eIF4E Availability Research Articles

Cap-independent translation and a precisely located RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response.

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5′UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5′UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5′UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

An eIF3d-dependent switch regulates HCMV replication by remodeling the infected cell translation landscape to mimic chronic ER stress.

SUMMARYRegulated loading of eIF3-bound 40S ribosomes on capped mRNA is generally dependent upon the translation initiation factor eIF4E; however, mRNA translation often proceeds during physiological stress, such as virus infection, when eIF4E availability and activity are limiting. It remains poorly understood how translation of virus and host mRNAs are regulated during infection stress. While initially sensitive to mTOR inhibition, which limits eIF4E-dependent translation, we show that protein synthesis in human cytomegalovirus (HCMV)-infected cells unexpectedly becomes progressively reliant upon eIF3d. Targeting eIF3d selectively inhibits HCMV replication, reduces polyribosome abundance, and interferes with expression of essential virus genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. This reveals a strategy whereby cellular eIF3d-dependent protein production is hijacked to exploit virus-induced ER stress. Moreover, it establishes how switching between eIF4E and eIF3d-responsive cap-dependent translation can differentially tune virus and host gene expression in infected cells.

Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution.

Chronic non-healing wounds fail to progress beyond the inflammatory phase, characterized by a disorder of inflammation resolution. PD-1/PD-L1, a major co-inhibitory checkpoint signaling, plays critical roles in tumor immune surveillance and the occurrence of inflammatory or autoimmune diseases, but its roles in wound healing remains unclear. Here, we described a novel function of PD-L1 in fibroblast-like cells as a positive regulator of wound healing. PD-L1 dynamically expressed on the fibroblast-like cells in the granulation tissue during wound healing to form a wound immunosuppressive microenvironment, modulate macrophages polarization from M1-type to M2-type, and initiates resolution of inflammation, finally accelerate wound healing. Loss of PD-L1 delayed wound healing, especially in mice with LPS-induced severe inflammation. Furthermore, the mainly regulatory mechanism is that combination of FGF-2 and TGF-β1 promotes PD-L1 translation in fibroblasts through enhancing the eIF4E availability regulated by both PI3K-AKT-mTOR-4EBP1 and p38-ERK-MNK signaling pathways. Our results reveal the positive role of PD-L1 in wound healing, and provide a new strategy for the treatment of chronic wounds.

Deep computational analysis details dysregulation of eukaryotic translation initiation complex eIF4F in human cancers.

eIF4F plays diverse roles in human cancers, which complicate the development of an overarching understanding of its functional and regulatory impacts across tumor types. Typically, eIF4F drives initiation from the mRNA 5' end (cap) and is composed of eIF4G1, eIF4A1, and cap-binding eIF4E. Cap-independent initiation is possible without eIF4E, from internal ribosomal entry sites (IRESs). By analyzing large public datasets, we found that cancers selectively overexpress EIF4G1 more than EIF4E. That expression imbalance supports EIF4G1 as a prognostic indicator in patients with cancer. It also attenuates "housekeeping" pathways that are usually regulated in a tissue-specific manner via cap-dependent initiation in healthy tissues and reinforce regulation of cancer-preferred pathways in cap-independent contexts. Cap-independent initiation is mechanistically attributable to eIF4G1 hyperphosphorylation that promotes binding to eIF4A1 and reduced eIF4E availability. Collectively, these findings reveal a novel model of dysregulated eIF4F function and highlight the clinical relevance of cap-(in)dependent initiation in cancer.

Upregulation of eIF4E, but not other translation initiation factors, in dendritic spines during memory formation.

Local translation can provide a rapid, spatially targeted supply of new proteins in distal dendrites to support synaptic changes that underlie learning. Learning and memory are especially sensitive to manipulations of translational control mechanisms, particularly those that target the initiation step, and translation initiation at synapses could be a means of maintaining synapse specificity during plasticity. Initiation predominantly occurs via recruitment of ribosomes to the 5' mRNA cap by complexes of eukaryotic initiation factors (eIFs), and the interaction between eIF4E and eIF4G1 is a particularly important target of translational control pathways. Pharmacological inhibition of eIF4E-eIF4G1 binding impairs formation of memory for aversive Pavlovian conditioning as well as the accompanying increase in polyribosomes in the heads of dendritic spines in the lateral amygdala (LA). This is consistent with a role for initiation at synapses in memory formation, but whether eIFs are even present near synapses is unknown. To determine whether dendritic spines contain eIFs and whether eIF distribution is affected by learning, we combined immunolabeling with serial section transmission electron microscopy (ssTEM) volume reconstructions of LA dendrites after Pavlovian conditioning. Labeling for eIF4E, eIF4G1, and eIF2α-another key target of regulation-occurred in roughly half of dendritic spines, but learning effects were only found for eIF4E, which was upregulated in the heads of dendritic spines. Our results support the possibility of regulated translation initiation as a means of synapse-specific protein targeting during learning and are consistent with the model of eIF4E availability as a central point of control.

Translational downregulation of Twist1 expression by antiproliferative gene, B-cell translocation gene 2, in the triple negative breast cancer cells

Twist1, a key transcription factor regulating epithelial–mesenchymal transition and cancer metastasis, is highly expressed in invasive cancers in contrast to the loss of BTG2/TIS21 expression. Based on our observation that forced expression of BTG2/TIS21 downregulated Twist1 protein expression without altering mRNA level, we investigated molecular mechanisms of the BTG2/TIS21-inhibited Twist1 translation in the triple negative breast cancer (TNBC) cells and in vivo BTG2/TIS21-knockout (KO) mice and human breast cancer tissues. (1) C-terminal domain of Twist1 and Box B of BTG2/TIS21 interacted with each other, which abrogated Twist1 activity. (2) BTG2/TIS21 inhibited translational initiation by depleting eIF4E availability via inhibiting 4EBP1 phosphorylation. (3) Expression of BTG2/TIS21 maintained p-eIF2α that downregulates initiation of protein translation, confirmed by eIF2α-AA mutant expression and BTG2/TIS21 knockdown in MEF cells. (4) cDNA microarray analysis revealed significantly higher expression of initiation factors-eIF2A, eIF3A, and eIF4G2-in the BTG2/TIS21-KO mouse than that in the wild type. (5) BTG2/TIS21-inhibited translation initiation lead to the collapse of polysome formation and the huge peak of 80s monomer in the BTG2/TIS21 expresser, but not in the control. (6) mRNAs and protein expressions of elongation factors were also downregulated by BTG2/TIS21 expression in TNBC cells, but much higher in both TIS21-KO mice and lymph node-positive human breast cancers. (7) BTG2/TIS21-mediated Twist1 loss was not due to the protein degradation by ubiquitination and autophagy activation. (8) Twist1 protein level was significantly higher in various organs of TIS21-KO mice compared with that in the control, indicating the in vivo role of BTG2/TIS21 gene in the regulation of Twist1 protein level. Altogether, the present study support our hypothesis that BTG2/TIS21 is a promising target to combat with metastatic cancers with high level of Twist1 without BTG2/TIS21 expression.

Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans

SummaryAging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma.

Translational control in the tumor microenvironment promotes lung metastasis: Phosphorylation of eIF4E in neutrophils

The translation of mRNAs into proteins serves as a critical regulatory event in gene expression. In the context of cancer, deregulated translation is a hallmark of transformation, promoting the proliferation, survival, and metastatic capabilities of cancer cells. The best-studied factor involved in the translational control of cancer is the eukaryotic translation initiation factor 4E (eIF4E). We and others have shown that eIF4E availability and phosphorylation promote metastasis in mouse models of breast cancer by selectively augmenting the translation of mRNAs involved in invasion and metastasis. However, the impact of translational control in cell types within the tumor microenvironment (TME) is unknown. Here, we demonstrate that regulatory events affecting translation in cells of the TME impact cancer progression. Mice bearing a mutation in the phosphorylation site of eIF4E (S209A) in cells comprising the TME are resistant to the formation of lung metastases in a syngeneic mammary tumor model. This is associated with reduced survival of prometastatic neutrophils due to decreased expression of the antiapoptotic proteins BCL2 and MCL1. Furthermore, we demonstrate that pharmacological inhibition of eIF4E phosphorylation prevents metastatic progression in vivo, supporting the development of phosphorylation inhibitors for clinical use.

Hyperactive mTOR and MNK1 phosphorylation of eIF4E confer tamoxifen resistance and estrogen independence through selective mRNA translation reprogramming.

