Translation Initiation Factor 4E Research Articles

Bacillus subtilis Produces Amino Acids to Stimulate Protein Synthesis in Ruminal Tissue Explants via the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta-Serine/Threonine Kinase-Mammalian Target of Rapamycin Complex 1 Pathway.

BackgroundBacillus subtilis is a probiotic strain that is widely used as a feed supplement for ruminants. In this study, one B. subtilis strain isolated from the ruminal fluid of Holstein dairy cows was used for an ex vivo study with ruminal tissue explants. The main goal was to assess the potential endosymbiotic links between B. subtilis and the ruminal epithelium using molecular analyses and amino acid profiling. The explant culture protocol was first optimized to determine the ideal conditions in terms of tissue viability before performing the actual experiments involving active and inactive bacteria with or without protein synthesis inhibitors, such as LY294002 (phosphatidylinositol 3-kinase inhibitor) or rapamycin [mammalian target of rapamycin (mTOR) inhibitor].ResultsThe mRNA levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), serine/threonine kinase (AKT), mTOR, P70S6K1, and eukaryotic translation initiation factor 4E binding protein 1 were the highest (p < 0.01), while those of programmed cell death 4 were the lowest when the tissue was incubated with 107 of B. subtilis. Compared with the inactivated bacteria, the expression levels of PIK3CB and AKT, and overall changes in mTOR and P70S6K1 were greater in rumen explants with living bacteria (p < 0.05). With an increase in B. subtilis concentration, the trends of protein and corresponding gene changes were consistent. There were differences in the concentrations of individual amino acids in the supernatants of living and inactivated bacterial culture groups, with most amino acids enriched in pathways, such as aminoacyl tRNA biosynthesis, cyanoamino acid metabolism, monobactam biosynthesis, or glycine, serine, and threonine metabolism. The addition of psilocybin upregulated the expression levels of PIK3CB and AKT. A significant decrease (p < 0.05) in PIK3CB and mTOR protein expression levels was detected after the addition of LY294002 and rapamycin. In addition, These responses were associated with the downregulation (p < 0.05) of AKT and P70S6K protein expression levels.ConclusionsWe confirmed that the in vivo ruminal tissue culture system is a suitable model for studying probiotic-induced alterations in tissue function. As such, this study provides a means for future mechanistic studies related to microbial regulation and the dietary supply of proteins. In addition, living and inactivated B. subtilis can promote protein synthesis in ruminal tissue explants by altering the expression levels of related factors in the PIK3CB–AKT–mTORC1 pathway, which could further aid in optimizing the feed efficiency and increasing the use of inactivated bacteria as additives in dairy cow farming.

Abstract 3281: DNMT targeting enhances vulnerability of glioblastoma cells to MNK inhibition

Abstract Many factors complicate therapeutic strategies for glioblastoma (GBM), including the existence of the blood brain barrier and a heterogenous population of difficult to treat glioma stem cells. Innovative strategies targeting novel pathways alone or in combination are needed for sustainable therapeutic improvements. The MAPK pathway has been implicated in many cancers. MAPK interacting kinases (MNK1 and MNK2) are downstream of MAPKs and phosphorylate the eukaryotic translation initiation factor 4E (eIF4E), a protein involved in translation of oncogenic mRNAs. We have previously established pharmacological MNK inhibition as a promising strategy for GBM. However, most currently available MNK inhibitors lack specificity and exhibit off-target effects. We developed novel selective MNK inhibitors that show MNK inhibition specificity in GBM established cell lines as well as patient-derived cell lines propagated under stem cell permissive conditions as 3-D neurospheres. MNK inhibitors reduced cell viability and neurosphere growth. Our previous work with MNK inhibitors showed involvement in negative feedback loops activated with treatment of other pharmacological agents, so we conducted a high-throughput screening to identify potential targets for combination treatment. One of the top hits was a DNA methyltransferase (DNMT) inhibitor that enhanced MNK inhibitor antineoplastic effects in GBM cells. Dual MNK and DNMT inhibition synergistically reduced neurosphere growth in 3-D glioma stem-like cells. The combination promoted apoptosis in the mesenchymal glioma stem-like cells as shown through flow cytometry and increased expression of cleaved PARP, cleaved caspase 3, and Bax. Also, DNMT targeting enhanced the viability reduction effects of siRNA mediated MNK1 knockdown in GBM cells. This combination of our novel MNK inhibitor with DNMT inhibition elicited antineoplastic benefits in both 2-D cultures and 3-D glioma stem cell-like populations, demonstrating a potential novel therapeutic strategy in GBM. Citation Format: Candice Mazewski, Frank Eckerdt, Aneta Baran, Mariafausta Fischietti, Purav P. Vagadia, Ricardo E. Perez, Charles D. James, Gary E. Schiltz, Leonidas C. Platanias. DNMT targeting enhances vulnerability of glioblastoma cells to MNK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3281.

