mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy.
Rapamycin, a fungal macrolide, was first isolated over four decades ago . This macrolide produced by Streptomyces hygroscopicus bacteria was found to possess immunosuppressive properties as well as having an ability to inhibit proliferation of mammalian cells [2,3]. Genetic screening in budding yeast first identified TOR (target of rapamycin) 1 and TOR2 as the mediators of rapamycin's inhibitory effects on yeast [4–6]. Later biochemical studies on mammals were able to purify mTOR (originally known as mammalian target of rapamycin, but now officially mechanistic target of rapamycin) and led to its discovery as the physical target of rapamycin [7–9]. mTOR plays a critical role in regulating cell metabolism, proliferation and survival in response to environmental cues [2,10–12]. Evidence linking deregulation of mTOR signalling to cancer has prompted significant interest in developing anti-cancer therapies that target mTOR, namely rapamycin and its analogues (rapalogues), including everolimus. Currently, everolimus is the only mTOR inhibitor approved by the U.S. FDA (Food and Drug Administration) for the treatment of breast cancer, papillary renal carcinoma, pancreatic neuroendocrine tumour, some types of breast cancer and subependymal giant-cell astrocytoma associated with tuberous sclerosis [13–17]. In the present review, we summarize our current understanding of the cellular and molecular effects of the mTOR inhibitor everolimus.
mTOR is a 289 kDa conserved serine/threonine protein kinase that belongs to the PI3K (phosphoinositide 3-kinase)-related protein kinase family (Figure 1). Structurally, the ~550-residue mTOR kinase domain forms a two-lobe structure consisting of an N-lobe (N-terminal lobe) and a larger C-lobe (C-terminal lobe). The two lobes are linked to one another by a cleft present between them, which contains a binding site for ATP. The mTOR kinase domain structure starts with a long kα1 helix (‘k’ denoting kinase domain), which is integral to the N lobe as it packs in the concave surface of the N-lobe β-sheet . The ~100-residue FRB [FKBP12 (FK506-binding protein 12)–rapamycin-binding] domain (containing 2021–2118 residues) is located immediately after the kα1 helix. This is followed by a β-strand and two short helices that pack with the base of the FRB. The C-domain, apart from containing a ~40-residue insertion that forms the binding site [LBE (mLST8-binding element)] for mLST8 (mammalian lethal with SEC-13 protein 8), also contains four structural insertions in the PI3K core structure [LBE, kαAL, kα9b and FATC, where FATC is C-terminal of FRAP (FKBP12–rapamycin-associated protein)–ATM (ataxia telangiectasia mutated)–TTRAP (transactivation/transformation-domain-associated protein) complex]. These four structures present in the vicinity of the catalytic cleft, form a spine of interactions centred on a ~30-residue activation loop. This activation loop is well ordered, with one side packed with the kα9 insertion (residues 2425–2436), the middle portion containing the kαAL helix insertion and the other side packed with the FATC domain. Whereas the four-helix FRB domain extends the N-lobe side of the catalytic cleft, the LBE and mLST8 extend the C-lobe side, giving the cleft a deep ‘V’ shape and thereby restricting access to the substrate binding site at the bottom of the cleft. This structural arrangement in turn causes the active site of the mTOR complex to be highly recessed, indicative that this kinase is controlled via restrictive access . The same authors, by superimposing the FRB domain of FKBP12–rapamycin and their mTOR crystalline structure, demonstrated that the FKBP12 extension from the FRB to the mLST8 on the C-lobe nearly caps the entire catalytic cleft; this suggests that the inhibitory effects of FKBP12–rapamycin occurs in part by drastically reducing the accessibility of the already constricted catalytic cleft. This is consistent with the evidence that mTORC (mTOR complex) 1 inhibition is substrate- and phosphorylation-site-dependent .
Structure and conformation of mTOR
mTOR EXISTS IN COMPLEXES
mTOR interacts with several proteins to form two structurally large and functionally distinct complexes: mTORC1 and mTORC2 (Figure 2). Both mTOR complexes share the catalytic mTOR subunit deptor (DEP domain-containing mTOR-interacting protein), mLST8, and the Tti1 (Tel2-interacting protein 1)–Tel2 complex [21–24] (Figure 2a). However, each complex has its own specific components . Both raptor (regulatory-associated protein of mTOR) and PRAS40 (proline-rich Akt substrate of 40 kDa) are two components that are specific to mTORC1. Raptor has many functions, which include regulating mTORC1 assembly, recruiting kinase substrates such as 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] [25,26], determining the subcellular localization of mTORC1 and in sensing amino acids . The role of the mTORC1 core component mLST8 remains unclear as its deletion has been shown to have no effect on either in vivo or in vitro mTORC1 activity . PRAS40 and deptor act as components as well as substrates of mTORC1, and appear to repress mTORC1 activity when dephosphorylated. When active, the mTOR component of mTORC1 phosphorylates both PRAS40 and deptor, weakening their association with the remainder of mTORC1 and thereby promoting the kinase activity of the complex [23,29–33] (Figure 2b).
mTOR complexes: mTORC1 and mTORC2
Rictor (rapamycin-insensitive companion of mTOR) is the main component of mTORC2 and is mutually exclusive with raptor for binding mTOR. Other unique components of mTORC2 include mSin1 [mammalian stress-activated MAPK (mitogen-activated protein kinase)-interacting protein 1] and protor (protein observed with rictor)-1/2. Rictor is essential for the catalytic activity of mTORC2  and, together with mSin1, stabilizes and may provide the structural integrity to mTORC2 [35,36]. Whereas protor-1 binds rictor, it is not required for mTORC2 catalytic function [31,37]. Unlike with mTORC1, mLST8 is essential for mTORC2 functioning in vivo and for its kinase activity in vitro  (Figure 2b).
The mTOR signalling pathway serves as a central regulator of cell metabolism, growth, proliferation and survival (Figure 3).
mTOR signalling pathway
Most of the knowledge regarding mTORC1 functions stems from the discovery and use of rapamycin. mTORC1 is the better characterized of the two mTOR complexes, and apart from being acutely sensitive to rapamycin, responds to amino acids, stress, oxygen, energy and growth factors. Yip et al.  revealed the 3D structure of mTORC1as an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. The main upstream regulators of mTORC1 are the heterodimers TSC1/2 (tuberous sclerosis complex 1/2), which also function as a GAP (GTPase-activating protein) for the Rheb (Ras homologue enriched in brain) GTPase. GTP-bound Rheb interacts directly with mTORC1, which strongly stimulates its kinase activity. TSC1/2 as a Rheb GAP negatively regulates mTORC1 by converting Rheb into its inactive GDP-bound state [39,40]. In response to growth factors, effector kinases including Akt/PKB (protein kinase B), ERK (extracellular-signal-regulated kinase) 1/2 and RSK1 (ribosomal S6 kinase) phosphorylate the TSC1/2 complex, thereby inactivating it and activating mTORC1 [41–45]. These kinases phosphorylate and inhibit TSC2 by binding 14-3-3 protein directly to TSC2 phosphoserine residues . Apart from growth factors, other stresses including low energy, low oxygen levels and DNA damage also mediate mTORC1 activity at least in part via TSC1/2 . Hypoxia or low energy status also induces AMPK (AMP-activated protein kinase) expression which in turn phosphorylates TSC2 and increases its GAP activity towards Rheb . AMPK can also directly target mTORC1, by phosphorylating raptor and promoting 14-3-3 protein binding, leading to allosteric inhibition of mTORC1 . Hypoxia also affects mTORC1 in an AMPK-independent manner by inducing REDD1 (regulated in development and DNA damage response 1), also known as DDIT4 (DNA damage-inducible transcript 4), which suppresses mTORC1 by releasing TSC2 from the association with inhibitory 14-3-3 proteins [48–50]. DNA damage inhibits mTORC1 activity in a p53-dependent manner by up-regulating the activation of p53 targets genes including REDD1, AMPK, PTEN (phosphatase and tensin homologue deleted on chromosome 10), IGF-BP3 (insulin-like growth factor-binding protein 3), TSC2, and sestrins 1 and 2 [51–55]. Activation of IGF-BP3 prevents the binding of IGF-1 (insulin-like growth factor 1) to its receptors, which shuts down IGF-1/Akt signalling. Similarly PTEN, a PIP3 (phosphatidylinositol 3,4,5-trisphosphate) phosphatase, results in the degradation of PIP3 to PIP2 (phosphatidylinositol 4,5-bisphosphate), and prevents the subsequent activation of PDK1 (pyruvate dehydrogenase kinase isoenzyme 1), mTORC2 and Akt. These two events in turn lead to the suppression of mTORC1 activity through the direct phosphorylation of TCS2 by Akt . p53 also promotes the interaction between AMPK subunits and sestrins 1 and 2, resulting in the activation of AMPK and subsequent inhibition of mTORC1 activity [54–57]. The AMPK metabolic pathway is also activated following DNA damage, by the ATM protein kinases, which leads to suppression of mTORC1 pathway activity .
mTORC1 controls and mediates major processes including cell growth, proliferation, autophagy, and protein and lipid synthesis. It controls protein synthesis by phosphorylation of 4E-BP1 and S6K1 (p70 ribosomal S6 kinase 1). Phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E to promote cap-dependent translation. mTORC1 stimulation of S6K1 activity results in increased mRNA biogenesis, cap-dependent translation and elongation, and the translation of ribosomal proteins through regulation of the activity of many proteins, such as SKAR (S6K1 Aly/REF-like target), PDCD4 (programmed cell death 4), eEF2K (eukaryotic elongation factor 2 kinase) and ribosomal protein S6 (reviewed in ). mTORC1 positively regulates various transcriptional factors including SREBP1/2 (sterol-regulatory-element-binding protein 1/2), PPARγ (peroxisome-proliferator-activated receptor γ), Myc and HIF-1α (hypoxia-inducible factor 1α) [60–64], which in turn control the expression of genes encoding proteins involved in glycolysis, and lipid and cholesterol homoeostasis. mTORC1 regulation of SREBP function occurs via several mechanisms and involves S6K1 [62,65,66]. Inhibition of mTORC1 subsequently decreases SREBP1/2 expression, consequently impairing their processing and lowering the expression of lipogenic genes [60,62,65,66].
mTORC1 increases mitochondrial DNA content and the expression of genes involved in oxidative metabolism, in part by mediating the nuclear association between PGC1α (PPARγ co-activator 1α) and the transcription factor Yin–Yang 1, which positively regulates mitochondrial biogenesis and oxidative function . Csibi et al.  demonstrated that the mTORC1 pathway regulates glutamine metabolism by promoting GDH (glutamate dehydrogenase) activity in an mTORC1-dependent proteasome-mediated degradation of CREB2 (cAMP-response-element-binding protein 2), the transcriptional regulator of the GDH suppressor sirtuin 4. mTORC1 enhances glycolytic flux by activating the transcription and translation of HIF-1α, which is a positive regulator of many glycolytic genes [62,64]. Recently, Dodd et al.  revealed that mTORC1 drives HIF-1α synthesis in a multifaceted manner involving 4E-BP1/eIF4E, S6K1 and STAT3 (signal transducer and activation of transcription 3). Furthermore, they showed that STAT3 was both a direct substrate of mTORC1 and also essential for the latter's regulation of HIF-1α transcription. STAT3, like HIF-1α, is well known to mediate several oncogenic cellular processes including cell survival, proliferation and metastasis [69,70]. Dodd et al.  also demonstrated that inhibition of 4E-BP1, but not S6K1, dramatically decreased VEGF (vascular endothelial growth factor)-A levels, indicating that mTORC1/4E-BP1 signalling drives VEGF-A expression via both HIF-1α-dependent and -independent mechanisms.
