Oncogene
SEARCH     advanced search my account e-alerts subscribe register
Journal home
Advance online publication
Current issue
Archive
Press releases
For authors
For referees
Contact editorial office
About the journal
For librarians
Subscribe
Advertising
naturereprints
Contact NPG
Customer services
Site features
NPG Subject areas
Access material from all our publications in your subject area:
Biotechnology Biotechnology
Cancer Cancer
Chemistry Chemistry
Dentistry Dentistry
Development Development
Drug Discovery Drug Discovery
Earth Sciences Earth Sciences
Evolution & Ecology Evolution & Ecology
Genetics Genetics
Immunology Immunology
Materials Materials Science
Medical Research Medical Research
Microbiology Microbiology
Molecular Cell Biology Molecular Cell Biology
Neuroscience Neuroscience
Pharmacology Pharmacology
Physics Physics
Browse all publications
 
10 September 2001, Volume 20, Number 40, Pages 5747-5754
Table of contents    Previous  Article  Next   [PDF]
Biology of EWS/ETS fusions in Ewing's family tumors
Afsane Arvand3 and Christopher T Denny1,2,4

1Molecular Biology Institute, Gwynne Hazen Cherry Memorial Labs, University of California at Los Angeles, California, USA

2Jonsson Comprehensive Cancer, Gwynne Hazen Cherry Memorial Labs, University of California at Los Angeles, California, USA

3Department of Pathology and Laboratory Medicine, Gwynne Hazen Cherry Memorial Labs, University of California at Los Angeles, California, USA

4Department of Pediatrics, Gwynne Hazen Cherry Memorial Labs, University of California at Los Angeles, California, USA

Correspondence to: T Denny, Molecular Biology Institute, Gwynne Hazen Cherry Memorial Labs, University of California at Los Angeles, California, USA

Abstract

Tumor-associated chromosomal translocations lead to the formation of chimeric fusions between the EWS gene and one of five different ETS transcription factors in Ewing's family tumors (EFTs). The resultant EWS/ETS proteins promote oncogenesis in a dominant fashion in model systems and are necessary for continued growth of EFT cell lines. EWS belongs to a family of genes that encode proteins that may serve as adapters between the RNA polymerase II complex and RNA splicing factors. EWS/ETS fusions have biochemical characteristics of aberrant transcription factors and appear to promote abnormal cellular growth by transcriptionally modulating a network of target genes. Early evidence suggests that EWS/ETS proteins may also impact gene expression through alteration in RNA processing. Elucidation of EWS/ETS target gene networks in the context of other signaling pathways will hopefully lead to biology based therapeutic strategies for EFT. Oncogene (2001) 20, 5747-5754.

Keywords

EWS/ETS; Ewing's family tumors; aberrant transcription factors

A karyotypic abnormality is key to defining Ewing's family tumors (EFTs)

Until the first discovery of the t(11;22) chromosomal rearrangement 16 years ago, the unambiguous diagnosis of Ewing's family tumors (EFTs) was fraught with uncertainty (Aurias et al., 1984; Turc-Carel et al., 1984). As a group we now recognize that EFTs include both Ewing's sarcomas and primitive neuroectodermal tumors, and afflict young adults in their first and second decade of life (Triche et al., 1987). EFTs typically present as destructive masses associated with bone though about 15% of patients show only soft tissue masses without apparent bone involvement.

A fundamental impediment to basic understanding of EFTs is that the cell of origin is unknown. On histologic section, EFTs consist of sheets of small round cells with few distinguishing characteristics. The current majority opinion is that EFTs derive from neural crest progenitors. This hypothesis is based on particular cellular features observed in some EFTs including: (i) expression of catachol acetyl transferase, an enzyme involved in neurotransmitter biosynthesis in cholinergic nerves; (ii) expression of neuron-specific enolase; and (iii) propensity for certain EFT tumor-derived cell lines to form primitive dendrites and express neural associated proteins in response to differentiating agents (Cavazzana et al., 1987). The finding of peripheral neural crest progenitors late in development that can differentiate along neural, glial and mesenchymal lineages suggests a possible cellular source for EFTs (Morrison et al., 1999).

Because of their uncertain lineage and nonspecific histology, the diagnosis of EFTs had been one of a process of elimination. If a particular tumor displayed a small round cell histology but lacked specific markers, it was frequently deemed an EFT. The recognition of a specific 11;22 chromosomal translocation associated with EFTs represented a major step towards prospectively defining EFTs as a distinct clinicopathologic entity.

Scrambled subdomains-EFT chromosomal translocations result in chimeric fusion genes

Approximately 85% of EFTs carry a tumor-associated t(11;22)(q24;q12) rearrangement that is detectable on karyotypic analysis (Delattre et al., 1994). This results in juxtaposition of the EWS gene on chromosome 22 with FLI1 on chromosome 11 (Figure 1) (Delattre et al., 1992). As a consequence chimeric transcripts and proteins are produced that consist of the N-terminus of EWS fused to the C-terminal portion of FLI1. Variant EFT translocations have been also described that join EWS to one of four additional ETS family transcription factors (Table 1). The observation that individual EFTs have only a single EWS fusion suggests that in spite of their structural differences, all five EWS/ETS fusions are playing similar oncogenic roles.

In contrast to adult epithelial cancers which can harbor a similar mutation in many different tumor types, EWS/ETS fusions appear to be specific to EFTs. There are scattered reports finding EWS/ETS fusions in a minority of neural crest and atypical mesenchymal derived tumors (Burchill et al., 1997; Scotlandi et al., 2000b; Sorensen et al., 1995; Thorner et al., 1996). Whether these exceptions represent the extremes of the EFT category or rare clinicopathologic entities themselves is still unclear.

