Silencing urokinase-type plasminogen activator in prostate cancer cells reduces their intraosseous proliferation and bone degradation Free

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Expression and activity of urokinase-type plasminogen activator (uPA) have been implicated in prostate cancer (PC) bone metastasis. To study the role of tumor-derived uPA in metastatic PC establishment in bone, we performed in vivo experiments using the cell line PC3, which expresses uPA and was originally initiated from human PC bone metastasis. PC3 cells were injected into human fetal bone xenografts in SCID mice, and the bones were harvested three weeks later. Histology showed 100% intraosseous tumor incidence with trabecular bone degradation. Immunoblot of bone lysates revealed uPA expression in PC3 bone tumors but not in the control bones injected with culture medium. uPA activity was consistent with uPA expression levels in PC3 bone tumors, but was minimal in control bones. Immunostaining showed that uPA was predominantly expressed in PC3 bone tumor cells. To establish the role of PC3-derived uPA, PC3 cells were stably transfected with vectors expressing either a specific uPA siRNA (uPAsi) or a scrambled siRNA (SCsi). Silencing uPA not only reduced uPA expression and activity but also hindered PC3 cell in vitro growth. The transfectants were inoculated into fetal bone xenografts in SCID mice, and the bone implants were harvested three weeks later. Histomorphometric analysis showed that skeletal tumor burden in bones injected with uPAsi PC3 cells was significantly reduced compared to bones injected with SCsi PC3 cells. Furthermore, trabecular bone degradation in the uPAsi PC3 bone tumor was dramatically inhibited compared to that in the SCsi PC3 bone tumor. Immunoblot analysis confirmed uPA expression only in bones injected with SCsi PC3 cells. Since plasminogen activator inhibitor-1 (PAI-1) has been found to be upregulated in many cancers despite its inhibitory activity on uPA, we also analyzed PAI-1 protein expression in the samples. Bone tumors produced by both parental and SCsi PC3 cells revealed induction of PAI-1 protein, which was localized to the bone stroma by immunohistochemistry. However, PAI-1 levels were much lower in uPAsi PC3 bone tumors. In vitro, interactions of both uPAsi and SCsi PC3 cells with bone marrow stromal cells (BMSC) led to an increase in PAI-1 protein expression. BMSC cells treated with PC3 conditioned medium also showed significant PAI-1 mRNA induction. When interacting with BMSC cells, PC3 cells showed an enhanced cell motility, which was almost completely abolished when PC3 cells were interacting with BMSC cells treated with PAI-1 siRNA. Taken together, these findings suggest that both tumor-derived uPA and tumor-stroma induced PAI-1 play important roles in intraosseous metastatic PC development by facilitating bone matrix turnover and promoting cancer cell growth and migration.