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Biochemical Society Transactions (2013) Volume 41, part 4
Adaptation to chronic mTOR inhibition in cancer
and in aging
Rebecca Gilley, Kathryn Balmanno, Claire L. Cope and Simon J. Cook1
Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.
Abstract
The mTOR [mammalian (or mechanistic) target of rapamycin] protein kinase co-ordinates catabolic and
anabolic processes in response to growth factors and nutrients and is a validated anticancer drug target.
Rapamycin and related allosteric inhibitors of mTORC1 (mTOR complex 1) have had some success in specific
tumour types, but have not exhibited broad anticancer activity, prompting the development of new ATPcompetitive mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2. In common with other targeted
kinase inhibitors, tumours are likely to adapt and acquire resistance to mTOR inhibitors. In the present article,
we review studies that describe how tumour cells adapt to become resistant to mTOR inhibitors. mTOR is a
central signalling hub which responds to an array of signalling inputs and activates a range of downstream
effector pathways. Understanding how this signalling network is remodelled and which pathways are
invoked to sustain survival and proliferation in the presence of mTOR inhibitors can provide new insights
into the importance of the various mTOR effector pathways and may suggest targets for intervention to
combine with mTOR inhibitors. Finally, since chronic mTOR inhibition by rapamycin can increase lifespan and
healthspan in nematodes, fruitflies and mice, we contrast these studies with tumour cell responses to mTOR
inhibition.
The mTOR signalling hub
mTOR [mammalian (or mechanistic) target of rapamycin]
is an atypical serine/threonine protein kinase that integrates
an array of signals from mitogens, growth factors, nutrients,
energy levels and stress in order to co-ordinate cellular catabolic and anabolic processes, cell growth and proliferation
[1]. In mammalian cells, mTOR is found in two distinct
multiprotein complexes: mTORC1 and mTORC2. These
share some components such as mTOR, mLST8 (mammalian
lethal with sec-13 protein 8) and deptor (DEP domaincontaining mTOR-interacting protein), but also unique
components that promote the assembly of complexes and the
binding of substrates and regulators. Notable among these
unique components are raptor (regulatory-associated protein
of mTOR), found in mTORC1, and rictor (rapamycininsensitive companion of mTOR), which is found in
mTORC2. mTORC2 also contains mSIN1 (mammalian
stress-activated protein kinase-interaction protein 1), which
Key words: acquired resistance, cap-dependent translation, eukaryotic initiation factor 4E (eIF4E),
eukaryotic initiation factor 4E-binding protein (4E-BP), mammalian target of rapamycin (mTOR),
rapamycin.
Abbreviations used: AMPK, AMP-dependent protein kinase; DKO, double knockout; 4E-BP,
eukaryotic initiation factor 4E-binding protein; eIF4E, eukaryotic initiation factor; ERK1/2,
extracellular-signal-regulated kinase 1/2; MEF, mouse embryonic fibroblast; MNK, MAPK
(mitogen-activated protein kinase)-interacting kinase; mSIN1, mammalian stress-activated
protein kinase-interaction protein 1; mTOR, mammalian (or mechanistic) target of rapamycin;
mTORC, mTOR complex; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein
kinase C; PTEN, phosphatase and tensin homologue deleted on chromosome 10; raptor,
regulatory-associated protein of mTOR; rictor, rapamycin-insensitive companion of mTOR; RTK,
receptor tyrosine kinase; S6K, S6 kinase; SGK, serum- and glucocorticoid-induced protein kinase;
5 -TOP, 5 -terminal oligopyrimidine tract; TOR, target of rapamycin; TSC, tuberous sclerosis
complex; WT, wild-type.
To whom correspondence should be addressed (email [email protected]).