At the genomic level the t(11;22) breakpoints are relatively tightly clustered within a 8 Kb region in the EWS locus but the FLI1 breakpoints are dispersed over approximately 35 Kb (Delattre et al., 1992). This can result in different EWS and FLI1 exons being incorporated into the fusion genes found in EFT tumor specimens. There have been 12 different EWS/FLI1 fusions described each with variable combinations of exons flanking the fusion point (Zoubek et al., 1994, 1996; Zucman et al., 1993b). More recently there have been two retrospective studies suggesting that structurally different EWS/FLI1 fusions may have prognostic significance (de Alava et al., 1998; Zoubek et al., 1996). It appears that patients with EFTs harboring a type 1 fusion (EWS exon 7 juxtaposed to FLI1 exon 6) have an increased chance of survival over those EFT patients with alternate EWS/FLI1 fusions. These observations are currently being confirmed in prospective studies but they do suggest that different EWS/FLI1 fusion proteins may have different biologic potencies.

EWS/ETS fusions are dominant acting oncoproteins

The high prevalence of EWS/ETS fusions in EFTs suggested that these chimeric products are important to the genesis and perhaps maintenance of these tumors. Work in animal model systems supports the view that EWS/ETS fusions act as dominant oncogenes. Forced expression of EWS/FLI1 promotes anchorage independent growth of NIH3T3 murine fibroblasts (May et al., 1993a). Polyclonal NIH3T3 populations stably expressing EWS/ETS fusion genes form tumors in immunodeficient mice at an accelerated rate (Thompson et al., 1999). Instead of displaying spindle cell histology that is typical for transformed fibroblasts, EWS/ETS NIH3T3 tumors have a small round cell morphology that is reminiscent of that seen in human EFTs (Teitell et al., 1999). These data suggest that in addition to actively promoting oncogenesis, EWS/ETS fusions appear also to dictate some of the histologic characteristics found in EFTs.

Studies in human EFT tumor derived cell lines also indicate that EWS/ETS fusions are playing crucial biologic roles. Transduction of a dominant negative FLI1 construct into EFT cell lines results in growth inhibition (Kovar et al., 1996). Multiple independent groups have now shown that treatment of EFT cell lines with antisense EWS/FLI1 oligonucleotides or cDNAs results in decreased in vivo tumor growth in immunodeficient mice (Ouchida et al., 1995). These effects are associated in vitro with a shift in cell cycle profile towards G0/G1 and are apparent in vivo even if antisense oligonucleotides are given exogenously after establishment of tumor growth (Lambert et al., 2000; Tanaka et al., 1997). These data indicate that continued expression of EWS/ETS fusion genes is necessary for maintaining EFT tumor growth. In trying to understand how EWS/ETS genes are mediating these biologic effects, investigators have looked to the normal functions of both translocation partners.

Adoption into the TET family - putative RNA binding proteins with far reaching biologies

Since the EWS gene was found in the context of fusion to FLI1 in EFTs, the normal functions of EWS are still being elucidated. With the discovery of the related TLS/FUS, a gene fused to the CHOP transcription factor by chromosomal translocation in myxoid liposarcoma, certain structural features became more apparent (Crozat et al., 1993; Rabbitts et al., 1993). TLS/FUS, EWS and TAFII68, a TBP-associated factor present in some transcription complexes, have now been grouped into the TET family based on a distinctive 87 amino acid RRM/RNP-CS domain that is thought to mediate protein-RNA binding (Bertolotti et al., 1996). TET proteins have variable numbers of RGG (arginine-glycine-glycine) repeats that also seem to facilitate binding to RNA. The drosophila SARFH/Cabezza gene contains both RRM and RGG motifs and is thought to be a TET family member (Immanuel et al., 1995; Stolow and Haynes, 1995).

In addition to containing putative RNA binding domains at their C-termini, mammalian TET family members share glutamine rich N-termini consisting of a series of degenerate repeats. Not only are these N-termini fused to ETS genes in EFTs but they are also involved in fusions with a variety of transcription factors in other human cancers (Table 1). Interestingly with one exception, EWS and TLS chimeras are found only in sarcomas and not malignancies deriving from other lineages.

Based on their structural features, TET proteins are thought to be involved in RNA transcription and/or processing. Both EWS and TLS proteins have been shown to bind RNA in vitro (Ohno et al., 1994; Prasad et al., 1994). TAFII68 and EWS have been shown to be complexed with both the TFIID and RNA polymerase II fractions in nuclear cell lysates (Bertolotti et al., 1996, 1998). The association with RNA polymerase II appears to involve the N-terminus of EWS (Petermann et al., 1998). TLS has been shown to co-localize in cells with known RNA-binding proteins and to bind RNA in vivo (Zinszner et al., 1994, 1997). Inhibition of RNA polymerase II results in subcellular redistribution of TLS from the nucleus to the cytoplasm. Finally the C-terminus of TLS has been shown to bind members of a serine-arginine family of RNA splicing factors (Yang et al., 1998). A current hypothesis is that TET proteins act as adapters between transcription and mRNA processing by interacting with components of the transcription apparatus and splicing factors.

