Glioblastoma multiforme (GBM), a high-grade astrocytoma, is the most common primary brain tumor in adults. GBM is a highly lethal cancer associated with neurological morbidity, and median survival of GBM patients is between 12 to 15 months. Previous work in our laboratory demonstrated that the EphA2 receptor, a tyrosine kinase protein receptor, but not its preferred ligand, ephrinA1, is over expressed in GBM specimens and cell lines, and its expression is linked to malignancy grade and patient survival. Binding of ephrinA1 to EphA2 results in receptor downregulation and internalization, which mediates reversal of malignant properties in GBM cells. Moreover, ectopic expression of ephrinA1 in GBM cells results in its release into the extracellular environment as a soluble, monomeric protein which mediates its effects in a paracrine manner. Thus, we have begun studying the structure-function relationship of ephrinA1 with the goal of developing an optimal soluble ligand for EphA2. The region selected was the GH loop of ephrinA1, a highly conserved region known to mediate high-affinity binding of ephrinA5 to EphA3 and very recently confirmed to mediate ephrinA1 to EphA2 binding. Alanine substitution mutants of all amino acids within the ephrinA1 GH loop (H-108FQRFTPFTLGKEFKE123G-OH) were generated by site-directed mutagenesis. Sequence-verified ephrinA1-mutant plasmids were then transfected into U-251 MG GBM cells. Individual clones were selected and high-expressing clones were further cultured. Mutant ephrinA1 expression in the media was verified by western blotting (WB). Conditioned media (CM) was assayed for ephrinA1 content using a sandwich ELISA. Standardized wild-type and mutant CM was used in subsequent experiments. Functional activity of each mutant was assessed by EphA2 down-regulation in U-251 cells. Alanine substitutions at positions P113 and T115 enhanced, while mutants of ephrinA1 at positions F111, T112, F114, L116, K118, E119, and F120 had a deleterious effect on EphA2 down-regulation when compared to that induced by wild type ephrinA1. In addition, those mutations that enhanced receptor down-regulation also caused dramatic changes in cell morphology in GBM cells within 15 min of treatment. Furthermore, treatment using P113A and T155A CM decreased phosphorylation of ERK within 30 min, indicative of suppression of intracellular signaling survival pathways. Being that ephrinA1 is a tumor-suppressing factor in GBM, finding its optimal genetically engineered variant(s) will have an important impact on the design and production of novel anti-tumor agents. These molecularly-targeted agents will represent a novel approach to GBM treatment.

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 5465.