Role of Brachyury in regulating notochord development and neoplasia

Brachyury 在调节脊索发育和肿瘤中的作用

• 批准号: 9343811
• 负责人: Susan Mackem
• 金额: $ 13.46万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9343811
• 关键词: Affect; Alleles; Animals; Apoptosis; Behavior; Brachyury protein; Cell Communication; Cell Survival; Cells; Child; Chordoma; Development; Developmental Process; Embryo; Embryonic Development; Forelimb; Genes; Genetic; Genetic Models; Growth; Human; Infant; Intercept; Knowledge; Left; Life; Limb structure; Malignant Neoplasms; Manuscripts; Mesoderm; Mesoderm Cell; Methods; Modeling; Morbidity - disease rate; Movement; Mus; Muscle; Neoplasm Metastasis; Neoplasms; Pathogenesis; Pathway interactions; Phenotype; Play; Primitive Streaks; Process; Production; Proliferating; Protein p53; Reporting; Role; Signal Transduction; Spinal Cord; Structure; Subfamily lentivirinae; TSC1/2 gene; Testing; Therapeutic Intervention; Time; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Tuberous sclerosis protein complex; Tumor Biology; Vertebral column; base; bone; cancer cell; cell behavior; cell growth; cell motility; cellular targeting; embryo tissue; in vivo; insight; knock-down; mortality; mouse development; mouse model; mutant; notochord; notochord development; novel; novel therapeutics; null mutation; progenitor; recombinase-mediated cassette exchange; skull base; small hairpin RNA; therapeutic target; tool; transcription factor; tumor; tumorigenesis; young adult

We recently developed a novel method to control the production of the transcription factor Brachyury/T in cells by using a lentivirus that produces an shRNA directed against Brachyury and, when introduced into cells (or animals), causes the loss of Brachyury/T. This novel tool will enable us to alter Brachyury function during mouse development to understand how it regulates cell fate and behavior, and then to determine whether its function is necessary for notochord cell survival. It has been hypothesized that Brachyury plays a central role in the formation of cancers that arise from notochord (chordomas), and this tool may also provide the impetus for devising new therapies for these very difficult to treat cancers. We have introduced this shRNA construct into transgenic mice and employed Cre-lox technology to selectively activate its expression in different embryonic tissues at specific times. We have shown that early activation of this knock-down construct in mouse embryos that are heterozygous for the Brachyury null mutation (T+/-) reproduces the null embryonic lethal phenotype with loss of the body axis caudal to the forelimb level. Now that we have shown that Brachyury can be effectively removed from cells and animals using this approach, we are using it to study the normal function of Brachyury in regulating growth and cell fate during notochord formation, in comparison to its role in primitive streak. The activation of Brachyury expression has been proposed to be essential for the genesis of chordomas, cancers that arise in notochord remnants. The gene knock-down tools we have developed to study Brachyury function have allowed us to test this hypothesis and unravel the possible mechanisms by which this gene may promote tumor formation, and may also give new insights on developing potential therapeutic interventions for chordoma. Surprisingly, our results indicate that Brachyury function is essential for maintaining notochord cell fate, but dispensible for the survival and proliferation of notochord progenitors. A manuscript reporting these results is ccurrently under review. We are also developing a genetic mouse model for chordoma based on activated canonical Wnt pathway in conjunction with p53 tumor suppressor inactivation in the notochord lineage. Such a genetic model will allow us to introduce and evaluate the effects of mutant alleles for tuberous sclerosis complex genes (TSC1,2) which are know to predispose to chordoma in humans, but not in mice, suggesting other factors are also necessary in the pathogenesis of TSC-related chordoma. We also plan to use the shRNA transgene approach to knock-down brachyury expression in this mouse chordoma model, to assess the potential of brachyury inactivation as an approach in treating this tumor.

我们最近开发了一种新方法，通过使用慢病毒来控制细胞中转录因子 Brachyury/T 的产生，该慢病毒产生针对 Brachyury 的 shRNA，并且当引入细胞（或动物）时，会导致 Brachyury/T 的丢失。这种新工具将使我们能够在小鼠发育过程中改变 Brachyury 功能，以了解它如何调节细胞命运和行为，然后确定其功能是否对于脊索细胞生存是必要的。据推测，Brachyury 在脊索（脊索瘤）引起的癌症的形成中发挥着核心作用，并且该工具也可能为为这些非常难以治疗的癌症设计新疗法提供动力。我们已将这种 shRNA 构建体引入转基因小鼠中，并利用 Cre-lox 技术在特定时间选择性激活其在不同胚胎组织中的表达。我们已经证明，在 Brachyury 无效突变 (T+/-) 杂合的小鼠胚胎中，这种敲低构建体的早期激活会复制无效胚胎致死表型，并导致前肢水平尾部的体轴丧失。现在我们已经证明，使用这种方法可以有效地从细胞和动物中去除 Brachyury，我们正在使用它来研究 Brachyury 在脊索形成过程中调节生长和细胞命运的正常功能，并与其在原条中的作用进行比较。 Brachyury 表达的激活被认为对于脊索瘤（脊索残余物中出现的癌症）的发生至关重要。我们开发的用于研究 Brachyury 功能的基因敲除工具使我们能够检验这一假设，并揭示该基因促进肿瘤形成的可能机制，并且还可能为开发脊索瘤的潜在治疗干预措施提供新的见解。令人惊讶的是，我们的结果表明 Brachyury 功能对于维持脊索细胞的命运至关重要，但对于脊索祖细胞的生存和增殖却是可有可无的。报告这些结果的手稿目前正在审查中。我们还在开发一种脊索瘤遗传小鼠模型，该模型基于激活的经典 Wnt 通路以及脊索谱系中 p53 肿瘤抑制因子的失活。这样的遗传模型将使我们能够引入和评估结节性硬化症复合体基因（TSC1,2）的突变等位基因的影响，已知这些基因在人类中易患脊索瘤，但在小鼠中则不然，这表明其他因素在发病机制中也是必要的TSC 相关脊索瘤。我们还计划使用 shRNA 转基因方法来敲低该小鼠脊索瘤模型中的 brachyury 表达，以评估 brachyury 失活作为治疗该肿瘤的方法的潜力。