NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma

Kinsey, Michelle; Smith, Richard A.; Lessnick, Stephen L.

doi:10.1158/1541-7786.mcr-06-0090

Cited by 184 publications

(243 citation statements)

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“…One of the challenges, however, for identifying which EWS/FLI targets participate in cancer-relevant phenotypes is the complexity of assays required to assess many of these phenotypes, which precludes the development of high-throughput screening approaches. An alternate approach, then, is to limit the number of gene products to be assessed in a lower-throughput screening assays, for example, by comparing microarray profiles across multiple different experiments (Kinsey et al, 2006;Hancock and Lessnick, 2008), or by identifying which genes are 'direct' EWS/FLI targets .…”

Section: Formationmentioning

confidence: 99%

“…We recently showed that EWS/FLI uses a GGAAmicrosatellite to regulate NR0B1, a gene that is required for oncogenic transformation in Ewing's sarcoma cells (Kinsey et al, 2006;. During the course of these studies, we identified thousands of GGAA-microsatellite-containing genes, suggesting that at least a portion of these might be regulated by direct binding of EWS/FLI to their promoters.…”

Section: Formationmentioning

confidence: 99%

“…These data sets included (i) the transcriptional profile of EWS/FLI in Ewing's sarcoma cells (produced by comparing patient-derived Ewing's sarcoma cells with retroviral-mediated RNA interference (RNAi) constructs targeting EWS/FLI with those harboring control constructs; Kinsey et al, 2006;Smith et al, 2006), (ii) genome-wide localization data of EWS/ FLI in Ewing's sarcoma cells (produced by 'ChIP-chip' experiments; and (iii) comprehensive mapping data of GGAA-microsatellites in the human genome (produced computationally; . Through these cross-platform comparisons, we identified genes that have all of the characteristics we sought.…”

Section: Formationmentioning

confidence: 99%

“…To evaluate the role of EWS/FLI in regulating GSTM4, we first extracted the GSTM4 data from our transcriptional profiling studies (Kinsey et al, 2006;Smith et al, 2006). In these studies, two different retroviral RNAi constructs targeting the 3 0 -UTR of endogenous EWS/FLI (EF-2-RNAi and EF-4-RNAi) were introduced into patient-derived Ewing's sarcoma Luciferase assays using a pGL3-promoter luciferase vector (Promega Corporation, Madison, WI, USA) containing the GSTM4 GGAA-microsatellite, or an empty control vector, co-transfected into TC71 Ewing's sarcoma cells with the EWS/FLI RNAi construct (EF-2-RNAi), or a negative control construct (ERG-RNAi).…”

Section: Formationmentioning

confidence: 99%

“…This has important implications for the design of molecularly targeted agents. We would predict that agents targeting EWS/FLI (such as cytarabine or RNAi-based approaches; Dubois et al, 2008;Hu-Lieskovan et al, 2005;Kinsey et al, 2006;Owen et al, 2008;Stegmaier et al, 2007) might be most effective when combined with standard cytotoxic agents. In this way, the loss of the key oncoprotein will reduce both oncogenic transformation and drug resistance, thereby allowing the tumor to be more effectively treated with standard chemotherapies.…”

Section: Formationmentioning

confidence: 99%

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GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

et al. 2009

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Ewing's sarcoma is a malignant bone-associated tumor of children and young adults. Most cases of Ewing's sarcoma express the EWS/FLI fusion protein. EWS/FLI functions as an aberrant ETS-type transcription factor and serves as the master regulator of Ewing's sarcoma-transformed phenotype. We recently showed that EWS/FLI regulates one of its key targets, NR0B1, through a GGAAmicrosatellite in its promoter. Whether other critical EWS/FLI targets are also regulated by GGAA-microsatellites was unknown. In this study, we combined transcriptional analysis, whole genome localization data, and RNA interference knockdown to identify glutathione S-transferase M4 (GSTM4) as a critical EWS/FLI target gene in Ewing's sarcoma. We found that EWS/FLI directly binds the GSTM4 promoter, and regulates GSTM4 expression through a GGAA-microsatellite in its promoter. Reduction of GSTM4 levels caused a loss of oncogenic transformation. Furthermore, reduction of GSTM4 resulted in an increased sensitivity of Ewing's sarcoma cells to chemotherapeutic agents, suggesting a role for this protein in drug resistance. Consistent with this hypothesis, patients with Ewing's sarcoma whose tumors had higher levels of GSTM4 expression had worse outcomes than those with lower expression levels. These data show that GSTM4 contributes to the cancerous behavior of Ewing's sarcoma and define a wider role for GGAA-microsatellites in EWS/FLI function than previously appreciated. These data also suggest a novel therapeutic resistance mechanism, in which the central oncogenic abnormality directly regulates a resistance gene.

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Section: Formationmentioning

confidence: 99%

Section: Formationmentioning

confidence: 99%

Section: Formationmentioning

confidence: 99%

Section: Formationmentioning

confidence: 99%

Section: Formationmentioning

confidence: 99%

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GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

et al. 2009

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Screening for DAX1/EWS‐FLI1 functional inhibitors identified dihydroorotate dehydrogenase as a therapeutic target for Ewing's sarcoma

et al. 2023

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Objective EWS‐FLI1 is the most common oncogenic fusion protein in Ewing's sarcoma family tumors (ESFTs). DAX1, an orphan member of the nuclear receptor superfamily, is up‐regulated by EWS‐FLI1 and plays a key role in the transformed phenotype of ESFTs. Methods To discover a functional inhibitor of DAX1 and EWS‐FLI1, we screened small‐molecular inhibitors using a DAX1 reporter assay system. Results K‐234 and its derivatives, which were dihydroorotate dehydrogenase (DHODH) inhibitors, showed inhibitory effects in the reporter assay. K‐234 inhibited the growth of Ewing's sarcoma with various fusion types, and K‐234 derivatives altered the expression of EWS‐FLI1‐regulated genes. The DAX1 expression had no effect on the growth inhibitory effect of the K‐234 derivatives, while DHODH overexpression or uridine treatment attenuated their inhibitory effects, suggesting that inhibition by K‐234 derivatives occurs through DHODH inhibition. An in vivo study showed that a K‐234 derivative clearly inhibited tumor growth in an Ewing's sarcoma xenograft mouse model. Conclusion Taken together, the present results suggest that DHODH inhibitors can inhibit the function of DAX1/EWS‐FLI1 in ESFTs and might be a therapeutic agent with potent anti‐tumor activity for Ewing's sarcoma patients.

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Cancer biology functional genomics: From small RNAs to big dreams

Rajan

Ludwig

Hall

et al. 2020

Molecular Carcinogenesis

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The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function-from a single gene to the whole genome-in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi-based methods to study carcinogenesis. We discuss how RNAi-based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome-wide scale efforts to identify cancer or cancer-type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer-relevant molecular targets and the implications for cancer of the first RNAi-based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.

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NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma

Cited by 184 publications

References 32 publications

GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

GSTM4 is a microsatellite-containing EWS/FLI target involved in Ewing's sarcoma oncogenesis and therapeutic resistance

Screening for DAX1/EWS‐FLI1 functional inhibitors identified dihydroorotate dehydrogenase as a therapeutic target for Ewing's sarcoma

Cancer biology functional genomics: From small RNAs to big dreams

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