mTORC1 also promotes growth by negatively regulating autophagy, the central degradative process in cells. In mammals, mTORC1 directly phosphorylates and suppresses unc-51-like kinase 1 (ULK1)/mammalian autophagy-related gene 13/focal adhesion kinase family-interacting protein of 200 kDa, a kinase complex required to initiate autophagy [71–73]. AMPK activity has been shown to be important for ULK1-mediated autophagy, by recruiting 14-3-3 to raptor and thereby inhibiting mTORC1 activity . Alternatively, other studies demonstrated that ULK1 negatively regulates S6K1 [75,76]. This was corroborated by Dunlop et al. , who demonstrated that ULK1 directly phosphorylates and inhibits substrate binding to raptor, thereby preventing optimal phosphorylation of mTORC1 downstream substrates 4E-BP1 and S6K1. When mTORC1 is inhibited, autophagosomes form, which then engulf cytoplasmic proteins and organelles, and fuse with lysosomes, leading to the degradation of cell components and the recycling of cellular building blocks. The degradation of macromolecules and the release of their constituents into the cytosol can in turn reactivate mTORC1, which terminates autophagy . Activation of mTORC1 requires its translocation to the surface of lysosomes, which is facilitated by Rag GTPases where it interacts with Ragulator (a protein complex at the lysosome) [27,79,80]. Apart from serving as a docking station for the mTORC1, the lysosomal V-ATPase (vacuolar ATPase) complex is also essential for sensing amino acids inside the lysosomal lumen, which is required for mTORC1 activation . Recent studies have demonstrated that lysosomal reformation in response to mTORC1-dependent autophagy is mediated via TFEB (transcription factor EB) [82,83]. Abundance of nuclear TFEB has been previously shown to regulate genes encoding proteins important for autophagy [83–85]. mTORC1 facilitates the Ser211 phosphorylation-dependent binding of 14-3-3 proteins to TFEB in the cytoplasm. Inactivation of mTORC1 results in TFEB's Ser211 dephosphorylation, subsequent loss of 14-3-3 interaction and localization of TFEB into the nucleus , indicative of a negative-feedback loop linking mTORC1, the lysosome and TFEB.
In comparison with mTORC1, knowledge regarding mTORC2 is still limited. Although mTORC2 signalling is insensitive to nutrients, it responds to growth factors such as insulin via PI3K signalling. However, these mechanisms are still poorly defined. Zinzalla et al.  demonstrated one possible mechanism that involves a new role for ribosomes, where they reported that mTORC2 binds ribosomes that are essential for its activation in a PI3K-dependent manner. mTORC2 also controls several members of the AGC subfamily of kinases including Akt, SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKCα (protein kinase Cα). mTORC2 activates Akt directly by phosphorylating its hydrophobic motif (Ser473), a site required for its maximum activation . Akt in turn regulates numerous cellular processes including metabolism, survival, apoptosis, growth and proliferation via phosphorylation of other effectors. Defective Akt Ser473 phosphorylation has been associated with mTORC2 depletion and subsequent impairment of Akt targets such as FOXO1/3a (forkhead box O1/3a). However, other Akt targets, including TSC2 and GSK3β (glycogen synthase kinase 3β) have been shown to remain unaffected [28,36]. Interestingly, these results can be explained by the fact that Akt activity is not completely lost in cells that lack mTORC2. mTORC2 also directly activates SGK1, a kinase that controls ion transport and growth . In contrast with Akt, SGK1 activity is completely blocked by the loss of mTORC2. As SGK1 phosphorylates FOXO1/3a at the same residues as Akt, it is probable that the decrease in phosphorylated FOXO1/3a in mTORC2-depleted cells stems more from the corresponding decrease of SGK1 activity than of Akt. Along with other effectors, including paxilin and Rho GTPases, mTORC2 also regulates actin cytoskeletal organization by promoting phosphorylation of PKCα [21,34]. However, these molecular mechanisms are yet to be completely elucidated.
mTOR INHIBITOR: EVEROLIMUS
Rapamycin and its analogues (allosteric irreversible inhibitors of raptor-bound mTOR) have been approved by the FDA for many years as immunosuppressants to prevent organ transplant rejections. Observations of their anti-proliferative properties, as well as the growing awareness of the crucial role of mTORC1 in cellular homoeostasis, prompted the development of these agents as anti-cancer therapies that target the oncogenic PI3K/mTOR signalling pathway [89–91]. Currently, the FDA has approved the rapamycin analogues temsirolimus (CCI-779) and everolimus (RAD001, Afinitor) for the treatment of advanced-stage RCC (renal cell carcinoma) and sarcoma respectively [92–94]. However, the efficacy of rapamycin and rapalogues as broad-based monotherapies for cancer appears more limited than initially expected [92,95–97].
Everolimus, an orally administrated inhibitor of mTOR was initially developed as an immunosuppressant for solid-organ transplantation [98,99]. Everolimus blocks the cytokine-driven activation responses of T- and B-cells, preventing their proliferation and differentiation [100,101]. Pre-clinical studies have demonstrated the anti-proliferative effect of everolimus in cancer cell lines and in animal models [102–105]. This led to its development and subsequent approval by the FDA for the treatment of various cancers [106–108]. Its success in the treatment of these cancer types led to the evaluation of its effects on other cancers known to deregulate the PI3K/Akt/mTOR pathway. Pre-clinical studies demonstrated that inhibition of mTOR, which is well known to be up-regulated in a large proportion of HCC (hepatocellular carcinoma) cells, by rapamycin and everolimus yielded a positive outcome [109–113]. Subsequent Phase I/II studies also found that administration of everolimus to patients with advanced HCC was well tolerated and delayed disease progression . Furthermore, Kneteman et al.  showed that median progression-free survival was 3.8 months, suggestive of a modest anti-tumour effect of everolimus. However, the follow-up global Phase III randomized EVOLVE-1 trial utilizing everolimus as a second-line therapy failed to improve the overall survival of patients with advanced HCC for whom sorafenib was discontinued owing to disease progression or drug intolerance .
