eIF4E Gene Research Articles

Effects of metformin and pioglitazone combination on apoptosis and AMPK/mTOR signaling pathway in human anaplastic thyroid cancer cells.

Anaplastic cancer constitutes 1% of thyroid cancers, and it is one of the most aggressive cancers. Treatment options are external radiation therapy and/or chemotherapy. The success rate with these treatment modalities is not satisfactory. We aimed to evaluate the effects of metformin (MET) and pioglitazone (PIO) combination on apoptosis and AMP-activated protein kinase/mammalian target of rapamycin (mTOR) signaling pathway in human anaplastic thyroid cancer cells. In this study, we evaluated the effects of MET and PIO individually and the combination of the two drugs on the cellular lines SW1736 and C643 ATC. Genes contained in the mTOR signaling pathway were examined using human mTOR Signalization RT2 Profiler PCR Array. In C643 and SW1736 cell lines, IC50 doses of MET and PIO were found out as 17.69 mM, 11.64 mM, 27.12 µM, and 23.17 µM. Also, the combination of MET and PIO was determined as an additive according to isobologram analyses. We have found the downregulation of the expression levels of oncogenic genes: AKT3, CHUK, CDC42, EIF4E, HIF1A, IKBKB, ILK, MTOR, PIK3CA, PIK3CG, PLD1, PRKCA, and RICTOR genes, in the MET and PIO combination-treated cells. In addition, expression levels of tumor suppressor genes, DDIT4, DDIT4L, EIF4EBP1, EIF4EBP2, FKBP1A, FKBP8, GSK3B, MYO1C, PTEN, ULK1, and ULK2, were found to have increased significantly. The MET + PIO combination was first applied to thyroid cancer cells, and significant reductions in the level of oncogenic genes were detected. The decreases, particularly, in AKT3, DEPTOR, EIF4E, ILK, MTOR, PIK3C, and PRKCA expressions indicate that progression can be prevented in thyroid cancer cells and these genes could be selected as therapeutic targets.

Transcriptomic evidence that insulin signalling pathway regulates the ageing of subterranean termite castes

Insulin is a protein hormone that controls the metabolism of sugar, fat and protein via signal transduction in cells, influencing growth and developmental processes such as reproduction and ageing. From nematodes to fruit flies, rodents and other animals, glucose signalling mechanisms are highly conserved. Reproductive termites (queens and kings) exhibit an extraordinarily long lifespan relative to non-reproductive individuals such as workers, despite being generated from the same genome, thus providing a unique model for the investigation of longevity. The key reason for this molecular mechanism, however, remains unclear. To clarify the molecular mechanism underlying this phenomenon, we sequenced the transcriptomes of the primary kings (PKs), primary queens (PQs), male (WMs) and female (WFs) workers of the lower subterranean termite Reticulitermes chinensis. We performed RNA sequencing and identified 33 insulin signalling pathway-related genes in R. chinensis. RT-qPCR analyses revealed that EIF4E and RPS6 genes were highly expressed in WMs and WFs workers, while mTOR expression was lower in PKs and PQs than in WMs and WFs. PQs and PKs exhibited lower expression of akt2-a than female workers. As the highly conserved insulin signalling pathway can significantly prolong the healthspan and lifespan, so we infer that the insulin signalling pathway regulates ageing in the subterranean termite R. chinensis. Further studies are recommended to reveal the biological function of insulin signalling pathway-related genes in the survival of termites to provide new insights into biomolecular homeostasis maintenance and its relationship to remarkable longevity.

Prediction of secondary, tertiary protein structure and interaction eIF4E from Capsicum annum with Rep Geminivirus

The eIF4E gene is a gene that plays a role in the initiation of protein translation. This gene is relatively conserved and is present in all organisms. In humans, this gene functions as a proto-oncogene, its expression and activation are associated with transformation and tumorigenesis. The eIF4E protein takes a role in the translation initiation by recruiting ribosomes to the 5’-cap structure. In some conditions, it functions as an initiator in the process of translation and co-factor of the plant’s defense system from virus attacks. The eIF4E protein also can bind with the genome linked viral protein to turnip mosaic virus [TuMV] in Capsicum annum. Their interaction is effective for virus infectivity and upregulated genome amplification. Understanding the tertiary structure of the eIF4E protein and its ligands will help in elucidating its interactions with viruses. So that it can be used to avoid spreading the virus in chili plants.

