title: Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Copenhagen, Denmark.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Botkjaer
authors.forename: Elena I
authors.initials: EI
authors.lastname: Deryugina
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Dupont
authors.forename: Henrik
authors.initials: H
authors.lastname: Gårdsvoll
authors.forename: Erin M
authors.initials: EM
authors.lastname: Bekes
authors.forename: Cathrine K
authors.initials: CK
authors.lastname: Thuesen
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Zhou
authors.initials: Z
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Ploug
authors.forename: James P
authors.initials: JP
authors.lastname: Quigley
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000235:genetics* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002474:Cell-Free System; D002642:Chick Embryo; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D058990:Molecular Targeted Therapy; D009361:Neoplasm Invasiveness; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000628:therapy*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D010958:Plasminogen; D057057:Serine Proteases; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase