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ISBN: 978-0-12-803367-8
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CONTRIBUTORS
Khaled Alawam
Forensic Medicine Department, Ministry of Interior, Kuwait City, Kuwait
Daniela Amicizia
Department of Health Sciences (DISSAL), Via Antonio Pastore 1, University of Genoa,
Genoa, Italy
Claudia Andrieu
INSERM, U1068, CRCM; Institut Paoli-Calmettes; Aix-Marseille University, and CNRS,
UMR7258, Marseille, France
Martin R. Berger
German Cancer Research Center, Toxicology and Chemotherapy Unit, Heidelberg,
Germany
Nicola Luigi Bragazzi
Genoa, Italy
Rossen Donev
Biomed Consult Ltd., Swansea, United Kingdom
Roberto Gasparini
Genoa, Italy
Nadya V. Ilicheva
Institute of Cytology RAS, St. Petersburg, Russia
Ilinka Ivanoska
Faculty of Computer Science and Engineering, University Ss. Cyril and Methodius, Skopje,
Macedonia
Slobodan Kalajdziski
Macedonia
Sara Karaki
Ljupco Kocarev
Faculty of Computer Science and Engineering, University Ss. Cyril and Methodius;
Macedonian Academy of Sciences and Arts, Skopje, Macedonia, and BioCircuits Institute,
University of California, San Diego, California, USA
Aneliya Kostadinova
Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia,
Bulgaria
ix
x Contributors
Claudio Larosa
Department of Surgical Sciences and Integrated Diagnostics, University of Genoa,
Genoa, Italy
Xi Liu
Bioinformatics Section, School of Basic Medical Sciences, Southern Medical University,
Guangzhou, China
Albena Momchilova
Bulgaria
Donatella Panatto
Genoa, Italy
Olga I. Podgornaya
Institute of Cytology RAS; Cytology and Histology Chair, Biological Faculty, St. Petersburg
State University, St. Petersburg, Russia, and FEF University, Vladivostok
Emanuela Rizzitelli
Genoa, Italy
Palma Rocchi
Ruixian Song
Guangzhou, China
Biljana Risteska Stojkoska
Macedonia
Tanya Topouzova-Hristova
Faculty of Biology, Cytology, Histology and Embryology, Sofia University, Sofia, Bulgaria
Daniela Tramalloni
Genoa, Italy
Kire Trivodaliev
Macedonia
Rumiana Tzoneva
Bulgaria
Ivana Valle
SSD “Popolazione a rischio,” Health Prevention Department, Local Health Unit ASL3
Genovese, Genoa, Italy
Contributors xi
Alex P. Voronin
Institute of Cytology RAS, and Cytology and Histology Chair, Biological Faculty,
St. Petersburg State University, St. Petersburg, Russia
Nan Wu
Guangzhou, China
Jingwen Yang
Guangzhou, China
Hao Zhu
Guangzhou, China
Hajer Ziouziou
CHAPTER ONE
The Eukaryotic Translation

Initiation Factor 4E (eIF4E) as a
Therapeutic Target for Cancer
Sara Karaki*,†,{,}, Claudia Andrieu*,†,{,}, Hajer Ziouziou*,†,{,},
Palma Rocchi*,†,{,},1
*INSERM, U1068, CRCM, Marseille, France
†
Institut Paoli-Calmettes, Marseille, France
{
Aix-Marseille University, Marseille, France
}
CNRS, UMR7258, Marseille, France
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 2
2. eIF4E's Structure and Expression 2
2.1 Structure 2
2.2 eIF4E's Expression and Regulation 4
3. eIF4E's Functions 9
3.1 mRNA Translation Initiation 9
3.2 Nuclear Export 11
4. eIF4E: A Therapeutic Target in Cancer 14
4.1 eIF4E in Cancers 14
4.2 EIF4E’s Mechanisms in Cancer 15
4.3 Targeting eIF4E in Cancers 16
5. Conclusion 20
References 21
Abstract
Cancer cells depend on cap-dependent translation more than normal tissue. This
explains the emergence of proteins involved in the cap-dependent translation as tar-
gets for potential anticancer drugs. Cap-dependent translation starts when eIF4E binds
to mRNA cap domain. This review will present eIF4E's structure and functions. It will also
expose the use of eIF4E as a therapeutic target in cancer.
Advances in Protein Chemistry and Structural Biology, Volume 101 # 2015 Elsevier Inc. 1
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/bs.apcsb.2015.09.001
2 Sara Karaki et al.
1. INTRODUCTION
When eIF4E was discovered, it was considered as an isolated protein,
not belonging to any known protein family. Research of the last decade
showed that all eukaryotes have several members of the eIF4E family.
Joshi et al. (2005) identified, through sequence analysis, 411 eIF4E family
members, in 230 species. Three isoforms (eIFF-1, 4EHP, and eIF4E-3)
are present in mammals ( Joshi, Cameron, & Jagus, 2004). Not all proteins
from eIF4E’s family bind to 7 methylguanosine mRNA cap (m7GDP) and
to the same ligand ( Joshi et al., 2004; Robalino et al., 2004; Rosettani et al.,
2007), which give them different physiological functions. Hernandez and
Vazquez-Pianzola (2005) suggested that in each organism, there is one
member of the eIF4E family expressed that intervenes in translation and that
other members have other functions (development, translation repression,
specific mRNA nuclear transport). This hypothesis is being confirmed since
eIF4E’s isoforms are thought to be involved in many functions such as sper-
matogenesis, oogenesis, aging, and other functions (Amiri et al., 2001;
Dinkova et al., 2005; Evsikov & Marin de Evsikova, 2009; Minshall
et al., 2007; Syntichaki, Troulinaki, & Tavernarakis, 2007). Cap-dependent
translation starts when eIF4E binds to the mRNA cap domain. Cancer cells
depend on cap-dependent translation more than normal tissues ( Jia et al.,
2012). This review will expose eIF4E’s structure and functions and will
expose the use of eIF4E as an anticancer target.
2. eIF4E'S STRUCTURE AND EXPRESSION

2.1 Structure
eIF4E’s primary structure (Fig. 1A) is highly conserved in all eukaryotes
because of the important role they play in the cell. In the N-terminal
end, sequences are variable between different organisms, but this end does
not seem to be involved in the initiation to translation function. The tertiary
structure was characterized in mice, men, yeast, and wheat (Monzingo et al.,
2007; Tomoo et al., 2002). This structure is composed of eight antiparallel β
strands and three helices on the convex side (Fig. 1B). eIF4E binds to the
m7GDP of the mRNA cap to allow the translation initiation. eIF4E tridi-
mensional structures that interact with cap analogs were identified, allowing
to identify the interaction site (Gross et al., 2003; Niedzwiecka et al., 2002;
Tomoo et al., 2003). The cap interaction happens in a hydrophobic pocket
eIF4E and Cancer 3
A
MATVEPETTPTPNPPTTEEEKTESNQEVANPEH
YIKHPLQNRWALWFFKNDKSKTWQANLRLISK
FDTVEDFWALYNHIQLSSNLMPGCDYSLFKDGI
EPMWEDEKNKRGGRWLITLNKQQRRSDLDRF
WLETLLCLIGESFDDYSDDVCGAVVNVRAKGDK
IAIWTTECENREAVTHIGRVYKERLGLPPKIVIGY
QSHADTATKSGST TKNRFVV
B N-term
human elF4E 4E-BP1

C-term N-term
Convex side
cap-binding
pocket dorsal
Trp 102 surface
Trp 56
7
m GpppA
C-term
Concave side
Figure 1 (A) Human eIF4E's primary structure. (B) eIF4E's structure. Crystal structure of
the human protein eIF4E (blue; dark gray in the print version) linked to the mRNA
m7GDP cap (light pink; light gray in the print version) and to its ligand 4E-BP1 (green;
gray in the print version) (http://atlasgeneticsoncology.org). The eIF4E interaction with
the cap occurs on the concave side and requires two highly conserved tryptophan res-
idues (Trp). The interaction between eIF4E and its ligands 4E-BPs, eIF4G, and PML occurs
on the convex side.
on eIF4E’s concave side, due to the interaction with two highly conserved
tryptophan residues (56 and 102 in mice) (Fig. 1B). This interaction is sta-
bilized by three hydrogen bonds. The interaction with partner proteins
involved in translation regulation, such as eIF4G or 4E binding proteins
(4E-BP), takes place in a hydrophobic region on the convex side, and it
involves two conserved tryptophan residues (43 and 73 in mice)
(Fig. 1B). These proteins interact with eIF4E through a bonding pattern,
which consensus sequence is: Y(X) 4LΦ, with X being any amino acid
and Φ being a hydrophobic residue. The eIF4G or the 4E-BPs’ binding
to eIF4E causes conformational changes which increases eIF4E’s affinity
to the cap (Niedzwiecka et al., 2002; von Der Haar, Ball, & McCarthy,
2000). The PML protein (promyelocytic leukemia protein) and the viral
protein Z (VPZ) represent a second class of eIF4E regulators that intervene

in the mRNA nuclear export function. These proteins bind to eIF4E’s con-
vex side using their RING domain, which, in contrast to the bond to eIF4G
and 4E-BP, decreases the affinity of eIF4E to the cap (Cohen et al., 2001;
Kentsis et al., 2001; Volpon et al., 2010). Structural studies show that eIF4E
has different conformations and different ligand binding affinities depending
on whether it is binding to the cap or not (Niedzwiecka et al., 2002;
Niedzwiecka, Darzynkiewicz, & Stolarski, 2004; Volpon et al., 2006;
Tomoo et al., 2002).
2.2 eIF4E's Expression and Regulation

2.2.1 Expression
Cell and tissue growth depend on protein synthesis. eIF4E’s expression is
significantly higher in human malignant tissues than in normal tissues. For
cells to be viable, it is important for protein translation to be closely regulated
to prevent malignant transformation and cancer development. The transla-
tion control is rather at initiation, even though there are controls during
elongation phase. eIF4E’s activity is controlled by several mechanisms
described below (Van Der Kelen et al., 2009). Although eIF4E is well stud-
ied for its role in the translation initiation and for its involvement in tumor-
igenesis, little is known about its expression regulation. Surprisingly, eIF4E’s
overexpression does not lead to a global increase in the proteins’ translation,
but it leads to a selective increase in the translation of mRNAs that have a
structure called “sensible elements to eIF4E” and that are involved in
tumorigenesis.
2.2.2 Regulation
Studies show that the eIF4E inhibition can lead to HeLa cancer cell death
and its absence is lethal for Saccharomyces cerevisiae. When overexpressed,
eIF4E can act like an oncogene, by promoting malignant transformation
and lymphomagenesis in rodent cells. An overproduction of eIF4E causes
uncontrollable cell growth or oncogenesis, which indicates its importance
in protein synthesis (Andrieu et al., 2010).
Given the important function of this protein, it is not surprising to find its
activity highly regulated.
2.2.3 Transcription Levels

Serum, growth factors, and the immunologic activation of T lymphocyte
lead to an increase in the gene transcription (Schmidt, 2004). There are also
eIF4E and Cancer 5
consensus binding sites to transcription factors (such as c-Myc and

hnRNPK) that are involved in the control of the gene transcription in
response to stimuli (Lynch et al., 2005). For example, 4E-BP1 has at least
seven phosphorylation sites among which four are known to be regulated
by signaling pathways such as mTOR (Gingras, Raught, & Sonenberg,
2001; Heesom et al., 2001; Wang et al., 2005). When c-Myc is over-
expressed, due to growth factors, eIF4E’s expression rises.
2.2.4 Protein Level

2.2.4.1 Phosphorylation
In mammals, eIF4E is phosphorylated at the 209th serine residue located in a
C-terminal motif which is conserved in all species except for plants and
S. cerevisiae. The Mnk1 and Mnk2 kinases (MAPK-integrating kinases)
(Ueda et al., 2004) bind to the C-terminal end of eIF4G, to be close to eIF4E
to phosphorylate it. These kinases are themselves activated by phosphoryla-
tion realized by the Erk kinase (extracellular signal-regulated kinase) and by
the p38 MAP kinase (Fig. 2) (Scheper et al., 2001). Growth factors, phorbol
esters, and insulin can activate the Mnk kinases via the Erk pathway
(Tschopp et al., 2000). Cytokines and some stress conditions can activate
the p38 MAP kinase pathway. Phosphorylation can also be regulated during
viral infection. For example, during an adenovirus infection, eIF4E is
dephosphorylated because the 100K viral protein binds to eIF4G and moves
the Mnk kinases from the eIF4F complex. The same phenomenon was
observed during an influenza virus infection (Cuesta, Xi, & Schneider,
2000). However, a coronavirus infection activates Mnk1 and increases
eIF4E’s phosphorylation via the p38 MaP kinase pathway (Banerjee et al.,
2002). Although eIF4E’s phosphorylation mechanism is known, the conse-
quences of this phosphorylation on translation initiation are still unclear and
depend on the cellular context (Scheper & Proud, 2002). By a modulation of
the Mnk–eIF4G interaction, eIF4E’s phosphorylation is controlled: eIF4G
binding is controlled by MAPK-mediated phosphorylation of the Mnk1
active site. Furthermore in the absence of MAPK signaling, eIF4E phos-
phorylation is prevented by the C-terminal domain of Mnk1 that restricts
its interaction with eIF4G (Shveygert et al., 2010).
2.2.5 4E-BP
The protein family 4E-BP regulates eIF4E capacity to form the cap-binding
complex (eIF4F). Currently, three 4E-BPs are known in mammals:
4E-BP1, 4E-BP2, and 4E-BP3. Their interaction strength is regulated by
Serum, growth factors, Transcription factors

lymphocyte T activation (cMyc, hrRNPK), Stimulis
eIF4e transcription
eIF4e overexpression
translation of mRNA having « sensible to eIF4e

elements »
Tumorigenesis
Figure 2 eIF4E's expression regulation and its implication in tumorigenesis. Serum,

growth factors, and T-lymphocyte immunologic activation lead to an increase of eIF4E's
transcription. There are also consensus binding sites to transcription factors (such as
c-Myc and hnRNPK) that are involved in the control of the gene transcription in
response to stimuli. When c-Myc is overexpressed, eIF4E's expression rises. eIF4E's over-
expression leads to a selective increase in the translation of mRNAs that have a structure
called “sensible to eIF4E elements” and that are involved in tumorigenesis.
phosphorylation. The 4E-BPs are phosphorylated in response to growth fac-

