RESUMO
Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of vimentin, while epithelial-associated genes like E-cadherin are lost. This enables cells to be more metastatic. Factors that are able to induce EMT include growth factors such as transforming growth factor-ß (TGF-ß) and epidermal growth factor, and transcription factors such as Snail. Snail-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by the activity of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). LNCaP, 22Rv1 and ARCaP human prostate cancer (CaP) cells stably transfected with empty vector control (Neo) or constitutively active Snail exhibited increased cell invasion. Superarray analysis revealed an upregulation in uPA and uPAR RNA expression in Snail-transfected ARCaP cells compared with that of a Neo control. In addition, the protein expression levels of Snail, uPA and uPAR were measured by western blot analysis which showed that overexpression of Snail increased uPA and uPAR protein levels. The activity of uPA in conditioned media was measured using an ELISA which revealed that uPA activity was elevated in LNCaP, 22Rv1 and ARCaP cells overexpressing Snail. Additionally, transient silencing of uPAR in ARCaP cells overexpressing Snail using short interfering RNA resulted in abrogation of Snail-mediated invasion. Snail overexpression was associated with increased extracellular-signal-regulated kinase activity, and antagonism of this activity with mitogen-activated protein (MAPK) inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human CaP cells may occur via the regulation of uPA/uPAR and the MAPK signaling pathway.
RESUMO
Cell- and receptor-specific regulation of cell migration by Gi/oα-proteins remains unknown in prostate cancer cells. In the present study, oxytocin (OXT) receptor was detected at the protein level in total cell lysates from C81 (an androgen-independent subline of LNCaP), DU145 and PC3 prostate cancer cells, but not in immortalized normal prostate luminal epithelial cells (RWPE1), and OXT-induced migration of PC3 cells. This effect of OXT has been shown to be mediated by Gi/oα-dependent signaling. Accordingly, OXT inhibited forskolin-induced luciferase activity in PC3 cells that were transfected with a luciferase reporter for cyclic AMP activity. Although mRNAs for all three Giα isoforms were present in PC3 cells, Giα2 was the most abundant isoform that was detected at the protein level. Pertussis toxin (PTx) inhibited the OXT-induced migration of PC3 cells. Ectopic expression of the PTx-resistant Giα2-C352G, but not wild-type Giα2, abolished this effect of PTx on OXT-induced cell migration. The Giα2-targeting siRNA was shown to specifically reduce Giα2 mRNA and protein in prostate cancer cells. The Giα2-targeting siRNA eliminated OXT-induced migration of PC3 cells. These data suggest that Giα2 plays an important role in the effects of OXT on PC3 cell migration. The Giα2-targeting siRNA also inhibited EGF-induced migration of PC3 and DU145 cells. Expression of the siRNA-resistant Giα2, but not wild type Giα2, restored the effects of EGF in PC3 cells transfected with the Giα2-targeting siRNA. In conclusion, Giα2 plays an essential role in OXT and EGF signaling to induce prostate cancer cell migration.