Introduction cancerous cells. For instance, high levels of

 

Introduction (involvement
of translation, metabolism and drugs eg KI that affect these processes)

In normal
cells, protein synthesis is a carefully coordinated process involving the
interaction of factors, ribosomes, mRNA, tRNAs and amino acids. Hence, it is
necessary that the process is regulated because different cellular processes (proliferation,
growth, differentiation and development) are affected by it (Hershey,J. W.B et al . Cold Spring Habour 2012, Protein synthesis
and Translational control). It is no surprise that deregulated mRNA translation
is a common feature of cancer because it is implicated in most of the hallmarks
of cancer such as deregulated cell proliferation, survival, angiogenesis and deregulated
cellular energetics (Refer to Bhat Review). It is not a recent observation that
aberrant protein synthesis has been found in malignant cells. Malignant cells
were observed to have enlarged and abnormally shaped  nucleoli over a century ago (Pianese, G. Beitrag, Beitr. Pathol. Anat.
Allgem. Pathol., 1896). In fact, a positive correlation has been observed
between cancer cell proliferation and rates of protein synthesis (Johnson, L.
F., et al J. Cell Biol). Furthermore, several mRNA translation initiation
factors have been found to be amplified and deregulated in transformed cells
(Ruggero D. Cold Spring Harbour Perspect, 2013). Also, several oncogenes and
tumour suppressors that are driver mutations in different types of cancers converge
signalling on the translation machinery (Ruggero D. Cold Spring Harbour
Perspect, 2013) (Vogelstein Bert et al., Science, 2013). Overall, the aberrant
functioning or signalling to the components of the translational machinery
results in translational reprogramming to favour metabolic reprogramming,
angiogenesis, survival, proliferation and metastasis of cancerous cells.  For instance, high levels of eukaryotic
translation initiator factor eIF4E have been linked to chemoresistance and its
levels affect cell cycle progression and neoplastic growth (Larson
O et al., Cancer Res, 2007).  

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            The
mammalian target of rapamycin (mTOR) is the key regulator of translation
initiation (Sonenberg N and Hinnebusch AG, Cell 2009). mRNA translational
control plays a profound role in the regulation of gene expression as it leads
to immediate and effective changes in protein levels (Sonenberg N and
Hinnebusch AG, Cell 2009). This immediate adaptive response to environmental
cues such as stress and nutrient deprivation is essential for cells to survive
(Spriggs KA et al. Mol Cell 2010). The importance of gene expression regulation
at the translational level is evident as steady-state mRNA levels have low
concordance with the proteome (Schwanhausser, B et al., Nature, 2011).

mRNA translation is one of the most energy demanding
cellular processes as it consumes approximately 20 percent of cellular ATP (F
Buttgereit and M.D. Brand, Biochem J 1995). Thus, in order to sustain the rapid
growth and proliferation of cells that correlate with elevated levels of mRNA
translation in malignant cells, the corresponding
adjustments to energy metabolism has to be made. AMP-activated protein kinase (AMPK) acts as the cellular
energy censor which is activated when nutrients or oxygen is in short supply
and results in elevated cellular AMP levels (Kahn et al. Cell Metab 2005, Shaw
Acta Physiol (Oxf) 2009). In effect, activated AMPK results in the
downregulation of cell growth and proliferation via mTORC1 signalling pathway
(Shaw et al. Cancer cell 2004a). Consequently, AMPK/mTORC1 signalling pathway
links cellular metabolic energy to mRNA translation.