EWS and TLS are ubiquitously expressed in growing mammalian cells. Subcellular localization studies indicate that these proteins are primarily nuclear but can shift with different physiologic states. Addition of serum to previously starved cells results in EWS dissociation from Pyk2, a cytoplasmic protein tyrosine kinase, and transit from the cytoplasm to nucleus (Felsch et al., 1999). In addition, there is evidence to suggest that EWS and TLS may be post-translationally modified. EWS has been shown to bind calmodulin and targeted for phosphorylation by protein kinase C (PKC) in vitro (Deloulme et al., 1997). PKC phosphorylation of TLS is associated with increased proteosome-mediated degradation in cells (Perrotti et al., 2000). Finally there is early evidence that EWS may interact with Bruton's tyrosine kinase and TLS with the nuclear hormone receptors as well as certain ETS transcription factors (Guinamard et al., 1997; Hallier et al., 1998; Powers et al., 1998). The physiologic significance of these wide ranging associations is still under investigation.

Conservation across species suggests that TET proteins have important physiologic roles. In somatic cells, TLS/FUS is up regulated in response to the BCR/ABL oncoprotein and potentiates growth factor independence (Perrotti et al., 1998). On a developmental level, TLS has been successfully knocked out in the mouse by two independent groups and the resulting phenotypes have been surprising (Hicks et al., 2000; Kuroda et al., 2000). Inbred TLS -/- mice have high neonatal death rate, a developmental block in B-lymphocyte development and profound chromosomal instability. Outbred TLS null mice live into adulthood but have defective spermatogenesis in addition to the genomic instability and increased cellular sensitivity to ionizing radiation. Together these data indicate that TLS plays a dominant role in genomic maintenance and stability.

FLI1 is a member of the ever expanding ETS transcription factor family

All the EWS fusion partners in EFTs belong to the well characterized ETS family of transcription factors (for reviews see Graves and Petersen, 1998; Watson and Seth, 2000). All ETS members are defined by an 87 amino acid domain that is both necessary and sufficient for site-specific DNA-binding (DBD) in vitro. Flanking the ETS DBD are protein-protein interaction domains that mediate transcriptional activation and/or repression. Certain ETS factors also possess auto-inhibitory domains that block DNA binding in the absence of cofactors.

ETS factors are thought to act by binding to promoter and/or enhancer elements of target genes and result in transcriptional activation or repression. Identification of physiologic direct target genes of ETS factors has been difficult. First, most ETS factors bind a small and degenerate core DNA sequence (GGAA/T). While there are preferences, the in vitro DNA-binding specificities of different ETS factors can be nonstringent and a single DNA site could potentially be occupied by multiple ETS factors. Given that many different ETS factors can be simultaneously expressed in cells, the ability of a particular ETS protein to bind to a specific gene's regulatory sequence in vitro does not necessarily mean that the ETS factor is actually regulating the gene in vivo.

Protein-protein interactions have a dominant impact on ETS function (for review see Li et al., 2000). For example, while the optimum in vitro DNA binding sequences have been determined for many ETS proteins, their in vivo binding specificities may be hard to predict. ETS factors typically form heteromeric complexes with non-ETS transcription factors that coordinately bind to target gene sites. Such synergy with other transcription factors can result in ETS factors binding to non-optimal sites (Fitzsimmons et al., 1996). In addition to facilitating DNA binding at regulatory elements, protein-protein interactions can be important for recruiting additional factors necessary for transcriptional modulation of specific target genes. Together these data indicate that the intrinsic physiologic specificity of any particular ETS protein depends on both its protein-DNA and protein-protein interactions.

FLI1 has been linked to normal and malignant hematopoietic cell growth

FLI1 is the predominant EWS fusion partner in EFTs and contains a typical ETS DBD located towards the C-terminus (for overview see Truong and Ben-David, 2000). It contains an N-terminal transcriptional activation though there are data suggesting that the C-terminal 89 amino acids may have transcriptional activation capability as well (Rao et al., 1993). Several isoforms of FLI exist that differ slightly at their N-termini (Dhulipala et al., 1998; Sarrazin et al., 2000). These FLI1 species arise from differences in transcription initiation at alternative promoters and also from multiple translation sites that are present in most transcripts. On a primary amino acid level, FLI1 is very similar to ERG the second most common ETS gene involved in EFT EWS translocations.

Normal FLI1 is primarily a nuclear protein. Murine, Quail, Zebrafish and Xenopus FLI1 orthologues are expressed early in embryologic development primarily in mesenchymal cells of either neural crest or mesodermal origin (Brown et al., 2000; Mager et al., 1998; Mélet et al., 1996; Meyer et al., 1993). Later in development FLI1 is also found in vascular endothelia and blood cell precursors. In adult tissues, FLI1 expression is restricted mainly to hematopoietic cells. Levels are highest in thymus and spleen due to high level expression in lymphocytes as well as erythroid and megakaryocyte precursors (for review see Maroulakou and Bowe, 2000).

Several FLI1 interacting proteins have been defined but their physiologic significance is still being investigated. Both FLI1 and EWS/FLI1 have been shown to form in vitro ternary complexes with serum response factor (SRF) at promoter regions from serum response genes (Dalgleish and Sharrocks, 2000; Magnaghi-Jaulin et al., 1996; Watson et al., 1997). These interactions are mediated by FLI1 sequences that flank either side of the ETS DBD. The ETS gene TEL can inhibit FLI1 induction of model reporter gene constructs and TEL/FLI1 complexes can be immunoprecipitated from cells overexpressing these two genes (Kwiatkowski et al., 1998). Overexpression of TEL antagonizes FLI1 induced megakaryocytic differentiation in the K562 leukemia cell line suggesting a biological correlate to the apparent physical interaction of these two proteins (Kwiatkowski et al., 2000). Finally forced expression of FLI1 both antagonizes and is antagonized by a variety of nuclear hormone receptors in a hormone dependent manner but direct physical interaction between these two transcription factor classes has not yet been documented (Darby et al., 1997).

