A major goal of cancer biology research is to uncover molecular changes that occur dur-ing the transformation process. These include genetic changes, such as mutations in proto-oncogenes and tumor suppressors, and epi-genetic abnormalities, such as alterations in DNA methylation. Recent advances in genom-ics have contributed to a greater understanding of the genetic mutations and signaling pathways involved in oncogenesis. This, in turn, has led to the development of a new therapeutic para-digm, known as molecular targeted therapeutics – the development of drugs/agents that target specific gene products driving disease. These agents take advantage of the fact that cancer cells often become dependent on the initial genetic reprogramming that occurred for their survival, a concept known as ‘oncogene addic-tion’ [1,2]. Thus, inhibition of the particular oncogene driving tumor progression selectively eliminates cancer cells while sparing normal ones. In addition, transformed cells may also become dependent on gene products that are not inherently oncogenic, but whose activity is required to a much greater extent than in normal cells. In this ‘non-oncogene addiction’ model [3], these proteins become rate limiting for the growth and survival of cancer cells and, therefore, also represent attractive drug targets. A well characterized example of such a depen-dency, which arises presumably because of the need for tumor cells to handle increased levels of abnormally high folded proteins, are compo-nents of the molecular chaperone network [4].
In recent years, there has accumulated much evidence that the normal checks and balances ordinarily imposed on components of the trans-lation apparatus are frequently deregulated in tumor cells, due to the need of transformed cells to support their altered proteome. In this review, we discuss strategies for targeting a key regulator of eukaryotic translation, the eukary-otic initiation factor 4F (eIF4F) complex, as an antineoplastic approach.
Translational initiation & its regulation Protein synthesis occurs in three phases: initia-tion, elongation, and termination. Translation initiation, during which elongation-competent 80S ribosomes are assembled on mRNA tem-plates, is the rate-limiting step [5], and therefore, regulation of translation primarily occurs at this stage. This step is stimulated by eIF4F, a hetero-trimeric complex composed of eIF4E, eIF4A, and eIF4G (FiguRe 1). The 5́ end of the mRNA, which is marked by a 7-methylguanosine residue linked via a 5´–5 -́pyrophosphate residue to the rest of the mRNA template (m7GpppN; where N is any nucleotide), provides the distinguish-ing mark for recognition by eIF4E [6]. Here, it is not clear whether eIF4E binds directly to the cap structure, then associates with eIF4G/eIF4A or if the eIF4F complex binds directly to the cap structure. The ATP-dependent DEAD-box helicase, eIF4A, is delivered to the 5’ cap-proximal mRNA template via its interactions with eIF4G [7,8]. eIF4F also interacts with the poly(A)-binding protein (PABP) located at the
Eukaryotic initiation factor 4F: a vulnerability of tumor cells
Protein synthesis is a complex, tightly regulated process in eukaryotic cells and its deregulation is a hallmark of many cancers. Translational control occurs primarily at the rate-limiting initiation step, where ribosomal subunits are recruited to template mRNAs through the concerted action of several eukaryotic initiation factors (eIFs). One factor that interacts with both the mRNA and ribosomes, and appears limiting for translation is eIF4F, a complex composed of the cap-binding protein, eIF4E; the scaffold protein, eIF4G; and the ATP-dependent DEAD-box helicase, eIF4A. eIF4E appears to play an important role in tumor initiation and progression since its overexpression can cooperate with oncogenes to accelerate transformation in cell lines and animal models, and its levels are elevated in many human cancers. This, therefore, represents a vulnerability for transformed cells, and presents an opportunity for therapeutic intervention. In this review, we discuss approaches for targeting eIF4F activity.
Teresa Lee1 & Jerry Pelletier*1,2
1Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada 2The Rosalind & Morris Goodman Cancer Research Center, McGill University, Montreal, QC H3G 1Y6, Canada *Author for correspondence: Tel.: +1 514 398 2323 Fax: +1 514 398 7384 E-mail: [email protected]
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Key Terms
Non-oncogene addiction: Growth or survival dependency by cancer cells on gene products or non-oncogenic pathways.
eIF4F: Heterotrimeric complex composed of the cap-binding protein, eIF4E; the scaffold protein, eIF4G; and the DEAD-box helicase, eIF4A.
mRNA discrimination: Phenomenon that occurs during translation initiation that preferentially leads to translation of one mRNA template over another.
3’ end of the mRNA, inducing circularization of the mRNA template [9]. eIF4G interacts with the 43S complex (40S ribosome and associated initiation factors) via eIF3, thereby recruiting the latter to the mRNA and forming the 48S ribosomal complex [10]. The bound pre-initia-tion complex is then thought to scan the mRNA until it reaches the appropriate initiation codon – resulting in recruitment of the 60S ribosomal subunit and formation of an 80S ribosome competent for peptide bond formation. An alternative initiation strategy involves recruit-ment of 40S ribosomes to internal locations in the mRNA (e.g., internal ribosome entry sites [IRESes]). Although this latter mecha-nism is 5’ cap-independent, components of the eIF4F complex can participate in this process depending on the nature of the IRES [11].
In addition to interacting with eIF3, eIF4G also interacts with eIF4E, eIF4A, PABP and the mRNA [12–14]. The binding of eIF4G causes a conformational change in the active site of eIF4E, leading to enhanced cap-binding [15]. In addition, eIF4G enhances the helicase activ-ity of eIF4A [16]. There are two homologues of eIF4G – eIF4GI and eIF4GII (sharing 46% identity), with eIF4GI being more abundant [17]. The exact role of eIF4A in translation is not clear. It may be required to unwind 5’-proxi-mal secondary structure to facilitate 40S ribo-some recruitment, participate in pre-initia-tion complex scanning of the 5´ untranslated region (UTR), and/or may hydrolyze ATP to re-arrange protein–protein or protein-mRNA complexes [18]. eIF4A helicase activity is aided by interacting with the RNA chaperones, eIF4B and eIF4H [19]. There are two eIF4A isoforms, eIF4AI and eIF4AII, which participate in trans-lation initiation. They share 90% sequence identity and are thought to be functionally indistinguishable [20].
eIF4F formation is tightly regulated. Under normal cellular conditions, in which transla-tion is low, eIF4E is prevented from binding to eIF4G by the eIF4E-binding proteins (4E-BPs) (FiguRe 1). There are three functionally equiva-lent isoforms: 4E-BPI, 4E-BP2 and 4E-BP3, which compete with eIF4G for binding to eIF4E. One of the major signaling pathways regulating translation is the PI3K/AKT/mTOR pathway, which responds to mitogenic signals such as growth factors, cytokines, hormones, and nutrient levels. Stimulation of PI3K/AKT/mTOR signaling results in phosphorylation of 4E-BP on four residues (serine 37, threonine 46,
threonine 70 and serine 65), which, in turn, releases eIF4E from being bound to eIF4E-BP, allowing it to associate with eIF4G and form the eIF4F complex [21].
Activated mTOR also phosphorylates and activates ribosomal S6 kinase, which, in turn, phosphorylates the tumor-suppressor gene prod-uct, programmed cell death protein 4 (PDCD4) (FiguRe 1) [22]. PDCD4 associates with both eIF4A and eIF4G [23]. This inhibits eIF4A helicase activity and prevents its binding to eIF4G, leading to translation downregulation. Phosphorylation of PDCD4 marks it for ubiq-uitin-mediated degradation, thereby preventing sequestration of eIF4A [23]. S6 kinase also phos-phorylates eIF4B, an event thought to increase eIF4B association with eIF4A and enhance eIF4A’s helicase activity [24]. eIF4A availability is also regulated by a small noncoding RNA, BC1, found almost exclusively in dendritic cells [25]. BC1 represses translation in these cells by binding to eIF4A and blocking its helicase activ-ity while increasing its ATPase activity, thereby uncoupling the two properties [26,27].
