF4E and allows eIF4E binding to eIF4G, 24–26 functions, with TORC1 primarily acting as regulator of cell growth, so initiation of translation can occur. protein synthesis and autophagy, whereas TORC2 controls cellular mTORC1 activity can be controlled by multiple growth 12,27–31 events important for cytoskeletal rearrangement, cell survival, factor and cytokine pathways. AKT activates mTORC1 via cell-cycle progression and metabolism.15 phosphorylation and inhibition of tuberous sclerosis 1/2 (TSC1/2), which leads to RAS homolog enriched in brain (RHEB) activation.27,30 Extracellular signal-related kinase (ERK) and 90 kDa ribosomal protein S6 kinase 1 (RSK1), have been also REGULATION AND FUNCTION OF mTORC1 AND mTORC2 shown to phosphorylate/inactivate the TSC1/2 complex, mTORC1 suggesting that they may also regulate mTORC1.29,31 The WNT mTORC1 in mammals is composed of regulatory-associated pathway can also control mTORC1 activity by inhibition of GSK-3b, 32 protein of mTOR (RAPTOR), 40 kDa proline-rich AKT substrate which inhibits mTORC1 via phosphorylation/activation of TSC2.

1Robert H Lurie Comprehensive Cancer Center, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and 2Department of Medicine, Jesse Brown VA Medical Center, Chicago, IL, USA. Correspondence: Dr LC Platanias, Robert H Lurie Comprehensive Cancer Center, 303 East Superior Street, Lurie 3-107, Chicago, IL 60611, USA. E-mail: [email protected] Received 7 September 2012; revised 8 October 2012; accepted 8 October 2012; published online 17 December 2012 Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3924 Also, mTORC1 can be regulated by cellular energy through AMP- S6K.51,54 Whether VPS34 directly activates S6K or mTORC1 is not activated protein kinase (AMPK).33,34 AMPK can inhibit mTORC1 by known, but mTORC1 has been co-immunoprecipitated with phosphorylating and activating TSC2.32,35 AMPK has been also VPS34,48 suggesting a regulatory effect at the mTORC1 level. shown to directly phosphorylate RAPTOR, and affect mTORC1 Under energy stress, AMPK is also able to inhibit VPS34, which complex formation.36 The AMPK phosphorylation sites in RAPTOR indicates that both amino acids and energy levels can regulate are highly conserved in all eukaryotes, even in those lacking the mTORC1 through VPS34.54 The role of VPS34 in autophagy and TSC complex, indicating it was probably the first mechanism to mTORC1 signaling seems counterintuitive as mTORC1 is link AMPK to TOR.36 It should be noted that a direct interaction established as a key negative regulator of autophagy48 between RAPTOR and AMPK, however, has only been shown in (Figure 1). Thus, the precise regulation and engagement of mammals.36 mTORC1 can be inhibited by DNA damage or other VPS34 in both processes remains to be investigated. It should be cellular stress signals through the p53 response by upregulating noted, that both mTORC1 and mTORC2 can regulate autophagy, the expression of negative regulators of PI3K/mTOR.37 Also p53 but only mTORC1 does so directly (Figure 1). Whereas AMPK can has been shown to upregulate the genes for Sestrin1 and Sestrin2, phosphorylate ULK1 to activate autophagy, mTORC1 can directly which activate AMPK, thus inhibiting mTORC1.38 Another potential phosphorylate ULK1 to inhibit autophagy.55,56 On the other hand, regulator of mTORC1 is PRAS40. This protein was originally mTORC2 has been reported to inhibit autophagy indirectly, via discovered as a substrate for AKT,39,40 and then subsequently repression by the AKT/forkhead box protein O (FOXO) axis of shown to be a component and substrate of mTORC1.41–44 There certain genes encoding for elements of the autophagic have been studies to show that it can act as either a negative or machinery.57 positive regulator of mTOR signaling, likely depending on the context of engagement and the activation or interference of other signaling elements.41,42,44–47 Therefore, its specific role in mTOR mTORC2 signaling remains to be clarified and precisely defined. The mTORC2 complex is composed of mammalian stress-activated The precise mechanism of mTORC1 regulation by amino acids is map kinase-interacting protein 1 (mSIN1), protein observed with not known. However, it has been established that the amino acid RICTOR (PROTOR), RICTOR, DEPTOR and mLST8, where the unique levels can affect mTORC1 activity through VPS34 and RAG complex components are mSIN1, PROTOR and RICTOR.15 mTORC2 GTPases.15,48 Amino acids cause a switch of RAGs to their active phosphorylates AGC kinases, such as AKT, serum and conformation by promoting their conversion to their proper glucocorticoid-regulated kinase (SGK) and protein kinase GTP/GDP-bound state.49,50 The active RAGs then bind to RAPTOR C-a (PKCa).58–61 A defining function of the mTORC2 complex is and target mTORC1 to the endosome and lysosome where it can its ability to phosphorylate AKT on the Ser473 (Ser505 in flies) be activated by RHEB in response to amino acids.50 RHEB is residue, priming AKT for further phosphorylation by required for activation of mTORC1 by amino acids independent of 3-phosphoinositide-dependent protein kinase 1 (PDK1) at the the TSC complex, unlike insulin, which requires both RHEB and Thr308 residue.58,60,61 The integrity of RICTOR is essential for such TSC1/2.27,51 AKT phosphorylation and RICTOR knockout cells exhibit defective VPS34 is a class III PI3K that was recently shown to be critical for phosphorylation of AKT at Ser473 in response to induction by a the process of autophagy, by forming a complex with BECLIN1 variety of stimuli.13,61,62 Loss of phosphorylation at the Ser473 site, and UV radiation resistance-associated gene protein (UVRAG)52,53 however, only affects some of its substrates such as the FOXOs but (Figure 1). Two independent studies have also established that not TSC2, in response to growth factor signaling.62 However, it amino acids stimulate VPS34 activity, which in turn activates may have different implications in signaling for certain cytokines, as in the IFN-system it appears that phosphorylation of certain mTOR effectors such as S6K and 4E-BP1 is impaired in the absence of RICTOR, suggesting differential effects on AKT Ser473 AMPK phosphorylation on downstream mTORC1 signaling by IFNs, as 13 Vps34 compared with growth factor signals. mSIN1 is required for the function and activity of mTORC2.63–65 mSIN1 is also directly phosphorylated by mTOR, which prevents its mTORC2 mTORC1 degradation by the lysosome.66 mSIN1 exists in multiple splice 67 ULK1 variants. Only three of them can be incorporated into mTORC2 63 Vps34 (Sin 1.1, 1.2 and 1.5) and regulate AKT phosphorylation. The S6K ATG13 FIP200 Beclin1 UVRAG different spliced forms make up distinct mTORC2 complexes as only two of them can be stimulated by insulin, indicating that AKT these distinct complexes respond differently to upstream stimuli.63 The identity of the physiological activator of mTORC2 complexes containing Sin 1.5 remains elusive at this time. FOXO Autophagy The precise role of PROTOR in mTORC2 structure and function is not well known. There are two isoforms of this protein, Chloroquine PROTOR1 and PROTOR2.43,68 PROTOR knockout had no obvious Bafilomycin1 effects on mTORC2 assembly or function.43,68,69 PROTOR1 Figure 1. Role of mTOR in autophagy. mTORC1 inhibits autophagy knockdown in cells impairs platelet-derived growth factor by directly phosphorylating ULK1, while AMPK activates autophagy (PDGF)-dependent induction of AKT and S6K phosphorylation, by directly phosphorylating ULK1. ULK1 forms a complex with with modest effects on insulin or epidermal growth factor (EGF)- FIP2000 and ATG13, leading to induction of autophagy. AMPK can dependent phosphorylation, due to a PDGF receptor b leading to inhibit mTORC1 directly or through VPS34. VPS34 can lead to an effect on cell proliferation rates.68 PROTOR2 knockdown in cells activation of S6K; however, it is unknown whether it does so directly leads to a resistance to apoptosis and PROTOR2 dissociates from or via mTORC1 engagement. VPS34 activates autophagy by binding 43 to Beclin 1 and UVRAG. mTORC2 can indirectly inhibit autophagy by mTORC2 when TSC1/2 is knocked down. Knockout mice for both engaging AKT which inhibits FOXOs. Autophagy inhibitors such as the Protor1 and Protor2 genes were recently generated, chloroquine or bafilomycinA1 could be conceivably used in in an attempt to better elucidate their function. Surprisingly, combination with mTOR inhibitors to more effectively target the only defect was a decrease in activation of SGK1 and malignant cells. its downstream effector, N-myc downstream regulated 1 (NDRG1),

