EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function

Fujimura, Yasuo; Siddique, H.; Lee, Leo; Rao, V. N.; Reddy, E. S. P.

doi:10.1038/sj.onc.1204684

Cited by 44 publications

(37 citation statements)

References 45 publications

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“…35 The EWS-ATF-1 36 whose mechanism of action appears to be targeted repression of the CBP/p300 transcriptional coactivator, resulting in loss of function of p53. 37 Although not diagnostically helpful, polysomy of chromosome 8 is another common cytogenetic abnormality in clear cell sarcoma. 11,31 Other molecular characteristics which may have diagnostic utility in the future include activating mutations in the BRAF gene, which have been demonstrated in a significant percentage of malignant melanoma, but not clear cell sarcoma, 38 and the protein product of the epidermal growth factor receptor ERBB3 gene, which has been reported to be a marker of clear cell sarcoma.…”

Section: Discussionmentioning

confidence: 99%

Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

et al. 2005

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Clear cell sarcoma of soft tissue (malignant melanoma of soft parts) is a soft tissue sarcoma with melanocytic differentiation that typically occurs in the tendons and aponeuroses of young adults. As demonstrated by cytogenetics and reverse-transcriptase polymerase chain reaction, between 70% and over 90% of clear cell sarcomas have a t(12;22) translocation, fusing the EWS and ATF1 genes on chromosomes 22q12 and 12q13, respectively. Identification of this translocation distinguishes clear cell sarcoma from histologic mimics, most importantly conventional malignant melanoma. We report our experience with a commercially available, dualcolor, break-apart fluorescence in situ hybridization (FISH) probe, which allows detection of EWS (22q12) gene rearrangement in formalin-fixed, paraffin-embedded tissues. Histologically and immunophenotypically wellcharacterized cases of clear cell sarcoma (n ¼ 10) and malignant melanoma (n ¼ 32) were evaluated with a 22q12 dual-color, break-apart probe (Vysis, Downer's Grove, IL, USA), which spans the known common breakpoints in the EWS gene on chromosome 22 (introns 7-10). Signals from tumor cell nuclei were counted under a fluorescence microscope and the presence of red-green break-apart signals was recorded. Of the clear cell sarcoma cases, seven of 10 showed evidence of an EWS gene rearrangement with a mean of 81.6% positive cells per sample (range: 60-95%). All cases of malignant melanoma (n ¼ 32) showed virtually absent break-apart signals in the EWS gene (less than 4% cells per case). FISH detects EWS gene rearrangement in a substantial proportion of clear cell sarcomas, with excellent specificity. Importantly, EWS FISH is negative in malignant melanoma, a clinically dissimilar tumor, which may closely mimic clear cell sarcoma histologically and immunohistochemically. As the studied probe can be utilized in routinely processed tissue, FISH provides an excellent alternative to reverse-transcriptase polymerase chain reaction in cases where fresh tissue is unavailable. Modern Pathology ( Clear cell sarcoma of soft tissue (malignant melanoma of soft parts) is a rare soft tissue sarcoma with melanocytic differentiation first described by Enzinger 1 in 1965. The tumor originates predominantly in the tendons, aponeuroses, and fascial structures of the extremities of young adults. [1][2][3][4][5] Clear cell sarcoma shares morphologic, immunohistochemical, ultrastructural, and molecular features with malignant melanoma. 2,[6][7][8][9][10] In contrast to malignant melanoma, a unique and recurrent t(12;22)(q13;q12) involving the EWS gene has been identified in 70% to over 90% of clear cell sarcomas by cytogenetics, reverse-transcriptase polymerase chain reaction (RT-PCR) looking for the resultant EWS-ATF1 fusion protein, and by fluorescence in situ hybridization (FISH). [11][12][13][14][15][16][17][18][19] Clear cell sarcoma is rare and the true incidence of this neoplasm is not well established; however, the increased utilization of molecular studies for the

