Abstract Background: We have previously shown that the EphA2 receptor is overexpressed in pancreatic cancer. Silencing of EphA2 receptor in combination with gemcitabine decreases tumor growth and metastases in a mouse model of gemcitabine resistant pancreatic cancer. However, regulation of EphA2 expression in pancreatic cancer is unknown. The objective of this study was to determine a possible relationship between Ras/MAPK pathway and EphA2. Methods: Pancreatic cancer cell lines, MPanc96 and MiaPaca-2, were used in this study. Ras was silenced with 2 different siRNA constructs; EphA2 protein and RNA expression levels were measured. ERK involvement in EphA2 regulation was analyzed by treating cells with 2 inhibitors for MEK (U0126 and PD 98059) and siRNA specific for either MEK1 or MEK2. Effect of MEK inhibition on EphA2 mRNA stability was examined by blocking mRNA synthesis with actinomycin D and measuring mRNA degradation by Q-PCR. In order to investigate the role of subcellular localization of ERK in regulation of EphA2 expression, we overexpressed cytoplasmic anchor protein PEA-15, which binds to ERK and sequesters it to the cytoplasm. Results: Silencing of mutant K-Ras decreased EphA2 mRNA and protein levels. EphA2 mRNA and protein levels decreased in a time- and dose-dependent manner after treatment with MEK chemical inhibitors. Inhibition of MEK activity did not affect EphA2 mRNA stability. Silencing of MEK2 but not MEK1 decreased EphA2 mRNA levels. Overexpression of PEA-15 depleted nuclear ERK, indicated by the absence of phosphorylated Fra-1. Depletion of the nuclear ERK did not affect EphA2 transcription, suggesting that EphA2 is regulated through the cytoplasmic ERK. Conclusions: These data suggest that EphA2 levels are regulated by the RAS / MEK2 / cytoplasmic ERK pathway via regulation of EphA2 mRNA transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3920.