Regulation of urokinase receptor expression in colon cancer

项目摘要 大量的研究表明，细胞膜糖脂连接尿激酶受体（u-PAR）参与了 促进肿瘤生长和进展。u-PAR以两种不同的方式促进这些效应。首先，它与 与α 5 β 1整合素结合，从而增加活化的ERK/p38的比率，产生增加的生长分数。 其次，u-PAR结合丝氨酸蛋白酶尿激酶，从而增加纤溶酶形成和细胞外 基质降解、肿瘤细胞迁移和侵袭。然而，u-PAR的表达调控较差， 明白我们已经做了有趣的观察，在一些结肠癌细胞中，克隆群体振荡 在u-PAR中，在高和低细胞表面密度之间显示具有降低的致瘤性， 后者有趣的是，改变的u-PAR细胞表面展示在本质上是翻译后的，反映了细胞内蛋白质的转移。 u-PAR贩运。在具体目标#1中，我们将确定u-PAR显示可塑性在结肠癌中的患病率。 以及改变的致瘤性是否反映了细胞表面u-PAR密度的这种振荡。而且还要 确定振荡u-PAR展示下游激活的ERK/p38比率的变化是否指示肿瘤发生 在这些亚稳态细胞群体中的潜力。最后，我们将确定是否u-PAR分泌或 脱落是u-PAR显示可塑性的机制。如果是后者，我们将使用表达 分析和siRNA/shRNA实验以鉴定GPI锚定或脱落机制中的蛋白质 负责调制的u-PAR显示。迄今为止，有几个已知的生物调节u-PAR的表达。 在具体目标#2中，我们将采用无偏倚的表达克隆策略来鉴定新的调节因子， u-PAR表达。候选调节因子将通过组织中的过表达/敲低实验进行验证 培养，并通过使用小鼠基因捕获的调节剂，以确定是否u-PAR表达的调节， 野生型动物中共表达候选调节子和u-PAR的组织。最后，我们将通过免疫组织化学， 在切除的结肠癌切片中调节剂和u-PAR的量以获得进一步的证据 在这种恶性肿瘤中，调节因子在驱动u-PAR表达中的作用。之前我们描述了1469 bp的上游序列作为u-PAR表达的调控序列。然而，根据我们的转染实验， 报告基因构建体，并对携带LacZ报告基因的转基因小鼠进行了研究， 然而，我们得出的结论是，这种顺序虽然是必要的，但不足以 忠实地反映内源基因表达。我们在此描述了一种新的增强子，其位于染色质化的 u-PAR基因内含子1（+665/+2068）区域，有助于结肠癌中的最佳表达。 这些发现导致了特异性目标#3，其中使用染色质免疫沉淀测定和显性的 阴性表达技术，我们将鉴定控制u-PAR表达的增强子结合的共调节子。 此外，我们将确定这种内含子增强子是否赋予组织特异性（胎盘，受伤的皮肤） 小鼠中u-PAR表达。

Project SummaryAbstract A strong body of work has implicated the cell membrane glycolipid-linked urokinase receptor (u-PAR) in promoting tumor growth and progression. u-PAR promotes these effects in two distinct ways. First, it interacts with a5¿1 integrin thereby increasing the ratio of activated ERK/p38 yielding an increased growth fraction. Second, the u-PAR binds the serine protease urokinase thus increasing plasmin formation and extracellular matrix degradation, tumor cell migration and invasion. However, the regulation of u-PAR expression is poorly understood. We have made the intriguing observation that in some colon cancer cells, clonal populations oscillate in u-PAR display between high and low cell surface density with reduced tumorigenecity segregating with the latter. Interestingly, the altered u-PAR cell surface display is post-translational in nature reflecting a shift in u-PAR trafficking. In Specific Aim #1 we will determine the prevalence of u-PAR display plasticity in colon cancer and whether altered tumorigenecity mirrors this oscillation in cell surface u-PAR density. Moreover, we will determine if a shift in the ratio of activated ERKs/p38 downstream of oscillating u-PAR display dictates tumorigenic potential in these metastable cell populations. Finally, we will determine whether u-PAR secretion or shedding is the mechanism responsible for u-PAR display plasticity. If the latter, we will employ expression profiling and siRNA/shRNA experiments to identify protein(s) in the GPI anchoring or shedding machineries responsible for modulated u-PAR display. To date, there are few known biological regulators of u-PAR expression. In Specific Aim # 2, we will employ an unbiased expression cloning strategy to identify novel regulators of u-PAR expression. Candidate regulators will be validated by over-expression/knockdown experiments in tissue culture and by employing mice gene-trapped for the regulator to determine if u-PAR expression is modulated in tissues that in wt animals co-express the candidate regulator and u-PAR. Finally, we will correlate, by immunohistochemistry, the amounts of regulator and u-PAR in resected colon cancer sections to accrue further evidence for a role of the regulator in driving u-PAR expression in this malignancy. Previously, we described 1469 bp of upstream sequence as regulatory for u-PAR expression. However, based on our transfection experiments with reporter constructs and studies with transgenic mice harboring a LacZ reporter driven by this upstream sequence, we have concluded that this sequence while necessary for is, nevertheless, insufficient to faithfully mirror endogenous gene expression. We describe herein a novel enhancer residing in a chromatinized region of intron 1 (+665/+2068) of the u-PAR gene, that contributes to optimal expression in colon cancer. These findings lead to Specific Aim # 3 where using chromatin immunoprecipitation assays and dominant negative expression technology we will identify enhancer-bound co-regulator(s) controlling u-PAR expression. Additionally, we will determine whether this intronic enhancer confers tissue-specific (placenta, wounded skin) u-PAR expression in mice.