Seminars in Cancer Biology xxx (xxxx) xxx–xxx

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Seminars in Cancer Biology

journal homepage: www.elsevier.com/locate/semcancer

Review

mTOR: Role in cancer, metastasis and drug resistance

Avaniyapuram Kannan Murugan
Department of Molecular Oncology, King Faisal Specialist Hospital & Research Centre, PO Box 3354, Research Center (MBC 03), Riyadh, 11211, Saudi Arabia

A R T I C LE I N FO A B S T R A C T

Keywords: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that gets inputs from the amino acids,
Cancer nutrients, growth factor, and environmental cues to regulate varieties of fundamental cellular processes which
mTOR include protein synthesis, growth, metabolism, aging, regeneration, autophagy, etc. The mTOR is frequently
Mutation deregulated in human cancer and activating somatic mutations of mTOR were recently identified in several types
Oncogene
of human cancer and hence mTOR is therapeutically targeted. mTOR inhibitors were commonly used as im-
Molecular target
munosuppressors and currently, it is approved for the treatment of human malignancies. This review briefly
Resistance
Metastasis focuses on the structure and biological functions of mTOR. It extensively discusses the genetic deregulation of
Precision medicine mTOR including amplifications and somatic mutations, mTOR-mediated cell growth promoting signaling,
Inhibitors therapeutic targeting of mTOR and the mechanisms of resistance, the role of mTOR in precision medicine and
other recent advances in further understanding the role of mTOR in cancer.

1. Introduction including lysosome biogenesis and autophagy and it is a master reg-

Rapamycin is a natural macrolide with antifungal, antiproliferative,
and immunosuppressive properties which was originally discovered
from the bacteria Streptomyces hygroscopicus in soil samples by Surendra
Nath Sehgal and his group when they traveled to Easter Island (it was
known as Rapa Nui by locals) in 1964 [1]. Three decades later, two
genes (TOR1 and TOR2) were identified as a direct target of the im-
munosuppressant, rapamycin toxicity in yeast cells by MN Hall and his
colleagues in 1991 [2]. After three years, the mTOR protein was dis-
covered as a direct target of rapamycin-FKBP12 (12 kDa FK506-binding
protein) complex in mammalian cells, called mammalian target of ra-
pamycin (mTOR). This discovery came independently from three dif-
ferent labs, SL Schreiber and SH Snyder’s lab demonstrated in mid-1994
while RT Abraham lab reported in early 1995 [3–5]. These studies
confirmed the previously-identified yeast TOR to be the homolog of
mTOR. Since then pioneering discoveries in this area were made by
many labs from all over the world. In particular, the molecular and
biochemical approach uncovered various binding partners, down-
stream/upstream effectors, signaling mediators of mTOR and its role in
the biological process which was recently reviewed by DM Sabatini [6].
In a normal cell, mTOR gets various environmental stimuli from amino
acids, growth factors, oxygen, stress, redox sensors, and energy. In re-
sponse to these diverse environmental cues, the active mTOR promotes
cellular anabolism to generate various macromolecules such as nucleic
acids, proteins, lipids which build cellular biomass, ribosome biogen-
esis, and protein translation. mTOR also blocks catabolic processes
ulator of cellular metabolism. The mTOR integrates these wide ranges
of environmental cues-induced anabolic processes to modulate meta-
bolic pathways for cell proliferation, growth, and metabolism. The
mTOR also plays a central role in the regulation of autophagy [7].
Cancer is a multifactorial and complex disease and known to be the
most leading causes of human deaths worldwide. Phosphatidylinositol
3-kinase (PI3K)-Akt-mTOR pathway is one of the most deregulated
signaling pathways in human cancer. The mTOR is considered as a
master regulator of this signaling pathway and recent findings reported
that the mTOR has a pivotal role in human cancer when it is activated.
Further, mTOR signaling is often reported to be hyperactivated in the
majority of human cancers particularly implicated in the cell transfor-
mation, growth, survival, etc [7–9].
2. Structure of mTOR
mTOR is a 288.892 kDa serine/threonine protein kinase that com-
prises 2550 amino acids (including stop codon) which are encoded by
7650 nucleotides and the mTOR gene is located in chromosome 1
(1p36.22). The mTOR basically belongs to PI3K-related kinases (PIKK)
and most of the PIKK members have conserved domain architecture
[10,11]. The mTOR protein molecule has a large N-terminal α-solenoid
(HEAT repeats), a FAT (FRAP, ATM, TRRAP) domain, KD (a protein
kinase domain), and an FRB (FKBP12-rapamycin binding) domain and
a FATC (C-terminal FAT) domain. The mTOR plays an essential role as a
core component of two functionally different multi-subunit protein

E-mail address: akmurugan@gmail.com.

https://doi.org/10.1016/j.semcancer.2019.07.003
Received 16 March 2019; Received in revised form 14 June 2019; Accepted 3 July 2019
1044-579X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Avaniyapuram Kannan Murugan, Seminars in Cancer Biology, https://doi.org/10.1016/j.semcancer.2019.07.003
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 1. Functions of mTORC1 and mTORC2 and their cellular signaling. A. mTORC1 and its signaling pathway. The capsulated diagram shows the schematic picture of
mTOR protein and its various multiprotein subunits of mTORC1 and related binding sites on the mTOR. The lower panel shows the 3D ribbon view of the cryo-EM
structure of human mTOR complex1 with 4.4-Aº resolution (PDB ID: 5H64). Arrow indicates mTORC1-mediated various vital cellular function-based pathways and
their signaling proteins. B. mTORC2 and its signaling pathway. The capsulated diagram shows the schematic picture of mTOR protein and its various multiprotein
subunits of mTORC2 and related binding sites on the mTOR. The arrow indicates the mTORC2-mediated vital cellular function-based pathways and their signaling
proteins. Lower panel shows the 3D ribbon view of the cryo-EM structure of human mTOR complex 2 with 4.9-Aº resolution (PDB ID: 5ZCS).

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A.K. Murugan
complexes named as mTORC1 (mTOR complex 1) and mTORC2 (mTOR
complex 2) [12,13].
2.1. mTORC1
As illustrated in Fig. 1A, in addition to the mTOR - a core protein,
the mTORC1 contains Raptor (regulatory protein associated with
mTOR) and mLST8 (mammalian lethal with sec 13 protein 8, otherwise
called as GβL). Apart from these three proteins, mTORC1 also consists
of an additional two inhibitory protein subunits including PRAS40
(proline-rich Akt substrate of 40 kDa) and DEPTOR (DEP domain con-
taining mTOR interacting protein) [13]. The Raptor helps to recruit
substrate to mTORC1 via binding to the TOR signaling motif and also
essential for the subcellular localization of mTORC1. The mLST8 sta-
bilizes the kinase activation loop by associating with the kinase domain
of mTORC1. When the FKBP12-rapamycin complex binds to the FRB
domain, the substrate entry could be blocked that results in inhibition
of mTORC1 kinase activity [14]. Recent three-dimensional structural
studies have revealed various structural inputs, assembly, and functions
of mTORC1. The cryo-EM structures of mTORC1 and TORC1 found this
complex as a dimer with a “lozenge” shape [14–17]. A recent report on
the atomic resolution structure of mTORC1 showed many structural
insights of Rheb-mediated activation of mTORC1 and inhibition by
PRAS40 [17].
2.2. mTORC2
The mTORC2 constitutes an mTOR, mLST8, and Rictor (rapamycin-
insensitive companion of mTOR). The Rictor is a unique component of
the mTORC2 while the Raptor is a part of mTORC1 [18]. In addition to
these basic protein subunits, mTORC2 has DEPTOR, mSin1 (stress-ac-
tivated map kinase interacting protein 1), a regulatory subunit and
Protor1/2. Although the PH domain of mSin1 inhibits the kinase ac-
tivity of mTORC2, availability of the PIP3 (phosphatidylinositol 3,4,5-
triphosphate) releases the auto-inhibition [19]. Nevertheless, the me-
chanism is not clearly understood how the PH domain of mSin1 inhibits
the mTORC2 activity. The mTORC2 is insensitive to rapamycin
[12,20,21,7] however it is not known why the FKBP12-rapamycin
complexes do not abrogate the mTORC2 activity. Further, a prolonged
rapamycin treatment was shown to inhibit the mTORC2 signaling that
may be because of the binding of rapamycin-FKBP12 complex to “free
mTOR” (mTOR, unbound with Raptor and Rictor), could cause deple-
tion of the “free mTOR” as all the synthesized mTOR molecules are
gradually trapped by rapamycin-FKBP12 complex which prohibits
binding of mTOR with Rictor and this process inhibits assembly of
mTORC2. Particularly, more than 24 h treatments make this saturation
of rapamycin-FKB12 and newly-synthesized mTOR binding, which re-
sults in inhibition of mTORC2-mediated Akt signaling. Still, this effect
has been observed as a differential effect among various cell types that
may be due to the cross-talk between the two mTOR complexes [22,23].
Moreover, it has been shown that the downstream effector of mTORC1,
p70S6K (p70 ribosomal protein S6 kinase 1) could phosphorylate Rictor
to inhibit mTORC2 [24]. Structural analyses that include the negative
stain and cryo-EM (electron microscopy) structures of yeast TORC2
discovered various conformational patterns and subunit architectures
of the TORC2 (TOR complex2) [25,26]. More recently, a cryo-EM
structure of human mTOR complex was reported that displayed
mTORC2 as a dimerized form with hollow rhombohedral fold and ex-
hibited a 2-fold symmetry which is compact than mTORC1. Further, the
study also revealed the mechanism for rapamycin insensitivity of
mTORC2 showing how Rictor and mSin1 jointly make steric hindrance
for inhibiting the binding of FKBP12-rapamycin to mTOR (Fig. 1B)
[27].
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
3. Upstream and downstream signaling of mTORC1 and mTORC2
Growth factor-mediated RTKs (receptor tyrosine kinases)/PI3K/Akt
signaling pathway is an important upstream signaling pathway of the
mTOR protein molecule [28]. In response to various extracellular sti-
muli such as growth factors, nutrients, and amino acids, mTOR strongly
associates to various protein molecules and to form two distinct mul-
tiprotein molecular complexes such as mTORC1 and mTORC2, and
regulates various growth signals by directly phosphorylating the im-
mediate substrates [29]. In a normal cell, various RTKs, such as epi-
dermal growth factor receptor (EGFR), insulin receptor (IR), and G-
protein coupled receptor (GPCR) avails extracellular stimuli from var-
ious growth factors. Upon the growth factor stimulation, the RTKs
trigger input signals to PI3K. Generally, PI3K activity is tightly con-
trolled to a basal level in normal condition. Then, the PI3K catalyzes the
production of phosphatidylinositol 3,4,5-triphosphate (PIP3) by phos-
phorylating phosphatidylinositol 4,5-bisphosphate (PIP2). This activity
is antagonized by PTEN (phosphatase and tensin homolog deleted on
chromosome 10), the tumor suppressor which reverses the PIP3 to PIP2
[30]. Accumulation of the PIP3, the intracellular second messenger, at
the plasma membrane recruits the protein serine-threonine kinases, Akt
(a cellular homolog of the murine thymoma virus Akt8 oncogene),
SGK1 (serum and glucocorticoid-induced kinase 1), and PDK1/2 (3-
phosphatidylinositol dependent kinase-1 and 2), the PH (pleckstrin-
homology) domain-containing proteins to the membrane [31] where,
PDK1 phosphorylates Akt at threonine 308 that results in the activation
of Akt and its signaling pathway [32]. Further, increased protein
synthesis and cell proliferation signals from Akt reach the GTPase-ac-
tivating protein TSC1/2 (tuberous sclerosis complex1/2) and active Akt
directly phosphorylates that results in inactivation of TSC2 (known also
as tuberin), which makes an enhanced Rheb (Ras homolog enriched in
brain) activity [33]. Activated Rheb (GTP-bound) directly switches on
the mTOR thus mTOR assembles its components of mTORC1. The
mobilized mTORC1 then relays signaling by phosphorylating two key
substrate protein molecules, p70S6K, and 4EBP1 (4E-binding protein
1), resulting in activation of p70S6K and inactivation of 4EBP1 [34].
This signaling results in enhanced S6 kinase activity that promotes
translation, and protein synthesis. The mTORC1 is reported to regulate
many downstream effectors including the transcriptional activity of
various protein molecules such as ATF4 (activating transcription factor
4), SREBP (sterol regulatory element-binding proteins), HIF1α (hy-
poxia-inducible factor 1α), PGC1α (peroxisome proliferator-activated
receptor gamma coactivator 1-alpha), and TFEB (Transcription factor
EB), which promotes metabolic homeostasis, lipid synthesis, glycolysis,
mitochondrial biogenesis, lysosome biogenesis, respectively [35]. Fur-
ther, mTORC1-mediated signaling to HIF1α, LIPIN1, p70S6K, and ATF4
enhance glucose metabolism, lipid synthesis, nucleotide synthesis, re-
spectively. Moreover, various inhibitory signaling to ERK5 (extra-
cellular-signal-regulated kinase 5), TFEB, ULK1 (unc-51 like autophagy
activating kinase 1), and UVRAG (UV radiation resistance associated
gene) promotes proteasome assembly, lysosome biogenesis, and in-
hibits autophagy, respectively. The RAS is an important member of the
RTK-mediated PI3K/Akt and MAPK (mitogen-activated protein kinase)
signaling. Growth factor-mediated activation of RTKs enhances RAS
signaling thus result in activation of PI3K/Akt and MAPK pathway. In
addition, accumulation of PIP3 was also shown to stimulate RAS ac-
tivity [36–38].
On the other hand, an activated mTOR associates with its compo-
nents of protein subunits and forms mTORC2. Active mTORC2 phos-
phorylates Akt at the hydrophobic motif (HM) site serine 473 and turn
motif (TM) site threonine 450 when the mTORC2 is activated by growth
factors and associated with translating ribosomes, respectively. Besides,
the mTORC2 also phosphorylates SGK at HM site serine 422. In addi-
tion, mTORC2 phosphorylates cPKC (conventional PKC) and nPKC
(novel PKC) isotypes. It phosphorylates at the TM site threonine 638/
641 of PKCα/βII and at the HM site serine 657/660 of all cPKC) and
A.K. Murugan
some nPKC also requires mTORC2. These phosphorylations of the key
substrate protein molecules critically modulate cellular apoptosis, au-
tophagy, glucose metabolism, cytoskeleton rearrangement and motility
[39]. Moreover, Akt either directly or indirectly regulates the tran-
scription factors that control many vital cellular processes. It has been
demonstrated that mTOR inhibition could induce IRS-1 (insulin re-
ceptor substrate-1) expression and abolishes feedback inhibition of the
pathway, thus results in the activation of Akt. Inhibition of IGF-I re-
ceptor suppressed rapamycin-induced Akt activation and tumor cells
become sensitive to mTOR inhibition suggests a rationale for combi-
nation therapy [40]. The mTOR was delineated to function as a tyrosine
protein kinase because it could activate IRs (insulin receptors) and IGF-
IR1 (insulin-like growth factor receptor 1) when it is a core component
of mTORC2 [41]. In order to turn on the downstream signaling, the Akt
directly phosphorylates FOXO proteins (forkhead box proteins) for
downregulating the transcriptional activity whenever the transcrip-
tional activity is enhanced [42]. Akt indirectly regulates p53 in-
dependent of mTOR through MDM2 (murine double minute) and hy-
peractivates c-Myc and β-catenin, thereby inhibits GSK3β and this
cascade provides an advantage for cell proliferation and regulating
apoptosis [35.43]. Recently, BioID (proximity-dependent biotin la-
beling)-mediated analysis identified known interactors for wild-type
and mutant RAS (H, K, and NRAS) which includes RAF and PI3K and a
set of 130 unknown novel proximal proteins. Further CRISPR-based
screen of these 130 proteins for RAS dependence identified mTOR. The
oncogenic RAS was shown to bind directly with two components of
mTORC2 which includes mTOR and MAPKAP1 and that resulted in
enhanced mTORC2 kinase activity at the cell membrane. The mTORC2
promoted RAS-induced pro-proliferative cascade of the cell and in-
hibition of RAS-mTORC2 interaction impaired RAS-mediated tumor-
igenesis in vivo [44]. Identification of mTORC2 as a direct RAS effector
paves an alternative way for RASopathy for the undruggable oncogenic
RAS mutants in human cancer that could be achieved by mTOR-
mediated inhibitors mainly in combination with BRAF/ERK inhibitors
as RAS also activates the MAPK pathway (Fig. 1).
4. Biological functions of mTOR
The mTOR is a multifaceted protein molecule and is a catalytic core
component of Raptor-mTOR (mTORC1) and Rictor-mTOR (mTORC2)
complexes known to function both as a serine/threonine kinase and
tyrosine kinase, respectively [41]. The mTOR is demonstrated to play a
key role in early embryonic development particularly in pluripotency
and T-cell transdifferentiation [45,46]. As seen in Fig. 1, the mTORC1
plays a central role in metabolic homeostasis, protein, and lipid
synthesis, glycolysis, mitochondrial biogenesis, lysosome biogenesis by
regulating various transcription factors and directly controls translation
[39,7,9]. It modulates glucose metabolism and nucleotide synthesis
through metabolic pathway proteins. It also regulates the proteasome
assembly and autophagy [47]. The mTORC2 mainly regulates cell
proliferation, survival, cytoskeletal rearrangement, cell migration,
glucose metabolism, and apoptosis [39,7,9,48].
5. Deregulated mTOR in human diseases
The mTOR signaling has a broader impact on basic vital cellular
functions and its deregulated signaling alters normal physiological
functions that result in pathogenesis in humans. Moreover, deregulated
mTOR is displayed to be implicated in human growth and metabolic
diseases including neuronal degeneration, obesity, type 2 diabetes, and
cancer [7,9,49].
5.1. mTOR and brain diseases
Recently, various advanced next-generation sequencing technolo-
gies paved the way for identifying many disease-associated mutations
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
in human samples. Researches on human genetics identified several de
novo heterozygous mutations in the mTOR gene in Smith-Kingsmore
syndrome. mTOR mutations were detected in many different functional
codons, for example, C1483F 49, E1799K [50–53] F1888C and M2327I
[54] and the majority of them were harbored in the FAT domain.
Structural analysis based on molecular modeling identified that the
amino acid residue E1799 of FAT domain binds on the kinase domain
that results in negative regulation of mTOR. Genetic mutations which
alter this critical amino acid residue (E1799) causes destabilization of
the mTOR protein molecule thus the protein could switch to an active
state as the negative regulation machinery is functionally impaired
[51]. Consistent with this observation, subsequent functional analyses
of all these mutations exhibited a gain-of-function effect [49–51,54,53].
These observations suggested that mutation in these amino acid re-
sidues may critically impair the protein conformation that may activate
the mTOR activity thus resulting in enhanced protein synthesis during
the early stages of brain development and this may likely to ignite an
inflammatory reaction in the brain.
In addition to whole-exome sequencing, deep sequencing, conven-
tional PCR amplification-based Sanger sequencing of mTOR gene in
diverse neurological diseases found mTOR mutations in 15.6% (12/77)
of patients in focal cortical dysplasia, type II (FCD type II). These mTOR
mutations were found to be somatic origin as the brain tissue-derived
mutations were not detected in the blood samples of the same patients.
Many mTOR mutations such as W1456G 55, L1460P 56, L2427P [57],
S2215Y [56,54,52] were identified in FCD type II and those mutations
were mostly distributed both in the FAT and kinase domain of mTOR.
Inoculation of three of these mutations into the HEK293T cells ex-
hibited constitutive activation of mTOR kinase activity while compared
to that of wild-type control cells. Treatment of mice carrying these
mutants with rapamycin inhibited cytomegalic neuron and seizures
[57]. Consistent with these findings, another study also found de novo
somatic missense mTOR mutations in 46% (6/13) of FCD type IIb [56].
Further, functional studies on these mutations observed hyperactivation
of mTOR resulting in phosphorylation of its immediate substrate,
4EBP1.
5.2. mTOR and human cancer
Activated mTOR signaling is a key for growth promotion and is
implicated in human cancer, playing various roles in cell survival, cy-
toskeleton rearrangement, invasion, metastasis, anti-apoptosis and in-
hibition of autophagy (Fig. 2). The mTOR signaling pathway is altered
by a variety of mechanisms. Firstly, as a mediator of the PI3K signaling
pathway, it gets hyperactivated by upstream RTKs/PI3K/Akt signaling.
Secondly, deregulation of closely related and its immediate signal
transducers of mTOR such as TSC1/2, Rheb and p70S6K/4EBP1 acti-
vate mTOR signaling. Thirdly, recent research revealed that mTOR itself
was found to be genetically deregulated in human cancers by amplifi-
cation, downregulation, and somatic mutation. These genetic altera-
tions cause hyperactivation of mTOR that results in activation of its
signaling pathway and these mechanisms are detailed below.
6. Mechanisms of mTOR activation in human cancer
6.1. Activation of mTOR by upstream signaling molecules in human cancer
The RTKs-mediated PI3K activation phosphorylates and switches
PIP2 to PIP3 (directly antagonized by the tumor suppressor, PTEN by
dephosphorylating the PIP3 to PIP2) that drives PI3K signaling cascade
which feeds onto both mTORC1 and mTORC2 complexes.
Amplifications and genetic mutations are the common genetic changes
that constitutively activate the protein molecules. Either activation of
RTKs or the other upstream members such as PIK3CA, RAS (H, K, and
NRAS), Akt and loss of PTEN molecules could activate this signaling
axis that in turn trigger mTOR which result in activation of the mTOR
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 2. mTOR-mediated signaling pathways in cancer. Growth factors, stress, amino acids, energy, and oxygen give inputs to mTOR which activate the mTORC1.
Activated mTORC1 regulate stress/DNA damage, enhance nucleotide synthesis, protein synthesis and metabolism that promotes, cell proliferation, cell survival
metastasis, etc. Growth factor-induced RTKs activate mTORC2 which then increase cell proliferation, survival, and cytoskeleton rearrangements by phosphorylating
various downstream effectors.