Oncogene (2013) 3923 – 3932 & 2013 Macmillan Publishers Limited Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3925 in kidney cells of Protor 1-deficient mice only.69 Why PROTOR1 cycle progression from the G1 phase.78 SCH9 is an AGC kinase that only affects mTORC2 in the kidney remains to be definitively is an important downstream effector of TORC1 in S. cerevisiae that established, but it may simply reflect a dependence of kidney cells functions similarly to S6K in mammals.79 TORC1 in yeast, like on SGK1 signaling.69 However, it is also possible that PROTOR mammals, can also inhibit the induction of autophagy.80 TORC2 exhibits effects on other mTORC2 substrates and tissues not controls the polarization of the actin cytoskeleton during cell-cycle investigated in that study.69 progression.81 It should be also noted that TORC2 in yeast mSIN1 and RICTOR associate with each other to form a regulates the actin cytoskeleton through PKC1.74,82 heterodimer that then binds mTOR.63,64 PROTOR also binds RICTOR, but does so independently of the mTOR–RICTOR Caenorhabditis elegans 43,68 interaction. Recent evidence has shown that a specific A characteristic feature of the mTOR pathway in C. elegans is the residue, Gly934, is required for mTORC2 complex assembly and 83 70 absence of the TSC complex. In this case, AKT activity and TORC1 the mSIN1, but not PROTOR, interaction with RICTOR. are linked by AKT’s control on DAF-16 (FOXO)-dependent Mutation of the analogous residue in Caenorhabditis elegans, transcriptional regulation of DAF-15 (RAPTOR).83 Deletion of Gly1120, leads to a loss of function of RICTOR, indicating the ceTOR leads to developmental arrest at L3 larval stage, intestinal importance of this residue for RICTOR, whose function is highly 84 71,72 atrophy, and shares some characteristics with starved L3 larvae. conserved. In addition, the function of RICTOR is regulated by DAF-15 (RAPTOR) mutants have a similar phenotype with ceTOR multisite acetylation, and p300-mediated acetylation increases 83 73 mutants, while knockdown of ceTOR or DAF-15 in adulthood mTORC2 activity, as measured by AKT phosphorylation. leads to an increased lifespan.83,85 In addition, knockdown of S6K The domain required for acetylation is adjacent to a stability or eIF4G leads to increased lifespan, which indicates a role for domain required for the interaction of RICTOR with mSIN1 and 73 protein translation as an important downstream TOR effector in mLST8. aging of C. elegans.86,87 Loss of TORC1 leads to the induction of autophagy and autophagy is required for an increase in lifespan.88 ceRICTOR mutants that are associated with a loss of TORC2, are EVOLUTION OF THE TOR PATHWAY IN EUKARYOTES characterized by high-body fat, developmentally delayed growth, 71,72 Saccharomyces cerevisiae decreased fecundity and a shorter lifespan. Importantly, effects of RICTOR were mediated via SGK1 engagement and not Unlike in mammals where there is only one mTOR gene, in yeast 71 2 through the AKT/Daf-16 (FOXO) axis. PKC was also not involved, there are two TOR encoding genes, TOR1 and TOR2. Either TOR1 71 or TOR2 can be incorporated into TORC1, whereas TOR2 can only as PKC mutants do not mimic ceRICTOR mutants. Interestingly, 74 C10H11.8 (LST8) mutants mimic ceRICTOR mutants even though be incorporated into TORC2. TOR1 mutants are viable, while 71 TOR2 mutants are lethal.75,76 TORC1 is composed of TOR1 or TOR2, LST8 is present in both complexes. Unlike VPS34’s regulatory LST8, KOG1 (RAPTOR) and TCO89.74,77 Notably, TCO89 does not effects on mTORC1 signaling, siRNA against VPS34 did not have any homologs in higher eukaryotes34 and at this time its recapitulate the phenotype of DAF-15 or TOR mutations, indicating that it does not play a role in TOR signaling in functional equivalent, if any, is unknown. TORC2 is composed of 89 TOR2, AVO1, AVO2, AVO3, LST8 and BIT61.74,77 AVO3 is the yeast C. elegans. homolog of RICTOR, while AVO1 shares limited homology to the mammalian protein mSIN1.34 AVO2 and BIT61 seem to exist only Drosophila melanogaster in S. cerevisiae.34 Another unique feature of the TOR pathway in In flies, TOR knockouts have a similar phenotype to amino acid S. cerevisiae is the lack of both RHEB and the TSC complex.1 The deprived larvae, characterized by developmental arrest and lipid similarities and differences in components of the pathway and vesicle aggregation.90,91 In the adult fly, inhibition of the TOR regulatory mechanisms in various eukaryotic species are shown in pathway results in the extension of lifespan.92 4E-BP1 is an Figure 2. TORC1 controls protein translation and subsequent cell- important downstream TOR effector for this process.93 Like in

S.cerevisiae C.elegans D. melanogaster Mammals

glucose TSC growth factors growth factors amino acids glucose 1/2 growth factors growth factors PI3K 1 nutrients AGE-1 PI3K 1 Nutrients ? AMPK amino acids TSC RAGs AMPK 1/2 Rheb mTORC2 TORC1 TORC2 TORC1 TORC2 TORC2 mSIN1 PROTOR TORC1 Rheb KOG1 BIT61 AVO2 VPS34/ PRAS40 RICTOR TC089 DEPTOR TOR1/ LST8 AVO3 AVO1 DAF-15 C10H11.8 RAGs PI3K 3 mLST8 LST8 ceRICTOR C10H11.8 dRICTOR CG30004 DEPTOR RAPTOR mTOR TOR2 TOR2 ceTOR dRAPTOR CG30004 mLST8 ceTOR dTOR mTOR dTOR PDK PDK mTORC1 autophagy PDK SCH9 autophagy PKC SGK SGK α PKC1 VPS34/ S6K 4EBP-1 S6K SGK PI3K 3 S6K autophagy AKT AKT 4EBP-1 AKT protein synthesis actin cytoskeleton actin protein synthesis protein synthesis cytoskeleton Cell cycle Fat metabolism DAF-16 autophagy protein synthesis progressoion dFOXO cell cycle progression Growth Cell survival FOXO Feeding behavior cell survival cell metabolism Figure 2. Similarities and differences in TOR signaling among eukaryotes. Despite many similarities in patterns of expression and functional outcomes, there are several differences in the TOR pathway among different eukaryotes. The same shape and color indicates that a given element is homologous throughout the different organisms. (a) In the yeast S. cerevisiae, the unique components are TCO89, AVO2 and BIT63 and they lack the TSC complex and RHEB. Yeast also does not have a PI3K axis as part of TORC2. S. cerevisiae has two TOR genes, TOR1 and TOR2, both of which can be part of TORC1, while TOR2 can only be part of TORC2. (b)InC. elegans, TORC1 includes RAPTOR and LST8 homologs, while TORC2 includes RICTOR and LST8 homologs. The TSC complex is missing, but TORC1 activity can be controlled by AKT through DAF-16 transcriptional regulation of DAF-15 (RAPTOR) expression. (c) The D. melanogaster TOR pathway is quite similar to mammals; however, it lacks some of the components of the mTORC1 and mTORC2 complexes like in C. elegans. VPS34 in D. melanogaster does not regulate TORC1 but is placed downstream of TORC1. TORC1 inhibits it through ATG1 (ULK1) to inhibit autophagy. (d) The mTOR pathway in mammals is by far the most complex with multiple unique components and signaling inputs.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3923 – 3932 Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3926

C. elegans, VPS34 is not linked to TOR signaling in D. melanogaster, PI3K 1 as VPS34 null flies have no defects in TOR signaling.94 Recruitment Dual EGFR PI3K/mTOR of VPS34 in autophagosome formation requires inhibition of TOR inhibitors mTORC2 and activation of ATG1 (ULK1 in flies) signaling, indicating that 94 AKT VPS34 is downstream of TOR. It should be noted that VPS34 IRS1 seems to play a conserved role in autophagy and vesicle Catalytic 94,95 mTOR trafficking in mammals, yeast and flies. The role of VPS34 in RAS inhibitors MEK TSC1/2 TOR signaling is not conserved in lower eukaryotes and may have inhibitors evolved as a mechanism of regulation later on. D. melanogaster IR has only one sestrin gene, and like in mammals, was shown to be a MEK DEPTOR 96 regulator of AMPK/TOR signaling. mTORC1 FOXO

ERK MNK S6K Mammals inhibitors The DEPTOR, PRAS40 and PROTOR components of the pathway Rapalogs Sesn3 Grb10 AMPK are only found in mammals. Knockout of mTOR in mice is MNK embryonic lethal.97–99 The knockout of certain components of the mTORC2 complex is also embryonic lethal.62,100 It should be noted eIF4E 4E-BP1 that S6K mutants in worms, flies and mice show inhibition of Figure 3. Feedback loops in the mTOR pathway. S6K can inhibit growth, reduced body size and slightly delayed development, but insulin/PI3’K signaling through inhibition of IRS1. mTORC1 activates unlike TOR, S6K mutants can survive embryogenesis, indicating 84,101–103 GRB10 which inhibits both MAPK and PI3K pathways through effects S6K does not play as critical a role in development. In fact, on growth factor receptors such as the IR. mTORC1 can inhibit MNK/ in S6K1/2 double knockout mice, it was shown that some of these eIF4E, although the exact mechanism is unknown; however, MAPK mice die at or shortly after birth, while others make it to signaling could be effected through the S6K/IRS1 feedback loop. adulthood, and those that died had no gross developmental The MAPK pathway can inhibit PI3K and downstream signaling defects.102 Like in the worm, mLST8 mutant mice embryos display through inhibition of either EGFR or IRS1. ERK can activate mTORC1 the same phenotype of RICTOR knockout embryos, whereas via inhibition of the TSC complex, while S6K can inhibit mTORC2 RAPTOR and mTOR knockout mouse embryos share similar through direct phosphorylation of RICTOR by S6K. The TSC complex 62 while it inhibits mTORC1 can activate mTORC2. AKT inhibits FOXOs, characteristics. This indicates that mLST8 is more crucial for and FOXOs can activate AMPK by promoting Sesn3 expression, mTORC2 assembly and function than it is for mTORC1, but the thereby inhibiting mTORC1. FOXOs can also activate mTORC2 via reasons for this are unknown at this time and this requires further increased expression of RICTOR; however, this mechanism is not investigation. through FOXO1’s ability to bind DNA. AKT can activate mTORC1 via The role of mTORC2 in cytoskeletal rearrangement and motility in inhibition of the TSC complex. DEPTOR can inhibit mTORC1 and mammals is still not well understood. Knockdown of mTOR, mLST8 thus relieve the S6K/IRS1 feedback loop. Various classes of drugs are or RICTOR inhibits cell spreading and actin polymerization.104,105 shown in green color that can be used to target the pathway as well Expression of active RAS-related C3 botulinum toxin substrate 1 as feedback loops, and potentially enhance the therapeutic efficacy (RAC1) was able to restore the actin cytoskeletal defects in mTOR, of mTOR effectors. mLST8 or RICTOR knockdown cells.104 Interestingly, RAC1 seems to be required both