Genomic targets of oncoproteins and tumor suppressors

描述（由申请人提供）：癌症以细胞生长受到异常调控为特征，这一过程最终依赖于大量基因被转录因子正确表达和调控，这些转录因子可以作为癌蛋白和肿瘤抑制因子。在之前的授权期内，我们开发了一个涉及src诱导的乳腺上皮细胞系（MCF-10A）的实验模型，该模型可以动态跟踪细胞转化过程，包括癌症干细胞和乳房微球的形成。此外，我们发现非转化细胞和转化细胞之间的表观遗传转换是由一个正反馈回路介导的，该回路涉及炎症转录因子NF-:B、Lin28 microRNA调节剂、Let-7 microRNA和白细胞介素6。本研究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回路和潜在的分子机制。首先，利用分子生物学方法，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、microRNA和microRNA靶点的有序步骤）。我们将讨论成纤维细胞模型和其他非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，以了解表观遗传开关发生在MCF-10A中，而不在其他细胞中发生的原因和方式。此外，已经证明miR-335 microRNA在许多方面与Let-7相似，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）如何促进炎症与细胞转化的过程。其次，我们将获得mrna、microRNAs、转录因子结合位点和染色质特征的全基因组图谱（通过chip测序），并将对转录因子对全基因组基因表达和转化的影响进行基因测试。由此产生的描述将提供转化过程的全基因组分子视图，确定与转化相关的转录因子的直接靶标，提供后续详细机制实验的关键信息。第三，使用相同的逻辑和实验方法，我们将确定基因、microrna和参与癌症干细胞的产生和乳房微球形成的调控途径。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，以阐明转录回路。例如，我们将从机制上继续我们最近的发现，即miR-200 microRNA家族在癌症干细胞中下调，并直接靶向多梳复合体的组成部分SUZ12。总的来说，这套紧密结合的功能基因组学和定向机制实验将有助于阐明与人类癌症有关的转录调控回路。

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.