Non-transformed cells predominantly depend on mitochondrial
oxidative phosphorylation under aerobic conditions to produce energy in the
form of ATP to fuel various cellular processes. On the other hand, under anaerobic
conditions, glycolysis is heavily relied on by the cells instead of oxidative
phosphorylation for its energy source. However, it was discovered in the 1920s
that cancer cells reprogram their metabolism to depend on glycolysis for their
energy production even in the presence of oxygen (Warburg, 1930). It was
observed, that tumor cells had elevated glucose uptake as well as lactate production
when compared to normal tissues in the presence of oxygen (Warburg 1956a). This
metabolic anomaly is referred to as the Warburg effect or “aerobic glycolysis”.
The metabolic reprogramming has been shown to be accompanied by the
upregulation of glucose transporters such as GLUT1 to increase glucose uptake
into the cytoplasm and compensate for the relatively inefficient utilization of
glycolysis to produce ATP (DeBeradinis, R.J. et al Cell Metab, 2008; Hsu, P.P
and Sabatini, D.M. (2008) Cell). Although, glycolysis is a relatively
inefficient process for producing ATP, ATP is produced at a faster rate compared
to oxidative phosphorylation to fuel the rapid proliferation of cancer cells.
Glycolysis also fuels neoplastic growth through increased biosynthesis of
lipids, nucleotides, NADPH and amino acids (Lunt SY Annu Rev Cell Dev Biol
2011). Furthermore, the lactic acid produced as the end product of aerobic
glycolysis has been found to favor cancel cell invasion (Smallbone K., et al J
Theor Biol 2005). Due to the universality of this feature in
malignant cells, the reprogramming of cellular energy metabolism in order to
facilitate sustained growth and cell proliferation is indeed one of the
hallmarks of cancer (Hanahan and Weinberg, Cell 2011). Furthermore, epigenetic changes and
modifications in the expression of various genes that encode metabolic enzymes
and transporters have been linked to different types of cancers. In vivo, a
tumour microenvironment (such as blood flow, oxygen and nutrient supply) can
cause metabolic plasticity (Dang CV, J Mol Med,2011; Hsu PP and Sabatini DM,
Cell, 20008; Jessani N et al., Prot Natl Acad Sci USA, 2004) and alterations in
the expression of numerous genes encoding metabolic enzymes, transporters and
regulatory effectors have been associated with cancer.

            Intratumour
heterogeneity that exists between malignant cells within a given primary tumour
makes targeted therapeutics inefficient. This is because such therapeutics only
target cancer cells having a specific kind of lesion. Cancer cells without said
mutation survive and thrive. However, since most aberrant signalling pathways
lead to deregulated mRNA translation, which is common to all cancer cells, it
has been thought that targeting the translational machinery not only provides a
better therapeutic window but also bypasses the issue of intratumor
heterogeneity. Although mRNA translation is a series of highly regulated steps:
initiation, elongation, termination and ribosome recycling) the rate limiting
step is initiation (Hershey, J. W. et sl, 2012 Cold Spring Habour 2012). mRNA
translation initiation is a highly coordinated process involving the formation
of the eIF4F complex on the mRNA cap and the formation of the 43S preinitiation
complex (PIC) (Sonenberg and Hinenbusch, Cell 2009). Thus, there
are a lot of therapeutics that exist targeting the initiation phase of
translation. Lots of therapeutic agents prevent the assembly of the eIF4F
complex. There are 4E
antisense oligonucleotides (ASOs) that effectively target 4E mRNAs and break it
down in cancers where the eIF4E is hyperactivated (Graff et al., J Clin Invest, 2007). Also, the assembly of the eIF4F can be targeted indirectly
through the use of inhibitors that block the signal cascades that eventually
lead to protein synthesis such as mTOR kinase inhibitors (rapalogs) (Roux and
Topisirovic, Cold Spring Harbour Perspective Biol 2012).

An alternate/accompanying
approach to treat different types of cancers involves targeting the cellular
metabolic processes. The metabolic processes and rewiring of malignant cells are
different from normal cells. Hence, targeting these processes offers an attractive route to treat cancers.
A lot of drugs designed exploit
the fact that most cancer cells are more dependent on aerobic glycolysis,
fatty acid synthesis and glutaminolysis for energy, generating building blocks
for neoplastic growth and proliferation (Vander Heiden, Cantley and Thompson,
Science 2009). For example, WZB117 is an inhibitor of GLUT1  glucose transporter that results in reduced
glucose uptake, downregulation of glycolysis and reduction in cellular growth
(Liu  Y et al Mol Cancer Therapeutics
2012). Overall, targeting two major reprogramming processes in cancer cells
simultaneously may be monumental in the treatment of certain types of cancers.

 

In this review, we highlight
various instances whereby kinase inhibitors impinged on the translational and
metabolic reprogramming in malignant cells via the mTORC1/4EBP1 axis.