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming.

Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation

Picornaviruses use internal ribosome entry sites (IRESs) to translate their genomes into protein. A typical feature of these IRESs is their ability to bind directly to the eukaryotic initiation factor (eIF) 4G component of the eIF4F cap-binding complex. Remarkably, the hepatitis A virus (HAV) IRES requires eIF4E for its translation, but no mechanism has been proposed to explain this. Here we demonstrate that eIF4E regulates HAV IRES-mediated translation by two distinct mechanisms. First, eIF4E binding to eIF4G generates a high-affinity binding conformation of the eIF4F complex for the IRES. Second, eIF4E binding to eIF4G strongly stimulates the rate of duplex unwinding by eIF4A on the IRES. Our data also reveal that eIF4E promotes eIF4F binding and increases the rate of restructuring of the poliovirus (PV) IRES. This provides a mechanism to explain why PV IRES-mediated translation is stimulated by eIF4E availability in nuclease-treated cell-free extracts. Using a PV replicon and purified virion RNA, we also show that eIF4E promotes the rate of eIF4G cleavage by the 2A protease. Finally, we show that cleavage of eIF4G by the poliovirus 2A protease generates a high-affinity IRES binding truncation of eIF4G that stimulates eIF4A duplex unwinding independently of eIF4E. Therefore, our data reveal how picornavirus IRESs use eIF4E-dependent and -independent mechanisms to promote their translation.

Abstract B33: Translational control pathways form a molecular hub that regulates tamoxifen resistance in ER+ breast cancer

Abstract Currently, ER+ breast cancer is treated with adjuvant antiestrogen therapies (i.e. tamoxifen and raloxifene). Despite maintaining expression of the estrogen receptor, many women treated with adjuvant therapy develop resistance several years after curative care. In fact, resistance to tamoxifen therapy is responsible for a vast majority of deaths resulting from metastatic disease. Our lab and others have shown that upregulation of the abundance and/or activity of translation initiation factors eIF4E and eIF4G1 occurs widely in advanced breast cancer, selectively upregulating translation of certain mRNAs required for survival, proliferation, and metastasis. Therefore, we hypothesized that tamoxifen resistance in ER+ breast cancer involves selective mRNA translation mediated by hyperactivation of the mTORC1 translational control pathway via regulating the availability and activity of eIF4E. Tamoxifen resistant (R) and sensitive (S) ER+ cell lines have been developed by several labs by long-term cultivation in tamoxifen and are used in my work. We define here, using the aforementioned cell lines, a molecular hub consisting of the two translational control pathways, PI3K/Akt/mTOR and MAPK/ERK/MNK which converge on translation initiation factor 4E (eIF4E) increasing its availability and activity. We show that blocking eIF4E-mediated translation was able to resensitize drug resistant cells to tamoxifen; conversely, overexpression of eIF4E, without concurrent mTORC1 hyperactivity, was not sufficient to generate resistance. Furthermore, knockdown of two genes identified from our genome-wide translation state analysis, TEX14 and RUNX2, significantly impacted drug resistance and serve as a major regulators in disease progression. Our findings demonstrate that eIF4E is a key player regulating tamoxifen resistance in ER+ disease. Citation Format: Phillip A. Geter, Robert J. Schneider. Translational control pathways form a molecular hub that regulates tamoxifen resistance in ER+ breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B33.

