Src requirement for u-PAR expression by HGF and hypoxia

HGF 和缺氧对 u-PAR 表达的 Src 要求

• 批准号: 6922046
• 负责人: Douglas D. Boyd
• 金额: $ 25.1万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6922046
• 关键词: athymic mouse; biological signal transduction; cell surface receptors; clinical research; enzyme linked immunosorbent assay; gel mobility shift assay; genetic transcription; hepatocyte growth factor; human tissue; hypoxia; inhibitor /antagonist; neoplastic process; northern blottings; prostate neoplasms; protein tyrosine kinase; ribozymes; southern blotting; tissue /cell culture; transfection; urokinase; western blottings

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.

描述（由申请人提供）：前列腺癌患者的高死亡率反映了其疾病向远处的扩散。纤溶酶原激活剂尿激酶通过将纤溶酶原转化为纤溶酶，后者降解细胞外基质来促进这一过程。尿激酶与细胞表面受体（u-PAR）特异性结合，受体结合酶以比液相反应物更快的速度将纤溶酶原转化为纤溶酶。既然前列腺癌的侵袭性需要u-PAR，那么该结合位点的上调是如何实现的呢？先前的报道表明，HGF/SF增加了u-PAR mRNA/蛋白。然而，u-PAR蛋白增加是由于转录增加还是mRNA稳定性增加尚不清楚。在Specific Aim # 1中，我们将确定HGF/SF导致的前列腺癌u-PAR蛋白升高是由于转录增加还是mRNA周转减少。我们之前报道了AP-2alpha相关因子结合到u-PAR启动子的足迹区域（跨越-148/124的区域II）在pma依赖的u-PAR表达调控中的作用。因此，如果HGF/ sf依赖性u-PAR蛋白的增加是由于转录的升高，我们还将确定ii区结合的ap -2alpha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依赖性基因对c-Src的需求不同，使用显性阴性和反义技术以及c-Src抑制剂（PD 173955），将在Specific Aim # 2中确定c-Src在HGF/ sf诱导的u-PAR表达中的作用。先前的研究表明，缺氧促进肿瘤进展，上调u-PAR mRNA/蛋白。此外，前列腺癌中c-Src活性因缺氧而升高。由于c-Src在缺氧介导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Specific Aim # 3中，我们将使用显性阴性和反义表达构建体和c-Src抑制剂来确定缺氧诱导u-PAR表达是否依赖于c-Src。前列腺癌中u-PAR合成升高是否完全由c-Met活化引起尚不清楚。为了回答这个问题，我们将在Specific Aim # 4中，利用前列腺癌的体内和体外侵袭性作为生物学终点，确定一种用于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产生升高的真正可能性也可能是由于c-Met以外的因素，我们还将确定将抗c-Met核酶与药物拮抗剂（A6）联合使用以减弱前列腺癌的侵袭性的能力。