Genomic targets of oncoproteins and tumor suppressors

 描述(申请人提供)：癌症的特征是细胞生长的异常调节，这个过程最终依赖于大量基因的正确表达和转录因子的调节，这些转录因子可以作为癌蛋白和肿瘤抑制因子。我们开发了一个可诱导的转化模型，在该模型中，瞬时的炎症信号导致稳定的未转化的人乳腺上皮细胞株经历表观遗传学转换到稳定的转化状态，其中包括一群癌症干细胞(CSCs)。表观遗传开关是由由转录因子、miRNAs和作为癌基因或肿瘤抑制因子的靶基因组成的炎症反馈环介导的；这一途径与许多形式的人类癌症有关。我们发现转化群体中的CSCs和它们的非干细胞癌细胞对应物在表观遗传学上并不是截然不同的，而是存在于一种涉及白细胞介素6和整合转录调控的动态平衡中。 充当双稳开关的电路。最后，我们证明了Wnt、Hippo和钙信号通路的终极靶标--3个转录共激活因子(？)这项建议的中心目标是在全基因组范围内阐明参与细胞转化和CSC形成的转录调控电路，这两个方面都没有以这种方式进行研究。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失去功能，然后是RNA-seq)和芯片序列实验，以确定这些因子在基因组中结合的位置以及它们调节的基因。结果将被整合到转录调控电路中。其次，我们将确定共激活子(？)的直接和间接靶标(？)，并将结果与DNA结合转录因子的结果相结合。此外，我们还鉴定了WDR77和精氨酸甲基酶PRMT5与B-连环蛋白的相互作用，以及多聚腺苷化机制的组成部分与YAP/TAZ的相互作用；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，使用新的概念性方法，基于由致癌和非致癌蛋白衍生物或不同信号分子介导的mRNA图谱，我们将识别与肿瘤相关的靶点。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。识别出的基因将被整合到AIMS 1和2中阐明的转录电路中。 根据这些结果得出的调节电路将在其他类型的癌细胞中得到验证，并通过癌症患者样本中的基因表达模式进行验证。总之，这套紧密结合的关于转化和CSC形成的诱导模型的遗传和功能基因组实验将在分子水平上揭示癌症进展的基本问题，并可能确定新的治疗途径和靶点。

 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.