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mTOR and Regulation of Translation

Chapter · October 2014


DOI: 10.1007/978-94-017-9078-9_15

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Chapter 15
mTOR and Regulation of Translation

Yoshinori Tsukumo, Mathieu Laplante, Armen Parsyan, Davide Ruggero and


Bruno Fonseca

Contents

15.1 Introduction ������������������������������������������������������������������������������������������������������������������ 308
15.2 Mechanistic and Functional Aspects of mTOR and Its Signaling ������������������������������� 308
15.3 mTORC1 Pathway and Translational Control ������������������������������������������������������������� 312
15.3.1 4E-BPs ������������������������������������������������������������������������������������������������������������ 312
15.3.2 S6 Kinase ������������������������������������������������������������������������������������������������������� 314
15.3.3 eIF4G �������������������������������������������������������������������������������������������������������������� 315
15.3.4 eEF2K ������������������������������������������������������������������������������������������������������������ 316
15.4 Translation and mTORC1 in Cell Proliferation and Growth ��������������������������������������� 316
15.5 Translation and mTORC1 in Invasion and Metastasis ������������������������������������������������ 317
15.6 Translation and mTORC1 in Apoptosis and Tumor Survival �������������������������������������� 318
15.7 mTOR Pathway and Translational Regulation in Etiology and
Pathogenesis of Cancer ������������������������������������������������������������������������������������������������ 319
15.8 Targeting the mTOR Pathway in Cancer ��������������������������������������������������������������������� 320
15.8.1 Renal Cell Carcinoma ������������������������������������������������������������������������������������ 321
15.8.2 Pancreatic Neuroendocrine Tumors ��������������������������������������������������������������� 321
15.8.3 Tuberous Sclerosis Complex and Subependymal Giant-Cell Astrocytomas ��� 322
15.8.4 Mantle Cell Lymphoma ���������������������������������������������������������������������������������� 322
15.8.5 Sarcoma ���������������������������������������������������������������������������������������������������������� 323
15.8.6 Diffuse Large B-Cell Lymphoma ������������������������������������������������������������������� 323
15.8.7 Breast Cancer ������������������������������������������������������������������������������������������������� 323

Y. Tsukumo () · B. Fonseca
Department of Biochemistry, Rosalind and Morris Goodman Cancer Research Centre, McGill
University, Montreal, QC, Canada
e-mail: yoshinori.tsukumo@mcgill.ca
M. Laplante
Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec
(CRIUCPQ), Faculté de Médecine, Université Laval, Quebec City, QC, Canada
A. Parsyan
Division of General Surgery, Department of Surgery, Faculty of Medicine, McGill University,
Montreal, QC, Canada
D. Ruggero
Helen Diller Family Comprehensive Cancer Center, Department of Urology, School of Medicine,
University of California San Francisco, San Francisco, CA, USA
A. Parsyan (ed.), Translation and Its Regulation in Cancer Biology and Medicine, 307
DOI 10.1007/978-94-017-9078-9_15, © Springer Science+Business Media Dordrecht 2014
308 Y. Tsukumo et al.

15.9 New Compounds Targeting the mTOR Pathway ��������������������������������������������������������� 324


15.9.1 Reasons for Low Therapeutic Efficacy of Rapalogs �������������������������������������� 324
15.9.2 ATP-Competitive mTOR Inhibitors ��������������������������������������������������������������� 325
15.9.3 Dual mTOR/PI3K Inhibitors �������������������������������������������������������������������������� 326
15.9.4 Other Compounds ������������������������������������������������������������������������������������������ 327
15.10 mTOR Inhibition and Therapeutic Resistance and Radioresistance �������������������������� 327
15.11 Conclusions and Perspectives ������������������������������������������������������������������������������������ 330
References ������������������������������������������������������������������������������������������������������������������������������� 330

Abstract  The PI3K/AKT/mTOR pathway is implicated in various cellular events


including translation, cell proliferation, growth and metabolism, and is frequently
dysregulated in cancer. In this chapter, we unveil how the mTOR pathway regu-
lates the translation machinery and how dysregulation of translation via this path-
way participates in cancer biology. We also discuss the clinical and therapeutic
importance of the control of translation by the mTOR signaling in various types of
cancers.

15.1 Introduction

Mammalian target of rapamycin (mTOR), also known as mechanistic target of ra-


pamycin, is a well-conserved serine/threonine kinase that, as a key component of
the phosphoinositide 3-kinase (PI3K)/v-Akt murine thymoma viral oncogene ho-
molog (AKT, also known as PKB or protein kinase B)/mTOR signaling pathway
(hereinafter mTOR pathway), regulates a variety of cellular processes, including
protein and lipid synthesis, transcription and ribosome biogenesis, impacting on
cell proliferation, growth, motility, apoptosis and autophagy in normal physiology
and a variety of diseases, such as cancer, obesity, type 2 diabetes and neurological
disorders (see Laplante and Sabatini 2009, 2012). Notably, the mTOR pathway is
frequently dysregulated in human cancers (Guertin and Sabatini 2005). A descrip-
tion of the mTOR pathway that contains dozens of proteins and hubs deserves a
separate publication. Importantly, however, this pathway converges on the control
of translation. This chapter will introduce the role of mTOR in regulation of protein
synthesis in physiology and cancer.

15.2 Mechanistic and Functional Aspects of mTOR and


Its Signaling

As reflected by its abbreviated name, TOR is a target of a compound named


rapamycin, a macrolide produced by the bacterial species Streptomyces hygro-
scopicus, which was isolated from a soil sample collected on the Easter Island
(Rapa Nui) in the 1970s (Vezina et al. 1975). mTOR is a large (290 kDa) protein
and a member of the PI3K-related kinase (PIKK) family. Despite its homology to
15  mTOR and Regulation of Translation 309

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Fig. 15.1   TOR structure. The N-terminus of target of rapamycin (TOR) contains tandem HEAT
repeats that are important for protein–protein interactions. The FAT and FATC domains modulate
the activity of the kinase domain. The FRB domain is the docking site of the FKBP12–rapamycin
complex.

PI3Ks and PI4Ks, mTOR has not been demonstrated to have lipid kinase activ-
ity, and it essentially functions as a Ser/Thr protein kinase (Benjamin et al. 2011).
mTOR contains multiple domains, which are required for a large functional com-
plex formation (Fig. 15.1). The N-terminus contains up to 20 tandem HEAT re-
peats (a protein–protein interaction domain). These are followed by a FAT (FRAP,
ATM, TRRAP) domain. The C‑terminal portion of mTOR contains the kinase do-
main and the FKBP12–rapamycin-binding (FRB) domain. The C-terminus also
contains a FATC (FRAP, ATM, TRRAP C-terminal) domain that is paired with
the upstream FAT domain in all PIKKs to modulate kinase activity in an unknown
manner (Fig. 15.1).
mTOR interacts with many proteins to form two distinct multiprotein complex-
es: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Laplante and
Sabatini 2012). Both mTOR complexes share a catalytic mTOR subunit, as well as
mammalian lethal with sec-13 protein 8 (mLST8), DEP domain-containing mTOR-
interacting protein (DEPTOR) and the TTI1/TEL2 complex (Laplante and Saba-
tini 2012). In contrast, the regulatory-associated protein of mTOR (RAPTOR) and
proline-rich AKT substrate 40 kDa (PRAS40) are specific to mTORC1, whereas
rapamycin-insensitive companion of mTOR (RICTOR), mammalian stress-activat-
ed MAPK-interacting protein 1 (mSIN1), and protein observed with RICTOR 1 and
2 (PROTOR1/2) are only part of mTORC2 (Laplante and Sabatini 2012). Impor-
tantly, each mTOR complex has different sensitivities to rapamycin and regulates
distinct biological processes (Fig. 15.2) (Laplante and Sabatini 2012).
The activity of the mTORC1 complex is governed by five major signals: (1)
growth factors, (2) stress, (3) energy level, (4) oxygen and (5) amino acids. These
signals aim at regulating many processes involved in cell growth and proliferation
(Fig. 15.2). With an exception of amino acids, these signals regulate mTORC1 by
modulating the activity of TSC1/2. TSC1/2 negatively regulates mTORC1 activ-
ity by converting a GTPase, RAS homolog enriched in brain (RHEB) (Inoki et al.
2003a), into its inactive GDP-bound form. Growth factor stimuli inhibit TSC1/2
function by phosphorylation through the upstream signaling pathways PI3K/AKT or
RAS/MAPK, resulting in the activation of mTORC1 (Fig. 15.2). Like growth factor
inputs to mTORC1, many stress stimuli, including low oxygen or energy levels or
presence of DNA damage, signal to mTORC1 through TSC1/2. In response to hy-
310 Y. Tsukumo et al.