Translational Control by 4E-BP1/2 Suppressor Proteins Regulates Mitochondrial Biosynthesis and Function during CD8+ T Cell Proliferation

CD8+ T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E-dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8+ T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2-deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8+ T cell expansion.

Abstract 1037: Targeting the RBM38-EIF4E complex to increase wild-type p53 as a novel cancer therapeutic strategy

Abstract To date, dysregulation of more than 450 RNA-binding proteins (RBPs) across a multitude of cancers has been implicated in tumorigenesis and chemoresistance. In fact, emerging evidence suggests RBPs are principal modulators of every hallmark of cancer progression. Functionally, RBPs are believed to modulate the stability, localization, and translation of transcripts encoding for virtually all known oncogenes and tumor suppressor genes. Consequently, aberrant expression of RBPs may contribute to the malignant transformation of cancer cells through enhancing the expression of oncogenes or decreasing the expression of tumors suppressor genes, such as TP53. As a potent transcription factor, TP53 is a leading regulator of numerous signaling pathways involved in tumor suppression. While mutation or deletion of TP53 contributes to tumorigenesis in more than half of all human cancers, suppression of wild-type TP53 expression also drives tumorigenesis. Thus far, multiple RBPs have been implicated in the repression of wild-type TP53, such as RNA-binding motif protein 38 (RBM38). Specifically, RBM38 interacts with eukaryotic translation initiation factor 4E (EIF4E) on the TP53 transcript, preventing EIF4E from binding to TP53 m7G cap, thus inhibiting TP53 translation. We previously demonstrated that Pep8, an 8 amino acid peptide derived from RBM38, disrupts the RBM38-EIF4E complex enhancing TP53 expression. Moreover, Pep8 alone or in combination with doxorubicin, significantly decreased tumor xenograft growth. As activation of wild-type TP53 by targeting non-genetic mechanisms that inhibit TP53 expression is a promising therapeutic tactic for malignancies that carry wild-type TP53, we are developing more efficacious Pep8 derivatives as a novel therapeutic approach for cancers that express wild-type TP53. Citation Format: Chris August Lucchesi, Jin Zhang, Xinbin Chen. Targeting the RBM38-EIF4E complex to increase wild-type p53 as a novel cancer therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1037.

Nuclear-targeted 4E-BP1 is dephosphorylated, induces nuclear translocation of eIF4E, and alters mRNA translation

Mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates and inhibits eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). This leads to the release of eIF4E from 4E-BP1 and the initiation of eIF4E-dependent mRNA translation. In this study, we examined the expression of a 4E-BP1-based reporter (mTORC1 activity reporter; TORCAR) with various localization signal tags to clarify the relationship between the localization of 4E-BP1 and its phosphorylation. Phosphorylation of 4E-BP1 at threonine 37/46 and serine 65 was efficient at lysosomes and the plasma membrane, whereas it was significantly decreased in the nucleus. In addition, the localization of endogenous eIF4E shifted from the cytoplasm to the nucleus only when nuclear-localized TORCAR was expressed. Nuclear-localized TORCAR decreased cyclin D1 protein levels and altered cell cycle distribution. These data provide an experimental tool to manipulate the localization of endogenous eIF4E without affecting mTORC1 and highlight the important role of nuclear-cytoplasmic shuttling of eIF4E.

The Translational Regulation in mTOR Pathway.