From its first discovery, FLI1 has been biologically linked to oncogenesis. In lower mammals, FLI1 is a target for up regulation by retroviral insertion by four different leukemia/lymphoma inducing viruses (for review see Blair and Athanasiou, 2000). In this context, FLI1 expression inhibits differentiation and promotes proliferation of erythroid progenitor cell lines (Pereira et al., 1999; Tamir et al., 1999). However, FLI1 is not simply an inhibitor of differentiation and its biologic effects can be cell specific. For example, FLI1 induces megakaryocytic differentiation of the K562 human leukemia cell line (Athanasiou et al., 1996).

Consistent with in vivo expression and oncogenic data, genetic animal model systems also confirm a significant role for FLI1 in normal hematopoietic development. Transgenic mice having forced expression of normal FLI1 demonstrated immune dysregulation (B-lymphocyte hyperplasia and hypergammaglobulinemia) and immune complex related renal disease (Zhang et al., 1995). In the reciprocal experiment, two independent groups created mice with homozygous germline deletions within the FLI1 locus (Hart et al., 2000; Spyropoulos et al., 2000). Both mouse strains displayed embryonic lethality, intracranial hemorrhage and defective hematopoiesis particularly in the megakaryocyctic lineage.

Like most ETS factors, the elucidation of the target genes that mediate these physiologic effects is still in progress. There is strong evidence that the tumor suppressor RB is directly down regulated by FLI1 during erythropoietin induced differentiation in an erythroleukemia cell line (Tamir et al., 1999). FLI1 can bind promoter sequences and activate reporter gene constructs from the glycoprotein IX and MPL genes that are normally expressed primarily in megakaryocytes and platelets (Bastian et al., 1999; Deveaux et al., 1996). These observations coupled with the defective megakaryocytic development in FLI1 null mice, suggests that direct interaction of FLI1 at these target genes may be physiologically important.

EWS/ETS fusions can function as aberrant transcription factors

Considering that EFT associated chromosomal translocations result in fusions between TET and ETS gene families, it is logical to think that the chimeric products could biochemically function as aberrant TET and/or ETS proteins. A current hypothesis is that EWS/ETS fusions can act as abnormal transcription factors (Figure 2). EWS/ETS proteins display several biochemical attributes supporting this theory: (i) both in EFT cell lines and in transduced NIH3T3 cells, EWS/ETS proteins localize to the nucleus; (ii) EWS/ETS fusions can bind DNA in a site-specific manner; (iii) the N-terminal EWS domain that is present in EWS/ETS fusions can act as a potent transcriptional activation domain in model reporter assays (Bailly et al., 1994; Mao et al., 1994; May et al., 1993b; Ohno et al., 1993). In fact it appears that the N-terminus of EWS encodes a stronger transcriptional activation domain than the native FLI1 domain that is displaced by the t(11;22). This suggests that EWS/FLI1 could function as a dominant active FLI1 protein.

Sequence analysis of EFT fusions and mutagenesis studies support the theory that EWS/ETS proteins can act as aberrant transcription factors. In spite of their structural heterogeneity across different tumors, all EWS/ETS fusions contain an intact ETS DNA-binding domain. Site-directed mutagenesis within the ETS DBD results in EWS/ETS proteins with reduced or absent transformation potential (Jaishankar et al., 1999; May et al., 1993a). Similarly, deletion of EWS transcriptional activation domains reduces the biological potency of EWS/ETS proteins (Lessnick et al., 1995). However other strong transcriptional activation domains can functionally replace the N-terminus of EWS in model systems. Expression of chimeric constructs containing the herpes virus VP16 transcriptional activation domain fused to the FLI1 C-terminus renders NIH3T3 cells anchorage independent. These data indicate that both EWS and ETS portions of the fusion are necessary for full biologic activity and suggests that transcriptional activation is a primary function of the EWS domain in EWS/ETS fusions.

Two major inherent biochemical properties dictate the specificity of transcription factors: (i) protein-DNA interaction specificity defined in the case of EWS/ETS fusions by the ETS DBD; and (ii) protein-protein interaction specificity at transcriptional activation domains. Through somatic genomic rearrangement the normal ETS transcriptional activation domains are replaced with the EWS N-terminus, thereby generating transcription factors with novel biochemical and genetic specificities. It therefore appears that a major mechanism through which EWS/ETS fusions promote oncogenesis is by transcriptionally modulating a repertoire of target genes that is qualitatively and/or quantitatively different than the normal ETS gene partners.

Several comparative and functional strategies have been used to identify EWS/ETS target genes. By comparing gene expression patterns in NIH3T3 populations with and without EWS/ETS fusions, a number of putative target genes have been identified. At this point, three EWS/ETS target genes appear to have biologic impact. Manic fringe (m-FNG) a gene coding for a glycosyltransferase important in normal limb development, is up regulated by EWS/ETS genes (May et al., 1997). Furthermore, forced expression of m-FNG accelerates NIH3T3 tumorigenesis in immunodeficient mice. Very recently it has been shown that PDGF-C, a novel PDGF ligand, is up regulated by EWS/ETS proteins and forced expression of PDGF-C accelerates tumorigenic and anchorage independent growth of NIH3T3 cells (Zwerner and May, 2001). By contrast EWS/ETS fusions appear to down regulate the endogenous TGFbetaII receptor (TGFbetaIIR) and a TGFbetaIIR model reporter gene construct in NIH3T3 cells (Hahm, 1999; Im et al., 2000). Overexpression of TGFbetaIIR receptor in an EFT tumor-derived cell line significantly reduced its tumorigenic potential.