upstream and downstream of mTORC2, as it A relatively recent study highlighted a new regulatory loop that regulates both mTORC2 localization and the downstream actin exists between AKT, FOXO1 and mTOR.112 In a TSC2-dependent polymerization effects.104,106 Knockdown of RICTOR or mTOR leads 105 manner, FOXO1 was shown to activate AMPK via transcriptional to a decrease in phosphorylation of PKCa, implicating this upregulation of Sestrin3, leading to a decrease in mTORC1 activity member of the PKC family of proteins in mTORC2 signaling. and activation of AKT via suppression of the S6K feedback loop.112 It should be also noted that a recent study suggested that RICTOR Then via a TSC2-independent mechanism, FOXO1, independent of may have effects on the cytoskeleton outside of mTORC2 as RICTOR its ability to bind DNA, was found to increase the levels of RICTOR, knockdown, but not mSIN1 knockdown, suppressed RhoGDI2, a 107 leading to an increase in mTORC2 formation and activation of negative regulator of cell migration. AKT.112 Other feedback loops with regulatory effects on mTOR functions involve the TSC/RHEB axis. RHEB activates mTORC1, FEEDBACK LOOPS AND NEGATIVE REGULATORS OF THE mTOR but has the opposite effect on mTORC2 and its activity results in PATHWAY inhibition of AKT.113 Most likely this involves the S6K feedback The regulation of the TOR pathway is highly complex due to the loop. TSC1/2 has the opposite effect where it traditionally inhibits existence of multiple negative feedback regulatory loops RHEB/mTORC1, but it can activate mTORC2, possibly by relieving (Figure 3). Several studies have shown a negative effect of the negative feedback loop.113 However, another study has mTORC1 on the insulin-PI3K-AKT pathway axis.103,108,109 S6K1 can demonstrated that TSC2 can directly interact with mTORC2 suppress insulin receptor substrate 1 (IRS1) function through through RICTOR to activate the complex independently direct phosphorylation of the protein at multiple sites,103,108 and it of its effects on mTORC1.114 mTORC1 can also inhibit mTORC2 can also inhibit IRS1 expression at the transcriptional level.108,109 in a negative feedback loop through S6K, where S6K The resulting impaired adaptor function of IRS1 leads to inhibitory directly phosphorylates RICTOR at Thr1135.115 Importantly, effects on the insulin- PI3K-AKT pathway. Recently, a new the phosphorylation on Thr1135 only effects mTORC2-mediated substrate of mTOR, growth factor receptor-bound protein 10 phosphorylation of AKT but not SGK or PKCa.115 This (GRB10), was discovered, independently by two groups, using a phosphorylation site is only found in mammals, and therefore it phospho-proteomic analysis approach.110,111 These studies is not evolutionally conserved.115 demonstrated that GRB10 is an mTORC1 effector that DEPTOR is a negative regulator of mTOR and can inhibit both suppresses insulin/growth factor-receptor generated-signals and mTORC1 and mTORC2.116 It is well established from previous work negatively controls mitogenic pathways.110,111 After its that mTORC1 positively regulates adipogenesis.117 Recent phosphorylation by mTOR, GRB10 is activated and inhibits the evidence has shown that DEPTOR, though it negatively regulates insulin receptor (IR), thus blocking PI3K-AKT signaling.110,111 mTOR, also positively regulates adipogenesis by suppressing the

Oncogene (2013) 3923 – 3932 & 2013 Macmillan Publishers Limited Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3927 mTORC1/S6K feedback loop on the insulin pathway.118 This activation of the mTORC1 pathway and inhibition of autophagy, leads to peroxisome proliferator-activated receptor-g activation leading to liver damage and inflammation.138 TSC1 knockout in through AKT to increase adipogenesis, and it is associated with this case may promote tumorigenesis because inhibition of increased obesity in mice and humans when DEPTOR is autophagy by mTOR may be enhancing tumor progression in overexpressed.118 the liver, whereas in other tumor types it may be protective.139 Mitogen-activated protein kinase (MAPK)-interacting kinases Recently, a third member of the TSC complex has been discovered (MNKs) are downstream effectors of MAPK pathways and are called TBC1D7 and was shown to be required for inhibition of known to phosphorylate eIF4E on Ser209, a site whose mTORC1.140 Since B15% of TS patients do not have mutations in phosphorylation correlates with induction of cell growth and either TSC1 or TSC2, it was proposed that TBC1D7 could be a third proliferation.119,120 There is evidence for a feedback loop induced TSC gene.140,141 However, after genetic analysis no changes were during mTOR inhibition that results in activation of the MNK/eIF4E observed in the TBC1D7 gene, indicating it is not mutated in TS.140 pathway. It has been shown that in cells treated with the mTORC1 Outside of the familiar cancer syndromes, there is evidence that inhibitor rapamycin, eIF4E is phosphorylated in a MNK-dependent deregulation of other elements of the mTOR pathway play manner.121,122 This indicates that the mTOR pathway inhibits MNK important roles in the growth and survival of many malignancies. and eIF4E via a negative feedback loop. The inhibition of MNK by Activating mutations in PI3K or deletion of phosphatase and mTOR may be dependent on PI3K, as in PI3K-deficient cells there tensin homolog (PTEN) occur frequently in malignancies and was no activation of MNK/eIF4E when they were treated with result in activation of AKT and mTOR.142 RHEB is overexpressed in rapamycin.122 A proposed mechanism of MAPK inhibition by some human lymphomas and leads to the development of mTORC1 was through the S6K-IRS1 feedback loop.123 The recent aggressive lymphomas in a transgenic mouse model.143 The discovery of GRB10110,111 may also shed some light on the eukaryotic initiation factor eIF4E, which is downstream of the mechanism. It is possible that the inhibitory effects of activated mTOR pathway can act as an oncogene by transforming cells GRB10 on growth factor receptors may account for its suppressive in vivo,144,145 and its overexpression has been associated with a effects on MNK/eIF4E phosphorylation, via modulation of MAPK poor prognosis in several human malignancies.126 In addition, loss pathways. Such engagement of MAPK pathways may have clinical of 4E-BP1, which suppresses eIF4E function, leads to increased implications, as in a phase I clinical trial, 50% of cancer patients tumorigenesis,146 while overexpression of constitutively activated treated with the rapalog everolimus had activation of the MAPK 4E-BP1 suppresses the growth of tumors in vivo.147,148 RICTOR pathway as measured by ERK phosphorylation in biopsy samples, expression is required for the growth of multiple cancer cell types indicating that a feedback loop between mTORC1 and the MEK/ in vitro and in vivo.149–151 There is a frequent loss of heterozygosity ERK pathway is induced in vivo.123 Similarly, MAPK pathways can in the gene region of PROTOR-1 in human breast cancer and also negatively regulate the PI3K-mTOR-AKT pathway, as PROTOR-1 is downregulated in a subset of breast tumors and cell treatment with MEK inhibitors leads to activation of AKT.124,125 lines.152 As mentioned above, DEPTOR inhibits mTORC1 but can One feedback loop that exists involves suppression of MEK- also activate PI3K/AKT signaling by relieving the S6K/IRS1 negative dependent inhibition of the epidermal growth factor receptor feedback loop.116,118 Therefore, DEPTOR can be pro-tumorigenic (EGFR) and associated suppression of EGF-dependent PI3K/AKT by promoting cell survival through PI3K/AKT, and is overexpressed activity.125 An additional mechanism involves suppression of ERK- in multiple myeloma, hepatocellular carcinoma and thyroid dependent inhibition of IRS1, thus effecting signaling through carcinoma.116,153–155 The mTOR pathway has also been shown insulin-like growth factor activation of PI3K/AKT.124 to be activated in myeloid leukemia, including BCR-ABL transformed cells, and plays a role in their growth and survival.156–161 mTOR and cancer Medulloblastoma is a pediatric brain tumor of the cerebellum There has been extensive evidence that mTOR plays an important that can be classified into four distinct molecular subtypes based role in tumorigenesis, and aberrant activation of the mTOR upon mutations/pathway activation, Hedgehog (Hh), WNT, pathway promotes the growth and survival of various types of Group 3 and Group 4.162–166 The PI3K/mTOR pathway has been malignant cells.9,15,126 There are multiple familial cancer implicated in the tumorigenesis of 3 of the 4 subtypes, Hh, WNT syndromes that lead to hyperactivation of the mTOR pathway. and Group 3. In the Hh pathway subtype, there is evidence for the The LKB1 tumor suppressor is lost in Peutz–Jegher’s syndrome, extensive involvement of PI3K/mTOR pathways in tumorigenesis leading to intestinal polyps and an increased risk for the of this subtype of medulloblastoma. In a Hh-driven mouse model development of certain cancers.127,128 Approximately 50% of of medulloblastoma metastasis, activation of the PI3K signaling NSCLC (non-small cell lung cancer) tumors harbor mutations in pathway was associated with dissemination of the primary tumor LKB1.129 It is well established that LKB1 is the key kinase into the spinal column.167 PI3K/mTOR activation was also responsible for activating AMPK.130 Inactivating mutations in associated with resistance to Hh pathway inhibitors in Hh-driven LKB1 result in a lack of AMPK activation with an associated mouse models of medulloblastoma.168 Combined