The mechanistic/mammalian target of rapamycin (mTOR) plays a master role in cell proliferation and growth in response to insulin, amino acids, energy levels, and oxygen. mTOR can coordinate upstream signals with downstream effectors, including transcriptional and translational apparatuses to regulate fundamental cellular processes such as energy utilization, protein synthesis, autophagy, cell growth, and proliferation. Of the above, protein synthesis is highly energy-consuming; thus, mRNA translation is under the tight and immediate control of mTOR signaling. The translational regulation driven by mTOR signaling mainly relies on eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP), ribosomal protein S6 kinase (S6K), and its downstream players, which are significant in rapid cellular response to environmental change. mTOR signaling not only controls the general mRNA translation, but preferential mRNA translation as well. This means that mTOR signaling shows the stronger selectivity to particular target mRNAs. Some evidence has supported the contribution of 4E-BP and La-related proteins 1 (LARP1) to such translational regulation. In this review, we summarize the mTOR pathway and mainly focus on mTOR-mediated mRNA translational regulation. We introduce the major components of mTOR signaling and their functions in translational control in a general or particular manner, and describe how the specificity of regulation is coordinated. Furthermore, we summarize recent research progress and propose additional ideas for reference. Because the mTOR pathway is on the center of cell growth and metabolism, comprehensively understanding this pathway will contribute to the therapy of related diseases, including cancers, type 2 diabetes, obesity, and neurodegeneration.

MTOR INHIBITOR IMPROVES TESTOSTERONE-INDUCED MYOCARDIAL HYPERTROPHY IN HYPERTENSIVE RATS

Objective: Compelling evidence have described the incidence of hypertension and left ventricular hypertrophy (LVH) in postmenopausal women is significantly increased worldwide. Our team’s previous research identified that androgen was an underlying factor contributing to increased blood pressure and LVH in postmenopausal women. However, little is known about how androgens affect LVH in postmenopausal hypertensive women. The purpose of this study was to evaluate the role of mTOR signaling pathway in myocardial hypertrophy in androgen-induced postmenopausal hypertension and whether mTOR inhibitors can protect the myocardium from androgen-induced interference to prevent and treat cardiac hypertrophy. Design and method: For that, ovariectomized (OVX) spontaneously hypertensive rats (SHR) aged 12 weeks were used to study the effects of testosterone (T 2.85 mg/kg/weekly im) on blood pressure and myocardial tissue. On the basis of antihypertensive therapy (chlorthalidone 8 mg/kg/day ig), the improvement of blood pressure and myocardial hypertrophy in rats treated with different dose gradients of rapamycin (0.8 mg/kg/day Vs 1.5 mg/kg/day Vs 2 mg/kg/day ip) in OVX+ estrogens (E 9.6 mg/Kg/day, ig)+T group was further evaluated. Results: After T intervention, the OVX female rats exhibited significant increments in the heart weight / tibial length (TL), cross-sectional area of cardiomyocytes and the mRNA expressions of atrial natriuretic peptide, B- myosin heavy chain and matrix metalloproteinase 9 accompanied by a significant reduction in the uterine weight/TL and issue inhibitor of metalloproteinase 1. Mammalian rapamycin receptor (mTOR), ribosomal protein S6 kinase (S6K1), 4E-bindiong protein 1(4EBP1) and eukaryotic translation initiation factor 4E in myocardial tissue of OVX+E+T group were expressed at higher levels than those of the other four groups. On the other hand, rapamycin abolished the effects of T-induced cardiac hypertrophy, decreased the systolic and diastolic blood pressure of SHR, and inhibited the activation of mTOR/ S6K1/4EBP1 signaling pathway in a concentration-dependent manner. Conclusions: Collectively, these data suggest that the mTOR/S6K1/4EBP1 pathway is an important therapeutic target for the prevention of LVH in postmenopausal hypertensive women with high T levels. Our findings also support the standpoint that the mTOR inhibitor, rapamycin, can eliminate T-induced cardiomyocyte hypertrophy.

Circadian activities of the brain MNK-eIF4E signalling axis contribute to diurnal rhythms of some cognitive functions.

Although it is well recognized that the circadian timing system profoundly influences cognitive performance, the underlying molecular mechanisms remain poorly defined. Our previous work has found that the mitogen‐activated protein kinase‐interacting kinase (MNK)‐eukaryotic translation initiation factor 4E (eIF4E) axis, a conserved cellular signalling pathway regulating mRNA translation, modulates the function of the suprachiasmatic nucleus (SCN), the master circadian clock. Here, with the use of a combination of genetic, biochemical and behavioural approaches, we investigated the distribution and temporal regulation of eIF4E phosphorylation in the brain and its role in regulating the diurnal oscillations of some aspects of cognition in mice. We found that activities of the MNK‐eIF4E axis, as indicated by the level of eIF4E phosphorylation at Ser209, exhibited significant circadian oscillations in a variety of brain regions, including but not limited to the prefrontal cortex, the hippocampus, the amygdala and the cerebellum. Phosphorylated eIF4E was enriched in neurons but not in astrocytes or microglia. Mice lacking eIF4E phosphorylation (eIF4E S209A/S209A ) or the MNKs (Mnk 1−/−,2−/−), the kinases that phosphorylate eIF4E, exhibited impaired diurnal variations of novel object recognition, object location memory, Barnes maze learning and ambulatory activities. Together, these results suggest that circadian activities of the MNK‐eIF4E axis contribute to the diurnal rhythms of some cognitive functions, highlighting a role for rhythmic translational control in circadian regulation of cognitive performance.