In the face of this encouraging data, it is important to bear two perspectives in mind. First, no single target gene has been identified that fully recapitulates the phenotype of an EWS/ETS transformed cell. For example, while m-FNG does increase the tumorigenic rate of NIH 3T3 cells, the resulting tumors do not display the small round cell histology that is invariably seen in tumors derived from EWS/ETS NIH3T3 cells and EFT tumor derived cell lines (Thompson et al., 1999). This suggests that EWS/ETS fusions promote oncogenesis through a gene network where modulation of multiple target genes is necessary for complete biologic impact. Second, efforts to identify EWS/ETS gene targets thus far have not distinguished between direct target genes, those whose genomic regulatory sequences are presumably bound by EWS/ETS proteins and indirect target genes, those whose transcription is modulated through intermediate genes. Identification of physiologic direct targets will be crucial to better understand how EWS/ETS fusions work at a biochemical level.

EWS/ETS fusions may impact RNA processing

While a major focus has been on how EWS/ETS fusions may be subverting normal ETS functions, there is recent data suggesting that these fusions might be acting as aberrant TET factors as well (Figure 2). This concept stemmed from the observation that both EWS and TLS bind TASR proteins, a family of putative RNA splicing factors. Considering that TASR binding domains are lost in EWS/ETS fusions, it is possible that the fusions could be interfering with normal RNA processing. It has recently been shown that both EWS/FLI1 and TLS/ERG fusions can antagonize splicing of model mRNA constructs in cells (Yang et al., 2000a,b).

Additional evidence that EWS/ETS fusions may operate through mechanisms different than simply binding promoter/enhancer sites at specific target genes, comes from the finding that an artificial EWS/FLI1 DBD mutant that has no apparent in vitro DNA-binding activity still retains some transformation potency (Jaishankar et al., 1999). It has recently been shown that EWS/FLI1 binds the U1C a member of the small nuclear ribonucleoprotein family involved in RNA splicing and that these interactions can inhibit EWS/FLI1 transactivation of model reporter gene constructs (Knoop and Baker, 2000).

While the overall picture is still unresolved, these data may be indicating that EWS/ETS fusions may be promoting oncogenesis by modulating gene expression at the RNA processing stages of transcription control. The relative contributions of each biochemical mechanism to EWS/ETS biologic activity is an exciting area of ongoing investigation.

Additional interacting oncogenic pathways in EFTs

EWS/ETS fusions do not play out their biologic effects in isolation and there are almost certainly other significant oncogenic events in EFTs. Homozygous deletion of the p16 locus is found in 25-30% of EFT tumor specimens and represents the most common EFT genomic mutation besides EWS/ETS causing translocations (Kovar et al., 1997). Retrospective analysis suggests that this mutation is associated with a subgroup of patients with poor prognosis (Wei et al., 2000). In addition a 1;16 chromosomal translocation and loss of chromosomes 8 and 12 have been described in EFTs. The physiologically important genes that are affected by these genomic rearrangements remain to be defined.

In addition signaling pathways have been identified that when modulated affect EFT cell growth in model systems. The NF-kappaB transcription factor is expressed in EFT cell lines and appears to exert an anti-apoptotic effect. Inhibition of NF-kappaB sensitized these cells to TNFalpha induced killing (Javelaud et al., 2000). CD99 (MIC2) a cell surface protein expressed at high levels on hematopoietic and EFT cells presents another possible therapeutic target (Kovar et al., 1990). Exposure of EFT cell lines to an anti-CD99 monoclonal antibody induces apoptosis via a non-calcineurin dependent pathway (Scotlandi et al., 2000a; Sohn et al., 1998). It has been shown that both FAS and FAS ligand are expressed on EFT tumor specimens (Kontny et al., 1998). A subset of EFT cell lines are susceptible to FAS mediated apoptosis through inhibition of matrix metalloproteinases (Mitsiades et al., 1999). Finally there is evidence that interferon-beta can induce apoptosis in some EFT cell lines (Sancéau et al., 2000).

At this point there are no established connections between these signaling pathways and those mediated by EWS/ETS oncoproteins. Future challenges will be towards not only better defining EWS/ETS biology but integrating it into the context of EFT physiology. It is hoped that this work will eventually lead to the development of effective biology based therapeutic strategies to supplant the empirically derived methods used today.

Acknowledgements

This work is supported through the National Institutes of Health grant RO1-87771.