4E-BP1 as an oncotarget

The cap-dependent mRNA translation process that enables post-transcriptional control of gene expression and protein synthesis is tightly regulated in eukaryotes. Deregulation of this process, particularly in the formation of the eIF4F translation initiation complex—comprising the eIF4E mRNA cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase—is associated with cancer development and progression. It is widely believed that translation of certain key oncogenic mRNAs is strongly dependent on the level of eIF4E. Consequently, expression of these oncogenic mRNAs is preferentially and dis-proportionately affected by eIF4E availability and is sensitive to alteration of its level. Free eIF4E level can be increased substantially in cancer cells by a number of mechanisms, including elevated eIF4E expression, decreased expression of eIF4E inhibitory binding proteins, 4E-BPs, and release of eIF4E from 4E-BPs by inactivating phosphorylation of 4E-BPs results from the oncogenic activation of PI3K/AKT and RAS/RAF/MEK/ERK signaling pathways. Our recent studies have revealed that 4E-BP1 is a critical regulatory node that integrates oncogenic signals of the P3K/AKT and RAS/RAF/MEK/ERK pathways for cancer progression and metastasis [1-3]. Mutations in genes that encode components of these two pathways occur at high frequency in cancer. Moreover, the AKT and ERK pathways are often concurrently activated by separate mutations in many human tumors. For instance, KRAS and PIK3CA mutation occur simultaneously in colorectal carcinoma; BRAF and PIK3CA mutation in thyroid carcinoma; and BRAF and PTEN mutation occur simultaneously in melanoma. A number of small molecule inhibitors targeting components of these two pathways have been tested for the treatment of cancer in preclinical and clinical studies, but have shown only limited activity as a single agent. Our work demonstrated that in tumors such as colorectal cancer (CRC) with coexistent activation of both PI3K/AKT and RAS/ERK pathways, drug resistance, following inhibition of either pathway, is associated with redundant activation of eIF4E-initiated cap-dependent translation through convergent phosphorylation of the translational repressor 4E-BP1 mediated by the AKT and ERK pathways. We found that combined inhibition of both pathways is required to maximally inhibit 4E-BP1 phosphorylation and subsequent induction of 4E-BP1 binding to eIF4E to prevent cap-dependent translation of oncogenic mRNAs (e.g. survivin) and ultimately achieve antitumor activity. Furthermore, we showed that a genetically-modified non-phosphorylated 4E-BP1 mutant that binds constitutively to eIF4E and represses eIF4E-dependent oncogene expression exerts similar inhibitory effects on tumor growth and metastasis as the combined pharmacological inhibition of AKT and ERK pathways, whereas loss of 4E-BP1 expression induces epithelial-mesenchymal transition through translational control of snail expression and its activity to promote metastatic progression of CRC and reduce CRC dependence on AKT/ERK signaling for translation, survival and motility. Collectively, these findings support the conclusion that deregulation of 4E-BP1 phosphorylation-mediated assembly of eIF4F translation initiation complex causes CRC resistance to its upstream kinase targeted therapies and thus maintain the transformed phenotype for CRC progression and metastasis. Of notice, several lines of evidence have also established incomplete inhibition of 4E-BP1 phosphorylation, amplification of eIF4E or increased eIF4E/4E-BP ratio as a mechanism of primary resistance to anti-PI3K/AKT/mTOR and anti-BRAF/MEK/ERK pathway inhibitors in a variety of cancers [4-6]. Our data and others strongly suggest that 4E-BP1 phosphorylation is an important biomarker and therapeutic target in cancer. It is well know that mTOR kinase complex 1 (mTORC1) phosphorylates 4E-BP1, which in turn, activates cap-dependent translation and promotes protein synthesis. Our recent studies demonstrated that the effects of AKT and ERK activation on translational regulation of CRC progression are largely mediated by mTORC1 [2], suggesting that targeting mTOR is an attractive strategy for cancer therapy. Nevertheless, incomplete inhibition of 4E-BP1 phosphorylation and a concomitant activation of AKT via loss of a negative feedback mechanism by mTOR inhibitors, including rapalogs and mTOR kinase inhibitors, have been implicated in reduction of their therapeutic efficacy. Our most recent findings revealed a critical role of PRAS40, a mTORC1 inhibitor, as a key integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor progression, and indicated that the redundant phosphorylation of PRAS40 on Ser183 and Thr246 sites by both mTORC1 and AKT signaling is a novel mechanistic basis for the acquired resistance to mTOR inhibitors in cancer cells [7]. Our findings further suggest that directly targeting the convergence of multiple oncogenic signals on eIF4F translation initiation complex may provide a promising strategy for improving cancer therapy. Several translation initiation inhibitors, including eIF4E antisense-oligonucleotides and the eIF4A inhibitor silvestrol, have recently produced encouraging anti-tumor effects with limited toxicity. Future works require additional clinical correlative studies and await the development of clinically effective translation initiation inhibitors for cancer treatment, either as single agents or as part of combination therapy.