CRISPR/Cas genome editing perspectives for barley breeding.

The CRISPR/Cas9 technology shows potential to improve crop breeding efficiency and antiviral defense. The interest in DNA editing in crops has grown due to the possibility of increasing the resistance of different plants to many viruses. Our aim was to create an elite disease-resistant local barley cultivar using CRISPR/Cas9 biotechnology. For this purpose, we used CRISPR/Cas 9-eIF4E with the eukaryotic translation initiation factor 4E (eIF4E) barley gene to edit the genomes of five local Kazakhstan barley cultivars. After identifying the single guide RNA (sgRNA) target sequences, they were synthesized and cloned into the CRISPR-plant vector before being introduced into barley cells via our own patented Agrobacterium germ-line transformation technique. Barley plants eIF4E-modified were successfully obtained and were resistant to virus infection. Based on our research, the CRISPR/Cas9 system for plant genome editing could be a prospect for applying this breakthrough biotechnology in barley breeding.

Preliminary observation on the differential expression of metformin in preventing noise-induced hearing loss in inner ear protein group of rats

Objective: To study the protective effects of metformin on noise-induced hearing loss (NIHL) and its differential protein omics expression profile. Methods: In January 2021, 39 male Wistar rats were randomly divided into control group, noise exposure group and metformin+noise exposure group, with 13 rats in each group. Rats in the noise exposure group and metformin+noise exposure group were continuously exposed to octave noise with sound pressure level of 120 dB (A) and center frequency of 8 kHz for 4 h. Rats in the metformin+noise exposure group were treated with 200 mg/kg/d metformin 3 d before noise exposure for a total of 7 d. Auditory brainstem response (ABR) was used to test the changes of hearing thresholds before noise exposure and 1, 4, 7 d after noise exposure in the right ear of rats in each group. Tandem mass tag (TMT) quantitative proteomics was used to identify and analyze the differentially expressed protein in the inner ear of rats in each group, and it was verified by immunofluorescence staining with frozen sections. Results: The click-ABR thresholds of right ear in the noise exposure group and metformin+noise exposure group were significantly higher than those in the control group 1, 4, 7 d after noise exposure (P<0.05) . The click-ABR threshold of right ear in the metformin+noise exposure group were significantly lower than that in the noise exposure group (P<0.05) . Compared with the noise exposure group, 1035 up-regulated proteins and 1145 down-regulated proteins were differentially expressed in the metformin+noise exposure group. GO enrichment analysis showed that the significantly differentially expressed proteins were mainly involved in binding, molecular function regulation, signal transduction, and other functions. Enrichment analysis of KEGG pathway revealed that the pathways for significant enrichment of differentially expressed proteins included phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, focal adhesion, diabetic cardiomyopathy, mitogen, and mitogen-activated protein kinase (MAPK) signaling pathway. Immunofluorescence experiments showed that compared with the noise exposure group, the fluorescence intensity of insulin-like growth factor 1 receptor (IGF1R) in the metformin+noise exposure group was increased, and the fluorescence intensity of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1) was decreased. Conclusion: Noise exposure can lead to an increase in rat hearing threshold, and metformin can improve noise-induced hearing threshold abnormalities through multiple pathways and biological processes.

