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Elucidation of the Proteases Responsible for and the Form of Soluble, Monomeric EphrinA1 Released From Cancer Cells

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abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor. Despite the current standard of care, consisting of surgery, radiotherapy, and chemotherapy, the prognosis for GBM patients remains dismal, with a median survival of only 14.5 months. Therefore, there is great need for novel therapeutics with the ability to target GBM cells and spare normal brain tissue. In a search for novel tumor-specific targets, ephA2 was identified as an up-regulated gene in GBM patients. Further analysis demonstrated the overexpression of the receptor at the protein level in tumor specimens and cell lines while being absent in normal brain, making EphA2 an attractive target for novel therapeutics and imaging agents. EphrinA1 is a 182-amino acid, GPI-linked ligand for the EphA2 receptor. Activation of the receptor by ephrinA1 leads to a decrease in the oncogenic properties of GBM cells. We recently uncovered that a monomeric functional form of ephrinA1 is released from GBM cells. We thus explored the mechanism of release from the cell membrane, as well as the primary sequence of this monomeric form. We demonstrate here that multiple matrix 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 within the C-terminus, ending in amino acids valine-175, histidine-177, and serine-178. Moreover, ephrinA1 truncated at amino acid 175 but still GPI linked (ephrinA1-175) is not released, underlining the necessity of amino acids 175-181 for proteolysis. Furthermore, recombinant ephrinA1-175 retains activity towards the EphA2 receptor. These findings define the mechanism of release and the isoforms of monomeric ephrinA1. Moreover, in attempts to design ephrinA1-based recombinant anti-cancer therapeutics or imaging agents in the form of protein fusion or conjugates to the C-terminal end, ephrinA1 should only retain through amino acid 175 in order to prevent inevitable cleavage within the tumor microenvironment.
subject
EphA2
EphrinA1
Glioblastoma
MMP
contributor
Beauchamp, Amanda Susan (author)
Debinski, Waldemar (committee chair)
Lively, Mark (committee member)
Seeds, Michael (committee member)
Kridel, Steve (committee member)
Mintz, Akiva (committee member)
date
2012-06-12T08:35:56Z (accessioned)
2012-12-12T09:30:07Z (available)
2012 (issued)
degree
Molecular Medicine and Translational Science (discipline)
embargo
2012-12-12 (terms)
identifier
http://hdl.handle.net/10339/37276 (uri)
language
en (iso)
publisher
Wake Forest University
title
Elucidation of the Proteases Responsible for and the Form of Soluble, Monomeric EphrinA1 Released From Cancer Cells
type
Dissertation

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