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper “Micro-Pep”. This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING.

Enhancement of resistance to PVY in intragenic marker-free potato plants by RNAi-mediated silencing of eIF4E translation initiation factors

Among various defence strategies used to protect potato plants from a viral infection, a deficiency of the eukaryotic translation initiation factors (eIF) provided by the host-mediated translational suppression has been characterized as a promising approach in modern crop breeding. The strategy used in the present study relies on knocking down the genes of the eIF4E-encoding family in a susceptible potato cultivar by the RNAi silencing mechanism. Taking into account the high concern regarding the potential effects of biotech crops on product safety and public acceptance, no bacterial/viral sequences and selectable marker genes were used. A hairpin construct carrying the fragment of potato eIF4E1 gene was created under control of potato Lhca3 (photosystem I 24 kDa light-harvesting protein) gene promoter and terminator. As a result of marker-free Agrobacterium-mediated transformation, two independent events were identified; one was confirmed to be clean from vector-backbone DNA sequences. Due to high homology between the genes encoding eIF4E1 and eIF4E2 potato factors, the silencing of both genes was achieved. During the two seasonable experiments, the intragenic line with higher production of small RNAs due to the expression of hpRNAi cassette showed a strong level of resistance after the mechanical inoculation with agriculturally significant PVYNTN strain and produced tubers phenotypically similar to non-infected control. Overall, the results obtained here indicate that RNAi-mediated transcriptional regulation of targeted eIF4E gene family using plant tissue-specific promoters in marker-free intragenic potato plants is a promising strategy for improving the viral resistance in clonally propagated crops without affecting plant phenotype and productivity. This study is a proof-of-concept for successful generation of viral resistance in potato by the RNAi-mediated transcriptional regulation of targeted eIF4E gene family using intragenic approach.

Camellia (Camellia oleifera Abel.) seed oil promotes milk fat and protein synthesis-related gene expression in bovine mammary epithelial cells.

Camellia (Camellia oleifera Abel.) seed oil is a commonly used edible oil of China. In ancient Chinese literature, it is mentioned to be helpful for postpartum repair and lactation in women. Research on camellia seed oil (CO) as a feed additive for dairy cattle is less. We investigated the effect of CO on the expression of milk fat and protein syntheses‐related genes in differentiated bovine mammary epithelial cells (MAC‐T) using soybean oil (SO) as the control. The results showed that CO increased the expression of genes related to de novo synthesis of fatty acids including sterol regulatory element‐binding protein 1 (SREBP1), acetyl‐CoA carboxylase 1 (ACC), fatty acid synthase (FASN), lipoprotein lipase (LPL), and stearoyl‐CoA desaturase (SCD) (p < .05). Among the milk protein genes analyzed, CO increased β‐casein mRNA expression (p < .05) and decreased αS1‐casein mRNA expression (p < .05) in MAC‐T cells. CO upregulated the pathways related to milk protein synthesis with increased mRNA levels of phosphoinositide 3‐kinase (PI3K), RAC‐alpha serine/threonine‐protein kinase (AKT1), and mammalian target of rapamycin (mTOR) (p < .05) in MAC‐T cells. Ribosomal protein S6 kinase beta‐1 (S6K1) gene was upregulated, and eukaryotic initiation factor 4E (eIF4E) gene (p < .05) was downregulated with CO treatment. The mRNA expression levels of janus kinase 2 (JAK2), activator of transcription 5‐β (STAT5‐β), and E74‐like factor 5 (ELF5) were elevated in MAC‐T cells treated with CO (p < .05). Meanwhile, the protein expression levels of S6K1, STAT5‐β, phosphorylated mTOR (p‐mTOR), p‐S6K1, and p‐STAT5‐β increased in MAC‐T cells treated with CO (p < .05). In summary, CO promoted β‐casein synthesis by regulating PI3K‐mTOR‐S6K1 and JAK2‐STAT5 signaling pathways and influenced fatty acid synthesis by regulating SREBP1‐related genes in MAC‐T cells. We need to further confirm the function of CO using in vivo models.