Everolimus, like rapamycin, acts through allosteric binding to its intracellular receptor FKBP12 which lies adjacent to the catalytic site of mTORC1 [117–119] (Figure 4). This binding weakens the interaction between mTORC1 and raptor, subsequently inhibiting downstream functions including cell metabolism, growth and proliferation [26,120,121]. Interestingly, Yip et al.  revealed that, in contrast with previous assumptions, raptor is dispensable for mTORC1 to phosphorylate S6K1 in a rapamycin-sensitive fashion in vitro. Various studies have reported that everolimus treatment significantly decreased cell proliferation in a dose- and time-dependent manner, and this effect was correlated with inactivation of S6K1 and decreased phosphorylation of 4E-BP1 [105,122,123]. Although mTORC2 was initially thought to be resistant to rapamycin, Shor et al.  demonstrated that high concentrations of rapamycin and its derivatives could inhibit mTOR directly through an FKBP12-independent mechanism, suppressing both mTORC1 and mTORC2. Sarbassov et al.  proposed that prolonged rapamycin treatment may interfere with the de novo assembly of mTORC2. They proposed that the binding of rapamycin/FKBP12 to mTOR impedes the subsequent binding of the mTORC2-specific components mSin1 and rictor, which are required for its downstream signalling. This was further corroborated by Rosner and Hengstschlager  who observed that rapamycin treatment led to the dephosphorylation and subcellular relocalization of mSin1 and rictor in non-immortalized human diploid fibroblasts and NIH 3T3 cells. However, these effects are cell-type-specific, and the factors that render mTORC2 sensitive to rapamycin still need to be determined [34,87].
Everolimus inhibitory action on the mTOR signalling pathway
CELLULAR EFFECTS OF EVEROLIMUS
Everolimus and other rapalogues were initially developed to function like rapamycin in supressing cell growth and proliferation (via inhibition of mTORC1), but possessing improved pharmacokinetic properties. Effects of everolimus reported include inhibition of cell proliferation, migration, invasion and angiogenesis in some human tumours, as well as promoting apoptosis in some tumour cell lines (Figure 4).
Some of the mechanisms responsible for this apoptotic effect have been already characterized and include mTOR targets such as Myc . Mabuchi et al.  demonstrated that everolimus attenuated the expression of Myc as well as the phosphorylation of both 4E-BP1 and S6K1, resulting in significant reduction of proliferation of ovarian cancer cell lines in vitro. Costa et al.  observed that stimulating bone-marrow-derived endothelial cells with growth factors or associating them with leukaemia cells resulted in the activation of mTOR and phosphorylation of its substrates 4E-BP1 and S6K1. Subsequent addition of rapamycin significantly inhibited cell proliferation by causing G0/G1 cell cycle arrest via the down-regulation of cyclin D1 and up-regulation of CDK (cyclin-dependent kinase) inhibitors p27kip1 and p21cip1. The accumulation of cells in G1-phase following rapamycin treatment is consistent with the fact that the drug inhibits ribosome biogenesis and global translation, in part by blocking the phosphorylation of S6K1 and 4E-BP1 . In contrast, Juengel et al.  reported that everolimus resistance in vitro was characterized by elevated expression of Akt and S6K1, as well as cell-cycle-activating proteins CDK2 and cyclin A, which allowed for the subsequent increased number of G2/M-phase cells. In a follow-up study, they reported that everolimus evoked a strong response in sunitinib-resistant RCC cells by inducing cell growth delay in G0/G1 and diminishing expression levels of p-Akt, p-raptor and p-rictor . Everolimus also counteracted chronic sunitinib-induced CDK1 and CDK2 up-regulation. This study was corroborated by Larkin et al.  who showed that sequential therapy with sunitinib, followed by everolimus in their mice model resulted in significant reduction of primary tumour and metastatic burden. As an activating component of mTORC1, Akt contributes to its downstream events such as GTP cap-dependent regulation or IRES (internal ribosome entry site)-dependent translation of Myc [133–135]. Akt is also known to phosphorylate pro-apoptotic proteins such as BAD (Bcl-2-associated death promoter), GSK3 and the FOXO family [136–138]. For example, phosphorylation of BAD by Akt allows for its sequestration by 14-3-3 protein and blocking of its apoptotic function . Tsaur et al.  observed increased chemotactic activity of prostate cancer cells resistant to everolimus, a process that was characterized by significant alterations of integrin α2, α5 and α1 expression profiles as well as the reactivation of Akt, whereas treatment of sensitive prostate cancer cells with everolimus resulted in decreased tumour cell chemotaxis, migration and invasion. Similarly, Ierano et al.  demonstrated that the effect of CXCR (CXC chemokine receptor) 4, CXCR7 and mTOR inhibitors was additive in impairing migration and cell growth. Additionally, they observed that everolimus inhibited CXCL12 (CXC chemokine ligand 12)-induced migration, wound healing and cell growth. Wedel et al.  observed that everolimus inhibited prostate cancer cell adhesion to the vascular endothelium and extracellular matrix proteins, thereby diminishing their migratory potential. However, the same study showed that everolimus also increased membranous β3 integrin expression levels. Given that β3 integrins induce the metastatic spread of prostate carcinoma to the bone [143,144], everolimus up-regulation of β3 could reflect an undesired feedback loop that counteracts the adhesion-diminishing properties of this drug. As mentioned earlier, prolonged rapamycin treatment has been shown to inhibit p-Akt (Ser473) in some cells , whereas reducing phosphatidic acid levels in renal 786-O cells conferred on them sensitivity to p-Akt (Ser473) suppression by rapamycin . Jin et al.  demonstrated that everolimus when compared with rapamycin was more effective in inhibiting endothelial functional changes. Additionally, they showed that everolimus effectively blocked HLA I-stimulated cell proliferation by inhibiting mTORC1 and ERK1/2 signal transduction pathways. HLA I has been previous shown to promote cell proliferation and protein synthesis via activation of mTORC1 downstream target S6K1 [148,149].