treatment with upregulation of mTORC1 activity, as mTORC1 signaling has been a dual PI3K/mTOR inhibitor and an inhibitor of the hedgehog shown to be hyperactivated in LKB1-deficient mouse embryonic pathway was able to overcome this resistance.168 Activation of fibroblasts and livers as well as in hamaratomas from LKB1 PI3K signaling was also associated with survival of the stem cells heterozygous mice.131 after radiation exposure in Hh-driven mouse models of Tuberous sclerosis (TS), another familial cancer syndrome that is medulloblastoma, suggesting its involvement in relapse of characterized by hamartomas and benign tumors in various medulloblastoma patients after radiation treatment.169 Within organs, is caused by mutations in TSC1 or TSC2.132–135 the WNT subtype of medulloblastoma, crosstalk between the Interestingly, while double knockout of TSC1 or TSC2 is PI3K and WNT pathways has been shown to be important as PI3K embryonic lethal, heterozygous mice are viable and develop inhibition of GSK-3b enhances b-catenin activity, and treatment renal tumors, hemangiomas or leiomyomas.136,137 Loss of the TSC with a PDK inhibitor reduced the growth of medulloblastoma complex leads to a decrease in mTORC2 signaling, indicating that xenografts in mice.170 PIK3CA gene mutations have been found in the benign nature of TS tumors may be due to only mTORC1 multiple subtypes of medulloblastoma patients.165,171 When the activation.114 However, specific knockout of TSC1 in the liver leads PIK3C(E545K) gene mutation was combined with a WNT-driven to the development of hepatocellular carcinoma in mice and mouse model of medulloblastoma, the mice showed accelerated follows a similar progression as seen in human hepatocellular tumor development and enhanced mTOR activity, as measured by carcinoma.138 These tumors are characterized by chronic rpS6 and 4EBP-1 phosphorylation.165 These studies indicate that

& 2013 Macmillan Publishers Limited Oncogene (2013) 3923 – 3932 Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3928 the PI3K/mTOR pathway activation promotes WNT-driven mRNA translation in leukemia cells, as compared with medulloblastoma tumorgenesis. A majority of the group 3 rapamycin.189 The drawback with dual catalytic inhibitors, like tumors are characterized by c-MYC overexpression and have the rapalogs, is that the mTORC1 feedback loops can still be relieved worst prognosis.164,172,173 A recent mouse model of this subtype leading to activation of PI3K or MAPK signaling. AKT can also still was developed by transplanting c-MYC and dominant negative be activated at the Thr308 residue despite mTORC2 inhibition.185 p53 expressing cells into the cerebellum of donor mice.174 These As in the case of rapalogs, catalytic inhibitors can also induce tumors had activation of the PI3K pathway and were sensitive to autophagy. In BCR-ABL positive leukemia, the co-treatment of the dual PI3K/mTOR inhibitor.174 OSI-027 with the autophagy inhibitor chloroquine enhanced the anti-leukemic effects of OSI-027 alone.156 Therefore, combined treatment of certain cancers with a catalytic inhibitor and an TARGETING THE mTOR PATHWAY autophagy inhibitor may ultimately prove to be a better Rapamycin and rapalogs therapeutic strategy. Rapamycin was the first mTOR inhibitor that was originally discovered and purified in 1970s.175 Despite its antitumor activity in preclinical models, rapamycin has not been Dual PI3K/mTOR inhibitors successfully developed as an anticancer agent, among other The concerns over mTOR inhibitors led to the development of reasons due to poor solubility and stability.126 Other analogs of dual PI3K/mTOR kinase inhibitors. As mentioned previously, PI3K rapamycin have been developed and in phase II and III trials, and mTOR have high homology in their kinase domains, so 15 temsirolimus and everolimus showed efficacy in treating RCC development of dual inhibitors was possible. Two inhibitors (renal cell carcinoma), neuroendocrine tumors and mantle cell PI-103 or NVP-BEZ235 were found to strongly suppress both S6K and lymphoma.176–179 Currently, everolimus and temsirolimus are AKT and to induce apoptosis in gliomas, breast tumors and leukemia 157,190–193 approved for the treatment of RCC.180 Recently, everolimus was cells. PI3K-mTOR dual inhibitors inhibit all input signals to also approved by the FDA for the treatment of progressive AKT; however, they may not affect RAS mutant tumors as they can 194 advanced hormone receptor-positive, HER2-negative, breast signal alternatively through the MEK-ERK pathway. Co-treatment cancer in combination with examestane, based on the results of with a MEK inhibitor and a PI3K inhibitor was required for anti- 194 a pivotal trial that showed important clinical activity.181 tumorigenic responses in mutant RAS lung tumors in mice. The Due to the prevalent activation of mTOR in cancer, one would BRAFV600E mutation that is found frequently in melanomas was expect rapalogs to be more effective in inhibiting a wide variety of shown to negatively regulate AKT via mTORC2, as it required RICTOR tumors, but this does not appear to be the case. In glioblastoma, for inhibition of AKT activity, but independently of BRAF kinase 195 in both preclinical models and clinical trials, PTEN mutations did activity and downstream MEK-ERK engagement. Subsequent PTEN not predict response to rapalogs.182,183 In NSCLC, LKB1 mutations loss, however, was able to override the BRAFV600E inhibitory effects 195 in lung cancer cell lines also did not predict response to on AKT. Therefore, melanomas with just BRAFV600E may respond rapamycin.129 Traditionally, only mTORC1 is sensitive to well to MEK inhibition alone, whereas melanomas that have both rapamycin, as FKBP12-rapamycin cannot bind to mTORC2.104,105 PTEN loss and the BRAFV600E will likely require a combination of 195 There is also evidence for rapamycin-insensitive mTORC1 both PI3K and MEK inhibition for optimal results. In LKB1 or LKB1/ (RI-mTORC1) in BCR-ABL expressing leukemia cell lines.156 Other KRAS mutant tumors, the triple combination of a SRC inhibitor, Dual studies have also demonstrated that rapamycin only partially PI3K/mTOR inhibitor and MEK inhibitor was required to achieve 129 inhibits mTORC1, as measured by an incomplete block of 4E-BP1 significant anti-tumor effects. phosphorylation,184–186 suggesting the existence of RI-mTORC1 signals. Therefore, the relative limitations in clinical value of rapalogs may reflect activation of the pathway at other nodes due CONCLUSIONS to non-effectiveness, or by relieving negative feedback loops. Another issue that may affect the activity of rapalogs is that Because of its central role in cell metabolism and other important inhibition of mTOR can lead to induction of autophagy. In cancer functions, the mTOR pathway has emerged as a major target for cells, autophagy can be both anti- and pro-tumorigenic the treatment of malignancies. There is evidence for deregulation depending on the context and stimulus.139 Autophagy was of mTOR, or upstream and/or downstream effectors of the recently shown to be a mechanism of resistance to cell death pathway, in several human malignancies, and this has led to in RCC cell lines treated with the rapalog temsirolimus.187 extensive efforts towards the development of mTOR targeting Co-treatment with an autophagy inhibitor increased cell death antineoplastic agents. The complexities of the mTOR pathway and suppressed xenograft growth in mice.187 This indicates that warrant further investigation in order to tease out the intricacies combining rapalogs with autophagy inhibitors in RCC may further and inherent contradictions of the specific pathway member’s increase their therapeutic potential and clinical efficacy, but this roles and their regulation. The existence of multiple feedback remains to be directly examined in future trials. loops and crosstalk with other pathways makes it difficult to target it effectively in cancer. Based on what we know now, it is unlikely that mTOR inhibitors as single agents will be proven curative in a Catalytic mTOR inhibitors wide array of patients. Multimodal treatment strategies will likely Due to the limited success of rapalogs, mTOR catalytic inhibitors be required, where mTOR pathway inhibitors will be combined have been developed that target both mTORC1 and mTORC2. with other targeted agents. Such approaches warrant further These inhibitors show superior anti-tumorigenic effects compared development and clinical evaluation. However, one should also with rapalogs in preclinical evaluations, due to their ability to keep in mind that combinations of multiple agents may lead to block both mTORC1 as well as mTORC2.188 PP242 and OSI-027 additional toxicities. Another important clinical goal will be to both showed superior anti-leukemic effects compared with identify potential responders to mTOR inhibitors or combinations rapamycin in BCR-ABL expressing cell lines,156,157 as well as a based on the presence of genetic mutations and or other BCR-ABL positive leukemia mouse model.157 Treatment of acute evidence for altered signaling pathways within a given tumor. myeloid leukemia cells with OSI-027 also showed superior anti- With the rapid advances in the fields of genomics and proteomics, leukemic effects against cell lines as well as primary patient there is optimism that among other areas, personalized samples.189 OSI-027 was able to completely block 4E-BP1 approaches for the treatment of cancer patients will be able to phosphorylation and was much more effective in blocking successfully incorporate drugs that target mTOR.

