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: 7058227
• 负责人: Douglas D. Boyd
• 金额: $ 25.8万
• 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/7058227
• 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(申请人提供)：尿激酶受体(u-PAR)有助于结肠癌的侵袭和转移，部分是通过加速蛋白质分解，u-PAR转录在浸润性结肠癌中高出10倍，我们有兴趣确定转录上游调节其表达的分子。由于转移性结肠癌中蛋白酪氨酸激酶Src的活性升高了8倍，而且由于将Src导入结肠上皮细胞使其具有侵袭性，我们假设(特定目标1)，u-PAR的表达受该蛋白酪氨酸激酶的调节。这将通过确定(A)Src抑制物(PP2)(B)结构性活性或(C)显性负性Src对培养的或遗传诱导的(粘蛋白2-/-)结肠癌中u-PAR表达的影响，以及在培养和切除的结肠癌中u-PAR水平与Src活性的相关性来进行测试。我们的初步研究发现，一个足迹区域(-148/-124)与Sp1/Sp3结合，调控结构性和Src诱导的u-PAR表达。为了确定Src通过这个Sp1/Sp3结合区调节u-PAR表达的机制(特定目的#2)，我们将确定Src(A)增加Sp1的表达(B)改变Sp1的磷酸化以增加其DNA结合(C)增加这个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的反式作用活性。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的表达。在特定的目标#3中，我们将在翻译研究中利用这些信息，通过确定双珊瑚环素WP631(它阻断Sp1/SP3与-148/-124区域的结合)单独或与Src抑制剂(PP2)联合抑制u-PAR表达和结肠癌体外侵袭力的能力。到目前为止，对u-PAR表达的研究采用了体外技术，这些技术没有提供关于组织特异性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。在特定的目标#4中，含有受5‘缺失的u-PAR启动子片段调控的LacZ报告的转基因小鼠将被用来确定(A)最小的启动子序列和(B)在胎盘和遗传诱导的结肠癌(以其高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。