Src requirement for u-PAR expression by HGF and hypoxia

描述（由申请人提供）：前列腺癌患者的高死亡率反映了他们的疾病向远处扩散。纤溶酶原激活剂尿激酶通过将纤溶酶原转化为纤溶酶来促进这一过程，后者降解细胞外基质。尿激酶特异性地与细胞表面受体 (u-PAR) 结合，受体结合酶将纤溶酶原转化为纤溶酶，其速度比液相反应物更快。既然u-PAR是前列腺癌侵袭性所必需的，那么这个结合位点的上调是如何实现的呢？之前的报告表明 HGF/SF 会增加 u-PAR mRNA/蛋白质。然而，u-PAR 蛋白增加是否是由于转录增加或 mRNA 稳定性增加所致尚不清楚。在具体目标#1中，我们将确定 HGF/SF 引起的前列腺癌中 u-PAR 蛋白的增加是否是由于转录增加或 mRNA 更新减少所致。我们之前报道了与 u-PAR 启动子足迹区（跨 -148/124 的 II 区）结合的 AP-2α 相关因子在 u-PAR 表达的 PMA 依赖性调节中的作用。因此，如果 u-PAR 蛋白的 HGF/SF 依赖性增加是由于转录升高所致，我们还将确定 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 HGF/SF 依赖性基因表现出不同的 c-Src 需求，使用显性失活和反义技术以及 c-Src 抑制剂 (PD 173955)，c-Src 在 HGF/SF 诱导的 u-PAR 表达中的作用将在特定目标 #2 中确定。先前的研究表明，促进肿瘤进展的缺氧会上调 u-PAR mRNA/蛋白。此外，前列腺癌中缺氧会增加 c-Src 活性。由于c-Src在介导缺氧引起的u-PAR表达增加中的作用尚未被报道，并且考虑到其他缺氧诱导基因表现出不同的c-Src敏感性，在特定目标#3中，使用显性失活和反义表达构建体以及c-Src抑制剂，我们将确定u-PAR表达的缺氧诱导是否是c-Src依赖性的。前列腺癌中 u-PAR 合成增加是否完全是由于 c-Met 激活尚不清楚。为了回答这个问题，我们将在具体目标#4中，使用前列腺癌的体外和体内侵袭性作为生物学终点，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 产生升高也可能是由 c-Met 以外的因素造成的，因此我们还将确定抗 c-Met 核酶与药理学拮抗剂 (A6) 组合以减弱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.