Ribavirin inhibits the growth and ascites formation of hepatocellular carcinoma through downregulation of type I CARM1 and type II PRMT5.

Type I co-activator-associated arginine methyltransferase 1 (CARM1) and type II protein arginine methyltransferase 5 (PRMT5) are highly expressed in multiple cancers including liver cancer and their overexpression contributes to poor prognosis, thus making them promising therapeutic targets. Here, we evaluated anti-tumor activity of ribavirin in hepatocellular carcinoma (HCC). We found that ribavirin significantly inhibited the proliferation of HCC cells in a time- and dose-dependent manner. Furthermore, ribavirin suppressed the growth of subcutaneous and orthotopic xenograft of HCC in mice, decreased vascular endothelial growth factor (VEGF) and peritoneal permeability to reduce ascites production, and prolonged the survival of mice in HCC ascites tumor models. Mechanistically, ribavirin potently down-regulated global protein expression of CARM1 and PRMT5, and concurrently decreased accumulation of H3R17me2a and H3R8me2s/H4R3me2s. However, ribavirin did not affect the activity and mRNA levels of both CARM1 and PRMT5 in vivo and in vitro HCC cells. In addition, our ChIP results shown that ribavirin inhibited CARM1 which in turn decreased the H3R17me2a, binds to the eukaryotic translation initiation factor 4E (eIF4E) and VEGF promoter region, and reduced the relative mRNA expression level of eIF4E and VEGF in HCC cells. Our findings suggested a potential therapeutic strategy for patients with HCC through inhibition of the abnormal activation/expression of both CARM1 and PRMT5.

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.

High EIF4E2 expression is an independent prognostic risk factor for poor overall survival and recurrence-free survival in uveal melanoma.

Uveal melanoma (UM), as the most common primary intraocular carcinoma, is a relatively rare but lethal tumor. Upregulated eukaryotic translation initiation factor 4E family member 2 (EIF4E2) promotes the progression of multiple human carcinomas. However, its role remains unclear in UM. To identify the prognostic value of EIF4E2 in UM, we downloaded RNA-sequencing data along with clinical information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. EIF4E2 mRNA was significantly increased in three different subgroups in the TCGA-UM dataset. High mRNA expression was correlated with shorter overall survival (OS) and shorter recurrence-free survival (RFS). Moreover, we constructed a prognostic signature using Cox regression analyses in our training cohort TCGA-UM dataset as follows: risk score = 0.04335 × Age +0.49639 × expression of EIF4E2. Based on the risk score, each patient was classified as high-risk or low-risk. Additional survival analyses suggested that patients in the high-risk score group had an unfavorable OS compared with patients in the low-risk score group, which was validated in two external GEO datasets, including GSE84976 and GSE22138. Functional enrichment analysis demonstrated that UM was correlated with hypoxia-related functions. Gene set enrichment analysis (GSEA) indicated significant enrichments of the p53 and Notch pathways. In addition, EIF4E2 was genetically altered in 12.5% (10/80) of UM patients. Epigenetically, higher expression of cg03852847 was correlated with longer OS and longer RFS. In conclusion, our findings demonstrated that high EIF4E2 expression is an independent prognostic risk factor for UM patients. EIF4E2 might play an important role in hypoxia-related signaling pathways during UM progression. Both genetic and epigenetic alterations may contribute to UM pathogenesis. These findings could offer individualized clinical prognostication and potential novel treatment targets for UM patients.

Acute alcohol prevents the refeeding-induced decrease in autophagy but does not alter the increased protein synthetic response in heart.

Ethanol produces a state of anabolic resistance in skeletal muscle; however, whether the heart displays a similar defect is unknown. Hence, the purpose of this study was to determine the impact of acute ethanol administration on the major signal transduction pathways in the heart that are responsible for regulating the protein synthetic and degradative response to refeeding. Adult male C57Bl/6 mice were fasted for 12 h. Mice were then either refed normal rodent chow for 30 min or a separate group of mice remained food deprived prior to administration of 3-g/kg ethanol. Cardiac tissue and blood were collected 1 h thereafter and analyzed. Acute ethanol prevented the nutrient-induced stimulation of S6K1 phosphorylation in heart, but did not alter the phosphorylation of S6, eIF4B, and eEF2, known downstream substrates for this kinase. The refeeding-induced redistribution of eIF4E into the active eIF4F complex was also not changed by acute ethanol. Consistent with the above-mentioned changes in signaling proteins, ethanol did not impair the refeeding-induced increase in cardiac protein synthesis. Proteasome activity was not altered by alcohol and/or refeeding. In contrast, ethanol antagonized the refeeding-induced increase in ULK1 phosphorylation and p62 as well as the reduction in LC3B-II and Atg5/12 complex proteins. These data indicate that acute ethanol prevents the normally observed inhibition of autophagy seen after refeeding, while the mTOR-dependent increase in protein synthesis remains largely unaltered by alcohol.

