Mechanism of Constitutive NF-kappa B Activity

描述(申请人提供)：癌症通常是通过破坏对细胞外信号的适当反应而发展的。因此，了解正常细胞如何感知并对环境变化做出适当的反应是重要的。正常情况下，细胞外信号转化为细胞反应涉及一系列生化事件，最终诱导基因表达。这些事件是由转录因子(Tf)介导的，Tf是一类DNA结合蛋白，决定了所表达基因的性质。通过基因和表观遗传异常解除对TF活性的调控，可以通过不受控制的基因表达来放大被破坏的细胞反应。细胞外信号对一种关键的转铁蛋白--核因子-kappaB的激活通常是短暂的，因为核因子-kappaB激活其自身的抑制物IkappaBalpha的合成，该抑制物进入细胞核，将核因子-kappaB从DNA结合部位移除，并将其输出到细胞质以终止核因子-kappaB的功能。相反，在人类癌症中常见的对核因子-kappaB活性的解除调控，必须抵消这种反馈机制，以维持结构性(恒定)的核因子-kappaB活性，以维持生存并诱导化疗/放射耐药。本实验室的研究表明，小鼠B细胞通过降解新合成的IkappaBalpha来维持这种活性，这是一种罕见的非病理结构性核因子-kappaB激活的机制。我们的初步数据还表明，为了发生IkappaBalpha的持续降解，必须将新形成的核NF-kappaB/IkappaBalpha复合体输出到小鼠B细胞和人类癌细胞的细胞质中。因此，拟议的研究将检验这样的假设，即结构性的NF-kappaB激活需要一种机制来抵消其抑制剂IkappaBalpha施加的自身抑制因子反馈调节。在目标1中，将使用突变分析来描述新的IkappaBalpha降解机制。根据目标2，IkappaBalpha的核输出在维持组成性的NF-kappaB激活和人类癌细胞存活方面的功能作用将被确定。目的3将通过产生携带N-NES点突变的IkappaBalpha基因座的小鼠，在体内测试IkappaBalpha核输出在B细胞发育中的作用。这项研究计划将有助于确定在B细胞发育和人类恶性肿瘤中对结构性核因子-kappaB激活至关重要的基本机制。它们还可能揭示N-NES介导的IkappaBalpha核输出是一个合理的治疗靶点，通常可以破坏结构性的NF-kappaB激活，从而诱导人类癌症的细胞死亡或化疗/放射增敏。

DESCRIPTION (provided by applicant): Cancer often develops through the disruption of proper responses to extracellular signals. Thus, understanding how normal cells sense and generate proper responses to changes in environment is important. Normally, transduction of extracelluar signals into cellular responses involves a cascade of biochemical events that eventually induce gene expression. These events are mediated by transcription factors (TF), a class of DNA-binding proteins that dictate the nature of genes expressed. Deregulation of TF activities by genetic and epigenetic anomalies can lead to amplification of disrupted cellular responses via uncontrolled gene expression. Activation of a critical TF, NF-kappaB, by extracellular signals normally occurs only transiently since NF-kappaB activates synthesis of its own inhibitor, IkappaBalpha, which enters the nucleus, removes NF-kappaB from DNA binding sites, and exports it out to the cytoplasm to terminate NF-kappaB function. By contrast, deregulation of NF-kappaB activity, frequently seen in human cancers, must counteract this feedback mechanism to maintain constitutive (constant) NF-kappaB activation to sustain survival and induce chemo/radioresistance. Research in this laboratory has demonstrated that murine B cells, a rare example with non-pathological constitutive NF-kappaB activation, maintain such activity by degrading newly synthesized IkappaBalpha via a previously uncharacterized mechanism. Our preliminary data also suggest that in order for continual degradation of IkappaBalpha to occur, there is a requirement that newly formed, nuclear NF-kappaB/IkappaBalpha complexes must be exported out to the cytoplasm in both murine B cells and human cancer cells. Thus, the proposed research will test the hypothesis that constitutive NF-kappaB activation requires a mechanism to counteract the autoinhibitorv feedback regulation imposed by its inhibitor IkappaBalpha. In Aim 1, mutational analysis will be employed to delineate novel IkappaBalpha degradation mechanisms. Under Aim 2, the functional role of the nuclear export of IkappaBalpha will be determined in both maintenance of constitutive NF-kappaB activation and survival of human cancer cells. Aim 3 will test the in vivo roles of nuclear export of IkappaBalpha in B cell development by the generation of mice harboring IkappaBalpha loci with N-NES point mutations. This research program will help define fundamental mechanisms critical for constitutive NF-kappaB activation in B cell development and human malignancies. They may also reveal N-NES-mediated nuclear export of IkappaBalpha as a rational therapeutic target to generally disrupt constitutive NF-kappaB activation to induce cell death or chemo/radiosensitization in human cancer.