Synthesis of novel S-linked dihydroartemisinin derivatives and evaluation of their anticancer activity

We report herein the synthesis and anticancer activity of a set of novel S-linked artemisinins bearing an aliphatic/aromatic/heterocyclic nucleus as a substituent on the sulfur. The compounds were prepared from artemisinin via its lactol-form by an acid-catalyzed condensation of the desired thiol with the lactol. Both the C-10-α- and β-configured thiol ethers were synthesized with a view to making them available for the anticancer activity evaluation using a variety of cell lines. The results show that many of the synthetic derivatives studied possessed good potential as anticancer agents. In order to draw more information on the origin of the anticancer activity, one of the compounds (9a), that showed a broad-spectrum activity in terms of reducing the viability of most of the cell lines studied, in particular proven to be most effective against Prostate (PC-3) cells, was studied in detail to find the underlying mechanism of its action by MTT assay, immunoblotting, flow cytometry and microscopy. Pretreatment of the PC-3 cells with N-acetyl cysteine affected the efficacy of 9a, suggesting the role of reactive oxygen species in reducing their viability. Cell cycle analysis showed increase in G1 phase that was indicative of G1 cell cycle arrest. Wound healing assay revealed anti-migratory effect of 9a Quantitative PCR and western blot analysis showed changes in the gene expression of PCNA, E2F1, Pin1, cyclinD1, phospho-c-jun, c-Myc, eIF4E and other genes involved in proliferation and maintaining the transformed phenotype of prostate cancer cells. Here we report the anti–proliferative property of 9a with a vital and potent target(s) in prostate cancer cells with one of such targets being Pin1 belonging to the parvulin family of PPIases. The results suggest that 9a could be a promising agent in combating prostate cancer.

A complex eIF4E locus impacts the durability of va resistance to Potato virus Y in tobacco.

SummaryMany recessive resistances against potyviruses are mediated by eukaryotic translation initiation factor 4E (eIF4E). In tobacco, the va resistance gene commonly used to control Potato virus Y (PVY) corresponds to a large deletion affecting the eIF4E‐1 gene on chromosome 21. Here, we compared the resistance durability conferred by various types of mutations affecting eIF4E‐1 (deletions of various sizes, frameshift or nonsense mutations). The ‘large deletion’ genotypes displayed the broadest and most durable resistance, whereas frameshift and nonsense mutants displayed a less durable resistance, with rapid and frequent apparition of resistance‐breaking variants. In addition, genetic and transcriptomic analyses revealed that resistance durability is strongly impacted by a complex genetic locus on chromosome 14, which contains three other eIF4E genes. One of these, eIF4E‐3, is rearranged as a hybrid gene between eIF4E‐2 and eIF4E‐3 (eIF4E‐2‐3) in the genotypes showing the most durable resistance, while eIF4E‐2 is differentially expressed between the tested varieties. RNA‐seq and quantitative reverse transcriptase‐polymerase chain reaction experiments demonstrated that eIF4E‐2 expression level is positively correlated with resistance durability. These results suggest that besides the nature of the mutation affecting eIF4E‐1, three factors linked with a complex locus may potentially impact va durability: loss of an integral eIF4E‐3, presence of eIF4E‐2‐3 and overexpression of eIF4E‐2. This latter gene might act as a decoy in a non‐productive virus–plant interaction, limiting the ability of PVY to evolve towards resistance breaking. Taken together, these results show that va resistance durability can in large part be explained by complex redundancy effects in the eIF4E gene family.

Microplitis bicoloratus bracovirus modulates innate immune suppression through the eIF4E–eIF4A axis in the insect Spodoptera litura

Eukaryotic initiation factor 4E (eIF4E) is regulated during the innate immune response. However, its translational regulation under innate immune suppression remains largely unexplored. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus harbored by the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we generated eIF4E dsRNA and used it to silence the eIF4E gene of S. litura, resulting in a hallmark immunosuppressive phenotype characterized by increased apoptosis of hemocytes and retardation of head capsule width development. In response to natural parasitism, loss of eIF4E function was associated with similar immunosuppression, and we detected no significant differences between the response to parasitism and treatment with eIF4E RNAi. Under MbBV infection, eIF4E overexpression significantly suppressed MbBV-induced increase in apoptosis and suppressed apoptosis to the same extent as co-expression of both eIF4E and eIF4A. There were no significant differences between MbBV-infected and uninfected larvae in which eIF4E was overexpressed. More importantly, in the eIF4E RNAi strain, eIF4A RNAi did not increase apoptosis. Collectively, our results indicate that eIF4E plays a nodal role in the MbBV-suppressed innate immune response via the eIF4E–eIF4A axis.

