Mechanisms of Delta-N-p63-alpha transcriptional repression and its role in squamo

描述（由申请人提供）：p53相关蛋白Np631是一种有效的致癌基因，可驱动各种来源的鳞状细胞癌（SCC）的发生和进展。虽然该因子的生理水平是上皮干细胞维持所必需的，但Np631过表达可促进癌细胞增殖和存活，并抑制细胞周期阻滞和凋亡。该项目的总体目标是确定Np631影响基因转录和细胞行为的机制。我们的初步研究结果表明，在H226 SCC细胞中，Np631是促凋亡因子NOXA的一种有效的基因特异性抑制因子，其机制独立于其在p53应答元件上结合染色质的能力，以及在p53介导的组蛋白乙酰化后的调节步骤。然而，在不同的SCC细胞系中，其他研究人员已经表明，Np631缺失会抑制其他p53/p73应答基因。本项目的第一个Specific Aim将确定其他家族成员p53和p73对Np631缺失在H226细胞和其他细胞系中的转录和细胞效应的相对贡献。将使用稳定表达的shRNA构建缺乏p53和/或p73的等基因H226细胞系，并确定Np631缺失对细胞存活、增殖、凋亡和bbb30p53 /p73靶基因表达的影响。此外，NOXA和Np631将在H226细胞中同时被敲低，以确定NOXA上调是否对Np631敲低后细胞增殖丧失至关重要。这些研究将使我们能够确定p53/p73、Np631和NOXA之间的上位性关系。第二个特异性目标将确定Np631抑制特定p53/p73靶基因表达的分子机制。通过对Np631基因敲除前后NOXA和p21位点发生的转录事件进行详尽的染色质免疫沉淀（ChIP）分析，在基础和激活p53的条件下，Np631影响的精确调控步骤将被确定。最终的特异性目的将确定生物学相关的Np631突变对基因特异性抑制的影响。体内结构功能分析将通过挽救Np631敲低的细胞来进行，这些细胞要么是野生型Np631，要么是在DNA结合域、SAM域和其他区域中含有自然发生突变的构建体。这些实验将确定Np631的关键功能区域，并进一步阐明Np631的作用机制。预计在这个项目中产生的知识将极大地提高我们对Np631的理解，它的作用机制，它在正常上皮维持和肿瘤发生中的作用，以及帮助制定由Np631过表达驱动的各种癌症的治疗控制策略。

DESCRIPTION (provided by applicant): The p53-related protein, Np631, is a potent oncogene driving the development and progression of squamous cell carcinomas (SCC) of various origins. While physiologic levels of this factor are required for epithelial stem cell maintenance, Np631 overexpression promotes cancer cel proliferation and survival, and inhibits cel cycle arrest and apoptosis. The overall goal of this project is to define the mechanisms by which Np631 impacts gene transcription and cell behavior. Our initial findings indicate that in H226 SCC cells, Np631 is a potent gene-specific repressor of the pro-apoptotic factor NOXA, by mechanisms independent of its ability to bind chromatin at p53 responsive elements, and at regulatory steps that follow p53-mediated histone acetylation. Yet in different SCC cell lines, other investigators have shown that Np631 depletion de-represses other p53/p73-responsive genes. The first Specific Aim of this project will define the relative contributions of the other family members, p53 and p73, to the transcriptional and celular efects of Np631 depletion in H226 cells and other cell lines. A panel of isogenic H226 cell lines lacking p53 and/or p73 will be created using stably-expressed shRNA constructs, and the effect of Np631 depletion on cel survival, proliferation, apoptosis, and the expression of >30 p53/p73 target genes will be determined. Furthermore, NOXA and Np631 will be concomitantly knocked down in H226 cells to determine whether NOXA upregulation is crucial for the loss of cell proliferation upon Np631 knockdown. These studies will enable us to define the epistatic relationship between p53/p73, Np631, and NOXA. The second Specific Aim will identify the molecular mechanism by which Np631 represses the expression of specific p53/p73 target genes. By performing an exhaustive chromatin immunoprecipitation (ChIP) analysis of the transcriptional events taking place at the NOXA and p21 loci before and after Np631 knockdown under conditions of both basal and activated p53, the precise regulatory steps affected by Np631 will be identified. The final Specific Aim will determine the impact of biologically-relevant Np631 mutations on gene-specific repression. An in vivo structure-function analysis will be performed by rescuing Np631 knockdown cells with either wild type Np631 or constructs containing naturally occurring mutations in the DNA binding domain, SAM domain, and other regions. These experiments will identify crucial functional regions of Np631 and will further illuminate the Np631 mechanism of action. It is anticipated that the knowledge generated during this project will vastly improve our understanding of Np631, its mechanism of action, and its role in normal epithelial maintenance and oncogenesis, as well as to help enable strategies for the therapeutic control of the various cancers driven by Np631 overexpression. PUBLIC HEALTH RELEVANCE: This project will investigate the role of the oncogenic protein, Np631, in the development and progression of squamous cell carcinomas. We will determine the molecular mechanisms by which Np631 promotes cancer cell proliferation, and will identify the means by which Np631 suppresses the expression of genes crucial for cell cycle arrest (cessation of proliferation) and apoptosis (cell death). Ultimately, this research may help to identify novel targets for the development of therapeutics to treat squamous cell carcinomas of various origins.