Abstract B22: Identification of novel drug combinations to target molecular pathways involved in breast cancer

Abstract Introduction: At a global level breast cancer is the second most common cancer, and the most frequent in women. Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors. HMG-CoA reductase is a rate-limiting enzyme in the mevalonate pathway, making it important in the pathology of cancer. The serine/threonine kinase, mammalian target of rapamycin (mTOR) regulates cell survival, proliferation and growth via translational initiation control, and this pathway is deregulated in many types of cancer which are associated with phosphatidylinositol-3-kinase (PI3K)/Akt signaling activation. HMG-CoA reductase expression is dependent on eukaryotic translation initiation factor 4E (eIF4E), and requires high translation initiation efficiency for polysome recruitment. eIF4E availability is reduced by mTOR inhibitors such as rapamycin and metformin, leading to a reduction in HMG-CoA reductase expression. Objectives: This research aims to study the proposed combinatory treatments of rapamycin or metformin with HMG-CoA reductase inhibitors, in order to identify breast cancer subtypes sensitive to the proposed drug combinations. The specific mechanisms of action will also be studied. Methodology: The breast cancer cell lines of Hs587T, MCF-7 and MDA-MB-468 were seeded in 96-well plates. Following 24 hours, triplicate wells of each cell line were treated with 0, 2, 4, 6, 8, 10, 12, and 14mM metformin. MTT assays were carried out 24, 48, and 72 hours post treatment. Successively, similar procedures for each cell line were carried out using 0, 10, 25, 35, 50, 65, 75, 100ng/mL rapamycin. For every cell line the time point and the dose at which a suppression of cell viability (not reaching IC50) occurred for 3 consecutive doses, were defined as the sensitization time point (Ts) and the sensitization dose (Ds) respectively. Ongoing research: The same cell lines are currently being pre-treated with Ds of metformin and rapamycin separately, and at time Ts simvastatin is added at a range of µM concentrations. Following these conditions MTT assays are being carried out, and compared to control experiments using simvastatin alone. Annexin V FITC / propidium iodide flow cytometry based apoptosis assays, as well as Western Blot analysis for the quantification of HDJ2, HMG-CoA reductase, and eukaryotic translation initiation factor 4E binding protein 1 proteins are being planned to be carried out at a later stage. Results: Time Ts and dose Ds for MCF-7 cells treated with rapamycin were found to be 72 hours and 12.5ng/mL respectively. MCF-7 cells were found to exhibit sensitivity to metformin that was too high to be considered for further combinatory treatment with simvastatin, since IC50 was reached post 48 hours of treatment with 10mM. Substantial sensitivity of all breast cancer cell lines studied was also observed for rapamycin or metformin treatment alone. In addition, previous work published by our group shows that the breast cancer cell lines MDA-MB-436 and BT-20 are sensitive to rapamycin. Conclusion: Cell viability assays have shown that response to mTOR modulators is time, cell type and dose dependent. This project will determine optimum conditions for the combinatory treatment described above. The targeting of these two specific mechanisms converging onto a common target is novel, and expected to result in a higher efficacy, as well as decreased unwanted effects in an in vivo situation due to the lower individual doses used. Drug combinations would thus be superior to using either drug alone. Citation Format: Vanessa Petroni, Marie Therese Camilleri Podesta, Anthony George Fenech, Godfrey Grech. Identification of novel drug combinations to target molecular pathways involved in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B22.

Ligustrazine Suppresses the Growth of HRPC Cells through the Inhibition of Cap- Dependent Translation Via Both the mTOR and the MEK/ERK Pathways.

Ligustrazine (TMP) has recently been used for the treatment of various cancers. However, its exact mechanisms of action, particularly the functions and the mechanisms of Ligustrazine in human hormone-refractory prostate cancer (HRPC), have not yet been extensively studied. Recently, our findings suggest that Ligustrazine dose- and time-dependently inhibits the growth of HRPC cells by reducing their proliferation and promoting apoptosis. Interestingly, the treatment of hormone-refractory prostate cancer (PC-3) cells with Ligustrazine results in a significant inhibition of the activation of mTOR and related downstream targets, which are critical for cell growth. Furthermore, pull-down assays with 7-methyl- GTP Sepharose 4B beads indicate that Ligustrazine reduces the available eIF4E for translation initiation. Accordingly, the results from the translation assay using a luciferase reporter system further demonstrate that Ligustrazine indeed inhibits cap-dependent translation. In addition, the transient overexpression of eIF4E or MNK1 prevents the Ligustrazine-induced inhibition of proliferation and confers significant protection against Ligustrazine-induced apoptosis. Therefore, the present study provides evidences that Ligustrazine may be a candidate for therapeutic reagent for the treatment of HRPC and certifies that Ligustrazine modulates the availability of eIF4E mainly through the mTOR and MEK/ERK signaling pathways to inhibit cap-dependent translation. Taken together, our results indicate that the inhibition of cap-dependent translation is likely an essential mechanism in Ligustrazine-induced apoptosis.