Micro RNA-1298 opposes the effects of chronic oxidative stress on human trabecular meshwork cells via targeting on EIF4E3.

OBJECTIVE: This study aimed to investigate the effect and potential mechanism of miR-1298 in the progression of human trabecular meshwork (HTM) cells. MATERIAL AND METHODS: Expression of miR-1298 was assessed by quantitative real time PCR (qRT-PCR), as well as in HTM-1 and HTM-2 cells. Mature miR-1298 mimic, miR-1298 inhibitor, and si-EIF4E3 and their corresponding controls were transfected into HTM-1 and HTM-2 to obtain stable HTM cells. Luciferase reporter assay was used to verify regulation between miR-1298 and EIF4E3. Cytotoxicity and Oxidative damage were assessed using commercial kits, and apoptosis was determined using flow cytometry. ECM and apoptosis related factors were determined using qRT-PCR and western blotting, as well as the pathway related factors. RESULTS: The expression of miR-1298 was significantly decreased both in glaucoma and HTM cells. MiR-1298 mimic could significantly inhibit the increase of cytotoxicity, apoptosis, accumulation of carbonylated proteins and ECM induced by COS, but miR-1298 inhibitor could obviously promote the increase effects caused by COS in HTM cells. EIF4E3 was a downstream target of miR-1298. Sliced EIF4E3 could significantly inhibit the increase effects induced miR-1298 inhibitor in HTM cells under COS. The expression levels of TGF-ß2 and Smad4 were significantly increased, and Wnt3a and ß-cantenin were obviously decreased under COS, and miR-1298 inhibitor could markedly promote this increase effect, while sliced EIF4E3 could reverse the effect of miR-1298 under COS. CONCLUSIONS: miR-1298 could protect HTM cells to against damage caused by COS via inhibiting TGF-ß2/Smad4 pathway and activating canonical Wnt pathway.

Targeting BRD4 proteins suppresses the growth of NSCLC through downregulation of eIF4E expression.

Lung cancer is the leading cause of cancer-related death worldwide. Bromodomain and extraterminal domain (BET) proteins act as epigenome readers for gene transcriptional regulation. Among BET family members, BRD4 was well studied, but for its mechanism in non-small cell lung carcinoma has not been elucidated. eIF4E regulates gene translation and has been proved to play an important role in the progression of lung cancer. In this study, we first confirmed that BET inhibitors JQ1 and I-BET151 suppressed the growth of NSCLCs, in parallel with downregulated eIF4E expression. Then we found that knockdown of BRD4 expression using siRNAs inhibited the growth of NSCLCs as well as decreased eIF4E protein levels. Moreover, overexpression of eIF4E partially abrogated the growth inhibitory effect of JQ1, while knockdown of eIF4E enhanced the inhibitory effect of JQ1. Furthermore, JQ1 treatment or knockdown of BRD4 expression decreased eIF4E mRNA levels and inhibited its promoter activity by luciferase reporter assay. JQ1 treatment significantly decreased the binding of eIF4E promoter with BRD4. Finally, JQ1 inhibited the growth of H460 tumors in parallel with downregulated eIF4E mRNA and protein levels in a xenograft mouse model. These findings suggest that inhibition of BET by JQ1, I-BET151, or BRD4 silencing suppresses the growth of non-small cell lung carcinoma through decreasing eIF4E transcription and subsequent mRNA and protein expression. Considering that BET regulates gene transcription epigenetically, our findings not only reveal a new mechanism of BET-regulated eIF4E in lung cancer, but also indicate a novel strategy by co-targeting eIF4E for enhancing BET-targeted cancer therapy.

Overexpression of eIF4F components in meningiomas and suppression of meningioma cell growth by inhibiting translation initiation.

