Botkjaer, Kenneth A2; Deryugina, Elena I3; Dupont, Daniel Miotto4; Gårdsvoll, Henrik10; Bekes, Erin M3; Thuesen, Cathrine König6; Chen, Zhuo7; Chen, Zhou3; Ploug, Michael11; Quigley, James P12; Andreasen, Peter A13
1 Ploug Group, BRIC Research Groups, BRIC, Københavns Universitet2 Department of Molecular Biology and Genetics, Aarhus University, Copenhagen, Denmark.3 unknown4 Institut for Molekylærbiologi og Genetik - Molekylær intervention5 Behrendt Group, BRIC Research Groups, BRIC, Københavns Universitet6 Molekylærbiologisk Institut7 Det Teknisk-Naturvidenskabelige Fakultet, Aalborg Universitet8 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet9 Joint expenses, Centre m.v., Faculty of Science, Københavns Universitet10 Behrendt Group, BRIC Research Groups, BRIC, Københavns Universitet11 Ploug Group, BRIC Research Groups, BRIC, Københavns Universitet12 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet13 Joint expenses, Centre m.v., Faculty of Science, Københavns Universitet
Data accumulated over the latest two decades have established that the serine protease urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. When designing inhibitors of the proteolytic activity of serine proteases, obtaining sufficient specificity is problematic, because the topology of the proteases' active sites are highly similar. In an effort to generate highly specific uPA inhibitors with new inhibitory modalities, we isolated uPA-binding RNA aptamers by screening a library of 35 nucleotides long 2'-fluoro-pyrimidine RNA molecules using a version of human pro-uPA lacking the epidermal growth factor-like and kringle domains as bait. One pro-uPA-binding aptamer sequence, referred to as upanap-126, proved to be highly specific for human uPA. Upanap-126 delayed the proteolytic conversion of human pro-uPA to active uPA, but did not inhibit plasminogen activation catalyzed by two-chain uPA. The aptamer also inhibited the binding of pro-uPA to uPAR and the binding of vitronectin to the preformed pro-uPA/uPAR complex, both in cell-free systems and on cell surfaces. Furthermore, upanap-126 inhibited human tumor cell invasion in vitro in the Matrigel assay and in vivo in the chick embryo assay of cell escape from microtumors. Finally, upanap-126 significantly reduced the levels of tumor cell intravasation and dissemination in the chick embryo model of spontaneous metastasis. Together, our findings show that usage of upanap-126 represents a novel multifunctional mechanistic modality for inhibition of uPA-dependent processes involved in tumor cell spread.
Molecular Cancer Research : Mcr, 2012, Vol 10, Issue 12, p. 1532-43