P-mTOR, p-4EBP-1 and eIF4E expression in canine prostatic carcinoma

The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC.

Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease.

Cassava (Manihot esculenta) is an important tropical subsistence crop that is severely affected by cassava brown streak disease (CBSD) in East Africa. The disease is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Both have a (+)-sense single-stranded RNA genome with a 5’ covalently-linked viral protein, which functionally resembles the cap structure of mRNA, binds to eukaryotic translation initiation factor 4E (eIF4E) or its analogues, and then enable the translation of viral genomic RNA in host cells. To characterize cassava eIF4Es and their potential role in CBSD tolerance and susceptibility, we cloned five eIF4E transcripts from cassava (accession TMS60444). Sequence analysis indicated that the cassava eIF4E family of proteins consisted of one eIF4E, two eIF(iso)4E, and two divergent copies of novel cap-binding proteins (nCBPs). Our data demonstrated experimentally the coding of these five genes as annotated in the published cassava genome and provided additional evidence for refined annotations. Illumina resequencing data of the five eIF4E genes were analyzed from 14 cassava lines tolerant or susceptible to CBSD. Abundant single nucleotide polymorphisms (SNP) and biallelic variations were observed in the eIF4E genes; however, most of the SNPs were located in the introns and non-coding regions of the exons. Association studies of non-synonymous SNPs revealed no significant association between any SNP of the five eIF4E genes and the tolerance or susceptibility to CBSD. However, two SNPs in two genes were weakly associated with the CBSD responses but had no direct causal-effect relationship. SNPs in an intergenic region upstream of eIF4E_me showed a surprising strong association with CBSD responses. Digital expression profile analysis showed differential expression of different eIF4E genes but no significant difference in gene expression was found between susceptible and tolerant cassava accessions despite the association of the intergenic SNPs with CBSD responses.

Abstract IA29: Role of eIF4F in anticancer drug resistance

Abstract Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the hallmarks of cancer. Specific RBPs allow the selective regulation of cancer genes at multiple post-transcriptional levels from pre-mRNA splicing/polyadenylation to mRNA stability/translation. These multiple activities are mediated by RBP binding to mRNAs. In recent years, we have studied how the eIF4F translation initiation factor bound to the 7-methylguanosine cap structure present at the 5'-end of all cellular mRNAs contributes to tumorigenesis with a focus on its role in chemoresistance as well as the promising use of new small molecule inhibitors of the complex, including flavaglines/rocaglates, hippuristanol and pateamine A. We have specifically shown that, due to its location downstream of the the PI(3)K/AKT/mTOR pathway and the RAS-RAF-MEK-ERK-MNK mitogen-activated protein kinase (MAPK) signal transduction pathways, eIF4F is a nexus of resistance to anti-BRAF and anti-MEK therapies in both BRAF-mutated (melanoma, colon, thyroid) and NRAS-mutated (melanoma) cancer cells. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a novel flavagline (FL3) that is insensitive to P-glycoprotein-mediated multidrug resistance, synergizes with inhibiting BRAF and MEK to kill BRAF-mutant cancer cells and synergizes with inhibiting MEK to kill NRAS-mutant cancer cells in melanoma. In parallel, while investigating a set of data based on high-throughput sequencing of polyadenylated transcripts 3'-ends (3'-Seq), we have found that a short eIF4E mRNA isoform is generated through alternative use on an intronic polyadenylation (IPA) site. The global generation of transcripts with shorter 3' untranslated region (3'UTR) is known to occur during enhanced cell proliferation and transformation into cancer cells. However, the involvement of alternative polyadenylation in the response to targeted therapies and in the spreading of cancer cells to metastatic sites is unknown. IPA site usage is known to be widely inhibited by the U1 small ribonucleoprotein particle (U1) bound to an adjacent 5' splice site (5'ss). We have found that targeting U1 with an antisense oligonucleotide (U1-ASO) leads to activation of the use of IPA sites in the eIF4E gene but also in several genes with functions enriched in cell movement and involved in the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) and PI(3)K-AKT-mTOR signal transduction pathways. We have also demonstrated that the specificity of IPA regulation by U1 during tumor cell spreading is provided by the regulation of its binding to specific 5'ss by the RBP and U1-interacting splicing factor TIA1. The significance of these findings in tumor cell spreading and in the response to targeted therapies will be discussed. Citation Format: Galina Boldina, Maricarmen Vallejos, Delphine Allard, Isabelle Girault, Hélène Malka-Mahieu, Laurent Désaubry, Martin Dutertre, Caroline Robert, Stéphan Vagner. Role of eIF4F in anticancer drug resistance. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA29.

