Effect of EphA2 knockdown on melanoma metastasis depends on intrinsic ephrinA1 level.

Abstract

Upregulation of receptor tyrosine kinase EphA2 has been found to be associated with a poor prognosis in many types of cancer and is considered an attractive therapeutic target. As yet, few efforts have been focused on its tumor suppressive activity triggered by its ligand, ephrinA1. Here, we aimed to determine the potential of ephrinA1 as an important player in melanoma metastasis. Data from the Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) were analyzed to explore the expression and prognostic implications of EphA2 and ephrinA1 in melanoma. Western blotting, shRNA, colony formation and immunofluorescence assays, as well as two in vivo xenograft models (subcutaneous and metastatic) were used to evaluate the role of EphA2 in melanoma progression. Akt inhibition and ephrinA1-Fc were used to confirm the influence of Akt activation and ephrinA1 levels on the EphA2 effects. Immunohistochemistry (IHC) was performed on xenograft and patient melanoma tissues. We found that high levels of ephrinA1, but not EphA2, were negatively correlated with melanoma metastasis. The expression levels of EphA2 and ephrinA1 were not correlated. After EphA2 downregulation, colony forming abilities and lung metastatic growth were reduced in melanoma cell lines with a low ephrinA1 expression, but were increased in melanoma cell lines with a high ephrinA1 expression. EphA2-mediated colony formation in EphA2-high/ephrinA1-low cells was found to be Akt-dependent and to be inhibited by the addition of ephrinA1-Fc. IHC staining of primary melanoma specimens revealed that EphA2-high/ephrinA1-low patients exhibited poorer outcomes than EphA2-high/ephrinA1-high patients. From our data we conclude that evaluation of ephrinA1 levels may be helpful for the application of EphA2-targeted therapies and for prognostic predictions in melanoma patients.