Meningiomas frequently display activation of the PI3K/AKT/mTOR pathway, leading to elevated levels of phospho-eukaryotic translation initiation factor 4E binding proteins, which enhances protein synthesis; however, it is not known whether inhibition of protein translation is an effective treatment option for meningiomas. We found that human meningiomas expressed high levels of the three components of the eukaryotic initiation factor 4F (eIF4F) translation initiation complex, eIF4A, eIF4E, and eIF4G. The expression of eIF4A and eIF4E was important in sustaining the growth of NF2-deficient benign meningioma Ben-Men-1 cells, as shRNA-mediated knockdown of these proteins strongly reduced cell proliferation. Among a series of 23 natural compounds evaluated, silvestrol, which inhibits eIF4A, was identified as being the most growth inhibitory in both primary meningioma and Ben-Men-1 cells. Silvestrol treatment of meningioma cells prominently induced G2/M arrest. Consistently, silvestrol significantly decreased the amounts of cyclins D1, E1, A, and B, PCNA, and Aurora A. In addition, total and phosphorylated AKT, ERK, and FAK, which have been shown to be important drivers for meningioma cell proliferation, were markedly lower in silvestrol-treated Ben-Men-1 cells. Our findings suggest that inhibiting protein translation could be a potential treatment for meningiomas.

Matrine reduces the proliferation of A549 cells via the p53/p21/PCNA/eIF4E signaling pathway.

The aim of the present study was to investigate how matrine affects the proliferation of A549 human lung adenocarcinoma cells via the p53/p21/proliferating cell nuclear antigen (PCNA)/eukaryotic translation initiation factor 4E (eIF4E) signaling pathway. The effect of different concentrations of matrine on the proliferation of A549 cells was investigated using a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay. The migration of A549 cells following exposure to varied concentrations of matrine was detected using a Transwell cell migration assay. The effect of 240 mg/l matrine on the apoptotic rate of A549 cells was determined using flow cytometry. The change in the mRNA and protein expression levels of p53, p21, PCNA and eIF4E following exposure to matrine were detected using reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. The increase of matrine from 60­240 mg/l led to reduced cell migration and inhibition of A549 cell proliferation. The apoptotic rate of A549 cells when treated with 240 mg/l matrine was significantly different when compared with the untreated control. The mRNA expression levels of p53 and p21 in the group treated with 240 mg/l matrine were significantly higher compared with the control group. The mRNA expression levels of PCNA and eIF4E were significantly lower in the 240 mg/l matrine­treated group compared with the control. The protein expression levels of p53 and p21 were significantly higher in the 240 mg/l matrine group compared with the control group. Treatment with 240 mg/l matrine reduced the protein expression levels of PCNA and eIF4E. Matrine also reduced the migration ability of A549 cells and inhibited their proliferation, which may be associated with the overexpression of p53 and p21, and the reduction of PCNA and eIF4E expression levels.

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

BACKGROUND: Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. MATERIALS AND METHODS: To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. RESULTS: AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. CONCLUSION: The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation.

The Eukaryotic Translation Initiation Factor 4E (eIF4E) as a Therapeutic Target for Cancer.

Cancer cells depend on cap-dependent translation more than normal tissue. This explains the emergence of proteins involved in the cap-dependent translation as targets for potential anticancer drugs. Cap-dependent translation starts when eIF4E binds to mRNA cap domain. This review will present eIF4E's structure and functions. It will also expose the use of eIF4E as a therapeutic target in cancer.

LMP1 promotes nasal NK/T-cell lymphoma cell function by eIF4E via NF-κB pathway.

Nasal natural killer T-cell lymphoma (NKTL) is a highly malignant tumor that is closely associated with Epstein-Barr virus (EBV) infection. Latent membrane protein 1 (LMP1) is encoded by EBV and plays an important role in EBV-induced cell transformation. Therefore, we assessed the function of LMP1 as a stimulant of NKTL progression and the underlying mechanism. A human EBV-positive NKTL cell line (SNK-6) was transfected with pcDNA3.1-LMP1, LV-LMP1 shRNA or LV-eukaryotic translation initiation factor 4E (eIF4E)-shRNA. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the proliferation of SNK-6 cells, and cell migration and invasion were analyzed by transwell chamber assay. Flow cytometry was used to analyze the cell cycle and apoptosis. The results showed LMP1 was highly expressed in SNK-6 cells compared with control groups. Following pretreatment with LMP1 shRNA, the proliferation of SNK-6 cells was inhibited and resulted in a G0/G1 phase arrest. A reduction in invasion and migration was also observed. LMP1 silencing promoted cell apoptosis. Further mechanistic analysis suggested that LMP1 overexpression induced the expression of eIF4E, while eIF4E-shRNA dramatically attenuated the increase in cell proliferation, invasion, migration and the inhibition of apoptosis triggered by LMP-1 upregulation. Moreover, the effect of LMP1 on eIF4E expression was mediated by the NF-κB pathway. Therefore, this finding may provide a potential target against NKTL.