EIF4E Resistance: Natural Variation Should Guide Gene Editing.

eIF4E translation initiation factors have emerged as major susceptibility factors for RNA viruses. Natural eIF4E-based resistance alleles are found in many species and are mostly variants that maintain the translation function of the protein. eIF4E genes represent major targets for engineering viral resistance, and gene-editing technologies can be used to make up for the lack of natural resistance alleles in some crops, often by knocking out eIF4E susceptibility factors. However, we report here how redundancy among eIF4E genes can restrict the efficient use of knockout alleles in breeding. We therefore discuss how gene-editing technologies can be used to design de novo functional alleles, using knowledge about the natural evolution of eIF4E genes in different species, to drive resistance to viruses without affecting plant physiology.

Quantitative expression of the eukaryotic translation initiation factor 4E (eIF4E) in egyptian acute leukemia patients and its clinical significance

Purpose: The eukaryotic translation initiation factor eIF4E is part of the eIF4F protein complex, which includes, in addition to eIF4E, eIF4G (a scaffolding protein) and eIF4A (an ATP-dependent RNA helicase). The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers including leukemias. Methods: In this study, the expression level of eIF4E gene was analyzed in 20 normal healthy controls and 64 patients with de novo acute leukemia (33 Acute myeloid leukemia (AML) and 31 Acute lymphoblastic leukemia (ALL)) using a real-time quantitative reverse-transcriptase polymerase chain reaction (RTQ-PCR) to investigate a possible relation, association or correlation with the clinical features at diagnosis, such as age, gender, lineage, hemoglobin (Hb), total leucocytic count (TLC), platelet count and bone marrow (BM) blast cell infiltration as well as its effect on patients outcome. Results: Comparing AML and ALL patients as regards their clinical and laboratory data showed no statistical significance for TLC and hemoglobin ( p -0.838 and 0.920) respectively, but was of statistically significant difference for platelets ( p = 0.022) and bone marrow blasts percentage ( p = 0.007). Comparison between the 2 groups as regards eIF4E level was of no statistically significant difference, p -value being ( p = 0.257) but there was statistically significant difference between eIF4E expression level in AML/Control ( p = 0.002), ALL/Controls ( p = 0.025). Also analysis of overall survival (OS) time and disease free survival (DFS) in each group and its relation to eIF4E gene showed no statistical significance ( p = 0.843 and 0.310) respectively in AML group and ( p = 0.971 and no p -value for DFS in ALL as all cases remained alive except for one case while 3 cases were relapsed) in ALL group. Correlation studies showed no significant correlation between AML group and eIF4E gene level as regards age, TLC, hemoglobin and platelets (r = -0.064, p = 0.722; r = 0.062, p = 0.732; r = 0.068, p = 0.712; and r = -0.318, p = 0.071) respectively, while there was significant positive moderate correlation on comparing bone marrow blast% and eIF4E gene level (r = 0.545 and p = 0.001 ). There was no significant correlation between ALL group and Eif4e gene level as regards age, TLC, hemoglobin, platelets and bone marrow blasts% (r = -0.214, p = 0.248; r = 0.175, p = 0.347; r = -0.056, p = 0.766; r = -0.072, p = 0.700; and r = -0.0004, p = 0.983) respectively. Conclusion: eIF4E was found to be elevated in acute leukemia patients in relation to normal controls and its levels were more in myeloid than lymphoid leukemia and positively correlated with the blast percentage in AML thus its level may contribute to leukemogenesis. eIF4E levels and translation initiation may be an attractive target for anticancer therapeutics.

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines.

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.

Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology.

