Genomic targets of oncoproteins and tumor suppressors

 描述（由申请人提供）：癌症的特征是细胞生长的异常调节，这一过程最终取决于大量基因的正确表达和转录因子的调节，这些转录因子可作为癌蛋白和肿瘤抑制因子。我们开发了一种诱导型转化模型，其中瞬时炎症信号导致稳定的未转化的人乳腺上皮细胞系经历表观遗传转换到包括癌症干细胞（CSC）群体的稳定转化状态。表观遗传开关由转录因子、miRNA和作为致癌基因或肿瘤抑制因子的靶基因组成的炎症反馈环介导;该途径与许多形式的人类癌症相关。我们发现，在转化群体中，CSC和它们的非干细胞癌细胞对应物在表观遗传学上并不不同，而是存在于一种动态平衡中，涉及白细胞介素6和整合的转录调控因子。 用作开关的电路。最后，我们证明了分别是Wnt、Hippo和钙信号传导途径的最终靶标的3种转录共激活因子（β-连环蛋白、雅普/TAZ、S100 A9/A8）对于转化是关键的。这个建议的中心目标是阐明，在全基因组规模上，参与细胞转化和CSC形成的转录调控回路，这两者都没有以这种方式进行过研究。首先，我们将对我们已经确定的候选转录因子进行遗传（通过siRNA或CRISPR，然后是RNA-seq）和ChIP-seq实验，以确定这些因子在基因组中的结合位置以及它们调节的基因。结果将被整合到转录调控电路。其次，我们将鉴定共激活因子（β-catenin，雅普/TAZ，S100 A9/A8）的直接和间接靶点，并将结果与DNA结合转录因子的结果整合。此外，我们确定了WDR 77和精氨酸甲基化酶PRMT 5与β-连环蛋白相互作用，以及与雅普/TAZ相互作用的多聚腺苷酸化机制的组成部分;我们将研究这些相互作用的蛋白质介导其对基因表达和转化的影响的分子机制。第三，使用新的概念方法的基础上的致癌和非致癌蛋白衍生物或不同的信号分子介导的mRNA的档案，我们将确定致癌相关的目标。作为补充，我们将使用我们的高通量转化试验来进行基因组规模的遗传筛选，以寻找对转化重要的基因。所鉴定的基因将被整合到目标1和2中阐明的转录电路中。四是 从这些结果得到的调节回路将在其他癌细胞类型中以及通过癌症患者样品中的基因表达模式得到验证。总之，这一紧密整合的一套遗传和功能基因组实验的诱导模型的转化和CSC的形成将揭示新的光在分子水平上的癌症进展的基本问题，和新的途径和目标的治疗可能会被确定。

 DESCRIPTION (provided by applicant): Cancer is characterized by abnormal regulation of cell growth, a process that ultimately depends on the correct expression and regulation of a large number of genes by transcription factors that can act as oncoproteins and tumor suppressors. We developed an inducible transformation model in which a transient inflammatory signal causes a stable non-transformed human breast epithelial cell line to undergo an epigenetic switch to a stable transformed state that includes a population of cancer stem cells (CSCs). The epigenetic switch is mediated by an inflammatory feedback loop consisting of transcription factors, miRNAs, and target genes that are oncogenes or tumor suppressors; this pathway is relevant for many forms of human cancer. We showed that CSCs and their non-stem cancer cell counterparts in the transformed population are not epigenetically distinct, but rather exist in a dynamic equilibrium involving interleukin 6 and an integrated transcriptional regulatory circuit that acts as a bistable switch. Lastly, we demonstrated that 3 transcriptional co-activator (ß-catenin, YAP/TAZ, S100A9/A8) that, respectively, are the ultimate targets of the Wnt, Hippo, and calcium signaling pathways, are critical for transformation. The central goal of this proposal is to elucidate, on a whole-genome scale, the transcriptional regulatory circuits involved in cellular transformation and CSC formation, neither of which have been investigated in this fashion. First, we will perform genetic (loss of function via siRNA or CRISPR followed by RNA-seq) and ChIP-seq experiments on candidate transcription factors we have already identified to determine where the factors bind in the genome and what genes they regulate. The results will be integrated into transcriptional regulatory circuits. Second, we will identify direct and indirec targets of co-activators (ß-catenin, YAP/TAZ, S100A9/A8) and integrate the results with those of the DNA-binding transcription factors. In addition, we identified WDR77 and the arginine methylase PRMT5 as interacting with ß-catenin as well as components of the polyadenylation machinery as interacting with YAP/TAZ; we will examine the molecular mechanisms by which these interacting proteins mediate their effects on gene expression and transformation. Third, using novel conceptual approaches based on mRNA profiles mediated by oncogenic and non-oncogenic protein derivatives or by different signaling molecules, we will identify oncogenically relevant targets. As a complement, we will use our high-throughput transformation assay to perform genome-scale genetic screens for genes important for transformation. The identified genes will be integrated into the transcriptional circuitry elucidated in aims 1 and 2. Fourth, the regulatory circuits derived from these results will be validated in other cancer cell types and by gene expression patterns in cancer patient samples. In summary, this tightly integrated set of genetic and functional genomic experiments on an inducible model of transformation and CSC formation will shed new light on fundamental issues in cancer progression at the molecular level, and new pathways and targets for therapy might be identified.