Regulation of urokinase receptor expression in colon CA

结肠CA中尿激酶受体表达的调节

• 批准号: 6607838
• 负责人: Douglas D. Boyd
• 金额: $ 26.43万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607838
• 关键词: DNA binding protein; clinical research; colon neoplasms; enzyme inhibitors; enzyme linked immunosorbent assay; genetic transcription; genetically modified animals; human tissue; immunocytochemistry; laboratory mouse; neoplasm /cancer invasiveness; phosphorylation; plasminogen activator; polymerase chain reaction; protein tyrosine kinase; receptor expression; urokinase; western blottings

DESCRIPTION (provided by applicant): The urokinase receptor (u-PAR) contributes to colon cancer invasion and metastasis partly by accelerating proteolysis, u-PAR transcription is >10 fold higher in invasive colon cancer and we are interested in identifying molecules upstream of transcription that regulate its expression. Since the protein tyrosine kinase Src activity is elevated > 8 fold in metastatic colon cancer and because transfection of Src into colonic epithelial cells renders them invasive, we hypothesize (Specific Aim # 1), that u-PAR expression is regulated by this protein tyrosine kinase. This will be tested by determining the effect of (a) a Src inhibitor (PP2) (b) a constitutively active or (c) dominant negative Src on u-PAR expression in cultured or genetically-induced (mucin 2 -/-) colon cancer and correlating u-PAR levels with Src activity in cultured and resected colon cancers. Our preliminary studies implicate a footprinted region (-148/-124) bound with Sp1/Sp3 that regulates constitutive and Src-inducible u-PAR expression. To determine the mechanism by which Src regulates u-PAR expression via this Sp1/Sp3 -bound region (Specific Aim # 2), we will determine if Src (a) increases Sp1 expression (b) alters Sp1 phosphorylation to increase its DNA binding (c) increases histone acetylation at this footprinted region thereby promoting chromatin relaxation and Sp1/Sp3 binding or (d) increases the trans-acting activity of Sp1/Sp3. Our preliminary data indicate that Src regulates u-PAR expression in part through the Sp1/Sp3-bound -148/-124 region. In Specific Aim # 3, we will exploit this information in translational studies by determining the ability of a bisanthracycline WP631 (which blocks Sp1/Sp3 binding to the -148/-124 region) alone, or combined with a Src inhibitor (PP2), to suppress u-PAR expression and colon cancer invasiveness in vitro. Studies on u-PAR expression, to date, have employed in vitro techniques which provide no information on promoter requirements for tissue-specific u-PAR expression and ignore the role of chromatin in regulating this gene. In Specific Aim # 4, transgenic mice harboring a LacZ reporter regulated by 5' deleted u-PAR promoter fragments will be employed to determine (a) the minimal promoter sequence and (b) the role of the -148/-124 region required for u-PAR expression in the placenta and genetically-induced colon cancer (tissues characterized by their high u-PAR expression). Additionally, the sensitivity of transgene expression to Src inhibition will be determined.

描述（由申请人提供）：尿激酶受体（u-PAR）部分通过加速蛋白水解作用促进结肠癌侵袭和转移，u-PAR转录在侵袭性结肠癌中高出>10倍，我们有兴趣识别转录上游调节其表达的分子。由于蛋白酪氨酸激酶 Src 活性在转移性结肠癌中升高 > 8 倍，并且由于 Src 转染到结肠上皮细胞中使其具有侵袭性，因此我们假设（具体目标＃1），u-PAR 表达受该蛋白酪氨酸激酶调节。这将通过确定 (a) Src 抑制剂 (PP2) (b) 组成型活性或 (c) 显性失活 Src 对培养或遗传诱导的（粘蛋白 2 -/-）结肠癌中 u-PAR 表达的影响并将 u-PAR 水平与培养和切除的结肠癌中的 Src 活性相关联来进行测试。我们的初步研究表明，足迹区域 (-148/-124) 与 Sp1/Sp3 结合，调节组成型和 Src 诱导型 u-PAR 表达。为了确定 Src 通过该 Sp1/Sp3 结合区域调节 u-PAR 表达的机制（具体目标 # 2），我们将确定 Src 是否 (a) 增加 Sp1 表达 (b) 改变 Sp1 磷酸化以增加其 DNA 结合 (c) 增加该足迹区域的组蛋白乙酰化，从而促进染色质松弛和 Sp1/Sp3 结合或 (d) 增加 Sp1/Sp3。我们的初步数据表明，Src 部分通过 Sp1/Sp3 结合的 -148/-124 区域调节 u-PAR 表达。在具体目标#3中，我们将在转化研究中利用这一信息，确定双蒽环类 WP631（阻断 Sp1/Sp3 与 -148/-124 区域的结合）单独使用或与 Src 抑制剂 (PP2) 联合使用在体外抑制 u-PAR 表达和结肠癌侵袭性的能力。迄今为止，对 u-PAR 表达的研究采用了体外技术，这些技术没有提供有关组织特异性 u-PAR 表达的启动子要求的信息，并且忽略了染色质在调节该基因中的作用。在具体目标#4中，将使用携带由5'删除的u-PAR启动子片段调节的LacZ报告基因的转基因小鼠来确定（a）最小启动子序列和（b）胎盘和遗传诱导的结肠癌（以高u-PAR表达为特征的组织）中u-PAR表达所需的-148/-124区域的作用。此外，还将确定转基因表达对 Src 抑制的敏感性。