Integrin ß6 can be translationally regulated by eukaryotic initiation factor 4E: Contributing to colonic tumor malignancy.

It is well known that both eukaryotic initiation factor 4E (eIF4E) and integrin αvß6 can contribute to malignant behavior of colon cancer. We have found that integrin αvß6 and eIF4E were co-expressed and positively correlated in colon cancer tissues. Recently, deregulation of the protein synthesis apparatus has begun to gain attention as a major participant in cancer development and progression. However, the regulation of integrin ß6 expression at translational level has never been investigated before. In present study, gene-silencing technique for eIF4E by small interfering RNA (siRNA) was used in all the subsequent experiments, in order to investigate whether eIF4E could translationally regulate expression of integrin ß6 in colon cancer SW480 and HT-29 cell lines. Additionally, the subsequent effects of eIF4E knockdown on cellular malignant behavior were observed. siRNA in SW480 and HT-29 transfectants. Subsequently, protein expression of ß6 was markedly suppressed, while mRNA expression of ß6 showed no significant variation before and after eIF4E RNA interfering. Therefore, it could be seen that eIF4E could upregulate the expression of ß6, without effect on ß6 mRNA expression. More importantly, after treated with eIF4E siRNA, cellular migratory capacity on fibronectin of HT-29 and ß6-transfected SW480 as well as their survival to 5-FU was decreased distinctly. Expression of integrin ß6 could be translationally regulated by eIF4E, which subsequently contributed to tumor malignancy through enhancing cellular migration, survival, anti-apoptosis, and chemoresistance of colon cancer in vitro. Thus, targeting eIF4E in integrin αvß6 expressing tumors can be a potential therapeutic strategy for patients with colon cancer.

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor ß Early Protransforming Events in Breast Epithelial Cells.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor ß (TGF-ß) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin ß1 mRNA, which drives the translationally controlled assembly of a TGF-ß receptor signaling complex containing α3ß1 integrins, ß-catenin, TGF-ß receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-ß. TGF-ß can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-ß, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-ß. Overexpression of eIF4E may be a proinvasive facilitator of TGF-ß activity.

Control of the MYC-eIF4E axis plus mTOR inhibitor treatment in small cell lung cancer.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have anti-tumor effects against renal cell carcinoma, pancreatic neuroendocrine cancer and breast cancer. In this study, we analyzed the antitumor effects of mTOR inhibitors in small cell lung cancer (SCLC) cells and sought to clarify the mechanism of resistance to mTOR inhibitors. METHODS: We analyzed the antitumor effects of three mTOR inhibitors including everolimus in 7 SCLC cell lines by MTS assay. Gene-chip analysis, receptor tyrosine kinases (RTK) array and Western blotting analysis were performed to identify molecules associated with resistance to everolimus. RESULTS: Only SBC5 cells showed sensitivity to everolimus by MTS assay. We established two everolimus resistant-SBC5 cell lines (SBC5 R1 and SBC5 R10) by continuous exposure to increasing concentrations of everolimus stepwise. SPP1 and MYC were overexpressed in both SBC5 R1 and SBC5 R10 by gene-chip analysis. High expression levels of eukaryotic translation initiation factor 4E (eIF4E) were observed in 5 everolimus-resistant SCLC cells and SBC5 R10 cells by Western blotting. MYC siRNA reduced eIF4E phosphorylation in SBC5 cells, suggesting that MYC directly activates eIF4E by an mTOR-independent bypass pathway. Importantly, after reduction of MYC or eIF4E by siRNAs, the SBC5 parent and two SBC5-resistant cells displayed increased sensitivity to everolimus relative to the siRNA controls. CONCLUSION: These findings suggest that eIF4E has been shown to be an important factor in the resistance to everolimus in SCLC cells. Furthermore, a link between MYC and mTOR-independent eIF4E contribute to the resistance to everolimus in SCLC cells. Control of the MYC-eIF4E axis may be a novel therapeutic strategy for everolimus action in SCLC.

Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation.

Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.

eIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer.

A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

Expression of eukaryotic initiation factor 4 E in hypopharyngeal carcinoma.