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Fig. 15.2   mTORC1 and mTORC2 complexes. The mTOR kinase forms two distinct protein
complexes called mTORC1 and mTORC2. mTORC1 responds to growth factor stimuli, oxygen,
energy, stress and amino acids. mTORC1 regulates translation via the best characterized substrates
4E-BPs and S6Ks; lipid biogenesis by controlling function or expression of PPARγ and SREBP1/2
at the multiple levels (Duvel et al. 2010; Kim and Chen 2004; Li et al. 2010; Porstmann et al. 2008;
Wang et al. 2011a; Zhang et al. 2009); lysosome biogenesis via nuclear translocation of a transcrip-
tion factor TFEB, that controls many genes with key roles in lysosomal function (Settembre et al.
2013); energy metabolism via glucose metabolism by activating transcription and translation of
HIF-1α, which is a positive regulator of many glycolytic genes (Brugarolas et al. 2003; Duvel et al.
2010; Hudson et al. 2002; Laughner et al. 2001), or via mitochondrial oxidative metabolism, by
mediating the nuclear association between PPARγ coactivator 1α (PGC1α) and the transcription
factor Ying-Yang 1 (YY1), which positively regulate mitochondrial biogenesis and oxidative func-
tion (Cunningham et al. 2007); and autophagy via phosphorylation of its substrates, the ULK1/
ATG13/FIP200 complex and DAP1 (Ganley et al. 2009; Hosokawa et al. 2009; Jung et al. 2009;
Koren et al. 2010). mTORC2 responds to growth factors and regulates cell survival by phosphory-
lating and activating AKT and SGK1 (Garcia-Martinez and Alessi 2008; Sarbassov et al. 2005),
and cytoskeletal organization by affecting the actin cytoskeleton via activation of PKCα, as well as
other effectors, such as paxilin and RHO GTPases in cell type-specific fashion (Jacinto et al. 2004;
15  mTOR and Regulation of Translation 311

poxia or a low energy state, AMP-activated protein kinase (AMPK) phosphorylates


TSC2 and increases its GAP activity toward RHEB (Inoki et al. 2003b). Like AKT,
AMPK also communicates directly with mTORC1 through RAPTOR phosphoryla-
tion, leading to 14-3-3 binding and the allosteric inhibition of mTORC1 (Gwinn
et al. 2008). Hypoxia also induces the expression of REDD1 (regulated in develop-
ment and DNA damage responses 1), which activates TSC2 function in a still poorly
understood manner (Brugarolas et al. 2004; DeYoung et al. 2008; Reiling and Hafen
2004). DNA damage also signals to mTORC1 through p53-dependent induction of
TSC2 and PTEN, causing downregulation of the entire PI3K/mTORC1 axis (Feng
et al. 2005; Stambolic et al. 2001), and activates AMPK through a mechanism that
depends on the induction of sestrin 1/2 (Budanov and Karin 2008). Unlike other
stimuli, amino acids regulate mTORC1 independently of TSC1/2, via a mechanism
that depends on the RAS-related GTPase (RAG), a group of proteins composed
of RAG A, B, C and D (Kim et al. 2008; Sancak et al. 2008). Under amino acid
starvation conditions, inactive mTORC1 is diffusely dispersed throughout the cyto-
plasm. Amino acid stimulation signals to vacuolar H + -ATPase (V‑ATPase), which
is required to induce the GEF activity of RAGULATOR, a multisubunit complex
that activates the RAGs by promoting the GTP loading of RAG A/B (Sancak et al.
2010). When active, the RAG complex interacts with mTORC1, which promotes its
translocation from the cytoplasm to lysosomal membranes, where RHEB is thought
to activate mTORC1 (Laplante and Sabatini 2012; Zoncu et al. 2011).
mTORC1 regulates a variety of cellular events (such as translation, lipid synthe-
sis, lysosome biogenesis, autophagy and energy metabolism) via multiple down-
stream targets (Fig. 15.2). Activated mTORC1 promotes the translation initiation
process by phosphorylating two major targets: ribosomal protein S6 kinase (S6K)
and the translation regulator eIF4E-binding proteins (4E-BPs) (Fig. 15.2) (Gingras
et al. 2001; Laplante and Sabatini 2012; Ma and Blenis 2009; Silvera et al. 2010;
Sonenberg and Hinnebusch 2009). mTORC1 also induces lipid biogenesis by ac-
tivating sterol regulatory element-binding protein 1 (SREBP1) and PPARγ tran-
scription factors (Fig. 15.2) (Laplante and Sabatini 2009). In addition to promoting
anabolism, mTORC1 also inhibits catabolism by blocking autophagy through the
phosphorylation of the ULK1/ATG13/FIP200 complex (UNC-51-like kinase 1/au-
tophagy-related 13/FAK-interacting protein of 200 kDa) (Fig. 15.2) (Laplante and
Sabatini 2012; Settembre et al. 2013). Importantly, sustained mTORC1 activation
blocks growth factor signaling by activating various negative feedback loops that
inhibit PI3K signaling (Laplante and Sabatini 2009, 2012).
Compared to mTORC1, much less is known about mTORC2. mTORC2 is activated
by growth factors through a mechanism that is not well understood. mTORC2 has been
shown to regulate cell survival, metabolism, motility and cytoskeletal organization by

Sarbassov et al. 2004). Rapalogs inhibit mTORC1 activity, but not mTORC2. However, chronic
exposure to the drug can reduce mTORC2 activity. Active-site mTOR inhibitors (asTORi) inhibit
both mTORC1 and mTORC2 complexes more strongly than rapalogs. Specific components or
substrates of mTORC1 or mTORC2 are shown in red or green. Common components are shown
in purple. See List of Abbreviations.
312 Y. Tsukumo et al.

phosphorylating many AGC kinases, including AKT, serum/glucocorticoid regulated


kinase 1 (SGK1), and PKCα (Laplante and Sabatini 2012; Zoncu et al. 2011). In con-
trast to mTORC1, mTORC2 is insensitive to acute treatment with rapamycin. Never-
theless, reports indicate that prolonged exposure to rapamycin blocks mTORC2 func-
tion in certain cell lines (Sarbassov et al. 2006). Hence in this chapter and throughout
the book, unless otherwise specified, we refer to mTORC1 by referencing to mTOR.

15.3 mTORC1 Pathway and Translational Control

Components of the translation machinery are the well-studied targets of mTORC1.


Translation initiation is a rate-limiting step commencing with ribosome recruitment
to the 5′ end of an mRNA and ending with an elongation competent 80S ribosom-
al complex positioned at an initiation codon (Gingras et al. 1999b) (see Chap. 2).
Ribosome binding is facilitated by a number of translation initiation factors that
recruit the ribosome to the mRNA 5′ end. The 5′ end of mRNAs possesses a cap
structure (m7GpppN, in which “m” represents a methyl group and “N” represents,
any nucleotide) that is specifically recognized by eIF4E. eIF4E is a part of a tri-
meric complex referred to as eIF4F that also includes scaffolding protein eIF4G and
RNA helicase eIF4A (Gingras et al. 1999b; Parsyan et al. 2011; Sonenberg and Hin-
nebusch 2009). Following its binding to the 5′ cap, eIF4A activity within eIF4F is
thought to unwind the mRNA 5′ proximal secondary structure to facilitate the bind-
ing of the 40S ribosomal subunit and its subsequent 5′ to 3′ scanning towards the
initiation codon. The scanning process is thought to be enhanced by other proteins,
such as the eIF4A interacting partner eIF4B and a RNA helicase, DHX29 (Gingras
et al. 1999b; Parsyan et al. 2011; Sonenberg and Hinnebusch 2009) (see Chap. 5).
Importantly, mTORC1 directly and indirectly regulates several factors involved in
translation initiation, such as 4E-BPs, eIF4G, S6K, rpS6, eIF4E, eIF4B, and elonga-
tion factor eEF2 (see Fig. 15.3 and Sonenberg and Hinnebusch 2009).

