Abstract
The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.
Highlights
Proteolytic enzymes and their regulatory networks, including cofactors, activators, and endogenous inhibitors, are frequently dysregulated in tumors resulting in increased protease activities that contribute to progression of disease [1]
We took advantage of the requirement for anthrax toxin to be proteolytically activated on the cell surface to engineer a novel testisin-activated anthrax toxin, protective antigen (PrAg)-PCIS toxin
We established that the PrAg-PCIS toxin can be activated by tumor cell-expressed testisin and inhibit the growth of tumor cells in both cell culture and in testisin-expressing tumors in vivo
Summary
Proteolytic enzymes and their regulatory networks, including cofactors, activators, and endogenous inhibitors, are frequently dysregulated in tumors resulting in increased protease activities that contribute to progression of disease [1]. Anthrax toxins requiring proteolytic activation have been engineered to target tumor-overexpressed proteases. Anthrax toxin is a cytotoxic pore-forming exotoxin secreted by Bacillus anthracis. PrAg (83 kDa) bound to its cell-surface receptor(s) www.impactjournals.com/oncotarget is proteolytically cleaved and activated by furin (FURIN) or furin-like proprotein convertases in an exposed flexible loop to generate an active C-terminal 63-kDa PrAg fragment. The newly-generated 63-kDa fragment remains receptor bound and catalyzes the formation of a PrAg/ receptor oligomer that presents docking sites to enable up to 4 molecules of LF or EF to bind and translocate into the cytosol, through an endosomal PrAg-formed pore, where they have potent cytotoxic effects [7]
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