Abstract
BackgroundThe most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation.ResultsWe developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1’s ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E.ConclusionsWe show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance.
Highlights
The most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery
We have developed an eIF4E:eIF4G NanoBit biosensor that accurately measures the disruption of the eIF4F complex by endogenous 4EBP1 upon mTORC1 inhibition and allows us to delineate specific effects of small molecule inhibitors on the eIF4F complex
The eIF4E:eIF4G NanoBit biosensor unlike other reported assays in the literature that measure eIF4F complex disruption, such as an in vitro time-resolved – fluorescence resonance excitation transfer (TR-FRET)-based method [35] that relies on purified protein and a proximity ligation assay (PLA) [34] methodology that measures the integrity of endogenous eIF4F complex in fixed and permeabilised cells, can quantify eIF4F complex integrity in live cells
Summary
The most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. Mutational activation of mitogenic signalling is a common event in human cancer and is frequently found in genes that encode components of the PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK pathways [1, 2]. Activation of these pathways induces changes in transcription, metabolism, proliferative rate, protein synthesis and other processes that contribute to cellular transformation [1, 2] Both these pathways converge to regulate the formation of the eukaryotic translation initiation complex 4F (eIF4F). Structural studies have established the presence of a non-canonical binding motif in the 4EBPs downstream of their primary interaction site with eIF4E. Despite the non-canonical site not being required for the interaction of the 4EBPs with eIF4E, it does play a critical role in ensuring that the 4EBPs efficiently compete with eIF4G for binding to eIF4E and repress translation [10,11,12,13,14]
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