15.3.1  4E-BPs

The interaction between eIF4E and eIF4G is regulated by the 4E-BPs. These pro-
teins are translational repressors. In mammals, there are three 4E-BP variants: 4E-
BP1, 4E-BP2 and 4E-BP3, each encoded by a distinct gene (Pause et al. 1994; Pou-
lin et al. 1998). 4E-BPs are negatively regulated through phosphorylation at mul-
tiple sites by mTORC1. Hypophosphorylated 4E-BPs compete with eIF4G proteins
for an overlapping binding site on eIF4E and block the eIF4F initiation complex
formation, while the hyperphosphorylated 4E-BPs dissociate from eIF4E, resulting
in the eIF4F formation and promoting translation (Haghighat et al. 1995; Mader
et al. 1995; Marcotrigiano et al. 1999). Most of the studies on the roles of these
proteins in translation and their control were conducted with 4E-BP1, although this
15  mTOR and Regulation of Translation 313

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Fig. 15.3   Oncogenic signals promote translation of specific mRNAs and global changes in protein
synthesis. Hyperactivation of PI3K, AKT, c-MYC or loss of PTEN and TSC1/2 tumor suppresser
genes promote protein synthesis by coordinating the regulation of translation initiation, translation
elongation and ribosome biogenesis. Activated mTORC1 phosphorylates and inactivates 4E-BP1,
resulting in stimulating translation initiation by eIF4E. Furthermore, mTORC1 also phosphory-
lates S6K and activates translation initiation and elongation. c-MYC promotes protein synthe-
sis by increasing the transcription of multiple translational components including eIF4E mRNA
and by promoting rRNA synthesis (Barna et al. 2008; Hannan et al. 2003; Martin et al. 2004;
Mayer et al. 2004; Ruggero 2009). Together, oncogenic activation of the mTOR pathway as well
as MYC leads to promoting both translation of specific mRNAs and global changes in protein
synthesis. mTORC1 controls also many negative feedback loops that regulate RTK/PI3K signal-
ing. mTORC1 directly, or indirectly via S6K1, phosphorylates IRS-1 to promote its degradation,
resulting in reducing the ability of growth factors to signal downstream of RTK (Harrington et al.
2004; Tzatsos and Kandror 2006; Um et al. 2004). Additionally, mTORC1 directly phosphorylates
growth factor receptor-bound protein 10 (GRB10), which is a suppressor of RTK signaling (Hsu
et al. 2011; Yu et al. 2011). Owing to the existence of these negative feedback loops, mTORC1
inhibition can activate the RTK/PI3K signaling pathway and promote cancer cell survival. Nega-
tive regulators are shown in blue.
314 Y. Tsukumo et al.

isoform is not the most abundant one uniformly in all tissues. For example, 4E-BP2
is the major form in the mammalian brain and has an important role in long-lasting
synaptic plasticity, learning and memory (Banko et al. 2005; Gkogkas et al. 2013;
Tsukiyama-Kohara et al. 2001). 4E-BP3 appears to be expressed broadly but more
abundantly in the human skeletal muscle, kidney and pancreas (Poulin et al. 1998).
Differences in the kinetics and phosphorylation sites have been observed among the
three 4E-BPs (Tee and Proud 2002; Wang et al. 2005).
mTORC1 directly phosphorylates two priming sites on 4E-BP1, Thr37 and
Thr46 (Brunn et al. 1997; Gingras et al. 2001). This phosphorylation event is
required for subsequent phosphorylation of Thr70, followed by Ser65, ultimately
resulting in the release of 4E-BP1 from eIF4E (Gingras et al. 1999a; Heesom and
Denton 1999; Mothe-Satney et al. 2000). Biological and clinical importance of
mTORC1-mediated 4E-BPs phosphorylation has been supported by a variety of
studies (Alain et al. 2012; Dowling et al. 2010; Hsieh et al. 2010). 4E-BP1/2 DKO
MEFs continue to proliferate even under serum-starved conditions, which in wild-
type MEFs would result in growth arrest due to the severe inhibition of the mTOR
cascade (Dowling et al. 2010). Similarly, RAS-transformed 4E-BP DKO MEFs
become resistant to mTOR inhibitors in vivo and, in contrast, the overexpression
of mTOR-mediated phosphorylation-resistant 4E-BP1 strongly reduces tumorigen-
esis (Alain et al. 2012; Hsieh et al. 2010). These studies, as well as another study
(Thoreen et al. 2012), indicate that 4E-BPs are critical mediators of mTORC1 ac-
tion in vitro and in vivo.

15.3.2  S6 Kinase

Mammalian cells contain two similar rpS6 kinase proteins (S6K1 and S6K2), which
are direct targets of mTORC1 and regulate cell growth (Grove et al. 1991; Shima
et al. 1998) (see Chap. 16). S6K1 has been well-studied and its impact on cell
growth also has been better characterized than that of S6K2, partly because S6K2
was discovered much later than S6K1 (Shima et al. 1998). The activation of S6K1
promotes protein synthesis and cell growth through the phosphorylation of several
effectors (Ben-Sahra et al. 2013; Magnuson et al. 2012; Pende et al. 2004; Robitaille
et al. 2013; Ruvinsky et al. 2005). S6K1 was originally thought to control transla-
tion of an abundant subclass of mRNAs characterized by an oligopyrimidine tract
at the 5′ end (5′ TOP mRNAs), through the phosphorylation of rpS6 (Ruvinsky and
Meyuhas 2006). It appears that many proteins belonging to the translation machin-
ery are encoded by these 5′ TOP mRNAs (Ruvinsky and Meyuhas 2006). However,
inhibition of mTORC1 with rapamycin only mediates a partial inhibitory effect on
5′ TOP mRNA translation while causing complete dephosphorylation of S6K1 and
rpS6 (Stolovich et al. 2002; Tang et al. 2001). In addition, MEFs lacking S6K1/2
or knocked-in with an unphosphorylatable form of S6 still translate 5′ TOP mRNA
(Pende et al. 2004; Ruvinsky et al. 2005). Thus, it is likely that the S6K/rpS6 axis is
not the major signaling node regulating 5′ TOP mRNA translation. Interestingly, a
15  mTOR and Regulation of Translation 315

recent study has proposed that 4E-BPs may play a role in the control of translation
of 5′ TOP mRNAs (Thoreen et al. 2012).
PDCD4 is one of the targets of S6K1. PDCD4 was originally identified as a sup-
pressor of neoplastic transformation (Cmarik et al. 1999) (see Chap. 6). Dorrello
et al. found that PDCD4 inhibits eIF4A helicase (Dorrello et al. 2006). In response
to mitogens, PDCD4 is rapidly phosphorylated on Ser67 by S6K1 and subsequent-
ly degraded via β-TRCP (see Chap. 6). In cultured cells, expression of a stable
PDCD4 mutant that is unable to bind β-TRCP inhibits translation of mRNA with
a structured 5′ UTR, leading to a reduction in cell size and a decrease in cellular
proliferation (Dorrello et al. 2006; Schmid et al. 2008). Thus, S6K1-regulated
degradation of PDCD4 in response to mitogens can control protein synthesis and
cell growth downstream of mTORC1.
eIF4B is a physiologically relevant target of S6K1 that could explain its effect
on translation and cell growth. eIF4B is an RNA-binding protein that mediates effi-
cient recruitment of ribosomes to mRNA and stimulates the ATPase and RNA heli-
case activities of eIF4A (Rogers et al. 2002) (see Chap. 5). eIF4B is phosphorylated
in response to a variety of extracellular stimuli that promote cell growth and pro-
liferation, such as serum, insulin, and phorbol esters (Duncan and Hershey 1985).
Ser422, one of the phosphorylation sites in eIF4B, is specifically phosphorylated by
S6Ks (Raught et al. 2004). In addition, a recent report showed that eIF4B regulates
translation of proliferative and prosurvival mRNAs, which have complex structures
in their 5′ UTR (Shahbazian et al. 2010). Thus, eIF4B is an important mediator of
some of the effects of S6Ks on translation and cell growth.

15.3.3 eIF4G

eIF4G is a scaffolding protein that plays a key role in the assembly of the initiation
complex (see Chap. 7). All eukaryotes have two related eIF4G proteins, namely
eIF4G1 and eIF4G2. Both eIF4Gs are phosphoproteins (Raught et al. 2000; Tuazon
et al. 1989). Phosphorylation of eIF4G1, but not eIF4G2, is induced in an mTOR-
dependent manner in response to extracellular stimuli, such as serum, insulin, and
other factors that promote cell growth (Raught et al. 2000; Tuazon et al. 1989).
eIF4G1 has a cluster of serum-stimulated phosphorylation sites (Ser1148, Ser1188,
and Ser1232), which are sensitive to PI3K and mTOR inhibitors (Raught et al.
2000). The physiological significance of eIF4G1 phosphorylation has not been
determined because no evidence for changes in activity or association with other
initiation factors has been reported so far following its phosphorylation. On the
other hand, a recent study showed that the depletion of initiation factor eIF4G1
blocks cell proliferation and diminishes cell size, similarly to rapamycin treatment
or silencing of the mTORC1 component, RAPTOR (Ramirez-Valle et al. 2008).
Therefore, eIF4G1 may mediate some of the effects of mTORC1 on the promotion
of cell growth and proliferation.
316 Y. Tsukumo et al.

