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


METHODS
Retrospective chart review of PTC patients, followed by prospective study using previously obtained patient tissue and PTC cellular models.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Nowicki
Theodore S
TS
Department of Microbiology and Immunology, New York Medical College, Valhalla, New York 10595, USA.
Kummer
Nicolas T
NT
Iacob
Codrin
C
Suslina
Nina
N
Schaefer
Steven
S
Schantz
Stimson
S
Shin
Edward
E
Moscatello
Augustine L
AL
Tiwari
Raj K
RK
Geliebter
Jan
J
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002291:Carcinoma, Papillary / Q000378:metabolism* / Q000473:pathology*; D002364:Caseins / Q000032:analysis; D002460:Cell Line; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D011446:Prospective Studies; D055293:Receptors, Urokinase Plasminogen Activator / Q000037:antagonists & inhibitors / Q000502:physiology*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013964:Thyroid Neoplasms / Q000378:metabolism* / Q000473:pathology*; D046509:Tissue Culture Techniques; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000502:physiology*
D002364:Caseins; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator