Ribavirin targets eIF4E dependent Akt survival signaling

The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m⁷G) cap moiety on the 5′-end of mRNAs. We demonstrate that a physical mimic of the m⁷G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m⁷G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m⁷G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.     

N1-guanyl-1,7-diaminoheptane (GC7) enhances the therapeutic efficacy of doxorubicin by inhibiting activation of eukaryotic translation initiation factor 5A2 (eIF5A2) and preventing the epithelial–mesenchymal transition in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) cells undergo the epithelial–mesenchymal transition (EMT) during chemotherapy, which reduces the efficacy of doxorubicin-based chemotherapy. We investigated N1-guanyl-1,7-diaminoheptane (GC7) which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation; eIF5A2 is associated with chemoresistance. GC7 enhanced doxorubicin cytotoxicity in epithelial HCC cells (Huh7, Hep3B and HepG2) but had little effect in mesenchymal HCC cells (SNU387, SNU449). GC7 suppressed the doxorubicin-induced EMT in epithelial HCC cells; knockdown of eIF5A2 inhibited the doxorubicin-induced EMT and enhanced doxorubicin cytotoxicity. GC7 combination therapy may enhance the therapeutic efficacy of doxorubicin in HCC by inhibiting eIF5A2 activation and preventing the EMT.     

Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches

Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.     

Deciphering the mechanistic effects of eIF4E phosphorylation on mRNA‐cap recognition

The mRNA cap‐binding oncoprotein “eIF4E” is phosphorylated at residue S209 by Mnk kinases, and is closely associated with tumor development and progression. Despite being well‐established, mechanistic details at the molecular level of mRNA recognition by eIF4E due to phosphorylation have not been clearly elucidated. We investigated this through molecular modeling and simulations of the S209 phosphorylated derivative of eIF4E and explored the associated implication on the binding of the different variants of mRNA‐cap analogs. A key feature that emerges as a result of eIF4E phosphorylation is a salt‐bridge network between the phosphorylated S209 (pS209) and a specific pair of lysine residues (K159 and K162) within the cap‐binding interface on eIF4E. This interaction linkage stabilizes the otherwise dynamic C‐terminal region of the protein, resulting in the attenuation of the overall plasticity and accessibility of the binding pocket. The pS209‐K159 salt‐bridge also results in an energetically less favorable environment for the bound mRNA‐cap primarily due to electrostatic repulsion between the negative potentials from the phosphates in the cap and those appearing as a result of phosphorylation of S209. These observations collectively imply that the binding of the mRNA‐cap will be adversely affected in the phosphorylated derivative of eIF4E. We propose a mechanistic model highlighting the role of eIF4E phosphorylation as a regulatory tool in modulating eIF4E: mRNA‐cap recognition and its potential impact on translation initiation.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of Combination Treatment of Rapamycin and Isoflavones on mTOR Pathway in Human Glioblastoma (U87) Cells

Glioblastoma Multiforme (GBM) is a malignant primary brain tumor associated with poor survival rate. PI3K/Akt pathway is highly upregulated in gliomas due to deletion or mutation of PTEN and its activation is associated with tumor grade. mTOR is downstream from PI3K/Akt pathway and it initiates translation through its action on S6K and 4E-BP1. mTOR is an important therapeutic target in many cancers, including glioblastomas. Rapamycin and its analogues are known to inhibit mTOR pathway; however, they also show simultaneous upregulation of Akt and eIF4E survival pathways on inhibition of mTOR, rendering cells more resistant to rapamycin treatment. In this study we investigated the effect of combination treatment of rapamycin with isoflavones such as genistein and biochanin A on mTOR pathway and activation of Akt and eIF4E in human glioblastoma (U87) cells. Our results show that combination treatment of rapamycin with isoflavones, especially biochanin A at 50 μM, decreased the phosphorylation of Akt and eIF4E proteins and rendered U87 cells more sensitive to rapamycin treatment when compared to cells treated with rapamycin alone. These results suggest the importance of combining chemopreventive with chemotherapeutic agents in order to increase the efficacy of chemotherapeutic drugs.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1)

Background information. The translational inhibitor protein 4E‐BP1 [eIF4E (eukaryotic initiation factor 4E)‐binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E‐BP1. Phosphorylation of 4E‐BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E‐BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E‐BP1 to a truncated form. Unlike full‐length 4E‐BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full‐length 4E‐BP1. Inhibitor studies indicate that the p53‐induced cleavage of 4E‐BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full‐length 4E‐BP1. Measurements of the turnover of 4E‐BP1 indicate that the truncated form is much more stable than the full‐length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E‐BP1, which may exert a long‐term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth‐inhibitory and pro‐apoptotic effects of p53.     

Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.     

Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap

Ribavirin is a guanosine ribonucleoside analog that displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections. Ribavirin has recently been proposed to also be a mimic of the 7-methyl guanosine cap found at the 5' end of mRNAs. To obtain supporting functional data for this hypothesis, we assessed the ability of ribavirin triphosphate to interfere with the interaction between eIF4E and 7-methyl guanosine capped mRNA. In chemical cross-linking assays, cap-affinity chromatography, and cap-dependent translation assays, ribavirin was unable to function as a cap analog.     

Protein kinase C-related kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes

Phosphorylation of eIF4E is required for protein synthesis during starfish oocyte maturation. The activity of protein kinase C-related kinase 2 (PRK2) increases prior to the phosphorylation of eIF4E (G. Stapleton et al., 1998, Dev. Biol. 193, 34-46). We investigate here whether eIF4E is activated by PRK2. A 3.5-kb eIF4E clone isolated from starfish cDNA is 57% identical to human eIF4E and contains the putative phosphorylation site serine-209. The serine-209 environment (SKTGS(209)MAKSRF) is similar to the consensus sequence of the phosphorylation site of protein kinase C and related kinases. A starfish eIF4E fusion protein (GST-4E) was phosphorylated in vitro by PRK2 in the presence of 1,2-diolyl-sn-glycerol 3-phosphate. In contrast, replacing the GST-4E serine-209 with an alanine significantly reduced this phosphorylation. Analysis by two-dimensional phosphopeptide mapping reveals a major phosphopeptide in trypsin-digested GST-4E, but not in its serine-209 mutant. Importantly, this major phosphopeptide in GST-4E corresponds to a major phosphopeptide of eIF4E isolated from (32)P-labeled oocytes. Thus, PRK2 may regulate translation initiation during oocyte maturation by phosphorylating the serine-209 residue of eIF4E in starfish. We also demonstrate that high levels of cAMP inhibit the activation of PRK2, eIF4E, and the eIF4E binding protein during starfish oocyte maturation, while PI3 kinase activates these proteins.     

The eIF4E RNA regulon promotes the Akt signaling pathway

Eukaryotic initiation factor 4E (eIF4E) promotes cellular proliferation and can rescue cells from apoptotic stimuli such as serum starvation. However, the mechanisms underlying apoptotic rescue are not well understood. In this study, we demonstrate that eIF4E overexpression leads to enhanced survival signaling through Akt and that eIF4E requires Akt1 to rescue serum-deprived fibroblasts. Furthermore, a mutant form of eIF4E (W73A), which is messenger RNA (mRNA) export competent but does not promote translation, rescues cells as readily as wild-type eIF4E. We show that eIF4E mediates Akt activation via up-regulation of Nijmegen breakage syndrome 1 (NBS1), a phosphoinositide-3 kinase-Akt pathway upstream activator. Additionally, eIF4E coordinately up-regulates the expression of downstream effectors of the Akt pathway, thereby amplifying Akt signaling effects. A negative regulator of eIF4E, the promyelocytic leukemia protein (PML), suppresses Akt activation and apoptotic rescue. These PML activities likely arise, at least in part, through its inhibition of eIF4E-mediated NBS1 mRNA export. In summary, eIF4E coordinately regulates gene expression to potentiate Akt activation, an activity required for apoptotic rescue.     

Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.     

Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation?

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E can interact either with the scaffold protein eIF4G or with repressor proteins termed eIF4E-binding proteins (4E-BPs). High levels of expression can disrupt cellular growth control and are associated with human cancers. A fraction of the cellular eIF4E is found in the nucleus where it may play a role in the transport of certain mRNAs to the cytoplasm. eIF4E undergoes regulated phosphorylation (at Ser209) by members of the Mnk group of kinases, which are activated by multiple MAP kinases (hence Mnk = MAP-kinase signal integrating kinase). The functional significance of its phosphorylation has been the subject of considerable interest. Recent genetic studies in Drosophila point to a key role for phosphorylation of eIF4E in growth and viability. Initial structural data suggested that phosphorylation of Ser209 might allow formation of a salt bridge with a basic residue (Lys159) that would clamp eIF4E onto the mRNA and increase its affinity for ligand. However, more recent structural data place Ser209 too far away from Lys159 to form such an interaction, and biophysical studies indicate that phosphorylation actually decreases the affinity of eIF4E for cap or capped RNA. The implications of these studies are discussed in the light of other, in vitro and in vivo, investigations designed to address the role of eIF4E phosphorylation in mRNA translation or its control.     

The mitogen-activated protein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mammalian cells

The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.     

Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells

Background

mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival.

Methods

Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival.

Results

Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis.

Conclusions

These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.