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A.K. Murugan
signaling. Receptor tyrosine kinase, EGFR is frequently shown to be
genetically altered in human cancers. The EGFR member, HER2
(ERBB2) is found to be overexpressed in breast cancer in ∼10-30% of
patients and associated with poor survival [58]. EGFR gene mutation
results in overexpression of this gene and its mutations are often found
in human cancers. EGFR mutations are more frequently reported in
∼25% to 82% of colorectal cancers [59], ∼30-50% glioblastomas [60],
and in 5%–20% in non-small-cell lung cancer, etc [61]. Though these
tumors benefitted from the monoclonal antibody-based (Herceptin)
molecular targeted therapy, but only achieved for patients with the
wild-type RAS molecule. PDGFRα mutations were identified in ∼5% of
GIST (gastrointestinal stromal cancer) [62] and a 5–10% of amplifica-
tions were found in esophageal cancer, brain malignancies (glio-
blastoma multiforme and oligodendrocytoma), and artery intimal sar-
comas [63–67]. Some gene rearrangements have also been reported in
leukemia [68]. TK inhibitors were developed and used for the GIST-
positive KIT; despite a significant increase in overall survival, patients
develop resistance over time. FGFR mutations have been reported in a
wide spectrum of human cancers [69]. Somatic mutations of FGFR1/2/
3 were reported and shown to have constitutive activation of this
protein in head and neck, melanoma, brain, breast, lung, stomach,
uterus, and colon cancers [70–80]. Amplifications of FGFR1/2 have also
been reported in breast cancer [81–84]. The FGFR mutation in gastric
cancer was shown to be associated with poor prognosis [85]. Moreover,
chromosomal translocation in the FGFR produces oncogenic fusions and
this was frequently reported in several blood cancers including multiple
myelomas and myeloproliferative disorder syndrome [86–88]. FGFR
inhibitors are in clinical trials and these inhibitors (Brivanib) are ef-
fective both on wild-type and activated FGFR3 isoforms. Further,
PIK3CA is an oncogenic activator of mTOR signaling and shown to be
amplified and mutated in human cancers [89,43]. Otherwise, the loss-
of-function of PTEN also gives a similar effect on this signaling
pathway. The loss-of-function mutations/deletions of PTEN were often
found in human malignancies [90]. The RAS proto-oncogenes such as
HRAS, KRAS and NRAS were often altered in human cancers. In
sporadic cancer, activating KRAS mutations were more frequent at
21.6%, when compared to either NRAS (8%) or HRAS (3.3%) [91].
Activated RAS could directly turn on MAPK pathway and binds to
p110α via the RBD (RAS-binding domain) that results in activation of
PI3K/Akt pathway which has the capacity to inactivate TSC1/TSC2 via
phosphorylation of TSC2 by activated MAPK and PI3K downstream
members such as ERK (extracellular signal–regulated kinase) [92], RSK
(ribosomal s6 kinase) [93], and Akt [94], respectively thus results in the
conversion of Rheb to a GTP-bound form and activation of mTORC1.
Further, the Raptor is directly phosphorylated by RSK in order to fur-
ther enhance the activity of mTORC1 [95]. Gene amplification of
members of RTKs which are upstream of both PI3K and RAS are also
commonly detected in cancer that also ultimately leads to aberrant
signal transduction through both mTOR complexes (Fig. 2).
6.2. Activation of downstream members of the mTOR signaling pathway in
human cancer
The downstream effectors of mTOR including p70S6K, 4EBP1, and
eIF4 are reported to play a key role in cancer by regulating protein
synthesis, survival, and cell growth (Fig. 2). A microarray-mediated
analysis used 4209 complementary DNA clones from breast cancer cell
lines, primary tumors with matched normal tissues to determine the
amplification of p70S6K1. They found that the p70S6K1 was amplified
and highly overexpressed in MCF-7 cells. Moreover, compared with the
normal tissues, p70S6K1 was identified to be amplified in 8% (59/668)
of primary breast cancers and the amplification was significantly as-
sociated with poor prognosis (p = 0.0021) while combined with HER-2
found poor survival (p = 0.0001) thus suggested a crucial role in on-
cogenesis and prognosis of breast cancer patients [96]. Further, the
p70S6K1 was shown to be implicated in glial cell transformation and
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
oncogenesis [97]. Recently, it has been demonstrated that p70S6K1-
mediated phosphorylation of PIPKIγ90 is critical for the development of
focal adhesions and invadopodia formations, the key machinery for
migration and invasion [98]. Consistent with this finding, a subsequent
study also found that this molecule plays a leading role in motility of
cells, a preclinical testing of a newly developed small molecule inhibitor
of p70S6K1 (FS-115) showed that the compound could potentially in-
hibit the colony formation and anchorage-independent growth of breast
cancer cells suggesting that opportunity for repressing loco-regional
relapse and metastasis using p70S6K inhibitor in breast cancer [99].
Analyses of phosphorylated 4EBP1 (p-4EBP1) revealed that this protein
molecule was overexpressed and its expression was directly associated
with progressive malignancies and an unfavorable prognosis in-
dependent of the upstream oncogenic aberrations in major malig-
nancies including breast, ovary, and prostate cancers. This study sug-
gested that p-4EBP1 likely to function as a funnel factor for oncogenic
signaling particularly in oncogenic ability and self-sufficiency in pro-
moting proliferating signals and may be a prominent molecular marker
of malignant ability [100]. Moreover, it has been found that cell-type
specific 4EBP1 abundance level could determine the sensitivity or re-
sistance to inhibitors of the PI3K/Akt pathway [101]. A further study
also confirms that the overexpression of p-4EBP1 associates with poor
prognosis in human cancer [102]. A recent study found that the levels
of 4EBP1/eIF4e activation could readily predict early and late recur-
rence in renal cell carcinomas [103]. The eIF4E has been shown to be
implicated in cell proliferation, oncogenesis, and metastasis of human
cancer including prostate cancer [104,105]. Moreover, recently it is
revealed that the p-4EBP1 is a novel independent predictor of overall
and progression-free survival and suggested that it could be a potential
biomarker for prognostic stratification and treatment decisions in
ovarian cancer patients [106].
6.3. Genetic deregulation of mTOR in human cancer
Copy gain, amplification and/or mutation of a gene are the typical
mechanism for an oncogenic activation which exerts growth and shut
off the apoptosis and autophagy. The mTOR is shown to be deregulated
via different molecular mechanisms which ultimately lead to human
pathogenesis. The recent literature shows that mTOR is frequently ge-
netically deregulated that includes gene amplification and mutation.
6.3.1. Copy number alterations (CNAs): gain/loss of copy number
alterations
The mTOR gene is located at chromosome 1p36.22 and this locus is
often genetically altered in human cancers. The copy number variations
for this locus are clearly recorded in the COSMIC (Catalogue Of Somatic
Mutations in Cancer, UK) database. The prevalence of both the gain and
loss of copy number variation is not significantly high in the observed
cases as all the copy number variations shown in different types of
cancers invariably reveals a small fraction of percentage. For example,
though copy gain was observed in stomach cancer, 0.4% was the
highest prevalence among the various malignancies with the copy gain.
On the other hand, the highest prevalence of copy number loss was
observed in adrenal gland with a prevalence of 1.49% while the other
cancers exhibited even much lower than this tumor type. These ob-
servations clearly show that loss or gain of copy number alterations-
mediated genetic deregulation may contribute to only a small fraction
of human cancers and suggest the importance of obtaining a clear ge-
netic data before the therapeutic strategy which could facilitate ca-
tering the cases for precision medicine (Table 1).
6.3.2. Amplification: overexpression /underexpression of mTOR gene
A detailed analysis of the COSMIC database revealed a frequent
overexpression of mTOR gene in human cancer (Table 1). Over-
expression of mTOR gene was more prevalent in the skin cancers
(8.46%), urinary tract cancers (6.62%) followed by soft tissue cancers
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Table 1
Genetic alterations of the mTOR gene in human cancers.
S. No Primary tumor tissues Mutations (%)* Copy number variations (%)* Gene expression (%)*

Gain (Chro. 1p36.22) Loss (Chro. 1p36.22) High Low

1 Adrenal gland 0/657 (0%) – 4/268 (1.49%) 1/79 (1.27%) –

2 Anatomic ganglia 0/1221 (0%) – – – –
3 Biliary tract 19/1056 (1.8%) – – – –
4 Bone 3/734 (0.4%) – – – –
5 Breast 87/4641 (1.87%) – 2/1544 (0.13%) 39/1104 (3.53%) 37/1104 (3.35%)
6 Central nervous system 28/3160 (0.89%) 1/1093 (0.09%) – 21/697 (3.01%) 90/697 (12.91%)
7 Cervix 16/384 (4.17%) – – 17/307 (5.54%) 5/307 (1.63%)
8 Endometrium 59/942 (6.26%) – – 28/602 (4.65%) 9/602 (1.5%)
9 Eye 1/177 (0.56%) – – – –
10 Fallopian tube 1/3 (33.3%) – – – –
11 Gastrointestinal tract 0/1 (0%) – – – –
12 Genital tract 14/249 (5.62%) – – – –
13 Hematopoietic & lymphoid 38/4809 (0.79%) – – 5/221 (2.26%) 6/221 (2.71%)
14 Kidney 134/2879 (4.65%) – – 17/600 (2.83%) 7/600 (1.17%)
15 Large intestine 244/3839 (6.37%) – 1/773 (0.13%) 13/610 (2.13%) 27/610 (4.43%)
16 Liver 53/2274 (2.23%) – – 5/373 (1.34%) –
17 Lung 142/4448 (3.19%) 4/1185 (0.34%) – 53/1019 (5.2%) 21/1019 (2.06%)
18 Meninges 0/95 (0%) - – – –
19 Nervous system 24/382 (6.28%) – – – –
20 Oesophagus 31/1633 (1.9%) 1/546 (0.18%) – 6/125 (4.8%) –
21 Ovary 28/1373 (2.04%) 1/729 (0.14%) – – 28/266 (10.53%)
22 Pancreas 23/2447 (0.94%) – – – 12/179 (6.7%)
23 Parathyroid 4/45 (8.89%) – – – –
24 Penis 2/7 (28.57%) – – – –
25 Perineum 0/1 (0%) – – – –
26 Peritoneum 1/33 (3.03%) – – – –
27 Pituitary 0/60 (0%) – – – –
28 Pleura 0/463 (0%) – – – –
29 Prostate 26/2828 (0.92%) – 1/1037 (0.1%) – 15/498 (3.01%)
30 Salivary gland 7/388 (1.81%) – – – –
31 Skin 140/1844 (7.49%) 2/650 (0.31%) – 40/473 (8.46%) 24/473 (5.07%)
32 Small intestine 4/111 (3.64%) – – – –
33 Soft tissue 9/1322 (0.68%) – – 17/263 (6.46%) –
34 Stomach 44/1260 (3.49%) 2/501 (0.4%) – 18/285 (6.32%) 4/285 (1.4%)
35 Testis 5/476 (1.05%) – – – –
36 Thymus 0/172 (0%) – – – –
37 Thyroid 36/1846 (1.95%) – – 17/513 (3.31%) 7/513 (1.36%)
38 Upper aero-digestive tract 25/1703 (1.47%) – – 26/522 (4.98%) 7/522 (1.34%)
39 Urinary tract 58/1137 (5.11%) – – 27/408 (6.62%) –
40 Vagina 0/2 (0%) – – – –
41 Vulva 1/30 (3.33%) – – – –

1307/51,132 (2.55%) 11/4704 (0.233%) 8/3622 (0.22%) 350/8201 (4.26%) 299/7896 (3.78%)

*
Mutated samples/Total number of sample analyzed (%); -, not analyzed. The data is derived from COSMIC as on February 24, 2019.