Oncogene (2013) 3923 – 3932 & 2013 Macmillan Publishers Limited Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3929 CONFLICT OF INTEREST 28 Lekmine F, Uddin S, Sassano A, Parmar S, Brachmann SM, Majchrzak B et al. The authors declare no conflict of interst. Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons. J Biol Chem 2003; 278: 27772–27780. 29 Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and REFERENCES cancer pathogenesis. Cell 2005; 121: 179–193. 1 Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, Thomas EL et al. With TOR, less is 30 Potter CJ, Pedraza LG, Xu T. Akt regulates growth by directly phosphorylating more: a key role for the conserved nutrient-sensing TOR pathway in aging. Tsc2. Nat Cell Biol 2002; 4: 658–665. Cell Metab 2010; 11: 453–465. 31 Roux PP, Ballif BA, Anjum R, Gygi SP, Blenis J. Tumor-promoting phorbol esters 2 Heitman J, Movva NR, Hall MN. Targets for cell cycle arrest by the immuno- and activated Ras inactivate the tuberous sclerosis tumor suppressor complex suppressant rapamycin in yeast. Science 1991; 253: 905–909. via p90 ribosomal S6 kinase. Proc Natl Acad Sci USA 2004; 101: 13489–13494. 3 Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS et al. A mammalian 32 Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X et al. TSC2 integrates Wnt protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994; 369: and energy signals via a coordinated phosphorylation by AMPK and GSK3 to 756–758. regulate cell growth. Cell 2006; 126: 955–968. 4 Chen Y, Chen H, Rhoad AE, Warner L, Caggiano TJ, Failli A et al. A putative 33 Inoki K, Kim J, Guan KL. AMPK and mTOR in cellular energy homeostasis and drug targets. Annu Rev Pharmacol Toxicol 2012; 52: 381–400. sirolimus (rapamycin) effector protein. Biochem Biophys Res Commun 1994; 34 Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. 203: 1–7. Cell 2006; 124: 471–484. 5 Chiu MI, Katz H, RAPT1 Berlin V. a mammalian homolog of yeast Tor, 35 Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell interacts with the FKBP12/rapamycin complex. Proc Natl Acad Sci USA 1994; 91: growth and survival. Cell 2003; 115: 577–590. 12574–12578. 36 Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS et al. 6 Sabatini DM, Pierchala BA, Barrow RK, Schell MJ, Snyder SH. The rapamycin and AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity. J Biol Chem 2008; 30: 214–226. 1995; 270: 20875–20878. 37 Feng Z, Hu W, de Stanchina E, Teresky AK, Jin S, Lowe S et al. The regulation of 7 Sabers CJ, Martin MM, Brunn GJ, Williams JM, Dumont FJ, Wiederrecht G et al. AMPK beta1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, Isolation of a protein target of the FKBP12-rapamycin complex in mammalian and the role of these gene products in modulating the IGF-1-AKT-mTOR path- cells. J Biol Chem 1995; 270: 815–822. ways. Cancer Res 2007; 67: 3043–3053. 8 Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 38 Budanov AV, Karin M. p53 target genes sestrin1 and sestrin2 connect genotoxic 2012; 149: 274–293. stress and mTOR signaling. Cell 2008; 134: 451–460. 9 Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev 39 Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J et al. Large-scale Cancer 2006; 6: 729–734. characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA 10 Bhaskar PT, Hay N. The two TORCs and Akt. Dev Cell 2007; 12: 487–502. 2004; 101: 12130–12135. 11 Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. 40 Kovacina KS, Park GY, Bae SS, Guzzetta AW, Schaefer E, Birnbaum MJ et al. Nat Rev Mol Cell Biol 2009; 10: 307–318. Identification of a proline-rich Akt substrate as a 14-3-3 binding partner. J Biol 12 Kaur S, Sassano A, Dolniak B, Joshi S, Majchrzak-Kita B, Baker DP et al. Role of the Chem 2003; 278: 10189–10194. Akt pathway in mRNA translation of interferon-stimulated genes. Proc Natl Acad 41 Oshiro N, Takahashi R, Yoshino K, Tanimura K, Nakashima A, Eguchi S et al. The Sci USA 2008; 105: 4808–4813. proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of 13 Kaur S, Sassano A, Majchrzak-Kita B, Baker DP, Su B, Fish EN et al. Regulatory mammalian target of rapamycin complex 1. J Biol Chem 2007; 282: 20329– effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN 20339. responses. Proc Natl Acad Sci USA 2012; 109: 7723–7728. 42 Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E et al. 14 Chen CH, Shaikenov T, Peterson TR, Aimbetov R, Bissenbaev AK, Lee SW et al. ER PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell stress inhibits mTORC2 and Akt signaling through GSK-3beta-mediated phos- 2007; 25: 903–915. phorylation of rictor. Sci Signal 2011; 4: ra10. 43 Thedieck K, Polak P, Kim ML, Molle KD, Cohen A, Jeno P et al. PRAS40 and PRR5- 15 Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, like protein are new mTOR interactors that regulate apoptosis. PLoS One 2007 diabetes and ageing. Nat Rev Mol Cell Biol 2011; 12: 21–35. 2: e1217. 16 Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. 44 Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. Insulin signalling to Nat Rev Immunol 2005; 5: 375–386. mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 2007; 9: 316–323. 17 Schalm SS, Blenis J. Identification of a conserved motif required for mTOR sig- 45 Fonseca BD, Smith EM, Lee VH, MacKintosh C, Proud CG. PRAS40 is a target for naling. Curr Biol 2002; 12: 632–639. mammalian target of rapamycin complex 1 and is required for signaling 18 Schalm SS, Fingar DC, Sabatini DM, Blenis JTOS. motif-mediated raptor binding downstream of this complex. J Biol Chem 2007; 282: 24514–24524. regulates 4E-BP1 multisite phosphorylation and function. Curr Biol 2003; 46 Hong-Brown LQ, Brown CR, Kazi AA, Huber DS, Pruznak AM, Lang CH. Alcohol 13: 797–806. and PRAS40 knockdown decrease mTOR activity and protein synthesis via 19 Ma XM, Yoon SO, Richardson CJ, Julich K, Blenis J. SKAR links pre-mRNA splicing AMPK signaling and changes in mTORC1 interaction. J Cell Biochem 2010 to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs. Cell 109: 1172–1184. 2008; 133: 303–313. 47 Wang L, Harris TE, Roth RA, Lawrence Jr. JC. PRAS40 regulates mTORC1 kinase 20 Raught B, Peiretti F, Gingras AC, Livingstone M, Shahbazian D, Mayeur GL et al. activity by functioning as a direct inhibitor of substrate binding. J Biol Chem Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modu- 2007; 282: 20036–20044. lated by S6 kinases. EMBO J 2004; 23: 1761–1769. 48 Nobukuni T, Kozma SC, Thomas G. hvps34, an ancient player, enters a growing 21 Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Regulation of elon- game: mTOR Complex1/S6K1 signaling. Curr Opin Cell Biol 2007; 19: 135–141. gation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 2001; 20 49 Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag 4370–4379. GTPases in nutrient response. Nat Cell Biol 2008; 10: 935–945. 22 Wang Y, Ding Q, Yen CJ, Xia W, Izzo JG, Lang JY et al. The crosstalk of mTOR/S6K1 50 Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L et al. The and Hedgehog pathways. Cancer Cell 2012; 21: 374–387. Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 23 Haghighat A, Mader S, Pause A, Sonenberg N. Repression of cap-dependent 2008; 320: 1496–1501. translation by 4E-binding protein 1: competition with p220 for binding to 51 Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P et al. Amino acids eukaryotic initiation factor-4E. EMBO J 1995; 14: 5701–5709. mediate mTOR/raptor signaling through activation of class 3 phosphatidylino- 24 Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004 sitol 3OH-kinase. Proc Natl Acad Sci USA 2005; 102: 14238–14243. 18: 1926–1945. 52 Liang C, Feng P, Ku B, Dotan I, Canaani D, Oh BH et al. Autophagic and tumour 25 Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: suppressor activity of a novel Beclin1-binding protein UVRAG. Nat Cell Biol 2006; mechanisms and biological targets. Cell 2009; 136: 731–745. 8: 688–699. 26 Hara K, Yonezawa K, Kozlowski MT, Sugimoto T, Andrabi K, Weng QP et al. 53 Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N et al. Bcl-2 Regulation of eIF-4E BP1 phosphorylation by mTOR. J Biol Chem 1997; 272: antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122: 26457–26463. 927–939. 27 Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt 54 Byfield MP, Murray JT. Backer JM. hVps34 is a nutrient-regulated lipid kinase and suppresses mTOR signalling. Nat Cell Biol 2002; 4: 648–657. required for activation of p70 S6 kinase. J Biol Chem 2005; 280: 33076–33082.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3923 – 3932 Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3930 55 Egan D, Kim J, Shaw RJ, Guan KL. The autophagy initiating kinase ULK1 is 80 Noda T, Ohsumi Y. Tor a phosphatidylinositol kinase homologue, controls regulated via opposing phosphorylation by AMPK and mTOR. Autophagy autophagy in yeast. J Biol Chem 1998; 273: 3963–3966. 