tors, amino acids, or hormones such as insulin which activates the mTOR
pathway (molecular target of rapamycin) (Fig. 3) (Gingras et al., 2001;
Gingras, Raught, & Sonenberg, 2004; Kimball, 2001). For example,
4E-BP1 has at least seven phosphorylation sites, among which four are
known to be regulated by signaling pathways such as mTOR (Gingras
et al., 2001; Heesom et al., 2001; Wang et al., 2005). In contrast, hypoxia
induces a phosphorylation decrease in 4E-BP1 (Shenberger et al., 2005).
When 4E-BPs are hypophosphorylated, they can sequestrate eIF4E and
eIF4E and Cancer 7
Figure 3 eIF4E's implication in the mRNA translation initiation. The translation initiation
of most mRNAs occurs due to a cap-dependent mechanism that involves eIF4E. This
mechanism is regulated by eIF4E's phosphorylation by Mnk proteins, as well as by
4E-BP factors. ? ¼ activator or repressor role of eIF4E phosphorylation on translation.
prevent the interaction with eIF4G and inhibit the translation. When they
are hyperphosphorylated, they cannot bind to eIF4E, which is then released
to participate in the protein translation initiation (Fig. 3) (Gingras et al.,
2001). The 4E-BP proteins and eIF4G have the same binding site to eIF4E.
So there is a competition between these proteins. On the other hand, the
bond between eIF4E and 4E-BP does not prevent its bond to the cap. Oth-
erwise, some viruses can modulate eIF4E’s activity by acting on the 4E-BP
phosphorylation. For example, the picornaviruses induce 4E-BP’s dephos-
phorylation which inhibits protein synthesis. So the 4E-BPs work as inhib-
itors of the cap-dependent translation.
2.2.6 Ubiquitination
Another eIF4E’s posttranslational modification is ubiquitination. It has been
demonstrated that it does not prevent the eIF4E mRNA cap binding but it
prevents the eIF4G bond and thus eIF4E phosphorylation is reduced
(Murata & Shimotohno, 2006; Othumpangat, Kashon, & Joseph, 2005).
However, the ubiquitination consequences on the translation initiation
are still unknown.
eIF4E degradation depends on the proteasome and happens principally
when ligases such as Chip ubiquitinate the Lys-159 residue (Murata &
Shimotohno, 2006; Othumpangat et al., 2005). This ubiquitination does
not prevent the bond to the mRNA cap, but the bond with eIF4G and
eIF4E’s phosphorylation is reduced. Moreover, Hsp27 interacts directly
with eIF4E and regulates it. After Hsp27 knockdown, eIF4E is ubiquitinated
and degraded through the ubiquitin–proteasome pathway indicating that
cytoprotection induced by Hsp27 involves eIF4E. Andrieu et al. showed
in castrate-resistant prostate cancers that forced overexpression of Hsp27
increases the protein expression level of eIF4E without affecting its mRNA
expression level. They also showed that Hsp27 could exert an effect directly
on eIF4E and that the effect of Hsp27 on eIF4E level is independent of
4E-BP1. They showed that a decrease in eIF4E ubiquitination is associated
with resistance to androgen withdrawal and paclitaxel, concluding that
Hsp27 knockdown reduces eIF4E stability, enhancing its ubiquitination
and degradation, thereby reducing cell viability after androgen withdrawal
and/or chemotherapy (Andrieu et al., 2010). In pancreatic cancer cells,
Baylot et al. demonstrated that the C-terminal part of Hsp27 interacts with
eIF4E and that Hsp27 phosphorylation enhances this interaction and eIF4E
expression level and gemcitabine resistance. Hsp27 enhances eIF4E protein
expression by inducing a decrease of approximately 30% in the amount of
ubiquitinated eIF4E, thereby inhibiting its proteasomal degradation (Baylot
et al., 2011).
It has also been described that the DIAP1 protein of the IAP family
(inhibitor of apoptosis protein) interacts with eIF4E and leads to its
ubiquitination (Lee et al., 2007).
2.2.7 Poly-A
A new translation repression mechanism of some specific mRNAs has been
described by Richter and Sonenberg (2005). In Xenopus laevis, during oocyte
development, there is a translation regulation mechanism based on the
length of the poly-A tail. Some dormant but stable mRNAs have a short
eIF4E and Cancer 9
poly-A tail, unlike the majority of mRNAs who have a long tail. The CPEB
protein controls polyadenylation by interacting with the CPE element on
the mRNA 30 extremity. CPEB also binds to the Maskin protein which
sequestrates eIF4E and prevents the translation of these specific mRNAs.
When the oocytes are stimulated, a signaling cascade takes place and allows
the poly-A tail’s elongation by CPEB and the Maskin protein’s moving. The
translation can now start. A similar mechanism is observed in the drosophila
with the Bicoid and Cup proteins (Nakamura, Sato, & Hanyu-Nakamura,
2004; Niessing, Blanke, & Jackle, 2002) or during neurogenesis, where the
neuroguidin protein binds to eIF4E to prevent the translation ( Jung,
Lorenz, & Richter, 2006).
3. eIF4E'S FUNCTIONS
3.1 mRNA Translation Initiation
There are two types of mRNA translation initiation: the cap-dependent
translation initiation and the cap-independent translation initiation. Fur-
thermore, there is another mRNA category (10%) that is translated in a
cap- and eIF4E-independent manner. These mRNAs have a structure called
“IRES” (internal ribosome entry sites) that allows the ribosome’s 40S sub-
unit to bind directly. Originally identified as a translation mechanism of viral
genes, it is now identified as playing an important role during the death cell’s
process, mitosis, and stress conditions, where cap-dependent protein synthe-
sis is reduced (Stoneley & Willis, 2004).
3.1.1 The CaP-Dependent Translation Initiation Mechanism

The translation initiation of most cellular mRNAs takes place due to a cap-
dependent mechanism. The 7-methylguanosine (m7GDP) structure (also
called cap) is located on the 50 extremity of the cytoplasmic mRNAs that pro-
cess a cap-dependent translational process. It is a posttranscriptional modifica-
tion introduced by the successive action of several nuclear enzymes. The cap
has many roles. It protects the mRNA against degradation by ribonuclease, it
intervenes in the nuclear export, and it allows the ribosome recruitment. In
fact, this structure is specifically recognized by eIF4E, enabling recruitment of
the eIF4F complex to bind to the cap (Fig. 3). This complex is formed
by eIF4E associated to eIF4A and eIF4G and allows the recruitment of the
ribosome on the mRNAs. The protein eIF4A is a helicase that catalyzes
the separation of the paired strands of the RNA, in an ATP-dependent man-
ner. Its activity is slow and requires stimulation by eIF4B/eIF4H and eIF4G
(Rogers, Komar, & Merrick, 2002). The protein eIF4G acts like a scaffolding
protein by linking the mRNA to the 40S subunit of the ribosome through its
interaction with eIF3, which stabilizes the complex (Gross et al., 2003; Prevot,
Darlix, & Ohlmann, 2003). This step leads to the recruitment of the
preinitiation complex 43S (40S + eIF3 + eIF1 + eIF1A + eIF5 + eIF2–GTP–
Met-tRNAi) on the mRNA cap and to the formation of the initiation com-
plex 48S (ARNm + 43S + eIF4F) (Fig. 3). The mRNA is then scanned in the
50 –30 direction in order to find the start codon (Kozak, 2002). This is due to
the following initiation factors: eIF1, eIF1A, eIF5, and the complex eIF2–
GTP–Met-tRNAi. Once the initiation codon is located, eIF5 interacts with
eIF2 and promotes the intrinsic hydrolysis of the GTP associated to eIF2. This
hydrolysis leads to the detachment of the initiation factor from the ribosome’s
subunit 40S and to the recruitment of the 60S subunit resulting in the forma-
tion of the 80S complex (Fig. 3). The protein synthesis can now begin. The 50
and 30 UTR extremities (untranslated region) also play an important role in
the translation initiation mechanism. In fact, on the 50 extremity, the sequence
surrounding the start codon plays a role in the initiation site selection by the
48S complex and gives an indication about the translation efficiency that
might be weak or strong. In mammals, the Kozak sequence is the best
sequence to initiate translation. At the 30 extremity, the poly-A tail is capable
of interacting with the cap in 50 through the PABPs (polyadenylate-binding
proteins) (Fig. 3). This interaction promotes the ribosome’s 40S subunit
recruitment through direct interaction between PABPs and eIF4G. This
interaction gives a circular conformation to the mRNA which improves
the translation initiation. So the eIF4E protein plays a major role in mRNA
cap-dependent translation regulation (Sonenberg, 2008) and therefore in the
cell cycle progression (O’Farrell, 2001).
3.1.2 The CaP-Independent Translation Initiation Mechanism

It has to be noted that other modes of translation initiation are described.
The protein eIF4E is, for example, involved in the translation of viral
mRNAs that do not have a cap. In fact, it has recently been demonstrated
that the calicivirus mRNAs are linked covalently to a viral protein (VPg) that
acts like a substitute to the cap to recruit eIF4E (Chaudhry et al., 2006). The
VPg-binding site to eIF4E is different from the cap-binding site and from the
4E-BP protein binding site since the complex VPg/eIF4E/4E-BP1 has been
isolated (Goodfellow et al., 2005). This interaction is unique within known
virus in mammals, but we can find a similar interaction in the potyvirus that
infects plants (Dreher & Miller, 2006).
eIF4E and Cancer 11
3.2 Nuclear Export

It is described that eIF4E is mainly located into the cytoplasm where it fulfills
its role in the translation initiation, but it is also found in the nucleus.
Recently, it has been described that eIF4E has a function in the regulation
of translation but at a different level than that of the initiation (Strudwick &
Borden, 2002). Lejbkowicz et al. were the first one to describe eIF4E’s
expression in the nucleus’ small structures called nuclear bodies (Fig. 4A
and B). This was then observed in a variety of mammalian cell lines and
would be conserved among eukaryotes. We can find 10–20 nuclear bodies
per nucleus, and their size varies between 0.1 and 1 μm (Cohen et al., 2001;
Dostie, Lejbkowicz, & Sonenberg, 2000a; Lai & Borden, 2000). These bod-
ies are not affected by RNases or DNases, which indicates that these struc-
tures are not formed by nucleic acid (Cohen et al., 2001; Dostie et al.,
2000a). eIF4E is exported to the nucleus via importin active pathways
involving 4E-T protein (eIF4E-transporter) that binds to eIF4E in a similar
region than eiF4G and 4E-BP (Dostie et al., 2000b). About 68% of the
eIF4E proteins are found in the nuclear bodies where they are involved
in the export of an mRNA category from the nucleus to the cytoplasm
(Culjkovic, Topisirovic, & Borden, 2007). This mRNA category has a
structure called “sensible to eIF4E elements” 4E-SE that allows eIF4E to
recognize these mRNAs (Fig. 4C) (Culjkovic et al., 2005). Normally,
mRNAs are prepared for export through a process regulated by the nuclear
complex CBC (cap-binding complex), but for this category of mRNAs,
eIF4E nuclear bodies are able to regulate their own transport (Cohen
et al., 2001; Lai & Borden, 2000). These mRNAs “sensitive to eIF4E” also
have a long and complex 50 UTR end that it is hardly decondensed by heli-
cases eIF4A/4B (Zimmer, DeBenedetti, & Graff, 2000). Theoretically, since
eIF4E is a limiting factor for the helicase recruitment, an eIF4E increase
should rise the helicase activity and thus increase these specific mRNAs’
protein synthesis (Zimmer et al., 2000). However, these mRNAs
“sensitive to eIF4E” do not show an increase in their translation initiation
rate. This mRNA protein synthesis is regulated by their export from the
nucleus to the cytoplasm (Lai & Borden, 2000). Indeed, when eIF4E is over-
expressed, the cyclin D1 mRNA level does not change, but the level of
nuclear mRNA decreases and the level of cytoplasmic mRNA is increased.
These results show that an increased in eIF4E expression increases the export
of these mRNAs and thus the level of protein (Lai & Borden, 2000). This
export mechanism contributes to the oncogenic potential of eIF4E (Cohen
et al., 2001). It is therefore possible that there are, in the nucleus, negative
Figure 4 (A) eIF4E's implication in the mRNA nuclear export. (A) eIF4E is localized in the
nuclear bodies in the NIH3T3 cells. DAPI ¼ nuclear marker (Culjkovic et al., 2005).
(B) U2OS cell's nucleus showing the eIF4E expression in the nuclear bodies (Culjkovic
et al., 2006, JCB). (C) eIF4E's implication in the mRNA nuclear export. Diagram rep-
resenting the eIF4E role in the nuclear export of an mRNA category. This mechanism
is regulated by the PML protein.
regulators to this process identical to 4E-BPs in the cytoplasm. Several pro-

teins can associate with eIF4E nuclear bodies such as the ribosomal protein
L7 and P, eIF4G (Iborra, Jackson, & Cook, 2001), the PRH protein
(proline-rich homeodomain protein) (Topisirovic et al., 2003a), the
eIF4E and Cancer 13
homeodomain proteins, the Z protein, and the PML protein which is the
most studied (Campbell Dwyer et al., 2000; Cohen et al., 2001; Lai &
Borden, 2000). These proteins regulate the eIF4E–cap bond, a bond that
is necessary for the mRNA export (Dostie et al., 2000a). The majority of
eIF4E nuclear protein colocalizes with the PML protein (Lai & Borden,
2000) as a result of stress, viral infection, or an interferon treatment
(Regad & Chelbi-Alix, 2001). This protein interacts on the convex side
through its RING domain using the 73th tryptophan residue (Cohen
et al., 2001; Lai & Borden, 2000). Even though this interaction site is
far from the cap-binding site, this bond can inhibit the eIF4E–cap inter-
action (Culjkovic et al., 2007). The PML protein binds to eIF4E and
lowers its mRNA’s cap affinity (100 times), thereby changing its mRNA
export function (Fig. 4C) (Cohen et al., 2001; Culjkovic et al., 2007;
Kentsis et al., 2001; Lai & Borden, 2000). PML would have an antitumor
function. There are approximately 200 homeodomain proteins containing
potential binding sites for eIF4E and could therefore regulate it (Culjkovic
et al., 2007). So it seems that the ability to modulate eIF4E’s activity by
acting on its binding to the cap is conserved from the cytoplasm to the
nucleus. Finally, it has been suggested that eIF4E contributes to the
mRNA translation in the nucleus (Dostie et al., 2000a; Iborra et al.,
2001). This nuclear translating phenomenon has already been observed
in mammals’ cells (Iborra et al., 2001) and an increasing number of proteins
from the translation machinery are involved in nuclear processes. This
translation may be involved in aberrant transcript elimination, by an
mRNA quality control system: NMD (nonsense-mediated decay). Indeed,
this system requires an active protein synthesis in order to detect the
appearance of premature STOP codons leading to the synthesis of trun-
cated proteins.
All known data on eIF4E’s role in translation initiation and nuclear
export led to the hypothesis that there is an eIF4E regulon (Culjkovic
et al., 2007). The “regulons” are a set of genes regulated by the same pro-
tein. The hypothesis has suggested that mRNAs belonging to the eIF4E
regulon have a signal that allows its recruitment. The eIF4E protein is
considered as regulatory since it allows, on the one hand, the nuclear export
through the 4E-SE site recognition and, on the other hand, the protein
translation through another unknown signal. In some cases, eIF4E acts on
both mechanisms likely due to the presence of both of these signals. The
eIF4E protein can thus orchestrate genes’ expression and control the cell
cycle progression.
4. eIF4E: A THERAPEUTIC TARGET IN CANCER