References

Athanasiou M, Clausen PA, Mavrothalassitis GJ, Zhang XK, Watson DK, Blair DG. (1996). Cell Growth Differ. 7, 1525-1534. MEDLINE

Attwooll C, Tariq M, Harris M, Coyne JD, Telford N, Varley JM. (1999). Oncogene 18, 7599-7601. MEDLINE

Aurias A, Rimbaut C, Buffe D, Zucker JM, Mazabraud A. (1984). Cancer Genet. Cytogenet. 12, 21-25. MEDLINE

Bailly RA, Bosselut R, Zucman J, Cormier F, Delattre O, Roussel M, Thomas G, Ghysdael J. (1994). Mol. Cell. Biol. 14, 3230-3241. MEDLINE

Bastian LS, Kwiatkowski BA, Breininger J, Danner S, Roth G. (1999). Blood 93, 2637-2644. MEDLINE

Bertolotti A, Lutz Y, Heard DJ, Chambon P, Tora L. (1996). EMBO J. 15, 5022-5031. MEDLINE

Bertolotti A, Melot T, Acker J, Vigneron M, Delattre O, Tora L. (1998). Mol. Cell. Biol. 18, 1489-1497. MEDLINE

Blair DG, Athanasiou M. (2000). Oncogene 19, 6472-6481. MEDLINE

Brown LA, Rodaway AR, Schilling TF, Jowett T, Ingham PW, Patient RK, Sharrocks AD. (2000). Mech. Dev. 90, 237-252. Article MEDLINE

Burchill SA, Wheeldon J, Cullinane C, Lewis IJ. (1997). Eur. J. Cancer 33, 239-243. MEDLINE

Cavazzana AO, Miser JS, Jefferson J, Triche TJ. (1987). Am. J. Path. 127, 507-518. MEDLINE

Clark J, Benjamin H, Gill S, Sidhar S, Goodwin G, Crew J, Gusterson BA, Shipley J, Cooper CS. (1996). Oncogene 12, 229-235. MEDLINE

Crozat A, Aman P, Mandahl N, Ron D. (1993). Nature (Lond.) 363, 640-644. MEDLINE

Dalgleish P, Sharrocks AD. (2000). Nucleic Acids Res. 28, 560-569. MEDLINE

Darby TG, Meissner JD, Ruhlmann A, Mueller WH, Scheibe RJ. (1997). Oncogene 15, 3067-3082. MEDLINE

de Alava E, Kawai A, Healey JH, Fligman I, Meyers PA, Huvos AG, Gerald WL, Jhanwar SC, Argani P, Antonescu CR, Pardo-Mindan FJ, Ginsberg J, Womer R, Lawlor ER, Wunder J, Andrulis I, Sorensen PH, Barr FG, Ladanyi M. (1998). J. Clin. Oncol. 16, 1248-1255. MEDLINE

Delattre O, Zucman J, Melot T, Garau XS, Zucker JM, Lenoir GM, Ambros PF, Sheer D, Turc-Carel C, Triche TJ, Aurias A, Thomas G. (1994). N. Engl. J. Med. 331, 294-299. MEDLINE

Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, Kovar H, Joubert I, de Jong P, Rouleau G, Aurias A, Thomas G. (1992). Nature 359, 162-165. MEDLINE

Deloulme JC, Prichard L, Delattre O, Storm DR. (1997). J. Biol. Chem. 272, 27369-27377. Article MEDLINE

Deveaux S, Filipe A, Lemarchandel V, Ghysdael J, Roméo PH, Mignotte V. (1996). Blood 87, 4678-4685. MEDLINE

Dhulipala PD, Lee L, Rao VN, Reddy ES. (1998). Oncogene 17, 1149-1157. MEDLINE

Felsch JS, Lane WS, Peralta EG. (1999). Curr. Biol. 9, 485-488. MEDLINE

Fitzsimmons D, Hodsdon W, Wheat W, Maira SM, Wasylyk B, Hagman J. (1996). Genes Dev. 10, 2198-2211. MEDLINE

Graves BJ, Petersen JM. (1998). Adv. Cancer Res. 75, 1-55. MEDLINE

Guinamard R, Fougereau M, Seckinger P. (1997). Scand. J. Immunol. 45, 587-595. MEDLINE

Hahm KB. (1999). Nat. Genet. 23, 481. Article MEDLINE

Hallier M, Lerga A, Barnache S, Tavitian A, Moreau-Gachelin F. (1998). J. Biol. Chem. 273, 4838-4842. Article MEDLINE

Hart A, Melet F, Grossfeld P, Chien K, Jones C, Tunnacliffe A, Favier R, Bernstein A. (2000). Immunity 13, 167-177. MEDLINE

Hicks GG, Singh N, Nashabi A, Mai S, Bozek G, Klewes L, Arapovic D, White EK, Koury MJ, Oltz EM, Van Kaer L, Ruley HE. (2000). Nature Genet. 24, 175-179. Article MEDLINE

Ichikawa H, Shimizu K, Hayashi Y, Ohki M. (1994). Cancer Res. 54, 2865-2868. MEDLINE

Im YH, Kim HT, Lee C, Poulin D, Welford S, Sorensen PH, Denny CT, Kim SJ. (2000). Cancer Res. 60, 1536-1540. MEDLINE

Immanuel D, Zinszner H, Ron D. (1995). Mol. Cell. Biol. 15, 4562-4571. MEDLINE

Jaishankar S, Zhang J, Roussel MF, Baker SJ. (1999). Oncogene 18, 5592-5597. MEDLINE

Javelaud D, Wietzerbin J, Delattre O, Besancon F. (2000). Oncogene 19, 61-68. MEDLINE

Jeon IS, Davis JN, Braun BS, Sublett JE, Roussel MF, Denny CT, Shapiro DN. (1995). Oncogene 10, 1229-1234. MEDLINE

Kaneko Y, Yoshida K, Handa M, Toyoda Y, Nishihira H, Tanaka Y, Sasaki Y, Ishida S, Higashino F, Fujinaga K. (1996). Genes, Chrom. Cancer 15, 115-121. MEDLINE

