Genomic targets of oncoproteins and tumor suppressors

癌蛋白和肿瘤抑制因子的基因组靶标

• 批准号: 8433243
• 负责人: KEVIN STRUHL
• 金额: $ 65.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-18 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8433243
• 关键词: Address; Binding Sites; Biological; Breast; Cancer cell line; Cancerous; Cell Line; Cells; ChIP-seq; Chromatin; Complex; Development; Disease; Enhancers; Epigenetic Process; Epithelial; Epithelial Cells; Experimental Models; Family; Feedback; Fibroblasts; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genome; Genomics; Goals; Grant; Human; IL6 gene; Inflammation; Inflammatory; Inflammatory Response; Interleukin-6; Link; Logic; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic Diseases; MicroRNAs; Modeling; Molecular; Oncogene Proteins; Other Genetics; Pathway interactions; Polycomb; Population; Process; RNA; Regulation; Regulatory Pathway; Sampling; Series; Signal Transduction; Staging; System; Tenascin; Testing; Tissues; Transcription Coactivator; Transcription factor genes; Transformed Cell Line; Tumor Suppressor Proteins; cancer stem cell; cell growth regulation; cell transformation; cell type; follow-up; functional genomics; genetic analysis; genome-wide; metaplastic cell transformation; novel; promoter; public health relevance; research study; transcription factor; tumor; v-src Oncogenes

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.

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。在上一个授予期，我们开发了一种涉及Src诱导型乳腺上皮细胞系（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腺球的形成。此外，我们发现了非转化细胞和转化细胞之间的表观遗传开关，该开关由涉及炎性转录因子NF-：B、Lin 28 microRNA调节因子、Let-7 microRNA和白细胞介素6的正反馈环介导。该提案的中心目标是阐明参与细胞转化和癌症干细胞形成过程的转录调控回路和潜在分子机制。首先，使用分子生物学方法，我们将从功能上剖析调控途径（即涉及转录因子、直接靶基因、microRNA和microRNA靶点的有序步骤系列），炎症信号通过该途径导致细胞转化。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（例如Lin 28和IL 6）的启动子/增强子的机制分析，以了解表观遗传开关为什么以及如何发生在MCF-10A中，而不是在其他细胞中。此外，已经表明miR-335 microRNA在许多方面与Let-7的行为相似，我们将进行类似的机制实验以确定miR-335（及其靶点腱生蛋白C和Sox 4转录因子）如何有助于将炎症与细胞转化联系起来的过程。其次，我们将获得mRNA、microRNA、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），并将从遗传学上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，提供将通过详细的机理实验跟进的关键信息。第三，使用相同的逻辑和实验方法，我们将识别参与癌症干细胞产生和乳腺球形成的基因、微小RNA和调节途径。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的实验，以阐明转录电路。作为一个例子，我们将机械地追求我们最近的发现，即miR-200 microRNA家族在癌症干细胞中下调，并直接靶向SUZ 12，这是polycomb复合物的一个组成部分。总的来说，这一紧密整合的功能基因组和定向机制实验的同基因模型的细胞转化将有助于阐明参与人类癌症的转录调控回路。