169 research outputs found
Abstract 3509: EWS/FLI regulates transcriptional activation via length-dependent GGAA microsatellites
No full textAbstract
Objective: The purpose of this study is to investigate how EWS/FLI transcriptionally activates gene targets via polymorphic GGAA microsatellites. Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA microsatellites, which show length polymorphisms. These microsatellite polymorphisms may critically affect EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 20-25 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1 through clinical observation and in vitro studies. The mechanism underlying this optimal length is unknown.
Methods: We explored the stoichiometry and binding affinity of EWS/FLI for different repeat lengths through biochemical studies, including fluorescence polarization, and immunoprecipitation assays, combined with bioinformatics analysis. Additionally, use of EWS/FLI mutant constructs has been critical for elucidating particular binding behavior of EWS/FLI at different microsatellite repeat lengths.
Results: Fluorescence anisotropy studies demonstrate that FLI binding affinity is independent of GGAA microsatellite length. In contrast, the stoichiometry of protein to DNA binding increases in specific incremental patterns with increasing microsatellite repeats. EWS/FLI mutants inform on binding, suggesting a complex relationship between microsatellite length and transcriptional activity.
Conclusion: Overall our data suggests a model in which the DNA binding domain of multiple FLI monomers function as independent binding units to facilitate transcriptional activity in a length-dependent manner. The EWS portion appears to be critical for in vivo binding of genomic DNA globally. We propose that GGAA microsatellites are necessary and sufficient for EWS/FLI-mediated oncogenic transformation and that repeat length affects optimal DNA binding stoichiometry and transcriptional activity.
Citation Format: Kirsten M. Johnson, Cenny Taslim, Stephen L. Lessnick. EWS/FLI regulates transcriptional activation via length-dependent GGAA microsatellites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2017-3509</jats:p
Molecular Pathogenesis of Ewing Sarcoma: New Therapeutic and Transcriptional Targets
No full text Approximately one-third of sarcomas contain specific translocations. Ewing sarcoma is the prototypical member of this group of sarcomas; it was the first to be recognized pathologically as a singular entity and to have its signature translocation defined cytogenetically, which led to the identification of its key driver alteration, the EWS-FLI1 gene fusion that encodes this aberrant, chimeric transcription factor. We review recent progress in selected areas of Ewing sarcoma research, including the application of genome-wide chromatin immunoprecipitation analyses, to provide a comprehensive view of the EWS-FLI1 target gene repertoire, the identification of EWS-FLI1 target genes that may also point to therapeutically targetable pathways, and data from model systems as they relate to the elusive cell of origin of Ewing sarcoma and its possible similarities to mesenchymal stem cells. </jats:p
Abstract 5817: Protein phosphatase 1 regulatory subunit 1A promotes tumorigenesis and metastasis in Ewing sarcoma
No full textAbstract
Background: Ewing sarcoma (ES) is a highly invasive and metastatic pediatric soft tissue and bone tumor. Children with metastatic ES have a cure rate of less than 30% (De loris MA et al, 2013). Novel and specific therapeutic targets are urgently needed. ES is characterized by the oncogenic fusions, mostly EWS/FLI, which functions as an aberrant transcription factor to deregulate downstream targets and mediate ES pathogenesis. By comparing genes dysregulated by EWS/FLI across multiple model systems (Niedan S et al, 2014; Sankar S et al, 2013; Tirode F et al, 2007), we identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor upon PKA phosphorylation, as one of the core EWS/FLI targets (Luo W et al, 2016).
Objective: In the current study, we seek to define the role of PPP1R1A in ES pathogenesis.
Methods: Quantitative reverse transcription polymerase chain reaction and luciferase reporter assays were performed to investigate transcriptional regulation of PPP1R1A by EWS/FLI. In vitro functional assays and orthotopic injections in immune-deficient mice were conducted to investigate the effect of PPP1R1A on ES oncogenic transformation and cell migration, and tumorigenesis and metastasis, respectively. High-throughput sequencing and functional annotation were utilized to identify PPP1R1A regulated genes and cellular functions. Drug treatment was performed to test the effect of PKA inhibitors on ES cell proliferation and tumor development.
Results: We found that PPP1R1A is directly up-regulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A caused a significant decrease in oncogenic transformation (p&lt;0.05) and cell migration (p=0.009) in vitro and limited xenograft tumor growth (p=0.0009) and metastasis (p=0.009) in vivo. We also discovered that PPP1R1A regulates genes involved in various cellular processes including acting binding, cell adhesion, and differentiation. Interestingly, PPP1R1A regulated gene set significantly overlap with that of ZEB2 (p=2.67246E-33) and EWS (p=1.2559E-45), which regulates metastasis and neuronal differentiation in ES, respectively. Further, we demonstrated that PKA phosphorylation and activation of PPP1R1A, and subsequent PP1 binding and inhibition by activated PPP1R1A, is required for PPP1R1A mediated ES pathogenesis, likely by increasing the phosphorylation level of various PP1 substrates, such as RB and CREB, which are critical for fundamental cellular functions including proliferation and differentiation. Consistently, we found that PKA inhibitors impaired ES cell proliferation and xenograft tumor growth and metastasis.
Conclusion: Collectively, we identified an essential kinase and phosphatase pathway, PKA/PPP1R1A/PP1, that plays a critical role in ES tumorigenesis and metastasis, and thus is a potential therapeutic target in the treatment of primary and metastatic ES.
Citation Format: Wen Luo, Changxin Xu, Janet Ayello, Filemon De La Cruz, Jeremy Rosenblum, Stephen L. Lessnick, Mitchell S. Cairo. Protein phosphatase 1 regulatory subunit 1A promotes tumorigenesis and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5817. doi:10.1158/1538-7445.AM2017-5817</jats:p
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