Determining the Epigenetic Mechanisms Driving Tissue Factor (F3) Upregulation and its Role in Metastatic Osteosarcoma

确定驱动组织因子 (F3) 上调的表观遗传机制及其在转移性骨肉瘤中的作用

• 批准号: 9396464
• 负责人: Ian Matthew Bayles
• 金额: $ 4.4万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9396464
• 关键词: 3-Dimensional; Affect; Architecture; Automobile Driving; CRISPR interference; CRISPR/Cas technology; Cell Line; Cells; Cessation of life; Chromatin; Clinical; Coagulation Process; Coupled; DNA; DNA Sequence; Data; Development; Disease; Distant Metastasis; Elements; Enhancers; Epigenetic Process; F3 gene; Gene Activation; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Enhancer Element; Genome; Histones; Home environment; Impairment; Lead; Lung; Malignant - descriptor; Malignant Bone Neoplasm; Malignant Neoplasms; Mentors; Metastatic Neoplasm to the Lung; Metastatic Osteosarcoma; Modality; Molecular Conformation; Mus; Neoplasm Metastasis; Normal Cell; Patients; Phenotype; Play; Primary Neoplasm; Proliferating; Regulatory Element; Role; Route; Site; Techniques; Technology; Testing; Therapeutic; Thromboplastin; Untranslated RNA; Up-Regulation; base; bone; cancer cell; chromosome conformation capture; cohort; epigenomics; experimental study; in vivo; insight; lung metastatic; metastatic process; mortality; neoplastic cell; novel; osteosarcoma; promoter; small hairpin RNA; transcription activator-like effector nucleases; tumor progression; tumorigenesis

PROJECT SUMMARY/ABSTRACT Distant metastases cause >90% of cancer-related mortality. In osteosarcoma, metastatic progression typically occurs as the cancer spreads from the bones to the lungs. Forty percent of patients with osteosarcoma die from metastatic disease. There is an urgent clinical need to determine the factors responsible for lung metastasis in osteosarcoma so that these factors can be exploited therapeutically. Our lab discovered that epigenetic alterations at enhancer elements play a critical role in the transition of normal cells to malignant cells during tumorigenesis. More recently, the lab has gathered evidence that enhancer alterations also contribute to metastasis, specifically in osteosarcoma. Through epigenomic studies of non-metastatic and highly lung-metastatic osteosarcoma cell line pairs, combined with functional studies, our lab homed in on a gene, Tissue Factor III (F3), that appears to be a critical regulator of the metastatic process in osteosarcoma. The F3 locus is endowed with multiple active enhancer elements in metastatic cells, and these enhancers are almost completely absent in non-metastatic cells. Through functional studies in vivo, we show that F3 is switched on as the metastatic cells engage the lung microenvironment. Remarkably, blocking the activation of F3 via shRNA dramatically impairs the ability of the cells to colonize and proliferate in the lungs, suggesting F3 activation is required for metastasis. Our aims in this study are twofold. First we will test the hypothesis that the enhancers acquired in the metastatic cells at the F3 locus are responsible for activating F3 expression in the lung. This hypothesis will be tested via direct genetic and epigenetic manipulation of the F3 enhancers using TALENs, CRISPR-Cas9, and CRISPRi. Second, we will use chromatin conformation capture technology (4C-seq) to test if the mechanistic basis of F3 gene activation is physical looping between the acquired enhancers and the F3 promoter. As part of this aim we will also test whether the potential contacts between the F3 enhancers and promoters in metastatic cells are absent from non-metastatic cells. Successful completion of these experiments would provide new evidence that non-coding DNA elements are critical a critical driver of metastatic progression of cancer.

项目总结/摘要 远处转移导致>90%的癌症相关死亡率。在骨肉瘤中，转移进展通常 当癌症从骨骼扩散到肺部时就会发生。40%的骨肉瘤患者死亡 转移性疾病临床上迫切需要确定导致肺结核的因素。 骨肉瘤的转移，使这些因素可以利用治疗。我们的实验室发现， 增强子元件的表观遗传改变在正常细胞向恶性细胞的转变中起关键作用 肿瘤发生过程中的细胞。最近，该实验室收集到的证据表明，增强子的改变也 有助于转移，特别是在骨肉瘤中。通过对非转移性和 高肺转移性骨肉瘤细胞系对，结合功能研究，我们的实验室在一个 基因，组织因子III（F3），似乎是骨肉瘤转移过程的关键调节因子。 F3基因座在转移性细胞中被赋予多个活性增强子元件，并且这些增强子是 在非转移性细胞中几乎完全不存在。通过体内功能研究，我们表明F3是 当转移细胞进入肺部微环境时，就会打开。值得注意的是，阻止激活 F3通过shRNA显著削弱细胞在肺中定殖和增殖的能力，表明F3 转移需要活化。我们在这项研究中的目的是双重的。首先，我们将测试假设， 在转移性细胞中在F3基因座获得的增强子负责激活F3在转移性细胞中的表达。 肺。该假设将通过F3增强子的直接遗传和表观遗传操作进行测试， TALEN、CRISPR-Cas9和CRISPRi。其次，我们将使用染色质构象捕获技术 （4C-seq）以测试F3基因激活的机制基础是否是获得的F3基因之间的物理环。 增强子和F3启动子。作为这一目标的一部分，我们还将测试 转移性细胞中的F3增强子和启动子在非转移性细胞中不存在。成功 这些实验的完成将提供新的证据，表明非编码DNA元件是关键的， 癌症转移进展的关键驱动因素。