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING-induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh-eIF4E1(PI24)-pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re-establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss-of-function alleles to generate resistance may be counter-productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.

A Mutant eIF4E Confers Resistance to Potato Virus Y Strains and is Inherited in a Dominant Manner in the Potato Varieties Atlantic and Russet Norkotah

Potato Virus Y (PVY) is one of the most important pathogens affecting potato production worldwide. Here we tested the efficacy of engineered resistance against PVY in two popular susceptible potato varieties, Russet Norkotah and Atlantic. This resistance is based on the expression of a modified potato eIF4E gene containing mutations homologous to those in the recessive resistant allelic variant found in pepper. The expression of this eIF4E variant conferred differential resistance responses against three prevalent PVY strains including PVYO, PVYN:O and PVYNTN in the five tested transgenic lines. When resistance was established, the virus was not detectable in the inoculated leaves, the newly emerged leaves, nor in the sprouted tubers. Some strains were restrained to a low but detectable level in some of the potato lines. Crosses between transformed and wild type lines demonstrated that the engineered resistance gene was inherited in a dominant manner.

Knockdown of eIF4E suppresses cell proliferation, invasion and enhances cisplatin cytotoxicity in human ovarian cancer cells.

Eukaryotic initiation factor 4E (eIF4E) plays an important role in cap-dependent translation. The overexpression of eIF4E gene has been found in a variety of human malignancies. In this study, we attempted to identify the potential effects of eIF4E and explore the possibility of eIF4E as a therapeutic target for the treatment of human ovarian cancer. First the activation of eIF4E protein was detected with m7-GTP cap binding assays in ovarian cancer and control cells. Next, the eIF4E-shRNA expression plasmids were used to specifically inhibit eIF4E activity in ovarian cancer cells line A2780 and C200. The effects of knockdown eIF4E gene on cell proliferation, migration and invasion were investigated in vitro. Moreover, the changes of cell cycle and apoptosis of ovarian cancer cells were detected by flow cytometry. Finally, we investigated the effect of knockdown of eIF4E on the chemosensitivity of ovarian cancer cells to cisplatin in vitro. Our results show there is elevated activation of eIF4E in ovarian cancer cells compared with normal human ovarian epithelial cell line. The results of BrdU incorporation and FCM assay indicate that knockdown of eIF4E efficiently suppressed cell growth and induce cell cycle arrest in G1 phase and subsequent apoptosis in ovarian cancer cells. From Transwell assay analysis, knockdown eIF4E significantly decrease cellular migration and invasion of ovarian cancer cells. We also confirmed that knockdown eIF4E could synergistically enhance the cytotoxicity effects of cisplatin to cancer cells and sensitized cisplatin-resistant C200 cells in vitro. This study demonstrates that the activation of eIF4E gene is an essential component of the malignant phenotype in ovarian cancer, and aberration of eIF4E expression is associated with proliferation, migration, invasion and chemosensitivity to cisplatin in ovarian cancer cells. Knockdown eIF4E gene can be used as a potential therapeutic target for the treatment of human ovarian cancer.

HPV18 E7 induces the over-transcription of eIF4E gene in cervical cancer.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in cervical cancer (CC). However, the molecular mechanisms are unclear. This study aimed to investigate the molecular mechanism of eIF4E gene overexpression in CC. The human papillomavirus (HPV) type 18 E7 and eIF4E mRNAs were measured following knock down or overexpression of E7 gene by RT-PCR and real-time PCR. Cell counting kit-8 assay was used to determine the cell proliferation. Flow cytometry was used to analyze the cell cycle and apoptosis. Transwell system was employed to determine the cell migration. Overexpression of E7 gene increased eIF4E mRNA level by 24.3% (P<0.01) in HPV negative C33A cells. Knock down of E7 decreased markedly eIF4E mRNA by 73% (P<0.01) in HPV18 positive HeLa cells. Under the state of high expression of E7, 1) up-regulation of eIF4E drastically promoted the cell proliferation, cell cycle and cell migration, and inhibited the cell apoptosis. 2) down-regulation of eIF4E significantly inhibited the cell proliferation, cell cycle and the ability of cell migration, and also promoted the apoptosis of cervical cancer cells. HPV E7 induced eIF4E gene over transcription which might be a new marker for CC. The finding broadens the understanding of the CC carcinogenesis.