4.1 eIF4E in Cancers
Protein synthesis is a highly regulated process that controls mRNA transla-
tion. Alterations of this process are associated with the development and pro-
gression of cancer. As we described, the components of the translation
machinery are regulated by several fundamental signaling pathways that
are often disrupted in cancer. Thus, the protein translation process becomes
oncogenic. Sonenberg et al. were the first to show the involvement of eIF4E
in oncogenesis in 1990. Since then, the oncogenic potential due to eIF4E
hyperactivity has been widely described in vitro and in vivo. The over-
expression of eIF4E can induce primary epithelial cells and fibroblast trans-
formation. Similarly, an extended overexpression of eIF4E in NIH 3T3 and
CHO cell lines leads to an oncogenic transformation and to a metastatic phe-
notype (Avdulov et al., 2004; De Benedetti & Graff, 2004; Zimmer et al.,
2000). In vivo, an eIF4E overexpression leads to lymphoma, angiosarcoma,
and lung carcinoma development in transgenic mice (Ruggero et al., 2004).
In addition, it is described to be capable to increase cellular proliferation and
inhibit apoptosis (Li et al., 2004; Ruggero et al., 2004; Wendel et al., 2004).
It can act as a survival factor in serum-deprived cells or cells whose ras and
c-Myc oncogene expression is deregulated (Li et al., 2003; Polunovsky et al.,
2000; Tan et al., 2000). Upstream signaling pathways that are mutated or
amplified in cancers have a direct impact on eIF4E activity. For example,
the eIF4E promoter contains two domains that are the oncogene c-Myc’s
targets. The mTOR pathway’s activation, which occurs in many cancers,
also allows the 4E-BP1 phosphorylation and consequently eIF4E hyper-
activation. The 4E-BP1 hyperphosphorylation is also associated with malig-
nant progression of breast, ovarian, prostate, and colon cancer (Armengol
et al., 2007; Coleman et al., 2009; Graff et al., 2009). Finally, an eIF4E level
increase was observed in the following human tumors: breast, bladder,
colon, lung, skin, head and neck, ovarian, and prostate cancer, compared
to healthy tissues (Berkel et al., 2001; Coleman et al., 2009; Crew et al.,
2000; Graff et al., 2009; Holm et al., 2008; Matthews-Greer et al., 2005;
Nathan et al., 2004; Salehi, Mashayekhi, & Shahosseini, 2007;
Thumma & Kratzke, 2007; Wang et al., 2009). Although high eIF4E
expression levels seem to correlate with aggressive and metastatic tumors
and that this protein is given as a diagnostic marker for cancer (Berkel
et al., 2001; De Benedetti & Graff, 2004; DeFatta, Li, & De Benedetti,
eIF4E and Cancer 15
2002; Li et al., 2002), it is not found in some aggressive cancers (Yang et al.,
2007). In breast cancer, it was shown that patients who, after therapy, have
low eIF4E levels have a better survival rate (Hiller et al., 2009). However,
those who have high eIF4E levels have a higher risk of recurrence (Holm
et al., 2008). eIF4E overexpression also leads to the TLK1B protein over-
expression that induces resistance to doxorubicin treatment as well as to
radiotherapy (Li et al., 2001; Sillje & Nigg, 2001). In prostate cancer, immu-
nohistochemistry studies on 148 tissues showed that eIF4E’s and 4E-BP1’s
phosphorylated form expressions were significantly increased in the
advanced prostate cancer compared to benign hyperplasia (Graff et al.,
2009). In addition, it has been shown that phosphorylation of eIF4E is
required for the translation of several proteins involved in tumorigenesis.
Furthermore, phosphorylated eIF4E levels are correlated with pancreas
and prostate cancer progression (Baylot et al., 2011; Bianchini et al.,
2008; Furic et al., 2010). Moreover, we previously showed that Hsp27
knockdown leads to eIF4E ubiquitination and degradation by the
ubiquitin–proteasome pathway and that a decrease in eIF4E ubiquitination
and degradation is associated with resistance to androgen withdrawal and
paclitaxel in prostate cancer and gemcitabine in pancreatic cancers
(Andrieu et al., 2010; Baylot et al., 2011). In vivo studies show that blocking
eIF4E’s hyperactivity by inhibiting the mTOR pathway (PP242) causes an
inhibition of tumor growth after its formation in a transgenic mouse model
developing thymus lymphomas (Hsieh et al., 2010). All these works dem-
onstrate eIF4E’s oncogenic potential and the interest of therapeutically
targeting this protein’s activity.
4.2 EIF4E’s Mechanisms in Cancer

The exact mechanism by which eIF4E and the eIF4F complex induce onco-
genic transformation is highly debated, but it is described that it may partly
be mediated by an mRNA subset’s translation increase, rather than an overall
increase in the translation rate (Fig. 5). The classification and regression tree
(CART) divides the mRNAs according to their 50 UTR end (Davuluri
et al., 2000). The vast majority of mRNAs have a short, unstructured
50 UTR end and are strongly translated. These mRNAs encode the
“housekeeping” proteins. However, there are also mRNAs whose 50 UTR
end is long, structured, and rich in G/C nucleic acids and are poorly trans-
lated under normal cellular conditions. This 50 UTR end prevents an effec-
tive eIF4F activity and binding to ribosomes. In this second category,
the mRNAs encode proteins that have an important role in oncogenesis.
Figure 5 eIF4E's involvement in the cells' oncogenic transformation. Schematic repre-

sentation of one of eIF4E mechanisms of action inducing the oncogenic transformation.
Cellular mRNAs can be divided into two categories: the majority of mRNAs that are
"highly translated" even when eIF4E expression is limited and a minority of mRNAs
("weakly translated") which are translated when eIF4E is overexpressed like during can-
cer development. This second category includes genes involved in tumorigenesis.
Adapted from Graff et al. (2008).
Thus, there are proteins involved in proliferation (cyclin D1, c-Myc,

CDK2), apoptosis (survivin, Bcl-2, Mcl-1), angiogenesis (VEGF, FGF2),
and metastasis (MMP9, heparanase) (Mamane et al., 2004; Schmidt,
2004; Zimmer et al., 2000). Given that eIF4E is the limiting factor in the
translation initiation mechanism, mRNAs compete in normal cellular con-
ditions. However, if eIF4E’s level is increased like in cancers, the mRNAs
that are poorly translated are selected and translated disproportionately
(Fig. 5) (De Benedetti & Graff, 2004; Graff et al., 2008; Mamane et al.,
2004). Thus, the eIF4E factor governs cancer’s progression by coordinating
certain genes’ expression (Avdulov et al., 2004). In addition, it is described
that eIF4E overexpression increases specific mRNAs “sensitive to eIF4E”
transport and translation (Topisirovic et al., 2003b). Some of these mRNAs
encode proteins involved in cell proliferation and tumorigenesis, such as
cyclin D1. This transport mechanism would therefore contribute to eIF4E
oncogenic potential (Cohen et al., 2001).
4.3 Targeting eIF4E in Cancers

Due to eIF4E’s important involvement in the process of tumorigenesis, sev-
eral inhibitory strategies have been developed to block its functions.
eIF4E and Cancer 17
4.3.1 ASOs and siRNAs

The first of these strategies was the development of antisense oligonucleo-
tides (ASOs) that block eIF4E’s mRNA translation. Thus, Defatta et al. had
shown that eIF4E translation inhibition through ASOs eliminates tumori-
genic and angiogenic properties in FaDu human squamous carcinoma cell
(DeFatta, Nathan, & De Benedetti, 2000). More recently, a second-
generation ASO (4E-ASO4) was designed by Graff et al. to resist nuclease
(Fig. 6A) (Graff et al., 2007). Nanomolar concentrations of 4E-ASO4 are
Figure 6 eIF4E's inhibitors. Diagram showing the different strategies to inhibit eIF4E in
cancer therapy: inhibition of eIF4E's production by ASOs (e.g., 4E-ASO4). (A) Inhibition of
eIF4E's interaction with its ligands 4E-BPs and eIF4G through inhibitory molecules (e.g.,
4EGI-1, 4E1RCat) (B) and inhibition of the eIF4E/cap interaction through mRNA's cap
analogs (e.g., the ribavirin) (C).
able to reduce eIF4E level and thus induce apoptosis in several cancer cell
lines in vitro. In vivo models of breast cancer, 4E-ASO4 significantly inhibited
tumor growth without side effects or weight loss. eIF4E’s expression is
reduced by 64% in the observed tissues. Moreover, similar results were
observed in prostate cancer xenografts after treatment (Graff et al., 2007).
On the other hand, siRNAs targeting eIF4E have recently been described
for their ability to inhibit tumor growth, induce apoptosis, and enhance the
effect of chemotherapy with cisplatin in breast carcinomas in vitro and in vivo
(Dong et al., 2009). In prostate cancer models, in vivo, eIF4E knockdown
using siRNA reverses the cytoprotection to androgen withdrawal (serum-
free media) and paclitaxel treatment normally conferred by Hsp27 over-
expression. Moreover, eIF4E’s overexpression confers resistance to combine
treatment with paclitaxel and androgen withdrawal in LNCaP cells (Andrieu
et al., 2010).
4.3.2 Inhibition of the eIF4E/eIF4G Interaction

Another strategy for inhibition of the eukaryotic factor eIF4E is to target its
interaction with eIF4G, which prevents the formation of the eIF4F complex
and leads to inhibition of cap-dependent translation. For example, some
peptides able to disrupt eIF4E–eIF4G interaction (Hu4G, W4G, 4E-BP2)
are developed. These peptides are described to induce apoptosis in
MRC5 lung cells in a dose-dependent manner (Herbert et al., 2000). More
recently, a high-throughput screening was performed to identify inhibitors
of the eIF4E/eIF4G interaction. The compound 4EGI-1 has been identified
as a hit by binding to eIF4E and blocking its interaction with eIF4G (Moerke
et al., 2007). Although eIF4G and 4E-BPs share the same interaction site on
eIF4E, 4E-BPs seem to take a larger space because 4EGI-1 does not block
the eIF4E/4E-BP1 interaction. It has even been reported that 4EGI-1
increases the interaction between eIF4E and 4E-BP1, which results in the
inhibition of the cap-dependent translation (Fig. 6B). This compound has
been shown to reduce the c-Myc and Bcl-2 level, to induce apoptosis,
and to inhibit lung cancer cell proliferation (Fan et al., 2010). It would
be interesting to know this compound specificity to inhibit the eIF4E/
eIF4G interaction by determining all protein–protein interactions and sig-
naling pathways that are blocked. In fact, studies have shown that it can
induce apoptosis through an eIF4E/eIF4G interaction-independent mech-
anism, by degrading the antiapoptotic protein c-FLIP (Fan et al., 2010).
More recently, the 4E1RCat compound was characterized as an inhibitor
of the interaction of eIF4E with eIF4G and 4E-BP1 (Fig. 6B) (Cencic
eIF4E and Cancer 19
et al., 2011a). It has been reported that this compound may partially inhibit
the cap-dependent translation and restore the chemosensitivity in a lym-
phoma mouse model. Another compound from the same screen, 4E2Rcat,
inhibits the cap-dependent translation and the coronavirus 229E replication
which is dependent on complex eIF4F (Cencic et al., 2011b).
4.3.3 mRNA Cap Analogs

Another strategy is based on inhibition of the synthesis of mRNA cap ana-
logs that would compete with the eIF4FE/cap interaction and block it
(Quiocho, Hu, & Gershon, 2000). A series of cap analogs have been devel-
oped (Brown et al., 2007; Ghosh et al., 2005, 2009; Kowalska et al., 2009),
but only ribavirin is currently used (Fig. 6C). Indeed, using these analogs as
drugs is difficult because of the low membrane permeability, due to the
nature of the extremely charged phosphate groups, and the metabolic labil-
ity, due to the instability of the glycosidic bond. Ribavirin is a broad-
spectrum antiviral drug used for the treatment of hepatitis C. The similarities
between ribavirin structure and mRNA cap have suggested that this drug
can act as an eIF4E inhibitor by mimicking the cap. Later studies showed
that ribavirin interacts with eIF4E and prevents it from binding to the
mRNA cap. This inhibits the cap-dependent translation and cell transforma-
tion (Kentsis et al., 2004, 2005; Tan et al., 2008). However, questions arise as
to the specificity of action of ribavirin on eIF4E and studies are controversial
(Westman et al., 2005; Yan et al., 2005). Nevertheless, this molecule is cur-
rently in a clinical trial phase II in the treatment of acute myeloid leukemia
and the first clinical results show that it stabilized or at least partially cured
patients (Assouline et al., 2009). This study was the first to show that the cap-
dependent translation inhibition has a clinical utility in cancers that over-
express eIF4E (Borden & Culjkovic-Kraljacic, 2010).
4.3.4 eIF4E Upstream Pathway Inhibitors

As mentioned earlier, signaling pathway upstream of eIF4E is also involved
in tumorigenesis and represent therapeutic targets. Thus, several inhibitors
have been developed to target these components and indirectly eIF4E, such
as Mnk kinase inhibitors (cercosporamide) and mTOR pathway inhibitors
(rapamycin, temsirolimus, etc.) (Choo et al., 2008; Feldman et al., 2009;
Garcia-Martinez et al., 2009; Konicek et al., 2011; Yu et al., 2010). In
2007, temsirolimus was approved by the FDA for the treatment of patients
with advanced renal-cell cancer, as trials demonstrated that it had signifi-
cantly outperformed the standard of care in terms of progression-free
survival and overall survival by 2.4 and 3.6 months, respectively. Further-
more, preclinical evaluation of two TORKinibs (second-generation
small-molecule inhibitors), PP242 and PP30, demonstrates stronger inhibi-
tion of protein synthesis and cell proliferation than sirolimus (Blagden &
Willis, 2011).
4.3.5 Inhibition of the eIF4E/Hsp27 Interaction