Whereas the 4E-BPs regulate the availabil-ity of eIF4E, eIF4E activity is also modulated through phosphorylation. When bound to eIF4G, eIF4E may become phosphorylated on Serine 209 by the eIF4G-bound kinases, MNKI and MNKII. This phosphorylation event increases the affinity of eIF4E for capped mRNA 3–4 fold [28,29]. MNK1 and MNKII act downstream of the Raf/Mek/Erk pathway, a MAP kinase cascade activated by the GTPase Ras, with diverse roles in proliferation, cell cycle progression, differentiation and apoptosis [30]. The functional consequence of MNK-directed phosphorylation of eIF4E has most often been associated with stimulation of translation and contributes to the transformation potential of eIF4E ex vivo and in vivo [31–33].
Recently, eIF4E has been shown to undergo sumoylation on several lysine residues [34]. This modification has been attributed to HDAC2 [35] and is dependent on serine 209 phosphoryla-tion, promotes eIF4F complex formation, and is required for the anti-apoptotic and oncogenic activity of eIF4E [34]. Whether increased eIF4F levels/activity correlates with the elevated levels of HDAC2 in human cancers [36,37] remains to be established. It will be of interest to explore inhibiting this post-translational modification of eIF4E as a therapeutic avenue.
eIF4E (as well as eIF4AI and eIF4GI) lev-els are also under transcriptional regulation of
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c-Myc [38,39]. This regulation is exerted through the presence of E boxes in the promoters of eIF4E, eIF4GI and eIF4AI [39]. Since Myc is a major transcriptional activator of rRNA pro-duction, it makes good sense that when ribo-some production is increased [40], components of the translational apparatus are also upregu-lated. This link between ribosomal production and protein synthesis may also contribute to the pathological role that Myc plays in tumori-genesis, creating a dependency that offers a therapeutic opportunity.
Tumor cell addiction to translation Many lines of evidence support the idea that disruption of translational control has onco-genic consequences. It has been reported that transformed cells generally show higher rates of protein synthesis than normal cells [41]. Ectopic overexpression of some translation initiation factors is oncogenic in cell culture systems and transgenic animals – possibly reflecting increased translation rates of a subset of mRNAs that deregulate cell growth, survival and/or proliferation. This has been demonstrated for eIF4E [42], eIF4G [43], eIF2 [44,45], several eIF3 subunits [46,47], and increased eIF4F activity [48] in cell culture systems. Overexpression of eIF4E in genetically engineered mouse models accelerates tumorigenesis [49,50] and alters che-mosensitivity [49]. Consistent with this, eIF4E overexpression can suppress apoptosis [51–53], recapitulate key oncogenic functions of Akt [49], and antagonize the pro-apoptotic activ-ity of c-Myc [49,53,54]. In support of a role for eIF4E in oncogenesis, levels of eIF4E are an independent prognostic tumor marker in can-cers of the breast [55–57], colon [58], head and neck [59,60], lung [61,62] and prostate [63]. The levels of 4E-BP1, as well as its phosphorylation status, are sometimes inversely correlated with progression of various cancers [64,65], although in breast, ovarian, and prostate cancer phospho-4E-BP1 has been associated with disease pro-gression and adverse prognosis [66]. PDCD4 is a suppressor of transformation and it remains to be seen whether this feature is due to sequestra-tion of eIF4A [23]. Lastly, as indicated above, key tumor-suppressor genes (e.g., PTEN), onco-genes (e.g., Ras and Src) and signaling pathways (e.g., PI3K and Akt) impinge on the translation apparatus – in particular, on components of the eIF4F complex, and these can exert large-scale changes in gene expression at the translational level [67].
The mechanism by which increased eIF4F activity can lead to the transformation pheno-type is not completely understood. An attrac-tive hypothesis is that although increased eIF4F activity leads to only modest increases in global translation, it dramatically alters translation of a limited set of mRNAs. While it might seem surprising that upregulation of a general trans-lation factor can have mRNA class-specific effects, such mRNA discrimination was pro-posed over 25 years ago [68]. If eIF4E avail-ability is limiting for initiation, then mRNAs must compete for access. A strongly competi-tive (well-translated) mRNA will be translated better than a weakly competitive transcript. What defines competitive ability is not clear but appears to be complex and may be the result of a combination of features. Secondary structure within the 5’ UTR has been shown
eIF4A
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eIF4E mRNA
mTOR
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m7GpppN
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4EBPP
P P
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eIF4E eIF4E eIF4E
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4Ei-1
Figure 1. Eukaryotic initiation factor 4F-mediated cap recognition during translation initiation. The mTOR-dependent regulation of eukaryotic initiation factor 4F assembly is highlighted. Inhibitors acting at various steps of this process are shown within light gray rectangles. The 43S pre-initiation complex consists of the 40S ribosome and associated translation factors.
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to negatively regulate translation [69,70]; poly-pyrimidine tracks (so-called TOP sequences) adjacent to the cap structure are particularly responsive to changes in eIF4E levels [71] and RNA-binding proteins positioned near the 5’ end can dampen initiation [72].
It has been recognized that some ‘weakly competitive’ mRNAs encode proteins that reg-ulate cell growth and survival and that increas-ing eIF4F levels or activity favors translation of these weak mRNAs [73–75]. Disproportionate increases in the translation of these mRNAs may contribute to tumor progression and/or main-tenance. Examples of mRNAs whose transla-tion are responsive to eIF4F levels include c-Myc, ornithine decarboxylase, MMP-9; the growth factors FGF-2, VEGF and TGF-b; and the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. It is important to note that several of these eIF4E targets also possess short half-lives. In particular, Mcl-1, cyclin-D1 and ornithine decarboxylase possess half-lives of 30, 24, and 5–30 min, respectively [76–79]. This enhances cellular dependency on fluctuations in trans-lational activity, as the levels of these proteins will be rapidly depleted in the absence of robust translation. Hence, in addition to affecting the translational output of the transcriptome, a sec-ondary consequence of inhibiting translation initiation will be to lead to a rapid decrease of short-lived key regulatory proteins.
Breaking the addiction: targeting eIF4FInhibitors of mTOR and of the Raf/Mek/Erk pathway have been studied extensively and will not be reviewed in this article since it is not always clear if their effects are a consequence of influencing translation and/or other cellu-lar pathways under their governance. Given the aforementioned studies implicating eIF4E (and eIF4F) in supporting tumor initiation and maintenance, it should come as little surprise that several laboratories have sought to identify small molecules that could be used as tools to assess the antineoplastic potential of inhibiting eIF4F activity.
Early studies illustrated that overexpression of 4E-BP1 and 4E-BP2 in NIH3T3 cells trans-formed by eIF4E or v-Src, arrested cell growth and led to significant reversion of the trans-formed phenotype [80]. Miskimins and col-leagues generated a constitutively active form of 4E-BP1 by mutating five amino acids that are targets for phosphorylation by mTOR to alanines. When expressed in the human breast
cancer cell line, MCF7, this mutant, termed 4EBP-1–5A, inhibited proliferation [81]. In another study, Schmidt and colleagues gen-erated a similar constitutively active mutant, 4EBPµ, by introducing alanine substitu-tions at four phosphorylation sites in 4EBP1. Overexpression of 4EBPµ in Rat1 cells slowed cell proliferation and, when co-transfected with eIF4E or c-Myc expression vectors, inhibited transformation [82]. Furthermore, the effective-ness of 4EBPµ in blocking transformation was recapitulated in vivo, where it was found to inhibit formation of c-Myc-induced tumors in nude mice [82]. Experiments aimed at targeting eIF4E using antisense [83,84] or using peptides to interfere with eIF4E/eIF4G interaction [85] sup-pressed the oncogenic properties of transformed cell lines ex vivo. A clever approach, in which the eIF4E-binding segment of eIF4G1 was sta-bilized by an a-helical inducer and fused to the cell-permeable TAT peptide, inhibited cap-dependent translation and triggered apoptosis in cell lines [86]. Furthermore, fusing 4E-BP1 peptides to Gonadotropin-releasing hormone led to cellular uptake and anti-tumor activity in ovarian tumor xenograft models [87]. Taken together, these experiments provide validation for eIF4E as an anticancer target.