(6.46%) and stomach cancer (6.32%). Further, to date, analysis of the
PubMed database revealed no evidence of individual report on over-
expression of mTOR gene in human cancer.
As seen in Table 1, the mTOR gene was recorded to be down-
regulated in many human cancers such as breast, central nervous
system, cervix, endometrium, hematopoietic, lymphoid, kidney, etc.
The most prevalence of mTOR downregulation was detected in malig-
nancies of the central nervous system (12.91%) followed by the ovary
(10.53%), and pancreas (6.7%). As a result of upstream signaling ac-
tivation or genetic mutations in mTOR may achieve gain-of-function
and hyperactivate the mTOR signaling, thus it is clearly expected that
mTOR is more likely to show overexpression rather than under-
expression. These features show that mTOR has split personalities to-
wards triggering oncogenic signaling and warrant further studies on
how these downregulated gene expressions could contribute to the
oncogenesis. In normal physiological condition, downregulation of
mTOR has been shown in response to maternal nutrient restriction in
the placental tissues of the baboon [107]. In general, hypoxia, energy
stress or exposure to dopaminergic neurotoxins causes downregulation
of mTOR if the cell is under extreme stress and it is exclusively de-
pendent on intact TSC1/2 and the expression of RTP80 [108–110].
Although RTP801 requires the intact TSC1/2 to downregulate mTOR,
RTP801 shows no physical interaction with both the TSC1/2 [111].
Interestingly, it has been observed for the first time that down-
regulation of mTOR and P70S6Kβ2 expression is associated with poor
prognosis in T-cell acute lymphoblastic leukemia (T-ALL) [112]. These
observations warrant future studies on mTOR gene amplification in
human cancers.
7. Hyperactivating mutations of mTOR gene
Initially, the hyperactivation of mTOR was observed by Abraham
and his group. When the mTOR mutant (named as ΔTOR-deletion
mutant) was generated with unimpaired phosphorylation site for Akt
(2430–2450 amino acids close to the carboxyl terminus of the mTOR)
and overexpressed in HEK293 cells, they found that mutant could en-
hance a 3.5-fold of its basal protein kinase activity resulting in phos-
phorylation of p70S6K and mTOR-mediated in vivo signaling. This
group also found that mTOR is the direct target of the PI3K/Akt sig-
naling pathway [113]. Four years later, CB Thomson and his group
observed that expression of the ΔTOR-deletion mutant in the p53-/-
mouse embryonic fibroblasts (MEFs) could readily enhance the colony
formation efficiency and addition of adenoviral E1A protein further
increase the number of colony formation suggested that the deletion

7
A.K. Murugan
mutant of mTOR may contribute to cell survival and transformation
[114]. In line with these studies, two other activating mutations were
found when analyzed for caffeine target in yeast TOR complex1 [115].
Urano et al identified 22 single point mutations in the Tor2 protein
which could promote growth in the absence of rhb1 (the Rheb homolog
in yeast). It has also been found that mTOR harboring analogous to the
mutations identified in the yeast (L1460P or E2419K) exhibit con-
stitutive activation even in the nutrient-starved conditions though
sensitive to rapamycin. Moreover, it has been found that a heterodimer
comprising a mutant with a wild-type form of mTOR is active in nu-
trient-starved cells [116]. Subsequently, two other mTOR mutations
were shown to be hyperactive [117]. Even though these mTOR muta-
tions were derived from non-tumors, they were hyperactive, all mutants
activated the immediate substrate, p70S6K, and enhanced colony for-
mation. In 2010, for the first time, Sato et al reported that some cancer-
associated mTOR mutations derived from COSMIC database possessed
gain-of-function activities [118]. Nevertheless, till then, whether mTOR
mutation itself could transform the normal fibroblasts and make tumor
in vivo was completely unclear until M Xing and his colleagues de-
monstrated in 2013 [119]. When mutations were introduced in the
critical domains of mTOR and experimented both in vitro and in vivo,
found those mTOR mutations could increase protein kinase activities
many folds, activate mTOR/p70S6K and Akt signaling pathways. Fur-
ther those mutations induced cell transformation, invasion in NIH3T3
cells and remarkably rapid tumor formation in nude mice [119]. These
results suggested for the first time that mTOR is tumorigenic upon
mutation (Table 2).
8. Prevalence of mTOR mutations in human cancer
Analysis of the COSMIC database displayed that the incidence of
mTOR mutations greatly varies among the different types of cancer. The
highest frequency was detected in 28.57% (2/7) and 33.3% (1/3) of the
fallopian tube, penis cancer, respectively. Nonetheless, the number of
tumors analyzed for mTOR mutation was relatively very low and hence
raise the skepticism about the incidence of mutation. Several other
cancer types follow with a considerably higher incidence of mTOR
mutations consisting relatively more tumor samples particularly,
parathyroid, skin, large intestine, and endometrium having a pre-
valence of 8.89% (4/45), 7.49% (138/1830), 6.37% (244/3830) and
6.26% (58/938), respectively. The incidence observed in the later cases
shows higher reliability than the former cases which studied only a few
tumors. The mTOR mutations are only rarely found in several other
types of cancer. The mTOR mutational spectrum clearly reveals the
possible segregation towards a specific cell/tissue types. It seems that
mTOR mutations occur frequently in tumors of reproductive and ex-
cretory organs and their subset of tumors derived from cells/tissues
with high rates of self-renewal capacity. Further, a subset of the tumors
which are negative for mTOR mutations may have other genetic events
with mutual exclusivity that could give a similar downstream effect.
Moreover, mTOR mutations might have missed because of intra-tumor
heterogeneity, poor sensitivity of Sanger sequencing particularly when
malignant tumor dissected with more normal surrounding tissue, and
may be away from the analyzed hotspot exons. Whole-genome/exome
sequencing of human cancer has significantly identified the mTOR
mutations along with other somatic mutations suggesting that mTOR is
significantly mutated in human cancers and required to study and
analyze through more sensitive methods such as deep sequencing,
pyrosequencing, and next-generation sequencing including Illumina,
etc [120–123]. Whole-exome sequencing of 10 non-familial pancreatic
neuroendocrine tumors revealed that mutation harbored in the genes of
the mTOR pathway in 14% of tumors posing a potential treatment with
mTOR inhibitors [124]. Targeted sequencing analysis of 504 cancer-
related genes in 29 lymph node metastases of cutaneous squamous cell
carcinomas (SCC) found 6.8% (2/29) of mTOR mutations with other
somatic mutations and genetic alterations [125]. Initially, Sanger
8
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
sequencing of mTOR gene in thyroid cancer samples revealed no mTOR
mutation in differentiated thyroid cancer [126]. It is possible to spec-
ulate that mTOR mutation likely to exist in human cancer but they
might have been missed due to poor sensitivity of Sanger sequencing
and/or common intra-tumor heterogeneity of mTOR mutations in
human cancer [127]. Notwithstanding, whole-exome sequencing of the
ATC (anaplastic thyroid cancer), most aggressive and deadly thyroid
cancer samples revealed ∼10% (2/22) of mTOR mutations (R164Q and
M2327I), despite uncharacterized [128]. A recent study detected mTOR
somatic mutations in melanoma with a prevalence of ∼11%, (43/412)
which was associated with increased melanoma-specific mortality
[129]. The prevalence of mTOR mutation is profiled in Table 1 and the
frequently occurred mutations are shown in Fig. 3.
9. Oncogenic mutations of mTOR in human cancers
Although somatic mutations were earlier identified in the mTOR
gene, Sato et al were the first to functionally characterize the cancer-
associated mTOR mutation in vitro. Their study selected human cancer-
associated mTOR mutations (A8S, S2215Y, P2476L and R2505P) from
the COSMIC database and introduced them in the mTOR gene using
site-directed mutagenesis. Transient overexpression of each of these
mutants in HEK293T cells revealed enhanced protein kinase activity,
and hyperactivation of mTOR/p70S6K signaling in cells transfected
with S2215Y and R2505P mutants. Interestingly, even under nutrient-
starved conditions, the phosphorylation of mTORC1 substrates (p70S6K
and 4EBP1) was intact with higher phosphorylation. Further, compared
to the wild-type, enhanced focus formation was observed in Rat1 cells
when the mTOR mutant (S2215Y) was co-transfected with mutant
KRAS yet, it was insignificant when mTOR mutant alone expressed
[130]. Later, it has been demonstrated that intratumor heterogeneity
was identified for mTOR mutation which harbored within the auto-
inhibitory region. The renal clear cell carcinoma tumor-bearing mTOR
mutant (L2431P) could display enhanced staining of p-p70S6K and p-
4EBP1, but tumor regions with wild-type mTOR displayed no staining.
In addition, expression of this mutant in a clear cell carcinoma cell lines
showed increased phosphorylation of p70S6K even after serum-starved
condition and was sensitive to everolimus suggesting that the mTOR
mutant could constitutively activate mTOR signaling [127]. Functional
analyses of a large cell neuroendocrine carcinoma-associated mTOR
mutation (L2209V) with 28 other non-synonymous mutations identified
in different human cancer types revealed that 12 of 28 mutations in-
cluding the L2209V could activate p70S6K signaling and transform the
mouse 3T3 cell phenotypes. Structural analysis displayed that these
mutations were accumulated between the kinase and FAT domain. All
these transforming mTOR mutants were shown to accelerate cell sur-
vival in 3T3 cells, and they were sensitive to rapamycin inhibition
[131]. Furthermore, clear cell renal cell carcinoma (ccRCC)-associated
FAT domain mTOR mutations derived from the cBioPortal/COSMIC
database were demonstrated to be gain-of-function mutations as they
could activate both mTORC1 and mTORC2. Likewise, these mutations
enhanced the cell proliferation in HEK293T cells and shown to confer
relatively resistant to rapamycin-mediated mTOR inhibition. In addi-
tion, it has been found that FAT domain point mutations of mTOR
differentially activate the mTORC1 as it shows nutrient sensitive (p-
p70S6K at Thr389 and p-4EBP1 at Ser65) and resistant (p-4EBP1 at
Thr37/47) for the output of phosphorylation for different sites [132].
Analyses of cancer-associated recurrent and non-recurrent mTOR mu-
tations revealed that all the mTOR mutants tested were activating
mutations as they displayed enhanced p70S6K phosphorylation. Fur-
ther, functional characterization of recurrent mutations conferred ac-
tivation of respective substrates of both mTORC1 and mTORC2 sig-
naling such as p70S6K/4EBP1 and Akt at a different strength.
Moreover, it has been shown that some of those mutants (L1460P,
C1483F, S2215Y, and R2505P) preferably phosphorylated p70S6K/
4EBP1 while V2006I preferred the Akt and suggested that those
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Table 2
Functional characterization of activating/oncogenic mutations of mTOR gene.
Type of mutation Mutant Domain Artificial/Tumor* Function References

Missense H419R HEAT Tumor Tumorigenic Murugan et al. 2019 [230]