More recently, targeting Hsp27–eIF4E interaction has been described as an
interesting alternative strategy to target eIF4E. We previously found that
Hsp27 interacts directly with the eukaryotic translational initiation factor
eIF4E. Our work demonstrated that Hsp27 interaction protects eIF4E from
its degradation by the ubiquitin–proteasome pathways leading to Hsp27
cytoprotection in pancreas and CRPC (Andrieu et al., 2010; Baylot
et al., 2011). Using several Hsp27 deletion mutants, we found that eIF4E
interacts with the C-terminal domain of Hsp27. Inhibition of Hsp27–eIF4E
interaction using deletion mutants drives resistance to apoptosis induced by
gemcitabine in pancreatic cancers (Baylot et al., 2011) and androgen with-
drawal and docetaxel in castrate-resistant prostate cancers (unpublished
data). This experiment confirmed that this stress-induced cellular pathway
is involved in cell death blockade leading to therapy resistance in cancers.
Targeting the Hsp27–eIF4E interaction seems to be a promising therapeutic
strategy in advanced prostate and pancreatic cancers.
5. CONCLUSION
Tumorigenesis is highly affected by the regulation of the cap-
dependent translation. The cap-dependent translation consists of the
eukaryotic translation initiation factor 4F complex that can recognize the
50 end of cellular mRNAs at the 7-methylguanosine cap structure. eIF4E
is a component of this complex which makes it crucial to the cap-dependent
translation initiation and regulation of tumor cell apoptosis, proliferation,
and, potentially, metastasis. Indeed, since eIF4E’s inhibition induces cellular
death, we are entitled to ask about this inhibition’s consequence on normal
cells. It seems however that eIF4E’s residual and low levels after drug treat-
ment are tolerated and without adverse effects on normal tissues. In contrast,
eIF4E’s activity is so important in cancerous cells that its inhibitions have
a more visible effect (Graff et al., 2008). Many approaches over the years
have been used to try to inhibit eIF4E’s function, particularly by using
eIF4E and Cancer 21
small-molecule inhibitors that can disrupt the eIF4E–eIF4G interaction, the

use of cap analogs to directly target the eIF4E cap-binding site, or ASOs that
have been proved to be efficient in reducing the expression level of eIF4E
and have advanced to clinical trials in prostate cancer patients. More
recently, targeting Hsp27–eIF4E interaction has been described as an inter-
esting alternative strategy to target eIF4E. Taken together, these data seem to
show eIF4E to be a promising target for cancer therapy and new approaches
of inhibition deserve further studies.
REFERENCES
Amiri, A., et al. (2001). An isoform of eIF4E is a component of germ granules and is required
for spermatogenesis in C. elegans. Development, 128(20), 3899–3912.
Andrieu, C., et al. (2010). Heat shock protein 27 confers resistance to androgen ablation and
chemotherapy in prostate cancer cells through eIF4E. Oncogene, 29(13), 1883–1896.
Armengol, G., et al. (2007). 4E-binding protein 1: A key molecular “funnel factor” in human
cancer with clinical implications. Cancer Research, 67(16), 7551–7555.
Assouline, S., et al. (2009). Molecular targeting of the oncogene eIF4E in acute myeloid leu-
kemia (AML): A proof-of-principle clinical trial with ribavirin. Blood, 114(2), 257–260.
Avdulov, S., et al. (2004). Activation of translation complex eIF4F is essential for the genesis
and maintenance of the malignant phenotype in human mammary epithelial cells. Cancer
Cell, 5(6), 553–563.
Banerjee, S., et al. (2002). Murine coronavirus replication-induced p38 mitogen-activated
protein kinase activation promotes interleukin-6 production and virus replication in cul-
tured cells. Journal of Virology, 76(12), 5937–5948.
Baylot, V., et al. (2011). OGX-427 inhibits tumor progression and enhances gemcitabine
chemotherapy in pancreatic cancer. Cell Death and Disease, 2, e221.
Berkel, H. J., et al. (2001). Expression of the translation initiation factor eIF4E in the polyp-
cancer sequence in the colon. Cancer Epidemiology, Biomarkers & Prevention, 10(6),
663–666.
Bianchini, A., et al. (2008). Phosphorylation of eIF4E by MNKs supports protein synthesis,
cell cycle progression and proliferation in prostate cancer cells. Carcinogenesis, 29(12),
2279–2288.
Blagden, S. P., & Willis, A. E. (2011). The biological and therapeutic relevance of mRNA
translation in cancer. Nature Reviews. Clinical Oncology, 8(5), 280–291.
Borden, K. L., & Culjkovic-Kraljacic, B. (2010). Ribavirin as an anti-cancer therapy: Acute
myeloid leukemia and beyond? Leukemia & Lymphoma, 51(10), 1805–1815.
Brown, C. J., et al. (2007). Crystallographic and mass spectrometric characterisation of eIF4E
with N7-alkylated cap derivatives. Journal of Molecular Biology, 372(1), 7–15.
Campbell Dwyer, E. J., et al. (2000). The lymphocytic choriomeningitis virus RING protein
Z associates with eukaryotic initiation factor 4E and selectively represses translation in a
RING-dependent manner. Journal of Virology, 74(7), 3293–3300.
Cencic, R., et al. (2011a). Reversing chemoresistance by small molecule inhibition of the
translation initiation complex eIF4F. Proceedings of the National Academy of Sciences of
the United States of America, 108(3), 1046–1051.
Cencic, R., et al. (2011b). Blocking eIF4E-eIF4G interaction as a strategy to impair coro-
navirus replication. Journal of Virology, 85(13), 6381–6389.
Chaudhry, Y., et al. (2006). Caliciviruses differ in their functional requirements for eIF4F
components. Journal of Biological Chemistry, 281(35), 25315–25325.
Choo, A. Y., et al. (2008). Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate
cell-type-specific repression of mRNA translation. Proceedings of the National Academy
of Sciences of the United States of America, 105(45), 17414–17419.
Cohen, N., et al. (2001). PML RING suppresses oncogenic transformation by reducing the
affinity of eIF4E for mRNA. The EMBO Journal, 20(16), 4547–4559.
Coleman, L. J., et al. (2009). Combined analysis of eIF4E and 4E-binding protein expression
predicts breast cancer survival and estimates eIF4E activity. British Journal of Cancer,
100(9), 1393–1399.
Crew, J. P., et al. (2000). Eukaryotic initiation factor-4E in superficial and muscle invasive
bladder cancer and its correlation with vascular endothelial growth factor expression and
tumour progression. British Journal of Cancer, 82(1), 161–166.
Cuesta, R., Xi, Q., & Schneider, R. J. (2000). Adenovirus-specific translation by displace-
ment of kinase Mnk1 from cap-initiation complex eIF4F. The EMBO Journal, 19(13),
3465–3474.
Culjkovic, B., Topisirovic, I., & Borden, K. L. (2007). Controlling gene expression through
RNA regulons: The role of the eukaryotic translation initiation factor eIF4E. Cell Cycle,
6(1), 65–69.
Culjkovic, B., et al. (2005). eIF4E promotes nuclear export of cyclin D1 mRNAs via an ele-
ment in the 30 UTR. The Journal of Cell Biology, 169(2), 245–256.
Davuluri, R. V., et al. (2000). CART classification of human 50 UTR sequences. Genome
Research, 10(11), 1807–1816.
De Benedetti, A., & Graff, J. R. (2004). eIF-4E expression and its role in malignancies and
metastases. Oncogene, 23(18), 3189–3199.
DeFatta, R. J., Li, Y., & De Benedetti, A. (2002). Selective killing of cancer cells based on
translational control of a suicide gene. Cancer Gene Therapy, 9(7), 573–578.
DeFatta, R. J., Nathan, C. O., & De Benedetti, A. (2000). Antisense RNA to eIF4E sup-
presses oncogenic properties of a head and neck squamous cell carcinoma cell line.
Laryngoscope, 110(6), 928–933.
Dinkova, T. D., et al. (2005). Translation of a small subset of Caenorhabditis elegans mRNAs
is dependent on a specific eukaryotic translation initiation factor 4E isoform. Molecular
and Cellular Biology, 25(1), 100–113.
Dong, K., et al. (2009). Tumor-specific RNAi targeting eIF4E suppresses tumor growth,
induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells.
Breast Cancer Research and Treatment, 113(3), 443–456.
Dostie, J., Lejbkowicz, F., & Sonenberg, N. (2000). Nuclear eukaryotic initiation factor 4E
(eIF4E) colocalizes with splicing factors in speckles. The Journal of Cell Biology, 148(2),
239–247.
Dostie, J., et al. (2000). A novel shuttling protein, 4E-T, mediates the nuclear import of the
mRNA 50 cap-binding protein, eIF4E. The EMBO Journal, 19(12), 3142–3156.
Dreher, T. W., & Miller, W. A. (2006). Translational control in positive strand RNA plant
viruses. Virology, 344(1), 185–197.
Evsikov, A. V., & Marin de Evsikova, C. (2009). Evolutionary origin and phylogenetic anal-
ysis of the novel oocyte-specific eukaryotic translation initiation factor 4E in Tetrapoda.
Development Genes and Evolution, 219(2), 111–118.
Fan, S., et al. (2010). The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments
TRAIL-mediated apoptosis through c-FLIP down-regulation and DR5 induction
independent of inhibition of cap-dependent protein translation. Neoplasia, 12(4),
346–356.
Feldman, M. E., et al. (2009). Active-site inhibitors of mTOR target rapamycin-resistant
outputs of mTORC1 and mTORC2. PLoS Biology, 7(2), e38.
Furic, L., et al. (2010). eIF4E phosphorylation promotes tumorigenesis and is associated with
prostate cancer progression. Proceedings of the National Academy of Sciences of the United
States of America, 107(32), 14134–14139.
eIF4E and Cancer 23
Garcia-Martinez, J. M., et al. (2009). Ku-0063794 is a specific inhibitor of the mammalian