2011; 7: 643–644. 81 Schmidt A, Kunz J, Hall MN. TOR2 is required for organization of the actin 56 Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W et al. cytoskeleton in yeast. Proc Natl Acad Sci USA 1996; 93: 13780–13785. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects 82 Helliwell SB, Howald I, Barbet N, Hall MN. TOR2 is part of two related signaling energy sensing to mitophagy. Science 2011; 331: 456–461. pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics 1998; 57 Zhao J, Brault JJ, Schild A, Goldberg AL. Coordinate activation of autophagy and 148: 99–112. the proteasome pathway by FoxO transcription factor. Autophagy 2008; 4 83 Jia K, Chen D, Riddle DL. The TOR pathway interacts with the insulin signaling 378–380. pathway to regulate C. elegans larval development, metabolism and life span. 58 Facchinetti V, Ouyang W, Wei H, Soto N, Lazorchak A, Gould C et al. The Development 2004; 131: 3897–3906. mammalian target of rapamycin complex 2 controls folding and stability of Akt 84 Long X, Spycher C, Han ZS, Rose AM, Muller F, Avruch J. TOR deficiency in C. and protein kinase C. EMBO J 2008; 27: 1932–1943. elegans causes developmental arrest and intestinal atrophy by inhibition of 59 Garcia-Martinez JM, Alessi DR. mTOR complex 2 (mTORC2) controls hydrophobic mRNA translation. Curr Biol 2002; 12: 1448–1461. motif phosphorylation and activation of serum- and glucocorticoid-induced 85 Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Muller F. Genetics: influ- protein kinase 1 (SGK1). Biochem J 2008; 416: 375–385. ence of TOR kinase on lifespan in C. elegans. Nature 2003; 426: 620. 60 Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL. Essential function of TORC2 in PKC 86 Hansen M, Taubert S, Crawford D, Libina N, Lee SJ, Kenyon C. Lifespan extension and Akt turn motif phosphorylation, maturation and signalling. EMBO J 2008; 27: by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 2007; 1919–1931. 6: 95–110. 61 Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation 87 Pan KZ. Palter JE, Rogers AN, Olsen A, Chen D, Lithgow GJ et al. Inhibition of of Akt/PKB by the rictor-mTOR complex. Science 2005; 307: 1098–1101. mRNA translation extends lifespan in Caenorhabditis elegans. Aging Cell 2007; 62 Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J et al. 6: 111–119. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that 88 Hansen M, Chandra A, Mitic LL, Onken B, Driscoll M, Kenyon C. A role for mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Cell 2006; 11: 859–871. Genet 2008; 4: e24. 63 Frias MA, Thoreen CC, Jaffe JD, Schroder W, Sculley T, Carr SA et al. mSin1 is 89 Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino necessary for Akt/PKB phosphorylation, and its isoforms define three distinct acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab 2009; 296: mTORC2s. Curr Biol 2006; 16: 1865–1870. E592–E602. 64 Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY et al. SIN1/MIP1 maintains 90 Oldham S, Montagne J, Radimerski T, Thomas G, Hafen E. Genetic and bio- rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate chemical characterization of dTOR, the Drosophila homolog of the target of specificity. Cell 2006; 127: 125–137. rapamycin. Genes Dev 2000; 14: 2689–2694. 65 Yang Q, Inoki K, Ikenoue T, Guan KL. Identification of Sin1 as an essential TORC2 91 Zhang H, Stallock JP, Ng JC, Reinhard C, Neufeld TP. Regulation of cellular component required for complex formation and kinase activity. Genes Dev 2006; growth by the Drosophila target of rapamycin dTOR. Genes Dev 2000; 14: 20: 2820–2832. 2712–2724. 66 Chen CH. Sarbassov dos D. The mTOR (mammalian target of rapamycin) kinase 92 Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S. Regulation of lifespan maintains integrity of mTOR complex 2. J Biol Chem 2011; 286: 40386–40394. in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol 67 Schroder W, Cloonan N, Bushell G, Sculley T. Alternative polyadenylation and 2004; 14: 885–890. splicing of mRNAs transcribed from the human Sin1 gene. Gene 2004; 339: 93 Zid BM, Rogers AN, Katewa SD, Vargas MA, Kolipinski MC, Lu TA et al. 4E-BP 17–23. extends lifespan upon dietary restriction by enhancing mitochondrial activity in 68 Woo SY, Kim DH, Jun CB, Kim YM, Haar EV, Lee SI et al. PRR5, a novel component Drosophila. Cell 2009; 139: 149–160. of mTOR complex 2, regulates platelet-derived growth factor receptor beta 94 Juhasz G, Hill JH, Yan Y, Sass M, Baehrecke EH, Backer JM et al. The class III PI(3)K expression and signaling. J Biol Chem 2007; 282: 25604–25612. Vps34 promotes autophagy and endocytosis but not TOR signaling in Droso- 69 Pearce LR, Sommer EM, Sakamoto K, Wullschleger S, Alessi DR. Protor-1 is phila. J Cell Biol 2008; 181: 655–666. required for efficient mTORC2-mediated activation of SGK1 in the kidney. Bio- 95 Lindmo K, Stenmark H. Regulation of membrane traffic by phosphoinositide chem J 2011; 436: 169–179. 3-kinases. J Cell Sci 2006; 119: 605–614. 70 Aimbetov R, Chen CH, Bulgakova O, Abetov D, Bissenbaev AK, Bersimbaev RI 96 Lee JH, Budanov AV, Park EJ, Birse R, Kim TE, Perkins GA et al. Sestrin as a et al. Integrity of mTORC2 is dependent on the rictor Gly-934 site. Oncogene feedback inhibitor of TOR that prevents age-related pathologies. Science 2010; 2012; 31: 2115–2120. 327: 1223–1228. 71 Jones KT, Greer ER, Pearce D, Ashrafi K. Rictor/TORC2 regulates Caenorhabditis 97 Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, Spetz JF et al. Disruption elegans fat storage, body size, and development through sgk-1. PLoS Biol 2009; of the mouse mTOR gene leads to early postimplantation lethality and prohibits 7: e60. embryonic stem cell development. Mol Cell Biol 2004; 24: 9508–9516. 72 Soukas AA, Kane EA, Carr CE, Melo JA, Ruvkun G. Rictor/TORC2 regulates fat 98 Hentges KE, Sirry B, Gingeras AC, Sarbassov D, Sonenberg N, Sabatini D et al. metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev FRAP/mTOR is required for proliferation and patterning during embryonic 2009; 23: 496–511. development in the mouse. Proc Natl Acad Sci USA 2001; 98: 13796–13801. 73 Glidden EJ, Gray LG, Vemuru S, Li D, Harris TE, Mayo MW. Multiple site acet- 99 Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, Edenhofer F et al. mTOR is ylation of Rictor stimulates mammalian target of rapamycin complex 2 essential for growth and proliferation in early mouse embryos and embryonic (mTORC2)-dependent phosphorylation of Akt protein. J Biol Chem 2012; 287: stem cells. Mol Cell Biol 2004; 24: 6710–6718. 581–588. 100 Shiota C, Woo JT, Lindner J, Shelton KD, Magnuson MA. Multiallelic disruption of 74 Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D et al. Two the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth TOR complexes, only one of which is rapamycin sensitive, have distinct roles in and viability. Dev Cell 2006; 11: 583–589. cell growth control. Mol Cell 2002; 10: 457–468. 101 Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC, Thomas G. Drosophila S6 75 Helliwell SB, Wagner P, Kunz J, Deuter-Reinhard M, Henriquez R, Hall MN. TOR1 kinase: a regulator of cell size. Science 1999; 285: 2126–2129. and TOR2 are structurally and functionally similar but not identical phosphati- 102 Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J et al. S6K1(-/-)/S6K2(-/-) dylinositol kinase homologues in yeast. Mol Biol Cell 1994; 5: 105–118. mice exhibit perinatal lethality and rapamycin-sensitive 5’-terminal oligopyr- 76 Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, Hall MN. Target imidine mRNA translation and reveal a mitogen-activated protein kinase- of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog dependent S6 kinase pathway. Mol Cell Biol 2004; 24: 3112–3124. required for G1 progression. Cell 1993; 73: 585–596. 103 Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M et al. Absence of 77 Reinke A, Anderson S, McCaffery JM, Yates 3rd J, Aronova S, Chu S et al. TOR S6K1 protects against age- and diet-induced obesity while enhancing insulin complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with sensitivity. Nature 2004; 431: 200–205. Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem 104 Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A et al. Mammalian TOR 2004; 279: 14752–14762. complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell 78 Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, Hall MN. TOR controls Biol 2004; 6: 1122–1128. translation initiation and early G1 progression in yeast. Mol Biol Cell 1996; 7:25–42. 105 Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H et al. 79 Urban J, Soulard A, Huber A, Lippman S, Mukhopadhyay D, Deloche O et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell 2007; 26: raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004; 14: 663–674. 1296–1302.

