Eukaryotic translation elongation factor 1-alpha 1 inhibits p53 and p73 dependent apoptosis and chemotherapy sensitivity

Abstract

The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.

Figure 1. eEF1A1 interacts with p73 and p53.

Figure 1A , HeLa cells were transfected with plasmids encoding HA-TAp73α alone or together with T7-eEF1A1. Equal amounts of whole cell extracts were immunoprecipitated with anti-T7 antibody, resolved by SDS-PAGE and immunoblotted with the indicated antibodies (anti-HA Y-11). Figure 1B , SaOS-2 cells were transfected with plasmids encoding HA-p53 and T7-eEF1A1, and treated with camptothecin (0.2 µM) for 18 hours. Equal amounts of whole cell extracts were immunoprecipitated with anti-T7 antibody, resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Parallel sets of inputs (75 µg) were resolved in the same gel to immunoblot with anti-T7 or anti-HA (HA.11) antibodies.

Figure 2. Ectopic expression of eEF1A1 inhibits apoptosis.

Figures 2A–B , eEF1A1 inhibits p53 and 73-induced apoptosis. HeLa cells were transfected with constant amounts of plasmids encoding HA-p53 or HA-TAp73α and increasing amounts of plasmids encoding T7- or HA-tagged eEF1A1 (p53/73 to eEF1A1 ratios were 1∶1, 1∶5 and 1∶10). Cells were lysed and whole cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. In panel A the top anti-HA blot is p73 and the bottom anti-HA blot is eEF1A1. Figure 2C , eEF1A1 inhibits chemotherapy-induced apoptosis. HeLa cells were transfected with increasing amounts of plasmids encoding HA-eEF1A1 and treated with camptothecin (5 µM) or cisplatin (2 µM) for 18 hours. Whole cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Figure 3. Inhibition of eEF1A1 enhances chemotherapy-induced apoptosis.

Figure 3A , cells were transfected with siRNA oligonucleotides specific for eEF1A1 or control, and treated with cisplatin (2 µM) for 18 hours. Whole cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Figure 3B , HeLa and A549 cells were transfected with siRNA oligonucleotides specific for eEF1A1 or control, treated with cisplatin (2 µM) for 18 hours and subjected to caspase-3/7 activity assays. The activity of control-transfected cells was set to 1 and fold change was plotted. Results are representative of three independent experiments performed in triplicate. Inset: A fraction of the cells were lysed and 25 µg of whole cell extract were immunoblotted with the indicated antibodies.

Figure 4. eEF1A1 is a negative regulator of p53 and 73 dependent apoptosis.

Figure 4A–B , HeLa (panel A) or U2OS (panel B) cells were transfected with siRNA oligonucleotides specific for eEF1A1 and/or p53/73, and treated with cisplatin (2 µM) for 18 hours. Whole cell extracts were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Figure 4C , Inhibition of eEF1A1 increases p53 stability. Hela cells were transfected with siRNA oligonucleotides specific for eEF1A1 or control, and treated with cycloheximide (CHX). Results are representative of three independent experiments. Top panel: Whole cell extracts were collected at the indicated times following CHX treatment, resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Lower panel: The amount of p53 protein relative to vinculin protein was quantified by densitometry and plotted versus the time of cycloheximide treatment. The amount of p53/vinculin at time zero was set to 100%.