 

PI3K/AKT

The phosphotadylinositol-3-kinase
(PI3K)/AKT/mTOR signaling pathway is an essential regulator for many normal
physiological processes including cell growth, translation, proliferation,
survival, apoptosis and metabolism (Yao R, Cooper GM Science. 1995, Kauffmann-Zeh
A, Nature, 1997 and  Laplante M and
Sabatini DM Cell Science 2009). However, dysregulated signaling of this pathway
has been implicated in pathological conditions including diabetes and numerous types
of cancer whereby its hyperactivation results in the survival and proliferation
of malignant cells (Porta C, Paglino C., Mosca A. Front Oncol 2014 and Laplante
M and Sabatini DM 2012 Cell).

 

            The signal cascade is activated when receptor tyrosine
kinases bind to hormones such as insulin and/or growth factors (Ruggero D and
Sonenber N., Oncogene, 2005). The extracellular binding of the ligands results
in intracellular autophosphorylation process of tyrosine residues found within
the receptors. The phosphorylated tyrosine residues recruits PI3K, which is a
lipid kinase, to the membrane. At the membrane, PI3K phosphorylates
phosphatidyl inositol- 4,5-biphosphate (PIP2) to produce
phosphatidyl inositol-3,4,5-triphosphate (PIP3).  PIP3 then acts as a second messenger
and is responsible for translocating and activating downstream signaling proteins
such as Akt/protein kinase B (PKB) to the cell membrane (Fresno-Vara JA, Casado
E, De Castro J et al. Cancer Treat 2004). Akt is a serine/threonine protein
kinase known to regulate cell survival, growth and proliferation (Wan X,
Harkavy B et al. Oncogene 2007, Myers AP, Cantley LC, Sci Transl Med 2010). Its
hyperactivity due to genetic mutations and amplification, loss of upstream
regulators and mitogenic factors have been implicated in different types of
cancer (Cheng JQ et al., Oncogene 2005 and Malanga D et al, 2008 Cell Cycle).
As Akt is involved in many essential cellular process, it is able to carry out
its functions through various proteins including mTOR protein complexes that
are known to act both upstream and downstream of Akt (Slomovitz BM , Coleman RL
Clin Cancer Research 2012).

 

Considering that the PI3K/Akt
signaling pathway is used in many functions, there is need for the pathway to
be properly regulated. One of the major intrinsic negative regulator of this
pathway is phosphatase and tensin homologue deleted on
chromosome ten (PTEN). Although it has both lipid and protein phosphatase activities,
it represses the PI3K/Akt pathway through its lipid phosphatase activity. PTEN
catalyzes the conversion of PIP3  to PIP2. In
essence, the loss of PIP3
which is the second messenger in the PI3K/Akt signaling pathway results indeed
results in the repression of the pathway. Conversely, the loss of activity of
PTEN leads to hyperactivation of the signaling pathway. PTEN is widely regarded
as a tumour suppressor whereby the loss of its activity have been linked to
deregulation of cell growth and proliferation commonly observed in different
types of cancers (Simpson L, Parson R., Exp Cell Res 2001).

 

mTOR signaling (cross talk with
MAPK/ERK pathway) (Silvera
Nature Review)