15.3.4  eEF2K

eEF2K phosphorylates and inhibits translation elongation factor eEF2, which


mediates the translocation step of peptide-chain elongation on the ribosome (see
Chap. 12). mTORC1 indirectly promotes the phosphorylation of at least three eEF2K
sites (Ser366, Ser78 and Ser359). The phosphorylation of these sites downstream
of mTORC1 reduces the activity of eEF2K, which decreases the phosphorylation of
eEF2 and promotes translation elongation (Proud 2009). Ser366 is phosphorylated
directly by S6Ks. Phosphorylation of Ser78 more strongly inhibits eEF2K activity,
but the direct upstream kinase has not been identified. Recent work has shown that
the CDK1/cyclin B complex phosphorylates Ser359 in eEF2K, and that its activity
appears to be promoted by mTORC1 (Proud 2009). Thus, mTORC1 decreases the
eEF2K activity in different ways, resulting in regulation of the elongation step of
protein synthesis. eEF2K activity is increased in various cancers, including glioma
(Arora et al. 2003). In addition, the latest study revealed that AMPK-mediated
eEF2K activation is important for tumor cell adaptation to nutrient deprivation
(Leprivier et al. 2013). eEF2K may be a valid target for treatment of cancer cells
that adapt to poor nutrient conditions (Manning 2013).

15.4 Translation and mTORC1 in Cell Proliferation and


Growth

mTORC1 controls growth (increase in cell mass) and proliferation (increase in cell
number) by modulating mRNA translation through phosphorylation of 4E-BPs
(4E-BP1, 2 and 3) and S6K1 and 2 (Dowling et al. 2010; Ohanna et al. 2005).
Pharmacological or genetic inhibition of mTORC1 leads to decreased cell prolif-
eration and cell size (Dowling et al. 2010). The importance of the mTORC1/4E-BP
axis on cell proliferation has been demonstrated with the use of 4E-BPs KO MEFs
(Dowling et al. 2010). The cells lacking 4E-BPs sustained proliferation, but not cell
growth, under conditions of mTOR inhibition (Dowling et al. 2010). Consistently,
4E-BPs-deficient cells sustained translation of mRNAs that encode proprolifera-
tive and cell cycle progression proteins (Dowling et al. 2010). On the other hand,
S6Ks-deficient cells, but not 4E-BPs-deficient cells, exhibited smaller cell size than
wild-type counterparts and were resistant to reduction in cell size induced by mTOR
inhibition (Dowling et al. 2010). These phenotypes of 4E-BPs-deficient or S6Ks-
deficient cells were clearly rescued by re-expression of each protein via a recombi-
nant construct (Dowling et al. 2010). Together with other key reports (Alain et al.
2012; Deng et al. 2010; Larsson et al. 2012; Ohanna et al. 2005; Richardson et al.
2004), these studies provide evidence that mTORC1 regulates cell proliferation and
growth via translational control using distinct translational hubs. Owing to its role
in promoting cell growth and proliferation, it is not hard to imagine that dysregula-
tion of mTORC1 could play a fundamental role in cancer (Fig. 15.4).
15  mTOR and Regulation of Translation 317

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Fig. 15.4   Hyperactivated mTOR pathway leads to dysregulation of translational control and can-
cer development and progression. Upon receiving genetic insults, such as an activating mutation
of proto-oncogenes (RTK and PI3K) or inactivation of tumor suppressor genes (PTEN, TSC1/2 and
LKB1), the mTOR pathway becomes hyperactivated, leading to increased translation of specific
mRNAs coding prometastatic, proangiogenic, proproliferative and antiapoptotic proteins (such as
VEGF, MMP-9, YB-1, cyclin D, ODC, MCL-1, BCL-2 and survivin).

15.5 Translation and mTORC1 in Invasion and


Metastasis

Translational regulation by mTORC1 is implicated in many aspects of cancer biol-


ogy including invasion, metastasis and apoptosis (Del Bufalo et al. 2006; Hsieh et al.
2012; Zhou et al. 2004). Using ribosome profiling in a prostate cancer model, Hsieh
et al. showed that mTORC1 regulates translation of a large number of invasion- and
metastasis-related genes (Hsieh et al. 2012). Treatment of a human prostate cancer
cell line with an ATP-competitive mTOR inhibitor (INK128) reduced the expression
of several invasion-related proteins, such as CD44, vimentin and YB-1. Importantly,
4E-BPs depletion sustained the expression of these proteins (Hsieh et al. 2012). In
association with these observations, a previous clinical study of 165 invasive breast
cancers has shown that the phosphorylation of 4E-BP1, which activates eIF4E and
thus promotes translation, increased progressively from normal breast epithelium
to hyperplasia and from hyperplasia to invasive neoplasia (Zhou et al. 2004). Using
transgenic and allograft breast cancer models, Nasr et al. demonstrated that sup-
pressing mTOR activity or repressing its downstream translation regulators (eIF4E
and eIF4A) delayed breast cancer progression, onset of associated pulmonary metas-
tasis in vivo and breast cancer cell invasion and migration in vitro (Nasr et al. 2013).
Consistently, translation of VEGF, MMP-9 and cyclin D1, which encode products
associated with invasive and metastatic phenotypes, is inhibited upon eIF4E sup-
pression (Nasr et al. 2013). Another study has also shown that the mTORC1/eIF4E
axis appears to control translation of VEGF that is important for tumor angiogenesis.
318 Y. Tsukumo et al.

Indeed, Del Bufalo et al. (2006) showed that treatment of human breast cancer cell
line BT474 with mTOR inhibitor temsirolimus inhibited VEGF production in vitro
under both normoxic and hypoxic conditions. The specificity of this effect via the
mTORC1/eIF4E axis was further demonstrated by the decrease in VEGF production
upon mTOR or eIF4E knockdown. Consistent with reduced VEGF production, tem-
sirolimus treatment inhibited vessel formation in vitro and in vivo (Del Bufalo et al.
2006). These observations can partly explain the antiangiogenic effects of rapamycin
reported previously in preclinical studies (Guba et al. 2002). Overall, these studies
indicate that the mTORC1/4E-BPs pathway is an important contributor to tumor
invasion, angiogenesis and metastasis (Fig. 15.4). We refer the reader to chapters on
translational control and specific cancers presented in this book for more detailed
information on aberrations within the mTORC1/4E-BP axis and their clinical rel-
evance in various malignancies.

15.6 Translation and mTORC1 in Apoptosis and Tumor


Survival

The mTORC1/4E-BP axis plays an important role in tumor survival and apop-
tosis (Fig. 15.4) (Graff et al. 2007, 2009; Mamane et al. 2007; Mills et al. 2008).
mTORC1 inhibition by rapamycin leads to specific reduction in antiapoptotic
protein MCL-1 in MYC-driven mouse lymphoma and sensitizes the cells to che-
motherapy (Mills et al. 2008). eIF4E knockdown in a mouse cell line resulted in
reduced translation of specific mRNAs, including antiapoptotic protein survivin, and
induced apoptotic cell death (Mamane et al. 2007). In addition to these studies in
mouse cells, Graff et al. (2007, 2009) showed that the suppression of eIF4E by ASO
significantly induced apoptosis of human cancer cells and suppressed tumorigenesis
in vivo in a xenograft model. These ASOs repressed translation of eIF4E-sensitive
mRNAs, such as VEGF, cyclin D1, survivin, c-MYC and BCL-2 (Graff et al. 2007).
The effects of eIF4E knockdown also prevented endothelial cells from forming
vessel-like tube structures (Graff et al. 2007). Importantly, intravenous ASO ad-
ministration selectively and significantly reduced eIF4E expression and suppressed
tumorigenesis in the xenografts without major adverse effects (Graff et al. 2007). In
another study, Graff et al. (2009) also showed that eIF4E function is elevated in hu-
man prostate cancer, and that eIF4E ASO reduced expression of the eIF4E-regulated
proteins, including BCL-2 and c-MYC, and induced apoptosis. Collectively, these
data suggest that the 4E-BP/eIF4E axis is as an attractive target for cancer therapy.
15  mTOR and Regulation of Translation 319