MTOR inhibitor efficacy is determined by the eIF4E/4E-BP ratio

The mammalian/mechanistic target of rapamycin (mTOR) is a multifunctional serine/threonine kinase that is hyperactivated in cancer [1]. mTOR forms two distinct complexes, mTORC1 and 2. mTORC1 stimulates translation and perturbs energy metabolism to drive cell proliferation and growth, whereas mTORC2 regulates cytoskeletal organization and cell survival by stimulating AGC kinases (e.g. AKT). Therefore, suppressing mTOR's activity is widely considered as a very attractive anti-cancer therapy. Accordingly, mTOR inhibitors are increasingly used as anti-neoplastic agents in the clinic. Rapamycin is a naturally occurring allosteric inhibitor of mTORC1 and its analogs (rapalogs) are used as anti-cancer agents for the treatment of refractory renal cell carcinomas, mantle cell lymphomas, and pancreatic neuroendocrine tumors. However, success of rapalogs as anti-cancer monotherapies is limited [2]. This has been attributed to the activation of AKT signaling resulting from the loss of a negative-feedback mechanism, as well as rapamycin-resistant mTORC1 outputs, such as the phosphorylation of the eukaryotic initiation factor 4E-binding proteins (4E-BPs). To overcome these deficiencies a new generation of ATP-competitive mTOR inhibitors [also referred to as dual mTORC1/2 inhibitors or active-site mTOR inhibitors (asTORi)] was developed [2]. asTORi suppress AKT signaling by inhibiting mTORC2, and abrogate rapamycin-resistant mTORC1 outputs including the phosphorylation of 4E-BPs. Accordingly, asTORi exhibit stronger anti-proliferative and anti-tumorigenic effects as compared to rapalogs, and are currently in multiple clinical trials aiming to target aberrant mTOR signaling in cancer [2]. However, a major obstacle to applying asTORi to the clinic is a lack of predictive biomarkers that would facilitate the stratification of the patients that are most likely to respond to these drugs. Discovery of predictive biomarkers indicative of the efficacy of mTOR inhibitors is hampered by the complexity of the mTOR pathway, given that mTOR controls a variety of cellular processes via a multitude of substrates. 4E-BPs are a family of small translational repressors, which sequester the 5' mRNA cap-binding protein eukaryotic translation initiation factor (eIF)-4E (eIF4E), thereby impeding the assembly of the eIF4F complex. The eIF4F complex, which consists of eIF4E, scaffolding protein eIF4G and the DEAD-box helicase eIF4A, recruits the mRNA to the ribosome to initiate translation. mTORC1 phosphorylates and inactivates 4E-BPs, thereby facilitating the assembly of the eIF4F complex and translation initiation (See Figure ​Figure1).1). eIF4E acts as a general translation initiation factor, but a subset of mRNAs referred to as “eIF4E-sensitive” are particularly sensitive to changes in eIF4E activity. These mRNAs encode tumor-promoting factors, such as cyclin D3, ornithine decarboxylase and myc. The inability of rapalogs to completely suppress 4E-BP phosphorylation and translation of “eIF4E-sensitive” mRNAs is thought to be the reason for their relatively limited anti-tumorigenic efficacy in the clinic [2, 3]. Figure 1 Sensitivity of tumor to asTORi as a function of eIF4E/4E-BP ratio Overexpression of eIF4E and variations in 4E-BP levels and phosphorylation are frequently observed in tumors, thereby suggesting that eIF4E/4E-BP stoichiometry may significantly differ among patients, or even within a single tumor [4]. We demonstrated that the eIF4E/4E-BP ratio determines the ability of asTORi to suppress neoplastic growth [5]. Resistance of malignant cells with high eIF4E/4E-BP ratio to asTORi can be explained by their deficiency to inhibit eIF4F complex assembly and translation of “eIF4E-sensitive” mRNAs (Figure ​(Figure1).1). We also showed that cancer cells acquire resistance to asTORi by increasing eIF4E availability via downregulation of 4E-BP1 and 2. These data corroborate earlier findings showing that the amplification of the eIF4E gene underlies resistance to the dual PI3K/mTOR inhibitor BEZ235 [6], and that eIF4E translation activity can predict sensitivity to rapalogs [3]. Therefore, eIF4E/4E-BP ratio, rather than individual levels or phosphorylation status of these proteins, is more likely to serve as a prognostic biomarker to select the patients for clinical trials using asTORi and to tailor personalized mTOR targeted therapies. Recently, several mechanisms potentiating resistance to mTOR inhibitors emerged, including the activation of alternative signaling pathways such as the MAPK pathway [7]. Therefore, combined targeting of the mTOR and MAPK pathways has been suggested as a promising approach to overcome resistance to mTOR inhibitors in the clinic. In addition to this strategy, our findings suggest that combining therapeutic approaches that suppress eIF4E expression or activity (e.g. ISIS-EIF4ERx, 4EGI-1, or 4E1RCat [8]) with mTOR targeted therapies should be beneficial in patients bearing tumors with elevated eIF4E/4E-BP ratio.

