Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma

Malignant mesothelioma (MM) is a rare neoplasm associated with asbestos exposure. The prognosis of MM is poor because it is aggressive and highly resistant to chemotherapy. Using a rat model of asbestos-induced MM, we found elevated urokinase-type plasminogen activator receptor (uPAR; Plaur) expression in rat tissues, which was associated with poor prognosis. The proliferation, migration and invasion of MM cells were suppressed by uPAR knockdown and increased by overexpression experiments, irrespective of urokinase-type plasminogen activator (uPA; Plau) levels. More importantly, we found that uPAR expression is associated with sensitivity to cisplatin in MM through the PI3K/AKT pathway, which was demonstrated with specific inhibitors, LY294002 and Akti-1/2. uPAR knockdown significantly increased sensitivity to cisplatin whereas its overexpression significantly decreased cisplatin sensitivity. Furthermore, sera and tissues from MM patients showed significantly high uPAR levels, which suggested the pathogenic role of uPAR in the tumor biology of human MM. In conclusion, our findings indicate that uPAR levels are associated with malignant characteristics and cisplatin sensitivity of MM. In addition to the potential use of uPAR as a prognostic marker, the combination of uPAR abrogation and cisplatin may reveal a promising therapeutic approach for MM.


Western blot analysis
Cell lysates were prepared by ice-cold Lysis-M buffer (Roche, Switzerland) supplemented with cOmplete protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Switzerland). After centrifuge at 14,000 × g for 10 min, protein concentration of the supernatants were determined with BCA assay (Nacalai Tesque, Japan). Cell extracts were subjected to SDS-PAGE, and proteins were transferred to PVDF membranes (Merck Millipore, Germany) and probed with primary antibodies (1:1000). Subsequently, they were probed with HRP-conjugated secondary antibodies (1:2000), and signals were detected using ECL reagents (GE Healthcare, USA). Densitometric analysis was peformed using the ImageJ software (NIH, USA).

Expression vector
Total length of human uPAR cDNA (NM_002659) was cloned into pcDNA 3.1 vector (Invitrogen, USA). A pcDNA3 Myr HA Akt1 plasmid was obtained from Addgene, USA. Cells were transfected by Fugene HD (Promega, USA) and transfected cells were selected with G418 (Wako, Japan) at 800-3000 mg/ml at least for 2 weeks.

MTT assay
Same amount of cells suspension (100 μl) was added to a 96-well plate. Following incubation and treatment, 10 μl MTT stock solution (5 mg/ml in PBS; Sigma-Aldrich, USA) was added to each well. Cells were subsequently incubated at 37°C for 3 h, then supernatant was discarded and 100 μl dimethyl sulfoxide (Wako, Japan) was added and the plate was agitated in the dark. Finally, the optical density (OD) was detected using a microplate reader (Powerscan4; Bio-Tek Instruments, USA) at a wavelength of 540 nm with a reference wavelength of 630 nm.

Cell counting assay
Same amount of cells was plated to a 12-well plate. After different time of incubation, cells from each well were collected. Cell suspension was counted by trypan blue exclusion method by a standard hemocytometer counting chamber (ERMA, Japan).

Migration and invasion assay
Cell migration and invasion assays were performed using the transwell permeable supports (Boyden chamber inserts) with 8 mm pore filters uncoated (for migration) and coated with matrigel (for invasion) according to the manufacturer's protocol (BD Biosciences, USA). Cells were added to the upper chambers in medium with 1% FBS, and medium with 10% FBS was added to the lower chambers. After 24 h-incubation at 37°C, the cells that had not migrated were removed from the upper surface of the filters with cotton swabs. The cells that had migrated to the lower surface of the filters were fixed in 4% paraformaldehyde and stained with 0.2% crystal violet. Migration and invasion were quantified by counting cells in seven randomly selected fields on each filter under a microscope at 100 × magnification.