Downregulation of uPAR inhibits migration, invasion, proliferation, FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells

Abstract

Papillary thyroid carcinoma (PTC) is the most common endocrine and thyroid malignancy.  The urokinase plasminogen activator receptor (uPAR) plays an important role in cancer pathogenesis, including breakdown of the extracellular matrix, invasion, and metastasis.  Additionally, there is increasing evidence that uPAR also promotes tumorigenesis via the modulation of multiple signaling pathways.  BRAFV600E, the most common initial genetic mutation in PTC, leads to ERK1/2 hyperphosphorylation, which has been shown in numerous cancers to induce uPAR.  Treatment of the BRAFV600E-positive PTC cell line, BCPAP, with the MEK/ERK inhibitor U0126 reduced uPAR RNA levels by 90%.  siRNA-mediated down-regulation of uPAR in BCPAP cells resulted in greatly decreased activity in the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.  This phenomenon was concurrent with drastically reduced proliferation rates and decreased clonigenic survival, as well as demonstrated senescence-associated nuclear morphology and induction of b-galactosidase activity. uPAR-knockdown BCPAP cells also displayed greatly reduced migration and invasion rates, as well as a complete loss of the cells' ability to augment their invasiveness following plasminogen supplementation. Taken together, these data provide new evidence of a novel role for uPAR induction (as a consequence of constitutive ERK1/2 activation) as a central component in PTC pathogenesis, and highlight the potential of uPAR as a therapeutic target.

Acknowledgements

The authors wish to thank Dr. Radha Iyer for technical assistance with the qRT-PCR, as well as Melanie MacEwan for helpful discussions regarding the manuscript.

Financial Support

This work was supported by grants from the National Cancer Institute (1R01CA131946-01A2) and the Department of Otolaryngology, New York Medical College, Valhalla, NY USA.

Figures and Tables

Figure 1 Effect of the MEK inhibitor U0126 on ERK1/2 phosphorylation (A) and uPAR mRNA levels (B) in BCPAP cells. Cells were treated with 10 µM U0126 for 12 hours, at which point protein and RNA were collected for immunoblotting or qRT-PCR, respectively, as described in Materials and Methods. uPAR mRNA levels are expressed as a fraction relative to GAPDH expression (i.e., 2^−ΔCt). *indicates a significant (p < 0.05) decrease in uPAR mRNA levels relative to control BCPAP cells.

Figure 2 qRT-PCR (A) and western blot (B) analyses of uPAR expression in BCPAP control cells, non-targeting (NT) siRNA transfectants and uPAR siRNA transfectants. Cells were grown in Accell delivery media for at least 72 hours with the appropriate siRNA oligonucleotides as described in Materials and Methods. uPAR mRNA levels are expressed as a fraction relative to GAPDH expression (i.e., 2^−ΔCt). *indicates a significant (p < 0.05) decrease in uPAR mRNA levels relative to control or NT-siRNA-transfected BCPAP cells. Data shown are representative of three experiments.

Figure 3 Downregulation of uPAR leads to decreased levels of p-FAK^Tyr397, PI3K and p-Akt^Ser473. Protein lysates from control, non-targeting siRNA or uPAR siRNA-transfected BCPAP cells were subjected to SDS-PAGE and immunoblotted as described in Materials and Methods. Blots shown are representative of at least three experiments.

Figure 4 (A) Clonogenic assays demonstrating decreased ability of uPAR siRNA transfectant cells to form colonies compared to control or non-targeting (NT) siRNA transfectants. The accompanying graph summarizes (from independent replicate experiments) the colony-forming capacities of the various transfectants. *indicates a significantly lower (p < 0.05) number of colonies formed by the uPAR siRNA transfectants. (B) Effect of uPAR siRNA on BCPAP cell proliferation. Cells were grown in Accell delivery media with the appropriate siRNA for 72 hours, at which point the media was replaced with standard RPMI media (10% FBS) as described in Material and Methods. Cells counts were determined in triplicate for each time point via trypan blue exclusion. *indicates a significant difference (p < 0.05) in cell number for a given time-point.

Figure 5 (A) Representative LSC-assisted morphometric analysis of nuclear changes of BCPAP cells transfected with non-targeting siRNA or uPAR siRNA. Intensity of maximal (max) pixel of DNA/DAPI reports degree of chromatin condensation and in untreated cells has the highest value. In the senescing cells, while nuclear area increases, the intensity of maximal pixel decreases, likely due to the “flattening” of the cell. The insets in the top left panels show DNA frequency histograms of cells from the respective cultures. (B) The bar plots show the mean values (±SD) of DNA/DAPI maximal pixel intenxity, nuclear DNA/DAPI area (in pixels), and the ratio of maximal pixel to nuclear area. The ratio of maximal pixel/nuclear area of the uPAR-siRNA cells is expressed as a fraction of the average ratio of the respective controls (which is set at 1.0). *indicates a significant difference (p < 0.05) in DAPI maximal pixel, DAPI area or ratio of DAPI maximal pixel/DAPI area. (C) Senescence-associated β-galactosidase staining (blue-green) in BCPAP cells transfected with non-targeting siRNA (NT-siRNA) or uPAR-siRNA for 96 hours. 67.3% (±4.3%) of BCPAP cells treated with uPAR-siRNA displayed positive staining for senescence-associated β-galactosidase, compared to 3.2% (±1.1%) of the NT-siRNA tranfectants (p < 0.01). Figures are representative of three separate experiments, each with similar results.

Figure 6 Effect of uPAR downregulation on BCPAP cell migration (A) and invasion (B). Cells were grown in Accell delivery media with the appropriate siRNA oligonucleotides for 72 hours, at which point they were trysinized and re-seeded in migration/invasion transwell chambers (with or without supplemental plasminogen) as described in Materials and Methods. uPAR siRNA-transfected BCPAP cells had significantly lower (*) migratory potentials than the corresponding non-targeting (NT) siRNA-transfected controls (p < 0.05). The plasminogen-supplemented NT siRNA-transfected BCPAP cells' increased invasiveness was statistically significant (*) when compared to the control NT siRNA-transfected and both populations of uPAR siRNA-transfected cells (p < 0.05). uPAR siRNA-trasnfected cells' invasiveness was also significantly lower (#) than the NT siRNA-transfectants', regardless of the presence or absence of plasminogen. Figures are representative of two separate experiments with similar results. P = plasminogen.