1
C The
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Authors Journal compilation exists in five isoforms, three of which can form distinct
mTORC2 complexes with distinct specificity for PKB
(protein kinase B/Akt), SGK1 (serum- and glucocorticoidinduced protein kinase 1) and PKC (protein kinase C) [2].
mTORC1 is much the better understood of the two
mTORCs, in part because it is specifically inhibited
by rapamycin. mTORC1 is activated by growth factors
through the phosphorylation and inhibition of the TSC
(tuberous sclerosis complex) 1/2 complex by PKB, ERK1/2
(extracellular-signal-regulated kinase 1/2) or p90RSK (p90
ribosomal S6 kinase) [1]. TSC1/2 serves as a GAP
(GTPase-activating protein) for the small GTPase Rheb, so
TSC1/2 inhibition increases Rheb-GTP, which can activate
mTORC1. However, mTORC1 activation also requires an
adequate supply of nutrients such as amino acids. The Rag
GTPases are activated in the presence of amino acids, interact
with raptor and the ragulator complex and promote the
localization of mTORC1 to the lysosomal membrane where
Rheb is located, thereby ensuring appropriate activation of
mTORC1 [1]. DNA damage and nutrient deprivation lead to
activation of AMPK (AMP-dependent protein kinase) via
p53 and LKB1 respectively to inhibit mTORC1. AMPK
serves as a bioenergetic sensor, responding to changes in the
cellular AMP/ATP ratio and phosphorylating TSC2 directly;
this activates TSC2, thereby inactivating Rheb and inhibiting
mTORC1.
One of the key roles of mTORC1 is to promote protein
synthesis. mTORC1 phosphorylates 4E-BP [eIF (eukaryotic
initiation factor) 4E-binding protein] 1 and 2, inhibiting their
binding to eIF4E; this allows eIF4E to recruit eIF4G and
eIF4A to form the eIF4F translation initiation complex at
Biochem. Soc. Trans. (2013) 41, 956–961; doi:10.1042/BST20130080
Talks About TORCs: Recent Advances in Target of Rapamycin Signalling
Figure 1 The mTOR signalling hub
mTOR exists in two complexes within mammalian cells: mTORC1 and mTORC2. mTORC1 responds to growth factors and
is activated downstream of both the PI3K/PKB and Raf/MEK1/2/ERK1/2 pathways. It also senses amino acids, oxygen,
energy status and stress. Active mTORC1 has a key role in driving protein synthesis through its phosphorylation of key
substrates involved in the regulation of cap-dependent translation initiation and elongation. mTORC2 controls cell survival
and metabolism. Numerous components of these pathways are deregulated in cancer, depicted by the yellow stars,
leading to the hyperactivation of mTOR in up to 70 % of human cancers. Negative-feedback loops from mTORC1 or S6K,
indicated by red arrows, inhibit signalling from receptor tyrosine kinases. As a result, selective mTORC1 inhibition can actually
enhance RTK-dependent signalling to PI3K/PKB and may limit the efficacy of rapalogues. Deptor, DEP domain-containing
mTOR-interacting protein; eEF2, eukaryotic elongation factor 2; eEF2K, eEF2 kinase; Grb10, growth-factor-receptor-bound
protein 10; IGF1, insulin-like growth factor 1; IRS1, insulin receptor substrate 1; mLST8, mammalian lethal with sec-13
protein 8; PDK1, phosphoinositide-dependent kinase 1; PIP2 , phosphatidylinositol 4,5-bisphosphate; PIP3 , phosphatidylinositol
3,4,5-trisphosphate; PRAS40, proline-rich Akt substrate of 40 kDa; Protor, protein observed with rictor.
the 5 -cap of mRNAs, allowing cap-dependent translation to
progress [3]. mTORC1 can also promote protein synthesis
by phosphorylating S6K (S6 kinase) which activates eIF4B,
promoting the degradation of PDCD4 (programmed cell
death 4) to enhance the activity of the RNA helicase eIF4A.
S6K activation also promotes rRNA synthesis, ribosome
biogenesis and de novo pyrimidine synthesis to satisfy
the increased demand for RNA and DNA synthesis [4].
mTORC1 also promotes the synthesis of lipids required to
generate membranes in proliferating cells by regulating the
SREBP1/2 (sterol-regulatory-element-binding protein 1/2)
transcription factors [5]. Finally, mTORC1 phosphorylates
ULK1 (unc-51-like kinase 1) to repress autophagy [6].