15.7 mTOR Pathway and Translational Regulation in


Etiology and Pathogenesis of Cancer

The mTOR pathway is frequently dysregulated in human cancers (Figs. 15.3 and


15.4). For example, overexpression or constitutive active mutations of receptor
tyrosine kinases (RTKs), such as EGFR and HER2/neu, are frequently observed
in many cancers including lung and breast (Guertin and Sabatini 2005). In addi-
tion, amplification of the p110 catalytic subunit of PI3K, loss of PTEN (which is
a phosphatase that opposes activity of PI3K), amplification of AKT2, as well as
mutations of TSC1, TSC2 and TP53, are frequently observed in many cancers and
result in activation of mTORC1 (Cheng et al. 1996; Feng et al. 2005; Guertin and
Sabatini 2005; Inoki et al. 2003b; Laplante and Sabatini 2012). Furthermore, inacti-
vating mutations in serine/threonine kinase 11 (STK11) (also called liver kinase B1
(LKB1)) and TSC1/2, which are tumor suppressor proteins upstream of mTORC1,
occur in Peutz–Jeghers and tuberous sclerosis syndromes that engender predisposi-
tion for cancer development (Guertin and Sabatini 2005).
There is much evidence that dysregulation of translational control serves as a
common mechanism by which the PI3K/AKT/mTORC1 pathway promotes cellular
transformation and tumor development (Silvera et al. 2010). In a normal cell, this
pathway acts as a sensor for energy, stress, nutrient availability and growth factor
signaling. The pathway integrates these inputs to regulate ribosome production and
gene expression at the translational level. Importantly, this pathway becomes onco-
genic when hyperactivated or dysregulated. Although it remains unknown how the
PI3K/AKT/mTORC1 pathway alters translational control in cancer, recent studies
have shown that this signaling cascade directly modulates activity and expression of
specific components of the translation machinery (Alain et al. 2012; Dowling et al.
2010; Hsieh et al. 2010; Thoreen et al. 2012).
The activated PI3K/AKT/mTORC1 pathway leads to activation of translation
initiation and protein synthesis. mTOR phosphorylates 4E-BPs and leads to the
activation of eIF4E (Sonenberg and Hinnebusch 2009). The 4E-BPs/eIF4E axis
plays a primary role in translational control and cancer. As follows later in this
book, eIF4E overexpression or pathologic activation have been reported in a va-
riety of tumors including human breast, head and neck and other cancers (Berkel
et al. 2001; Rosenwald et al. 2001; Salehi and Mashayekhi 2006; Sorrells et al.
1998, 1999; Wang et al. 2009; Yoshizawa et al. 2010). The oncogenic potential
of eIF4E has been faithfully recapitulated both in vitro and in vivo (Avdulov et al.
2004; Furic et al. 2010; Lazaris-Karatzas et al. 1990; Ruggero et al. 2004). Over-
expression of eIF4E is sufficient to induce transformation of immortalized murine
fibroblasts and human epithelial cells (Avdulov et al. 2004; Lazaris-Karatzas et al.
1990). Transgenic mice, in which eIF4E expression is driven by the ubiquitous
β-actin promoter, have increased susceptibility to cancer (Ruggero et  al. 2004).
These mice develop lymphomas, angiosarcomas, lung carcinomas, and hepatomas
(Ruggero et al. 2004).
Activation of eIF4E either via inactivation of 4E-BPs or phosphorylation by the
MAPK pathway also plays an important role in cancer biology ((Alain et al. 2012;
320 Y. Tsukumo et al.

Dowling et al. 2010; Furic et al. 2010; Hsieh et al. 2010; Ueda et al. 2010) and
see Chaps. 4 and 17). Indeed, combined deficiency of the MAPK-interacting ki-
nases MNK1 and MNK2, which are upstream kinases that phosphorylate eIF4E at
Ser209, delays tumorigenesis in T-cell-specific PTEN-deficient mice (Ueda et al.
2010). In addition, eIF4ES209A knock-in mice, in which eIF4E cannot be phosphory-
lated on the 209th residue, are resistant to prostate cancer induced by the PTEN loss.
These results support a recent study showing that eIF4E phosphorylation is required
for translational upregulation of several proteins implicated in tumorigenesis (Furic
et al. 2010).
The most studied and, possibly the most fundamental mechanism supporting
cell growth and proliferation in cancer is the control of eIF4E activity via the
mTORC1/4E-BP axis. It has recently been shown (both in vitro and in vivo) that the
4E-BPs/eIF4E axis exerts significant control over cap-dependent translation, cell
proliferation, cancer initiation and progression downstream of mTORC1 hyperacti-
vation (Alain et al. 2012; Dowling et al. 2010; Hsieh et al. 2010). Most importantly,
restoring eIF4E oncogenic activity to normal levels downstream of AKT/mTORC1
hyperactivation results in blockage of tumor progression in a mouse model for
lymphomagenesis and associates with a drastic increase in overall survival (OS)
(Hsieh et al. 2010). Furthermore, using ribosome profiling, Hsieh et al. uncovered
specialized translation of the prostate cancer genome by oncogenic mTOR signal-
ing, revealing a remarkably specific repertoire of genes involved in cell prolifera-
tion, metabolism, and invasion (Hsieh et al. 2012). This signifies that translation,
downstream of the 4E-BPs/eIF4E axis, represents a key cellular process linking ab-
errant mTORC1 signaling to cancer etiology and pathogenesis. Specific examples
into the role of the activated mTORC1/4E-BPs/eIF4E axis in the promotion of
translation in different types of cancers can be found in other chapters of this book
(see Part IV).
Another mTORC1 substrate S6K1 is frequently amplified in a variety of tu-
mors including breast and ovarian cancer (Andersen et al. 2002; Barlund et al.
2000; Ip and Wong 2012). In breast cancer, both S6K1 and S6K2 are situated in
the commonly amplified chromosome regions 17q21-23 and 11q13 (Perez-Teno-
rio et al. 2011). Importantly, S6Ks amplification and protein overexpression have
been reported to be associated with a worse outcome in breast cancer (Perez-Teno-
rio et al. 2011).
Taken together, these reports indicate that the mTORC1-mediated translational
control has an essential role in maintaining the transformed phenotype and suggest
that inhibition of the mTOR pathway could be efficient for the treatment of a variety
of cancers.

15.8 Targeting the mTOR Pathway in Cancer

Several analogs of rapamycin called rapalogs have been designed in the past 10
years and have been approved for treatment of metastatic RCC, subependimal giant
cell astrocytoma and pancreatic neuroendocrine tumors (PaNETs) (Baselga et al.
15  mTOR and Regulation of Translation 321

2012; Hudes et al. 2007; Krueger et al. 2010; Motzer et al. 2010, 2008; Yao et al.
2011). These rapalogs include temsirolimus (CCI-779), everolimus (RAD001), and
ridaforolimus (AP-23573) and have therapeutic effects similar to rapamycin but
possess better pharmacokinetics. (Gabardi and Baroletti 2010; Mita et al. 2008;
Rini 2008). Currently, the National Institute of Health (NIH) website (clinicaltrials.
gov) lists a significant number of clinical trials testing rapalogs as single agent or in
combination therapy for the treatment of many cancer types.

15.8.1 Renal Cell Carcinoma

Renal cell carcinoma (RCC) is a cancer type in which activation of the mTOR path-
way correlates with tumor aggressiveness and poor prognosis (Pal and Quinn 2013)
(see Chap. 34). It has been shown that temsirolimus (Torisel) increases survival of
patients with previously untreated metastatic RCC with poor prognostic features
(Hudes et al. 2007). In a randomized phase II trial with 111 patients with treatment-
refractory metastatic RCC, the temsirolimus-treated group showed 7 % of overall
response rate and 6 months progression-free survival (PFS) (Atkins et al. 2004). In
the phase III trial, treatments with temsirolimus or IFN, or a combination of these
two drugs, were compared (Hudes et al. 2007). Of the three treatment options, the
group treated with temsirolimus alone showed the longest OS and PFS with mild-
est adverse effects. Based on these findings, temsirolimus was approved for first-
line treatment of metastatic RCC patients with poor prognosis (Hudes et al. 2007).
Everolimus has also been approved for treatment of RCC. In a phase III study with
410 RCC patients progressing after chemotherapy, median PFS (but not OS) of pa-
tients in the everolimus group was significantly longer than placebo (4.9 versus 1.9
months) (Motzer et al. 2010). Accordingly, everolimus was approved by the Food
and Drug Administration (FDA) for treatment of advanced RCC progressing after
chemotherapy (Gomez-Pinillos and Ferrari 2012).

15.8.2 Pancreatic Neuroendocrine Tumors

Pancreatic neuroendocrine tumors (PaNETs) represents about 1 % of all pancreatic


malignant neoplasms. Although these are rare, their incidence has doubled over the
past 20 years (Yao et al. 2008, 2011). mTOR pathway abnormalities in PaNET are
reported to be associated with increased aggressiveness and shortened PFS and OS
among patients with PaNET (Jiao et al. 2011; Yao et al. 2011) (see Chap. 31). These
clues from somatic or germline mutations and protein expression of the mTOR
pathway components supported the conduct of clinical studies of mTOR inhibitor
everolimus in patients with PaNET. A phase III study of everolimus named RADI-
ANT III was conducted in 410 patients with metastatic unresectable PaNETs (Yao
et al. 2011). Treatment with everolimus significantly prolonged the median PFS (11
versus 4.6 months), but not OS compared to placebo (Yao et al. 2011). Everolimus
has been recently approved by the FDA for the treatment of metastatic or unresect-
able PaNET (Yao et al. 2011).
322 Y. Tsukumo et al.

In addition to PaNETs, everolimus exhibited clinical benefit for patients with


advanced neuroendocrine tumors (NETs), which are neoplasms that arise from
cells of the endocrine and nervous systems. In the phase III RADIANT-2 trial
with 429 patients, the addition of everolimus to long-acting octreotide, which is
a somatostatin analog that blocks the actions of growth hormone, glucagon and
insulin, increased the median PFS to 16.4 compared to 11.3 months for place-
bo. Currently, everolimus is underway in the phase III RADIANT-4 trial among
patients with advanced nonfunctional neuroendocrine tumors of gastrointestinal
or lung origin (see clinicaltrials.gov identifier: NCT01524783, Novartis Protocol
CRAD001T2302).

