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Redefining the role of eIF4E dose in development, cancer, and protein synthesis

ABSTRACT: eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the requirement for eIF4E dose at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we surprisingly found that 50% reduction in eIF4E, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation and tumorigenesis. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for eIF4E dose specifically in translating a network of mRNAs enriched for a unique 5’UTR signature. In particular, we demonstrate that eIF4E dose is essential for translating mRNAs regulating reactive oxygen species (ROS) that fuel transformation and cancer cell survival in vivo. Therefore, mammalian cells have evolved surplus eIF4E levels that cancer cells hijack to drive a translational program supporting tumorigenesis

ORGANISM(S): Mus musculus

PROVIDER: GSE68238 | GEO | 2015/07/01

SECONDARY ACCESSION(S): PRJNA282121

REPOSITORIES: GEO

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Project description:Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease. To globally address the issue of which transcripts mediate the anti-apoptotic functions of eIF4E, we used a model in which NIH 3T3 cells ectopically overexpress translation initiation factor 4E (eIF4E) resulting in an apoptosis-resistant and tumorigenic phenotype. We performed a comprehensive study of apoptosis resistance in the NIH 3T3/4E model using an array containing 15k cDNAs and ESTs (NIH 15k collection printed at The Ontario Cancer Institute). To facilitate data analysis in the two-color competitive hybridization microarray approach employed, we used a universal standard mRNA [non-stratified poly(A) selected RNA derived from NIH 3T3 cells cultured in full growth medium]. We compared signals generated from this standard to both the light (fractions 4â6, <1 ribosome/transcript) and the heavy (fractions 7â10, >1 ribosomes/transcript) polyribosome fractions of RNA from NIH 3T3 and NIH 3T3/4E cells cultured either in complete growth medium or after 16 h of serum starvation, a time point before any evidence of cytochrome c release from the mitochondria, a signature step at the apex of the cell death program. We selected the 16 h time point in order to identify genes that might govern the decision to enter the apoptotic pathway, and exclude those involved in the actual process of cell execution.

Project description:Translation initiation factor eIF4E is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. Using immortalized human breast epithelial cells, we report that elevated expression of elF4E translationally activates the TGFβ pathway, promoting cell invasion, loss of cell polarity, increased cell survival and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGFβ receptor signaling complex containing α3β1 integrins, β-catenin, TGFβ receptor I, E-cadherin and phosphorylated Smads2/3. This receptor complex acutely sensitizes non-malignant breast epithelial cells to activation by typically sub-stimulatory levels of activated TGFβ. TGFβ can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGFβ, eIF4E confers selective mRNA translation, reprogramming non-malignant cells to an invasive phenotype by reducing the set-point for stimulation by activated TGFβ. Overexpression of eIF4E may be a pro-invasive facilitator of TGFβ activity. MCF10A cells were subjected to consitutive silencing with a non-silencing control shRNA or an shRNA to eIF4E. Total RNA, light polysomal RNA (2-3 ribosome fraction) and heavy polysomal RNA (greater than or equal to 4 ribosome fraction) were purified and used for hybridization to Affymetrix 133 2.0 Plus arrays. Data analysis was conducted using MicroArray Suite Software from Affymetrix. To remove probe sets with insignificant differences between perfect match and mismatch data, which creates a more robust data set of greater clarity without a high level of background noise, discrimination values for each probe pair were calculated for low intensity ratios using Wilcoxon signed rank test to assess significance and data re-assigned as either changed or unchanged mRNAs. Data sets were compared using Expressionist Suite software. Significance of mRNAs were assessed using fold changes and the FDRs estimated based on t-tests.

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Redefining the role of eIF4E dose in development, cancer, and protein synthesis

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