Inhibition of uPAR and uPA reduces invasion in papillary thyroid carcinoma cells

Abstract

We analyzed the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in papillary thyroid carcinoma (PTC) and normal thyroid tissue and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, and the functional consequences of inhibiting this system. Retrospective chart review of PTC patients, followed by prospective study using previously obtained patient tissue and PTC cellular models. uPA and uPAR RNA and protein levels were analyzed in PTC patient tissue samples, PTC and normal thyroid tissue culture cells, and conditioned media (CM) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and/or Western blotting. The plasminogen-activating ability of CM was examined using dark-quenched casein fluorimetry and casein-plasminogen gel zymography. The invasive potentials of the PTC and normal thyroid epithelial cell lines were assessed using an in vitro cellular invasion/migration system. uPA and uPAR RNA and protein levels were increased in PTC patient samples and PTC cells relative to controls. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and Western blotting demonstrated increased active uPA secreted by PTC cells compared with normal thyroid cells. Fluorimetric assays revealed that the PTC cells' CM was able to activate plasminogen, resulting in measurable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. Finally, the in vitro invasion phenotypes of PTC cells were augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.