Toxicological effects of tris(1,3-dichloro-2-propyl) phosphate in oyster Crassostrea gigas using proteomic and phosphoproteomic analyses

As a typical organophosphorus pollutant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been widely detected in aquatic environment. Previous studies showed that protein phosphorylation might be a vital way of TDCIPP to exert multiple toxic effects. However, there is a lack of high-throughput investigations on how TDCIPP affected protein phosphorylation. In this study, the toxicological effects of TDCIPP were explored by proteomic and phosphoproteomic analyses together with traditional means in oysters Crassostrea gigas treated with 0.5, 5 and 50 μg/L TDCIPP for 28 days. Integration of omic analyses revealed that TDCIPP dysregulated transcription, energy metabolism, and apoptosis and cell proliferation by either directly phosphorylating pivotal proteins or phosphorylating their upstream signaling pathways. The U-shaped response of acetylcholinesterase activities suggested the neurotoxicity of TDCIPP in a hormesis manner. What’s more, the increase in caspase-9 activity as well as the expression or phosphorylation alterations in eukaryotic translation initiation factor 4E, cell division control protein 42 and transforming growth factor-β1-induced protein indicated the disruption of homeostasis between apoptosis and cell proliferation, which was consistent with the observation of shedding of digestive cells. Overall, combination of proteomic and phosphoproteomic analyses showed the capability of identifying molecular events, which provided new insights into the toxicological mechanisms of TDCIPP.

Pharmacodynamic and therapeutic pilot studies of single-agent ribavirin in patients with human papillomavirus–related malignancies

ObjectivesRibavirin inhibits eukaryotic translation initiation factor 4E (eIF4E), thereby decreasing cap-dependent translation. In this two-part study, we assessed the pharmacodynamic effects and therapeutic potential of ribavirin in human papillomavirus (HPV)–related malignancies. MethodsIn the pharmacodynamic study, ribavirin (400 mg BID for 14 days) was evaluated in 8 patients with HPV-positive localized oropharyngeal carcinoma with phosphorylated-eIF4E (p-eIF4E) ≥ 30%. In the therapeutic study, ribavirin (1400 mg BID in 28-day cycles, continuously dosed) was evaluated in 12 patients with recurrent and/or metastatic HPV-related cancer. Dose interruptions or reductions were allowed according to prespecified criteria. Toxicities were assessed in accordance with National Cancer Institute Common Terminology Criteria for Adverse Events version 4; response was assessed using Response Evaluation Criteria in Solid Tumors version 1.1. Patients remained on study until disease progression or unacceptable toxicity. ResultsSix patients were evaluable in the pharmacodynamic study: 4 had decreased p-eIF4E after 14 days of ribavirin. In the therapeutic study, 12 patients were evaluable for toxicity, and 9 were evaluable for response. Among these, median follow-up was 3.5 months, and best overall response was stable disease in 5 patients and progression of disease in 4 patients. Median progression-free survival was 1.8 months. The most common treatment-related adverse events (grade > 2) were anemia, dyspnea, and hyperbilirubinemia. All patients had anemia (grades 1–3), with 33% having at least 1 dose reduction. ConclusionOral ribavirin decreases p-eIF4E levels and is well-tolerated. However, a clear signal of efficacy in patients with recurrent and/or metastatic HPV-related cancers was not observed. (NCT02308241, NCT01268579)

A Multi-Omics Pan-Cancer Analysis of 4EBP1 in Cancer Prognosis and Cancer-Associated Fibroblasts Infiltration.

Background: Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (4EBP1) involved in inhibition of protein translation and synthesis. However, the phosphoprotein of 4EBP1 (p-4EBP1) promotes the translation and synthesis of several proteins, including multiple classic oncogenic proteins. The prognostic significance of 4EBP1 mRNA, 4EBP1 protein, and p-4EBP1 in Pan-cancer are still unclear. Methods: In this study, we provided a multi-Omics investigation for the prognostic value of 4EBP1 mRNA, 4EBP1 protein, and different 4EBP1 phosphoproteins in a Pan-cancer manner based on the TCGA projects. We explored the correlation between 4EBP1 expression and the cancer-associated fibroblast (CAFs) infiltration, respectively using the EPIC, MCPCOUNTER, and TIDE algorithms. The functional states of 4EBP1 were explored using single-cell sequencing analysis in Pan-Cancer. Immunohistochemistry staining was used to detect and verify the expression of 4EBP1 in several cancers. Results: 4EBP1 mRNA was aberrantly overexpressed in most cancers, and was associated with the poor prognosis in ten cancers. Notably, increased 4EBP1 mRNA expression significantly correlated with tumor staging and worse prognosis in BRCA, KIRC, and KIRP, while having the opposite effect in STAD. 4EBP1 expression was associated with the CAFs infiltration level in ten cancer types. Interestingly, the correlation between 4EBP1 and CAFs infiltration had pronounced heterogeneity in digestive system tumors and urinary system tumors. In BLCA, KIRC, and ACC as well as BRCA, 4EBP1 was significantly positively correlated with CAFs infiltration and was associated with a poor prognosis. In STAD and COAD, 4EBP1 is negatively correlated with CAFs infiltration and was associated with a better prognosis. Lastly, the expression and prognostic significance of 4EBP1 protein and different p-4EBP1 varied enormously among cancers. Conclusion: Our multi-omics study indicates that 4EBP1-driven CAFs infiltration is associated with cancer prognosis and 4EBP1 mRNA, 4EBP1 protein, and p-4EBP1 proteins may serve as potential prognostic biomarkers and therapeutic targets in diverse cancer.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Targeted generation of Null Mutants in ZmGDIα confers resistance against maize rough dwarf disease without agronomic penalty.