Missense G2359E Kinase Tumor Tumorigenic Murugan et al, 2019 [230]
Missense Y1974H FAT Tumor Activating Rodríguez et al. 2017 [229]
Missense H1968Y FAT Tumor Activating Kong et al. 2016 [129]
Missense P2213S Kinase Tumor Activating Kong et al. 2016 [129]
Missense M2327I Kinase Tumor Activating Xu et al. 2016 [134]
Missense L2230V Kinase Tumor Activating Xu et al. 2016 [134]
Missense L2185A Kinase Artificial Activating Wu et al. 2015 [209]
Missense L2185C Kinase Artificial Activating Wu et al. 2015 [209]
Missense A1519T FAT Tumor Activating Ghosh et al. 2015 [132]
Missense Y1463S FAT Tumor Activating Ghosh et al. 2015 [132]
Missense K1452N FAT Tumor Activating Ghosh et al. 2015 [132]
Missense V2406A Kinase Tumor Transforming Yamaguchi et al. 2015 [131]
Missense V2006F FRB Tumor Transforming Yamaguchi et al. 2015 [131]
Missense T1971I FAT Tumor Transforming Yamaguchi et al. 2015 [131]
Missense D2512H Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense I2500F/M Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense A2226S Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense Q2223K Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense L2220F Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense R2217W Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense L2216P Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense S2215F/P Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense A2210P Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense L2209V Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense N2206S Kinase Tumor Hyperactive Grabiner et al. 2014 [133]
Missense V2006I FRB Tumor Hyperactive Grabiner et al. 2014 [133]
Missense T1977R/K FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense F1888I/L FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense E1799K FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense E1519T FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense C1483F/R/W/Y FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense A1459P FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense L1433S FAT Tumor Hyperactive Grabiner et al. 2014 [133]
Missense F2108L FRB Tumor Activating Wagle et al. 2014 [135]
Missense E2419K Kinase Tumor Activating Wagle et al. 2014 [135]
Missense E2014K FRB Tumor Activating Wagle et al. 2014 [135]
Missense E2288K Kinase Artificial Tumorigenic Murugan et al. 2013 [119]
Missense P2273S Kinase Artificial Tumorigenic Murugan et al. 2013 [119]
Missense T2294I Kinase Artificial Activating Murugan et al. 2013 [119]
Missense V2291I Kinase Artificial Activating Murugan et al. 2013 [119]
Missense V2284M Kinase Artificial Activating Murugan et al. 2013 [119]
Missense G1479N FAT Artificial Activating Murugan et al. 2013 [119]
Missense W1456R FAT Artificial Activating Murugan et al. 2013 [119]
Missense L2431P Kinase Tumor Activating Gerlinger et al. 2012 [127]
Missense R2505P Kinase Tumor Constitutive Sato et al. 2010 [210]
Missense S2215Y Kinase Tumor Constitutive Sato et al. 2010 [210]
Missense A2020V FRB Artificial Hyperactive Ohne et al. 2008 [117]
Missense I2017T FRB Artificial Hyperactive Ohne et al. 2008 [117]
Missense E2419K Kinase Artificial Constitutive Urano et al. 2007 [116]
Missense L1460P FAT Artificial Constitutive Urano et al. 2007 [116]
Missense A1957V FAT Artificial Enhanced Reinke et al. 2006 [115]
Missense I1954V FAT Artificial Enhanced Reinke et al. 2006 [115]
Deletion ΔTOR 2430-50 Kinase Artificial Transforming Edinger et al. 2004 [114]
Deletion ΔTOR 2430-50 Kinase Artificial Activating Sekulić et al. 2000 [113]

*Artificial, created artificially inserting mutation into the mTOR gene; Tumor, originally derived from primary malignant tumor; #, mTOR mutations without
significant substrate activation by Grabiner et al, 2014 (A41P/S/T, F1888V, T1977S, V2006L, S2215T, R2505Q/*, D2512G/Y). For each mutant, initial report is
listed in the table to avoid redundancy.

mutants selectively activate mTORC1 or mTORC2. These mTOR mu-
tations were shown to be dramatically sensitive to mTOR inhibitors
both in vitro and in vivo but partially resistant to nutrient deprivation
[133]. It has been shown that 22 kidney cancer-derived somatic mu-
tations of mTOR which clustered in FAT and kinase domains were
hyperactivating mutations by their increased kinase activity, increase
cell size, and resistant to glucose and serum starvation withal this does
not reflect in amino acid deprivation. Further, these mTOR mutations
were shown to be resistant to REDD1 (resistant to DNA damage-in-
ducible transcript 1)-mediated suppression, induce tumor formation
and those tumors were remarkably sensitive to rapamycin [134].
Functional characterization of melanoma-associated mTOR mutations
conferred gain-of-function, sensitive to PI3K-Akt-mTOR pathway in-
hibitors as these mutants (H1968Y and P2213S) expressing HEK293T
cells were more sensitive and proliferation was effectively inhibited by
LY294002 and AZD5363 rather than temsirolimus or BYL719 [129].
These results collectively suggest that mTOR mutations are oncogenic,
which strongly activate the p70S6K/4EBP1 and Akt, the downstream
effector signaling at different levels to promote tumorigenesis (Table 2).
Moreover, all the functionally characterized cancer-associated mTOR
mutations either identified in different types of cancer or derived from
the COSMIC/cBioPortal database were demonstrated to be oncogenic
and sensitive to the mTOR inhibitor, rapamycin except - an mTOR
mutation identified in an ATC. It was shown to confer resistance to

9
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 3. Genetic deregulation of mTOR in human cancers. A. Human cancer-associated hotspot somatic mutations and drug-resistant mutations of mTOR. Shown is the
schematic diagram of human mTOR protein structure which consists of various functional domains. Mutations plotted on top of mTOR indicate human cancer-
associated somatic mutational hotspots (frequency of 5 and more) and the mutations plotted in the bottom shows the drug-resistant mutations. Boxes indicated in the
bottom shows various mTOR inhibitors and their binding domains. B. Types of mTOR mutations. Tables show various types of mTOR mutations and different types of
nucleotide changes identified to date in human cancer, respectively. This data was derived from the COSMIC database. C.A 3D ribbon diagram of mTOR. It shows the
FRB domain with the drug-resistant mTOR mutations and other domain (PDB ID: 4jsv).

allosteric mTOR inhibitor but sensitive to kinase inhibitors [135]. conclusion that these mutants directly disturb the DEPTOR interacting
site on mTOR. This observation was also consistent with the other two
activating mTOR mutants localized in C1483 cluster of the FAT domain
10. Mechanisms of mutation-mediated activation of mTOR
(A1459P and C1483Y). These findings show that the possible me-
chanism of a subset of activating mutation particularly of FAT domain
The mechanism of mutant-induced mTOR activation is not ex-
and suggest other possibilities such as mutations in other domains may
clusively studied and clearly understood. However, it has been specu-
have different mechanisms to enhance the activity of kinase which
lated that mutations harboring in the kinase domain and close to the
could result in activation the mTORC1 [133].
activation loop are likely to alter the conformation that may result in
Xu et al divided 28 activating mutations of mTOR double mutants
enhanced kinase activities [119]. Further, Grabiner et al contemplated
into 6 groups and demonstrated that those mutants fall in the category
that mutation might distort the mTORC1 or mTORC2 assembly which
of synergistic Rheb independent mTORC1 activation. Moreover, it has
results in enhanced p70S6K1/Akt1 phosphorylation, respectively or
been shown that mTOR mutants were resistant to REDD1-mediated
mutant mTOR may mislay the binding efficiency to DEPTOR, the en-
inhibition suggesting that in the absence of the tumor suppressor, VHL
dogenous inhibitor of mTOR which binds to the FAT domain [136].
(von Hippel–Lindau) expression, activating mTOR mutations could es-
Experiments on protein-protein interactions revealed that mutant
cape from the negative feedback loop of mTORC1 and HIF1. Notably,
mTOR proteins showed reduced binding to DEPTOR compared with
activating mTOR mutants expressing VHL-deficient cells could grow
wild-type mTOR. On the other hand, like wild-type, all mTOR mutant
tumors which were inhibited by rapamycin. It additionally suggests that
proteins could bind to their binding partners including Raptor and
mTOR mutations harboring in cells with VHL loss and HIF activation is
Rictor towards the formation of the two mTOR complexes. These results
highly pathogenic in clear cell kidney cells [134].
suggested that mutations have no role in the complex formation and it
was consistent with the previous finding that even artificially created
mutants could activate both mTORC1 and mTORC2 [119]. These 11. The mTOR is a driver of invasion and metastasis
findings suggest that reduced DEPTOR binding could enhance mTOR
activity in mTOR mutants. Most of the cancer deaths occur because of the metastasis of the
Further, it has been demonstrated that two mTOR mutations in the primary tumor to the multiple organs. Acquiring the migratory and
FAT domain (L1460P and C1483Y) could activate mTORC1 and bind invasive potential is the initial step and that is the rate-limiting primary
relatively very low to DEPTOR compared to other mutants and draw the step in the metastasis cascade. Epithelial-mesenchymal transition

10
A.K. Murugan
(EMT) is found to be the critical mechanism in the primary step in the
metastatic process because during this molecular program epithelial
cells undergo various steps by losing polarized. Differentiated pheno-
types have many cell-cell junctions to mesenchymal phenotype which
includes non-polarized; loss of cell-cell junctions often results in in-
creased motility and invasive potential. Moreover, these phenotypes
exhibit more resistance to chemotherapy [137]. The flexibility of this
cascade is well known by the fact that EMT is reversible and the process
is named mesenchymal-epithelial transition (MET). In addition to this,
during the initial step of the metastasis cascade, small GTPases, that
includes RhoA and Rac1, are activated that results in actin cytoskeletal
rearrangement, cell migration, and invasion. Particularly, invasive
phenotypes are made when the RhoA protein gives the stimulus to form
actin stress fibers and cell-cell adhesions, and Rac1 induces the for-
mation of lamellipodia [138]. This programmed-change in the cell
phenotype enables the cell to be more invasive and that leads to being
involved in metastasis machinery. The contribution of mTOR and its
signaling members in the invasion and metastasis were demonstrated
when ribosome profiling was performed in prostate cancer [139].
Further studying the functional role of mTORC2 shed more lights on the
role of mTOR in the metastasis cascade.
Moreover, it has been found that TGF-β could induce enhanced cell
size and protein content during EMT that resulted in activation of
mTOR via the PI3K/Akt leading to the phosphorylation of the direct
regulators of translation initiation molecules, p70S6K1, and 4EBP1.
Rapamycin inhibited the TGF-β-induced EMT, translation signaling and
decreased the migratory and invasive behavior of cells. In addition, the
results revealed that the TGF-β-induced canonical transcription
pathway via Smads could have complemented by the mTOR-mediated
translation signaling [140]. The same group further delineated the role
of mTOR in the EMT transition and found that TGF-β-induced EMT
transition and invasion was driven solely by the mTORC2 and suggested
that mTORC2 is a necessary member in the downstream of TGF-β sig-
naling pathway [141]. Nevertheless, it has also been shown that both
the mTORC1 and mTORC2 were demonstrated to play a major role in
regulating EMT, migration, invasion, and metastasis mostly through
RhoA and Rac1 signaling mechanisms in colon cancer [142]. On the
other hand, this was further confirmed by a later study in prostate
cancer model that mTOR could regulate EMT through RhoA and Rac1
signaling pathways [143]. Furthermore, the direct substrate of mTOR,
p70S6K was shown to promote the IL-6-induced EMT and metastasis in
HNSCC (head and neck squamous cell carcinomas) [144]. Further, the
mTOR signaling pathway was reported to be involved in playing a
major role in regulating the growth and dissemination of metastatic
brain tumors through the classical EMT mechanisms [145]. Infiltrating
macrophages were shown to enhance the EMT and stem cell-like po-
pulations through Akt and mTOR signaling in renal cell carcinomas
[146]. Consistent with these reports, use of dual PI3K/mTOR inhibitor
NVP-BEZ235 was shown to strongly inhibit the EMT transition which
was induced by hypoxia and TGF-β1 in both ovarian (SKOV-3) and
prostate (PC-3) cancer cell lines suggest a role of mTOR in the EMT
transition [147]. Apart from the direct involvement of the mTOR and its
signaling, upregulation of various protein molecules such as TRIM29
(tripartite motif-containing protein 29), BMP2 (bone morphogenetic
protein 2), TRIM44 (tripartite motif containing protein 44), UBE2Q1
(ubiquitin conjugating enzyme E2 Q1), and PGAM1 (phosphoglycerate
mutase 1) were demonstrated to promote proliferation and metastasis
via mTOR and its signaling pathway in many different kinds of human
cancers that includes nasopharyngeal carcinoma, non-small cell lung
cancer, hepatocellular carcinomas, pancreatic-ductal adenocarcinomas,
respectively [148–152]. Downregulation of ROC-1 (regulator of cullins-
1) was demonstrated to inhibit malignant progression via mTOR/
DEPTOR signaling in muscle-invasive transitional cell carcinoma [153].
Fibrinogen-mediated EMT and malignant tumor phenotypes were also
shown to be regulated through the Akt/mTOR signaling in esophageal
cell carcinoma [154]. Inhibition of mTOR was demonstrated to
11
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
attenuate migration and invasion potentially suppressing EMT in gall-
bladder cancer [155]. In addition, inhibition of PI3K/mTOR and sonic
hedgehog (SHH) pathways could effectively inhibit the cancer stem cell
features and tumor progression in human pancreatic cancer stem cells
[156]. These reports collectively suggest that the mTOR and its sig-
naling pathways are critically involved in regulating EMT and the
metastasis cascade and treating with rapamycin potentially inhibit the
mTOR and its signaling partners and reverse the invasive phenotypes
represent an opportunity for rapamycin analogs in therapeutically tar-
geting various metastatic human cancers.
12. The mTOR inhibitors: rapamycin
The PI3K/Akt/mTOR signaling pathway is one of the most com-
monly deregulated pathways found in human cancers that supplied the
rationale for therapeutically targeting mTOR in cancer [47,39]. Sub-
sequent research activities led to synthesize and develop several small-
molecule inhibitors that could target many active molecules in the
pathway. Indeed, as of today, only the mTOR inhibitors were the first
small molecules which were translated from bench to bedside to target
the PI3K/Akt/mTOR pathway [157,158]. To date, three generations of
mTOR inhibitors were developed. In the early phase, rapamycin was
discovered with inhibiting the proliferation property in yeast [1], after
long time research demonstrated that rapamycin has im-
munosuppressive effects and many years later, FDA-approved for pre-
vention of transplant rejection mainly as an immunosuppressive agent
[159]. Currently, a high dose of rapamycin is clinically used (e.g.,
5 mg/day), and enrolled in many clinical trials for many types of can-
cers where mTOR and its signaling are hyperactivated and critical for
promoting and/or sustaining oncogenic transformation. It has been
discovered that rapamycin mechanistically acts by binding with the
protein FKBP12 [2], producing a complex that can bind the FRB region
of mTOR that could partially block the active site of mTOR resulting in
suppression of the mTORC1 activity [160]. This rapamycin-mediated
inhibition of mTORC1 results in suppression of protein synthesis, in-
hibition of cellular growth and enhances autophagy and these processes
facilitate tumor regression [161]. Although the rapamycin effectively
targets the mTORC1, it does not equally occlude the phosphorylation of
all its substrates. The p70S6K1 phosphorylation is exclusively inhibited
but the 4EBP1 phosphorylation is partially blocked [162]. On the other
hand, this phenomenon was also truly observed in activated mTOR
mediated phosphorylation as it phosphorylates more preferentially
p70S6K1 than 4EBP1. This effect was may be largely dependent on
species and cell-type [119]. Although differential substrate quality
could also be a cause, mTOR, rapamycin, and FKBP12 crystal structure
analysis predicted that this may be due to differential substrate access
to the active kinase site of mTOR which is exclusively controlled by the
mTOR FRB domain [163].
12.1. First generation mTOR inhibitors: allosteric inhibitors
Rapalogues, the structural and functional analogs of rapamycin
were developed particularly to improve bioavailability and pharmaco-
kinetics and these allosteric inhibitors were the first generation in-
hibitors. As seen in Table 3 and Fig. 4A, temsirolimus is a prodrug form
of rapamycin which inhibits mTORC1 by forming a complex with the
peptidyl-prolyl cis-trans isomerase FKBP12 that is integrated into
mTORC1 and suppress the mTOR. This drug was approved in 2007 as a
monotherapy for the treatment of renal cell carcinoma (RCC) with
advanced stage [157]. Everolimus also act allosterically by recruiting
the FKBP12 to mTORC1. Though the drug has a profound effect on
mTORC1, the mTORC2 does not show any sensitivity to inhibition as
observed in temsirolimus. This drug was approved by FDA as mono-
therapy for the therapeutic use of several human malignancies such as
hormone receptor-positive, and HER2-negative breast cancer in post-
menopause cases, selective neuroendocrine tumors, pediatric/adult
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

SEGA (subependymal giant cell astrocytoma) with TSC (tuberous

FDA approved in 2003-18

sclerosis complex), pancreatic neuroendocrine tumors, renal angio-

(for a series of diseases)

FDA approved in 2007-
FDA approved in 1999 myolipoma associated with TSC, advanced stage RCC [164–169]. Ri-
daforolimus, another analog of rapamycin progressed to the phase III
SUCCEED trial in metastatic soft-tissue sarcomas despite it was dropped
as no effective activity was observed [170]. Further, nab-rapamycin
Status

(nanoparticle albumin-bound rapamycin) named ABI-009 exhibited

2008

better bioavailability compared with oral rapamycin in RCC, NET, and

mesothelioma and it has progressed to phase II studies [171].
Advanced renal cell carcinoma, neuroendocrine tumors of

Rapalogs produced no effect on mTORC2 however, sustained

pancreatic, gastrointestinal, & tumors of lung origin as

treatment was shown to have an impact on mTORC2 as well [22]. This

may be via feedback mechanism through insulin signaling and/or via
the key subunit FKBP12-mediated binding competition, that could be
the limiting factor for the sensitivity that results in differential sensi-
tivity to rapalogues between the two complexes [172]. In human cancer
cell lines, the inhibitory effect of mTORC2 was found only after 24 h of
treatment. In practical, for human beings, it may be required for more
Acute lymphocytic leukemia
Cancer & Immuno rejection

than many weeks and months. The mTORC2 inhibition was demon-
strated to have a bigger role in dysregulated glucose metabolism, in-
sulin insensitivity which leads to diabetes. Various tissue-specific RNAi
studies on invertebrate animals particularly annelids assumed that it
Disease setting

monotherapy

was because of mTORC2 activity loss mainly in the intestinal region

that ultimately resulted in impaired glucose metabolism. [173]. It is
noteworthy that most of these studies evaluate based on phosphoryla-
tion of Akt on serine 473 by mTORC2 as a reflection of activity of
mTORC2 nevertheless this Akt phosphorylation site is may also be
PI3K/Akt/mTOR pathway
Activated mTOR pathway
PIK3CA mutation and/

targeted particularly by other kinases such as DNA-PK [174], IKKε, and

TBK1 suggesting an alternative mechanism for the mTORC2-specific
target site in Akt at serine 473 [175]. Nonetheless, compared with
amplifications

mTOR kinase inhibitors (TORKi), allosteric mTOR inhibitors (rapa-

alterations
Biomarker

mycin), partially suppress mTORC1 [176].

12.2. Second generation mTOR inhibitors: mTOR kinase inhibitors

Interaction is similar to rapamycin, hydrogen at C-40-O
position is replaced by dihydroxymethyl propionic acid

Similar to rapamycin, hydrogen at C-40-O position is

The mTOR has been reported to be hyperactivated in several types

Interacts with FKBP12 and blocks mTOR substrate

of human cancers and treatment of these cases with rapamycin caused

mTORC2-mediated activation of Akt as the rapamycin inhibits only the
mTORC1 thus leading to failure of treatment strategies [39,177–179].
This urged to develop second-generation mTOR inhibitors as anti-
replaced with hydroxyethyl group

cancer agents to target activated mTOR, particularly to inhibit both the

mTORC1 and mTORC2 [180]. As illustrated in Fig. 4B, the mTOR ki-
nase inhibitors potentially could inhibit both the mTORC1 and
mTORC2 and these inhibitors compete with ATP for binding to the
mTOR kinase active site. Most of these small molecules dramatically
exhibited high specificity and selectivity towards mTOR kinase. Among
Mode of action

various mTOR kinase inhibitors, vistusertib (AZD2014) was tested in

recognition

advanced stage malignancies including an acinar cell carcinoma [181].

mTOR inhibitors approved for the treatment of human cancers.

ester

Regardless of the unfavorable results, being clinically further evaluated

in multiple phase I and II studies [182]. AZD8055 has been reported to
have many hundred fold better inhibiting effect on the mTORC1 than
Generation

the effect of other PI3Ks [183]. Further, a dual inhibitory effect, both on
First

First

First

mTOR complex and PI3K was observed with the inhibitor, dactolisib
(BEZ235) [184] and exhibited a more than five-fold higher Kd for ATR,
mTORC1

mTORC1

mTORC1

a damage response kinase [185]. Notably, these inhibitors were shown

Class

to have a biphasic effect on Akt. Inhibiting mTORC2 results in depho-

sphorylation of Akt at serine 473 and expeditious yet transient inhibi-
tion of Akt phosphorylation at threonine 308 thus leading to a sup-
pression of Akt signaling. Nevertheless, mTOR inhibition using kinase
inhibitors also alleviate RTK-mediated feedback inhibition resulting in
Rapamycin (Sirolimus)

activation of PI3K leading to activation of Akt by rephosphorylating at

Everolimus (RAD001)
779,NSC683864)
Temsirolimus (CCI-

threonine 308. It clearly revealed that mTOR kinase inhibition could

lead to a different steady state profound mTORC1 abolishment still
Drug name

accumulate activated Akt phosphorylation on threonine 308 exempting

at serine 473. Besides, it was demonstrated that a combination of mTOR
Table 3

kinase inhibitors with RTK inhibitors could potentially suppress bi-

phasic Akt signaling that results in cell death and tumor regression in

12
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 4. mTOR inhibitors in human cancers. A. mTORC1 inhibitors (rapalogs). The mTORC1 inhibitors fail to inhibit the mTORC2 and that keep the activation of Akt
and mTORC2 signaling-mediated negative feedback loop intact. B. mTOR kinase inhibitors. The mTOR kinase inhibitors inhibit both the mTORC1 and mTORC2 that
exclusively interrupt the PI3K/Akt/mTOR pathway-mediated oncogenic signaling. C. Dual pan-PI3K and mTOR (mTORC1 and mTORC2) inhibitors. These inhibitors
potentially inhibit all four PI3K isoforms, mTORC1 and mTORC2 of the activated PI3K-Akt-mTOR signaling in all human cancers.

