eIF4E Expression Research Articles

Acetate regulates milk fat synthesis through the mammalian target of rapamycin/eukaryotic initiation factor 4E signaling pathway in bovine mammary epithelial cells

Acetate is a short-chain fatty acid (SFA) that is the major substrate for de novo fatty acid synthesis. The mammalian target of rapamycin/eukaryotic initiation factor 4E (mTOR/eIF4E) signaling pathway is involved in fat synthesis. However, the effect and mechanism of acetate on fatty acid synthesis by the mTOR/eIF4E signaling pathway is unclear in bovine mammary epithelial cells (BMECs). The objectives of this study were to investigate the effect of acetate on cell viability, triacylglycerol (TG), and mRNA expression of the genes related to lipid synthesis. The mechanism of acetate regulation milk fat synthesis through the mTOR/eIF4E signaling pathway was assessed by blocking the mTOR signaling pathway and silencing eIF4E in BMECs. Third-passage BMECs were allocated to 6 treatments including 0, 4, 6, 8, 10, and 12 mM acetate to evaluate the effect of acetate on lipid synthesis; the optimum concentration in the first study was selected for the subsequent study. Subsequently, cells were randomly allocated to 4 treatments, 1 control group and 3 treated groups, consisting of acetate (6 mM), rapamycin (100 nM), and acetate + rapamycin to test the role of mTOR signaling pathway response to acetate in milk lipid synthesis. Finally, eIF4E was silenced by small interfering RNA (siRNA) to detect the role of eIF4E in milk lipid synthesis. Treatments included control, eIF4E siRNA, acetate (6 mM), and acetate+ eIF4E siRNA. Results showed that acetate increased TG accumulation and the relative expression of fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACACA), fatty acid-binding protein 3 (FABP3), sterol regulatory element binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPARG), mTOR, eIF4E, P70 ribosomal protein S6 kinase-1 (S6K1), and 4E-binding protein-1 (4EBP1) in a dose-dependent manner. Rapamycin effectively inhibited the positive effect of acetate on the relative expression of mTOR, eIF4E, S6K1, 4EBP1, FASN, ACACA, FABP3, stearoyl-CoA desaturase (SCD1), SREBP1, and PPARG. The upregulation of acetate on the relative expressions of FASN, ACACA, SCD1, and SREBP1 was suppressed when eIF4E was knocked down. It suggested that acetate regulated milk fat synthesis through mTOR/eIF4E signaling pathway in BMECs.

Effects of DL-methionine and a methionine hydroxy analogue (MHA-Ca) on growth, amino acid profiles and the expression of genes related to taurine and protein synthesis in common carp (Cyprinus carpio)

In this trial, nine diets were formulated to investigate the effects of DL-methionine (DL-Met) and a methionine hydroxy analogue (MHA-Ca) on growth, amino acid profiles and the expression of genes related to taurine and protein synthesis in common carp. The basal diet was prepared without methionine supplementation and the other diets were formulated with 0.1%, 0.2%, 0.3% and 0.4% of DL-Met or MHA-Ca, respectively. After the eight-week feeding trial, fish fed with DL-Met or MHA-Ca supplemented diets had higher growth performance and feed utilization when compared with basal diet and two-way ANOVA showed the growth performance and feed utilization were significantly increased in fish fed DL-Met supplemented diets (P < 0.05). The concentrations of free amino acids in serum (Met, Leu, His, Lys, Val, Arg, Ile, Phe and Tau) and muscle (Tau) significantly increased as dietary DL-Met and MHA-Ca increased. Moreover, two-way ANOVA showed that taurine was higher in common carp fed with DL-Met diets compared with MHA-Ca diets. To find out how DL-Met and MHA-Ca affect taurine synthesis, genes involved in methionine degradation and taurine synthesis were further investigated. DL-Met or MHA-Ca supplementation decreased the expression of methionine adenosyltransferase II, alpha (MAT2A) and methionine synthase (MS) and increased the expression of the cysteine dioxygenase (CDO). Moreover, DL-Met supplementation significantly increased the expression of CDO and cysteine sulfinate acid decarboxylase (CSD) when compared with MHA-Ca supplementation. As for protein synthesis, the mRNA expression of mechanistic target of rapamycin (mTOR) in liver was not significantly different among all diet groups. But the expression of eukaryotic translation initiation factor 4E-Binding protein 1 (4EBP1), general control nonderepressible2 (GCN2) and asparagine synthetase (ASNS) in liver were significantly down-regulated as dietary DL-Met or MHA-Ca increased. Compared with MHA-Ca diets, the expression of S6 (ribosomal protein s6) and eIF4E (eukaryotic translation initiation factor 4E binding protein 2) were markedly elevated in DL-Met groups by the analysis of two-way ANOVA. This study indicated that dietary DL-Met or MHA-Ca supplement could increase free essential amino acids in serum, as well as improve taurine synthesis and protein synthesis in common carp. Multi-exponential regression analysis showed that MHA-Ca was less utilized by common carp than DL-Met with bioavailability values of 41% to 50% on weight-for-weight basis.

