Interleukin-8 Regulation by Proteasome and Nuclear IkB in Cancer and Inflammation

描述(申请人提供)：白介素8(IL-8)是一种炎性趋化因子，通过诱导肿瘤细胞增殖、肿瘤浸润性白细胞的募集和激活、血管生成和转移而在癌症进展中发挥关键作用。IL-8的表达以及许多其他炎性细胞因子的表达都是由核因子B在转录水平上进行调控的。然而，我们实验室的研究表明，IL-8的转录调控不同于其他依赖于核因子B的基因的转录调控。具体地说，我们已经发现了核I？B？不抑制刺激的白细胞中IL-8的表达，同时抑制其他依赖于核因子？B的基因的表达。此外，我们最近的数据显示，蛋白酶体抑制被用作抗癌治疗，因为它能够抑制NF？B依赖基因的表达，实际上增加了转移性前列腺癌、卵巢癌细胞和人巨噬细胞中IL-8的表达。然而，责任机制在很大程度上是未知的。这项研究解决了目前对蛋白酶体抑制和核I？B？对IL-8转录调控的认识不足。我们的长期目标是了解调节核因子B反应基因表达的转录机制。这项建议的目的是确定蛋白酶体和核I、B、B、C、B、C、C等。调节IL-8的转录，以及这种调节与其他依赖于核因子B的基因有何不同。核心假设是S536 p65的磷酸化、核因子B结合位点DNA序列的特异性和/或转录因子EGR-1使IL-8对I？B的抑制没有反应，而作为对蛋白酶体抑制的反应而增加IL-8的表达。根据我们的初步数据，该项目将测试两个相互非排他性的模型。在目标1中，我们将验证这样的假设，即IL-8启动子主要被S536上磷酸化的p65同源二聚体占据，并且蛋白酶体抑制增加了S536 p65的磷酸化，导致IL-8转录增加。在目标2中，我们将验证这样的假设，即IL-8启动子序列和EGR-1的参与与蛋白酶体抑制增加IL-8的表达和IL-8转录对I、B、B、C的抗性有关。抑制力。我们将使用转移性前列腺癌PC-3细胞、卵巢癌OVCAR-3细胞和刺激的U937巨噬细胞作为这两个目标的模型。由于IL-8促进肿瘤细胞生长、血管生成、转移以及激活白细胞，因此了解其通过蛋白酶体和核I、B、B？可能对以IL-8过度表达为特征的癌症和炎症性疾病具有重要的临床意义。此外，该项目将改善圣约翰大学的研究环境，为有动力的贫困学生提供大量学习生物医学研究基础知识的机会。

DESCRIPTION (provided by applicant): Interleukin-8 (IL-8) is an inflammatory chemokine that has a crucial role in cancer progression through its induction of tumor cell proliferation, recruitment and activation of tumor-infiltrating leukocytes, angiogenesis, and metastasis. The expression of IL-8, as well as expression of many other inflammatory cytokines is regulated at the level of transcription by NF?B. However, studies from our laboratory indicate that the transcriptional regulation of IL-8 differs from the regulation of other NF?B-dependent genes. Specifically, we have found that the nuclear I?B? does not inhibit IL-8 expression in stimulated leukocytes, while it inhibits expression of other NF?B-dependent genes. In addition, our recent data show that the proteasome inhibition that is used as an anti-cancer therapy for its ability to inhibit expression of NF?B-dependent genes actually increases IL-8 expression in metastatic prostate and ovarian cancer cells and in human macrophages. However, the responsible mechanisms are largely unknown. The proposed research addresses the lack of knowledge on the regulation of IL-8 transcription by the proteasome inhibition and by nuclear I?B?. Our long-term goal is to understand the transcriptional mechanisms regulating expression of NF?B-responsive genes. The objective of this proposal is to determine how the proteasome and nuclear I?B? regulate transcription of IL-8, and how this regulation differs from other NF?B-dependent genes. The central hypothesis is that S536 p65 phosphorylation, specificity of the DNA sequence of the NF?B binding site, and/or the transcription factor EGR-1 render IL-8 unresponsive to the inhibition by I?B?, and increase IL-8 expression in response to the proteasome inhibition. Based on our preliminary data, the project will test two mutually non-exclusive models. In Aim 1, we will test the hypothesis that the IL-8 promoter is occupied predominantly by p65 homodimers phosphorylated on S536, and that the proteasome inhibition increases S536 p65 phosphorylation, resulting in the increased IL-8 transcription. In Aim 2, we will test the hypothesis that the IL-8 promoter sequence and EGR-1 involvement are responsible for the proteasome inhibition increased IL-8 expression and resistance of IL-8 transcription to I?B? inhibition. We will use metastatic prostate cancer PC-3 cells, ovarian cancer OVCAR-3 cells and stimulated U937 macrophages as models in both Aims. Since IL-8 promotes tumor cell growth, angiogenesis, metastasis, as well as activates leukocytes, understanding its regulation by proteasome and nuclear I?B? may have important clinical implications in cancers and inflammatory disorders characterized by excessive IL-8 expression. In addition, this project will enhance the research environment at St. John's University by providing motivated underprivileged students with numerous opportunities to learn the fundamentals of biomedical research.