The mechanistic/mammalian
target of rapamycin (mTOR) is a conserved serine/threonine kinase that is part
of the phosphoinositide kinase related family of protein kinases. It functions
downstream of the PI3K/Akt signaling pathway by integrating extracellular and
intracellular signals in order to detect environmental cues, monitor nutrient
availability and cellular energetic status (Liu P et al.  Nat Rev Drug Discov. 2009 and Zhou H Huang S.
Curr Protein Pept Sci 2011). This is necessary to regulate cellular processes
including cell growth, proliferation, protein synthesis, survival, autophagy
and metabolism (Shimobayashi, M and Hall, M.N., Nat. Rev. Mol. Cell Biol 2014).
mTOR is the catalytic subunit of 2 functionally and structurally distinct
multiprotein complexes: mTORC1 and mTORC2. Both complexes contain three common
protein subunits which are: mTOR, DEP-domain-containing
mTOR-interacting protein (Deptor) and mammalian lethal with Sec13 protein 8
(mLST8, also referred to as G?L). Furthermore, Raptor (regulatory protein
associated with mTOR) and PRAS40 (proline-rich Akt substrate of 40 kDa)
are found as accessory proteins part of the mTORC1 complex while mTORC2 complex
contains Rictor (rapamycin insensitive companion of mTOR) and regulatory
subunits mSin1(stress-activated MAP
kinase-interacting protein 1) (Sabatini DM Nat Rev Cancer 2006 and Sabatini
DM Cell 2012). mTORC1 complex is primarily known to control cell growth and
proliferation through the regulation of protein synthesis (Hay N and Sonenberg
N, Genes Dev  2004). It is also involved
in regulating various other cellular metabolic processes and autophagy (Kim, J.
et al. Nat. Cell Biol 2011 and Duvel, K et al Mol Cell 2010).  As a result of the PI3K/Akt pathway
transmitting signals upstream of the mTORC1 complex, the activity of the
complex is predominantly modulated by the same regulators of the PI3K/Akt
pathway (Hay N and Sonenberg N, Genes Dev 
2004). Upon activation of the pathway, Akt catalyzes the phosphorylation
of Tuberous Sclerosis Complex (TSC) 2 (also referred to as tuberin) which
heterotrimerise with TSC1 and TBC1D7. Phosphorylation leads to the inhibition
of the protein complex. Since the TSC is a GTPase Activating Protein complex
for Ras homolog enriched in the brain (Rheb), which is a GTPase, the inhibition
of TSC2 results in Rheb being bound to GTP and active. GTP-bound Rheb directly
interacts with mTORC1 complex in order to activate it (Lon et al. 20005 and
Sancak et . al 2007). Signaling to the mTORC1 complex is abrogated once Rheb is
bound to GDP due to the upstream activity of TSC2. Hence, similar to the role
of PTEN in the PI3K/Akt pathway, TSC2 is regarded as a repressor of the mTORC1
signaling. Thus, genetic mutations in the TSC1/2 tumour suppressor
genes results in tuberous sclerosis which is associated with benign tumours
commonly found in the brain, kidneys, heart, lungs, and skin (Samy
L Habib et al. J Cancer 2016). Other than Akt which is indirectly activated by
growth factors, hormones and cytokines, the TSC1/TSC2 complex also integrates other
upstream signals from different proteins to mediate different cellular
processes via mTORC1 complex. One of such proteins is AMP-activated protein
kinase (AMPK) which monitors the intracellular energy level. (Hardie, D. G.
2007 Nat Rev Mol Cell Biol). Glucose deprivation leads to a high AMP:ATP ratio
which activates AMPK to phosphorylate TSC2 and indirectly shutting off mTORC1
signaling (Inoki K. et al. Cell 2006). However, AMPK can also directly inhibit
mTORC1 signaling by phosphorylating the RAPTOR subunit of the protein complex
(Gwinn D. M. et al. Mol. Cell 2008). Hypoxic conditions which impairs
mitochondrial functions leads to a high AMP:ATP ratio that results in the
repression of mTORC1 signaling through the activation of AMPK. The TSC2 complex
also mediates the effects of hypoxia in an AMPK-independent manner through its direct
activation by transcriptional regulation of DNA damage response 1 (REDD1)
(Brugarolas, J et al. Genes Dev 2004). Other important signal transduction
pathways also converge on the TSC2 complex to exert their effects on mTORC1.
The Ras-Raf-MEK-Erk signaling pathway which is activated by growth factors can
directly phosphorylate TSC2 and indirectly through p90 ribosomal S6 kinase 1
(RSK1) leading to stimulation of the mTORC1 complex (Memmott RM and Dennis
Cell Signalling 2009 and PA Roux et al 2004 Proc. Natl. Acad. Sci. USA). In fact, it has been shown that inhibition
of mTORC1 leads to the activation of the Ras-Raf-MEK-Erk signaling
pathway (Carracedo, A et al J. Clin Invest. 2008). Although
the TSC1/TSC2 complex integrates signals from different pathways to mTORC1
complex,  Akt activation can bypass it by
phosphorylating PRAS40 from the mTORC1 complex (Sancak Y et al. Mol Cell 2007).