Tumor-Derived Exosomal eIF4E as a Biomarker for Survival Prediction in Patients with Non-Small Cell Lung Cancer.

BackgroundThe aim of this study was to investigate the expression of tumor-derived exosomal RNA eIF4E (exo-eIF4E) in non-small cell lung cancer (NSCLC) and its correlation with prognosis.Material/MethodsThe Cancer Genome Atlas (TCGA) data was exacted to investigate the role of tissue eIF4E in NSCLC. We enrolled 99 NSCLC patients and 40 healthy volunteers with corresponding serum samples in this study. The levels of exo-eIF4E in the peripheral blood of each group were tested by quantitative polymerase chain reaction (PCR). The chi-squared test and the log-rank test were applied to analyze the correlation between the expression levels of exo-eIF4E and the patients’ clinical-pathological data, including the overall survival.ResultsTCGA data showed that increased eIF4E in NSCLC tissues was associated with late-stage disease (P=0.0497) and inferior overall survival (P=0.017). The expression of exo-eIF4E in the serum of the NSCLC group was significantly higher than that in healthy individuals (P<0.001). Furthermore, advanced TNM stage (P=0.003), distant metastasis (P=0.008), and serum positive cytokeratin fragment 19 (CYFRA21-1) (P=0.023) are more likely present in NSCLC patients with higher exo-eIF4E expression. Moreover, the multivariate combined with univariate analyses verified exo-eIF4E as an independent prognostic factor for shorter overall survival (P=0.01) and progression-free survival (P=0.005). Shorter overall survival (P=0.0005) and inferior progression-free survival (P=0.0017) are more likely present in NSCLC patients with higher exo-eIF4E.ConclusionsTumor-derived exo-eIF4E in serum can be a practical tool to predict the prognosis of NSCLC.

Translational regulation directs fate decisions involved in memory CD8 T cell differentiation

Abstract mTOR regulates the formation of memory CD8 T cells that circulate in the blood and are present in lymphoid organs. These memory CD8 T cells are the basis for long-lived T cell immunity and efficiently control re-infection to the previously encountered pathogens. Here, we show that the fate decisions between memory precursor and terminal effector CD8 T cells are regulated by the cap-dependent translation that is the downstream pathway of mTOR. Experiments using RNA interference to inhibit expression of eIF4E, which is a cap-binding protein and a translation initiation factor, in antigen-specific CD8 T cells revealed that high eIF4E expression induced terminal effector cells and low expression promoted memory precursor cells. This eIF4E dependent regulation of memory CD8 T cell differentiation was also confirmed by the overexpression of a constitutively active form of 4E-BP that inhibits cap-dependent translation by binding to eIF4E. RNA immunoprecipitation analysis with anti-eIF4E antibody between naïve and activated CD8 T cells showed that, rather than all mRNAs, the interaction of specific groups of mRNAs (i.e., translation and oxidative phosphorylation) with eIF4E was dynamically regulated during activation of the T cells. Transcripts related to translation and oxidative phosphorylation inefficiently bound to eIF4E in naïve CD8 T cells but the level of competence of these transcripts in binding to eIF4E was significantly improved in activated CD8 T cells. Thus, our data show that eIF4E-dependent translation is actively and selectively regulated during naïve to effector CD8 T cell differentiation and has an important role in fate decisions involved in the formation of memory CD8 T cells.

Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer

Eukaryotic translation initiation factor 4E (eIF4E) selectively promotes translation of mRNAs with atypically long and structured 5′-UTRs and has been implicated in drug resistance. Through genome-wide transcriptome and translatome analysis we revealed eIF4E overexpression could promote cellular activities mediated by ERα and FOXM1 signalling pathways. Whilst eIF4E overexpression could enhance the translation of both ERα and FOXM1, it also led to enhanced transcription of FOXM1. Polysome fractionation experiments confirmed eIF4E could modulate the translation of ERα and FOXM1 mRNA. The enhancement of FOXM1 transcription was contingent upon the presence of ERα, and it was the high levels of FOXM1 that conferred Tamoxifen resistance. Furthermore, tamoxifen resistance was conferred by phosphorylation independent eIF4E overexpression. Immunohistochemistry on 134 estrogen receptor (ER+) primary breast cancer samples confirmed that high eIF4E expression was significantly associated with increased ERα and FOXM1, and significantly associated with tamoxifen resistance. Our study uncovers a novel mechanism whereby phosphorylation independent eIF4E translational reprogramming in governing the protein synthesis of ERα and FOXM1 contributes to anti-estrogen insensitivity in ER+ breast cancer. In eIF4E overexpressing breast cancer, the increased ERα protein expression in turn enhances FOXM1 transcription, which together with its increased translation regulated by eIF4E, contributes to tamoxifen resistance. Coupled with eIF4E translational regulation, our study highlights an important mechanism conferring tamoxifen resistance via both ERα dependent and independent pathways.

Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato

BackgroundPotato virus Y (PVY) is a major pathogen of potatoes with major impact on global agricultural production. Resistance to PVY can be achieved by engineering potatoes to express a recessive, resistant allele of eukaryotic translation initiation factor eIF4E, a host dependency factor essential to PVY replication. Here we analyzed transcriptome changes in eIF4E over-expressing potatoes to shed light on the mechanism underpinning eIF4E-mediated recessive PVY resistance.ResultsAs anticipated, modified eIF4E-expressing potatoes demonstrated a high level of resistance, eIF4E expression, and an unexpected suppression of the susceptible allele transcript, likely explaining the bulk of the potent antiviral phenotype. In resistant plants, we also detected marked upregulation of genes involved in cell stress responses.ConclusionsOur results reveal a previously unanticipated second layer of signaling attributable to eIF4E regulatory control, and potentially relevant to establishment of a broader, more systematic antiviral host defense.

Borneol promotes apoptosis of Human Glioma Cells through regulating HIF-1a expression via mTORC1/eIF4E pathway.

The main reason for the failure of malignant glioma treatment is local tumor recurrence. Tumor cells in hypoxic microenvironment activate HIF-1 α transcription, and thus promoting tumor invasion and metastasis is one of the important reasons. In our previous study, we clearly established that borneol opens the blood-brain tumor barrier and its related mechanism. However, the effects of borneol itself on glioma proliferation have not yet been elucidated. Therefore, in this study, we evaluated the effect of borneol on glioma by constructing in vivo SD rat brain glioma model and in vitro human primary cultured glioma cell model. We found that borneol could suppress the proliferation of primary glioma cells and the tumor volume of SD rat brain glioma. Further, we measured the apoptosis effect induced by borneol in human primary cultured glioma cells. The results showed that the higher the concentration of borneol, the higher the apoptosis rate of human primary cultured glioma cells, but the effect was reversed after transfection of HIF-1 overexpression plasmid; In addition, borneol could downregulate the expression of Bcl-2 and upregulation the expression of Bax and caspase-3, similarly, the effect was also reversed after transfection of HIF-1 overexpression plasmid, suggesting that the apoptosis effect induced by borneol in human primary cultured glioma cells is mediated via HIF-1α. Moreover, the bioinformatics analysis of correlation between HIF-1α and apoptosis-related factors based on CGGA database showed that there was a positive correlation between the expression of eIF4E and HIF-1 α (P < 0.05), and in patients with high expression of eIF4E and HIF-1α had poor survival and prognosis (P<0.001). It was further discovered that in the human primary cultured glioma cells borneol regulated HIF-1a expression via mTORC1/eIF4E pathway. In conclusion, the findings of the present study suggest that HIF-1α may be a key factor in borneol induced apoptosis of glioma cells, and mTORC1 / eIF4E pathway is involved in the HIF-1α regulation by borneol in malignant glioma. Our results not only reveal the target and molecular mechanism and action of borneol leading to promote apoptosis in glioma cells, but also provide experimental basis and theoretical support for the clinical application of borneol.

AZGP1 suppresses the process of colorectal cancer after upregulating FASN expression via mTOR signal pathway.

Colorectal cancer (CRC) is the most common malignant gastrointestinal tumor. Obesity has been confirmed to be closely related to the occurrence of CRC, but the specific mechanism is not clear. This study mainly explored the roles of obesity-related genes, fatty acid synthase (FASN) and zinc-alpha-2-glycoprotein (AZGP1), in CRC. 30 cases of CRC tissues and adjacent normal colorectal tissues were obtained to quantify the levels of FASN and AZGP1 using qRT-PCR and Western blotting. Overexpression-AZGP1, overexpression-FASN and FASN shRNA were transfected into SW480 cells. CCK-8, wound healing and Transwell assays were used to evaluate the roles of FASN and AZGP1 on cell proliferation, migration as well as invasion. Western blot was performed to investigate the expression of MMP-2, MMP-9 and mTOR signaling-related proteins. AZGP1 expression was decreased in CRC tissues, which was negatively correlated with FASN expression. Overexpression-AZGP1 showed a significant inhibitory effect on cell proliferation, invasion and migration via inhibiting MMP-2 and MMP-9 expressions. Furthermore, up-regulation of AZGP1 suppressed the expression of mTOR pathway downstream proteins 4EBP and eIF4E through inhibiting FASN expression. Reintroduction of overexpression-FASN could partially reverse and inhibition of FASN further decrease the anti-tumor effect of AZGP1. AZGP1 suppresses CRC cellular activities by regulating FASN via mTOR pathway, suggesting that AZGP1 and FASN may be the targets for CRC therapy.

Ophiopogonin D inhibits proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating miR-519d-3p/EIF4E expression

Ophiopogonin D inhibits proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating miR-519d-3p/EIF4E expression

EIF4E‑related miR‑320a and miR‑340‑5p inhibit endometrial carcinoma cell metastatic capability by preventing TGF‑β1‑induced epithelial‑mesenchymal transition.

Endometrial cancer (EC) is a common form of cancer in women. Metastasis is the main cause of EC treatment failure. Eukaryotic translation initiation factor 4E (eIF4E) is an oncogene that is overexpressed in a variety of malignancies and their distant metastases. The present study analyzed microarray data from the Oncomine database and revealed that high eIF4E expression was associated with poor prognosis and high pathological grade of EC. The expression of eIF4E was higher in EC tissues compared with in adjacent normal tissues. In addition, microRNA (miR)-320a and miR-340-5p expression levels were downregulated in EC tissues compared with those in adjacent normal tissues, which suggested that these microRNAs may serve as EC tumor suppressor genes. miR-320a and miR-340-5p could bind to the 3′-UTR of eIF4E mRNA, thus downregulating the expression of eIF4E and phosphorylated (p)-eIF4E in EC cells. Overexpression of miR-320a or miR-340-5p effectively suppressed HEC-1A cell migration and invasion. The downregulation of eIF4E and p-eIF4E following miR-320a or miR-340-5p transfection reduced the invasiveness and metastatic capability of EC cells in a manner associated with decreased expression of matrix metallopeptidase (MMP)-3 and MMP-9. In addition, one of the effects of transforming growth factor β1 (TGF-β1), which is to induce the phosphorylation of eIF4E, was suppressed by miR-320a and miR-340-5p overexpression. These two microRNAs also attenuated the features of TGF-β1-induced epithelial-mesenchymal transition (EMT). In conclusion, the results of the present study demonstrated that eIF4E was upregulated in EC, whereas miR-320a and miR-340-5p were downregulated in EC compared with adjacent normal tissues. In vitro, miR-320a and miR-340-5p inhibited the migratory capability of EC cells by downregulating MMP-3 and MMP-9 and prevented TGF-β1-induced EMT through p-eIF4E.

EIF4E and 4EBP1 are prognostic markers of head and neck squamous cell carcinoma recurrence after definitive surgery and adjuvant radiotherapy.

There is high risk of metastasis and recurrence in head and neck squamous cell carcinoma (HNSCC) patients, especially for patient who received definitive surgery and adjuvant radiotherapy. Aberrant activation of PI3K/AKT/mTOR signaling occurs in approximately 80% of HNSCC, which has been indicated to serve as prognostic biomarkers for patients suffer from recurrence or metastasis. Therefore, in this study, we focus on the relationship between the expression level of signaling factors in PI3K/AKT/mTOR pathway and recurrence tumor from HNSCC patients. A tissue microarray (TMA) was constructed from 54 cases of HNSCC patients who received definitive surgery and adjuvant radiotherapy, are followed more than 5 years, and with no previous malignancy and synchronous tumor. Slides were scored and dichotomized by two pathologists and scores. Based on the TMA block with IHC staining, the results showed that PI3K/AKT/mTOR signaling was highly activated both in recurrence and non-recurrence patients. Particularly, in the recurrence population, the results showed the low expression phospho-eukaryotic initiation factor 4E (p-eIF4E) or high expression eIF4E, phospho-eIF4E binding protein 1 (p-4EBP1), phospho-ribosomal protein S6 kinase beta-1 (p-S6K1) and phospho-40S ribosomal protein S6 (p-S6R) exhibited worse overall survival. The expression level of eIF4E and p-4EBP1 were significantly associated with tumor recurrence and recurrence-free survival. Furthermore, high expression level of eIF4E and p-4EBP1 had worse recurrence-free survival. In conclusion, the expression of eIF4E and p-4EBP1 should be considered as predictive biomarkers for the HNSCC patients. This may contribute to potential predictive biomarkers for HNSCC patient who receive adjuvant radiotherapy.

Dietary non-protein energy source regulates antioxidant status and immune response of barramundi (Lates calcarifer)

This study evaluates the effects of different dietary sources of non-protein energy on growth performance, histological structure, antioxidant status and immune response of barramundi Lates calcarifer. Fish were fed with isoenergetic diets (18 kJ/g) with two types of non-protein energy in the experimental groups and a regular diet was used as the control for 56 days. The specific growth rate and survival of fish were not significantly different between experimental diets. Hepatic histology did not reveal significant differences between dietary treatments at cellular level. The activity of most antioxidant enzymes in the lipid group significantly increased, and the antioxidant capacity in the carbohydrate group was significantly higher than that in other treatments. In the TOR pathway, LST8 homolog (mLST8) expression in the high lipid group was downregulated, and the mechanistic target of rapamycin (mTOR) expression in the high carbohydrate group was downregulated and eIF4E expression was upregulated. The C-reactive protein (CRP) expression in the high lipid and high carbohydrate groups was upregulated. The expression levels of heat shock protein genes in the high lipid group and the high carbohydrate group were significantly downregulated. This study indicates that the lipid diet have less effect in fish immunity but is more suitable as a non-protein ingredient for energy supply for barramundi.

Upregulation of LncRNA Malat1 Induced Proliferation and Migration of Airway Smooth Muscle Cells via miR-150-eIF4E/Akt Signaling.

The increased proliferation and migration of airway smooth muscle cells (ASMCs) are critical processes in the formation of airway remodeling in asthma. Long non-coding RNAs (lncRNAs) have emerged as key mediators of diverse physiological and pathological processes, and are involved in the pathogenesis of various diseases, including asthma. LncRNA Malat1 has been widely reported to regulate the proliferation and migration of multiple cell types and be involved in the pathogenesis of various human diseases. However, it remains unknown whether Malat1 regulates ASMC proliferation and migration. Here, we explored the function of Malat1 in ASMC proliferation and migration in vitro stimulated by platelet-derived growth factor BB (PDGF-BB), and the underlying molecular mechanism involved. The results showed that Malat1 was significantly upregulated in ASMCs treated with PDGF-BB, and knockdown of Malat1 effectively inhibited ASMC proliferation and migration induced by PDGF-BB. Our data also showed that miR-150 was a target of Malat1 in ASMCs, and inhibited PDGF-BB-induced ASMC proliferation and migration, whereas the inhibition effect was effectively reversed by Malat1 overexpression. Additionally, translation initiation factor 4E (eIF4E), an important regulator of Akt signaling, was identified to be a target of miR-150, and both eIF4E knockdown and Akt inhibitor GSK690693 inhibited PDGF-BB-induced ASMC proliferation and migration. Collectively, these data indicate that Malat1, as a competing endogenous RNA (ceRNA) for miR-150, derepresses eIF4E expression and activates Akt signaling, thereby being involved in PDGF-BB-induced ASMC proliferation and migration. These findings suggest that Malat1 knockdown may present a new target to limit airway remodeling in asthma.