Oncogene (2013) 3923 – 3932 & 2013 Macmillan Publishers Limited Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3931 106 Saci A, Cantley LC, Carpenter CL. Rac1 regulates the activity of mTORC1 and 132 European Chromosome 16 Tuberous Sclerosis C. Identification and character- mTORC2 and controls cellular size. Mol Cell 2011; 42: 50–61. ization of the tuberous sclerosis gene on chromosome 16. Cell 1993; 75: 107 Agarwal NK, Chen CH, Cho H, Boulbes DR, Spooner E, Sarbassov DD. Rictor 1305–1315. regulates cell migration by suppressing RhoGDI2. Oncogene 2012 (epub ahead 133 van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S et al. of print 9 July 2012; doi:10.1038/onc.2012.287). Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 108 Harrington LS, Findlay GM, Gray A, Tolkacheva T, Wigfield S, Rebholz H et al. The 1997; 277: 805–808. TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS 134 Green AJ, Smith M, Yates JR. Loss of heterozygosity on chromosome proteins. J Cell Biol 2004; 166: 213–223. 16p13.3 in hamartomas from tuberous sclerosis patients. Nat Genet 1994; 109 Shah OJ, Wang Z, Hunter T. Inappropriate activation of the TSC/Rheb/mTOR/S6K 6: 193–196. cassette induces IRS1/2 depletion, insulin resistance, and cell survival defi- 135 Green AJ, Johnson PH, Yates JR. The tuberous sclerosis gene on chromosome ciencies. Curr Biol 2004; 14: 1650–1656. 9q34 acts as a growth suppressor. Hum Mol Genet 1994; 3: 1833–1834. 110 Hsu PP, Kang SA, Rameseder J, Zhang Y, Ottina KA, Lim D et al. The mTOR- 136 Kobayashi T, Minowa O, Kuno J, Mitani H, Hino O, Noda T. Renal carcinogenesis, regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhi- hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 bition of growth factor signaling. Science 2011; 332: 1317–1322. mutation in mice. Cancer Res 1999; 59: 1206–1211. 111 Yu Y, Yoon SO, Poulogiannis G, Yang Q, Ma XM, Villen J et al. Phosphoproteomic 137 Kobayashi T, Minowa O, Sugitani Y, Takai S, Mitani H, Kobayashi E et al. A germ- analysis identifies Grb10 as an mTORC1 substrate that negatively regulates line Tsc1 mutation causes tumor development and embryonic lethality that are insulin signaling. Science 2011; 332: 1322–1326. similar, but not identical to, those caused by Tsc2 mutation in mice. Proc Natl 112 Chen CC, Jeon SM, Bhaskar PT, Nogueira V, Sundararajan D, Tonic I et al. FoxOs Acad Sci USA 2001; 98: 8762–8767. inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and 138 Menon S, Yecies JL, Zhang HH, Howell JJ, Nicholatos J, Harputlugil E et al. Rictor. Dev Cell 2010; 18: 592–604. Chronic activation of mTOR complex 1 is sufficient to cause hepatocellular 113 Yang Q, Inoki K, Kim E, Guan KL. TSC1/TSC2 and Rheb have different effects on carcinoma in mice. Sci Signal 2012; 5: ra24. TORC1 and TORC2 activity. Proc Natl Acad Sci USA 2006; 103: 6811–6816. 139 White E. Deconvoluting the context-dependent role for autophagy in cancer. 114 Huang J, Wu S, Wu CL, Manning BD. Signaling events downstream of mam- Nat Rev Cancer 2012; 12: 401–410. malian target of rapamycin complex 2 are attenuated in cells and tumors 140 Dibble CC, Elis W, Menon S, Qin W, Klekota J, Asara JM et al. TBC1D7 is a deficient for the tuberous sclerosis complex tumor suppressors. Cancer Res 2009; third subunit of the TSC1-TSC2 complex upstream of mTORC1. Mol Cell 2012; 69: 6107–6114. 47: 535–546. 115 Dibble CC, Asara JM, Manning BD. Characterization of Rictor phosphorylation 141 Camposano SE, Greenberg E, Kwiatkowski DJ, Thiele EA. Distinct clinical char- sites reveals direct regulation of mTOR complex 2 by S6K1. Mol Cell Biol 2009; 29: acteristics of tuberous sclerosis complex patients with no mutation identified. 5657–5670. Ann Hum Genet 2009; 73: 141–146. 116 Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, Kuehl WM et al. 142 Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cancer. Nat Rev Cancer 2002; 2: 489–501. cells and required for their survival. Cell 2009; 137: 873–886. 143 Mavrakis KJ, Zhu H, Silva RL, Mills JR, Teruya-Feldstein J, Lowe SW et al. 117 Laplante M, Sabatini DM. An emerging role of mTOR in lipid biosynthesis. Curr Tumorigenic activity and therapeutic inhibition of Rheb GTPase. Genes Dev 2008; Biol 2009; 19: R1046–R1052. 22: 2178–2188. 118 Laplante M, Horvat S, Festuccia WT, Birsoy K, Prevorsek Z, Efeyan A et al. DEPTOR 144 Ruggero D, Montanaro L, Ma L, Xu W, Londei P, Cordon-Cardo C et al. The cell-autonomously promotes adipogenesis, and its expression is associated with translation factor eIF-4E promotes tumor formation and cooperates with c-Myc obesity. Cell Metab 2012; 16: 202–212. in lymphomagenesis. Nat Med 2004; 10: 484–486. 119 Mahalingam M, Cooper JA. Phosphorylation of mammalian eIF4E by Mnk1 and 145 Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan S et al. Survival Mnk2: tantalizing prospects for a role in translation. Prog Mol Subcell Biol 2001; signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 2004; 27: 132–142. 428: 332–337. 120 Pyronnet S. Phosphorylation of the cap-binding protein eIF4E by the MAPK- 146 Petroulakis E, Parsyan A, Dowling RJ, LeBacquer O, Martineau Y, Bidinosti M et al. activated protein kinase Mnk1. Biochem Pharmacol 2000; 60: 1237–1243. p53-dependent translational control of senescence and transformation via 121 Altman JK, Glaser H, Sassano A, Joshi S, Ueda T, Watanabe-Fukunaga R et al. 4E-BPs. Cancer Cell 2009; 16: 439–446. Negative regulatory effects of Mnk kinases in the generation of chemotherapy- 147 Lynch M, Fitzgerald C, Johnston KA, Wang S, Schmidt EV. Activated eIF4E- induced antileukemic responses. Mol Pharmacol 2010; 78: 778–784. binding protein slows G1 progression and blocks transformation by c-myc 122 Wang X, Yue P, Chan CB, Ye K, Ueda T, Watanabe-Fukunaga R et al. Inhibition of without inhibiting cell growth. J Biol Chem 2004; 279: 3327–3339. mammalian target of rapamycin induces phosphatidylinositol 3-kinase-depen- 148 She QB, Halilovic E, Ye Q, Zhen W, Shirasawa S, Sasazuki T et al. 4E-BP1 is a key dent and Mnk-mediated eukaryotic translation initiation factor 4E phosphor- effector of the oncogenic activation of the AKT and ERK signaling pathways that ylation. Mol Cell Biol 2007; 27: 7405–7413. integrates their function in tumors. Cancer Cell 2010; 18: 39–51. 123 Carracedo A, Ma L, Teruya-Feldstein J, Rojo F, Salmena L, Alimonti A et al. 149 Guertin DA, Stevens DM, Saitoh M, Kinkel S, Crosby K, Sheen JH et al. mTOR Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K- complex 2 is required for the development of prostate cancer induced by Pten dependent feedback loop in human cancer. J Clin Invest 2008; 118: 3065–3074. loss in mice. Cancer Cell 2009; 15: 148–159. 124 Buck E, Eyzaguirre A, Rosenfeld-Franklin M, Thomson S, Mulvihill M, Barr S et al. 150 Hietakangas V, Cohen SM. TOR complex 2 is needed for cell cycle progression Feedback mechanisms promote cooperativity for small molecule inhibitors of epi- and anchorage-independent growth of MCF7 and PC3 tumor cells. BMC Cancer dermal and insulin-like growth factor receptors. Cancer Res 2008; 68: 8322–8332. 2008; 8:282. 125 Mirzoeva OK, Das D, Heiser LM, Bhattacharya S, Siwak D, Gendelman R et al. 151 Masri J, Bernath A, Martin J, Jo OD, Vartanian R, Funk A et al. mTORC2 activity is Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback elevated in gliomas and promotes growth and cell motility via overexpression of signaling determine susceptibility of breast cancer cells to MEK inhibition. rictor. Cancer Res 2007; 67: 11712–11720. Cancer Res 2009; 69: 565–572. 152 Johnstone CN, Castellvi-Bel S, Chang LM, Sung RK, Bowser MJ, Pique JM et al. 126 Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev PRR5 encodes a conserved proline-rich protein predominant in kidney: analysis Cancer 2004; 4: 335–348. of genomic organization, expression, and mutation status in breast and color- 127 Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A et al. ectal carcinomas. Genomics 2005; 85: 338–351. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 153 Pei L, Xie P, Zhou E, Yang Q, Luo Y, Tang Z. Overexpression of DEP domain 1998; 391: 184–187. containing mTOR-interacting protein correlates with poor prognosis in differ- 128 Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R et al. Peutz-Jeghers entiated thyroid carcinoma. Mol Med Rep 2011; 4: 817–823. syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 154 Yen CH, Lu YC, Li CH, Lee CM, Chen CY, Cheng MY et al. Functional character- 1998; 18: 38–43. ization of glycine N-methyltransferase and its interactive protein DEPDC6/DEP- 129 Sanchez-Cespedes M. The role of LKB1 in lung cancer. Fam Cancer 2011; 10: TOR in hepatocellular carcinoma. Mol Med 2012; 18: 286–296. 