Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma

Smith, Richard A.; Owen, Leah A.; Trem, Deborah J.; Wong, Jenny; Whangbo, Jennifer; Golub, Todd R.; Lessnick, Stephen L.

doi:10.1016/j.ccr.2006.04.004

Cited by 303 publications

(416 citation statements)

References 43 publications

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“…This experiment further indicates that GLI1 expression in EFT cell lines is dependent on EWS/FLI1 expression. These data agree with findings recently published (in aggregate form) in which RNAi targeting a different region of EWS/FLI1 was used in another Ewing cell line (Smith et al, 2006). In that study, GLI1 was among the genes whose expression was both diminished by EWS/FLI1 RNAi and restored by reexpression of EWS/FLI1.…”

Section: Ews/fli1 Deregulates Gli1supporting

confidence: 93%

The EWS/FLI1 oncogenic transcription factor deregulates GLI1

et al. 2007

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Ewing family tumors (EFT), classically Ewing's sarcoma and peripheral primitive neuroectodermal tumor, share a common class of tumor-specific fusion genes thought to be key mediators of tumor biology. Here we demonstrate that the most common Ewing's fusion, EWS/FLI1, produces transcriptional upregulation of GLI1 and its direct transcriptional target PATCHED1 in a model transformation system. This deregulation of GLI1 is common to other EWS/ets chimera and depends on the functional transcriptional regulatory domains. Inhibition of GLI1 via RNAi or via overexpression of endogenous inhibitors results in a reduction of EWS/FLI1 transformation activity. Activation of GLI1 appears to occur in a Hedgehog-independent fashion as blockade of Hedgehog signaling has only a modest effect on EFT cells. We present evidence that EWS/FLI1 upregulation of cMYC may play a role in the upregulation of GLI1 in EWS/ FLI1-transformed NIH3T3 cells. Finally, we demonstrate that observations made in a model transformation system translate to an Ewing cellular background. EFT cell lines express GLI1 and PATCHED and this expression is EWS/FLI1 dependent. Inhibition of GLI1 expression via RNAi results in reduced anchorage-independent growth in an EFT cell line. GLI1 appears to be a transcriptionally deregulated target of EWS/FLI1 that mediates a portion of its tumorigenic phenotype.

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Section: Ews/fli1 Deregulates Gli1supporting

confidence: 93%

The EWS/FLI1 oncogenic transcription factor deregulates GLI1

et al. 2007

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“…EWS/FLI requires both its strong transcriptional activation domain (derived from EWS) and its ETS-type DNA-binding domain (derived from FLI) for oncogenic function (May et al, 1993a, b). A variety of studies have identified a large number of EWS/FLI-regulated genes (Prieur et al, 2004;Smith et al, 2006). However, only a handful of these genes have been shown to participate in Ewing's sarcoma oncogenesis.…”

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%

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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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“…7 NKX2-2, a homeodomain-containing transcription factor involved in neuronal development and glial/neuroendocrine differentiation, 8,9 is a downstream target of EWSR1-FLI1 oncogenic signaling in Ewing sarcoma. 10 In recent times, NKX2-2 has been reported as a diagnostically useful immunohistochemical marker for Ewing sarcoma. 11 In published studies on NKX2-2 expression in Ewing sarcoma, 11,12 only classic Ewing sarcomas with EWSR1-FLI1 rearrangement have been evaluated.…”

mentioning

confidence: 99%