target of rapamycin (mTOR). Biochemical Journal, 421(1), 29–42.
Ghosh, P., et al. (2005). Synthesis and evaluation of potential inhibitors of eIF4E cap binding
to 7-methyl GTP. Bioorganic & Medicinal Chemistry Letters, 15(8), 2177–2180.
Ghosh, B., et al. (2009). Nontoxic chemical interdiction of the epithelial-to-mesenchymal
transition by targeting cap-dependent translation. ACS Chemical Biology, 4(5), 367–377.
Gingras, A. C., Raught, B., & Sonenberg, N. (2001). Regulation of translation initiation by
FRAP/mTOR. Genes and Development, 15(7), 807–826.
Gingras, A. C., Raught, B., & Sonenberg, N. (2004). mTOR signaling to translation. Current
Topics in Microbiology and Immunology, 279, 169–197.
Goodfellow, I., et al. (2005). Calicivirus translation initiation requires an interaction between
VPg and eIF 4 E. EMBO Reports, 6(10), 968–972.
Graff, J. R., et al. (2007). Therapeutic suppression of translation initiation factor eIF4E
expression reduces tumor growth without toxicity. The Journal of Clinical Investigation,
117(9), 2638–2648.
Graff, J. R., et al. (2008). Targeting the eukaryotic translation initiation factor 4E for cancer
therapy. Cancer Research, 68(3), 631–634.
Graff, J. R., et al. (2009). eIF4E activation is commonly elevated in advanced human prostate
cancers and significantly related to reduced patient survival. Cancer Research, 69(9),
3866–3873.
Gross, J. D., et al. (2003). Ribosome loading onto the mRNA cap is driven by conforma-
tional coupling between eIF4G and eIF4E. Cell, 115(6), 739–750.
Heesom, K. J., et al. (2001). Cell cycle-dependent phosphorylation of the translational
repressor eIF-4E binding protein-1 (4E-BP1). Current Biology, 11(17), 1374–1379.
Herbert, T. P., et al. (2000). Rapid induction of apoptosis mediated by peptides that bind
initiation factor eIF4E. Current Biology, 10(13), 793–796.
Hernandez, G., & Vazquez-Pianzola, P. (2005). Functional diversity of the eukaryotic trans-
lation initiation factors belonging to eIF4 families. Mechanisms of Development, 122(7-8),
865–876.
Hiller, D. J., et al. (2009). Predictive value of eIF4E reduction after neoadjuvant therapy in
breast cancer. The Journal of Surgical Research, 156(2), 265–269.
Holm, N., et al. (2008). A prospective trial on initiation factor 4E (eIF4E) overexpression and
cancer recurrence in node-negative breast cancer. Annals of Surgical Oncology, 15(11),
3207–3215.
Hsieh, A. C., et al. (2010). Genetic dissection of the oncogenic mTOR pathway reveals
druggable addiction to translational control via 4EBP-eIF4E. Cancer Cell, 17(3), 249–261.
Iborra, F. J., Jackson, D. A., & Cook, P. R. (2001). Coupled transcription and translation
within nuclei of mammalian cells. Science, 293(5532), 1139–1142.
Jia, Y., et al. (2012). Cap-dependent translation initiation factor eIF4E: An emerging anti-
cancer drug target. Medicinal Research Reviews, 32(4), 786–814.
Joshi, B., Cameron, A., & Jagus, R. (2004). Characterization of mammalian eIF4E-family
members. European Journal of Biochemistry, 271(11), 2189–2203.
Joshi, B., et al. (2005). Phylogenetic analysis of eIF4E-family members. BMC Evolutionary
Biology, 5, 48.
Jung, M. Y., Lorenz, L., & Richter, J. D. (2006). Translational control by neuroguidin, a
eukaryotic initiation factor 4E and CPEB binding protein. Molecular and Cellular Biology,
26(11), 4277–4287.
Kentsis, A., et al. (2001). The RING domains of the promyelocytic leukemia protein PML
and the arenaviral protein Z repress translation by directly inhibiting translation initiation
factor eIF4E. Journal of Molecular Biology, 312(4), 609–623.
Kentsis, A., et al. (2004). Ribavirin suppresses eIF4E-mediated oncogenic transformation by
physical mimicry of the 7-methyl guanosine mRNA cap. Proceedings of the National Acad-
emy of Sciences of the United States of America, 101(52), 18105–18110.
Kentsis, A., et al. (2005). Further evidence that ribavirin interacts with eIF4E. RNA, 11(12),
1762–1766.
Kimball, S. R. (2001). Regulation of translation initiation by amino acids in eukaryotic cells.
Progress in Molecular and Subcellular Biology, 26, 155–184.
Konicek, B. W., et al. (2011). Therapeutic inhibition of MAP kinase interacting kinase
blocks eukaryotic initiation factor 4E phosphorylation and suppresses outgrowth of
experimental lung metastases. Cancer Research, 71(5), 1849–1857.
Kowalska, J., et al. (2009). Phosphorothioate analogs of m7GTP are enzymatically stable
inhibitors of cap-dependent translation. Bioorganic & Medicinal Chemistry Letters, 19(7),
1921–1925.
Kozak, M. (2002). Pushing the limits of the scanning mechanism for initiation of translation.
Gene, 299(1–2), 1–34.
Lai, H. K., & Borden, K. L. (2000). The promyelocytic leukemia (PML) protein suppresses
cyclin D1 protein production by altering the nuclear cytoplasmic distribution of cyclin
D1 mRNA. Oncogene, 19(13), 1623–1634.
Lee, S. K., et al. (2007). Translation initiation factor 4E (eIF4E) is regulated by cell death
inhibitor, Diap1. Molecules and Cells, 24(3), 445–451.
Li, Y., et al. (2001). A translationally regulated Tousled kinase phosphorylates histone H3 and
confers radioresistance when overexpressed. Oncogene, 20(6), 726–738.
Li, B. D., et al. (2002). Prospective study of eukaryotic initiation factor 4E protein elevation
and breast cancer outcome. Annals of Surgery, 235(5), 732–738. discussion 738–739.
Li, S., et al. (2003). Translation factor eIF4E rescues cells from Myc-dependent apoptosis by
inhibiting cytochrome c release. Journal of Biological Chemistry, 278(5), 3015–3022.
Li, S., et al. (2004). Translation initiation factor 4E blocks endoplasmic reticulum-mediated
apoptosis. Journal of Biological Chemistry, 279(20), 21312–21317.
Lynch, M., et al. (2005). hnRNP K binds a core polypyrimidine element in the eukaryotic
translation initiation factor 4E (eIF4E) promoter, and its regulation of eIF4E contributes
to neoplastic transformation. Molecular and Cellular Biology, 25(15), 6436–6453.
Mamane, Y., et al. (2004). eIF4E—From translation to transformation. Oncogene, 23(18),
3172–3179.
Matthews-Greer, J., et al. (2005). eIF4E as a marker for cervical neoplasia. Applied Immuno-
histochemistry & Molecular Morphology, 13(4), 367–370.
Minshall, N., et al. (2007). CPEB interacts with an ovary-specific eIF4E and 4E-T in early
Xenopus oocytes. Journal of Biological Chemistry, 282(52), 37389–37401.
Moerke, N. J., et al. (2007). Small-molecule inhibition of the interaction between the trans-
lation initiation factors eIF4E and eIF4G. Cell, 128(2), 257–267.
Monzingo, A. F., et al. (2007). The structure of eukaryotic translation initiation factor-4E
from wheat reveals a novel disulfide bond. Plant Physiology, 143(4), 1504–1518.
Murata, T., & Shimotohno, K. (2006). Ubiquitination and proteasome-dependent degrada-
tion of human eukaryotic translation initiation factor 4E. Journal of Biological Chemistry,
281(30), 20788–20800.
Nakamura, A., Sato, K., & Hanyu-Nakamura, K. (2004). Drosophila cup is an eIF4E binding
protein that associates with Bruno and regulates oskar mRNA translation in oogenesis.
Developmental Cell, 6(1), 69–78.
Nathan, C. O., et al. (2004). Overexpressed eIF4E is functionally active in surgical margins of
head and neck cancer patients via activation of the Akt/mammalian target of rapamycin
pathway. Clinical Cancer Research, 10(17), 5820–5827.
Niedzwiecka, A., Darzynkiewicz, E., & Stolarski, R. (2004). Thermodynamics of mRNA 50
cap binding by eukaryotic translation initiation factor eIF4E. Biochemistry, 43(42),
13305–13317.
Niedzwiecka, A., et al. (2002). Biophysical studies of eIF4E cap-binding protein: Recogni-
tion of mRNA 50 cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.
Journal of Molecular Biology, 319(3), 615–635.
eIF4E and Cancer 25
Niessing, D., Blanke, S., & Jackle, H. (2002). Bicoid associates with the 50 -cap-bound complex
of caudal mRNA and represses translation. Genes and Development, 16(19), 2576–2582.
O’Farrell, P. H. (2001). Triggering the all-or-nothing switch into mitosis. Trends in Cell Biol-
ogy, 11(12), 512–519.
Othumpangat, S., Kashon, M., & Joseph, P. (2005). Eukaryotic translation initiation factor
4E is a cellular target for toxicity and death due to exposure to cadmium chloride. Journal
of Biological Chemistry, 280(26), 25162–25169.
Polunovsky, V. A., et al. (2000). Translational control of the antiapoptotic function of Ras.
Journal of Biological Chemistry, 275(32), 24776–24780.
Prevot, D., Darlix, J. L., & Ohlmann, T. (2003). Conducting the initiation of protein syn-
thesis: The role of eIF4G. Biology of the Cell, 95(3-4), 141–156.
Quiocho, F. A., Hu, G., & Gershon, P. D. (2000). Structural basis of mRNA cap recognition
by proteins. Current Opinion in Structural Biology, 10(1), 78–86.
Regad, T., & Chelbi-Alix, M. K. (2001). Role and fate of PML nuclear bodies in response to
interferon and viral infections. Oncogene, 20(49), 7274–7286.
Richter, J. D., & Sonenberg, N. (2005). Regulation of cap-dependent translation by eIF4E
inhibitory proteins. Nature, 433(7025), 477–480.
Robalino, J., et al. (2004). Two zebrafish eIF4E family members are differentially expressed
and functionally divergent. Journal of Biological Chemistry, 279(11), 10532–10541.
Rogers, G. W., Jr., Komar, A. A., & Merrick, W. C. (2002). eIF4A: The godfather of the
DEAD box helicases. Progress in Nucleic Acid Research and Molecular Biology, 72, 307–331.
Rosettani, P., et al. (2007). Structures of the human eIF4E homologous protein, h4EHP, in
its m7GTP-bound and unliganded forms. Journal of Molecular Biology, 368(3), 691–705.
Ruggero, D., et al. (2004). The translation factor eIF-4E promotes tumor formation and
cooperates with c-Myc in lymphomagenesis. Nature Medicine, 10(5), 484–486.
Salehi, Z., Mashayekhi, F., & Shahosseini, F. (2007). Significance of eIF4E expression in skin
squamous cell carcinoma. Cell Biology International, 31(11), 1400–1404.
Scheper, G. C., & Proud, C. G. (2002). Does phosphorylation of the cap-binding protein
eIF4E play a role in translation initiation? European Journal of Biochemistry, 269(22),
5350–5359.
Scheper, G. C., et al. (2001). The mitogen-activated protein kinase signal-integrating kinase
Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mam-
malian cells. Molecular and Cellular Biology, 21(3), 743–754.
Schmidt, E. V. (2004). The role of c-myc in regulation of translation initiation. Oncogene,
23(18), 3217–3221.
Shenberger, J. S., et al. (2005). Hyperoxia alters the expression and phosphorylation of mul-
tiple factors regulating translation initiation. American Journal of Physiology. Lung Cellular
and Molecular Physiology, 288(3), L442–L449.
Shveygert, M., et al. (2010). Regulation of eukaryotic initiation factor 4E (eIF4E) phosphor-
ylation by mitogen-activated protein kinase occurs through modulation of Mnk1-eIF4G
interaction. Molecular and Cellular Biology, 30(21), 5160–5167.
Sillje, H. H., & Nigg, E. A. (2001). Identification of human Asf1 chromatin assembly factors
as substrates of Tousled-like kinases. Current Biology, 11(13), 1068–1073.
Sonenberg, N. (2008). eIF4E, the mRNA cap-binding protein: From basic discovery to
translational research. Biochemistry and Cell Biology, 86(2), 178–183.
Stoneley, M., & Willis, A. E. (2004). Cellular internal ribosome entry segments: Structures,
trans-acting factors and regulation of gene expression. Oncogene, 23(18), 3200–3207.
Strudwick, S., & Borden, K. L. (2002). The emerging roles of translation factor eIF4E in the
nucleus. Differentiation, 70(1), 10–22.
Syntichaki, P., Troulinaki, K., & Tavernarakis, N. (2007). eIF4E function in somatic cells
modulates ageing in Caenorhabditis elegans. Nature, 445(7130), 922–926.
Tan, A., et al. (2000). Inhibition of Myc-dependent apoptosis by eukaryotic translation ini-
tiation factor 4E requires cyclin D1. Oncogene, 19(11), 1437–1447.
Tan, K., et al. (2008). Ribavirin targets eIF4E dependent Akt survival signaling. Biochemical
and Biophysical Research Communications, 375(3), 341–345.
Thumma, S. C., & Kratzke, R. A. (2007). Translational control: A target for cancer therapy.
Cancer Letters, 258(1), 1–8.
Tomoo, K., et al. (2002). Crystal structures of 7-methylguanosine 50 -triphosphate (m(7)
GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-50 ,50 -triphosphate (m(7)GpppA)-
bound human full-length eukaryotic initiation factor 4E: Biological importance of the
C-terminal flexible region. Biochemical Journal, 362(Pt 3), 539–544.
Tomoo, K., et al. (2003). Structural features of human initiation factor 4E, studied by X-ray
crystal analyses and molecular dynamics simulations. Journal of Molecular Biology, 328(2),
365–383.
Topisirovic, I., et al. (2003a). The proline-rich homeodomain protein, PRH, is a tissue-
specific inhibitor of eIF4E-dependent cyclin D1 mRNA transport and growth. The
EMBO Journal, 22(3), 689–703.
Topisirovic, I., et al. (2003b). Aberrant eukaryotic translation initiation factor 4E-dependent
mRNA transport impedes hematopoietic differentiation and contributes to leukemo-
genesis. Molecular and Cellular Biology, 23(24), 8992–9002.
Tschopp, C., et al. (2000). Phosphorylation of eIF-4E on Ser 209 in response to mitogenic
and inflammatory stimuli is faithfully detected by specific antibodies. Molecular Cell Biol-
ogy Research Communications, 3(4), 205–211.
Ueda, T., et al. (2004). Mnk2 and Mnk1 are essential for constitutive and inducible phos-
phorylation of eukaryotic initiation factor 4E but not for cell growth or development.
Molecular and Cellular Biology, 24(15), 6539–6549.
Van Der Kelen, K., et al. (2009). Translational control of eukaryotic gene expression. Critical
Reviews in Biochemistry and Molecular Biology, 44(4), 143–168.
Volpon, L., et al. (2006). Cap-free structure of eIF4E suggests a basis for conformational reg-
ulation by its ligands. The EMBO Journal, 25(21), 5138–5149.
Volpon, L., et al. (2010). Structural characterization of the Z RING-eIF4E complex reveals a
distinct mode of control for eIF4E. Proceedings of the National Academy of Sciences of the
United States of America, 107(12), 5441–5446.
von Der Haar, T., Ball, P. D., & McCarthy, J. E. (2000). Stabilization of eukaryotic initiation
factor 4E binding to the mRNA 50 -Cap by domains of eIF4G. Journal of Biological Chem-
istry, 275(39), 30551–30555.
Wang, X., et al. (2005). Distinct signaling events downstream of mTOR cooperate to medi-
ate the effects of amino acids and insulin on initiation factor 4E-binding proteins. Molec-
ular and Cellular Biology, 25(7), 2558–2572.
Wang, R., et al. (2009). Overexpression of eukaryotic initiation factor 4E (eIF4E) and its
clinical significance in lung adenocarcinoma. Lung Cancer, 66(2), 237–244.
Wendel, H. G., et al. (2004). Survival signalling by Akt and eIF4E in oncogenesis and cancer
therapy. Nature, 428(6980), 332–337.
Westman, B., et al. (2005). The antiviral drug ribavirin does not mimic the 7-methylguanosine
moiety of the mRNA cap structure in vitro. RNA, 11(10), 1505–1513.
Yan, Y., et al. (2005). Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA
cap. RNA, 11(8), 1238–1244.
Yang, S. X., et al. (2007). Expression levels of eIF4E, VEGF, and cyclin D1, and correlation
of eIF4E with VEGF and cyclin D1 in multi-tumor tissue microarray. Oncology Reports,
17(2), 281–287.
Yu, K., et al. (2010). Beyond rapalog therapy: Preclinical pharmacology and antitumor activ-
ity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and
mTORC2. Cancer Research, 70(2), 621–631.
Zimmer, S. G., DeBenedetti, A., & Graff, J. R. (2000). Translational control of malignancy:
The mRNA cap-binding protein, eIF-4E, as a central regulator of tumor formation,
growth, invasion and metastasis. Anticancer Research, 20(3A), 1343–1351.
CHAPTER TWO
Antitumor Lipids—Structure,
Functions, and Medical
Applications
Aneliya Kostadinova*,1, Tanya Topouzova-Hristova†,
Albena Momchilova*, Rumiana Tzoneva*,1, Martin R. Berger{
*Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria
†
Faculty of Biology, Cytology, Histology and Embryology, Sofia University, Sofia, Bulgaria
{
German Cancer Research Center, Toxicology and Chemotherapy Unit, Heidelberg, Germany
1
Corresponding authors: e-mail address: [email protected]; [email protected]
Contents
1. Introduction 28
2. Development of Antitumor Lipids 29
3. Combining ATLs with Other Anticancer Approaches 29
3.1 Combining APLs with Other Anticancer Agents 30
3.2 Treatment with APLs and Radiation 31
3.3 Combined Treatment of APLs and Electroporation 32
4. Structure of Antitumor Lipids 32
5. Clinical Trials and Therapeutic Effect 37
6. Anticancer Mechanism of Action 40
6.1 Membrane Localization and Lipid Rafts 40
6.2 Targets of APLs in Leukemic Cells Versus Solid Tumor Cells 41
6.3 Major Biological Processes and Targets Affected by ATLs 49
6.4 Effect of ATLs on Cell Cycle and Mitosis 49
6.5 Interference with Phospholipid Metabolism 50
6.6 Signal Transduction Pathways Involved in the ATLs Action 52
6.7 Activation of SAPK/JNK AKT-mTOR Ras/Raf, PKC 53
7. Conclusion and Perspectives 55
Acknowledgments 56
References 56
Abstract
Cell proliferation and metastasis are considered hallmarks of tumor progression. There-
fore, efforts have been made to develop novel anticancer drugs that inhibit both the
proliferation and the motility of tumor cells. Synthetic antitumor lipids (ATLs), which
are chemically divided into two main classes, comprise (i) alkylphospholipids (APLs)
and (ii) alkylphosphocholines (APCs). They represent a new entity of drugs with distinct
antiproliferative properties in tumor cells. These compounds do not interfere with the
Advances in Protein Chemistry and Structural Biology, Volume 101 # 2015 Elsevier Inc. 27
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/bs.apcsb.2015.08.001
28 Aneliya Kostadinova et al.
DNA or mitotic spindle apparatus of the cell, instead, they incorporate into cell mem-
branes, where they accumulate and interfere with lipid metabolism and lipid-
dependent signaling pathways. Recently, it has been shown that the most commonly
studied APLs inhibit proliferation by inducing apoptosis in malignant cells while leaving
normal cells unaffected and are potent sensitizers of conventional chemo- and radio-
therapy, as well as of electrical field therapy. APLs resist catabolic degradation to a large
extent, therefore accumulate in the cell and interfere with lipid-dependent survival sig-
naling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthe-
sis. They are internalized in the cell membrane via raft domains and cause downstream
reactions as inhibition of cell growth and migration, cell cycle arrest, actin stress fibers
collapse, and apoptosis. This review summarizes the in vitro, in vivo, and clinical trials of
most common ATLs and their mode of action at molecular and biochemical levels.
1. INTRODUCTION
The development of antitumor drugs remains to be one of the most
significant challenges in modern medicine. Chemotherapeutic agents were
used for the first time in the early 1940s to repress tumor growth. Later,
researchers discovered and synthesized a variety of chemotherapeutic drugs
(e.g., mercaptopurine, fluorouracil, vincristine, and cisplatin), which dis-
played one or more of the following similar features: (i) inhibition of tumor
growth and proliferation as a consequence of inhibition of RNA and DNA
synthesis, (ii) inhibition of cell division via blockade of microtubule poly-
merization, and (iii) induction of apoptosis. However, the impact of con-
ventional chemotherapeutic agents affected not only tumor tissues but
also rapidly dividing cells of healthy organs (e.g., bone marrow, gastrointes-
tinal tract cells, gonads, and skin cells, especially hair follicles). Furthermore,
other organs, like the heart, liver, lungs, and kidneys, were also damaged. In
addition, one of the major obstacles in anticancer therapy was the presence
of multidrug resistance (MDR) mechanisms, which manifested by drug
efflux, drug inactivation, alterations in drug targeting, and evasion of apo-
ptosis (Wong & Goodin, 2009). Therefore, it was necessary to develop
novel strategies to overcome these significant problems. After more than
a half century of cancer research, it is evident that new antitumor drugs
should be metabolically stable, well adsorbed after oral administration,
and characterized by low toxicity at biologically effective doses, while gen-
erating limited effects to the bone marrow and intestinal epithelium.
Antitumor lipids represent a group of agents, which fulfill many of these
criteria. In the following, their discovery, development, and first clinical
use will be detailed.
Antitumor Lipids 29
2. DEVELOPMENT OF ANTITUMOR LIPIDS

