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.
Highlights
In this study we utilized small hairpin RNAs, referred of cancer cells through the digested extracellular matrix and contributes to as small interfering RNAs, to target human uPA and uPAR
Endogenous uPA and uPAR Protein Expression Is Associated with in Vitro Invasiveness of Human Prostate Cancer Cells—Previous studies have shown that PC3 cells are highly metastatic, whereas DU145 and LNCaP cells are moderately and poorly metastatic, respectively [45,46,47,48]
Because uPA and its receptor uPAR are involved in tumor invasion and metastasis, we first compared the levels of these proteins in the three human prostate cancer cell lines with different metastatic potentials
Summary
In this study we utilized small hairpin RNAs (shRNAs), referred of cancer cells through the digested extracellular matrix and contributes to as small interfering RNAs, to target human uPA and uPAR. Our results demonstrated that intratumoral injection with the multiple cells, binding of uPA to uPAR activates the extracellular-signal plasmid construct expressing shRNAs for uPA and uPAR almost regulated kinase 1/2 (ERK1/2) pathway, which controls cancer cell invacompletely inhibited established tumor growth and survival in an sion, proliferation, and survival [26, 27]. These results indicate that the simultaneous knockdown of uPA and uPAR using RNAi vectors is a promising tool for analysis of the function of downstream signaling pathways as well as the potential vectors for prostate cancer gene therapy.
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