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression in glioblastoma is driven by ETS1- and MYBL2-dependent transcriptional activation

Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms.

Identification of a dual FLT3 and MNK2 inhibitor for acute myeloid leukemia treatment using a structure-based virtual screening approach

Fms-like tyrosine kinase 3 (FLT3) is considered a promising therapeutic target for acute myeloid leukemia (AML) in the clinical. However, monotherapy with FLT3 inhibitor is usually accompanied by drug resistance. Dual inhibitors might be therapeutically beneficial to patients with AML due to their ability to overcome drug resistance. Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) phosphorylate eukaryotic translation initiation factor 4E (eIF4E), which brings together the RAS/RAF/ERK and PI3K/AKT/mTOR oncogenic pathways. Therefore, dual inhibition of FLT3 and MNK2 might have an additive effect against AML. Herein, a structure-based virtual screening approach was performed to identify dual inhibitors of FLT3 and MNK2 from the ChemDiv database. Compound K783-0308 was identified as a dual inhibitor of FLT3 and MNK2 with IC50 values of 680 and 406 nM, respectively. In addition, the compound showed selectivity for both FLT3 and MNK2 in a panel of 82 kinases. The structure-activity relationship analysis and common interactions revealed interactions between K783-0308 analogs and FLT3 and MNK2. Furthermore, K783-0308 inhibited MV-4-11 and MOLM-13 AML cell growth and induced G0/G1 cell cycle arrest. Taken together, the dual inhibitor K783-0308 showed promising results and can be potentially optimized as a lead compound for AML treatment.

Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis in non-small cell lung cancer

Everolimus is a kind of mammalian target of rapamycin (mTOR) inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). The eIF4E phosphorylation increased by mTOR inhibitors is mainly mediated by MNKs. However, the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposure. Our previous studies have confirmed that tumor suppressor miR-7-5p is decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. In this study, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. We confirmed that Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B-dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but a supplement of extra exosomal miR-7-5p could reverse it. Of note, both low expression of miR-7-5p and elevated MNK1 protein were associated with a poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus by inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and in vivo. The combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy through dual abrogation of MNK/eIF4E and mTOR in NSCLC. In conclusion, Everolimus decreases the intracellular miR-7-5p by releasing of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy by targeting MNK/eIF4E axis and mTOR. Besides, both low levels of miR-7-5p and positive expression of MNK1 act as independent poor prognostic biomarkers for NSCLC. Therefore, restoring miR-7-5p carried by exosome may be a promising novel combined therapeutic strategy with Everolimus for NSCLC.

Prothioconazole induces cell cycle arrest by up-regulation of EIF4EBP1 in extravillous trophoblast cells.

Prothioconazole (PTC) is a new broad-spectrum triazole antibacterial agent that is being widely used in agriculture. PTC has been linked to a number of reproductive outcomes including embryo implantation disorder; however, the exact mechanism underlying this relationship has yet to be determined. Proper trophoblast proliferation and migration is a prerequisite for successful embryo implantation. To elucidate the underlying molecular perturbations, we detect the effect of PTC on extravillous trophoblast cells proliferation and migration, and investigate its potential mechanisms. Exposure to different concentrations of PTC (0-500μM) significantly inhibited the cell viability and migration ability (5μM PTC exposure), and also caused the cell cycle arrest at the lowest dose (1μM PTC exposure). Transcriptome analysis revealed that PTC exposure disturbed multiple biological processes including cell cycle and apoptosis, consistent with cell phenotype. Specifically, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1, 4E-BP1) was identified as up-regulated in PTC exposure group and knockdown of EIF4EBP1, and attenuated the G1 phase arrest induced by PTC exposure. In summary, our data demonstrated that 4E-BP1 participated in PTC-induced cell cycle arrest in extravillous trophoblast cells by regulating cyclin D1. These findings shed light on the potential adverse effect of PTC exposure on the embryo implantation.

