Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
EGR1, one of the immediate-early response genes, plays an important role as a mediator for transmitting extracellular stimuli. EGR1 is down regulated in many cancers. Many studies show that it functions as a tumor suppressor gene in a variety of cancers. EGR1 also acts as an oncogene in number of cancers. We found that in rhabdomyosarcoma (RMS), which is a muscle derived pediatric cancer, EGR1 was expressed in both RMS subtypes, embryonal and alveolar, but with a much higher expression profile in embryonal RMS. This suggests different mechanisms of down regulation of EGR1 in these two subtypes. Molecular and cellular approaches were used to characterize the functional role of EGR1 in RMS. We found that over expression of EGR1 in alveolar RMS significantly decreased cell proliferation, mobility, and anchorage-independent growth. We showed that exogenous EGR1 up regulated the cell cycle regulator, p21, which is normally repressed in RMS. EGR1 also promoted differentiation in RMS cells by up regulating several genes involved in muscle differentiation including myosin heavy chain (MyHC), MyoD and myogenin. We found that EGR1 interacts with the oncogene TBX2 in RMS cells and that TBX2 inhibits EGR1 function. To understand how TBX2 inhibits EGR1, we depleted TBX2 in RMS and we found an up regulation of the EGR1 targeted tumor suppressor gene, PTEN, and the cysteine protease inhibitor gene, CST6. Also, we performed luciferase assays and found that TBX2 decreased the expression of luciferase constructs fused with the PTEN promoter when TBX2 was co-transfected with EGR1. Our novel findings on the EGR1 function in RMS highlights the significant role of EGR1 in muscle development and tumor growth. Significantly, our work also suggests the EGR1 could promote tumor regression in RMS through inducing programmed cell death, or apoptosis. We found that EGR1 induced apoptosis through triggering the intrinsic apoptosis pathway and activating caspase cascades involving caspase 3 and caspase 9, which are essential mitochondrial apoptotic factors. Also, we observed the activation of two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. Also, we found in our study that EGR1 is repressed by the catalytic subunit of PRC2 complex, EZH2, which mediates gene silencing through methylation of lysine 27 on histone 3 (H3K27me3). EGR1 also sensitized RMS cells to chemotherapeutic agents, which could be a future direction for improved therapeutic targeting. Therefore, this work provides a novel and powerful molecular therapeutic target for RMS cancer.
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