Several ex vivo experiments also highlighted the effectiveness of targeting eIF4A to cur-tail oncogenesis. Antisense RNA to eIF4AI decreased proliferation of melanoma cells [88] and introduction of PDCD4 into JB6 epider-mal cells blocked transformation ex vivo [89] and delayed tumor onset and progression in a chemically induced murine skin tumor model [90]. These experiments support the notion that interfering with the eIF4E:eIF4G interaction, reducing eIF4E or eIF4AI levels, or sequestra-tion of eIF4A, can exert antiproliferative effects in a possibly oncogene-dependent manner [82]. These studies set the stage for high-through-put screens and medicinal chemistry efforts to find small-molecule inhibitors of translation, to move beyond cell culture experiments and assess efficacy in vivo, as well as determine whether a therapeutic window exists for this type of approach.
� Blocking Mnk-dependent eIF4E phosphorylation MAP kinase signal-integrating kinases (Mnks, also known as MAP kinase-interacting kinases) are four proteins encoded from two genes (Mnk1 and Mnk2) and generated by alternative
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splicing that are dispensable for cell growth and mouse development [91,92]. The longer isoforms, Mnk1a and Mnk2a, are predominantly cyto-plasmic, whereas the shorter variants, Mnk1b and Mnk2b, are mainly nuclear [91]. The Mnks integrate input from the ERK and p38 MAP kinase pathways to exert effects on ERK signal-ing (Spry2), inflammatory responses (cPLA2), regulation of mRNA splicing, stability and trafficking (hnRNP A1 and PSF) and transla-tion initiation (eIF4E and eIF4G) [91]. Mnks phosphorylate eIF4E on Ser209 and this event is necessary for eIF4E’s transforming activ-ity [31–33]; it is also generally thought to be associated with increased translation initia-tion rates [33] due to reduced affinity for the cap structure [93]. Pharmacological inhibition of Mnk-directed phosphorylation of eIF4E reduced metastasis in a B16 melanoma model [94]. Consistent with at least part of this effect being mediated through eIF4E phosphoryla-tion, knock-in mice with a phosphorylation-resistant allele of eIF4E, S209A, are resistant to PTEN loss-induced prostate cancer [33].
� eIF4E-m7GpppN inhibitors One activity of eIF4F that has been extensively studied is the cap-binding properties of eIF4E. Indeed, details of the eIF4E-cap interaction are well characterized at the molecular level [95] and structural analogue inhibitors have been studied for approximately 30 years [96]. Extensive chem-Extensive chem-ical modifications to cap analogues and nucle-osides have identified novel structures with even greater inhibitory potential than the common synthetic cap-analogue precursor (m7GpppG and m7GTP) [97] but their use has been limited to in vitro studies since they are not readily cell permeable nor stable in cell culture [96,98]. In an attempt to circumvent these pitfalls, Ghosh et al. generated prodrug derivatives of 7-ben-prodrug derivatives of 7-ben-zyl GMP (containing a protecting group that becomes converted to the active 7-benzyl GMP form by native cytosolic enzymes) [99]. These compounds are relatively nontoxic, water-solu-ble and much more stable in blood plasma. One compound identified in this study, 4Ei-1, was capable of inhibiting cap- and eIF4E-dependent reporter constructs in vitro and ex vivo when injected into freshly fertilized zebrafish [99]. The in vivo properties and anti-tumor potential of 4Ei-1 remain to be established.
Since the cap structure is a point of regulation for gene expression at multiple levels, includ-ing pre-mRNA splicing, nucleo-cytoplasmic
transport and mRNA decay, and eIF4E may have a nuclear function [100], it will be important to distinguish whether results obtained with cap analogues on cellular growth or death are the consequence of inhibiting eIF4E-mediated translation, blocking eIF4E nuclear activity, or unrelated to eIF4E due to curtailing m7GpppN interaction with other cap-binding proteins (e.g., blocking CBP20/CBP80-m7GpppN or Dcp2-m7GpppN interaction).
Another nucleoside analogue, ribavirin, has been reported to be an inhibitor of eIF4E-m7GpppN recognition with anticancer activ-ity [101,102]. In Phase II monotherapy trials in patients with acute myeloid leukemia, one complete response, two partial remissions, two blast responses, four disease-stabilization responses were reported, while two patients did not respond [102]. Whether these results are a consequence of inhibiting eIF4E–cap recognition is not clear since two independent studies failed to confirm that ribavirin inhibits cap-dependent translation [103,104]. The exact mechanism of action of ribavirin is complex, with reports documenting its activity as an inhibitor of viral transcription or mRNA cap-ping to a more indirect method of promoting an interferon response [105].
� Targeting the eIF4E:eIF4G interaction Targeting the interaction between eIF4E and eIF4G is another area of interest that has been explored by several laboratories. The eIF4E-binding site on eIF4G is well defined, with the motif Y(X)
4LF (where X is any amino acid
and F is hydrophobic) being the site of rec-ognition [106]. Since this motif is also present in the 4EBPs, it is expected that inhibitors of the eIF4E:eIF4G interaction may also affect eIF4E:4EBP interaction.
To date, three small-molecule inhibitors of eIF4E:eIF4G interaction have been reported. One of these, 4EGI-1, inhibited cap-dependent translation and was toxic against several cancer cell lines [107]. Interestingly, whereas 4EGI-1 bound to eIF4E and disrupted eIF4E-eIF4G interaction, it enhanced eIF4E:4EBP-1 inter-action [107]. 4EGI-1 is able to induce apopto-sis in human lung cancer cells and cooperated with TRAIL in this event, although the effect appeared independent of inhibition of cap-dependent translation and rather depended on an endoplasmic reticulum stress, CHOP-dependent mechanism [108]. In a zebrafish model of engrafted AML tumor cells, 4EGI-1 showed
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teratogenic activity, precluding its assessment for antineoplastic activity in vivo [109]. Two other eIF4E:eIF4G inhibitors, 4E1RCat and 4E2RCat, block interaction of eIF4E with eIF4GI, eIF4GII and 4E-BP1 [110,111]. 4E1RCat and 4E2RCat were tested in Eµ-Myc mice for their antineoplastic activity. This is a trans-genic model in which the c-Myc oncogene is overexpressed in the B-cell compartment by the lymphoid-specific Ig heavy chain enhancer (Eµ), with mice developing predominantly pre-B- or B-cell lymphomas 4–6 months after birth [112]. The compounds were ineffective as single agents, but were able to synergize with doxo-rubicin to prolong lymphoma-free survival in this model (data not shown for 4E2RCat) [110]. We note that these compounds have also been identified in other high-throughput screens – for example, 4E1Rcat is active in 49 of 376 bioassays [201] and therefore the selectivity (and potencies) of these ‘hits’ awaits improvement through medicinal chemistry efforts.
� Suppressing eIF4E expressionRecently, Lilly Research Laboratories has tar-geted eIF4E expression using antisense oligo-nucleotides (ASOs) [113]. In their report, they demonstrated that although 80% knockdown of eIF4E marginally affected global protein synthesis (20% change), administration of eIF4E ASOs suppressed eIF4E expression and tumor growth in breast and prostate xenograft models [113]. In addition, the eIF4E ASOs also inhibited endothelial cell tube formation ex vivo, indicating a possible anti-angiogenic effect of inhibiting eIF4E [113]. A Phase I dose-escalation study demonstrated that one eIF4E ASO, LY2275796, could be safely administered to patients with advanced solid tumors [114]. LY2275796 was well tolerated with some minor toxicities noted (fatigue, nausea, fever, vomit-ing, prolongation of activated partial thrombo-plastin and prothrombin time, and thrombocy-topenia were the most frequently reported) and, importantly, resulted in quantifiable changes in tumor eIF4E mRNA levels [114]. Two targets of eIF4E were examined, VEGF and cyclin D1, and found to be reduced in some (53–60%) patients [114]. Although no tumor response was observed in this Phase I trial, stratification of patients based on specific tumor type in which eIF4E levels are often deregulated (e.g., prostate cancer), as well as combining LY2275796 with other therapies, will be important for assessing future potential clinical efficacy.