Manipulation of the host translation initiation complex eIF4F by DNA viruses

In the absence of their own translational machinery, all viruses must gain access to host cell ribosomes to synthesize viral proteins and replicate. Ribosome recruitment and scanning of capped host mRNAs is facilitated by the multisubunit eIF (eukaryotic initiation factor) 4F, which consists of a cap-binding protein, eIF4E and an RNA helicase, eIF4A, assembled on a large scaffolding protein, eIF4G. Although inactivated by many viruses to inhibit host translation, a growing number of DNA viruses are being found to employ diverse strategies to stimulate eIF4F activity in infected cells and maximize viral protein synthesis. These strategies include stimulation of cellular mTOR (mammalian target of rapamycin) signalling to inactivate 4E-BPs (eIF4E-binding proteins), a family of translational repressors that limit eIF4E availability and eIF4F complex formation, together with modulating the activity of the eIF4E kinase Mnk (mitogen-activated protein kinase signal-integrating kinase) in a variety of manners to regulate both host and viral mRNA translation. In some cases, specific viral proteins that mediate these signalling events have been identified, whereas others have been shown to interact with host translation initiation factors or complexes and modify their activity and/or subcellular localization. The present review outlines current understanding of the role of eIF4F in the life cycle of various DNA viruses and discusses its potential as a therapeutic target to suppress viral infection.

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr69-phosphorylated alone, Thr69- and Thr36/Thr45-phosphorylated, all these plus Ser64 phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr36/Thr45 phosphorylation alone was detected without Thr69 phosphorylation, and neither was Ser64 phosphorylation without Thr36/Thr45/Thr69 phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr69, Thr36/Thr45, and Ser64 residues, with 4E-BP1 remaining phosphorylated at Thr69 alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr36/Thr45 and Ser64, in addition to Thr69. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr69 is phosphorylated first followed by Thr36/Thr45 phosphorylation, and Ser64 is phosphorylated last. Thr69 phosphorylation alone allows binding to eIF4E, and subsequent Thr36/Thr45 phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.

Ribosomal Deficiencies Cause Translational Deregulation of Genes Crucial for Erythropoiesis.

