Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism

TY - JOUR

T1 - Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism

AU - Botkjaer, Kenneth A

AU - Deryugina, Elena I

AU - Dupont, Daniel Miotto

AU - Gårdsvoll, Henrik

AU - Bekes, Erin M

AU - Thuesen, Cathrine König

AU - Chen, Zhuo

AU - Chen, Zhou

AU - Ploug, Michael

AU - Quigley, James P

AU - Andreasen, Peter A

PY - 2012/12

Y1 - 2012/12

N2 - 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.

AB - 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.

KW - Animals

KW - Aptamers, Nucleotide

KW - Cell Line, Tumor

KW - Cell-Free System

KW - Chick Embryo

KW - Epidermal Growth Factor

KW - HEK293 Cells

KW - Humans

KW - Male

KW - Molecular Targeted Therapy

KW - Neoplasm Invasiveness

KW - Plasminogen

KW - Prostatic Neoplasms

KW - Recombinant Proteins

KW - Serine Proteases

KW - Urokinase-Type Plasminogen Activator

U2 - 10.1158/1541-7786.MCR-12-0349

DO - 10.1158/1541-7786.MCR-12-0349

M3 - Journal article

C2 - 23038812

SN - 1557-3125

VL - 10

SP - 1532

EP - 1543

JO - Molecular cancer research : MCR

JF - Molecular cancer research : MCR

IS - 12

ER -