� Targeting eIF4A with small-molecule inhibitors The enzymatic subunit of eIF4F is eIF4A. Compounds that have a dramatic effect on eIF4A activity have been identif ied from high-throughput in vitro translation screens utilizing a bicistronic mRNA that reported on cap-dependent and HCV IRES-mediated translation [115]. The sampling of 300,000 compounds by this screen identified three nat-ural products (pateamine A [Pat A], silvestrol and hippuristanol) that affected the helicase activity of eIF4A in different ways [116–118]. Hippuristanol is an allosteric inhibitor of eIF4A RNA binding that also leads to inhibition of ATPase (since this is RNA-stimulated) activ-ity and unwinding [117,119]. The binding site of hippuristanol on eIF4A has been identified and is not conserved among other members of the DEAD-box family, making hippuristanol a selective inhibitor of eIF4AI and eIF4AII, and, to a lesser extent (tenfold), eIF4AIII [119]. Hippuristanol has been shown to selectively inhibit the proliferation of adult T-cell leu-kemia cell lines, as well as being able to sup-press tumor growth in immunodeficient mice injected with HTLV-1-infected T cells [120]. Synthetic routes to hippuristanol have recently been reported paving the way for analogue gen-eration in an attempt to improve the solubility and potency of this molecule [121–124].
In contrast to hippuristanol, Pat A and silvestrol appear to act as chemical inducers of dimerization. Both compounds stimulate eIF4A activity, primarily by forcing sequence nonspecific binding of eIF4A to RNA on which much of its enzymatic activity relies [116,118,125–127]. This results in inhibition of translation initiation due to eIF4A depletion from the eIF4F complex [116,118,125]. Nonspecific bind-ing of eIF4A to mRNA may also interfere with the translation initiation or elongation processes by causing unscheduled unwind-ing of mRNA [116], as well as the formation of aberrant, eIF4F-independent ribosomal complexes involving eIF4A and eIF4B, which become loaded at random positions on the mRNA [116]. Pat A is an irreversible inhibitor of translation, and likely the consequence of a reactive Michael addition site [116]. Although the exact binding site and mechanism of Pat A is not known, mutational and domain dele-tion studies of eIF4A have indicated that it binds to the N-terminal ATP-binding domain [126]. Its inhibitory activity is dependent on the
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sequence of the linker region between the N- and C-terminal domains of eIF4A, and Pat A may relieve the negative regulation of eIF4A by the linker [127]. Pat A binds to free eIF4A exclusively and is unable to bind eIF4A that is already in the eIF4F complex suggesting that its binding site may overlap with that of eIF4G, or that its binding site is rendered inaccessible when eIF4A is eIF4G-bound [116]. It has been shown to induce stress granule formation [128] and inhibit nonsense mediated decay through its binding to eIF4AIII [129]. Pat A was origi-nally identified based on its cytotoxic activity against P388 murine leukemia cells [130], and subsequently shown to induce apoptosis in vari-ous cancer cell lines, particularly in Ras- or Bcr/Abl-transformed 32D myeloid cells [131]. Although the efficacy of Pat A has not been tested in in vivo preclinical models, a struc-a struc-turally simplified, chemically stabilized syn-thetic analogue, des-methyl, des-amino Pat A, displays anticancer activity in several human cancer xenograft models in nude mice [132].
Silvestrol is the most potent translation inhibitor among the rocaglamides tested to date [118]. Early experiments with rocaglamide family members indicated these to have insecticidal activity [133–135] and to be cytotoxic against can-cer cells [136–141]. Rocaglamides have also been shown to inhibit NF-kB activation [142,143] and block G2/M progression [144]. Whether these activities are related to the ability of rocaglam-ides to inhibit protein synthesis is not known. Silvestrol shows a favorable pharmacokinetic profile [145] and has been tested in several dif-ferent mouse cancer models with encouraging results. As a single agent, the compound does not cause distress, weight loss, liver damage, or immunosuppression in the mouse [146,147]. In some settings, silvestrol alone has therapeutic benefit as a single-agent chemotherapeutic. This is the case in xenograft studies of acute lympho-blastic leukemia [147]; prostate and breast cancer xenografts models [146]; in the Eµ-Tcl-1 mouse, a model of chronic lymphocytic leukemia (CLL); and primary human CLL samples [147]. In the latter study, it was noted that silvestrol was far more toxic towards B cells derived from CLL patients than from healthy individuals [147]. In the Eµ-Myc mouse model, silvestrol as a single agent showed no ability in inducing tumor remission, although this might be explained by the lower doses used [118]. However, when com-bined with doxorubicin it caused tumor remis-sion and significantly prolonged tumor-free
survival [118]. Moreover, this combination therapy was also effective against eIF4E-driven Eµ-Myc lymphomas, which do not respond to conventional monotherapy or to a combination chemotherapy regime [148]. This is also what is seen in AML cells, where silvestrol increases the cytotoxicity of daunorubicin, etoposide and cytarabine [149]. Treatment of tumor cells has been shown to lead to reductions in the levels of the pro-survival factor, Mcl-1 [118,149], as well c-FLIP; the latter being responsible for confer-ring resistance to receptor-mediated apoptosis [150]. Elucidation of a synthetic route to silvestrol [151,152] will afford extensive structure–activity relationship studies to better understand func-tional features of this class of compounds [153]. Resistance to silvestrol in acute lymphoblastic leukemia cells has been attributed to ABCB1/P-glycoprotein overexpression in some settings, an obstacle that must be taken into consideration for moving forward in developing silvestrol as an antineoplastic agent [154].
With the range of translation inhibitors available for targeting the various components of eIF4F, an issue of interest is their relative potency and effectiveness in repressing trans-lation. It has been observed that inhibiting eIF4E results in only small changes in global protein synthesis [113]. On the other hand, inhi- the other hand, inhi-bition of eIF4A causes a significant decrease in global translation [116–118], suggesting that inhibitors against eIF4A are more potent than those downregulating eIF4E, targeting either the eIF4E:cap or the eIF4E:eIF4G interac-tions, or affecting a slightly different event in the initiation process. A possible reason for this difference may be that cap-binding by eIF4E is not an absolute requirement for translation ini-tiation. Studies have shown that in the absence of eIF4E, eIF4A and the central domain of eIF4G, which binds eIF4A, are sufficient for recruitment of the 43S pre-initiation complex [155,156]. In addition, with the exception of arti-ficial, completely unstructured 5’UTR, eIF4A is required for ribosome recruitment to cellu-lar mRNA 5’ UTRs [157]. Furthermore, dele-tion of the N-terminal eIF4E-binding domain of eIF4G still allows translation of uncapped mRNA in a 5´ end-dependant manner [158–160]. This shows that upon eIF4E inhibition, recruit-ment of ribosomes to the 5´ end of the mRNA by the eIF4G:eIF4A complex likely still takes place, although much less efficiently. Hence, inhibition of eIF4E is unlikely to be equivalent to eIF4A inhibition.
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Future Med. Chem. (2012) 4(1)26 future science group
Future perspective The data summarized here indicate that inhibi- inhibi-inhibi-tors of translation initiation show much prom-ise as antineoplastic agents. The recent work in this field highlights the feasibility of curtailing cancer initiation and progression by targeting components of the eIF4F complex. From the ear-liest efforts in developing structural analogues to inhibit the eIF4E:cap interaction, to more recent efforts directed against eIF4A, eIF4E or the eIF4E:eIF4G interaction, these compounds have proven effective in killing cancer cells ex vivo, and, in many instances, in preclinical mouse models as well. Of significance is the fact that in many instances, selective tumor cell tox-icity is observed. Hence, although research on eIF4F inhibitors is still in its infancy, and much work needs to be done before they can make their way into the clinic, there is much optimism that they could lead to the development of potent anticancer drugs with good therapeutic indices. The next step will involve medicinal chemis-try efforts aimed at modifying and optimizing these compounds for efficacy and reduced toxic-ity in vivo, creating more ‘drug-like’ analogues, before moving onto clinical trials. Research in the next few years will likely also entail exploring the anti-tumor effects of inhibiting other trans-lation factors or initiation steps, such as AUG selection [157], thereby altering or restricting expression from a subset of mRNAs.
Future studies should continue to further investigate the molecular mechanisms involved in translation initiation – there remains sev-eral aspects of the functions and mechanistic details of the participants in translation that are not yet clear. Gaining a deeper fundamental
understanding of this process will also be impor-tant for clinical sample stratification. Ensuring that drugs are exerting their effects by targeting the translation apparatus, as opposed to off-target effects, will be paramount. Moreover, it is impor-tant to explore the significance and effectiveness of targeting translation in different types and stages of cancers. For example, elevated eIF4E levels is particularly significant in breast, prostate and colon cancers [55–58], and is used as a prog-nostic marker in these cases – hence, these types of cancers may be more amenable to treatment using eIF4E inhibitors. Lastly, the therapeutic context with which eIF4F inhibitors may be used remains to be determined. Questions to be addressed include the issue of whether they would be suitable as single agents, or if they would be better applied in conjunction with other drugs – and, in the case of the latter, what other agents may effectively be used in combination.
Financial & competing interests disclosureWe thank members of the Pelletier laboratory for their sup-port and scientific input. We apologize to those authors whose work is not cited here due to space constraints. T Lee was supported by the CIHR Strategic Training Initiative in Chemical Biology and the McGill Systems Biology Fellowship. Research in the Pelletier laboratory is funded by grants from the Canadian Institutes of Health Research (MOP-106530) and the Canadian Cancer Society Research Institute (#17099). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Executive summary � Translation deregulation is both a hallmark and vulnerability of cancer, as tumor cells often become ‘addicted’ to increased translational
activity.
� Regulation of translation occurs primarily at the rate-limiting initiation stage, where ribosomes are assembled on mRNA templates. This process is mediated by eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex composed of eIF4E, eIF4A and eIF4G, which associates with the 5’ cap of the mRNA and recruits 40S ribosomal subunits and other initiation factors.
� Understanding this process allows us to devise rational approaches to overcome this addiction.
� Increased eIF4F activity has been shown to promote tumorigenesis, due in part to differential translation of mRNAs encoding growth and survival factors. On the other hand, overexpression of endogenous negative regulators of eIF4E and eIF4A reverse or inhibit transformation.
� Targeting translation initiation via eIF4F inhibitors shows promise as an antineoplastic approach; these agents include the following classes of compounds:
� Blocking Mnk-dependent eIF4E phosphorylation; � eIF4E-m7GpppN cap mimics; � Inhibitors of the eIF4E:eIF4G interaction; � Blocking eIF4E production; � eIF4A inhibitors.
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ReferencesPapers of special note have been highlighted as:�� of considerable interest
2 Weinstein IB, Joe A. Oncogene addiction. Cancer Res. 68(9), 3077–3080 (2008).
3 Solimini NL, Luo J, Elledge SJ. Non-oncogene addiction and the stress phenotype of cancer cells. Cell 130(6), 986–988 (2007).
4 Dai C, Whitesell L, Rogers AB, Lindquist S. Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell 130(6), 1005–1018 (2007).
5 Nicholas T, Ingolia SG, John RS, Weissman JS. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924), 218–223 (2009).
6 Sonenberg N, Morgan MA, Merrick WC, Shatkin AJ. Polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5’-terminal cap in mRNA. PNAS 75(10), 4843–4847 (1978).
7 Edery I, Humbelin M, Darveau A et al. Involvement of eukaryotic initiation factor 4A in the cap recognition process. J. Biol. Chem. 258(18), 11398–11403 (1983).
8 Lindqvist L, Imataka H, Pelletier J. Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking. RNA 14(5), 960–969 (2008).
9 Bernstein P, Peltz, SW, Ross J. The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol. Cell Biol. 9(2), 659–670 (1989).
10 Lefebvre AK, Korneeva NL, Trutschl M et al. Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit. J. Biol. Chem. 281(32), 22917–22932 (2006).
11 Pestova TV, Kolupaeva VG, Lomakin IB et al. Molecular mechanisms of translation initiation in eukaryotes. Proc. Natl Acad. Sci. USA 98(13), 7029–7036 (2001).
12 Hinton TM Coldwell MJ, Carpenter GA Morley SJ, Pain VM. Functional analysis of individual binding activities of the scaffold protein eIF4G. J. Biol. Chem. 283(3), 1695–1708 (2007).
13 Imataka H, Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Mol. Cell Biol. 17(12), 6940–6947 (1997).
14 Yanagiya A, Svitkin YV, Shibata S, Mikami S, Imataka H, Sonenberg N. Requirement of RNA binding of mammalian eukaryotic
translation initiation factor 4GI (eIF4GI) for efficient interaction of eIF4E with the mRNA cap. Mol. Cell. Biol. 29(6), 1661–1669 (2009).
15 Haghighat A, Sonenberg N. eIF4G dramatically enhances the binding of eIF4E to the mRNA 5 -́cap structure. J. Biol. Chem. 272, 21677–21680 (1997).
16 Oberer M, Marintchev A, Wagner G. Structural basis for the enhancement of eIF4A helicase activity by eIF4G. Genes Dev. 19, 2212–2223 (2005).
17 Gradi A, Imataka H, Svitkin YV et al. A novel functional human eukaryotic translation initiation factor 4G. Mol. Cell. Biol. 18(1), 334–342 (1998).
18 Shibuya T, Tange TO, Sonenberg N, Moore MJ. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11(4), 346–351 (2004).
19 Rogers GW Jr, Richter NJ, Lima WF, Merrick WC. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F. J. Biol. Chem. 276(33), 30914–30922 (2001).
20 Yoder-Hill J, Pause A, Sonenberg N, Merrick WC. The p46 subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A. J. Biol. Chem. 268(8), 5566–5573 (1993).
21 Gingras AC, Raught B, Gygi SP et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 15(21), 2852–2864 (2001).
22 Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M. S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314(5798), 467–471 (2006).
23 Yang HS Jansen AP, Komar AA et al. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol. Cell. Biol. 23(1), 26–37 (2003).
24 Shahbazian D, Roux PP, Mieulet V, et al. The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J. 25(12), 2781–2791 (2006).
25 Tiedge H, Fremeau RT, Jr., Weinstock PH, Arancio O, Brosius J. Dendritic location of neural BC1 RNA. Proc. Natl Acad. Sci. USA 88(6), 2093–2097 (1991).
26 Wang H, Iacoangeli A, Popp S et al. Dendritic BC1 RNA: functional role in regulation of translation initiation. J. Neurosci. 22(23), 10232–10241 (2002).
27 Lin D, Pestova TV, Hellen CU, Tiedge H. Translational control by a small RNA:
28 Joshi B, Cai AL, Keiper BD et al. Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209. J. Biol. Chem. 270(24), 14597–14603 (1995).
29 Waskiewicz AJ Johnson JC, Penn B, Mahalingam M, Kimball Sr, Cooper JA. Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol. Cell. Biol. 19(3), 1871–1880 (1999).
30 Crews CM, Erikson RL. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell 74(2), 215–217 (1993).
31 Topisirovic I, Ruiz-Gutierrez M, Borden KL. Phosphorylation of the eukaryotic translation initiation factor eIF4E contributes to its transformation and mRNA transport activities. Cancer Res. 64(23), 8639–8642 (2004).
32 Wendel HG, Silva RL, Malina A et al. Dissecting eIF4E action in tumorigenesis. Genes Dev. 21(24), 3232–3237 (2007).
33 Furic L, Rong L, Larsson O et al. eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression. Proc. Natl Acad. Sci. USA 107(32), 14134–14139 (2010).
34 Xu X, Vatsyayan J, Gao C, Bakkenist CJ, Hu J. Sumoylation of eIF4E activates mRNA translation. EMBO Rep. 11(4), 299–304 (2010).
35 Xu X, Vatsyayan J, Gao C, Bakkenist CJ, Hu J. HDAC2 promotes eIF4E sumoylation and activates mRNA translation gene specifically. J. Biol. Chem. 285(24), 18139–18143 (2010).
36 Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat. Rev. Drug Discov. 5(9), 769–784 (2006).
37 Weichert W, Roske A, Gekeler V et al. Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy. Br. J. Cancer 98(3), 604–610 (2008).
38 Jones RM, Branda J, Johnston KA et al. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc. Mol. Cell. Biol. 16(9), 4754–4764 (1996).