OBJECTIVES: To investigate the expression of eukaryotic initiation factor 4 E (eIF4E) in hypopharyngeal carcinoma compared with benign lesions, and the relationships between eIF4E expression and various clinicopathological parameters. METHODS: Expression of eIF4E was analysed retrospectively in specimens from hypopharyngeal carcinomas and benign hypopharyngeal lesions using immunohistochemical staining and Western blotting. Tumours were classified using the tumour-node-metastasis staging system and the degree of histological differentiation was assessed. RESULTS: A total of 55 hypopharyngeal carcinoma and 20 benign specimens were analysed. All the hypopharyngeal carcinoma samples were positive for eIF4E expression on immunohistochemistry, whereas the benign lesions were negative or weakly positive. Semi-quantitative assessment (eIF4E score) showed that eIF4E expression was significatly higher in hypopharyngeal carcinoma than in benign lesions. On Western blotting, eIF4E expression assessed using integrated optical density (IOD) was significantly higher in hypopharyngeal carcinoma than in benign lesions. The eIF4E score and IOD value were significantly associated with tumour stage, lymphatic metastasis and degree of differentiation. The IOD value was significantly higher in recurrent compared with initial cases. CONCLUSIONS: eIF4E may play an important role in the development and metastasis of hypopharyngeal carcinoma;its expression may be helpful in establishing the diagnosis, stage and prognosis of this tumour type.

Eukaryotic translation initiation factor 4E (eIF4E) expression is associated with breast cancer tumor phenotype and predicts survival after anthracycline chemotherapy treatment.

Abnormal translation of mRNAs frequently occurring during carcinogenesis is among the mechanisms that can affect the expression of proteins involved in tumor development and progression. Eukaryotic initiation factor eIF4E is a key regulator of translation of many cancer-related transcripts and its expression is altered in various cancers and has been associated with worse survival. We determined the eIF4E protein levels using immunohistochemistry (IHC) in 1,233 breast tumors on tissue microarrays. We analyzed the effects of the IHC expression level on tumor characteristics and patient survival, also with stratification by adjuvant chemotherapy treatment. In 1,085 successfully stained tumors, high level of eIF4E protein expression was associated with features of aggressive tumor phenotype, namely grade, estrogen and progesterone receptor negativity, HER2 receptor positivity, and high expression of p53 and Ki67, and with triple negative subtype (p < 0.001). High eIF4E expression was associated with worse breast cancer-specific survival with a hazard ratio (HR) of 1.99 (95 % CI 1.32-3.00, p = 0.0008) and was in a multivariate analysis an independent prognostic factor. High eIF4E expression was associated with worse outcome also after detection of distant metastasis (HR = 1.88, 95 % CI 1.20-2.94, p = 0.0060). In the subgroup analysis the survival effect was strongest among patients treated with anthracycline chemotherapy (HR = 3.34, 95 % CI 1.72-6.48, p = 0.0002), whereas no such effect was seen among patients who had not received anthracycline with significant difference in heterogeneity between the two groups (p = 0.0358). High expression of eIF4E is associated with adverse tumor characteristics and predicts poor breast cancer-specific survival. This effect is emphasized in patients treated with anthracycline chemotherapy. eIF4E as a treatment predictive factor warrants further studies.

mTOR signaling pathway in penile squamous cell carcinoma: pmTOR and peIF4E over expression correlate with aggressive tumor behavior.

PURPOSE: Penile squamous cell carcinoma is a rare neoplasm associated with a high risk of metastasis and morbidity. There are limited data on the role of the mTOR signaling pathway in penile squamous cell carcinoma carcinogenesis and tumor maintenance. We assessed a possible role for mTOR signaling pathway activation as a potential predictive biomarker of outcome and a therapeutic target for penile cancer. MATERIAL AND METHODS: A cohort of 67 patients diagnosed with invasive penile squamous cell carcinoma from 1987 to 2010 who had known HPV status were selected for study. Tissue microarrays were constructed with 67 primary penile squamous cell carcinomas, matched normal tissues and 8 lymph node metastases. Immunohistochemical staining was performed for p53, pmTOR, pERK, p4E-BP1, eIF4E and peIF4E. Expression was evaluated using a semiquantitative H-score on a scale of 0 to 300. RESULTS: Expression of pmTOR, p4E-BP1, eIF4E and peIF4E was increased in penile tumors compared with matched adjacent normal tissues, indicating activation of the mTOR signaling pathway in penile tumorigenesis. Over expression of pmTOR, peIF4E and p53 was significantly associated with lymph node disease. peIF4E and p53 also correlated with a poor outcome, including recurrence, metastasis or disease specific death. In contrast, pERK and p4E-BP1 were associated with lower pT stages. pmTOR and intense p53 expression was associated with HPV negative tumors. CONCLUSIONS: Activation of mTOR signaling may contribute to penile squamous cell carcinoma progression and aggressive behavior. Targeting mTOR or its downstream signaling targets, such as peIF4E, may be a valid therapeutic strategy.