Role of Brachyury in regulating notochord development and neoplasia

我们最近开发了一种新方法，通过使用一种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，当引入细胞（或动物）时，导致Brachyury/T的丢失。这种新工具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，并确定其功能是否对脊索细胞存活是必要的。据推测，Brachyury在脊索瘤（脊索瘤）引起的癌症形成中起着核心作用，这一工具也可能为设计治疗这些非常难以治疗的癌症的新疗法提供动力。我们将该shRNA构建体导入转基因小鼠，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零突变（T+/-）杂合的小鼠胚胎中，这种敲低构建体的早期激活会再现零胚胎致死表型，身体轴尾端至前肢水平被截断。在证明Brachyury可以通过这种方法有效地从细胞和动物中去除后，我们使用该工具研究了Brachyury在脊索形成过程中调节生长和细胞命运的正常功能，并将其在原始条纹中的作用进行了比较。Brachyury表达的激活被认为是脊索瘤发生的必要条件，脊索瘤是在脊索残余中产生的癌症。我们开发的研究Brachyury功能的基因敲除工具使我们能够验证这一假设，并揭示该基因促进肿瘤形成的可能机制，也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，我们的研究结果表明，短肌腱功能对于维持脊索细胞的命运是必不可少的，但对于脊索祖细胞的生存和增殖是必不可少的，在缺乏短肌腱功能的情况下，脊索祖细胞采用替代的神经命运。我们目前正在开发一种基于激活的典型Wnt通路的脊索瘤遗传小鼠模型。我们对脊索谱系中典型Wnt激活与p53肿瘤抑制基因敲除的初步尝试表明，令人惊讶的是，p53突变并不是该肉瘤形成的重要驱动因素。目前的努力集中在利用遗传方法增强相邻椎体骨髓的脊索细胞播种，这似乎是人类脊索瘤形成的主要部位，可能提供一个有利的生态位，与Wnt激活相结合。Yamaguchi实验室已经开发出一种在多西环素治疗下有条件地错误表达转基因Brachyury的小鼠系，作为一种平行的方法，我们正在合作用这种转基因系建立脊索瘤模型，这将使我们能够测试Brachyury是否在脊索瘤中表现为传统的癌基因，并表现出“癌基因成瘾”（依赖于持续的癌基因功能作为驱动因素）。一个有效的小鼠遗传模型将使我们能够引入和评估突变等位基因对结节性硬化症复杂基因（tsc1,2）的影响，这些基因已知在人类中易患脊索瘤，但在小鼠中却没有，这表明其他因素在tsc相关脊索瘤的发病机制中也是必要的。根据多西环素激活Brachyury错误表达实验的结果，我们还计划使用shRNA策略来调节Brachyury在小鼠脊索瘤模型中的表达，并评估Brachyury失活作为治疗该肿瘤的一种方法的潜力。

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 enables us to alter Brachyury function during mouse development to understand how it regulates cell fate and behavior, and 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 truncation of the body axis caudal to the forelimb level. Having shown that Brachyury can be effectively removed from cells and animals using this approach, we used this tool 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 is dispensable for the survival and proliferation of notochord progenitors, which adopt an alternate neural fate in the absence of Brachyury function. We are now developing a genetic mouse model for chordoma based on activated canonical Wnt pathway. Our preliminary attempts to couple this canonical Wnt activation with p53 tumor suppressor knock-out in the notochord lineage suggest that, surprisingly, p53 mutation is not a significant driver for formation of this sarcoma. Current efforts are focused at using genetic approaches to enhance notochordal cell seeding of adjacent vertebral bone marrow, which appears to be the predominant site for chordoma formation in humans and may provide a favorable niche, in conjunction with Wnt activation. The Yamaguchi lab has developed a mouse line conditionally mis-expressing transgenic Brachyury upon doxycycline treatment, and as a parallel approach, we are collaborating to establish a chordoma model with this transgenic line which will enable us to test whether Brachyury behaves as a traditional oncogene in chordoma and displays "oncogene addiction" (dependence on continued oncogene function as a driver). An effective mouse genetic model will allow us to introduce and evaluate the effects of mutant alleles for tuberous sclerosis complex genes (TSC1,2) which are known to predispose to chordoma in humans, but not in mice, suggesting other factors are also necessary in the pathogenesis of TSC-related chordoma. Depending on the outcome of doxycycline-activated Brachyury mis-expression experiments, we also plan to use shRNA strategies to modulate Brachyury expression in mouse chordoma models, and assess the potential of Brachyury inactivation as an approach in treating this tumor.