Thus mTORC1 signalling promotes anabolic processes and
represses catabolic processes, programming the cell for
‘growth mode’ [1].
Compared with mTORC1, less is known about mTORC2
[2]. However, it is known to phosphorylate the hydrophobic
regulatory phosphorylation site on PKB, SGK and some
of the PKCs, thereby enhancing their activity to promote
cell survival and regulate metabolism. In addition, mTORC2
regulates paxillin and the small Rho GTPases to regulate
the cytoskeleton and cell migration. Thus it is apparent that
mTOR, through its two complexes mTORC1 and mTORC2,
regulates a wide range of cellular catabolic and anabolic
processes and thereby controls cell growth, cell proliferation
and cell survival (Figure 1).
Validation of mTOR as an anticancer drug
target
Deregulation of mTOR underlies many human diseases and
a wealth of evidence links it to cancer [1,7]. Up to 70 %
of all tumours exhibit hyperactivation of mTOR [7] and
many known oncogenic signalling pathways converge to
activate mTOR. Activation of oncogenes [RTKs (receptor
tyrosine kinases), Ras, B-Raf, PI3K (phosphoinositide 3kinase), PKB] or loss of tumour suppressors [LKB1, p53
or PTEN (phosphatase and tensin homologue deleted on
chromosome 10)] can inhibit TSC1/2 by PKB-, ERK1/2or RSK-dependent phosphorylation, thereby activating Rheb
and mTORC1, whereas Rheb itself is overexpressed in many
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Biochemical Society Transactions (2013) Volume 41, part 4
tumour cells and promotes carcinogenesis [8]. Mutations
in the tumour-suppressor genes TSC1/2, LKB1 and PTEN
promote tuberous sclerosis, Peutz–Jeghers syndrome or
Cowden’s syndrome, all characterized by benign tumours
and a predisposition to later malignancies [9]. Many
gliomas overexpress the mTORC2 component rictor, and its
enforced overexpression in glioblastoma cell lines increases
anchorage independent growth, proliferation, motility and
tumorigenicity [10]. These few examples highlight the
importance of mTOR signalling in cancer.
mTOR signalling helps to satisfy the increased demand for
glycolysis, lipid and nucleotide synthesis in tumour cells, but
there is growing evidence that mTORC1-dependent control
of protein synthesis plays a major role in cancer development,
with the mTORC1/4E-BP1/eIF4E pathway being especially
important. For example, loss of 4E-BP1/2 promoted cell
proliferation in culture [11], whereas phosphorylation and
inactivation of 4E-BPs and the resultant derepression of
eIF4E was essential in a PKB-driven lymphoma model
[12]. In this latter case, the mTOR kinase inhibitor PP242
inhibited the growth of these rapamycin-resistant tumours
and this was mediated by the loss of mTORC1 signalling
to 4E-BP/eIF4E and inhibition of cap-dependent mRNA
translation, whereas S6K signalling was dispensable in this
model [12]. These results are consistent with data showing
that reduced expression or increased phosphorylation of
4E-BPs or increased expression of eIF4E, often by gene
amplification, is seen in a wide range of cancers and
is linked to poor prognosis [3,13,14]. Increased eIF4E
expression correlates with higher-grade tumours and might
serve as a biomarker for progression, increased recurrence and
reduced sensitivity to mTOR inhibitors (see below), whereas
overexpression of eIF4E transforms fibroblasts and increases
cancer susceptibility in mice [15]. Finally, knockin of an eIF4E
mutant with reduced activity inhibits tumour growth in a
Pten-loss mouse model of prostate cancer [16] suggesting
that tumours acquire a dependency on eIF4E function.
This ‘eIF4E addiction’ appears to reflect its role in driving
the translation of key ‘eIF4E-sensitive’ mRNAs. These encode proteins that promote cell proliferation (cyclin D1), cell
survival (Mcl-1 and Bcl-2), invasion (matrix metalloproteases)
and angiogenesis (vascular endothelial growth factor) [17]
and their expression is thought to enhance tumorigenicity
of cancers with increased eIF4E. Many of these mTORdependent eIF4E-sensitive mRNAs share a common feature:
a 5 -TOP [5 -terminal oligopyrimidine tract], 5 -TOP-like
or pyrimidine-rich sequence in their 5 -UTR (untranslated
region) [18,19]. Many of these mRNAs are involved in protein
synthesis, but also cell invasion and metastasis; indeed,
mTOR kinase inhibitors reduce the invasive potential of
tumours in a Pten-loss mouse model of prostate cancer [18].