 

Unlike the mTORC1 complex,
the functions of mTORC2 complex have not been fully characterized in both
normal and pathophysiological states. Nonetheless, mTORC2 is known to regulate
cell survival, cytoskeletal organization and cell migration (Oh W. J.and
Jacinto E., Cell Cycle. 2011, Populo H, et al. Int J Mol Sci. 2012). The
complex is able to control some of these cellular process through the
phosphorylation of Akt at Ser 473 in order to activate it (Sarbassov D.D. et
al. Science 2005). Akt belongs to the AGC family of kinases and shares homology
with another member of the family  serum- and
glucocorticoid-induced protein kinase 1 (SGK1) that is also directly
phosphorylated and regulated by the mTORC2 complex (Garcia Martinez J.M and
Alessi D.R. 2008 Biochem. J. ). Protein Kinase C (PKC) is also an AGC kinase
that is a known downstream target of the mTORC2 complex. PKC has been
implicated in actin reorganization upon phosphorylation by the mTORC2 complex
(Sarbassov D.D. Curr. Biol 2004).

 

mTORC1 (downstream of mTORC1
complex) in controlling mRNA translation/Protein synthesis independently via
4ebp1 and S6K- 4e
sensitive mRNA and TOP mRNAs

mRNA translation is one of the major anabolic cellular
processes regulated by the mTORC1 complex. It is divided into 4 main steps: initiation,
elongation, termination and ribosome recycling (Hershey et al. Cold Spring
Harb. Perspect. Biol 2012, and Mathews et al. Cold Spring Harbor
Laboratory Press 2007).
There is evidence that implicates mTORC1 complex as a regulator of both
translation initiation and elongation processes. Active mTORC1 complex
phosphorylates translation suppressors
eukaryotic translation initiation factor 4E-binding proteins at various
sites (4EBP1-3) (Thoreen C.C. et al. Nature 2012 and Burnett P.E. et al. Proc
Natl Acad Sci 1998). Unphosphorylated 4EBPs sequester translational eukaryotic initiation factor 4E (eIF4E) (mRNA
5′ cap-binding protein) and prevents it from associating with other initiation
factors (eIF4A- A DEAD box helicase and eIF4G-a scaffold protein) to assemble
into the eIF4F complex that is required for recruiting
mRNA to the ribosome which is essential for initiating cap-dependent mRNA translation (Thoreen C.C. et al.
Nature, Pause et al., 1994. Nature,
Gingras A.C. et al. Genes Dev 1999). Hence, when 4EBPs are phosphorylated,
eIF4E is released to form the active eIF4F complex (Sonenberg N and Hinnebusch
A.G. Cell 2009).

The mTORC1 complex is also
known to control protein synthesis through the phosphorylation and activation
of ribosomal S6 kinases (S6Ks- S6K1 and S6K2) (Roux and Topisirovic 2012 Cold
Spring Harb Perspect). Activated S6Ks results in the phosphorylation of ribosomal
protein S6 (RPS6-a component of the 40S ribosomal subunit), eIF4B (an activator
of the eIF4F complex) and programmed cell death 4 (PDCD4-a negative regulator
of the eIF4F complex) (Holz et al. Cell 2005, Chauvin C et al. Oncogene 2014
and Dorrello, N.V. et al Science 2006). Although the function of RPS6 is mostly
unknown, S6Ks influence mRNA translation initiation by activating eIF4A through
two ways: activating its binding partner eIF4B and phosphorylating and
degrading its negative regulator to release it from PDCD4-eIF4F complex
(Dorrello, N.V. et al Science 2006 and Dennis M.D. 2012 J. Biol Chem.). S6Ks
not only mediate the effects of mTORC1 on translation initiation, they also
influence translation elongation. S6Ks phosphorylate and inactivate eukaryotic
elongation factor (eEF2) kinase. This prevents the phosphorylation and
repression of eEF2 on the Thr56 residue, thus enhancing translation elongation
by stimulatin ibosome association and translocation (Wang, X et al, 2001 EMBO
J., Carlberg et al., 1990 Eur. J. Biochem).

 

 

Protein synthesis TOP mRNAs,
TISU? (how inhibition of mRNA translation affect these?)