In the early 1960s, it was observed that the generation of lysolecithin
(2-lysophosphatidylcholine, LPC) by phospholipase A2 induced the phago-
cytic activity of peritoneal macrophages in vitro and in vivo (Munder &
Modolell, 1973; Snyder & Wood, 1969). Since LPC is not stable and
becomes biologically inactivated either by the action of acyltransferase into
lecithin (phosphatidylcholine, PC) or by lysophospholipase into
glycerophosphocholine (Mulder & van Deenen, 1965), subsequent efforts
were made to synthesize metabolically stable LPC analogs for translational
research and clinical trials. Some synthetic phospholipid analogs not only
worked as effective immune modulators (Modolell, Andreesen, Pahlke,
Brugger, & Munder, 1979) but also possessed selective antineoplastic activ-
ities in vitro and in vivo (Andreesen et al., 1978; Modolell et al., 1979;
Tarnowski et al., 1978). Until now, compounds like edelfosine, ilmofosine,
miltefosine, and perifosine have been tested for their antitumor activity in
clinical phase I and phase II trials for a variety of tumors. Furthermore,
miltefosine was the first antitumor lipid (ATL) to be used clinically for
the treatment of cutaneous metastases of breast cancer (Eibl & Unger,
1990). Encouraging results have been obtained with these compounds, pri-
marily in the treatment of leukemic malignancies (van Blitterswijk &
Verheij, 2008). Their antineoplastic effect is manifested by suppressing
malignant cell proliferation, stimulating apoptosis, inhibiting the action of
a series of enzymes, and activating macrophages. These lipids possess both
antitumor and antiviral effects and, unlike many anticancer drugs, cause
no serious side effects.
3. COMBINING ATLs WITH OTHER ANTICANCER

APPROACHES
For the clinical application, ATLs are most promising in combination
with other anticancer drugs that have different molecular targets in the cell.
For example, many conventional therapies target the DNA of proliferating
tumor cells, whereas ATLs act upon their cell membranes and interfere with
signaling pathways starting from these structures. Therefore, combining
these different principles of antineoplastic activity may have at least an addi-
tive, sometimes a synergistic therapeutic effect (Richardson, Eng, Kolesar,
Hideshima, & Anderson, 2012; Ruiter, Verheij, Zerp, & van Blitterswijk,
2001; Ruiter, Zerp, Bartelink, van Blitterswijk, & Verheij, 1999; Vink, van
Blitterswijk, Schellens, & Verheij, 2007). ATLs may also disturb signaling
pathways implicated in mediating chemo- and radioresistance of tumor cells
and are, also for this reason, attractive compounds in combination therapies
(Belka et al., 2004; Richardson et al., 2012; van Blitterswijk & Verheij,
2008; Vink et al., 2007).
3.1 Combining APLs with Other Anticancer Agents

Progress has been made in studies combining the Akt inhibitor perifosine
with anticancer mTOR inhibitors. The rationale behind this successful
combination is that suppression of mTOR signaling by single treatment
with rapamycin (or its analogs CCI 779 or temsirolimus) is associated with
upregulation of Akt phosphorylation/activation in a positive feedback loop.
This loop is suppressed by the Akt inhibitor perifosine. Cirstea et al. (2010)
first reported that perifosine and rapamycin together induced synergistic
cytotoxicity (apoptosis) and autophagy in multiple myeloma (MM) cells.
They also showed that nanoparticle albumin-bound rapamycin and peri-
fosine together enhanced the in vivo antitumor activity and prolonged sur-
vival in a MM mouse xenograft model (Cirstea et al., 2010). The success of
this combination of inhibitors was subsequently confirmed in other studies.
Perifosine in combination with mTOR inhibitors effectively killed platelet-
derived growth factor-driven mouse glioblastomas (Pitter et al., 2011) and
non-small-cell lung cancer cells (Ma et al., 2012) in vitro and in vivo. Clinical
trials for this combination therapy in MM and neuroblastoma patients are
underway (Rossi et al., 2012). Perifosine could be a potent sensitizer of con-
ventional chemotherapy, for example, in MM (Hideshima et al., 2006), gli-
oma (Momota, Nerio, & Holland, 2005), medulloblastoma (Kumar et al.,
2009), endometrial cancer (Engel et al., 2008), osteosarcoma (Yao et al.,
2013), and leukemias (Nyakern, Cappellini, Mantovani, & Martelli, 2006;
Papa et al., 2008). Phase II clinical trials with perifosine in combination with
bortezomib and dexamethasone in MM patients (Richardson et al., 2012),
or with capecitabine in patients with metastatic colorectal cancer (Bendell
et al., 2011) were promising. Perifosine synergized with TRAIL by inducing
expression of the respective death receptors (DRs) in human lung cancer
cells (Elrod et al., 2007) and in acute myelogenous leukemia cells (Tazzari
et al., 2008), and with histone deacetylase inhibitors in myeloid and lym-
phoid leukemia cells (Rahmani et al., 2005) to induce apoptosis. Further-
more, low-dose perifosine significantly enhanced the induction of
Antitumor Lipids 31
apoptosis by UVB light/oxidative stress in skin cells, with possible implica-

tion for a novel skin cancer prevention strategy ( Ji et al., 2012). Finally, in
combination with curcumin, a natural, unconventional anticancer agent,
perifosine demonstrated significant inhibition of colon cancer cell growth
in vitro and in vivo, by affecting multiple signaling pathways, including
Akt, SAPK/JNK, and ER stress (Chen et al., 2012). Next to the over-
whelming number of perifosine papers, there is one recent report on
miltefosine as sensitizer of paclitaxel therapy in glioblastoma (Thakur,
Joshi, Shanmugam, & Banerjee, 2013). Interestingly, in this study
miltefosine was provided in lipid nanovesicles encapsulating paclitaxel,
which were able to cross the blood–brain barrier.
3.2 Treatment with APLs and Radiation

ATLs represent a group of compounds that are attractive for use in combina-
tion with radiotherapy, since they enhance radiation-induced apoptosis (Belka
et al., 2004; van Blitterswijk & Verheij, 2008; Vink et al., 2007). The first APC
that displayed in vitro radiosensitizing potential was miltefosine (Bruyneel et al.,
1993). However, this effect was only observed in cell lines expressing an acti-
vated Ras oncogene. Berkovic et al. were among the first to show that
miltefosine and edelfosine affected clonogenic survival after radiation in KB
squamous cell carcinoma (Berkovic, 1998). These ATLs also strongly increased
radiation-induced apoptosis in two human leukemic cell lines (Ruiter et al.,
1999). Studies on the combination of ilmofosine and radiation are scarce.
Using the colony-forming assay as readout, only additive effects were found
in human K562 leukemic cells and murine MethA fibrosarcoma
(Neumann, Lichtinghagen, Borchardt, & Kissler, 1991). Perifosine has been
shown to enhance radiation-induced cytotoxicity in both short-term and
long-term assays. In a wide range of different human cancer cell lines from both
solid and leukemic origin, perifosine was found to strongly increase radiation-
induced apoptosis and—like classical radiosensitizers—reduce clonogenic sur-
vival at clinically relevant doses (Neumann et al., 1991; Ruiter et al., 1999;
Vink et al., 2006). More recently, enhanced radiation-induced apoptosis
and elimination of clonogenic tumor cells by erucylphosphocholine (ErPC)
was demonstrated in malignant glioma (Handrick et al., 2006; Rubel et al.,
2006). ErPC-induced inhibition of Akt-mediated antiapoptotic signaling
appeared instrumental in this combined response (Handrick et al., 2006).
Perifosine has predominantly been used to study the combination of radiation
and APCs in vivo. While increasing the dose of radiation or perifosine alone,
only induced growth delay of KB carcinomas in mice, the combination of both

modalities resulted in complete and sustained tumor response at clinically
achievable plasma levels (Vink et al., 2006). Although the cytotoxic mechanism
of action remains unclear, immunohistochemical analysis of tumor tissue after
treatment revealed a prominent apoptotic response, as measured by staining of
active caspase 3. Similar results were observed in a human prostate carcinoma
xenograft model, in which the combination of perifosine and radiation had a
significantly stronger effect on tumor growth than single modality treatment
(Gao et al., 2011).
3.3 Combined Treatment of APLs and Electroporation

In vitro experiments of our group, treating breast cancer cell lines with
erufosine alone and in combination with electrical field therapy (electropo-
ration, electrical field intensity: 500–1000 V cm1) showed an additive
effect of this treatment to that of erufosine regarding the inhibition of
cell migration, initiation of actin alteration, apoptosis induction, and cell
cycle arrest in the G2/M phase (R. Tzoneva, I. Ugrinova, V. Uzunova,
A. Momchilova, and M.R. Berger, unpublished data).
4. STRUCTURE OF ANTITUMOR LIPIDS

The first ATLs were synthesized as LPC (1-acyl-sn-glycero-3-
phosphocholine) analogs in a search for immune modulators. In the early
1960s, Herbert Fischer and Paul Gerhard Munder (Max-Planck-Institut
für Immunbiologie in Freiburg, Germany) found phospholipase A2-
mediated formation of LPC in macrophages during phagocytosis of
silicogenic quartz particles and in response to substances with adjuvant activ-
ity that exogenous LPC strongly enhanced the phagocytic activity of peri-
toneal macrophages both in vitro and in vivo (Burdzy, Munder, Fischer, &
Westphal, 1964; Munder, Ferber, Modolell, & Fischer, 1969; Munder,
Modolell, Ferber, & Fischer, 1966). This suggested an immune-modulatory
role for LPC in the defense mechanisms of the immune system, but the nat-
urally occurring LPC was rapidly metabolized by acyltransferase to PC or by
lysophospholipase to glycerophosphocholine. Thus, LPC analogs with lon-
ger half-life in vivo were synthesized in the following years by a joint effort of
different groups led by Herbert Fisher, Otto Westphal, Hans Ulrich
Weltzien, and Paul Gerhard Munder in Freiburg (Houlihan, Lohmeyer,
Workman, & Cheon, 1995; Munder & Westphal, 1990). Particular
Antitumor Lipids 33
emphasis was placed on changes in the positions C1 and C2 of the glycerol

backbone in the LPC molecule, replacing ester bonds for ether linkages in
order to render analogs unable to be metabolized by either acyltransferases or
lysophospholipases. A number of these new ether analogs of LPC turned out
to be potent immune modulators, but surprisingly Munder and coworkers
found that some of these ether lipids exerted strong antitumor activities
in vitro and in vivo in a rather selective way. According to their chemical
structure, the currently used ATLs can be divided into two main classes,
i.e., (i) alkylphospholipids (APLs) and (ii) alkylphosphocholines (APC).
APLs are compounds with aliphatic side-chains that are ether linked to a
glycerol backbone and are structurally derived from the platelet-activating
factor (PAF) (Fig. 1), which is a naturally occurring phospholipid and a
mediator of platelet aggregation and inflammation (Chignard, Le Couedic,
Tence, Vargaftig, & Benveniste, 1979; Edwards & Constantinescu, 2009;
Prescott, McIntyre, & Zimmerman, 1990; Snyder, 1995; Wolf et al., 2006).
The prototype of this class is 1-O-octadecyl-2-O-methyl-rac-glycero-
3-phosphocholine (Et-18-OCH3, edelfosine; Fig. 2), which presents an
18-C long alkyl chain at the sn-1 position and a methoxy group at the sn-2
position of the glycerol backbone. In the alkyl-lysophospholipid-prototype
edelfosine and its thio-ether derivative ilmofosine, the glycerol backbone
is maintained (Fig. 2). Edelfosine manifests pronounced anticancer acti-
vity in vitro and in vivo (Berger, Munder, Schmahl, & Westphal, 1984;
Mollinedo et al., 1997; Munder & Westphal, 1990; Scherf, Schuler,
Berger, & Schmahl, 1987).
However, it was established (Berdel, Fink, & Rastetter, 1987) in clinical
tests that edelfosine as an independent drug is poorly suitable for treatment of
tumors because of its high hemolytic activity. The concentration of
edelfosine causing lysis of 50% platelets is 16 μmol L1. In fact, the only clin-
ically relevant application of edelfosine at this moment is for purging of bone
marrow in acute leukemia patients (Vogler et al., 1996).
Figure 1 Structure of platelet-activating factor (PAF). From wikimedia.org/.

CH3
CH3 CH3 CH3 CH3 CH3 N+
H3C CH3
H3C N+ CH3 H3C N+ CH3 H3C N+ CH3 H3C N+ CH3 H3C N+ CH3 CH2
H3C + CH3
N CH2
CH2 CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2 CH2 CH2
O O− O O− O O− O O − O O− O O− O O−
P P P P P P P
O O O O O O O O O O O O O O
CH2 CH2 CH2
HO CH2 H3C O CH2 H3C O CH2
CH2 CH2 CH2
O O S
C O
CH3 CH3
CH3 CH3
Miltefosine Perifosine
Erucylphos- Erufosine
CH3 CH3 phocholine
CH3
LysoPC Edelfosine Ilmofosine (ErPC) (ErPC3)

Figure 2 Chemical structures and commercial names of synthetic clinically relevant alkylphospholipids (APLs), metabolically stable analogs
of natural lysophosphatidylcholine (LysoPC). According to van Blitterswijk and Verheij (2013). With permission from Elsevier.
Antitumor Lipids 35
Efforts were made to incorporate edelfosine in liposomes to dimi-

nish this clinically unfavorable property (Busto, Del Canto-Janez,
Goni, Mollinedo, & Alonso, 2008). When forming these liposomes,
DOPE (dioleoylphosphatidyl-ethanolamine), cholesterol, and DOPC
(dioleoylphosphatidyl-choline) were used as lipid helpers. In this case,
liposomes-containing cholesterol (50% hemolysis at 661 μmol L1 of
edelfosine) turned out to be most stable and least toxic for blood cells
(Mayhew et al., 1997) (Table 1).
Ilmofosine (1-hexadecylthio-2-methoxymethyl-1,3-propanediol-
phosphocholine) is another representative of ATLs with antitumor activity
(Giantonio, Derry, McAleer, McPhillips, & O’Dwyer, 2004). The studies
showed a high antineoplastic activity of this compound for different types
of tumors. Like edelfosine, ilmofosine causes cancer cell apoptosis. There
are published data on the cytotoxicity of ilmofosine in submicromolar con-
centrations toward a series of cell lines (Giantonio et al., 2004; Table 2).
It was found that the optical isomers exhibit differential cytotoxicity toward
the lines MCF7 and А549 for the R isomer with respect to the chiral center at
the second carbon atom of the glycerol skeleton. For the S isomer, the concen-
tration causing 50% cell death is by 10 μmol L1 higher (Bittman, Byun,
Reddy, Samadder, & Arthur, 1997). Unlike edelfosine and its analogs man-
ifesting significant cytotoxicity for a broad set of cell lines as a racemic mixture
(Duclos et al., 1994; Goekjian & Jirousek, 2001; Principe & Braquet, 1995),
Table 1 Cytotoxicity Indices (IC50) of Edelfosine in Human Cancer Cells Based on

