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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

Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)
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pone.0123352.g001: Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)

Mentions: Phosphorylation of eIF4E Ser-209 by Mnk1/2 is known to affect cell proliferation and tumor malignancy [27]. To further understand the role of eIF4E phosphorylation in these processes, we infected immortalized and malignant cell lines with retrovirus to overexpress GFP (control) or Myc-tagged phosphomimetic (S209D) or phospho-dead (S209A) versions of eIF4E. The levels of protein expression between the two mutants were similar, and were approximately the same as that of endogenous eIF4E in MDA-MB-231 and HaCaT cells, indicating a physiologically relevant expression system (Fig 1A). Analysis of cell proliferation did not reveal any statistically significant differences between the three expression constructs (Fig 1B).


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)

Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)
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pone.0123352.g001: Overexpression of phosphomimetic eIF4E-S209D does not affect proliferation but increases clonogenic cell survival.A, Western blot analysis showed similar, moderate levels of Myc-tagged exogenous eIF4E-S209D or-S209A and endogenous eIF4E expression in MDA-MB-231 and HaCaT cell lines. B, MTT cell proliferation assays revealed no significant difference in cell growth between GFP—or eIF4E-mutant–expressing cells under normal conditions. C, Crystal violet staining of clonogenic colony formation assays clearly indicate an increase in both colony number and size upon eIF4E-S209D expression compared with GFP control cells, which is reflected by a statistically significant increase in the total number of stained cells as measured by overall crystal violet absorbance. A moderate but not statistically significant increase in eIF4E-S209A–expressing cells was noted. Graphs depict the overall cell growth as measured in a crystal violet absorption assay. The survival and growth advantage after expression of eIF4E-S209D is highly significant in both HaCaT and MDA-MB-231 cells treated with arsenite for 90 minutes to induce oxidative stress before being plated. D, Graphs representing the percentage of the number of colonies after arsenite treatment compared to normal conditions in each case. Arsenite treatment clearly decreases the number of colonies in both cell lines. * = P<0.05, ** = P<0.01 and *** = P<0.001, compared to control, n = 3. (AU: Absorbance Units)
Mentions: Phosphorylation of eIF4E Ser-209 by Mnk1/2 is known to affect cell proliferation and tumor malignancy [27]. To further understand the role of eIF4E phosphorylation in these processes, we infected immortalized and malignant cell lines with retrovirus to overexpress GFP (control) or Myc-tagged phosphomimetic (S209D) or phospho-dead (S209A) versions of eIF4E. The levels of protein expression between the two mutants were similar, and were approximately the same as that of endogenous eIF4E in MDA-MB-231 and HaCaT cells, indicating a physiologically relevant expression system (Fig 1A). Analysis of cell proliferation did not reveal any statistically significant differences between the three expression constructs (Fig 1B).

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