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Section: Discussionmentioning

confidence: 99%

Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

et al. 2005

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“…The EAD is known to contact a limited number of transcriptional components including the coactivator CBP (38,39), multiple TAFs (40), and the RNA polymerase II subunits rpb7 (40,41) and rpb5 (40). rpb7 and its partner rpb4 are required for EAD-mediated transactivation in yeast (42), suggesting that the EAD might flexibly wrap around rpb7/4 via multiple contacts, a mode of recognition that is used by known IDPs (7).…”

Section: Effect Of Ead Mutations On Transformation By Ews/fli1mentioning

confidence: 99%

Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins

Potikyan

Savene

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

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Chromosomal translocations involving the N-terminal Ϸ250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.Ewings sarcoma oncogene ͉ EWS/ATF1 ͉ intrinsically disordered proteins ͉ EWS activation domain ͉ molecular recognition feature T he Ewings sarcoma (EWS) protooncogene together with TLS/FUS and hTAF II 68 form a subgroup (the TET family; ref. 1) within the RNP family of RNA-binding proteins. Aberrant chromosomal translocations involving EWS produce EWS fusion proteins (EFPs) that cause human cancers (2, 3) and EFPs share the following common features (Fig. 1A). First, all EFPs contain at least the N-terminal 7 exons of EWS (residues 1-264) encoding the EWS activation domain (EAD). Second, the EWS fusion partner is one of several cellular transcription factors containing a sequence-specific DNA-binding domain that specifies the tumor type. Third, EFPs are potent transcriptional activators that depend on both the EAD and the fusion partner. Gene-specific transcriptional deregulation is likely to be a primary route for tumorigenesis (2, 3). However, some biological effects of EFPs might occur by alternative mechanisms including perturbation of pre-mRNA splicing (3).Progress in determining structure/function relations for the EAD has been limited for several reasons. The EAD has a highly repetitive primary sequence (Fig. 1B) containing multiple copies of a degenerate hexap...

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“…CBP/p300 is shown to have a pivotal role in apoptotic pathway. Recently, we have found CBP to interact with EWS-ATF-1 and interfere with the transcriptional activity of p53, which requires CBP/p300 as a coactivator for transcriptional activation (Fujimura et al, 2001). These results have driven us to test whether EWS-Fli-1 or Fli-1 associates with CBP.…”

Section: Ews-fli-1 and Fli-1 Mutants That Are Deficient In Transcriptmentioning

confidence: 99%

“…It is proposed that E1A downregulates oncogene HER2/neu through the p300 pathway. We have demonstrated that EWS-ATF-1 in MMSP interacts with CBP and inhibits the transcriptional activity of target genes (Fujimura et al, 2001). EWS is also found to be associated with CBP/p300 (Rossow and Janknecht, 2001;Araya et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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Role of protein–protein interactions in the antiapoptotic function of EWS-Fli-1

et al. 2004

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In the majority of Ewing's family tumors, chromosomal translocation t(11;22) leads to aberrant fusion of RNAbinding protein EWS with DNA-binding ETS transcriptional factor Fli-1. EWS-Fli-1 has altered the transcriptional activity and modulating its downstream target genes through this transcriptional activity is thought to be responsible for this tumor. We have previously shown that both EWS-Fli-1 and Fli-1 have antiapoptotic activity against several apoptotic inducers. Here, we show that the transcriptional activity of EWS-Fli-1 and Fli-1 is not essential for its antiapoptotic activity. We also demonstrate that EWS-Fli-1 and Fli-1 interact with CBP through its amino-terminal region and inhibit the CBPdependent transcriptional activity of RXR. This activity appears to be independent of DNA-binding activity of EWS-Fli-1. Introduction of the dominant-negative form of CBP into Ewing's sarcoma cells sensitizes these cells against genotoxic or retinoic-acid induced apoptosis. These results suggest that the ability of EWS-Fli-1/Fli-1 to target transcriptional cofactor(s) and modulate apoptotic pathways may be responsible for its antiapoptotic and tumorigenic activities.

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EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function

Cited by 44 publications

References 45 publications

Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins

Role of protein–protein interactions in the antiapoptotic function of EWS-Fli-1

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