15.8.3  T
 uberous Sclerosis Complex and Subependymal Giant-
Cell Astrocytomas

Tuberous sclerosis is a genetic disorder characterized by the growth of numerous


benign tumors in many organs including the skin, brain, lungs and kidneys (Krueger
and Franz 2008). Tuberous sclerosis is caused by a mutation of either of two genes,
TSC1 and TSC2, which are negative regulators of mTORC1, as mentioned pre-
viously. Subependymal giant-cell astrocytomas (SEGAs) occur in up to 20 % of
people with tuberous sclerosis and are more likely to develop during childhood
and adolescence (Krueger and Franz 2008). The phase III clinical trial in tuberous
sclerosis patients showed that 35 % of subjects treated with everolimus had a 50 %
or greater reduction in the size of SEGAs compared to no response in the placebo
group (Krueger et al. 2010). Recently the FDA approved everolimus for the treat-
ment of SEGA in patients with tuberous sclerosis who were not candidates for cura-
tive surgical resection.

15.8.4  Mantle Cell Lymphoma

The mTOR pathway is an important therapeutic target in mantle cell lymphoma be-
cause the commonly observed t(11;14) translocation induces overexpression of cy-
clin D1 mRNA, whose translation is highly regulated by the mTORC1/4E-BP/eIF4E
axis (Bertoni et al. 2004; Ohanna et al. 2005). In a phase III trial with 162 patients
with relapsed mantle cell lymphoma, two doses of temsirolimus monotherapy
(175 mg weekly for three weeks followed by either 75 or 25 mg weekly) had
significantly better effects on median PFS than the investigator’s choice protocol
(4.8 versus 1.9 months). However, there was no significant difference in the median
OS between the temsirolimus treatment group and the investigator’s choice proto-
col treatment group (Hess et al. 2009). These results established temsirolimus as a
new therapeutic option for relapsed/refractory mantle cell lymphoma and led to its
inclusion in the National Comprehensive Cancer Network (NCCN) Clinical Prac-
tice Guidelines in Oncology for non-Hodgkin’s lymphomas (Zelenetz et al. 2010).
15  mTOR and Regulation of Translation 323

15.8.5 Sarcoma

The mTOR pathway is dysregulated in most sarcoma subtypes, including


leiomyosarcoma, rhabdomyosarcoma and perivascular epithelioid cell tumors
(Dobashi et al. 2009; Hernando et al. 2007; Pan et al. 2008; Rivera et al. 2011)
(see Chap. 22). Ridaforolimus, one of the rapalogs, prevents cell growth and pro-
liferation in various sarcoma cell lines and exhibits antitumor activity in sarcoma
xenograft models (Rivera et al. 2011; Squillace et al. 2011). Ridaforolimus showed
promising activity in advanced sarcoma patients in a phase I trial. In a subsequent
phase II trial involving 212 patients with advanced soft-tissue or bone sarcomas
with no restrictions on prior chemotherapy, ridaforolimus produced clinical benefit
in 29 % of patients with mild adverse effects (Chawla et al. 2012). Based on these
results, a double-blind phase III trial randomized 711 patients with metastatic sar-
coma was initiated. In this trial, ridaforolimus reduced the risk of progression or
mortality by 28 % in patients with advanced soft tissue and bone sarcomas (Keedy
2012). However, it has not yet been approved by the FDA due to the small improve-
ment in disease control.

15.8.6  Diffuse Large B-Cell Lymphoma

Recent studies have highlighted dysregulation of the mTOR pathway and its role
in controlling tumor cell proliferation and survival in DLBCL (Uddin et al. 2006;
Wanner et al. 2006). In a phase II trial with everolimus including 77 patients with
aggressive non-Hodgkin’s lymphoma (of which, eight follicular lymphoma, 47
DLBCL and 19 mantle cell lymphoma patients) who were heavily pretreated, the
overall response rate was 30 % (14/47) in DLBCL (Witzig et al. 2011). Current-
ly, everolimus is being investigated in a phase III trial as maintenance therapy for
DLBCL in patients with a complete response after R-CHOP chemotherapy (see
clinical trial, NCT00790036).

15.8.7  Breast Cancer

As indicated in preclinical breast cancer models, aberrant activation of the mTOR


pathway through growth factor signaling confers resistance to hormonal therapy,
while mTOR inhibition restores this response (deGraffenried et al. 2004; Kurokawa
and Arteaga 2003; Kurokawa et al. 2000) (see Chap. 26). In a phase III trial with
rapalogs combined with hormone therapy in metastatic breast cancer, temsiroli-
mus showed no benefit in median PFS, while everolimus showed a significant im-
provement in the median PFS (Ellard et al. 2009). Everolimus, in combination with
hormonal therapy (exemestane), is approved by the FDA for advanced hormone
receptor-positive, HER2-negative, breast cancer (Baselga et al. 2012).
324 Y. Tsukumo et al.

In addition to the cancers mentioned above, several clinical trials are presently
testing the effect of rapalogs in other malignancies including lung, endometrial and
gastric cancers (Doi et al. 2010; Gomez-Pinillos and Ferrari 2012; Oza et al. 2011;
Pandya et al. 2007; Slomovitz et al. 2010; Soria et al. 2009; Tarhini et al. 2010;
Yoon et al. 2012). However, while monotherapy with these mTOR inhibitors shows
some benefits, feedback activation of other pathways that lead to upregulation of
translational control (see below) represents a limitation of rapalogs in achieving
high efficacy of the therapy. We also refer the reader to the Part IV of this book
for more information on the role of the mTOR pathway and its targeting in various
cancers.

15.9 New Compounds Targeting the mTOR Pathway

15.9.1  Reasons for Low Therapeutic Efficacy of Rapalogs

As described in the previous section, rapalogs have exhibited clinical efficacy


against a variety of malignancies and have been approved for treatment of several
cancers (Benjamin et al. 2011; Dancey 2010). However, despite observed antitu-
mor responses, rapalogs showed modest clinical responses as a single-agent therapy
(Benjamin et al. 2011; Dancey 2010). There could be several reasons for the de-
creased performance of rapalogs in certain situations. First, it is well known that
mTORC1 activation downregulates IRS-1 via several signaling routes and induces
the retro-inhibition of PI3K/AKT signaling (Fig. 15.3) (Laplante and Sabatini 2012).
Previous studies have shown that inhibition of mTORC1 with rapamycin relieves
the negative feedback loops and massively promotes PI3K/AKT signaling (Har-
rington et al. 2004; Hsu et al. 2011; Tzatsos and Kandror 2006; Um et al. 2004; Yu
et al. 2011). In addition, several preclinical and clinical studies reported that rapalog
treatment resulted in higher levels of activated AKT (Floc’h et al. 2012; O’Reilly
et al. 2006; Tabernero et al. 2008; Wan et al. 2007; Wang et al. 2008a). Considering
that PI3K/AKT signaling engenders prosurvival and antiapoptotic functions, it is
believed that the aforementioned could contribute to reduced efficacy of rapalogs.
Another possible reason for the limited efficacy of rapalogs in cancer treatment
would be that these molecules only partially inhibit the phosphorylation of 4E-
BP1. Indeed, studies indicate that although rapalogs are very potent in inhibiting
S6K phosphorylation, these molecules incompletely inhibit the phosphorylation of
4E-BP1 (Choo et al. 2008; Feldman et al. 2009; Thoreen et al. 2009). Inability to
sustain 4E-BP1 dephosphorylation can lead to the persistence of protein synthe-
sis and cancer cell proliferation even in the presence of rapamycin (Feldman et al.
2009; Thoreen et al. 2009). Finally, rapamycin is largely ineffective in inhibiting
mTORC2, which is involved in cell motility and invasion (Jacinto et al. 2004; Masri
et al. 2007; Sarbassov et al. 2004).
15  mTOR and Regulation of Translation 325

Rapamycin is an allosteric inhibitor of mTORC1. New generation mTOR inhibi-


tors, which inhibit mTOR kinase activity by competing with ATP for binding to the
kinase domain of mTOR, have been designed and reported to have stronger anti-
tumor effects compared to rapalogs (Benjamin et al. 2011; Dancey 2010; Feldman
et al. 2009; Thoreen et al. 2009). Whereas rapamycin inhibits only mTORC1, the
active-site mTOR inhibitors (asTORi) inhibit both mTORC1 and mTORC2 (Feld-
man et al. 2009; Thoreen et al. 2009). mTOR and PI3Ks have similarities between
each kinase domains and, therefore, some of these compounds inhibit both kinases,
which are known as dual mTOR/PI3K inhibitors (Baumann et al. 2009; Manara
et al. 2010; Molckovsky and Siu 2008). Recent efforts lead to the development
of compounds that selectively inhibit both mTORC1 and mTORC2. Overall, the
anticancer efficacy of the ATP-competitive inhibitors in preclinical settings has
generally been superior to that of the rapalogs, as shown by effective inhibition of
mTORC2 activity and complete inhibition of 4E-BP1 phosphorylation (Feldman
et al. 2009; Janes et al. 2010).

