RNA Interference-directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion, Survival, and Tumorigenicity in Vivo

Abstract

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.

Inhibition of Histone Deacetylase Activity Promotes Invasion of Human Cancer Cells through Activation of Urokinase Plasminogen Activator

“…The finding, that repressed uPA gene in human cancer cells can be re-activated by the inhibition of HDACs, will not only enhance our understanding in HDAC-mediated uPA gene expression but will also have negative implications for the therapeutic use of HDAC inhibitors in the treatment of cancer. The role of uPA in tumor cell invasion and metastasis is well established (5,9,14,15), and we found that HDAIs enhance tumor cell invasion through induction of uPA expression. Therefore, it is noteworthy that the use of HDAI-based cancer therapies in patients may paradoxically establish metastasis through reactivation of uPA.…”

“…Ample evidence indicated that increased levels of uPA are crucial for tumor cell invasion and metastasis (5,9,14,15). Our in vitro invasion assays showed that HDAI-induced uPA activation might stimulate cancer cell invasion.…”

“…Cell Invasion-Previous studies by our group (5,14,30) and others (9,15) established that uPA expression is closely associated with the invasive properties of tumor cells. To determine whether HDACI-induced uPA functionally contributes to the metastatic activity, we examined invasive activity in vitro.…”

“…In a parallel experiment, we also examined whether the presence of uPA is sufficient to confer invasion of cells that are normally either low or non-invasive. The uPA-silenced prostate cancer cell line, LNCaP, which has a very low invasive potential (14), was transfected with either vector alone (pCMV) or pCMV-uPA. LNCaP cells stably expressing uPA exhibited a significant increase in invasion through Matrigel as compared with their empty vector-transfected counterparts (Fig.…”

“…Additionally, binding of uPA with its receptor uPA receptor activates the Ras/extracellular signal-regulated kinase pathway, which in turn, leads to cell proliferation, migration, and invasion (13). Several studies using uPA inhibitors (9) or uPA gene-silencing approaches (14,15) have confirmed the important role of uPA in the processes of tumor invasion and metastasis. Because uPA is crucial for invasion and metastasis, we are interested in understanding how its transcriptional activity is regulated by epigenetic mechanisms in human cancer cells.…”