vivo. These results reveal the adaptive capabilities of oncogenic sig-
naling and suggest that the importance of combination therapy to es-
cape from the biphasic effect [186]. In the majority of the in vitro stu-
dies, compared with rapalogues, the ATP-competitive inhibitors
showed significantly higher inhibitory effects. Although various such
small molecules were tested in clinical trials for safety, large scale trials
were not yet conducted to show greater and convincing efficacy than
the currently available best treatment options and hence, the inhibitors
such as WYE354, AZD2014, and AZD8055 were still could not get ap-
proval from FDA (Table 4). Despite their beneficial effects, many side
effects were also observed when these drugs were chronically ad-
ministered which includes ulceration of mucosal tissues, hematological
abnormalities, induction of insulin insensitivity, obesity, and diabetes,
howbeit these adverse side effects may mainly be dependent on dose
and case dependent [180].
12.3. Third generation mTOR inhibitors: RapaLink
The RapaLink-1, the TORKi linked to rapamycin is a third-genera-
tion mTOR inhibitor. This molecule exploits the unique juxtaposition of
the first and second-generation inhibitors-binding pockets to create a
bivalent interaction which exclusively allows inhibiting the drug-re-
sistant mTOR mutants that exhibited resistance to the mTOR kinase
inhibitors (TORKi). Various experiments were conducted and compared
the inhibiting potency of rapamycin, RapaLink-1, and MLN0128 in
LN229 and U87MG cells. Overall, RapaLink-1 showed the more potent
inhibiting effect on mTOR than the first and second-generation
inhibitors. This drug was shown effectively to reduce the levels of p-
4EBP1 and cell growth. Compared with rapamycin or MLN0128,
RapaLink-1 inhibited the growth and arrested the cell at G0/G1 phase
more potentially upon RapaLink-1 treatment. Moreover, in vivo anti-
tumor efficacy of RapaLink-1 was more significant. Interestingly,
RapaLink-1 treated tumors presented initial regression and followed by
tumor size stabilization in xenograft mice nonetheless vehicle, rapa-
mycin, or MLN0128 treated tumors could steadily grow. This drug ef-
fectively blocked mTORC1 and further associated with mTOR-inter-
acting protein, FKBP12, and enabled accumulation of RapaLink-1.
Furthermore, it inhibited cancer-derived, activating mutants of mTOR
with better efficacy than rapamycin or TORKi [187]. In glioblastoma,
PI3K-Akt-mTOR signaling is deregulated yet inhibition of PI3K or Akt
displayed a modest effect on downstream mTOR activity. A compara-
tive analysis of the drug effect of RapaLink-1 to the earlier generation
mTOR inhibitors such as rapamycin and TORKi - produced poor dur-
ability of TORKi compared with rapamycin and Rapalink-1. As Rapa-
Link-1 bound FKBP12 enable accumulation of RapaLink-1 molecules in
the cell, it could show better efficacy than earlier generation mTOR
inhibitors thus potently could block cancer-associated activating mTOR
mutants (Fig. 4A and B) [176]. The dual pan-PI3K inhibitors could
potentially inhibit all four PI3K isoforms (α, β, γ, and δ), mTORC1 and
mTORC2 of the activated PI3K-Akt-mTOR signaling pathway (Fig. 4C).
13. Drug resistance in human cancers: the pivotal role of mTOR
Precision medicine facilitates molecular targeted therapy and eases

13
A.K. Murugan Seminars in Cancer Biology xxx (xxxx) xxx–xxx

the therapeutic management. Tumors which tend to develop drug re-

Rodrik-Outmezguine et al. 2016

sistance during the course of treatment that complicates the subsequent
management. Understanding and resolving the resistance mechanism is
more complex as most resistance arises due to the activation of up-
stream/downstream molecules upon molecular targeted therapy.
ClinicalTrials.gov*

Further, resistance also arises due to the multi-clonal origin of tumors

NCT00499486

NCT01595009
NCT02352844
NCT02244463

NCT02091531

NCT03166904

NCT02549989

NCT03127020
and heterogeneity in targeted mutation. Tumor cell acquires this sur-
vival mechanism by inducing mutations during the course of the evo-

[187]
lution of malignancies and metastasis. Gene set enrichment analyses
identified activation of mTOR signaling in oxaliplatin-treated early
passaged cell lines and patient-derived xenografts of colon cancer and a
Phase IV
Phase II

Phase II
Phase II

Phase II

Phase II

Phase II

Phase II
Status

combination of oxaliplatin with an mTOR inhibitor in the in vitro and in

vivo experiments demonstrated a synergistic effect [188]. It has been
–

reported that AT406, the inhibitor of apoptosis proteins (IAPs) an-

Thyroid cancer, Metastatic anaplastic thyroid cancer, Head and
Solid cancers with advanced stage. Advanced pancreatic cancer.

Recurrent or persistent endometrial cancer Endometrial cancer.

tagonist could effectively induce cytotoxicity and pro-apoptotic cascade

on primary as well as established hepatocellular carcinoma cells
Advanced Castration-Resistant Prostat Cancer (CRPC).

(HepG2 and SMMC-7721). Notwithstanding, activation of mTOR in

neck neoplasms, and Endocrine gland neoplasms.

these cells caused resistance to the inhibitor AT406 and silencing the
mTOR or inhibition of mTOR by OSI-027 or by kinase-dead-mutant
Cancers with drug resistant mTOR mutations.
Neuroendocrine tumors. Neuroepithelioma.

dramatically increased the sensitivity in HCC cells suggesting that

mTOR is a likely resistance factor for AT406-induced therapy [189]. In
Non-Hodkin lymphoma Lymphoma.

non-small cell lung cancer (NSCLC) cells harboring KRAS mutation,

acquired resistance to Hsp90 inhibitor (Ganetespib) was shown to be
Advanced solid malignancies.

mediated by ERK-p90RSK-mTOR signaling [190]. Identification of

differentially expressed proteins in a trastuzumab-sensitive (NCI N87)
and resistant NCI N87 (T-R) HER2 positive gastric cancer cell lines by
parallel quantitative proteomics screening revealed the frequent acti-
Disease setting

Solid cancers

vation of mTOR signaling in resistant cells suggesting the need of an

mTOR inhibitor for these trastuzumab-resistant HER-positive gastric
cancers [191]. Tankyrase belongs to the poly [ADP-ribose]polymerase
(PARP) family and it positively regulates the Wnt/β-catenin signaling.
Nonetheless, the Wnt signaling-directed colon cancer cells were not
Mutations in TSC1/2.Activation of PI3K/Akt/mTOR

Mutations in PIK3CA, AKT1, PIK3R1, and PIK3R2,

sensitive to the tankyrase inhibitor and detailed dissection of various

Mutations in PI3K/Akt/mTOR pathway genes.
Amplification and/mutations of PIK3CA gene.
Activation of PI3K/Akt/mTOR/S6K pathway.

pathways revealed upregulation of mTOR signaling. Inhibition of

Drug resistant mTOR FRB & KD mutations.

mTOR reversed the resistant cells to be sensitive for tankyrase in-

Mutations in TSC1/2, NF1/2, or STK11

hibitors and suggested the combination of mTOR inhibitor with tan-

kyrase inhibitors targeting this colon cancer subset [192]. It has been
shown that resistance to sorafenib in acute myeloid leukemia (AML)
was due to hyperactivation of mTOR signaling [193]. A recent phase I
clinical trial that combined the everolimus and capecitabine on HER2-
Rictor amplification

Rictor amplification

metastatic breast cancer showed a better tolerated and enhanced pro-

Mutations TSC1/2

gression-free survival and overall survival [194]. A recent review by

mTOR gene.

MN Hall and his colleague detailed the mTOR-mediated resistance to

Biomarker

pathway.

various molecular targeted cancer therapeutic agents [195]. Collec-

tively all these studies show that mTOR plays a pivotal role in resistance
to major targeted therapies in human cancers regardless of the in-
Generation

hibitors and cancer types and suggest a generous combination of mTOR

Second

Second

Second

inhibitor.
Third

Next

Next
First

First
First

In addition, the mTOR causes self-resistance where it induces re-

sistance to various mTOR inhibitors that act on it. It is noteworthy that
mTOR inhibitors in clinical trials for human cancers.

malignancies harboring genetic alterations in a gene of a certain

pathway can exhibit its growth and survival dependency on that and
Dual PI3K &

Dual PI3K &

mTORC1/2

mTORC1/2

mTORC1/2

mTORC1/2

mTORC1/2
Drug class

likely to show enhanced sensitivity when treated with the related

mTORC1

mTORC1
mTORC1

pathway inhibitors. The hamartoma syndromes and perivascular epi-

mTOR

thelioid-cell cancers harboring alteration of genes of mTOR pathway

such as TSC1/2, and STK11 resulted in activation of mTOR signaling
Sapanisertib (TAK-228, MLN0128

which was targeted by mTOR inhibitors [196–199]. In line with these

studies, an urothelial carcinoma and a metastatic urothelial carcinoma
harboring loss of function mutation in TSC1 and activating mutations in
Rapamycin (Sirolimus)

mTOR gene, respectively exhibited exquisite sensitivity to the mTOR

Everolimus (RAD001)
Everolimus (RAD001)

PQR309 (Bimiralisib)

inhibitor, everolimus [200,164]. Recently, whole exome sequencing of

& INK128)

an everolimus resistant malignant tumor derived from an anaplastic

Sapanisertib

LY3023414
RapaLink-1
Drug name

thyroid cancer patient in parallel with pretreated tumor showed a

Vistusertib

nonsense loss of function mutation (Q1178*) in TSC2 in both pretreated

Table 4

and resistant cases. Yet, the resistant tumor was mutated in the mTOR
gene (F2108L) and it was consistent with immunohistochemical

