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。在之前的授权期，我们开发了一个涉及Src诱导的乳腺上皮系(MCF-10A)的实验模型，使其能够动态跟踪细胞转化的过程，包括癌症干细胞和乳房的形成。此外，我们还发现了未转化细胞和转化细胞之间的表观遗传转换，该转换是由一个正反馈环介导的，该环涉及炎症转录因子NF-：B、Lin28microRNA调节器、let-7microRNA和IL-6。这一建议的中心目标是阐明细胞转化和癌症干细胞形成过程中涉及的转录调控电路和潜在的分子机制。首先，使用分子生物学方法，我们将从功能上剖析炎症信号导致细胞转化的调控途径(即涉及转录因子、直接靶基因、microRNAs和microRNA靶标的有序步骤)。我们将讨论成纤维细胞模型和其他未转化细胞系中炎症反应和转化之间的关系，以及关键靶基因(如Lin28和IL6)的启动子/增强子的机制分析，以了解为什么和如何在MCF-10A中发生表观遗传转换，而不是在其他细胞中。此外，在证明miR-335 microRNA在许多方面与let-7相似后，我们将进行类似的机制实验，以确定miR-335(及其靶标tenascin C和Sox4转录因子)如何在炎症与细胞转化之间的过程中发挥作用。其次，我们将获得mRNAs、microRNAs、转录因子结合部位和染色质特征的全基因组图谱(通过芯片测序)，并将从遗传学角度测试转录因子对全基因组基因表达和转化的影响。由此产生的描述将提供转化过程的全基因组分子视图，识别与转化相关的转录因子的直接靶标，提供关键信息，随后将进行详细的机制实验。第三，使用相同的逻辑和实验方法，我们将确定与癌症干细胞的产生和乳房的形成有关的基因、microRNAs和调控途径。这些过程中涉及的关键基因和途径将受到遗传和其他机制实验的影响，以阐明转录电路。作为一个例子，我们将机械地继续我们最近的发现，即miR-200 microRNA家族在癌症干细胞中下调，并直接针对SUZ12，多梳复合体的一种成分。总体而言，这套紧密集成的功能基因组和定向的细胞转化等基因模型的机械实验将有助于阐明与人类癌症有关的转录调控电路。 公共卫生相关性：癌症是一个过程，最终依赖于大量基因和microRNAs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。癌症与炎症性和代谢性疾病有关，我们发现了一种由炎症信号激活的基因调控途径，该途径介导了从正常细胞到癌细胞的表观遗传转换。通过对细胞转化的等基因模型进行一系列紧密集成的功能基因组和定向机制实验，我们将阐明涉及人类癌症的转录调控电路，这将对对疾病的基本理解产生影响，并对治疗产生影响。

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. In the previous granting period, we developed an experimental model involving a Src-inducible breast epithelial line (MCF-10A) that makes it possible to kinetically follow the process of cellular transformation, including the formation of cancer stem cells and mammospheres. Furthermore, we discovered an epigenetic switch between non-transformed and transformed cells mediated by a positive feedback loop that involves the inflammatory transcription factor NF-:B, Lin28 microRNA regulator, Let-7 microRNA, and interleukin 6. The central goal of this proposal is to elucidate the transcriptional regulatory circuits and underlying molecular mechanisms involved in the processes of cellular transformation and the formation of cancer stem cells. First, using molecular biological approaches, we will functionally dissect the regulatory pathway (i.e. the ordered series of steps involving transcription factors, direct target genes, microRNAs, and microRNA targets) by which an inflammatory signal leads to cellular transformation. We will address the relationship between the inflammatory response and transformation in the fibroblast model and other non-transformed cells lines as well as mechanistic analysis of promoters/enhancers of key target genes (e.g. Lin28 and IL6) in order to understand why and how the epigenetic switch occurs in MCF-10A, but not in other cells. In addition, having shown that miR-335 microRNA behaves similarly to Let-7 in many respects, we will perform similar mechanistic experiments to determine how miR-335 (and its targets tenascin C and Sox4 transcription factor) contributes to the process linking inflammation to cellular transformation. Second, we will obtain whole-genome profiles of mRNAs, microRNAs, transcription factor binding sites and chromatin features (via ChIP-sequencing), as well will genetically test transcription factors for their effects on gene expression genome-wide and on transformation. The resulting description will provide a whole-genome molecular view of the transformation process, identify direct targets of transcription factors relevant for transformation, provide critical information that will be followed up with detailed mechanistic experiments. Third, using the same logic and experimental approaches, we will identify genes, microRNAs, and regulatory pathways involved in the generation of cancer stem cells and the formation of mammospheres. Key genes and pathways implicated in these processes will be subjected to genetic and other mechanistic experiments to elucidate transcriptional circuitry. As an example, we will mechanistically pursue our recent finding that the miR-200 microRNA family is down-regulated in cancer stem cells and directly targets SUZ12, a component of the polycomb complex. Overall, this tightly integrated set of functional genomic and directed mechanistic experiments on isogenic models of cellular transformation will help elucidate transcriptional regulatory circuits involved in human cancer. PUBLIC HEALTH RELEVANCE: Cancer is a process that ultimately depends on the correct expression and regulation of a large number of genes and microRNAs by transcription factors that can act as oncoproteins and tumor suppressors. Cancer is linked to inflammatory and metabolic diseases, and we discovered a gene regulatory pathway activated by an inflammatory signal that mediates an epigenetic switch from normal to cancerous cells. By performing a tightly integrated set of functional genomic and directed mechanistic experiments on isogenic models of cellular transformation, we will elucidate transcriptional regulatory circuits involved in human cancer, which will have an impact on the basic understanding of the disease with implications for treatment.