Although the activation of
the mTORC1 complex correlates with increased global protein synthesis, its
influence on the translation of mRNAs is not equally distributed. Different
elements of mRNAs tend to affect their translation efficiencies and degree of influence
by mTORC1 complex. Some mRNAs are commonly referred to as “eIF4E-sensitive”
because they are highly dependent on the formation of the eIF4F complex
composed of the mRNA 5′ cap-binding protein and a helicase (Sonenburg N and
Hinnebusch 2009 Cell). These mRNAs mostly have long and highly structured,
G/C-rich 5′ UTRs and require the helicase activity of eIF4A to unravel them
(Silvera D., et al Nat Rev Cancer 2010, Koromilas AE, et al., EMBO J., 1992,
Svitkin Y.V. et al, RNA 2001)). Therefore, eIF4A allows efficient recruitment
of the pre-initiation complex to the mRNAs. Some of these mRNAs encode cell
cycle regulators such as cyclins (cyclin D1) and cyclin dependent kinase (CDK-2),
anti-apoptotic and pro-survival proteins (B-cell lymphoma 2 (Bcl-2), Myeloid
cell Leukemia 1 (Mcl-1), Bcl-xL and survivin), oncogenes (c-Myc, Pim1  signal activator and
transducer of transcription 3 (STAT3) and other proteins critical
to cell proliferation (osteopontin and ornithine decarboxylase.
(Martelli A.M.,
et al., Leukemia 2011, Martelli A.M., et al Oncotarget. 2012, De Benedetti A.,
Graf J.R.,Oncogene 2004 and Mamane Y., et al Oncogene, 2004). Furthermore, it has been shown that
mTOR inhibitors reduced the translation efficiency of some of the previously
mentioned eIF4E-sensitive transcripts compared to mRNAs of “housekeeping
genes” that have relative short 5′ UTR structures (Larsson O., et al., Proc
Natl Acad Sci., 2012).

 

1ncrease
in eIF4E availability is thought to selectively stimulate translation of those
mRNAs that critically depend on the dissolution of 5?UTR secondary structures
by eIF4A. (Feoktistova K., Tuvshintogs E., Do A., Fraser CS.. Human
eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc Natl Acad Sci U S A 2013;

)

 

Other than eIF4E sensitive mRNAs, mTOR preferentially
enhances the translation of another subset of mRNAs bearing a series of 4-14
pyrimidines following the C nucleotide found immediately
after the 5′ 7 methyl guanosine cap structure (Meyuhas, O and Dreazen, A.,
Prog. Mol. Biol. Transl. Sci 2009). This motif is referred to as the 5?
terminal oligopyrimidine (5? TOP) motif. Some of the TOP mRNAs encode
proteins important for translation such as ribosomal proteins, eEF2 and poly (A)-binding
protein (PABP) and are very sensitive to
mTOR inhibitors (Meyuhas, O and Dreazen, A., Prog. Mol. Biol. Transl. Sci 2009 Hsieh,
A. C. et al., Nature 2012 ). Initially, it was proposed that S6, S6Ks mediated
the regulatory effects of mTOR on the translation of TOP mRNAs (Jefferies et
al. 1994, 1997). Subsequently, it was found that there was no difference in the
translation of TOP mRNAs when cells deficient in S6K and S6 were compared to
wild type cells (Pende M., et al. Mol Cell Biol. 2004 and RuvinskyI., et al.
Genes Dev 2005). However, there are different studies that implicate several factors
as important mediators of mTOR regulation on the translation of TOP mRNA
transcripts such as La-related protein 1 (LARP1), TIA(T-cell-restricted
Intracellular Antigen-1

)/TIAR-1(TIA-1-related
protein) and 4EBPs (Thoreen
C.C. et al, Nature 2012, Hsieh A.C. et al., Nature 2012 and Damgaard C.K. et
al. Genes Dev 2011). Furthermore, the context in which translation takes place
is known to affect the translation of TOP mRNAs (Miloslavski et al., J Mol Cell
Biol 2014).

 

Write
about TISU? (No established direct connection between TISU and mTOR yet)

 

mTOR as a regulator of metabolism

Talk about the various metabolic processes controlled by
mTOR (amino acids, autophagy, glucose)

Talk about the effect of mTOR on mitochondria
(mitochondria biogenesis and vice versa (mitochondria as a power house and
producer of ATP, AMPK as a censor AMP levels which is required for fueling translation).

x

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