447–453. 155 Zhao Y, Xiong X, Sun Y. DEPTOR an mTOR inhibitor, is a physiological substrate 130 Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D et al. LKB1 of SCF(betaTrCP) E3 ubiquitin ligase and regulates survival and autophagy. Mol is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol Cell 2011; 44: 304–316. 2003; 13: 2004–2008. 156 Carayol N, Vakana E, Sassano A, Kaur S, Goussetis DJ, Glaser H et al. Critical roles 131 Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA et al. The for mTORC2- and rapamycin-insensitive mTORC1-complexes in growth and LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 2004; survival of BCR-ABL-expressing leukemic cells. Proc Natl Acad Sci USA 2010 6: 91–99. 107: 12469–12474.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3923 – 3932 Evolution of TOR pathway and its role in cancer EM Beauchamp and LC Platanias 3932 157 Janes MR, Limon JJ, So L, Chen J, Lim RJ, Chavez MA et al. Effective and selective therapy for the treatment of relapsed or refractory mantle cell lymphoma. J Clin targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat Med 2010 Oncol 2009; 27: 3822–3829. 16: 205–213. 177 Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A et al. Temsir- 158 Ly C, Arechiga AF, Melo JV, Walsh CM, Ong ST. Bcr-Abl kinase modulates the olimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous 2007; 356: 2271–2281. leukemia cells via the mammalian target of rapamycin. Cancer Res 2003; 63: 178 Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S et al. Efficacy of 5716–5722. everolimus in advanced renal cell carcinoma: a double-blind, randomised, pla- 159 Mayerhofer M, Aichberger KJ, Florian S, Krauth MT, Hauswirth AW, Derdak S et al. cebo-controlled phase III trial. Lancet 2008; 372: 449–456. Identification of mTOR as a novel bifunctional target in chronic myeloid leuke- 179 Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E et al. Everolimus for mia: dissection of growth-inhibitory and VEGF-suppressive effects of rapamycin advanced pancreatic neuroendocrine tumors. N Engl J Med 2011; 364: 514–523. in leukemic cells. FASEB J 2005; 19: 960–962. 180 Khokhar NZ, Altman JK, Platanias LC. Emerging roles for mammalian target of 160 Mohi MG, Boulton C, Gu TL, Sternberg DW, Neuberg D, Griffin JD et al. Combi- rapamycin inhibitors in the treatment of solid tumors and hematological nation of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treat- malignancies. Curr Opin Oncol 2011; 23: 578–586. ment of leukemias caused by oncogenic PTKs. Proc Natl Acad Sci USA 2004; 101: 181 Baselga J, Campone M, Piccart M, Burris 3rd HA, Rugo HS, Sahmoud T et al. 3130–3135. Everolimus in postmenopausal hormone-receptor-positive advanced breast 161 Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leu- cancer. N Engl J Med 2012; 366: 520–529. kemia cells requires PI3 kinase activation. Blood 2003; 102: 972–980. 182 Galanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J et al. Phase II 162 Jones DT, Jager N, Kool M, Zichner T, Hutter B, Sultan M et al. Dissecting the trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North genomic complexity underlying medulloblastoma. Nature 2012; 488: 100–105. Central Cancer Treatment Group Study. J Clin Oncol 2005; 23: 5294–5304. 163 Northcott PA, Shih DJ, Peacock J, Garzia L, Morrissy AS, Zichner T et al. Subgroup- 183 Yang L, Clarke MJ, Carlson BL, Mladek AC, Schroeder MA, Decker P et al. PTEN specific structural variation across 1,000 medulloblastoma genomes. Nature loss does not predict for response to RAD001 (Everolimus) in a glioblastoma 2012; 488: 49–56. orthotopic xenograft test panel. Clin Cancer Res 2008; 14: 3993–4001. 164 Pugh TJ, Weeraratne SD, Archer TC, Pomeranz Krummel DA, Auclair D, Bochic- 184 Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Rapamycin differentially inhibits chio J et al. Medulloblastoma exome sequencing uncovers subtype-specific S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. somatic mutations. Nature 2012; 488: 106–110. Proc Natl Acad Sci USA 2008; 105: 17414–17419. 165 Robinson G, Parker M, Kranenburg TA, Lu C, Chen X, Ding L et al. Novel muta- 185 Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D et al. Active-site tions target distinct subgroups of medulloblastoma. Nature 2012; 488: 43–48. inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. 166 Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC et al. PLoS Biol 2009; 7: e38. Molecular subgroups of medulloblastoma: the current consensus. Acta Neuro- 186 Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y et al. An ATP-competitive pathol 2012; 123: 465–472. mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions 167 Wu X, Northcott PA, Dubuc A, Dupuy AJ, Shih DJ, Witt H et al. Clonal selection of mTORC1. J Biol Chem 2009; 284: 8023–8032. drives genetic divergence of metastatic medulloblastoma. Nature 2012; 482: 187 Bray K, Mathew R, Lau A, Kamphorst JJ, Fan J, Chen J et al. Autophagy suppresses 529–533. RIP kinase-dependent necrosis enabling survival to mTOR inhibition. PLoS One 168 Buonamici S, Williams J, Morrissey M, Wang A, Guo R, Vattay A et al. Interfering 2012; 7: e41831. with resistance to smoothened antagonists by inhibition of the PI3K pathway in 188 Gentzler RD, Altman JK, Platanias LC. An overview of the mTOR pathway as a medulloblastoma. Sci Transl Med 2010; 2: 51ra70. target in cancer therapy. Expert Opin Ther Targets 2012; 16: 481–489. 169 Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, 189 Altman JK, Sassano A, Kaur S, Glaser H, Kroczynska B, Redig AJ et al. Dual Manova-Todorova K, Holland EC. PI3K pathway regulates survival of cancer stem mTORC2/mTORC1 targeting results in potent suppressive effects on acute cells residing in the perivascular niche following radiation in medulloblastoma myeloid leukemia (AML) progenitors. Clin Cancer Res 2011; 17: 4378–4388. in vivo. Genes Dev 2008; 22: 436–448. 190 Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, Lane H et al. Specific 170 Baryawno N, Sveinbjornsson B, Eksborg S, Chen CS, Kogner P, Johnsen JI. Small- apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 molecule inhibitors of phosphatidylinositol 3-kinase/Akt signaling inhibit Wnt/ amplified and PIK3CA mutant breast cancer cells. Proc Natl Acad Sci USA 2009; beta-catenin pathway cross-talk and suppress medulloblastoma growth. Cancer 106: 22299–22304. Res 2010; 70: 266–276. 191 Chiarini F, Fala F, Tazzari PL, Ricci F, Astolfi A, Pession A et al. Dual inhibition of 171 Broderick DK, Di C, Parrett TJ, Samuels YR, Cummins JM, McLendon RE et al. class IA phosphatidylinositol 3-kinase and mammalian target of rapamycin as a Mutations of PIK3CA in anaplastic oligodendrogliomas, high-grade astro- new therapeutic option for T-cell acute lymphoblastic leukemia. Cancer Res 2009; cytomas, and medulloblastomas. Cancer Res 2004; 64: 5048–5050. 69: 3520–3528. 172 Cho YJ, Tsherniak A, Tamayo P, Santagata S, Ligon A, Greulich H et al. Integrative 192 Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, Stokoe D et al. A dual PI3 genomic analysis of medulloblastoma identifies a molecular subgroup that kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 2006 drives poor clinical outcome. J Clin Oncol 2011; 29: 1424–1430. 9: 341–349. 173 Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S et al. 193 Liu TJ, Koul D, LaFortune T, Tiao N, Shen RJ, Maira SM et al. NVP-BEZ235, a novel Medulloblastoma comprises four distinct molecular variants. J Clin Oncol 2011; dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, 29: 1408–1414. elicits multifaceted antitumor activities in human gliomas. Mol Cancer Ther 2009; 174 Pei Y, Moore CE, Wang J, Tewari AK, Eroshkin A, Cho YJ et al. An animal model of 8: 2204–2210. MYC-driven medulloblastoma. Cancer Cell 2012; 21: 155–167. 194 Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R et al. Effective 175 Sehgal SN. Sirolimus: its discovery, biological properties, and mechanism of use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R action. Transplant Proc 2003; 35: 7S–14S. murine lung cancers. Nat Med 2008; 14: 1351–1356. 176 Hess G, Herbrecht R, Romaguera J, Verhoef G, Crump M, Gisselbrecht C et al. 195 Chen B, Tardell C, Higgins B, Packman K, Boylan JF, Niu H. BRAFV600E negatively Phase III study to evaluate temsirolimus compared with investigator’s choice regulates the AKT pathway in melanoma cell lines. PLoS One 2012; 7: e42598.

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