Abstract 179Diamond Blackfan anemia (DBA) is a rare congenital pure red cell aplasia in which mutations in distinct ribosomal proteins (RPs) have been identified in 50% of patients. Mutated genes include Ribosomal Protein Small 19 (RPS19), RPS24 and RPS17, components of the 40S ribosomal subunit, and Ribosomal Protein Large 11 (RPL11), RPL5 and RPL35A, components of the 60S subunit. It is not known why mutations in such ubiquitously expressed proteins cause such a strong erythroid phenotype. Previously, we showed that selective translation of transcripts with a complex RNA structure in the 5'untranslated region (5'UTR) is crucial in Stem Cell Factor (SCF) dependent expansion of the early erythroid compartment. Transcripts such as Immunoglobulin binding protein 1 (Igbp1) are actively recruited into translating polyribosomes upon SCF signaling which activates the PI3K-mTOR pathway leading to release of eukaryote initiation factor 4E (eIF4E), a limiting factor for assembly of the scanning complex. The scanning complex also involves the ribosomal subunits. We therefore investigated if polysome recruitment of specific mRNAs is affected in erythroblasts deficient for RPs. Mouse primary erythroblasts derived from p53 deficient and wild-type (wt) fetal livers were cultured in presence of erythropoietin, SCF and dexamethasone under serum free conditions. Downregulation of either Rps19 or Rpl11 by lentivirus-delivered shRNA resulted in severely reduced proliferation and inhibition of differentiation in comparison to nontransduced cells or cells expressing a scrambled control shRNA. Analysis of subpolysomal and polysome-bound RNA by sucrose gradient centrifugation showed a specific reduction of the 40S and 60S ribosomal subunit upon knock down of Rps19 and Rpl11, respectively. Subpolysomal and polysome-bound RNA fractions from 3 independent experiments were used for expression profiling. Surprisingly, the polysome recruitment of transcripts that require SCF and increased eIF4E availability to be translated was not affected by RP deficiency. The ratio of polysome association was calculated per gene for each experiment and datasets from wt, scrambled treated, Rps19 and Rpl11 deficient conditions were compared using F-test with random variance model (p<0.0005). This revealed a distinct set of mRNAs, including Fxc1 (fractured callus expressed-1), Siva1 (apoptosis inducing gene), Csde1 (cold shock domain containing E1) and Cdc25B (cell dividion cycle homolog B) as being down regulated from translating polyribosomes upon RP deficiency. This downregulation was independent from the presence of p53, or glucocorticoids and also occurred in cells deficient for other ribosomal proteins (Rps14, Rpl8). Currently we are analyzing selective translation of these genes in erythroblasts of DBA patients. Erythroblasts could be cultured from peripheral blood of DBA patients although expansion potential of the cultures was decreased compared to control cultures. Protein expression of several identified target genes (CSDE1, CDC25B) was down regulated in erythroblasts derived from DBA patients as compared to controls, whereas their transcript level on total mRNA remained unchanged, indicating a specific translation defect. Further investigation showed that loss of Csde1 perturbed cell cycle progression and erythroid differentiation. In conclusion, we identified transcripts whose translation is selectively affected in RP deficient erythroblasts cultured from mouse fetal livers or DBA patients. We propose that the erythroid phenotype in DBA patients is caused by defective translation of specific set of mRNAs, which are essential for erythroid development. Disclosures:Pospisilova:Grant NS9935-3 from Ministry of Health, Czech Republic: Research Funding.

EIF4E‐Binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs

Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap-binding protein eIF4E, which is released from its repressor 4E-BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E-BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E-BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E-BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E-BP and eIF4G.

Shiga Toxin 1-Induced Proinflammatory Cytokine Production Is Regulated by the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Pathway

Shiga toxin 1 (Stx1) transiently increases the expression of proinflammatory cytokines by macrophage-like THP-1 cells in vitro. Increased cytokine production is partly due to activation of the translation initiation factor eIF4E through a mitogen-activated protein kinase (MAPK)- and Mnk1-dependent pathway. eIF4E availability for translation initiation is regulated by association with eIF4E binding proteins (4E-BP). In this study, we showed that Stx1 transiently induced 4E-BP hyperphosphorylation, which may release eIF4E for translation initiation. Phosphorylation of 4E-BP at priming sites T37 and T46 was not altered by Stx1 but was transiently increased at S65, concomitant with increased cytokine expression. Using kinase inhibitors, we showed that 4E-BP phosphorylation was dependent on phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) activation but did not require MAPKs. Stx1 treatment resulted in increased levels of cytosolic Ca(2+). PI3K and Akt activation led to the phosphorylation and inactivation of the positive cytokine regulator glycogen synthase kinase 3alpha/beta (GSK-3alpha/beta). PI3K, Akt, and mTOR inhibitors and small interfering RNA knockdown of Akt expression all increased, whereas a GSK-3alpha/beta inhibitor decreased, Stx1-induced soluble tumor necrosis factor alpha and interleukin-1beta production. Overall, these findings suggest that despite transient activation of 4E-BP, the PI3K/Akt/mTOR pathway negatively influences cytokine induction by inactivating the positive regulator GSK-3alpha/beta.