Ephrin-A1 and its primary receptor, EphA2, are involved in numerous physiological processes and have been intensely studied for their roles in malignancy. Ephrin-Eph signalling is complex on its own and is also cell-type dependent, making elucidation of the exact role of ephrin-A1 in neoplasia challenging. Multiple oncogenic signalling pathways, such as MAP/ERK and PI3K are affected by ephrin-A1, and in some cases evidence suggests the promotion of a specific pathway in one cell or cancer type and inhibition of the same pathway in another type of cell or cancer. EphrinA1 also plays an integral role in angiogenesis and tumor neovascularization. Until recently, studies investigating ephrins focused on the ligands as GPI-anchored proteins that required membrane anchoring or artificial clustering for Eph receptor activation. However, recent studies have demonstrated a functional role for soluble, monomeric ephrin-A1. This review will focus on various forms of ephrin-A1-specific signalling in human malignancy.

EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.

Schizophrenia, a progressive disorder displaying widespread pathological changes, is associated with the loss of glutamatergic function and selective loss of cytoskeletal proteins, such as MAP2, in regions severely affected by this disease. As schizophrenia is associated with perinatal brain trauma, we monitored changes in several functionally different proteins following injury-promoting MK801 blockade of N-methyl-D-aspartate receptors in neonatal rats. Within the somatosensory cortex, MK801 triggered robust, caspase-3-dependent apoptotic injury, reduced expression of cytoskeletal proteins MAP2 and tau, and increased synapse associated protein SNAP25. Thus, both neuronal injury and loss of structural elements important for successful cell-cell contact may reorganize brain circuitry, which at later ages could promote similar behavioral changes observed in schizophrenia.

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Our goal is to therapeutically exploit Glioblastoma Multiforme (GBM) restricted biomarkers that are not expressed in normal brain. Since GBMs are highly heterogeneous tumors, targeting any single biomarker will likely not be relevant to all GBMs. Thus, we hypothesize that utilizing a combinatorial approach that targets two GBM-restricted biomarkers (IL13Rα2 and EphA2) can successfully deliver high doses of targeted therapy to nearly all GBM patients. IL13Rα2 and EphA2 are membrane-associated receptor biomarkers that are independently expressed and each present in the majority of GBMs. We therefore created high-affinity ligands that separately target each biomarker. To target IL13Rα2, we created a novel IL13Rα2-Targeted Quadruple Mutant of IL13 (TQM13; IL13.E13K.R66D.S69D.K105R) based on our prior work that identified functional “hotspot” mutations. Recombinant TQM13 was expressed in an E. coli expression system and purified via Nickel-based affinity chromatography. Binding to the tumor-associated IL13Rα2 was confirmed with Biacore binding analysis. Disruption of binding to the physiologically abundant IL13Rα1/IL4Rα heterodimer was confirmed by TF1 proliferation assay. To target EphA2, we genetically fused its high-affinity ligand, EphrinA1, to the constant domain of human IgG1 (Fc1) and expressed it in a CHO expression system. Binding to the EphA2 biomarker was confirmed via ELISA and autoradiography. Each ligand was separately radiolabeled utilizing the IODOGEN method. Binding of each ligand to 10 human GBM tumor specimens was measured via autoradiography. TQM13 demonstrated high affinity towards the GBM-restricted IL13Rα2 (KD∼2nM), but did not bind/activate the physiologically abundant IL13Rα1/IL4Rα heterodimer. We confirmed functionality of purified EphrinA1.Fc1 by eliciting cell rounding and EphA2 activation in U251MG cells. We investigated the potential of each ligand to bind a series of 10 human GBM specimens either alone or in combination by performing autoradiography with 125I-TQM13 and 125I-EphrinA1.Fc1. 125I-TQM13 demonstrated specific binding towards 8/10 specimens (5/10 moderate/strong binding). 125I-EphrinA1.Fc1 specifically bound 10/10 specimens (7/10 moderate/strong binding). When both ligands were co-incubated on the same specimens, there was an additive binding and all specimens bound the TQM13/EphrinA1 mixture (9/10 strong binding, 1/10 moderate binding). Neither ligand significantly bound normal brain. Importantly, we found that even in subpopulations of GBM cells that did not express IL13Rα2 still expressed EphA2. We successfully created two ligands that separately target attractive GBM-restricted biomarkers and demonstrated that utilizing them in combination targeted all tested GBM samples and subpopulations of GBM cells. We anticipate that these ligands can therefore be used to deliver targeted therapeutics selectively to GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2011-1650