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Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

Martínez A, Sesé M, Losa JH, Robichaud N, Sonenberg N, Aasen T, Ramón Y Cajal S - PLoS ONE (2015)

Bottom Line: Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1).We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required.Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

No MeSH data available.


Related in: MedlinePlus

Phospho-eIF4E/4E-T binding is necessary for recovery after stress.A, Cells stably expressing eIF4E-S209D or-S209A were transfected with scramble or specific short hairpins to knockdown 4E-T expression. eIF4E-S209D significantly enhanced cell recovery from arsenite stress in scramble-transfected cells, whereas knockdown of 4E-T completely abolished any protective effect of eIF4E-S209D. B, Expression of the eIF4E mutants W73A, W73A/S209D, both unable to bind 4E-T, and S209D; W73A and W73A/S209D did not confer any resistance to arsenite treatment compared with S209D. * = P<0.05 and ** = P<0.01 compared to control, n = 3.
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pone.0123352.g005: Phospho-eIF4E/4E-T binding is necessary for recovery after stress.A, Cells stably expressing eIF4E-S209D or-S209A were transfected with scramble or specific short hairpins to knockdown 4E-T expression. eIF4E-S209D significantly enhanced cell recovery from arsenite stress in scramble-transfected cells, whereas knockdown of 4E-T completely abolished any protective effect of eIF4E-S209D. B, Expression of the eIF4E mutants W73A, W73A/S209D, both unable to bind 4E-T, and S209D; W73A and W73A/S209D did not confer any resistance to arsenite treatment compared with S209D. * = P<0.05 and ** = P<0.01 compared to control, n = 3.

Mentions: We speculated that the association between 4E-T and phosphorylated eIF4E might regulate resistance to stress. We therefore analyzed the role of the eIF4E/4E-T complex in MDA-MB-231 cells by knocking down 4E-T and by using eIF4E mutants unable to bind 4E-T. 4E-T protein levels were downregulated by over 80% following transfection with shRNA plasmids targeting 4E-T (S7 Fig). Knockdown of 4E-T in MDA-MB-231 cells did not affect proliferation (Fig 5A). However, it completely abolished the added resistance to arsenite mediated by eIF4E-S209D (Fig 5A). It thus appears that 4E-T is crucial for the protection against cellular stress conferred by phosphorylated eIF4E. To further delineate the hypothesis that a specific interplay between phospho-eIF4E and 4E-T is required for stress resistance, we used the eIF4E-S209D-W73A mutant that does not bind 4E-T ([4] and S8 Fig). The inability of eIF4E to interact with 4E-T completely abolished the increased resistance to stress mediated by the S209D mutant (Fig 5B). It should be noted that this mutant also do not bind eIF4G and initiates translation however. On the other hand, it does retain its ability to transport mRNA from the nucleus to the cytoplasm, suggesting mRNA export is not the mechanism of enhanced resistance. Overall, the results from our 4E-T knockdown experiments, supported by result where the eIF4E-W73A mutants unable to bind 4E-T, strongly support the theory that an association between 4E-T and the eIF4E protein is crucial for stress resistance.


Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

Martínez A, Sesé M, Losa JH, Robichaud N, Sonenberg N, Aasen T, Ramón Y Cajal S - PLoS ONE (2015)

Phospho-eIF4E/4E-T binding is necessary for recovery after stress.A, Cells stably expressing eIF4E-S209D or-S209A were transfected with scramble or specific short hairpins to knockdown 4E-T expression. eIF4E-S209D significantly enhanced cell recovery from arsenite stress in scramble-transfected cells, whereas knockdown of 4E-T completely abolished any protective effect of eIF4E-S209D. B, Expression of the eIF4E mutants W73A, W73A/S209D, both unable to bind 4E-T, and S209D; W73A and W73A/S209D did not confer any resistance to arsenite treatment compared with S209D. * = P<0.05 and ** = P<0.01 compared to control, n = 3.
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pone.0123352.g005: Phospho-eIF4E/4E-T binding is necessary for recovery after stress.A, Cells stably expressing eIF4E-S209D or-S209A were transfected with scramble or specific short hairpins to knockdown 4E-T expression. eIF4E-S209D significantly enhanced cell recovery from arsenite stress in scramble-transfected cells, whereas knockdown of 4E-T completely abolished any protective effect of eIF4E-S209D. B, Expression of the eIF4E mutants W73A, W73A/S209D, both unable to bind 4E-T, and S209D; W73A and W73A/S209D did not confer any resistance to arsenite treatment compared with S209D. * = P<0.05 and ** = P<0.01 compared to control, n = 3.
Mentions: We speculated that the association between 4E-T and phosphorylated eIF4E might regulate resistance to stress. We therefore analyzed the role of the eIF4E/4E-T complex in MDA-MB-231 cells by knocking down 4E-T and by using eIF4E mutants unable to bind 4E-T. 4E-T protein levels were downregulated by over 80% following transfection with shRNA plasmids targeting 4E-T (S7 Fig). Knockdown of 4E-T in MDA-MB-231 cells did not affect proliferation (Fig 5A). However, it completely abolished the added resistance to arsenite mediated by eIF4E-S209D (Fig 5A). It thus appears that 4E-T is crucial for the protection against cellular stress conferred by phosphorylated eIF4E. To further delineate the hypothesis that a specific interplay between phospho-eIF4E and 4E-T is required for stress resistance, we used the eIF4E-S209D-W73A mutant that does not bind 4E-T ([4] and S8 Fig). The inability of eIF4E to interact with 4E-T completely abolished the increased resistance to stress mediated by the S209D mutant (Fig 5B). It should be noted that this mutant also do not bind eIF4G and initiates translation however. On the other hand, it does retain its ability to transport mRNA from the nucleus to the cytoplasm, suggesting mRNA export is not the mechanism of enhanced resistance. Overall, the results from our 4E-T knockdown experiments, supported by result where the eIF4E-W73A mutants unable to bind 4E-T, strongly support the theory that an association between 4E-T and the eIF4E protein is crucial for stress resistance.

Bottom Line: Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1).We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required.Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca, VHIR, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

No MeSH data available.


Related in: MedlinePlus