15.9.2  ATP-Competitive mTOR Inhibitors

Compared to rapalogs, PP242 is a stronger mTOR inhibitor of both mTORC1


(including rapamycin-resistant 4E-BP1 phosphorylation) and mTORC2 (Feld-
man et al. 2009; Thoreen et al. 2009). In a preclinical trial, PP242 sustained 4E-
BP1 dephosphorylation and strongly suppressed tumor growth in a mouse model
of AKT-driven lymphangiogenesis, whereas rapamycin was generally ineffective
(Hsieh et al. 2010). In another study, PP242 synergized with imatinib or dasatinib
to induce apoptosis in BCR-ABL-positive cells (Janes et al. 2010). A derivative
of PP242, INK128 (MLN0128), inhibited the three primary downstream mTOR
effectors 4E-BP1, S6K1/2 and AKT in human prostate cancer PC-3 cells (Hsieh
et al. 2012). INK128 also inhibited prostate cancer cell invasion in vitro and in an
in vivo mouse model (Hsieh et al. 2012). Furthermore, recent reports from preclini-
cal model studies show that INK128 was also effective against BCR-ABL-positive
B-cell acute lymphoblastic leukemia (B-ALL) and HER2-therapy-resistant breast
cancer (Garcia-Garcia et al. 2012; Janes et al. 2013). Currently, INK128 is under
study in an early clinical trial for advanced malignancy.
AZD8055 completely dephosphorylated phospho-4E-BP1 Thr37/46 compared
with rapamycin and has a greater effect on inhibition of cap-dependent translation
in cell-line experiments (Chresta et al. 2010). In addition, AZD8055 inhibited tumor
development more than rapamycin in the mouse xenograft model (Chresta et al.
2010). AZD8055 treatment induced HER3 phosphorylation in several HER2‑am-
plified breast cancer cells while combination of AZD8055 with the HER2 inhibitor
lapatinib induced synergistic cell death consistent with inhibition of feedback ac-
tivation of phospho-HER3 (Marshall et al. 2011). Similarly, AZD8055 synergized
with the MEK inhibitor AZD6244 in a NSCLC xenograft model, as evidenced by
AZD8055-driven increase in ERK phosphorylation (Holt et al. 2012; Marshall et al.
326 Y. Tsukumo et al.

2011). Thus, combining mTOR and MAPK pathway inhibition is a potentially ef-
fective strategy for cancer treatment.
Compared to rapamycin, OSI-027 (OSI Pharmaceuticals) exhibited stronger
translational inhibition activity and apoptotic effects against leukemic cell lines
expressing the T315I BCR-ABL mutation, which is refractory to all BCR-ABL
inhibitors currently in use (Carayol et al. 2010). OSI-027 had also similar and
suppressive effects on primitive leukemic progenitors from AML patients (Altman
et al. 2011).
Torin 1 is a highly potent and selective mTOR kinase inhibitor with strong bio-
logical activity in various preclinical settings (Thoreen et al. 2009). It potently in-
hibits mTORC1 (including sustained inhibition of 4E-BP1 phosphorylation) and
mTORC2. A medicinal chemistry program was initiated based on the original Torin
1 backbone to address some of its unfavorable properties, such as a low-yielding
synthetic route, poor water solubility and oral bioavailability and low stability (Liu
et al. 2011). This led to the production of Torin 2, which has better pharmacokinetic
properties and an improved synthetic route, allowing further in vivo evaluation of its
therapeutic potential (Liu et al. 2011). In a preclinical model, combination of Torin
2 with the MEK inhibitor AZD6244 yielded significant growth inhibition against
K-RAS-driven lung tumors (Liu et al. 2013).

15.9.3  Dual mTOR/PI3K Inhibitors

In a phase I clinical trial, dual mTOR/PI3K inhibitor BEZ235 was effective for mul-
tiple myeloma and sarcoma as a single agent and, even more so, in combination with
other drugs (bortezomib, vincristine, doxorubicin or melphalan) (Baumann et al.
2009; Manara et al. 2010). It is currently in several phase I and II trials of advanced
solid tumors and metastatic breast cancer, either as a monotherapy or a combination
therapy with paclitaxel, trastuzumab, BKM120 or BKM162. On the other hand, in a
mouse lung cancer model driven by mutant KRAS (G12D), BEZ235 was ineffective
as a single agent; however the combination with MEK inhibitor AZD6244 led to a
more significant tumor regression (Engelman et al. 2008). Similarly, a combination
of BEZ235 and AZD6244 has been reported to more effectively suppress growth
of CRC and melanoma (Aziz et al. 2010; Migliardi et al. 2012; Roberts et al. 2012;
Simmons et al. 2012). A clinical trial of BEZ235 combined with a MEK inhibitor
MEK162 has started for selected advanced solid tumors. Another dual mTOR/PI3K
inhibitor and the first orally available pharmaceutical of its class, XL765, is also
currently being investigated in clinical trials for several solid tumors (Molckovsky
and Siu 2008).
15  mTOR and Regulation of Translation 327

15.9.4 Other Compounds

SF1126 is a novel prodrug that consists of a well-characterized PI3K inhibitor


LY294002 conjugated to an RGDS (Arg-Gly-Asp-Ser) peptide fragment (Garlich
et al. 2008). The RGDS peptide binds integrin within the tumor compartment, re-
sulting in enhanced delivery of the active compound LY294002 to the tumor vascu-
lature and the tumor cells themselves, and inhibiting PI3K‑dependent angiogenesis
specifically in the tumor (Garlich et al. 2008). SF1126 was tested in a dose es-
calation study in patients with advanced solid tumors. This compound was well
tolerated with low-grade adverse events and 46 % of patients showed stabilization
of their disease (Mahadevan et al. 2012; Zhang et al. 2011).
Metformin is used for the treatment of type 2 diabetes. Epidemiological studies
have consistently shown a lower incidence of various cancer types among diabetic
patients taking metformin (Evans et al. 2005; Libby et al. 2009). Metformin in-
hibits mTORC1 through activation of AMPK, which activates mTORC1 negative
regulator TSC2, and subsequently decreases cap-dependent translation (Dowling
et al. 2007; Zakikhani et al. 2006). This compound consistently displays a strong
antiproliferative effect in a wide range of cancer cell lines in vitro and in xenograft
models (Zhou et al. 2004). A recent study revealed that metformin, as well as ca-
nonical mTOR inhibitors, affect translation of genes encoding cell-cycle regulators
via the mTORC1/4E-BP axis (Larsson et al. 2012). Since metformin is a safe and
remarkably well-tolerated drug, numerous clinical trials on its anticancer potential
are underway (Pollak 2012).
Farnesyltransferase inhibitors (FTI) block the activity of RHEB, which is a
GTPase that directly activates mTORC1 (McMahon et al. 2005). Like RAS, RHEB
activity requires farnesylation. RHEB activity is blocked by FTI. In PTEN-deficient
tumor cells, inhibition of RHEB by FTI is responsible for the specific antineoplastic
effects that are dampened in the farnesylation-independent mutant of RHEB (Ma-
vrakis et al. 2008). RHEB overexpression, observed in some human lymphomas,
results in mTORC1 activation and increased sensitivity to rapamycin and FTI treat-
ment (Mavrakis et al. 2008). The FTI tipifarnib is currently in phase I and II trials
for the treatment of AML in adults.

15.10 mTOR Inhibition and Therapeutic Resistance and


Radioresistance

Dysregulation of the PI3K/AKT/mTOR pathway in cancer not only contributes to


a tumorigenic phenotype, such as increased proliferation, motility and invasion, but
also leads to drug resistance. In this section, we review the role of the PI3K/AKT/
mTOR pathway in resistance to chemotherapy including conventional anticancer
medications, molecular targeted agents, hormonal therapy and radiation.
328 Y. Tsukumo et al.

