Phosphorylation of EPHA2 on S897 Promotes Cell Survival from Irradiation

Abstract

The EphA2 tyrosine kinase receptor is overexpressed in a variety of cancers that are also treated with radiation. Previously, we investigated the effect of radiation on the EphA2 receptor and showed that X-rays induced S897 EphA2 phosphorylation in a MEK/ERK/RSK dependent manner in lung, cervical, and pancreatic cell lines. Phosphorylation of EphA2 on S897 is correlated with increased cell motility, invasion and poor patient outcomes. However, the biological significance of S897 phosphorylation was not determined. In this project, we characterized stable cell lines that expressed either wild-type EphA2 or the S897A mutant to determine if S897 phosphorylation impacts the response to radiation to better understand mechanisms of radioresistance. To determine if S897 EphA2 phosphorylation plays a role in the radiation response, HEK293 cell lines were generated that stably expressed vector only, human recombinant wild-type EphA2 (WT-EphA2) or the S897A mutant of EphA2 (S897A-EphA2). Multiple, independent, stable cell lines were analyzed for growth rate, DNA damage signaling, and cell survival after treatment with radiation as measured by MTT assay, Western blotting, and clonogenic assay respectively. Statistical significance was determined using the Student t test. WT-EphA2 underwent constitutive S897 phosphorylation in HEK293 cells whereas, as expected, no phosphorylation of S897 was observed in cells expressing the S897A mutant. No significant difference in cell growth rate was observed between cell lines expressing vector, WT-EphA2 or S897A-EphA2. There was also no difference in the amount of radiation-induced DNA damage signaling as judged by Chk1 phosphorylation between the cell lines. However, cell lines expressing WT-EphA2, on average, had a ∼2 fold greater clonogenic survival than cells expressing the S897A mutant after treatment with 2 Gy of X-rays (p < 0.05 from 3 independent experiments). Previously we showed that irradiation of cancer cell lines resulted in the induction of S897 EphA2 phosphorylation in a growth factor pathway dependent manner. Here we show that S897 phosphorylation promotes cell survival from irradiation suggesting that S897 phosphorylation constitutes a cell survival mechanism. Further experiments are underway to determine how S897 phosphorylation promotes cell survival. These results suggest that it may be beneficial to target pathways that catalyze EphA2 S897 phosphorylation to reduce tumor cell survival and improve the therapeutic index.