МТТ Test Data Gathered After 72 h of Cell Incubation with Edelfosine, According
to Markova et al. (2014)
Cell Line Index IC50 for
Cell Line Edelfosine (μmol L21)
HL60 (promyelocytic leukemia) 3.2
HCT116 (colon cancer) 1.6
CEM (T cell leukemia) 1.5
HUT-78 (T cell lymphoma) 4.5
Namalwa (Burkitt lymphoma) 15.3
SKOV3 (ovarian adenocarcinoma) 4.9
SK-MEL-2 (melanoma) 5.4
XF498 (glioma) 4.9
Table 2 Cytotoxicity Indices (IC50) of Ilmofosine in Human Cancer Cells Based on

МТТ Test Data Gathered After 72 h of Cell Incubation with Ilmofosine, According
to Markova et al. (2014)
Cell Line Index IC50 for
Cell Line Ilmofosine (μmol L21)
MCF7 (mammary adenocarcinoma) 7.2 (R)
A427 (pulmonary adenocarcinoma) 20
A549 (pulmonary adenocarcinoma) 7.5 (R)
CCRF/CEM (T cell leukemia) 2.8
HeLa (cervical adenocarcinoma) 9.9
ilmofosine has not yet found wide use, for it acts at relatively high cytotoxic
concentrations only and for the necessity to perform a selective synthesis,
which is required to obtain the respective enantiomer with a satisfactory
anticancer effect.
In contrast, the glycerol backbone in APCs is absent, the alkyl chain
being linked directly to the phosphate group. These molecules consist of
a simple long-chain alcohol conjugated to the polar phosphocholine head
group. High antibacterial activity was revealed for these compounds along
with anticancer properties. Miltefosine (hexadecylphosphocholine) (Fig. 2)
represents the prototype of this class. Miltefosine exhibits antitumor effects
in different cell lines; however, also a strong hemolytic effect was observed
for this compound (Scherer & Stoffel, 1987). Therefore, the application of
miltefosine is restricted to per oral and local uses. Similar to edelfosine, acting
upon adhesion and suspension cells, tumor cells of epithelial and leukemic
origin are both sensitive to miltefosine (Konstantinov, Eibl, & Berger, 1998;
Konstantinov, Topashka-Ancheva, Benner, & Berger, 1998). Prospects of
using miltefosine for the treatment of cancer diseases and leishmaniasis are
being evaluated presently (Croft, Snowdon, & Yardley, 1996;
Konstantinov, Kaminsky, Brun, Berger, & Zillmann, 1997; McBride,
Mullen, Carter, & Roberts, 2007). Another well-studied and promising
new APC is perifosine (D-21266, octadecyl-(1,1-dimethyl-piperidino-4-
yl)-phosphate), in which the choline moiety of miltefosine is replaced by
a heterocyclic methylated piperidyl residue (Fig. 2). The presence of the
N,N dimethyl piperidinium fragment attached to the alkylphosphate chain
in the perifosine structure increased its stability under physiological condi-
tions and enhanced the anticancer efficiency. Like miltefosine, perifosine is
considered for per oral administration. Perifosine is well adsorbed from
Antitumor Lipids 37
gastrointestinal tract and is cytotoxic toward adhesion cultures, in particular,

toward cancer cells of gullet and large intestine (Vink, Schellens, van
Blitterswijk, & Verheij, 2005). Due to its metabolic stability, perifosine is
promising for the development of related drugs as an independent chemo-
therapeutic agent as well as in combined chemo- and radiotherapy (Lux,
Heise, Klenner, Hart, & Opperdoes, 2000; Vink et al., 2007). ErPC
(Fig. 2) differs from miltefosine not only by a longer chain length
(C16 ! C22) but also the introduction of an ω-9 cis-double bond. Erufosine
(ErPC3; erucylphosphohomocholine; Fig. 2) differs from ErPC by one
additional methyl group in the choline head group, yielding higher solubil-
ity in aqueous solutions. Erufosine is among the series of widely known and
studied lipid PAF antagonists. Because of their long 22 carbon chain and the
ω-9 cis-double bond, both agents lack hemolytic toxicity due to the forma-
tion of lamellar instead of micellar structures in aqueous solutions and are
suitable for intravenous administration. The pharmacodynamic properties
of ErPC and erufosine resulted in significant tumor remissions in experi-
mental rat gliomas and mammary carcinomas at relatively low doses and with
reduced gastrointestinal toxicity (Berger, Sobottka, Konstantinov, & Eibl,
1998; Jendrossek & Handrick, 2003). Importantly, erufosine and ErPC
are superior to other ATLs in their ability to cross the blood–brain barrier
can accumulate in brain tissue (Markova, Plyavnik, Morozova, Maslov, &
Shtil, 2014) and thus have a potential for application in the treatment of
patients with otherwise poorly responsive primary or metastatic brain
tumors. In view of the positive response of myeloma cells in vitro (Yosifov
et al., 2011) and of autochthonous rat mammary gland cancer in vivo
(Dineva, Zaharieva, Konstantinov, Eibl, & Berger, 2012), erufosine can be
considered as promising APC. A common property of erufosine with
miltefosine and perifosine is its clear activity toward cells of human pro-
myelocytic leukemia HL60 (Konstantinov, Topashka-Ancheva, et al.,
1998), which is comparable to that of miltefosine and perifosine. Erufosine
is considered as a compound for combinations with other conventionally used
anticancer drugs—cytarabine, etoposide, and idarubicine (Fiegl et al., 2008;
Georgieva, Konstantinov, Topashka-Ancheva, & Berger, 2002).
5. CLINICAL TRIALS AND THERAPEUTIC EFFECT

The main advantage of this class of drugs is the target. In contrast to
most anticancer drugs, which interfere at the DNA level with cell
proliferation, APCs act at the cell membrane, where they disturb several
pathways, among which is the PI3K/Akt/mTOR signal transduction path-
way (Fig. 9). Initial preclinical tests were promising, indicating a good anti-
cancer activity in chemically induced, autochthonous mammary carcinoma
(Berger et al., 1993; Berger, Muschiol, Schmahl, & Eibl, 1987; Muschiol
et al., 1987; Sobottka, Berger, & Eibl, 1993) and in several human tumor
xenograft models in the mouse (Arndt, Zeisig, Eue, Sternberg, &
Fichtner, 1997; Fichtner et al., 1994), including different breast cancer cell
lines like: MT-3 (Zeisig, Arndt, Stahn, & Fichtner, 1998), MDA-MB 435
and MDA-MB 231 (Sobottka & Berger, 1992), MaTu (Arndt, Zeisig,
Fichtner, Teppke, & Fahr, 1999), MT-1 (Naundorf et al., 1992), C3H,
Ca 755 (Zeisig, Fichtner, Arndt, & Jungmann, 1991), and also syngeneic
models like murine P388 leukemia, and B16 melanoma (Zeisig et al.,
1991). Preclinical experiments further demonstrated that APCs, if used in
liposomal form, are able to abolish MDR in human breast cancer xenografts
(Zeisig, Teppke, Behrens, & Fichtner, 2004) and inhibit metastasis if com-
bined with an aggregation inhibitor inside liposomes in murine syngeneic
(Wenzel, Zeisig, & Fichtner, 2010) and human xenograft breast cancer
models (Wenzel, Zeisig, & Fichtner, 2009). Perifosine in combination with
DOPE as a component of the liposome bilayer also enhances transport of
drugs across the blood–brain barrier and in this way improves the treatment
of intracerebral tumors and metastases (Orthmann et al., 2010). Miltefosine
was also tested as an alternative approach for treatment of patients with pro-
gressive cutaneous lesions from breast cancer in phase I and II studies, which
indicated that miltefosine (either used alone or in conjunction with other
therapies for distant metastases) is an effective and tolerable local treatment
for cutaneous breast cancer (Clive, Gardiner, & Leonard, 1999; Unger &
Eibl, 1991). Gills et al. (Gills & Dennis, 2009) summarized the clinical trials
with Perifosine as single agent until 2009. Seven phase 1 single agent studies
of perifosine have been completed. The trials demonstrated that perifosine
can be safely given to humans with a manageable toxicity profile. The dose-
limiting toxicity of the phase I studies were similar to that of miltefosine,
involving gastrointestinal symptoms like nausea, vomiting, and diarrhea.
Perifosine as single agent has been further evaluated in phase II studies for
the treatment of the most common cancers, including breast, prostate, head
and neck, pancreatic cancers, melanoma, renal cell carcinoma, advanced
brain tumors, soft-tissue sarcomas, hepatocellular carcinoma as well as in
hematological malignancies including MM and Waldenstrom’s macroglob-
ulinemia (WM). Potent activity with perifosine, given as single agent, has
Antitumor Lipids 39
been observed so far in sarcoma and WM patients. Erufosine has entered

clinical development, as well, but will not be mentioned here, as these clin-
ical trials are not yet sufficiently published.
The preclinical and clinical studies on the effect of APCs on radio-
sensitization of cancer show a discrepancy between in vitro and in vivo studies.
Although De la Pena and coworkers showed clear radiosensitization by peri-
fosine in vitro, subcutaneous gliomas did not show enhanced response to
radiation after treatment with perifosine (de la Pena et al., 2006). Because
only one dose schedule was used, it remains uncertain whether an increased
radiation response by perifosine might be obtained at optimal (i.e., clinically
relevant) dose scheduling. Today, few clinical studies on the combined use
of APC (perifosine) and radiotherapy are available. A phase I/pharmacoki-
netic trial was performed in patients with advanced solid tumors, who were
treated with fractionated radiotherapy concurrently with daily intake of
perifosine in a dose-escalation schedule (Vink et al., 2006). Daily adminis-
tration commenced 2 days before radiotherapy and was continued through-
out the radiation treatment. Pharmacokinetic sampling was performed
weekly. Twenty-one patients were entered; 81% had non-small-cell lung
carcinoma (NSCLC). Major drug-related toxicities were nausea (57%),
fatigue (48%), vomiting (38%), diarrhea (38%), and anorexia (19%). No
bone marrow toxicity was observed. Perifosine proved tolerable up to a dose
of 150 mg/day. Gastrointestinal toxicity was reported to be dose limiting.
Dose-dependent steady-state plasma levels were reached after 1 week. From
this study, it was concluded that perifosine could be safely combined with
fractionated radiotherapy. A dosage of 150 mg/day, to be started at least
1 week prior to radiotherapy, was recommended for phase II evaluation.
In a subsequent randomized, double-blind, placebo-controlled multicenter
phase II study, the efficacy of daily perifosine and radiotherapy was assessed
in patients with locally advanced NSCLC (Verheij, Vens, & van Triest,
2010). Patients with inoperable NSCLC were randomly allocated to receive
either daily perifosine (150 mg) or placebo commencing 1 week prior to the
start of radiotherapy (17 3 Gy; 4 fractions/week) and continued during
radiation. Patients were stratified according to prior versus no prior chemo-
therapy. Primary endpoint was local control at 1 year, secondary endpoints
included tumor response rate, time to local failure, and overall survival.
A total of 177 patients from 19 centers was entered and patients most had
inoperable stage III (86%). Of these patients, 95 (54%) received perifosine
and 82 (46%) placebo. 121 patients entered the study without prior chemo-
therapy and 56 had prior chemotherapy. So far, the following preliminary
conclusions have been reported. A high number of patients did not reach the
1-year follow-up, mainly due to systemic progression, which compromised
the power of the trial to detect an effect of the study treatment on the local
control rate. For the subgroup of patients without prior chemotherapy, time
to local failure was slightly better in the perifosine group (mean 230 vs.
165 days; median 206 vs. 126 days). Kaplan–Meier survival curves indicated
a trend toward an advantage for perifosine patients without prior chemo-
therapy (p ¼ 0.088). The most frequently observed toxicity in the perifosine
group was gastrointestinal (grade 3–4: 15% vs. placebo 5%). Plasma concen-
trations of perifosine on the first and last day of radiation were comparable
and in the same range as determined previously (Verheij et al., 2010). The
final analysis of this study is currently ongoing. Based on several preclinical
observations, ErPC and erufosine are considered attractive candidates to be
further evaluated in patients and, after establishing safety and feasibility, to be
combined with radiotherapy (Kaufmann-Kolle, Berger, Unger, & Eibl,
1996; Rubel et al., 2006; Vink et al., 2007). This is based on the following
considerations. First, in vitro efficacy studies showed favorable IC50 values
for different tumor cell lines, including gastrointestinal, cervical, brain,
and breast cancer cell lines. Second, unlike other ATLs, ErPC and erufosine
show no significant in vitro hemolysis up to micromolar concentrations.
Third, in vivo efficacy studies in rat mammary gland and intracerebral glioma
models show significant antitumor effects after repeated i.v. doses. Fourth,
pharmacokinetic parameters after i.v. administration have been established
which allow guiding the dosing schedule. Fifth, biodistribution studies have
shown that ErPC can cross the blood–brain barrier of healthy rats and accu-
mulate in brain and brain tumor tissue. Finally, both toxicology and safety
pharmacology reports demonstrate no concerns that preclude the initiation
of a phase I/II clinical trials.
6. ANTICANCER MECHANISM OF ACTION

6.1 Membrane Localization and Lipid Rafts
Anticancer mechanisms of ATLs have been described and extensively dis-
cussed in some recent reviews (Danker, Reutter, & Semini, 2010;
Gajate & Mollinedo, 2002; Gills & Dennis, 2009; van Blitterswijk &
Verheij, 2008). Early interest focused on immune stimulating activity of
ATLs. It has been demonstrated that miltefosine and other lipids of this class
are able to activate T cells and macrophages to express and release
chemokines like GM-CSF (Vehmeyer, Eibl, & Unger, 1992; Vehmeyer,
Antitumor Lipids 41
Liersch, Eibl, & Unger, 1992), IF-gamma (Hochhuth, Vehmeyer, Eibl, &
Unger, 1992), and/or nitric oxide (NO) (Zeisig, Rudolf, Eue, & Arndt,
1995). This effect was improved when the APCs were used in liposomal
form. Because of their amphiphilic structure, they are able to form lamellar
bilayers, if combined with lipids of opposite molecular shape. Liposomes
were reported to be taken up by macrophages much better than free, micel-
lar lipids and to induce, after cellular uptake, the release of IF-gamma and
NO (Eue, Zeisig, & Arndt, 1995). But their potency as immune stimulators
was limited because the chemokines were not released at amounts sufficient
to completely inhibit tumor cell proliferation. Due to their amphiphilic
nature, ATLs are easily incorporated into cell membranes in substantial
amounts and then spread among intracellular membrane compartments,
where they accumulate and interfere with a wide variety of key enzymes
(Unger, Fleer, Kotting, Neumuller, & Eibl, 1992; van Blitterswijk,
Hilkmann, & Storme, 1987). At lower, clinically relevant concentrations,
ATLs interfere with phospholipid turnover and lipid-based signal transduc-
tion pathways. In mouse S49 lymphoma cells, ATLs are accumulated in
detergent-resistant, sphingolipid- and cholesterol-enriched lipid raft
domains and are rapidly internalized by clathrin-independent, raft-mediated
endocytosis (van der Luit et al., 2007). APLs uptake in KB carcinoma cells,
however, appears to be raft-independent and mediated by a yet unidentified
ATP-dependent lipid transporter (Vink et al., 2007).
6.2 Targets of APLs in Leukemic Cells Versus Solid Tumor Cells