Rapalogues and mTOR kinase inhibitors
The first mTOR inhibitor to be tested as an anticancer
drug was rapamycin, the drug that has underpinned our
knowledge of mTOR signalling. Rapamycin, originally an
C The
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Authors Journal compilation antifungal agent, is an allosteric inhibitor that selectively
targets mTORC1; it binds to FKBP12 (FK506-binding
protein of 12 kDa), forming a gain-of-function complex that
binds adjacent to the kinase domain, although the exact
mechanism of inhibition is still unclear. Rapamycin has antiproliferative activity and has shown some clinical benefit in
renal cancer. Indeed, a number of ‘rapalogues’ with superior
pharmacodynamic properties have been licensed for the
treatment of various cancers, including temsirolimus and
everolimus [20,21]; however, their performance in the clinic
has not lived up to early expectations.
Two major shortcomings are thought to limit the efficacy
of rapalogues. First, selective mTORC1 inhibition and
loss of S6K activity by rapalogues abolishes feedback
phosphorylation of IRS1 (insulin receptor substrate 1)
[22] and Grb10 (growth-factor-receptor-bound protein 10)
[23,24], thereby enhancing RTK signalling to PI3K (which
provides survival and proliferation signals via PKB and
SGK) and ERK1/2. Secondly, for reasons that are still
not clear, rapalogues completely inhibit S6K activity, but
are very poor inhibitors of mTORC1-dependent 4E-BP1
phosphorylation [25–27]. As a result, eIF4E-driven capdependent mRNA translation, a mTOR-dependent driver
of cell proliferation and survival (see above), persists in the
presence of drug. These results and others have prompted
the development of new ATP-competitive inhibitors of
the mTOR kinase domain, which inhibit both mTORC1
and mTORC2, completely inhibit 4E-BP1 phosphorylation
and prevent ‘rebound’ activation of PKB. These include
AZD8055 [27], AZD2014, MLN0128 (INK128) and OSI027, all of which are undergoing clinical evaluation [28].
Tumour cell responses to chronic mTOR
inhibition
Acquired resistance to targeted therapeutics is recognized as
a significant clinical problem, but few studies have examined
how tumour cells adapt and acquire resistance to selective
mTOR inhibitors, whether rapamycin or the new mTOR
kinase inhibitors. The first study generated Rh30 rhabdosarcoma cells with acquired resistance to rapamycin [29].
These cells exhibited a marked reduction in 4E-BP1 levels
compared with parental cells, 10-fold less 4E-BP1 bound to
eIF4E and increased expression of Myc, an ‘eIF4E-sensitive’
mRNA. Both rapamycin resistance and the reduced 4E-BP1
expression were reversible upon drug withdrawal. Finally,
several colon carcinoma cell lines with intrinsic rapamycin
resistance also exhibited low 4E-BP/eIF4E ratios.
More recently, two studies have examined acquired
resistance to the new ATP-competitive kinase inhibitors.
Sonenberg and colleagues showed that acquired resistance
to the mTOR inhibitor, PP242, was due to a loss of 4EBP1 and 4E-BP2 expression in two liver cancer cell lines and
E1A + Ras-transformed (p53-null) MEFs (mouse embryonic
fibroblasts) [30]. 4E-BP1/2-DKO (double knockout) MEFs
were also less sensitive to PP242 and a reduction or increase in
Talks About TORCs: Recent Advances in Target of Rapamycin Signalling
eIF4E expression in WT (wild-type) MEFs either increased or
decreased respectively the sensitivity of these cells to PP242.
mTOR inhibition caused an overall reduction of polysome
formation that was increased further by eIF4E knockdown
and reduced in the 4E-BP1/2-DKO MEFs. Finally, mTOR
inhibition reduced the levels of the ‘eIF-4E-sensitive’ cyclin
D3 and E1 in WT MEFs; this was more pronounced when
eIF4E was knocked down, whereas mTOR inhibitors were
without effect in the 4E-BP1/2-DKO MEFs.
