Regulation of urokinase receptor expression in colon CA

描述（申请人提供）：尿激酶受体（u-PAR）部分通过加速蛋白水解来参与结肠癌的侵袭和转移，u-PAR的转录在侵袭性结肠癌中高10倍，我们对确定调节其表达的转录上游分子感兴趣。由于蛋白酪氨酸激酶Src活性在转移性结肠癌中升高了80倍，并且由于Src转染到结肠上皮细胞使其具有侵袭性，我们假设（Specific Aim # 1）， u-PAR的表达是由蛋白酪氨酸激酶调节的。这将通过确定(a) Src抑制剂（PP2） (b)组成活性或(c)显性阴性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。我们的初步研究表明，与Sp1/Sp3结合的足迹区（-148/-124）调节构成型和src诱导的u-PAR表达。为了确定Src通过Sp1/Sp3结合区域调节u-PAR表达的机制（Specific Aim # 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)增加该印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区调节u-PAR的表达。在Specific Aim # 3中，我们将在转化研究中利用这一信息，通过确定双蒽环类药物WP631（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表达和体外结肠癌侵袭的能力。迄今为止，关于u-PAR表达的研究采用了体外技术，这些技术没有提供组织特异性u-PAR表达所需启动子的信息，并且忽略了染色质在调节该基因中的作用。在Specific Aim # 4中，将使用含有5'缺失u-PAR启动子片段调控的LacZ报告子的转基因小鼠来确定(a)最小启动子序列和(b) u-PAR在胎盘和遗传诱导的结肠癌（以u-PAR高表达为特征的组织）中表达所需的-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。

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.