Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.
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