Knoop LL, Baker SJ. (2000). J. Biol. Chem. 275, 24865-24871. MEDLINE

Kontny HU, Lehrnbecher TM, Chanock SJ, Mackall CL. (1998). Cancer Res. 58, 5842-5849. MEDLINE

Kovar H, Aryee DN, Jug G, Henockl C, Schemper M, Delattre O, Thomas G, Gadner H. (1996). Cell Growth Differ. 7, 429-437. MEDLINE

Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF, Gadner H. (1990). Oncogene 5, 1067-10670. MEDLINE

Kovar H, Jug G, Aryee DN, Zoubek A, Ambros P, Gruber B, Windhager R, Gadner H. (1997). Oncogene 15, 2225-2232. MEDLINE

Kuroda M, Sok J, Webb L, Baechtold H, Urano F, Yin Y, Chung P, de Rooij DG, Akhmedov A, Ashley T, Ron D. (2000). EMBO J. 19, 453-462. MEDLINE

Kwiatkowski BA, Bastian LS, Bauer TR, Tsai S, Zielinska-Kwiatkowska AG, Hickstein DD. (1998). J. Biol. Chem. 273, 17525-17530. MEDLINE

Kwiatkowski BA, Zielinska-Kwiatkowska AG, Bauer TR, Hickstein DD. (2000). Blood Cells Mol. Dis. 26, 84-90. MEDLINE

Labelle Y, Zucman J, Stenman G, Kindblom LG, Knight J, Turc-Carel C, Dockhorn-Dworniczak B, Mandahl N, Desmaze C, Peter M, Aurias A, Delattre O, Thomas G. (1995). Hum. Mol. Gen. 4, 2219-2226. MEDLINE

Ladanyi M, Gerald W. (1994). Cancer Res. 54, 2837-2840. MEDLINE

Lambert G, Bertrand JR, Fattal E, Subra F, Pinto-Alphandary H, Malvy C, Auclair C, Couvreur P. (2000). Biochem. Biophys. Res. Commun. 279, 401-406. MEDLINE

Lessnick SL, Braun BS, Denny CT, May WA. (1995). Oncogene 10, 423-431. MEDLINE

Li R, Huiping P, Watson DK. (2000). Oncogene 19, 6514-6523. MEDLINE

Mager AM, Grapin-Botton A, Ladjali K, Meyer D, Wolff CM, Stiegler P, Bonnin MA, Remy P. (1998). Int. J. Dev. Biol. 42, 561-572. MEDLINE

Magnaghi-Jaulin L, Masutani H, Robin P, Lipinski M, Harel-Bellan A. (1996). Nucleic Acids Res. 24, 1052-1058. MEDLINE

Mao X, Miesfeldt S, Yang H, Leiden JM, Thompson CB. (1994). J. Biol. Chem. 269, 18216-18222. MEDLINE

Maroulakou IG, Bowe DB. (2000). Oncogene 19, 6432-6442. MEDLINE

Mastrangelo T, Modena P, Tornielli S, Bullrich F, Testi MA, Mezzelani A, Radice P, Azzarelli A, Pilotti S, Croce CM, Pierotti MA, Sozzi G. (2000). Oncogene 19, 3799-3804. MEDLINE

May WA, Arvand A, Thompson AD, Braun BS, Wright M, Denny CT. (1997). Nat. Genet. 17, 495-497. MEDLINE

May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, Zucman J, Thomas G, Denny CT. (1993a). Proc. Natl. Acad. Sci. USA 90, 5752-5756. MEDLINE

May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB, Hromas R, Denny CT. (1993b). Mol. Cell. Biol. 13, 7393-7398. MEDLINE

Mélet F, Motro B, Rossi DJ, Zhang L, Bernstein A. (1996). Mol. Cell. Biol. 16, 2708-2718. MEDLINE

Meyer D, Wolff CM, Stiegler P, Senan F, Befort N, Befort JJ, Remy P. (1993). Mech. Dev. 44, 109-121. MEDLINE

Mitsiades N, Poulaki V, Leone A, Tsokos M. (1999). J. Natl. Cancer Inst. 91, 1678-1684. MEDLINE

Morrison SJ, White PM, Zock C, Anderson DJ. (1999). Cell 96, 737-749. MEDLINE

Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN, Reddy ES. (1994). Oncogene 9, 3087-3097. MEDLINE

Ohno T, Rao VN, Reddy ES. (1993). Cancer Res. 53, 5859-5863. MEDLINE

Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy ES. (1995). Oncogene 11, 1049-1054. MEDLINE

Panagopoulos I, Hoglund M, Mertens F, Mandahl N, Mitelman F, Aman P. (1996). Oncogene 12, 489-494. MEDLINE

Pereira R, Quang CT, Lesault I, Dolznig H, Beug H, Ghysdael J. (1999). Oncogene 18, 1597-1608. MEDLINE

Perrotti D, Bonatti S, Trotta R, Martinez R, Skorski T, Salomoni P, Grassilli E, Lozzo RV, Cooper DR, Calabretta B. (1998). EMBO J. 17, 4442-4455. Article MEDLINE

Perrotti D, Iervolino A, Cesi V, Cirinna M, Lombardini S, Grassilli E, Bonatti S, Claudio PP, Calabretta B. (2000). Mol. Cell. Biol. 20, 6159-6169. MEDLINE

Peter M, Couturier J, Pacquement H, Michon J, Thomas G, Magdelenat H, Delattre O. (1997). Oncogene 14, 1159-1164. MEDLINE

Petermann R, Mossier BM, Aryee DN, Khazak V, Golemis EA, Kovar H. (1998). Oncogene 17, 603-610. MEDLINE

Powers CA, Mathur M, Raaka BM, Ron D, Samuels HH. (1998). Mol. Endocrin. 12, 4-18.

