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.
| Zhu, Jianjian; Kwan, Kin Ming; Mackem, Susan (2016) Putative oncogene Brachyury (T) is essential to specify cell fate but dispensable for notochord progenitor proliferation and EMT. Proc Natl Acad Sci U S A 113:3820-5 |