Interestingly, although Yan et al.  observed that in vitro and in vivo rapamycin administrations sensitized multiple myeloma cell lines to subsequent dexamethasone treatment by dramatically increasing the degree of apoptosis, further analysis revealed that this increased sensitivity of the cells was induced independently of the activity of BAD phosphorylation, stress pathways, PTEN or p53 status. Instead, this sensitivity was attributed to the overexpression of constitutively active mutants of 4E-BP1 that could not be phosphorylated by mTORC1, indicating that inhibition of mTORC1 translation was solely responsible for this anti-apoptotic effect. This study corroborated findings of an earlier study that had reported that tumour cell lines overexpressing eIF4E were able to partially overcome rapamycin-induced effects . However, it remains unclear as to why rapamycin causes complete deactivation of S6K1, yet only exerts a moderate effect on 4E-BP1 phosphorylation . One explanation could be that mTORC1 binds to 4E-BPs and S6K1s with different affinities or conformations; therefore, although the rapamycin-mediated structural changes in mTORC1 are sufficient to disrupt its association with S6Ks, they are not enough to do the same with 4E-BPs [20,153]. 4E-BP1 binding and inhibition of eIF-4E primarily occurs via phosphorylation of Thr37, Thr46, Thr70 and Ser65 . Whereas mTORC1 phosphorylates the rapamycin-insensitive sites Thr37/Thr46, it has little effect on the rapamycin-sensitive sites Ser65 and Thr70in vitro. Thoreen et al.  demonstrated that whereas rapamycin completely prevented S6K1 phosphorylation and slightly decreased phosphorylation of Ser65, it had little effect on phosphorylation of either Thr37/Thr46 or Thr70. Furthermore, Choo and Blenis  observed that the 4E-BP1 phosphorylation sites that were acutely sensitive to rapamycin became re-phosphorylated in some cell lines after long periods of rapamycin treatment. As the recovery of 4E-BP1 phosphorylation depends on the mTORC1 component raptor, Thoreen et al.  suggested that prolonged rapamycin treatment confers on mTORC1 the capacity to phosphorylate 4E-BP1 in a rapamycin-resistant fashion. An alternative explanation is that prolonged rapamycin treatment is known to hyperactivate the PI3K pathway, which is upstream of mTORC1, hence prolonged treatment leads to hyperactivation of the rapamycin-resistant functionality of mTORC1, effectively overcoming the partial inhibition caused by rapamycin [20,155] (summarized in Table 1).
|Target molecules .||.|
|Down-regulation .||Up-regulation .||Cellular effect .|
|Myc||CDK inhibitors p27kip1 and p21cip1||Decreased cell proliferation|
|Cyclins A and D1||GSK3||G0/G-1 cell cycle arrest|
|4E-BP1 and S6K||Autophagy proteins LC3 and Beclin-1|
|PPARα||Sensitizes cells to radiation therapy|
|PGC1α||Sensitizes cells to radiation therapy|
|Target molecules .||.|
|Down-regulation .||Up-regulation .||Cellular effect .|
|Myc||CDK inhibitors p27kip1 and p21cip1||Decreased cell proliferation|
|Cyclins A and D1||GSK3||G0/G-1 cell cycle arrest|
|4E-BP1 and S6K||Autophagy proteins LC3 and Beclin-1|
|PPARα||Sensitizes cells to radiation therapy|
|PGC1α||Sensitizes cells to radiation therapy|
Decreased autophagy appears to be another common hallmark of tumour cells. In yeast, rapamycin is a potent activator of autophagy ; however, in mammalian cells, rapamycin has been shown to be an inconsistent activator of autophagy and requires combination with other PI3K/mTOR inhibitors (such as LY294002) or with concomitant starvation of nutrients . Previous studies have shown that everolimus treatment enhanced radiation-induced apoptosis via activation of autophagy [157,158]. Yu et al.  observed that inhibition of mTOR by everolimus resulted in increased levels of the autophagy-associated protein LC3 (light chain 3). Additionally, other studies demonstrated that suppression of mTORC1 by rapamycin prevented autophagic lysosome reformation . A study by Pattingre et al.  demonstrated a relationship between autophagy and apoptosis, by suggesting that the anti-apoptotic protein Bcl-2 could function as a negative regulator of Beclin-1, an essential autophagy gene. Previously, Majumder et al.  reported that the overexpression of Bcl-2 in prostatic intraepithelial neoplastic cells was able to block the apoptotic effects, but not the inhibition of proliferation by everolimus. Taken together, these studies raise the question of whether the inhibition of Beclin-1 by Bcl-2 and the consequent inhibition of autophagy could be the contributing factor to inhibition of everolimus-mediated apoptosis. However, it is also plausible that the overexpression of Bcl-2 counteracts the activation of any pro-apoptotic proteins that might interact with Bcl-2, thereby promoting survival.
Blocking mTORC1 with rapamycin also reduces the expression and the transactivation activity of PPARγ . Additionally, rapamycin reduces the phosphorylation of lipin-1 , a phosphatidic acid phosphatase that is involved in glycero-lipid synthesis and in the co-activation of many transcription factors linked to lipid metabolism, including PPARγ, PPARα and PGC1α. The precise impact of lipin-1 phosphorylation on lipid synthesis remains to be established. Apart from exerting an anti-cancer effect, everolimus has also been reported to inhibit angiogenesis by decreasing tumour HIF-1α activity, VEGF production and VEGF-induced proliferation of endothelial cells . Given that rapamycin incompletely suppresses eIF4E , Dodd et al.  recently demonstrated that mTORC1 regulates VEGF-A expression via 4E-BP1. This could explain why in TSC2+/− mice, VEGF-A expression was less sensitive to rapamycin inhibition than both HIF-1α and pS6K1. These studies highlight the need to target eIF4E in combination with rapamycin to more effectively reduce VEGF-A expression when treating vascularized tumours.