Effects of replacing fish meal with corn gluten meal on growth performance, intestinal microbiota, mTOR pathway and immune response of abalone Haliotis discus hannai

Corn gluten meal (CGM) is used as a protein source in aquafeeds. However, the potential for replacing fish meal (FM) with CGM in abalone feeds has not been adequately evaluated. A 110-day feeding trial was conducted to evaluate effects of replacing FM with CGM on abalone Haliotis discus hannai (initial weight: 21.83 ± 0.25 g). The control diet (CGM0) was designed to contain 20% of FM. The other four isonitrogenous (about 30% of crude protein) and isolipidic (about 4% of crude lipid) experimental diets were formulated with CGM protein replacing 25%, 50%, 75% and 100% of dietary FM protein, respectively. The five experimental diets were named as CGM0, CGM25, CGM50, CGM75 and CGM100, respectively. Results showed that no significant differences were found in survival rate among all the groups. Weight gain rate (WGR) of abalone in the CGM100 group was significantly lower than that in the control group. There were no significant differences in WGR among the CGM25, CGM50, CGM75 and the control groups. The increasing dietary CGM inclusion level elevated the operational taxonomic units, ACE estimator and Chao1 estimator, and changed the intestinal microbiota composition in abalone. The activities of trypsin, alanine transaminase, aspartate transaminase and Na+, K+-ATPase in digestive gland were significantly decreased when replacement level over 50%. The increasing dietary CGM inclusion level significantly downregulated the expressions of ribosomal S6 protein kinase (s6k), mammalian target of rapamycin (mtor) and eukaryotic translation initiation factor 4E (eif4e) in digestive gland. The CGM75 and CGM100 groups had significant lower activities of catalase and lysozyme in cell-free hemolymph and digestive gland. In conclusion, CGM could replace 75% of dietary FM (equivalent to 16.2% of CGM inclusion level in the diet) without significantly affecting the growth of abalone. However, high inclusion levels of CGM in diets (> 10.8%) had negative effects on intestinal microbiota, mTOR pathway and immunity in abalone.

Cloning and Characterization of Eukaryotic Translation Initiation Factor 4E (eIF4E) Gene Family in &amp;lt;i&amp;gt;Ipomoea batatas&amp;lt;/i&amp;gt; L. (Lam) for Understanding Hexaploid Sweetpotato-Virus Interactions

Characterization of genes related to sweetpotato to viral disease resistance is critical for understanding plant-pathogen interactions, especially with feathery mottle virus infection. For example, genes encoding eukaryotic translation initiation factor (eIF)4E, its isoforms, eIF(iso)4E, and the cap-binding protein (CBP) in plants, have been implicated in viral infections aside from their importance in protein synthesis. Full-length cDNA encoding these putative eIF targets from susceptible/resistant and unknown hexaploid sweetpotato (Ipomoea batatas L. Lam) were amplified based on primers designed from the diploid wild-type relative Ipomoea trifida consensus sequences, and designated IbeIF4E, IbeIF(iso)4E and IbCBP. Comparative analyses following direct-sequencing of PCR-amplified cDNAs versus the cloned cDNA sequences identified multiple homeoalleles: one to four IbeIF4E, two to three IbeIF(iso)4E, and two IbCBP within all cultivars tested. Open reading frames were in the length of 696 bp IbeIF4E, 606 bp IbeIF(iso)4E, and 675 bp IbCBP. The encoded single polypeptide lengths were 232, 202, and 225 amino acids for IbeIF4E, IbeIF(iso)4E, and IbCBP, with a calculated protein molecular mass of 26 kDa, 22.8 kDa, and 25.8 kDa, while their theoretical isoelectric points were 5.1, 5.57, and 6.6, respectively. Although the homeoalleles had similar sequence lengths, single nucleotide polymorphisms and multi-allelic variations were detected within the coding sequences. The multi-sequence alignment performed revealed a 66.9% - 96.7% sequence similarity between the predicted amino acid sequences obtained from the homeoalleles and closely related species. Furthermore, phylogenetic analysis revealed ancestral relationships between the eIF4E homeoalleles and other species. The outcome herein on the eIF4E superfamily and its correlation in sequence variations suggest opportunities to decipher the role of eIF4E in hexaploid sweetpotato feathery mottle virus infection.