39 Lin CJ, Cencic R, Mills JR, Robert F, Pelletier J. c-Myc and eIF4F are components
Review | Lee & Pelletier
Future Med. Chem. (2012) 4(1)28 future science group
of a feedforward loop that links transcription and translation. Cancer Res. 68(13), 5326–5334 (2008).
40 Van Riggelen J, Yetil A, Felsher DW. MYC as a regulator of ribosome biogenesis and protein synthesis. Nat. Rev. Cancer 10(4), 301–309 (2010).
41 Heys SD, Park KG, Mcnurlan MA et al. Measurement of tumor protein synthesis in vivo in human colorectal and breast cancer and its variability in separate biopsies from the same tumor. Clin. Sci. 80(6), 587–593 (1991).
42 Lazaris-Karatzas A, Montine KS, Sonenberg N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5’ cap. Nature 345(6275), 544–547 (1990).
43 Fukuchi-Shimogori T, Ishii I, Kashiwagi K, Mashiba H, Ekimoto H, Igarashi K. Malignant transformation by overproduction of translation initiation factor eIF4G. Cancer Res. 57(22), 5041–5044 (1997).
44 Koromilas AE, Roy S, Barber GN, Katze MG, Sonenberg N. Malignant transformation by a mutant of the IFN-inducible dsRNA- dependent protein kinase. Science 257(5077), 1685–1689 (1992).
45 Donze O, Jagus R, Koromilas AE, Hershey JW, Sonenberg N. Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14(15), 3828–3834 (1995).
46 Mayeur GL, Hershey JW. Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e). FEBS Lett. 514(1), 49–54 (2002).
47 Zhang L, Pan X, Hershey JW. Individual overexpression of five subunits of human translation initiation factor eIF3 promotes malignant transformation of immortal fibroblast cells. J. Biol. Chem. 282(8), 5790–5800 (2007).
48 Avdulov S, Li S, Michalek V et al. 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 (2004).
49 Wendel HG, De Stanchina E, Fridman JS et al. Survival signaling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 428(6980), 332–337 (2004).
50 Ruggero D, Montanaro L, Ma L et al. The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat. Med. 10(5), 484–486 (2004).
51 Li S, Takasu T, Perlman DM et al. Translation factor eIF4E rescues cells from
Myc-dependent apoptosis by inhibiting cytochrome c release. J. Biol. Chem. 278(5), 3015–3022 (2003).
52 Li S, Perlman DM, Peterson MS et al. Translation initiation factor 4E blocks endoplasmic reticulum-mediated apoptosis. J. Biol. Chem. 279(20), 21312–21317 (2004).
53 Polunovsky VA, Rosenwald IBB, Tan AT et al. Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc. Mol. Cell. Biol. 16(11), 6573–6581 (1996).
54 Tan A, Bitterman P, Sonenberg N, Peterson M, Polunovsky V. Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1. Oncogene 19(11), 1437–1447 (2000).
55 Li BD, Liu L, Dawson M, De Benedetti A. Overexpression of eukaryotic initiation factor 4E (eIF4E) in breast carcinoma. Cancer 79(12), 2385–2390 (1997).
56 Li BD, Gruner JS, Abreo F et al. Prospective study of eukaryotic initiation factor 4E protein elevation and breast cancer outcome. Ann. Surg. 235(5), 732–738; discussion 738–739 (2002).
57 Coleman LJ, Peter MB, Teall TJ et al. Combined analysis of eIF4E and 4E-binding protein expression predicts breast cancer survival and estimates eIF4E activity. Br. J. Cancer 100(9), 1393–1399 (2009).
58 Berkel HJ, Turbat-Herrera EA, Shi R, De Benedetti A. Expression of the translation initiation factor eIF4E in the polyp-cancer sequence in the colon. Cancer Epidemiol. Biomarkers Prev. 10(6), 663–666 (2001).
59 Nathan CO, Amirghahri N, Rice C, Abreo FW, Shi R, Stucker FJ. Molecular analysis of surgical margins in head and neck squamous cell carcinoma patients. Laryngoscope 112(12), 2129–2140 (2002).
60 Salehi Z, Mashayekhi F. Expression of the eukaryotic translation initiation factor 4E (eIF4E) and 4E-BP1 in esophageal cancer. Clin. Biochem. 39(4), 404–409 (2006).
61 Khoury T, Alrawi S, Ramnath N et al. Eukaryotic initiation factor-4E and cyclin D1 expression associated with patient survival in lung cancer. Clin. Lung Cancer 10(1), 58–66 (2009).
62 Seki N, Takasu T, Sawada S et al. Prognostic significance of expression of eukaryotic initiation factor 4E and 4E binding protein 1 in patients with pathological stage I invasive lung adenocarcinoma. Lung Cancer 70(3), 329–334 (2010).
63 Graff JR, Konicek BW, Lynch RL et al. eIF4E activation is commonly elevated in advanced human prostate cancers and significantly related to reduced patient survival. Cancer Res. 69(9), 3866–3873 (2009).
64 Martin ME, Perez MI, Redondo C, Alvarez MI Salinas M, Fando JL. 4E binding protein 1 expression is inversely correlated to the progression of gastrointestinal cancers. Int. J. Biochem. Cell Biol. 32(6), 633–642 (2000).
65 O’Reilly KE, Warycha M, Davies MA et al. Phosphorylated 4E-BP1 is associated with poor survival in melanoma. Clin. Cancer Res. 15(8), 2872–2878 (2009).
66 Armengol G, Rojo F, Castellvi J et al. 4E-binding protein 1: a key molecular ‘funnel factor’ in human cancer with clinical implications. Cancer Res. 67(16), 7551–7555 (2007).
67 Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, Holland EC. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol. Cell 12(4), 889–901 (2003).
68 Bergmann JE, Lodish HF. A kinetic model of protein synthesis. Application to hemoglobin synthesis and translational control. J. Biol. Chem. 254(23), 11927–11937 (1979).
69 Svitkin YV, Pause A, Haghighat A et al. The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5’ secondary structure. RNA 7(3), 382–394 (2001).
70 Babendure JR, Babendure JL, Ding JH, Tsien RY. Control of mammalian translation by mRNA structure near caps. RNA 12(5), 851–861 (2006).
71 Tang H, Hornstein E, Stolovich M et al. Amino acid-induced translation of TOP mRNAs is fully dependent on phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and is independent of S6K1 and rpS6 phosphorylation. Mol. Cell. Biol. 21(24), 8671–8683 (2001).
72 Goossen B, Caughman SW, Harford JB, Klausner RD, Hentze MW. Translational repression by a complex between the iron-responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position-dependent in vivo. EMBO J. 9(12), 4127–4133 (1990).
73 Clemens MJ, Bommer UA. Translational control: the cancer connection. Int. J. Biochem. Cell Biol. 31(1), 1–23 (1999).
74 Watkins SJ, Norbury CJ. Translation initiation and its deregulation during
Eukaryotic initiation factor 4F: a vulnerability of tumor cells | Review
www.future-science.com 29future science group
tumorigenesis. Br. J. Cancer 86(7), 1023–1027 (2002).
75 Hershey JWB, Miyamoto S. Translational Control and Cancer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA (2000).
76 Yang T. The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2. J. Cell Biol. 128(6), 1173–1184 (1995).
77 Hue I, Dedieu T, Huneau D, Ruffini S, Gall L, Crozet N. Cyclin B1 expression in meiotically competent and incompetent goat oocytes. Mol. Reprod. Dev. 47(2), 222–228 (1997).
79 Iwami K, Wang JY, Jain R, Mccormack S, Johnson LR. Intestinal ornithine decarboxylase: half-life and regulation by putrescine. Am. J. Physiol. 258(2 Pt 1), G308–G315 (1990).
80 Rousseau D, Gingras AC, Pause A, Sonenberg N. The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth. Oncogene 13(11), 2415–2420 (1996).
81 Jiang H, Coleman J, Miskimins R, Miskimins WK. Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells. Cancer Cell Int. 3(1), 2 (2003).
82 Lynch M, Fitzgerald C, Johnston KA, Wang S, Schmidt EV. Activated eIF4E-binding protein slows G1 progression and blocks transformation by c-myc without inhibiting cell growth. J. Biol. Chem. 279(5), 3327–3339 (2004).
