Mechanism of Constitutive NF-kappa B Activity

描述（由申请人提供）：癌症通常通过破坏对细胞外信号的适当反应而发展。因此，了解正常细胞如何感知并对环境变化产生适当的反应是很重要的。正常情况下，细胞外信号转导到细胞反应涉及最终诱导基因表达的生化事件级联。这些事件是由转录因子（TF）介导的，TF是一类决定基因表达性质的DNA结合蛋白。由遗传和表观遗传异常引起的TF活性失调可通过不受控制的基因表达导致破坏的细胞反应的放大。细胞外信号对关键TF NF-κ B的激活通常仅短暂发生，因为NF-κ B激活其自身抑制剂IkappaB α的合成，IkappaB α进入细胞核，从DNA结合位点去除NF-κ B，并将其输出到细胞质以终止NF-κ B功能。相比之下，NF-κ B活性的失调，经常在人类癌症中看到，必须抵消这种反馈机制，以维持组成性（恒定）NF-κ B激活，以维持生存和诱导化疗/放射抗性。本实验室的研究已经证明，鼠B细胞（具有非病理性组成性NF-κ B活化的罕见实例）通过经由先前未表征的机制降解新合成的I κ B α来维持这种活性。我们的初步数据还表明，为了使IkappaB α持续降解，需要新形成的核NF-κ B/IkappaB α复合物必须输出到鼠B细胞和人癌细胞的细胞质中。因此，拟议的研究将测试这一假设，即组成性NF-κ B激活需要一种机制来抵消其抑制剂IkappaB α施加的自体神经反馈调节。在目标1中，将采用突变分析来描述新的IkappaB α降解机制。在目标2下，将确定IkappaB α的核输出在维持组成性NF-κ B活化和人癌细胞存活中的功能作用。目的3将通过产生具有N-NES点突变的IkappaB α基因座的小鼠来测试IkappaB α核输出在B细胞发育中的体内作用。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组成型NF-κ B活化的关键基本机制。它们还可能揭示N-NES介导的IkappaB α核输出作为合理的治疗靶点，通常破坏组成性NF-κ B活化，以诱导人类癌症中的细胞死亡或化疗/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.