Mechanism of Constitutive NF-kappa B Activity

描述（由申请人提供）：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和产生对环境变化的适当反应是很重要的。通常，细胞外信号转导为细胞反应涉及一系列最终诱导基因表达的生化事件。这些事件是由转录因子（TF）介导的，转录因子是一类dna结合蛋白，它决定了表达基因的性质。遗传和表观遗传异常对TF活性的调节可以通过不受控制的基因表达导致细胞反应的扩增。细胞外信号对关键TF NF-kappaB的激活通常只是短暂的，因为NF-kappaB激活其自身抑制剂IkappaBalpha的合成，IkappaBalpha进入细胞核，将NF-kappaB从DNA结合位点移除，并将其输出到细胞质以终止NF-kappaB的功能。相反，在人类癌症中经常看到的NF-kappaB活性的解除，必须抵消这种反馈机制，以维持组成（恒定）NF-kappaB激活，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细胞是一种罕见的非病理性组成型NF-kappaB激活的例子，通过一种以前未表征的机制降解新合成的IkappaBalpha来维持这种活性。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复合物输出到小鼠B细胞和人类癌细胞的细胞质中。因此，提出的研究将验证这样的假设，即组成型NF-kappaB激活需要一种机制来抵消其抑制剂IkappaBalpha施加的自抑制或反馈调节。在Aim 1中，突变分析将用于描述新的IkappaBalpha降解机制。在Aim 2下，IkappaBalpha核输出的功能作用将被确定为维持组成型NF-kappaB激活和人类癌细胞的存活。目的3将通过培养携带N-NES点突变的IkappaBalpha基因座的小鼠，测试IkappaBalpha核输出在B细胞发育中的体内作用。本研究计划将有助于确定B细胞发育和人类恶性肿瘤中组成型NF-kappaB激活的关键基本机制。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，通常会破坏构成性NF-kappaB激活，从而诱导人类癌症细胞死亡或化疗/放射致敏。

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.