In HER2-positive breast cancer, loss of PTEN protein expression (30–40 % of


tumors) and activating mutations in PIK3CA (E545K or H1047R; 20–25 % of tu-
mors) are amongst the genetic alterations that lead to the activation of the mTOR
pathway (Hernandez-Aya and Gonzalez-Angulo 2011). Loss of PTEN is associated
with resistance to trastuzumab, which is a monoclonal antibody that interferes with
the HER2 receptor, since trastuzumab’s antitumor activity requires PTEN activa-
tion (Nagata et al. 2004). As previously described, PTEN is a tumor suppressor that
inactivates PI3K signaling and mTOR activation. Recent clinical studies showed
that activation of the PI3K/AKT/mTOR pathway through genetic alterations was
associated with significantly shorter PFS (Berns et al. 2007; Wang et al. 2011b)
and poor OS (Gallardo et al. 2012) in patients receiving trastuzumab therapy.
The trastuzumab-resistance caused by activation of the PI3K/AKT/mTOR path-
way indicates that inhibition of this pathway can suppress proliferation in HER2-
positive resistant breast cancer cells. Indeed, the pan-PI3K inhibitors GDC-0941
and LY294002 and dual PI3K/mTOR inhibitor BEZ235 were able to significantly
inhibit PI3K/AKT/mTOR signaling and cell proliferation in trastuzumab-resistant
breast cancer cell lines in a dose-dependent manner (Eichhorn et al. 2008; Junttila
et al. 2009; Kataoka et al. 2010; Nagata et al. 2004). These findings indicate that
inhibition of the PI3K/AKT/mTOR pathway may be an effective approach to treat
tumors that are resistant to trastuzumab.
In the case of a lapatinib, which is a small compound that inhibits both EGFR
and HER2 activity, it has been reported that activation of PIK3CA through hotspot
E545K or H1047R mutations conferred resistance to lapatinib in HER2-overex-
pressing breast cancer cells (Eichhorn et al. 2008; O’Brien et al. 2010). Correspond-
ingly, BEZ235 reversed lapatinib resistance in PTEN-deficient or PIK3CA-acti-
vated breast cancer cell lines (Eichhorn et al. 2008). Furthermore, mTOR inhibi-
tion with ridaforolimus (mTORC1 inhibitor) or INK-128 (mTORC1/2 inhibitor) in
combination with lapatinib resulted in significantly higher rates of tumor response
than either agent given alone in cell-based trastuzumab/lapatinib-resistant xenograft
models (Garcia-Garcia et al. 2012; Gayle et al. 2012). These studies support clinical
evaluation of mTOR inhibition in patients with HER2-overexpressing breast can-
cers, which are resistant to both trastuzumab and single-agent lapatinib. In phase I
and I/II study, everolimus combined with weekly paclitaxel and trastuzumab was
generally well tolerated and had encouraging antitumor activity in patients with
trastuzumab-pretreated and trastuzumab-resistant metastatic HER2-overexpressing
breast cancer (Andre et al. 2010; Morrow et al. 2011). The recently completed phase
III study (BOLERO-3) showed that everolimus in combination with trastuzumab
plus vinorelbine, which is an antimitotic drug, improved PFS compared to trastu-
zumab plus vinorelbine alone in trastuzumab-resistant metastatic breast cancer pa-
tients (Sendur et al. 2013).
Preclinical studies indicate that inhibition of the PI3K/AKT/mTOR pathway
can also overcome or prevent resistance to hormonal therapy (Cavazzoni et al.
2012; Ghayad et al. 2008, 2010; Miller et al. 2010). Treatment of breast cancer
with the dual PI3K/mTOR inhibitor BEZ235 has been shown to prevent cells from
developing resistance to hormone therapy by inducing apoptosis in long-term
15  mTOR and Regulation of Translation 329

estrogen-deprived cells (Miller et al. 2010). Furthermore, BEZ235 increased hor-


mone sensitivity, consistent with restoring levels of estrogen receptor (ER) and ER-
inducible target genes, and resulting in enhancement of sensitivity to tamoxifen.
Similarly, both everolimus and BEZ235 were able to restore sensitivity to hormone
therapy in resistant cell lines (Cavazzoni et al. 2012; Ghayad et al. 2008). Inclu-
sion of a MEK1 inhibitor in this combination further increased this effect (Ghayad
et al. 2010). A randomized phase III study (BOLERO-2) evaluated the efficacy
of an aromatase inhibitor, exemestane, with or without everolimus in postmeno-
pausal women with ER-positive/HER2-negative locally-advanced or metastatic
breast cancer who were refractory to nonsteroidal aromatase inhibitors and had
documented disease recurrence or progression (Baselga et al. 2012). This study
revealed that everolimus, combined with an aromatase inhibitor, improved PFS in
patients with hormone receptor-positive advanced breast cancer previously treated
with nonsteroidal aromatase inhibitors. Everolimus in combination with exemes-
tane for the treatment of postmenopausal women with advanced hormone receptor-
positive/HER2-negative breast cancer was approved by the FDA and the European
Medicines Agency (EMA) in 2012. Other trials are also ongoing with the PI3K/
AKT/mTOR pathway inhibitors in combination with hormonal therapy in resistant
patients (Burris 2013) (see also clinicaltrials.gov).
Loss of PTEN and activation of the PI3K/AKT/mTOR pathway are also fre-
quent and well-established mechanisms of prostate cancer progression (Bertram
et al. 2006; Mulholland et al. 2006; Sircar et al. 2009), that, by crosstalk induction
of ligand-independent activation of androgen receptors, support the development
of resistance to androgen deprivation therapy (Lin et al. 2004; Pienta and Bradley
2006). In turn, although androgen receptors upregulate mTOR activity by increas-
ing the androgen receptor–dependent transcription of nutrient transporters (Xu et al.
2006), the inhibition of mTOR with rapamycin increases androgen receptor mRNA
and transcriptional activity (Cinar et al. 2005; Wang et al. 2008b). Therefore, in-
hibiting both pathways would be essential to achieve complete responses. A phase
II trial is currently studying bicalutamide, which is an oral non-steroidal antian-
drogen, and everolimus to see how well they work in combination compared with
bicalutamide alone in treating patients with recurrent or metastatic prostate cancer
(Gomez-Pinillos and Ferrari 2012).
In glioblastoma multiforme, radiosensitization is an important therapeutic op-
tion because standard therapy for glioblastoma multiforme comprises adjuvant ra-
diotherapy combined with the alkylating agent temozolomide (TMZ) (Stupp et al.
2007). Ionizing radiation damages DNA and leads to apoptosis, resulting in tumor
regression. However, activation of some signaling pathways can promote cell sur-
vival even after radiation-induced DNA damage, thus limiting efficacy. One such
pathway is the PI3K/AKT/mTOR cascade, which is widely hyperactivated in glio-
blastoma multiforme through multiple genetic aberrations, such as loss of PTEN
expression; activating mutations in the genes encoding PI3K (such as PIK3CA or
PIK3R1); and overexpression of RTKs (such as EGFR (HER1), HER2 (also known
as ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2)),
platelet-derived growth factor receptor (PDGFR) and MET) (The Cancer Genome
330 Y. Tsukumo et al.

Atlas Research Network 2008). The frequency of genetic alterations in RTK/RAS/


PI3K in glioblastoma multiforme has been estimated to be about 90 % (The Cancer
Genome Atlas Research Network 2008). Chakravarti et al. found that glioblastoma
multiforme patients with an activated PI3K/AKT/mTOR pathway showed signifi-
cantly worse survival than patients without oncogenic activation of the pathway
(Chakravarti et al. 2004). Several clinical studies have evaluated mTORC1 inhibi-
tors in combination with TMZ and radiation in glioblastoma multiforme (Sarkaria
et al. 2010, 2011). These trials showed different results. In a phase I trial, a com-
bination of temsirolimus, TMZ and radiation was associated with high infection
rates (Sarkaria et al. 2010). In contrast, a similar trial of everolimus in combination
with TMZ and radiation, followed by everolimus plus adjuvant TMZ, demonstrated
partial metabolic responses in 22 % of patients, and stable metabolic disease in 78 %
(Sarkaria et al. 2011). A phase I clinical trial is ongoing to examine whether the ad-
dition of the dual PI3K/mTOR inhibitor can sensitize tumors to TMZ (Burris 2013).

15.11 Conclusions and Perspectives

mTOR regulates translation of specific mRNAs responsible for cell proliferation,


survival and invasion through the phosphorylation of several downstream sub-
strates, such as 4E-BPs and S6K. Therefore, the dysregulation of the mTOR path-
way leads to transformation accompanied by aberrant cell growth and proliferation
and, moreover, contributes to tumor angiogenesis, invasion and metastasis. In fact,
the overactivation of the mTOR pathway is observed in a variety of human cancers.
Consequently, inhibition of mTOR activity in cancer cells has great therapeutic po-
tential. Rapalogs are FDA-approved for the treatment of several types of cancers.
Despite these promising data, single treatment with rapalogs is ineffective for the
treatment of most tumor types. Given that rapalogs have been shown to restore
sensitivity to many standard chemotherapeutic agents, they may be best utilized
in combination chemotherapy. Second-generation mTOR inhibitors, which target
both mTORC1 and mTORC2, as well as dual PI3K/mTOR inhibitors, have already
been developed. Importantly, compared to rapalogs, these inhibitors have shown
improved efficacy in preclinical studies and appear to have a potential to circum-
vent rapalog resistance in some cancer types. Some of these inhibitors are currently
in phase I trials.

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