Translation initiation factor eIF4E is a target for tumor cell radiosensitization

Abstract

A core component in the cellular response to radiation occurs at the level of translational control of gene expression. Because a critical element in translation control is the availability of the initiation factor eIF4E, which selectively enhances the cap-dependent translation of mRNAs, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E silencing enhanced the radiosensitivity of tumor cell lines but not normal cells. Similarly, pharmacologic inhibition of eIF4E with ribavirin also enhanced tumor cell radiosensitivity. eIF4E attenuation did not affect cell-cycle phase distribution or radiation-induced apoptosis, but it delayed the dispersion of radiation-induced γH2AX foci and increased the frequency of radiation-induced mitotic catastrophe. Radiation did not affect 4E-BP1 phosphorylation or cap-complex formation but it increased eIF4E binding to more than 1,000 unique transcripts including many implicated in DNA replication, recombination, and repair. Taken together, our findings suggest that eIF4E represents a logical therapeutic target to increase tumor cell radiosensitivity.

Figure 1:

Effect of eIF4E knockdown on clonogenic cell survival. Cultures were transfected with siRNA specific to eIF4E (eIF4E KD) or non-targeted siRNA (Scramble). A) Representative immunoblots from each cell line showing extent of eIF4E protein reduction 72h after transfection. B) 72h post-transfection cells were plated at specified densities and colony-forming efficiency was determined 10–14 days later. Surviving fractions for eIF4E KD cells were calculated after normalizing to the surviving fraction obtained for cells receiving the scrambled siRNA. Values shown represent the means ± SE for 3–4 independent experiments. *p < 0.04 according to Student’s t test (all tumor cell lines compared to HMEC).

Figure 2:

The effects of eIF4E knockdown on cellular radiosensitivity. A) MDA-MB-231, B) A549, C) DU145, D) MRC9, and E) HMEC cells were transfected with non-targeted siRNA (Scramble) or siRNA specific for eIF4E (eIF4E KD). 72h post-transfection cells were plated, allowed to attach for 6h, and irradiated. Colony-forming efficiency was determined 10–14 days later and survival curves were generated after normalizing for cell killing from siRNA alone. DEFs were calculated at a surviving fraction of 0.1. Values shown represent the mean ± SE for 3–4 independent experiments. * p < 0.05; ** p < 0.1 according to Student’s t test.

Figure 3:

Mechanism of radiosensitization by eIF4E knockdown. In the following experiments MDA-MB-231 cells were transfected with siRNA specific to eIF4E (eIF4E KD) or non-targeted siRNA (Scramble). All experiments were carried out 72 hours post-transfection. A) Cell cycle phase distribution was determined. Values represent the mean of three independent experiments. B) Cells were irradiated with 2 or 4Gy and collected at the specified time; γH2AX foci were counted in at least 50 cells per condition. Values shown represent the means ± SE for 3 independent experiments, *p < 0.04 according to Student’s t test (eIF4E KD compared to scramble). C) Cells were irradiated (2 Gy) and collected at the specified time points. Cells were classified as being in mitotic catastrophe by the presence of nuclear fragmentation, which was defined as a single cell containing two or more distinct nuclear lobes. At least 50 cells per condition were scored. Values represent the mean ± SE for 3 independent experiments. *p< 0.04

Figure 4:

The effect of radiation on eIF4E activation. A) MDA-MB-231 cells were irradiated (2 Gy) and collected at the specified times and subjected to immunoblot analysis. Actin was used as a loading control. B) m⁷-GTP affinity chromatography was performed on MDA-MB-231 cells that were irradiated and collected 1h after 2 Gy, and compared to unirradiated counterparts. m⁷-GTP bound and unbound proteins (flow through) were resolved via SDS-PAGE followed by immunoblot analysis. eIF4E was used as a loading control. Blots are representative of two independent experiments.

Figure 5:

Rip Chip analysis of the effects of radiation on eIF4E mRNA clients. MDA-MB-231 cells were irradiated (2 Gy) and collected 6 hours later. eIF4E was immunoprecipitated, RNA bound to eIF4E was isolated and subjected to microarray analysis and mRNAs whose binding to eIF4E after irradiation were classified using IPA. A) The top ten biological functions (containing 100 or more genes) of the mRNAs whose binding to eIF4E was increased by radiation. B) The biological functions of the mRNAs (with greater than 10 genes) within the DNA Replication, Recombination, and Repair category are further delineated. C) Network 4 and D) Network 6 are shown with dark red indicating not bound to bound and lighter red indicating fold increase ≥ 1.5. E) Immunoblot analysis of DNA Damage response related proteins predicted by RIP-Chip analysis to be induced by radiation. MDA-MB-231 cells were radiated (6 Gy) and collected at the specified times. Actin was used as a loading control. Blots are representative of two independent experiments.

Figure 5:

Rip Chip analysis of the effects of radiation on eIF4E mRNA clients. MDA-MB-231 cells were irradiated (2 Gy) and collected 6 hours later. eIF4E was immunoprecipitated, RNA bound to eIF4E was isolated and subjected to microarray analysis and mRNAs whose binding to eIF4E after irradiation were classified using IPA. A) The top ten biological functions (containing 100 or more genes) of the mRNAs whose binding to eIF4E was increased by radiation. B) The biological functions of the mRNAs (with greater than 10 genes) within the DNA Replication, Recombination, and Repair category are further delineated. C) Network 4 and D) Network 6 are shown with dark red indicating not bound to bound and lighter red indicating fold increase ≥ 1.5. E) Immunoblot analysis of DNA Damage response related proteins predicted by RIP-Chip analysis to be induced by radiation. MDA-MB-231 cells were radiated (6 Gy) and collected at the specified times. Actin was used as a loading control. Blots are representative of two independent experiments.

Figure 5:

Rip Chip analysis of the effects of radiation on eIF4E mRNA clients. MDA-MB-231 cells were irradiated (2 Gy) and collected 6 hours later. eIF4E was immunoprecipitated, RNA bound to eIF4E was isolated and subjected to microarray analysis and mRNAs whose binding to eIF4E after irradiation were classified using IPA. A) The top ten biological functions (containing 100 or more genes) of the mRNAs whose binding to eIF4E was increased by radiation. B) The biological functions of the mRNAs (with greater than 10 genes) within the DNA Replication, Recombination, and Repair category are further delineated. C) Network 4 and D) Network 6 are shown with dark red indicating not bound to bound and lighter red indicating fold increase ≥ 1.5. E) Immunoblot analysis of DNA Damage response related proteins predicted by RIP-Chip analysis to be induced by radiation. MDA-MB-231 cells were radiated (6 Gy) and collected at the specified times. Actin was used as a loading control. Blots are representative of two independent experiments.

Figure 6:

Effects of ribavirin on radiosensitivity. A) MDA-MB-231 cells were plated for clonogenic survival analysis and treated with 50 μM ribavirin for 1h, followed by radiation. Ribavirin was left on for the duration of the clonogenic assay. Values represent the mean ± SE for 3 independent experiments.