In vitro culture of secondary follicles and prematuration of cumulus-oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes.

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.

Positive Correlative over-Expression between eIF4E and Snail in Nasopharyngeal Carcinoma Promotes its Metastasis and Resistance to Cisplatin.

EIF4E is the rate-limiting factor in the mRNA translation of specific set of oncogenes. Snail is the core transcription factor of epithelial-mesenchymal transition (EMT), a key step of cancer metastasis. The connection between the two oncoproteins has not been well established in the human cancer tissues and in nasopharyngeal carcinoma (NPC). Here we showed that the positive correlative over-expression was seen between eIF4E and Snail in NPC tissues, and the expression was significantly higher in the metastatic NPC than in the un-metastatic NPC. In NPC cells, eIF4E knockdown significantly reduced Snail mRNA and protein levels, increased the mRNA level of E-cad (a direct downstream gene of Snail and a negative EMT marker), attenuated the invasive ability of the cells, and sensitized the cells to cisplatin in invasion. In contrast, enforced the expression of eIF4E significantly increased Snail mRNA and protein levels, and promoted the invasive ability in NPC cells. Under the condition of the high eIF4E expression, Snail knockdown significantly increased E-cad mRNA level and weaken the invasive ability of NPC cells. Finally, eIF4E directly bound Snail mRNA for translation initiation displayed by the RIP assay. Therefore, the results firstly suggested that eIF4E enhanced the Snail expression in both transcription and translation manner in human cancer tissues and targeting the eIF4E/Snail axis might intervene with the EMT and metastasis of NPC. This finding provided a new clue for further understanding the metastatic mechanism of human cancers and for preventing and treating NPC metastasis.

Upregulation of eukaryotic translation initiation factor 4E associates with a poor prognosis in gallbladder cancer and promotes cell proliferation in vitro and in vivo.

Eukaryotic translation initiation factor 4 (eIF4E) has been demonstrated to promote tumorigenesis in different types of cancer; however, whether eIF4E is involved in the development of GBC is unclear. The present study aimed to explore the biological function of eIF4E in gallbladder cancer (GBC) and identified that the expression level of eIF4E was significantly increased in GBC tissues compared with that in normal gallbladder tissues. The overall survival (OS) was also shorter in the group of patients with GBC with increased eIF4E expression. Increased eIF4E was correlated with advanced stage and higher histologic grade. Knockdown of eIF4E significantly inhibited cell proliferation, colony formation and cell cycle-associated protein expression levels in 2 GBC cell lines. The weight of the tumors in the eIF4E knockdown group was remarkably decreased compared with the control group. It also was revealed that knockdown of eIF4E is associated with upregulating cyclin-dependent kinase inhibitor 1B and down-regulating the expression levels of cyclin E1 and cyclin D1 in vitro and in vivo. These data demonstrated that eIF4E is a novel prognostic marker in GBC and may serve a critical role in the regulation of cell proliferation.

AEG-1 promotes the growth of gastric cancer through the upregulation of eIF4E expression.

Background: AEG-1 has been proven to be tumor enhancer in gastric cancer. However, its mechanism has not yet been fully clarified.Methods: Gain-of-function and loss-of-function experiments were conducted to determine the role of eIF4E in AEG-1-induced growth of gastric cancer cells and xenografts of a nude mouse model. Western blot analysis and SRB assay were used to determine the protein expression levels and survival cell numbers.Results: Silencing the expression of AEG-1 inhibited the growth of gastric cancer cells in parallel with a decreased eIF4E and cyclin D1 expression; however, the overexpression of AEG-1 promoted cell growth and increased eIF4E and cyclin D1 expression. Moreover, the overexpression of eIF4E partially reversed the AEG-1 silencing-induced reduction of cyclin D1 and the inhibition of cell growth. An eIF4E knockdown also partially reversed the AEG-1 overexpression-induced upregulation of cyclin D1 and cell growth. Notably, manipulating the expression of eIF4E did not affect the expression of AEG-1. Finally, the silencing of AEG-1 expression inhibited the growth of SGC-7901 xenografts in parallel with the downregulation of eIF4E and cyclin D1 expression in the nude mouse model.Conclusion: AEG-1 promoted the growth of gastric cancer through upregulation of eIF4E/cyclin D1 signaling pathway.

Abstract 2190: Repurposing the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma

Abstract Purpose: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is often diagnosed at an advanced stage, leading to poor disease-free and overall survival. Accumulating literature suggests that elevated protein expression of EZH2, eIF4E, and IMPDH correlates with poor prognosis in NPC. These three proteins are modulated by the FDA-approved antiviral drug ribavirin, which has recently been repurposed by our laboratory and others as a promising anti-cancer agent. Based on this intersection of molecular signature and drug targets, we investigated the potential use of ribavirin as a therapeutic agent for NPC. Experimental Design: We assessed anti-neoplastic efficacy of ribavirin on human NPC cell lines in vitro using cellular growth assays, flow cytometry, and scratch wound assays. Mechanistic pathways involved were investigated using genomic expression datasets, western blots, and enzymatic activity assays. The effects of combining ribavirin with radiation were assessed via clonogenic assays and flow cytometry. Finally, we evaluated the effects of ribavirin on tumor growth in vivo using human cell line-derived xenograft models. Results: Ribavirin significantly decreased NPC cellular proliferation and migratory capacity in addition to promoting cell cycle arrest and cell death. Modulation of the EZH2, Snail, eIF4E, and IMPDH pathways was observed in response to ribavirin treatment. Ribavirin significantly enhanced the cytotoxic effects of radiation therapy in NPC. Most importantly, ribavirin significantly reduced flank tumor growth in NPC xenograft models. Conclusions: Our work suggests that ribavirin has potent anti-cancer effects in NPC and could represent a safe and promising addition to current NPC treatment regimens. Citation Format: Andy S. Ding, Joshua Casaos, Sakibul Huq, Henry Brem, Nicolas Skuli, Betty Tyler. Repurposing the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2190.

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway.

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p < 0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G1 phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G1 phase by regulating the p53/p21/PCNA/eIF4E pathways.

Impact of eIF4E phosphorylation at Ser209 via MNK2a on tumour recurrence after curative surgery in localized clear cell renal cell carcinoma.

Background: We investigated the roles of eIF4E phosphorylation (Ser209) in tumour recurrence after curative nephrectomy for localized clear cell renal cell carcinoma (ccRCC).Methods: Expression of eIF4E, p eIF4E and MNKs (MAPK interacting kinases), was evaluated in surgical specimens obtained from consecutive non metastatic ccRCC patients (n = 290) by immunohistochemistry (IHC), immunoblotting, and qRT PCR at the protein and mRNA levels. In human RCC cell lines, the effects of eIF4E phosphorylation were examined using immunoblotting, proliferation, migration and invasion assays with pharmacological inhibitors (CGP57380 or ETP45835) and specific small interfering (si) RNAs against MNK1/2(a/b).Results: In postoperative follow-up (median, 7.9 y), 40 patients experienced metastatic recurrence. In multivariate Cox analyses, higher IHC expression of p eIF4E in ccRCC significantly predicted a longer recurrence-free interval. eIF4E is phosphorylated mainly by MNK2a in tumour specimens and cell lines. In 786-O and A-498 cell lines, pharmacological inhibition of MNKs decreased p-eIF4E and increased vimentin and N cadherin but did not influence proliferation. Similarly, MNK2 or MNK2a inhibition with siRNA reduced p-eIF4E and enhanced vimentin translation, cell migration and invasion in the cell lines.Conclusions: MNK2a-induced eIF4E phosphorylation may suppress metastatic recurrence of ccRCC, partially due to vimentin downregulation at the translational level, consequently leading to inhibition of epithelial–mesenchymal transition.