14
A.K. Murugan
staining of rpS6 reflecting activated mTOR pathway and pretreated
tumor revealed no mutation in mTOR. This mTOR mutation conferred
resistance to allosteric mTOR inhibitors in vitro this phenomenon re-
veals acquired resistance of mTOR to its allosteric inhibitors and sug-
gesting an aggressive relapse with resistant mutations after successive
treatment. Treating this resistant mutation (F2108L) with mTOR kinase
inhibitor showed that the mutation was sensitive to it [201]. Recently,
it has been found that single point mutation accounts for acquired re-
sistance to both the first (rapamycin) and second-generation (a TORKi,
AZD8055) mTOR inhibitors when MCF-7 cancer cell lines were exposed
to these inhibitors. A 3-month exposure to these inhibitors developed
resistant colonies, a kinase inhibitor-treated cell harbored a mutation in
the kinase domain (M2327I) and rapamycin-treated cells had a muta-
tion in the FRB domain A2034V (RR1 cells) and F2108L (RR2 cells). RR
and TKi-R clones which were derived from rapamycin and TORKi ex-
posed cells, respectively demonstrated significantly less sensitivity to
their respective drugs in a 72 h proliferation assay when compared to
the parental line. This result was consistently reflected also in another
cell line, MDA-MB-468 when these mutants were expressed and treated
with a related inhibitor. Rapamycin-resistant FRB domain mutations
have also been found in the yeast by random mutagenesis [202–205]
and also been reported in untreated patients in the databases. Similarly,
rapamycin-resistant mutants which were identified in this screen also
likely to disrupt the mTOR interaction with FKBP12–rapamycin com-
plex (Fig. 3).
As an analysis of mTOR kinase structure (PDB: 4JT5) displayed that
M2327 is > 15 Å away from the inhibitor, the author speculated that
this mutation causes resistance via either reduced TORKi affinity due to
an allosteric mechanism or a mechanism which is not require reduced
drug binding. In contrast, both wild-type and a mutant mTOR (M2327I)
bound AZD8055 with similar affinities. Moreover, M2327I mutant
showed > 3 fold enhanced kinase activity compared with the mTOR
wild-type and rapamycin-resistant mutant. Like M2327I, other mTOR
kinase domain mutants identified in untreated patient tumors also ex-
hibited insensitivity to mTOR kinase inhibitors (TORKi, AZD8055 and
MLN0128) when overexpressed in MDA-MB-468 cells and compared
with wild-type cells. These results suggest that untreated patient har-
boring hyperactivated mTOR with a single point mutation in the kinase
domain may likely to show low/no sensitivity to mTOR kinase in-
hibitors (ATP-competitive mTOR inhibitors). Further pave the way for
using third generation inhibitor which overrides the resistance to the
currently available first and second generation inhibitors, rapalogs and
TOTKis, respectively [187]. Wu et al predicted that the gatekeeper re-
sidue is likely to cause resistance to ATP-competitive kinase inhibitions
as it is widely observed in other kinases. For example, in NSCLCs,
T790M mutation, a “gatekeeper” site of EGFR which constrains the
erlotinib binding as the methionine is larger at this residual change
[206] and T315I resist the imatinib-mediated ABL kinase inhibition in
CML (chronic myeloid leukemia) [207]. Similarly, it has also been
predicted that the mTOR residue I2237 is the gatekeeper as evidenced
by the sequence alignment [208]. Nonetheless, after a series of ex-
periments with yeast system, they found that the mTOR's gatekeeper
position (I2237L) fails to endure any changes in the amino acid residue
except the leucine, a highly conserved mutation [209]. These results
suggested that mTOR may not have drug-resistant gatekeeper muta-
tions. The same group continued to find drug-resistant mTOR mutation
in the kinase domain. To make mTOR kinase mutants, a library of
mutant clones were generated by error-prone PCR and recombined with
a gapped TOR2 vector via “gap-repair” that results in the in-frame fu-
sions of TOR2-mTOR via homologous recombination in yeast.
Screening the mutants towards various drugs identified varieties of
mutants among them, two distinct mutants (L2185A and L2185C)
conferred more resistant to OSI-027, PF-04691502, PKI-587, INK128
and AZD8055 while other mutants such as L2185D, L2185N, and
L2185G were moderately resistant to AZD8055, PF-04691502, PKI-587
and AZD8055, INK128, PF-04691502, PKI-587, respectively. CRISPR/
15
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
Cas9-mediated genome editing and expression of these mutants in
colorectal (SW480) and lung cancer (H460) cell lines revealed that the
L2185A mutation confers tumor type-independent drug-resistance in
both colorectal and lung cancers [209].
14. The mTOR and precision medicine
Focusing on individual genetic variations, environment, and life-
style intending to prevent and treat various human diseases is a new
strategy called precision medicine or personalized medicine. Recent
genetic techniques particularly the next-generation sequencing tech-
nologies along with computational technology facilitated whole
genome, exome and transcriptome sequencing which gathered genomic
data on human diseases that rapidly altered drastic changes in the
treatment strategies which paved way for precision medicine. It has a
bigger role in molecular targeted therapy with better clinical efficacy
and pharmacokinetics in addition to diagnosis and prognosis [210]. The
mTOR axis including upstream RTKs to downstream p70S6K and 4EBP1
are the predictive biomarkers and it is activated by one or other me-
chanism such as mutation and/or overexpression. The PI3K/Akt/mTOR
signaling pathway is hyperactivated in more than 30–80% of human
cancers. Therefore, targeted therapies using single mTOR or dual PI3K/
mTOR or Akt/mTOR are being investigated in clinical trials. Everolimus
monotherapy has been shown to be very effective with advanced gastric
cancers with PIK3CA mutations and rpS6K aberrations suggesting that
the predictive biomarkers are prime important [211]. Further, PIK3CA
and PTEN mutation resulting in Akt activation was reported to be very
sensitive to allosteric mTOR inhibitors [212,213]. Renal cell carci-
nomas show enhanced dependence on metabolic processes such as
pentose phosphate shunt, aerobic glycolysis, augmented lipogenesis,
when metabolic pathways are deregulated. For example, oxygen sen-
sing (VHL/HIF), HIF-responsive genes (VEGF, PDGF, and EGF), glucose
transporters (GLUT1 and GLUT4), reduced activities of AMPK and
Krebs cycle and/or nutrient sensing (AMPK-TSC1/2-mTOR and PI3K-
Akt-mTOR), energy sensing (fumarase-deficient, SDH-deficient), and
HGF/MET mutations. These predictive markers assist in decision
making in the treatment of renal cell carcinomas using mTOR inhibitors
[214]. Next-generation sequencing and other advanced technologies
found new landscapes of genomics and epigenomics that facilitated
approval and use of antiangiogenic and mTOR-directed targeted
therapies for metastatic well-differentiated gastroenteropancreatic
neuroendocrine tumors [215]. Anaplastic thyroid cancer showed
∼10% of co-occurrence of MAPK and PI3K alterations and these tumors
exhibited a unique transcriptional signature. A case with PIK3CA and
BRAF mutations presented no sensitivity to inhibitors of combined
mTOR/PI3K or RAF/MEK. Nevertheless, combining both pathway in-
hibitors exhibited dramatic therapeutic response [216]. Activation of
both MAPK and Akt/mTOR signaling pathways were shown to be
concordantly maintained in the different regions of the same biopsy
[217]. Moreover, SPOP mutation was shown to activate prostate cancer
via PI3K/mTOR signaling [218]. A patient with bi-phenotypic breast
cancer having STK11 mutation was shown to have an exceptional re-
sponse to everolimus [219]. Genomic profiling identified ∼25% of
cases with genetic alteration of mTOR in esophageal squamous cell
carcinoma and exhibited the base for developing targeted therapy of
mTOR in precision medicine [220]. Recently, it has been demonstrated
that PI3K inhibitors could induce insulin feedback loop that reactivates
the PI3K-mTOR signaling axis in several tumor models and inhibition of
this feedback by a dietary and pharmaceutical approach that enhance
the efficacy of PI3K inhibitors suggesting the utility of this work in
enhancing treatment effect [221]. It has been found that oral squamous
cell carcinoma cell lines harboring HRAS mutation were not sensitive to
inhibitors of PI3K. Consistently, HRAS overexpression exhibited low
sensitivity to inhibition of PI3K, while silencing showed sensitivity.
Interestingly, in both wild-type and HRAS mutant cell lines, PI3K in-
hibition resulted in reduced p-Akt, but cell lines harboring HRAS
A.K. Murugan
mutations displayed intact rpS6 phosphorylation. Subsequently,
treating with mTOR inhibitor resulted in remarkable sensitivity in cells
carrying HRAS mutation. This group later found that ERK-TSC2-mTOR
axis is the mediator of PI3K inhibitor resistance [222]. Whole genome
sequencing of pediatric high-grade gliomas (HGG) found mutations in
TP53, TSC1, and amplification of MYCN. High throughput drug
screening revealed remarkable sensitivity to mTOR inhibitors while no
sensitivity was observed with SHH pathway inhibitors [223]. Recently,
platelet-derived growth factor receptor alpha (PDGFRA) extracellular
domain driver mutations were demonstrated to be resistant to PDGFRA
targeted therapies, but enhanced sensitivity to PI3K/mTOR and MEK
inhibitors [224]. Frequent activation of the mTOR signaling pathway is
obtaining immense popularity as its pathway genes are significantly
activated by somatic mutation and/or amplification and de novo over-
expression of proteins. These genetic aberrations ultimately lead to
deregulation of this pathway that results in malignant tumorigenesis
and necessitating targeted therapy for effective anticancer treatment.
Many mTOR inhibitors are currently in pre-clinical or clinical trials for
both hematopoietic and solid cancer treatment while some mTOR in-
hibitors are already in the clinic. As several inhibitors are under de-
velopmental process, advancing studies toward predictive biomarkers
are critically useful to determine the specificity of any kind of ther-
apeutic intervention.
Rapalogs (sirolimus, temsirolimus, and everolimus) are approved
for cancer treatments; nonetheless, rapalogs do not seem to be effective
for the majority of solid cancers, particularly in advanced cancer [225].
Alternatively, vorinostat may be combined with any one of those ra-
palogs for better efficacy in the advanced cancer therapeutics and both
drugs likely to be patient-friendly as the former one is toxic selectively
on cancer cells [226] and the later one (rapalog) is not a toxic drug at
relevant doses and rapalogs do not kill cells but rather they slow down
their proliferation, growth, and motility [227]. Vorinostat (rINN) or
suberoylanilide hydroxamic acid (SAHA) is a potent reversible new
class of pan-histone deacetylase (class I and II but not III HDAC) in-
hibitor. It functions by binding to the active site of histone deacetylases
and acting as a chelator of zinc ions. It is recently approved by the FDA
and available in the drug store for the therapeutic use of cutaneous T-
cell lymphoma (CTCL), a type of skin cancer. The combination of these
drugs for the treatment of different types of human cancer is currently
being investigated.
Though the temsirolimus exhibits promising anticancer activity for
the treatment of advanced RCCs, some cases develop survivin expres-
sion-mediated drug resistance. To this end, the anticancer activity of
temsirolimus was synergistically improved by vorinostat in a panel of
RCC cell lines in vitro and in vivo. The combination of these drugs
dramatically reduced survivin expression, induced apoptosis and
strongly suppressed angiogenesis while every single agent showed a
modest effect [228]. Currently, a phase I trial of sirolimus or everolimus
or temsirolimus in combination with vorinostat is clinically studied in
advanced cancer and yet to arrive any conclusion (NCT01174199). In
contrast, a phase I clinical trial that treated patients using vorinostat in
combination with temsirolimus on metastatic prostate cancers (adeno-
carcinoma of the prostate, hormone-resistant prostate cancer, recurrent
prostate cancer, and stage IV prostate cancer) revealed no value in
finding efficacy and hence it was terminated (NCT01174199). These
results suggest that the rapalog and vorinostat combination therapy
have differential effects on various types of human cancer and these
inhibitors to be tested also on other types of human cancer prior to
concluding the efficacy of this combinatorial therapy. In the future
precision medicine, a genetic-guided “three-drug” combination may
also be sought as anticancer therapy mainly for the metastatic cancers.
For example, patients are currently recruited for phase I study using a
combination of vorinostat and temsirolimus with conventional radia-
tion therapy in diffuse intrinsic pontine glioma (DIPG) of children
(NCT02420613). Similarly, sirolimus, vorinostat and hydroxy-
chloroquine combinations are being clinically studied by a phase I
16
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
clinical trial in advanced cancer (NCT01266057, https://clinicaltrials.
gov/). These therapeutic regimens might be very common in the future
clinic for advanced metastatic cancers.
15. Conclusions and future prospective
The mTOR and its signaling pathway are implicated in a wide range
of normal physiological functions. In a variety of human cancers, the
mTOR signaling pathway is commonly deregulated either due to the
genomic alterations resulting in hyperactivation of an upstream/
downstream signaling pathway or hyperactivation of mTOR that in turn
provides an analogous impact. mTOR mutations have essential im-
plication not only in the pathogenesis of cancer but also in inducing
resistance to the mTOR inhibitors. These spectra of activating me-
chanisms of mTOR cooperatively play a fundamental role in cell
transformation, growth, survival, increasing tumor volume, neovascu-
larization, and invasion and metastasis and drug resistance, etc.
Therefore, mTOR is an important therapeutic target. Clinical responses
to mTOR inhibitors as monotherapy have been modest and suggest
combination therapies to overcome the resistance mechanisms.
Although the role of mTOR overexpression is clearer as it could result in
a gain-of-function, how the downregulation of mTOR pave the way for
carcinogenesis is not yet well understood and warrant future studies to
uncover these hidden mechanisms. Moreover, the copy number al-
terations were very minimal while most of the studies explored only the
expression of mTOR gene. As treatment strategies are advancing and
continuously being developed towards precision medicine, it is very
crucial to identify various potential predictive biomarkers for treatment
deploying mTOR inhibitors. This would facilitate the effective design of
clinical trials that would accelerate the introduction of these com-
pounds to clinical practice. Hence, patients may benefit from a tailor-
made precision therapy by various mTOR inhibitors alone or in com-
bination with other molecular targeted therapies.
Funding
This study was not funded.
Declaration of Competing Interest
Author, AK Murugan has no conflict of interest to declare except
that he is the guest editor of this special issue, “PI3K/Akt Signaling in
Human Cancer” in Seminars in Cancer Biology.
Acknowledgments
I thank all the researchers in this field whose valuable work pro-
vided me a delightful reading experience and apologize to my collea-
gues whose work could not be cited primarily due to space constraints.
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