In our own studies, the SW620 colorectal cell line
was rendered resistant to the ATP-competitive mTOR
inhibitor AZD8055 [27]; resistant cells were also crossresistant to other mTOR kinase inhibitors (C.L. Cope, R.
Gilley, K. Balmanno and S.J. Cook, unpublished work).
Resistant cells showed an increase in expression of eIF4E
and increased expression of known ‘eIF4E-sensitive’ mRNA
products, Mcl-1 and cyclin D1, so that their expression was
resistant to AZD8055. Consistent with this, resistant cells
showed increased cap-dependent translation compared with
the parental cells, when assessed using a bicistronic dual
luciferase reporter that allowed simultaneous measurement
of cap-dependent and IRES (internal ribosome entry site)dependent translation. Knockdown of eIF4E in these cells
reversed the increase in cap-dependent translation and resensitized the resistant cells to the anti-proliferative effects of
AZD8055. Finally, inducible expression of eIF4E in HEK
(human embryonic kidney)-293 cells reduced sensitivity
to AZD8055, whereas inducible expression of a nonphosphorylatable 4E-BP1 mutant that sequesters eIF4E was
sufficient to inhibit cell proliferation even in the absence of
AZD8055. Notably, AZD8055-resistant cells did not exhibit
increased S6K signalling; rather, S6K signalling was lost,
suggesting that it is dispensable for resistance to mTOR
kinase inhibitors.
These studies all reveal a common theme in which
tumour cells adapt to mTOR inhibition by maintaining
or even increasing cap-dependent mRNA translation. Since
mTORC1 and mTORC2 can regulate a wide array of
downstream effectors, it is remarkable that the 4E-BP/eIF4E
pathway is frequently targeted in this way to allow resistance
to mTOR kinase inhibitors. However, this is consistent with
other recent studies. For example, PP242 and torin exert
their anti-proliferative effects through mTORC1 and this
is maintained in mSIN1- or rictor-deficient MEFs that are
defective for mTORC2 [25,26]. Furthermore, downstream
of mTORC1, loss of 4E-BP1 and 4E-BP2 renders cells
insensitive to the anti-proliferative effects of PP242 and torin
by maintaining cap-dependent translation [11].
Taken together, these studies suggest that it is the 4EBP/eIF4E ratio that predicts tumour cell susceptibility to rapalogues or mTOR kinase inhibitors and highlight the importance of maintaining cap-dependent translation downstream
of mTORC1 to support tumour cell proliferation. This in
turn suggests that targeting eIF4E or other components of the
eIF4F complex may provide a rational basis for overcoming
resistance to mTOR kinase inhibitors. Strategies could include inhibiting the eIF4A RNA helicase or inhibiting MNK
[MAPK (mitogen-activated protein kinase)-interacting
kinase] 1 and/or MNK2, the kinases that phosphorylate and
thereby enhance the transforming activity of eIF4E [31].
Chronic mTOR inhibition and lifespan
extension
It is interesting to contrast studies in tumour cells with the
lifespan extension observed upon chronic mTOR inhibition
by rapamycin [32]. It has been known for some time that
dietary restriction can extend lifespan across a range of species
(nematodes, fruitflies and mice). More recently, studies have
shown that selective inhibition of TOR (target of rapamycin)
with rapamycin can also extend lifespan in yeast, nematodes
and fruitflies and also in mice, even when administered late in
life [33]. However, in contrast with tumour cells, the chronic
mTOR inhibition that drives lifespan extension is associated
with a reduction in anabolic processes such as protein
synthesis in favour of catabolic processes such as autophagy,
which favour cell maintenance and stress resistance.
In Caenorhabditis elegans, knockdown or knockout of
S6K, components of the mRNA cap-binding complex
(including eIF4E or eIF4G), general translation initiation
factors or ribosomal proteins all reduced protein synthesis but
increased lifespan and increased tolerance to stress [34–36].