MOLECULAR EFFECTS OF EVEROLIMUS
As mentioned above, the inhibitory effect of rapamycin and rapalogues occurs via a two-step process that involves the allosteric binding of everolimus to mTORC1 (Figure 4). Yip et al.  demonstrated that extended incubation with FKBP12–rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leaving no detectable intact mTORC1 particles and only small fragments (suggested to be representing free mTOR or its subcomplexes). They showed further that, whereas the FKBP12–rapamycin effect on the structural integrity of mTORC1 rapidly blocked S6K1 phosphorylation, inhibition of 4E-BP1 phosphorylation by mTORC1 occurred only after extended incubation (60 min) with the drug. The specificity of these drugs for their mTORC1 target suggests that these drugs seem to perform a surgical ‘hit’ on mTORC1 signalling in the cancer cells. However, it is now widely recognized that rapalogue inhibition of mTORC1 perturbs a dynamic signalling transduction network, with a variable response profile in different cell types . In general, these unanticipated effects reflect the operation of homoeostatic mechanisms that strive to return signal flow through mTOR to its normal pre-drug level. Although rapamycin and rapalogues have shown clinical efficacy in a subset of clinical tumours, their use as broad-based monotherapies is limited due to development of resistance. Resistance can occur due to the presence of KRAS or BRAF mutations, loss of PTEN, low cellular levels of p27 or 4E-BP1 and overexpression of eIF4E, mTORC2, activation of feedback loops promoting survival pathways including PI3K/Akt, ERK/MAPK, PIM kinases and PDK .
Rapamycin-mediated up-regulation of PI3K/Akt phosphorylation results from the loss of the negative-feedback loop from S6K1 to IRS-1 (insulin receptor substrate 1) [92,95,166,167]. In normal settings, when activated, mTORC1 stimulates S6K1 activity, which phosphorylates inhibitory sites (i.e. Ser636/Ser639) on IRS-1, thereby suppressing IRS-1-mediated activation of the PI3K/Akt pathway . Conversely, rapamycin-mediated inhibition of mTORC1 disrupts this negative feedback to IRS-1 [168,169] and/or up-regulation of receptor tyrosine kinases (or its substrates) such as PDGFR (platelet-derived growth factor receptor) [170,171], leading to increased Akt activity. Complete activation of Akt requires its phosphorylation at two sites, Ser473 and Thr308, which lie within its activation loop . Studies have shown that inhibition of mTORC1 induces phosphorylation of Akt at Ser473 in various cancer cell lines and patient tumours [173,174]. Recent studies have implicated mTORC2 as playing a role in stimulating Akt activity by phosphorylating it at its Ser473 site [87,175]. mTORC1 is located downstream of Akt; however, mTORC2 is located upstream of the latter, hence it has been postulated that long-term application of everolimus could induce a feedback activation of Akt via mTORC2 signalling . Taken together, these findings indicate that, at the molecular level, rapamycin treatment leads to hyperactivation of Akt through loss of the mTORC1/S6K1/IRS-1/PI3K negative-feedback loop, which in some types of cancer is reinforced by the inability of rapamycin to efficiently suppress mTORC2 signalling towards Akt. Interestingly, phosphorylation of Akt has been reported to be increased in tumours derived from patients treated with everolimus . Alternatively, Carracedo et al.  demonstrated that everolimus inhibition of mTORC1 resulted in the activation of the Ras and subsequent ERK cascade. Furthermore, they showed that this activation was mediated via the S6K1/PI3K/Ras pathway, and was independent of the up-regulation of mTORC2, Akt or its downstream components. This is supported further by studies that have reported the involvement of MEK1/2 (MAPK/ERK kinase 1/2), ERK1/2 and mTOR pathways in a mutual negative-feedback loop mediated via S6K1, in which each pathway can inhibit or activate the other, affecting cellular proliferation or differentiation . Furthermore, Friedman et al.  demonstrated that utilizing other agents such as PI3K (LY294002) and MEK1/2 (U0126) inhibitors in combination with inhibitors of mTOR (rapamycin) had a synergistic effect in preventing cell migration of glioblastoma cells. This echoes a previous study by Bresccia et al.  who reported that dual inhibition with PI3K and MAPK inhibitors was superior in efficacy to inhibition of a single pathway alone. Recently, Ierano et al.  described a novel method to resensitize RCC to everolimus treatment via inhibition of the CXCR4–CXCL12–CXCR7 axis. The cross-talk between CXCR4–CXCL12 and PI3K/mTOR has been well established in various cancer cells [26,27,29].
Various studies have reported that tumours exhibiting a dependency on the mTOR pathway demonstrated an enhanced sensitivity to mTOR inhibition with drugs such as everolimus. This dependency may occur through genomic alterations involving the mTOR pathway. For example, both TSC and Peutz–Jeghers syndromes that present inactivating mutations in the tumour-suppressor genes TSC1, TSC2 and STK11 (LKB1) result in mTOR pathway activation and are targetable by TOR inhibitors [180–182]. Iyer et al.  reported a complete remission from chemotherapy-refractory urothelial carcinoma following everolimus treatment in one patient having somatic TSC1 mutation. Additionally, other patients with TSC1 mutations also demonstrated decreased tumour size following everolimus treatment. Similarly, Wagner et al.  observed a significant clinical response to sirolimus by patients who lacked expression of TSC2. Together, these studies suggest that TSC1/2 loss may predict sensitivity to mTOR inhibition. Ohne et al.  reported that two hyperactivating FRB mutations, I2017T and A2020V, enhanced kinase activity and caused hyperactivation of the mTOR pathway. Additionally, mutant mTOR with both E2419K and I2017T exhibited higher activity of mTOR when compared with individual mutation. Recently, Wagle et al.  reported that activating mTOR mutations, such as mTORE2419K and mTORE2014K, could confer clinically significant sensitivity to mTOR inhibition. The mTORE2014K mutation occurs in the FKBP–rapamycin-binding domain of mTOR and is thought to be present in urothelial cancer cell lines .
