Abstract 1218: Investigation of the mechanism and form of ephrinA1 released from cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction. EphrinA1 is a GPI-linked ligand for the EphA2 receptor, which is overexpressed in Glioblastoma Multiforme (GBM). Activation of the receptor by ephrinA1 leads to a decrease in the oncogenic properties of GBM cells. We have recently uncovered that a monomeric functional form of ephrinA1 is released from GBM cells; however, the exact mechanism of release from the cell membrane, as well as the exact primary structure of this monomeric form need to be unraveled. Methods. To confirm the functionality of monomeric ephrinA1, GBM cells were treated with recombinant monomeric ephrinA1 or recombinant ephrinA1 homodimer-Fc and cell rounding assessed by phase contrast microscopy. EphA2 down-regulation was assessed by Western blot analysis for EphA2 in whole cell lysates. Due to a lack of canonical protease consensus sequences, with the exception of a thrombin cleavage site downstream of the GPI anchor, we first used broad spectrum inhibitors of serine proteases and matrix metalloproteinases, in addition to a thrombin inhibitor. Cells were treated with each inhibitor and media collected for Western blot analysis for ephrinA1. Furthermore, cytoplasmic and membrane associated proteins were separated using the ProteoJET membrane protein extraction kit (Fermentas). Additionally, an acellular cleavage assay was performed in which ephrinA1-Fc was incubated with active MMP's (Enzo Life Sciences) and cleavage products visualized by Western blot. GBM cells were also treated with MMP-13 or an MMP-13 inhibitor, respectively. Results. Monomeric recombinant ephrinA1 was able to induce morphological changes in GBM cells as well as lead to the down-regulation of the EphA2 receptor. GM-6001, a broad range MMP inhibitor and AEBSF, a serine protease inhibitor both prominently decreased the release of ephrinA1 from GBM cells in a dose-dependent manner, and increased ephrinA1 was retained on the membrane of MMP inhibitor-treated cells versus control cells. However, a thrombin inhibitor increased the amount of ephrinA1 released into the media. These results are indicative of proteolytic cleavage being responsible for ephrinA1 release into the extracellular environment. In an acellular cleavage assay, MMP-13 was able to cleave ephrinA1-Fc among the 10 MMPs tested. Furthermore, MMP-13 was also able to cleave ephrinA1 from the surface of ephrinA1-transfected GBM cells. Conclusions. Monomeric ephrinA1 induces similar EphA2 activation within GBM cells compared to a clustered form and thus could contribute to the tumor suppressing properties of ephrinA1. The release of ephrinA1 from cancer cells takes place in response not only to an MMP, but also to serine protease(s). We have provided, for the first time, evidence suggesting that MMP-13 specifically is involved in ephrinA1 cleavage. This new knowledge is indispensable in the design of potential therapeutics/imaging reagents based on ephrinA1. 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 1218. doi:10.1158/1538-7445.AM2011-1218