Src requirement for u-PAR expression by HGF and hypoxia

描述(申请人提供)：前列腺癌患者的高死亡率反映了他们的疾病扩散到遥远的地方。纤溶酶原激活剂，尿激活剂，通过将纤溶酶原转化为纤溶酶，后者降解细胞外基质，参与了这一过程。尿激酶与细胞表面受体(u-PAR)特异性结合，受体结合酶将纤溶酶原转化为纤溶酶的速度比液相反应物更快。既然前列腺癌的侵袭性需要u-PAR，那么这个结合部位的上调是如何实现的呢？以前的报道表明，HGF/SF增加了u-PAR的mRNA/蛋白。然而，u-PAR蛋白的增加是由于转录增加还是由于mRNA稳定性增加尚不清楚。在特定的目标1中，我们将确定HGF/SF引起的前列腺癌中u-PAR蛋白的增加是由于转录增加还是由于mRNA周转减少。我们先前报道了AP-2α相关因子与u-PAR启动子的足迹区域(区域II跨越-148/124)结合在PMA依赖的u-PAR表达调控中的作用。因此，如果依赖于HGF/SF的u-PAR蛋白的增加是由于转录增加，我们也将确定II区结合的AP-2α相关因子在这种上调中的作用。蛋白酪氨酸激酶c-Src在HGF/SF调节u-PAR表达中的作用尚不清楚。考虑到其他依赖于肝细胞生长因子/SF的基因表现出不同的c-Src需求，利用显性的负性和反义技术以及c-Src抑制剂(PD 173955)，c-Src在肝细胞生长因子/SF诱导的u-PAR表达中的作用将在特定的目标2中确定。先前的研究表明，缺氧促进肿瘤进展，上调u-PAR的mRNA/蛋白。此外，在前列腺癌中，c-Src活性因缺氧而增加。由于c-Src在介导低氧诱导的u-PAR表达增加中的作用尚未见报道，并且考虑到其他低氧诱导基因表现出不同的c-Src敏感性，在特定的目标#3中，使用显性的负义和反义表达构建体以及c-Src抑制剂，我们将确定低氧诱导u-PAR表达是否依赖于c-Src。前列腺癌的u-PAR合成增加是否完全是由于c-Met的激活尚不清楚。为了回答这个问题，我们将在特定的目标#4中，以前列腺癌的体外和体内侵袭性为生物学终点，确定针对c-Met合成的核酶下调u-PAR合成的能力。由于我们认识到u-PAR产生增加的真正可能性也可能是由c-Met以外的因素引起的，我们也将确定抗c-Met核酶与药理拮抗剂(A6)相结合以减轻前列腺癌侵袭性的能力。

DESCRIPTION (provided by applicant): The high mortality rate of prostate cancer patients reflects the spread of their disease to distant sites. The plasminogen activator, urokinase, contributes to this process by converting plasminogen to plasmin, the latter which degrades extracellular matrix. Urokinase binds to a cell surface receptor (u-PAR) specifically and receptor-bound enzyme converts plasminogen into plasmin at a faster rate than fluid-phase reactants. Since the u-PAR is required for the invasiveness of prostate cancer, how is the up-regulation of this binding site achieved? Previous reports have shown that HGF/SF increases u-PAR mRNA/protein. However, whether increased u-PAR protein is due to increased transcription or increased mRNA stability is unknown. In Specific Aim # 1, we will determine if increased u-PAR protein in prostate cancer brought about by HGF/SF is due to increased transcription or decreased mRNA turnover. We previously reported the role of an AP-2alpha related factor bound to a footprinted region (region II spanning -148/124) of the u-PAR promoter in the PMA-dependent regulation of u-PAR expression. Thus, if the HGF/SF-dependent increase in u-PAR protein is due to elevated transcription, we will also determine the role of region II-bound AP-2alpha-related factor in this up-regulation. The role of the protein tyrosine kinase c-Src in the regulation of u-PAR expression by HGF/SF is not known. Considering that other HGF/SF-dependent genes show differing c-Src requirements, using dominant negative and antisense technology and a c-Src inhibitor (PD 173955), the role of c-Src in the HGF/SF-induced expression of u-PAR will be determined in Specific Aim # 2. Previous studies have shown that hypoxia, which promotes tumor progression, up-regulates u-PAR mRNA/protein. Further, c-Src activity is increased by hypoxia in prostate cancer. Since the role of c-Src in mediating increased u-PAR expression by hypoxia has not been reported and considering that other hypoxic-inducible genes demonstrate differing c-Src-sensitivities, in Specific Aim # 3, using dominant negative and antisense expression constructs and a c-Src inhibitor we will determine if the hypoxic induction of u-PAR expression is c-Src-dependent. Whether elevated u-PAR synthesis in prostate cancer is entirely due to c-Met activation is unclear. To answer this question, we will in Specific Aim # 4, determine the ability of a ribozyme directed at c-Met synthesis to downregulate u-PAR synthesis using the in vitro and in vivo invasiveness of prostate cancer as a biological endpoint. Since we recognize the real possibility that elevated u-PAR production may also be due to factors other than c-Met, we will also determine the ability of combining the anti-c-Met ribozyme with a pharmacological antagonist (A6) to attenuate the invasiveness of prostate cancer.