83 DeFatta RJ Nathan CO, De Benedetti A. Antisense RNA to eIF4E suppresses oncogenic properties of a head and neck squamous cell carcinoma cell line. Laryngoscope 110(6), 928–933 (2000).
84 Rinker-Schaeffer CW, Graff JR, De Benedetti A, Zimmer SG, Rhoads RE. Decreasing the level of translation initiation factor 4E with antisense RNA causes reversal of ras-mediated transformation and tumorigenesis of cloned rat embryo fibroblasts. Int. J. Cancer 55(5), 841–847 (1993).
85 Herbert TP, Fahraeus R, Prescott A, Lane DP, Proud CG. Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E. Curr. Biol. 10(13), 793–796 (2000).
86 Brown CJ Lim JJ Leonard T et al. Stabilizing the eIF4G1 alpha-helix increases its binding
affinity with eIF4E: implications for peptidomimetic design strategies. J. Mol. Biol. 405(3), 736–753 (2011).
87 Ko SY, Guo H, Barengo N, Naora H. Inhibition of ovarian cancer growth by a tumor-targeting peptide that binds eukaryotic translation initiation factor 4E. Clin. Cancer Res. 15(13), 4336–4347 (2009).
88 Eberle J, Fecker LF, Bittner JU, Orfanos CE, Geilen CC. Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1. Br. J. Cancer 86(12), 1957–1962 (2002).
89 Yang HS, Knies JL, Stark C, Colburn NH. Pdcd4 suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation. Oncogene 22(24), 3712–3720 (2003).
90 Jansen AP, Camalier CE, Colburn NH. Epidermal expression of the translation inhibitor programmed cell death 4 suppresses tumorigenesis. Cancer Res. 65(14), 6034–6041 (2005).
91 Buxade M, Parra-Palau JL, Proud CG. The Mnks: MAP kinase-interacting kinases (MAP kinase signal-integrating kinases). Front Biosci. 13, 5359–5373 (2008).
92 Ueda T, Watanabe-Fukunaga R, Fukuyama H, Nagata S, Fukunaga R. Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development. Mol. Cell. Biol. 24(15), 6539–6549 (2004).
93 Scheper GC, Van Kollenburg B, Hu J, Luo Y, Goss DJ, Proud CG. Phosphorylation of eukaryotic initiation factor 4E markedly reduces its affinity for capped mRNA. J. Biol. Chem. 277(5), 3303–3309 (2002).
94 Konicek BW, Stephens Jr, Mcnulty AM et al. Therapeutic inhibition of MAP kinase interacting kinase blocks eukaryotic initiation factor 4E phosphorylation and suppresses outgrowth of experimental lung metastases. Cancer Res. 71(5), 1849–1857 (2011).
95 Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK. Cocrystal structure of the messenger RNA 5’ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89(6), 951–961 (1997).
96 Jemielity J, Kowalska J, Rydzik AM, Darzynkiewicz E. Synthetic mRNA cap analogs with a modified triphosphate bridge – synthesis, applications and prospects. N. J. Chem. (34), 829–844 (2010).
�� Description of recent developments in the synthesis and application of chemically modified cap analogues.
97 Cai A, Jankowska-Anyszka M, Centers A et al. Quantitative assessment of mRNA cap
analogues as inhibitors of in vitro translation. Biochemistry 38(26), 8538–8547 (1999).
98 Wagner CR, Iyer VV, Mcintee EJ. Pronucleotides: toward the in vivo delivery of antiviral and anticancer nucleotides. Med. Res. Rev. 20(6), 417–451 (2000).
99 Ghosh B, Benyumov AO, Ghosh P et al. Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation. ACS Chem. Biol. 4(5), 367–377 (2009).
�� Report on the synthesis and characterization of 4Ei-1, the first cell-permeable m7GpppN cap analogue that was relatively nontoxic and characterized ex vivo.
100 Lejbkowicz F, Goyer C, Darveau A, Neron S, Lemieux R, Sonenberg N. A fraction of the mRNA 5’ cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus. Proc. Natl Acad. Sci. USA 89(20), 9612–9616 (1992).
101 Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap. Proc. Natl Acad. Sci. USA 101(52), 18105–18110 (2004).
102 Assouline S, Culjkovic B, Cocolakis E et al. Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin. Blood 114(2), 257–260 (2009).
103 Yan Y, Svitkin Y, Lee JM, Bisaillon M, Pelletier J. Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap. RNA 11(8), 1238–1244 (2005).
104 Westman B, Beeren L, Grudzien E et al. The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro. RNA 11(10), 1505–1513 (2005).
105 Tam Rc, Lau JY, Hong Z. Mechanisms of action of ribavirin in antiviral therapies. Antivir. Chem. Chemother. 12(5), 261–272 (2001).
106 Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK. Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3(6), 707–716 (1999).
107 Moerke NJ, Aktas H, Chen H et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128(2), 257–267 (2007).
�� Identification, characterization and anticancer properties of 4EGI-1, the first reported inhibitor of eukaryotic initiation factor (eIF)4E–eIF4G interaction.
Review | Lee & Pelletier
Future Med. Chem. (2012) 4(1)30 future science group
108 Fan S, Li Y, Yue P, Khuri FR, Sun SY. The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments TRAIL-mediated apoptosis through c-FLIP downregulation and DR5 induction independent of inhibition of cap-dependent protein translation. Neoplasia 12(4), 346–356 (2010).
109 Pruvot B, Jacquel A, Droin N et al. Leukemic cell xenograft in zebrafish embryo for investigating drug efficacy. Haematologica 96(4), 612–616 (2011).
110 Cencic R, Hall DR, Robert F et al. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F. Proc. Natl Acad. Sci. USA 108(3), 1046–1051 (2011).
111 Cencic R, Desforges M, Hall DR et al. Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication. J. Virol. 85(13), 6381–6389 (2011).
112 Adams JM, Harris AW, Pinkert CA et al. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318(6046), 533–538 (1985).
113 Graff JR, Konicek BW, Vincent TM et al. Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity. J. Clin. Invest. 117(9), 2638–2648 (2007).
�� Report on the development of eIF4E-specific antisense oligonucleotides, the first eIF4E inhibitor to show anti-tumor effects in preclinical mouse models.
114 Hong DS Kurzrock R, Oh Y et al. A Phase I dose-escalation, pharmacokinetic and pharmacodynamic evaluation of eIF-4E antisense oligonucleotide LY2275796 in patients with advanced cancer. Clin. Cancer Res. (2011).
115 Novac O, Guenier AS, Pelletier J. Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res. 32(3), 902–915 (2004).
116 Bordeleau ME, Matthews J, Wojnar JM et al. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. Proc. Natl Acad. Sci. USA 102(30), 10460–10465 (2005).
�� Report on the identification and characterization of Pateamine A, the small-molecule inhibitor of eIF4A.
117 Bordeleau M-E, Mori A, Oberer M et al. Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. Nat. Chem. Biol. 2, 213–220 (2006).
�� Report on the identification and characterization of the eIF4A inhibitor hippuristanol.
118 Bordeleau M-E, Robert F, Gerard B et al. Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model. J. Clin. Invest. 118(7), 2651–2660 (2008).
�� Identification and characterization of silvestrol, the first eIF4A inhibitor to show anti-tumor activity in a preclinical mouse model.
119 Lindqvist L, Oberer M, Reibarkh M et al. Selective pharmacological targeting of a DEAD box RNA helicase. PLoS ONE 3(2), e1583 (2008).
120 Tsumuraya T, Ishikawa C, Machijima Y et al. Effects of hippuristanol, an inhibitor of eIF4A, on adult T-cell leukemia. Biochem. Pharmacol. 81(6), 713–722 (2011).
121 Li W, Dang Y, Liu Jo, Yu B. Expeditious synthesis of hippuristanol and congeners with potent antiproliferative activities. Chemistry 15(40), 10356–10359 (2009).
122 Li W, Dang Y, Liu Jo, Yu B. Structural and stereochemical requirements of the spiroketal group of hippuristanol for antiproliferative activity. Bioorg. Med. Chem. Lett. 20(10), 3112–3115 (2010).
123 Ravindar K, Reddy MS, Lindqvist L, Pelletier J, Deslongchamps P. Efficient synthetic approach to potent antiproliferative agent hippuristanol via Hg(II)-catalyzed spiroketalization. Org. Lett. 12(19), 4420–4423 (2010).
124 Ravindar K, Reddy MS, Lindqvist L, Pelletier J, Deslongchamps P. Synthesis of the antiproliferative agent hippuristanol and its analogues via Suarez cyclizations and Hg(II)-catalyzed spiroketalizations. J. Org. Chem. 76(5), 1269–1284 (2011).
125 Bordeleau ME, Cencic R, Lindqvist L et al. RNA-mediated sequestration of the RNA helicase eIF4A by pateamine A inhibits translation initiation. Chem. Biol. 13(12), 1287–1295 (2006).
126 Low WK, Dang Y, Schneider-Poetsch T et al. Inhibition of eukaryotic translation initiation by the marine natural product pateamine A. Mol. Cell 20(5), 709–722 (2005).
127 Low WK, Dang Y, Bhat S, Romo D, Liu Jo. Substrate-dependent targeting of eukaryotic translation initiation factor 4A by pateamine A: negation of domain-linker regulation of activity. Chem. Biol. 14(6), 715–727 (2007).
128 Mazroui R, Sukarieh R, Bordeleau M-E et al. Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation. Mol. Biol. Cell 17(10), 4212–4219 (2006).
129 Dang Y, Low WK, Xu J et al. Inhibition of nonsense-mediated mRNA decay by the natural product pateamine A through eukaryotic initiation factor 4AIII. J. Biol. Chem. 284(35), 23613–23621 (2009).
130 Northcote PT, Blunt JW, Munro MHG. Pateamine: a potent cytotoxin from the New Zealand marine sponge, Mycale sp. Tetrahedron Lett. 32, 6411–6414 (1991).
131 Hood KA, West LM, Northcote PT, Berridge MV, Miller JH. Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine. Apoptosis 6(3), 207–219 (2001).
132 Kuznetsov G, Xu Q, Rudolph-Owen L et al. Potent in vitro and in vivo anticancer activities of des-methyl, des-amino pateamine A, a synthetic analogue of marine natural product pateamine A. Mol. Cancer Ther. 8(5), 1250–1260 (2009).
133 Güssregen R, Fuhr M, Nugroho BW, Wray V, Witte L, Proksch P. New Insecticidal rocaglamide derivatives from flowers of Aglaia odorata. Z. Naturforsch. 52C, 334–339 (1997).
134 Schneider C, Bohnenstengel FI, Nugroho BW et al. Insecticidal rocaglamide derivatives from Aglaia spectabilis (Meliaceae). Phytochem. 54, 731–736 (2000).
135 Greger H, Pacher T, Brem B, Bacher M, Hofer O. Insecticidal flavaglines and other compounds from Fijian Aglaia species. Phytochem. 57, 57–64 (2001).
136 Bohnenstengel FI, Steube KG, Meyer C et al. Structure activity relationships of antiproliferative rocaglamide derivatives from Aglaia Species (meliaceae). Z. Naturforsch. 54, 55–60 (1999).
137 Wu T-S, Liou M-J, Kuoh C-S, Teng C-M, Nagao T, Lee K-H. Cytotoxic and antiplatelet aggregation principles from Aglaia elliptifolia. J. Nat. Prod. 60, 606–608 (1997).
138 Lee Sk, Cui B, Mehta RR, Kinghorn AD, Pezzuto JM. Cytostatic mechanism and antitumor potential of novel 1H-cyclopenta[b]benzofuran lignans isolated from Aglaia elliptica. Chemicobiol. Interact. 115, 215–228 (1998).
139 Hwang BY, Su BN, Chai H et al. Silvestrol and episilvestrol, potential anticancer rocaglate derivatives from Aglaia silvestris. J. Org. Chem. 69(10), 3350–3358 (2004).
140 Kim S, Hwang BY, Su BN et al. Silvestrol, a potential anticancer rocaglate derivative from Aglaia foveolata, induces apoptosis in LNCaP cells through the mitochondrial/apoptosome pathway without activation of executioner caspase-3 or -7. Anticancer Res. 27, 2175–2183 (2007).
Eukaryotic initiation factor 4F: a vulnerability of tumor cells | Review
www.future-science.com 31future science group
141 Ohse T, Ohba S, Yamamoto T, Koyano T, Umezawa K. Cyclopentabenzofuran lignan protein synthesis inhibitors from Aglaia odorata. J. Nat. Prod. 59, 650–652 (1996).
142 Baumann B, Bohnenstengel F, Siegmund D et al. Rocaglamide derivatives are potent inhibitors of NF-kB activation in T-cells. J. Biol. Chem. 277, 44791–44800 (2002).
143 Salim AA, Chai HB, Rachman I et al. Constituents of the leaves and stem bark of Aglaia foveolata. Tetrahedron 63(33), 7926–7934 (2007).
144 Mi Q, Su BN, Chai H et al. Rocaglaol induces apoptosis and cell cycle arrest in LNCaP cells. Anticancer Res. 26(2A), 947–952 (2006).
145 Saradhi UV, Gupta SV, Chiu M et al. Characterization of silvestrol pharmacokinetics in mice using liquid chromatography–tandem mass spectrometry. AAPS J. 13(3), 347–356 (2011).
146 Cencic R, Carrier M, Galicia-Vazquez G et al. Antitumor activity and mechanism of action of the cyclopenta[b]benzofuran, silvestrol. PLoS ONE 4(4), e5223 (2009).
147 Lucas DM, Edwards RB, Lozanski G et al. The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo. Blood 113(19), 4656–4666 (2009).
148 Wendel HG, Malina A, Zhao Z et al. Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo. Cancer Res. 66(15), 7639–7646 (2006).
149 Cencic R, Carrier M, Trnkus A, Porco JA Jr, Minden M, Pelletier J. Synergistic effect of inhibiting translation initiation in combination with cytotoxic agents in acute myelogenous leukemia cells. Leuk. Res. 34(4), 535–541 (2009).
150 Bleumink M, Kohler R, Giaisi M, Proksch P, Krammer PH, Li-Weber M. Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression. Cell Death Differ. 18(2), 362–370 (2011).
151 Gerard B, Cencic R, Pelletier J, Porco JA Jr. Enantioselective synthesis of the complex rocaglate (-)-silvestrol. Angew. Chem. Int. Ed. Engl. 46(41), 7831–7834 (2007).
152 Adams TE, El Sous M, Hawkins BC et al. Total synthesis of (-) episilvestrol and (-) silvestrol. Angew. Chem. Int. Ed. Engl. 46(41), 7835–7838 (2007).
153 Roche SP, Cencic R, Pelletier J, Porco JA Jr. Biomimetic photocycloaddition of 3-hydroxyflavones: synthesis and evaluation of rocaglate derivatives as inhibitors of eukaryotic translation. Angew. Chem. Int. Ed. Engl. 49(37), 6533–6538 (2010).
154 Gupta SV, Sass EJ, Davis ME et al. Resistance to the translation initiation inhibitor silvestrol is mediated by ABCB1/P-glycoprotein overexpression in acute lymphoblastic leukemia cells. AAPS J. 13(3), 357–364 (2011).
155 Pestova TV, Shatsky IN, Hellen CU. Functional dissection of eukaryotic initiation
factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol. Cell. Biol. 16(12), 6870–6878 (1996).
156 Pestova TV, Shatsky IN, Hellen CU. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol. Cell. Biol. 16(12), 6870–6878 (1996).
157 Takacs JE, Neary TB, Ingolia NT et al. Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery. RNA 17(3), 439–452 (2011).
158 Pestova TV, Kolupaeva VG. The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection. Genes Dev. 16(22), 2906–2922 (2002).
159 De Gregorio E, Preiss T, Hentze MW. Translational activation of uncapped mRNAs by the central part of human eIF4G is 5’ end-dependent. RNA 4(7), 828–836 (1998).
160 De Gregorio E, Preiss T, Hentze MW. Translation driven by an eIF4G core domain in vivo. EMBO J. 18(17), 4865–4874 (1999).