Alternatively, pre-clinical studies have demonstrated that mutational activation of the PI3K pathway through loss of PTEN or activation of the serine/threonine kinase Akt sensitized tumour cells to the anti-proliferative activity of mTOR inhibitors [161,188–193]. A Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma whose tumours lacked PTEN expression revealed that rapamycin sufficiently inhibited mTORC1 in tumour cells and decreased tumour cell proliferation. However, rapamycin treatment also induced Akt activation in some patients, presumably due to loss of negative feedback, and this activation was associated with shorter time to progression during post-surgical maintenance rapamycin therapy . Furthermore, inactivation of PTEN in advanced tumours has been speculated to increase variations in mTOR inhibitor sensitivity as it gives the cancer cells the opportunity to shape their oncogenic signalling networks around pathways other than the PI3K/Akt/mTOR cascade . mTORC2 has been shown to play a role in PTEN-dependent tumorigenesis. Guertin and Sabatini  reported that mTORC2 signalling is necessary for the development of prostate cancer caused by PTEN deletion. Intriguingly, mTORC2 activity was dispensable in the normal function of prostate epithelial cells. Other mutations, such as BRAF, have been shown to block LBK1/AMPK-driven TSC1/2 activation resulting in constitutive activation of mTORC1 [195,196]. In accordance, Di Nicolantonio et al.  showed that either PI3K activation or PTEN loss predicted enhanced susceptibility to everolimus in cancer cell lines, but this was abrogated if activating KRAS or BRAF mutations were also present. This was validated further in a small cohort of patients with advanced solid tumours that had both PTEN loss and KRAS activation, resulting in minimal benefit from everolimus treatment.
METABOLIC SIDE EFFECTS OF mTOR INHIBITION WITH EVEROLIMUS
Although everolimus and other mTOR inhibitors are generally well tolerated, they produce side effects that include rashes, stomatitis and metabolic effects (glucose and lipid metabolism). Occurrence of rashes and stomatitis are fairly common, with the latter occurring concurrently with oral Candida and herpes simplex infections . Insulin plays essential roles in regulating metabolism, clearance and storage of glucose and lipids. Furthermore, the insulin signalling pathway is closely associated with the PI3K/Akt/mTOR pathway . Di Paolo et al.  also showed that renal transplant recipients treated with chronic rapamycin had decreased basal and insulin-stimulated Akt phosphorylation, which corresponded to their increased insulin resistance. Additionally, rapamycin has been suggested to also interfere with insulin signalling both upstream and downstream of Akt via other mechanisms . mTOR inhibitors have been known to impair insulin metabolism, increase insulin resistance and reduce β-cell function, leading to the development of hyperglycaemia [163,199,200]. Patients with underlying diabetes should be carefully controlled for baseline blood glucose levels before starting treatment, as everolimus could induce hyperglycaemia in patients with elevated baseline levels . In general, plasma blood glucose levels should be monitored in all patients being treated with everolimus . Another side effect of mTOR inhibitors involves their interference with lipid metabolism. Rapamycin has been reported to increase the total non-esterified (‘free’) fatty acid pool, cholesterol, low-density lipoprotein cholesterol and triacylglycerol levels of patients who had undergone renal transplantation . This has been suggested to occur because of impaired lipid clearance and hyperlipidaemia, possibly caused by inhibition of insulin-stimulated LPL (lipoprotein lipase) . Insulin, as a lipogenic hormone, plays an important role in regulating LPL activity. LPL mediates the uptake of fatty acids into adipose tissue and muscle by hydrolysing the triacylglycerol component of circulating lipoprotein particles. Hyperglycaemia results in part due to a fasted metabolic state and is characterized by decreased utilization of glucose and increased use of fatty acids as metabolic fuel. Although metabolic switching between glucose and fatty acids for energy production is a normal physiological response, increased fatty acid oxidation and diminished glucose uptake is characteristic of a fasted metabolic state. Studies have shown that rapamycin seems to induce a fasting metabolic phenotype regardless of the circumstances [202,204]. Accordingly, Sipula et al.  demonstrated a 60% increase in fatty acid oxidation along with increased activation of carnitine palmitoyltransferases (the primary intracellular regulatory enzyme of the fatty acid oxidation pathway). In addition, they also observed a 40% reduction in glucose transport, glycogen synthesis and glycolysis.
FUTURE RESEARCH DIRECTIONS
Although inhibition of the mTOR pathway possesses significant therapeutic value, the recognition that rapamycin and its rapalogues have limited substrate-specific efficacy and activate several negative oncogenic feedback loops has fuelled the development of new strategies to address these limitations. One strategy to overcome these limitations has been to test combinations of rapalogues with other known inhibitory agents. For example, both Juengel et al.  and Larkin et al.  demonstrated the potential of using everolimus as a second-line therapy following failure of first-line sunitinib treatment. Yao et al.  showed improved efficacy of everolimus and octreotide combination to treat neuroendocrine tumours compared with everolimus alone. Similarly, combining exemestane with everolimus has shown promise, as has combining HER2 (human epidermal growth factor receptor 2) blockade by trastuzumab and everolimus in HER2+ breast cancer in early clinical studies . Recently, Ierano et al.  demonstrated that using antagonists (AMD3100 or Peptide R and anti-CXCR7) that block the CXCL12–CXCR4–CXCR7 axis resensitized cancer cells to everolimus therapy. Other strategies include the development of several TOR-KIs (kinase inhibitors) and PI3K/TOR-KIs, which not only target mTORC1 and mTORC2, but can also minimize the feedback activation of PI3K and/or ERK/MAPK signalling. Thoreen et al.  demonstrated the efficacy of Torin1 (a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes), which impaired cell growth and proliferation to a far greater degree than rapamycin. Interestingly, these effects are independent of mTORC2 inhibition and are instead caused by suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. Hsieh et al.  demonstrated that short-term treatment of prostate cancer cells with PP242, an mTOR ATP-site inhibitor, significantly inhibited the activity of the three primary downstream mTOR effectors 4E-BP1, S6K1/2 and Akt, when compared with rapamycin, which only blocked S6K1/2 activity. Similarly, Yu et al. [209,210] demonstrated the efficacy of various ATP-competitive mTOR inhibitors that acutely blocked substrate phosphorylation by mTORC1 and mTORC2. Although these agents have been effective in their blocking of mTOR signalling, it is imperative to investigate further their ability to function synergistically with other targeted therapies in clinical settings. The lysosome has been well established to play a critical role in facilitating amino-acid-mediated mTORC1 signalling. Cancer cells often exhibit increased lysosomal biogenesis, enhanced exocytosis and lysosomal hydrolase activity to enhance their ability to develop multidrug resistance and invasiveness. However, cancer cell lysosomes are more vulnerable to rupture, causing leakage of lysosomal contents and activation of apoptosis [211,212], posing a new option for therapeutic developments. El Gaafary et al.  demonstrated that treatment with both αATA(8,24) (3α-acetyloxy-tir-8,24-dien-21-oic acid) and rapamycin resulted in accumulation of acidic vesicular organelles and inhibition of mTOR/Akt signalling. Interestingly, and in contrast with rapamycin, αATA(8,24) alone was able to destabilize lysosomal and mitochondrial membranes, and induce ROS (reactive oxygen species) production. In another study, Loos et al.  presented a new avenue in controlling mTOR activation by using nanoparticles surface-functionalized with amino or carboxy groups to target lysosome–mTORC1 interactions.