Molinedo and coworkers (Nieto-Miguel, Gajate, & Mollinedo, 2006)
reported differential targets and subcellular localization of edelfosine in leu-
kemic and solid tumor cells. In leukemic cells, APL is mainly located in lipid
rafts of the plasma membrane and induces the formation of membrane raft
aggregates containing Fas/CD95 DR and the adaptor molecule Fas-
associated death domain-containing protein (FADD), which are critical in
triggering of apoptosis (Fig. 3; Gajate, Gonzalez-Camacho, & Mollinedo,
2009). There is convincing evidence that apoptosis may be induced by both
intracellular and extracellular factors. Initiation of intracellular mechanisms
of apoptosis occurs due to binding of certain ligands (death ligands) with
their specific receptors or due to deficit of exogenous ligands (e.g., factors
required for cell survival, extracellular matrix components, etc.) and lack
of activation of receptors responsible for transduction of signals required
for cell survival. Binding of “death ligands” belonging to the family of tumor
necrosis factor (TNF) or other families (TRAIL, FAS) with plasma
FasL
Fas
Drug preparation
Fas
Fas FasL
B
FasL Fas Fas cluster and sequential
activation of signaling
molecules resulting to
apoptosis
Fas FasL
FasL Fas
Figure 3 Involvement of signaling molecules in the mechanism of apoptosis induction

via the receptor-mediated (Fas/FasL) endocytosis.
membrane receptors is the most common and, consequently, the most stud-
ied mechanism of receptor-mediated initiation of apoptosis. In the case of
edelfosine, it has been demonstrated that receptor activation occurs via
the classical pathway of ligand binding to corresponding receptor; in this
case, the biologically active lipid acts as a ligand (Fig. 3). Triggering of
the extrinsic apoptotic pathway is associated with activation of caspase
8 by Fas/CD95 receptors, which form the death-inducing signaling com-
plex (DISC) (Fig. 3; Wei et al., 2013). DRs such as CD95 directly induce
activation of caspase-8 inside the DISC complex, which consists of the
receptor cluster, the molecular adaptor FADD, and procaspase-8
(Mollinedo, Gajate, Martin-Santamaria, & Gago, 2004). In many cases, cell
caspase-8 activates a cascade of effector caspases by means of proteolytic
cleavage. However, in cells with low initial level of activated caspase-8 a par-
allel process may occur, involving proteolytic cleavage of the proapoptotic
proteins Bcl-2 and Bcl-XL. These proteins delay onset of apoptosis by for-
ming apoptosomes and the cascade of effector caspases is triggered later and
this is known as the phenomenon of late apoptosis. The formation of these
complexes was based on the redistribution of Fas in lipid rafts (Eramo et al.,
Antitumor Lipids 43
2004). Flotillin, a raft-associated protein, prevents formation of DISC and

activation of apoptosis. Flotillin is shifted by caveolin after prolonged oxi-
dative stress, which promotes apoptosis. Caveolins are involved in
receptor-independent endocytosis and a member of this protein family,
caveolin1, is able to promote Fas cooperation and formation of DICS.
Recently, it has been shown that APLs were accumulated in lipid rafts in
HeLa cells via raft- and dynamin-mediated endocytosis (Van Der Luit,
Budde, Verheij, & Van Blitterswijk, 2003). It is very possible that the redis-
tribution of Fas and activation of DISC caused by the accumulation of ATLs
is related to their interaction with the caveolin protein family. In human leu-
kemic and glioblastoma cell lines, induction of apoptosis by liposomal
edelfosine and ErPC, respectively, is associated with cytochrome c release
(Verheij, Moolenaar, & Blitterswijk, 2014). It can be modulated by Bcl-2
family members and is related to intrinsic apoptotic activation.
It was also found that edelfosine can actively decrease the sensitivity
threshold of tumor cells; in addition, its action is selective, as edelfosine does
not influence normal cells in damaged tissues (Gajate & Mollinedo, 2002;
Mollinedo et al., 1997) and is characterized by a high degree of accommo-
dation in tumor tissues.
There is a strong correlation between the cellular uptake of edelfosine
and its cytotoxic activity. For example, incubation of various tumor cell lines
with edelfosine (3 μg mL1) resulted in various degrees of apoptosis induc-
tion, which correlated with the cellular uptake of edelfosine (Mollinedo
et al., 2004).
The most pronounced cell uptake of edelfosine was found in the case of
the U937 cells. In the case of normal cells of human blood (peripheral blood
leukocytes), the intracellular uptake was minimal and the apoptosis inducing
ability was basically absent (Mollinedo et al., 1997). The selectivity of the
effect of ether lipids is attributed to a higher uptake capacity of neoplastic
cells as compared to normal cells (Mollinedo et al., 2004). For instance, nor-
mal cells are unable to incorporate significant amounts of the ether lipid and
are spared, whereas most tumor cells incorporated edelfosine and thus were
triggered to subsequently undergo apoptosis (Gajate et al., 2000; Mollinedo
et al., 1997). Nontransformed 3T3 cells were resistant to the apoptotic
action of edelfosine and incorporated only small amounts of the ether lipid,
while upon transformation with SV40, these cells took up high amounts of
the lipid and became sensitive to it (Mollinedo et al., 1997). Conversely,
human leukemic HL-60 cells incorporated high amounts of edelfosine
and were sensitive to its apoptotic action, whereas following DMSO
treatment, HL-60 cells were differentiated toward cells with features of nor-
mal nontransformed mature neutrophils, and in this differentiated state the
uptake of the ether lipid was dramatically decreased as well as the sensitivity
to the drug (Alonso et al., 1997; Heesbeen et al., 1993; Mollinedo et al.,
1997; Vallari, Smith, & Snyder, 1988). These data indicate that the action
of the ether lipids is specific for tumor cells and that both cellular uptake
and edelfosine-induced apoptosis are dependent on the malignant state of
the cells. Another interesting finding is that normal human fibroblasts are
resistant to the exogenous addition of edelfosine because they do not take
up significant amounts of the ether lipid. But when edelfosine was micro-
injected the fibroblasts rapidly underwent apoptosis (Gajate et al., 2000),
indicating that the cell surface acts as a barrier to the ether lipid in normal
cells. In addition, the microinjected edelfosine induced apoptosis in a
dose-dependent way (Gajate et al., 2000), suggesting that a threshold for
intracellular edelfosine concentration must be reached in order to trigger
apoptosis. The mechanism of edelfosine uptake from cells involves two crit-
ical steps: (a) selective incorporation of edelfosine into the cell, likely to the
inner leaflet of the plasma membrane triggering of Fas/CD95 oligomeriza-
tion and (b) capping in membrane rafts, independent of FasL/CD95L and
activation of a Fas/CD95-mediated signaling route. Three major scenarios
can be found following incubation of edelfosine with distinct cell types
A B C
ET-18-OCH3
No uptake Uptake Uptake
Fas/CD95
A B C
No apoptosis No apoptosis
Apoptosis
Figure 4 Selective uptake of edelfosine by tumor cells. According to Mollinedo et al.

(2004). With permission from Bentham Science Publishers.
Antitumor Lipids 45
(Fig. 4): (A) Cells are unable to take up edelfosine, and therefore they are
spared after edelfosine treatment, despite their expression of Fas/CD95
(nontransformed, normal cells and resting T cells). (B) Cells are able to
incorporate edelfosine, but they do not undergo apoptosis following
edelfosine treatment because of Fas/CD95 absence (some resistant tumor
cells). (C) Fas/CD95-expressing cells are able to take up edelfosine and, once
inside the cell, edelfosine triggers Fas/CD95 oligomerization and capping
into membrane rafts (black dots) leading to apoptosis (cancer cells and acti-
vated T cells).
In summary, the selective action of ether lipids (for instance edelfosine)
to different cells can be explained by the degree of their transformation and
the specific chemical structure of the drug. Therefore, the membrane of nor-
mal cells is considered as a barrier for ether lipids (Gajate & Mollinedo,
2002). It is known that the composition of tumor membranes differs from
those of normal cells. These differences involve changes in the concentration
of major lipids (cholesterol (Chol), sphyngolipids and phosphatidylcholine
(PC)) and the proportion of saturated to unsaturated acyl chains. All these
modifications change the properties of tumor cells membranes, which are
more fluid as compared to normal cells (Ashkenazi & Dixit, 1998;
Evan & Littlewood, 1998; Green & Reed, 1998; Hengartner, 2000). The
fact that edelfosine and miltefosine are of higher affinity to unsaturated phos-
pholipids ( Jendrossek & Handrick, 2003; Jendrossek, Muller, Eibl, & Belka,
2003) and affect more strongly model membranes of higher fluidity
(Green & Kroemer, 1998; Igney & Krammer, 2002) explains an easier inser-
tion of these drug molecules into tumor membranes. In addition, it was
shown that the uptake of the ether lipids is strongly related to their interac-
tion with cholesterol in the lipids rafts (Wiecek, Covic, Locatelli,
Macdougall, & ORAMA Study Group, 2008). But this cannot yet explain
the active uptake of APLs into tumor cells, as in most cancer cells the amount
of cholesterol is at a lower concentration than in normal cells (Berra et al.,
1994). However, other factors should also be taken into consideration, like
the presence of specific molecules in tumor membranes, which are either
absent in normal cells or present at trace amounts, only. For instance, a char-
acteristic feature of tumor cells is the overexpression of gangliosides, the
concentration of which in normal cells is very low. In this regard, Hac-
Wydro and Dynarowicz-Latka (2010) have shown that edelfosine has tight
binding to molecules of the GM1 ganglioside. That fact gives reason to
hypothesize that gangliosides are those molecules which attract edelfosine
in membrane rafts and thus facilitate its inclusion in cancer cells (Fig. 3;
Hac-Wydro & Dynarowicz-Latka, 2010).
It has been shown (Gomide et al., 2013) that APCs (e.g., 10-(octyloxy)
decyl-2-(trimethylammonium) ethyl phosphate, ODPC, and perifosine)
can disrupt membrane raft domains in giant vesicles (Fig. 5) as the process
depends on the lipid composition. For instance, the presence of hospho-
lipids and cholesterol in homogeneous fluid lipid bilayers protects the
membrane from disruption. It was observed that ATLs have a tendency
to partitioning preferentially to the domain boundaries and lowering the
tendency for domain formation which later leads to membrane dissociation.
Therefore, the initial stage of lipid raft disruption by both ODPC and peri-
fosine, and maybe other APCs, by promoting lipid mixing, may be corre-
lated with their toxicity toward neoplastic cells, since selective (dis)
association of essential proteins within lipid raft microdomains must take
place in the plasma membrane. Accordingly, the changes in the lipid bilayer
biophysical properties and the selective (dis)association of essential proteins
within lipid bilayers have been demonstrated to trigger signaling pathways
(Cremesti, Goni, & Kolesnick, 2002). For instance, AKT/PI3K cell signal-
ing disruption by APCs has been observed (Kapoor, Zaharieva, Das, &
Berger, 2012), which is probably due to a primary action of the ATLs on
the organization of lipids in rafts and raft-associated proteins. For example,
Figure 5 Proposed mechanisms of alkylphospholipids (here represented by ODPC) that

induce raft disorganization: (A) Assembly of lipid rafts containing domains of varying
orders and compositions (TM-protein, transmembrane protein). (B) Treatment with
ODPC disrupts the assembly of lipid rafts, resulting in a displacement of raft-associated
proteins ODPC-induced modifications in the plasma membrane, by which lateral orga-
nization and fluidity are compromised, may lead to cell death. According to Gomide et al.
(2013).
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The Eukaryotic Translation

2. eIF4E'S STRUCTURE AND EXPRESSION

human elF4E 4E-BP1

protein Z (VPZ) represent a second class of eIF4E regulators that intervene

2.2 eIF4E's Expression and Regulation

2.2.3 Transcription Levels

consensus binding sites to transcription factors (such as c-Myc and

2.2.4 Protein Level

Serum, growth factors, Transcription factors

translation of mRNA having « sensible to eIF4e

Figure 2 eIF4E's expression regulation and its implication in tumorigenesis. Serum,

phosphorylation. The 4E-BPs are phosphorylated in response to growth fac-

3.1.1 The CaP-Dependent Translation Initiation Mechanism

3.1.2 The CaP-Independent Translation Initiation Mechanism

3.2 Nuclear Export

regulators to this process identical to 4E-BPs in the cytoplasm. Several pro-

4. eIF4E: A THERAPEUTIC TARGET IN CANCER

4.2 EIF4E’s Mechanisms in Cancer

Figure 5 eIF4E's involvement in the cells' oncogenic transformation. Schematic repre-

Thus, there are proteins involved in proliferation (cyclin D1, c-Myc,

4.3 Targeting eIF4E in Cancers

4.3.1 ASOs and siRNAs

4.3.2 Inhibition of the eIF4E/eIF4G Interaction

4.3.3 mRNA Cap Analogs

4.3.4 eIF4E Upstream Pathway Inhibitors

4.3.5 Inhibition of the eIF4E/Hsp27 Interaction

small-molecule inhibitors that can disrupt the eIF4E–eIF4G interaction, the

Garcia-Martinez, J. M., et al. (2009). Ku-0063794 is a specific inhibitor of the mammalian

2. DEVELOPMENT OF ANTITUMOR LIPIDS

3. COMBINING ATLs WITH OTHER ANTICANCER

3.1 Combining APLs with Other Anticancer Agents

apoptosis by UVB light/oxidative stress in skin cells, with possible implica-

3.2 Treatment with APLs and Radiation

only induced growth delay of KB carcinomas in mice, the combination of both

3.3 Combined Treatment of APLs and Electroporation

4. STRUCTURE OF ANTITUMOR LIPIDS

emphasis was placed on changes in the positions C1 and C2 of the glycerol

Figure 1 Structure of platelet-activating factor (PAF). From wikimedia.org/.

CH2 CH2 CH2 CH2 CH2 CH2

CH2 CH2 CH2

HO CH2 H3C O CH2 H3C O CH2

CH2 CH2 CH2

LysoPC Edelfosine Ilmofosine (ErPC) (ErPC3)

Efforts were made to incorporate edelfosine in liposomes to dimi-

Table 1 Cytotoxicity Indices (IC50) of Edelfosine in Human Cancer Cells Based on

Table 2 Cytotoxicity Indices (IC50) of Ilmofosine in Human Cancer Cells Based on