Inhibition of mTOR extended lifespan further in nematodes
lacking ife-2 (an eIF4E orthologue), indicating that inhibition
of TOR or loss of ife-2/eIF4E can extend lifespan through
distinct mechanisms or pathways [36]. Studies in Drosophila
have also demonstrated that lifespan extension resulting from
mTORC1 inhibition requires inhibition of S6K signalling
and protein synthesis [37,38]. In the C. elegans studies [34–
36], protein synthesis was not completely inhibited; this
would hardly be compatible with lifespan extension. Rather,
despite a global reduction in translation, the expression of
certain mRNAs encoding stress-response genes and longevity
factors was preferentially maintained [39], suggesting that
gene expression was remodelled to favour maintenance of
somatic cells.
This shift to ‘maintenance mode’ is also consistent with
autophagy serving as one of the major pathways for
lifespan extension following mTOR inhibition. For example,
RNAi (RNA interference) knockdown of atg-7 [40] or
loss of function mutations in bec-1, unc-51 and atg-18
inhibited autophagy [41], accelerated tissue aging and reduced
lifespan in in C. elegans. Most significantly, inhibiting genes
required for autophagy prevented dietary restriction and
mTOR inhibition from extending lifespan [41,42]. Thus
induction of autophagy contributes to, and is required for,
lifespan extension following mTOR inhibition. Since aging
is accompanied by, and arguably driven in part by, the
progressive accumulation of cellular damage throughout
life, these results are consistent with the role of autophagy
in removing and recycling damaged lipids, proteins and
organelles to maintain a healthy cellular environment.
The contrast with tumour cell responses to chronic mTOR
inhibition is striking (Figure 2) and probably reflects the
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Biochemical Society Transactions (2013) Volume 41, part 4
Figure 2 Adaptation to chronic mTOR inhibition in cancer and aging
The activation of mTORC1 inhibits autophagy and promotes protein synthesis, whereas inhibitors of mTOR, whether
mTORC1-selective rapalogues or pan-mTOR kinase inhibitors, block protein synthesis and induce autophagy. In tumour cells,
depicted in the right-hand panel, acquired resistance to these drugs can develop through loss of 4E-BP1 or amplification of
eIF4E. The net effect being increased eIF4E to drive cap-dependent translation of mRNAs encoding proteins involved in cell
survival, proliferation, metastasis and angiogenesis, thus promoting tumorigenesis. In aging cells (lower panel), cumulative
oxidative damage can cause the accumulation of protein aggregates and damaged/dysfunctional organelles. Rapamycin
delays aging by inducing autophagy to remove and recycle damaged lipids, proteins and organelles to maintain a healthy
cellular environment. FIP200, 200 kDa FAK (focal adhesion kinase) family-interacting protein; ULK1, unc-51-like kinase 1.
contrasting ways in which mitotic and post-mitotic cells
deal with cellular damage. Owing to defects in apoptotic
pathways, tumour cells are often able to withstand the
accumulation of damaged DNA, proteins and organelles.
Indeed, tumour cells typically accumulate significant and
varied genetic and epigenetic changes and this, together with
their high rate of cell division, will produce daughter cells with
varying capacities to adapt to mTOR inhibition; those with
attributes favourable for survival and proliferation (e.g. low
4E-BP or high eIF4E) will be selected for and predominate. In
contrast, because of the high demand for translation during
development, the studies of aging fruitflies and nematodes
were typically performed on young adults whose cells were
post-mitotic. Such ‘normal’ somatic cells may be exquisitely
dependent upon autophagy to remove and recycle damaged
proteins and organelles to ensure their lifelong fitness [43] so
that autophagy is favoured during the chronic treatment with
rapamycin that drives lifespan extension.
Funding
This work was funded by a Biotechnology and Biological Sciences
Research Council CASE Ph.D. studentship in collaboration with
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(to R.G., K.B. and S.J.C.).
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Received 13 May 2013
doi:10.1042/BST20130080
C The
C 2013 Biochemical Society
Authors Journal compilation 961