The identification of reliable biomarkers to assist in patient selection and to monitor treatment response is another ongoing strategic effort. The identification and standardization of biomarkers of rapalogue response is tedious and largely unsuccessful owing to the complexity of the PI3K/mTOR signalling network and its multiple feedback loops. However, recent studies have highlighted the potential of a systemic functional analysis of mutations in mTOR and its pathway derivatives that may help to predictive sensitivity to mTOR inhibition. It is important, when identifying potential alterations, to not only characterize their functional significance, but also ensure that the tumours are dependent on these alterations in situ. Additionally, routine screening of cancer patients for mutational alterations could help to better identify patient subsets who may respond well to targeted therapies against mTOR, including everolimus and other rapamycin analogues. Alternatively, identifying patient tumours having mutations, such as KRAS or BRAF, could help to select other therapeutic agents apart from rapalogues to obtain better results. A recent study  reporting that combined rapamycin and selective MEK inhibitor treatment effectively decreased expression of VEGF and MMP-9 (matrix metallopeptidase 9) in tumour tissues harbouring KRAS and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) mutations validates this theory further. Although genomic methods to identify mutations and activators of pathways are promising, they still warrants refinement for clinical application.
The dysregulation of mTOR signalling is implicated in a number of human diseases including cancer. Recent studies have yielded a greater understanding of the complexity of PI3K/mTOR signalling and the potential of mTOR-targeted anti-cancer therapies. Although the clinical efficacy of rapalogues has been modest, particularly in treatment of RCC and TSC-related tumours, their success in other cancer types has been limited. More importantly, their limited actions have prompted a more in-depth analysis of the mechanism underlying their limited clinical impact, particularly their partial inhibition of mTORC1 and activation of feedback loops. These insights in turn have given us valuable information when developing newer agents such as TOR-KIs and PI3K/TOR-KIs, which to date have demonstrated greater potency than the rapalogues. However, it remains to be seen whether their efficacy remains potent when tested in combination with other pathway inhibitors. Another avenue that has seen significant progress in recent years has been the understanding of molecular mechanisms regulating lysosomal functioning. It has been well established that lysosomes play a critical role in amino acid–mTORC1 activation, and therefore targeting lysosomes presents a novel therapeutic option to control mTOR signalling. Preliminary evidence utilizing αATA(8,24) and nanoparticles has been promising and provides a basis for potential future therapeutic development. It also remains to be seen whether these novel anti-mTOR therapeutics present any toxic side effects, have equal success in many cancer types and work in synergy with other drugs. Despite their limitations, recent studies have shown that rapalogues can still be effective when used as second-line treatment, for treatment of genomic screened tumours with TSC1/2 mutations and in combination with other pathway inhibitors.
We thank Dr Laurence Zulianello for preparing the Figures and Dr Simon Rhead for proofreading the paper before submission.
This study was supported by the Swiss Science Foundation [Sinergia, grant number CRSII-3_141798], Oncosuisse [grant number KFS-3506-08-2014], the Swiss Foundation against Liver Cancer and the Sander Foundation.
AMP-activated protein kinase
24), 3α-acetyloxy-tir-8,24-dien-21-oic acid
ataxia telangiectasia mutated
Bcl-2-associated death promoter
CXC chemokine ligand
CXC chemokine receptor
DEP domain-containing mTOR-interacting protein
eIF4E-binding protein 1
eukaryotic initiation factor 4E
C-terminal of FRAP–ATM–TTRAP (transactivation/transformation-domain-associated protein) complex
Food and Drug Administration
FK506-binding protein 12
forkhead box O
glycogen synthase kinase 3
human epidermal growth factor receptor 2
hypoxia-inducible factor 1α
insulin-like growth factor 1
insulin-like growth factor-binding protein 3
insulin receptor substrate 1
mitogen-activated protein kinase
mammalian lethal with SEC-13 protein 8
mammalian stress-activated MAPK-interacting protein 1
mechanistic target of rapamycin
pyruvate dehydrogenase kinase
PPARγ co-activator 1α
protein kinase Cα
proline-rich Akt substrate of 40 kDa
protein observed with rictor
phosphatase and tensin homologue deleted on chromosome 10
regulatory-associated protein of mTOR
renal cell carcinoma
regulated in development and DNA damage response 1
Ras homologue enriched in brain
rapamycin-insensitive companion of mTOR
p70 ribosomal S6 kinase 1
serum- and glucocorticoid-induced protein kinase 1
signal transducer and activation of transcription 3
transcription factor EB
target of rapamycin
tuberous sclerosis complex
Tel2-interacting protein 1
unc-51-like kinase 1
vascular endothelial growth factor