Prasad DD, Ouchida M, Lee L, Rao VN, Reddy ES. (1994). Oncogene 9, 3717-3729. MEDLINE

Rabbitts TH, Forster A, Larson R, Nathan P. (1993). Nature Gen. 4, 175-180.

Rao VN, Ohno T, Prasad DD, Bhattacharya G, Reddy ES. (1993). Oncogene 8, 2167-2173. MEDLINE

Rauscher FJ, Benjamin LE, Fredericks WJ, Morris JF. (1994). Cold Spring Harb. Symp. Quant. Biol. 59, 137-146. MEDLINE

Sancéau J, Hiscott J, Delattre O, Wietzerbin J. (2000). Oncogene 19, 3372-3383. MEDLINE

Sarrazin S, Starck J, Gonnet C, Doubeikovski A, Melet F, Morle F. (2000). Mol. Cell. Biol. 20, 2959-2969. MEDLINE

Scotlandi K, Baldini N, Cerisano V, Manara MC, Benini S, Serra M, Lollini PL, Nanni P, Nicoletti G, Bernard G, Bernard A, Picci P. (2000a). Cancer Res. 60, 5134-5142.

Scotlandi K, Chano T, Benini S, Serra M, Manara MC, Cerisano V, Picci P, Baldini N. (2000b). Intl. J. Cancer 87, 328-335.

Sjögren H, Meis-Kindblom J, Kindblom LG, Aman P, Stenman G. (1999). Cancer Res. 59, 5064-5067. MEDLINE

Sohn HW, Choi EY, Kim SH, Lee IS, Chung DH, Sung UA, Hwang DH, Cho SS, Jun BH, Jang JJ, Chi JG, Park SH. (1998). Am. J. Path. 153, 1937-1945. MEDLINE

Sorensen PH, Lessnick SL, Lopez-Terrada D, Liu XF, Triche TJ, Denny CT. (1994). Nat. Genet. 6, 146-151. MEDLINE

Sorensen PH, Shimada H, Liu XF, Lim JF, Thomas G, Triche TJ. (1995). Cancer Res. 55, 1385-1392. MEDLINE

Spyropoulos DD, Pharr PN, Lavenburg KR, Jackers P, Papas TS, Ogawa M, Watson DK. (2000). Mol. Cell. Biol. 20, 5643-5652. MEDLINE

Stolow DT, Haynes SR. (1995). Nucleic Acids Res. 23, 835-843. MEDLINE

Tamir A, Howard J, Higgins RR, Li YJ, Berger L, Zacksenhaus E, Reis M, Ben-David Y. (1999). Mol. Cell. Biol. 19, 4452-4464. MEDLINE

Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y. (1997). J. Clin. Invest. 99, 239-247. MEDLINE

Teitell MA, Thompson AD, Sorensen PH, Shimada H, Triche TJ, Denny CT. (1999). Lab. Invest. 79, 1535-1543. MEDLINE

Thompson AD, Teitell MA, Arvand A, Denny CT. (1999). Oncogene 18, 5506-5513. MEDLINE

Thorner P, Squire J, Chilton-MacNeil S, Marrano P, Bayani J, Malkin D, Greenberg M, Lorenzana A, Zielenska M. (1996). Am. J. Pathol. 148, 1125-1138. MEDLINE

Triche TJ, Askin FB, Kissane JM. (1987). Major Problems in Pathology Vol. 18. Feingold M. and Bennington JC (eds). Saunders: Philadelphia, pp. 145-195.

Truong AHL, Ben-David Y. (2000). Oncogene 19, 6482-6489. MEDLINE

Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM. (1984). Cancer Gen. Cytogen. 12, 1-19.

Urano F, Umezawa A, Hong W, Kikuchi H, Hata J. (1996). Biochem. Biophys. Res. Commun. 219, 608-612. MEDLINE

Watson DK, Robinson L, Hodge DR, Kola I, Papas TS, Seth A. (1997). Oncogene 14, 213-221. MEDLINE

Watson DK, Seth A. (2000). Oncogene 55, 6393-6547.

Wei G, Antonescu CR, de Alava E, Leung D, Huvos AG, Meyers PA, Healey JH, Ladanyi M. (2000). Cancer 89, 793-799. Article MEDLINE

Yang L, Chansky HA, Hickstein DD. (2000a). J. Biol. Chem. 275, 37612-37618.

Yang L, Embree LJ, Hickstein DD. (2000b). Mol. Cell. Biol. 20, 3345-3354. MEDLINE

Yang L, Embree LJ, Tsai S, Hickstein DD. (1998). J. Biol. Chem. 273, 27761-27764. MEDLINE

Zhang L, Eddy A, Teng YT, Fritzler M, Kluppel M, Melet F, Bernstein A. (1995). Mol. Cell. Biol. 15, 6961-6970. MEDLINE

Zinszner H, Albalat R, Ron D. (1994). Genes Dev. 8, 2513-2526. MEDLINE

Zinszner H, Sok J, Immanuel D, Yin Y, Ron D. (1997). J. Cell. Sci. 110, 1741-1750. MEDLINE

Zoubek A, Dockhorn-Dworniczak B, Delattre O, Christiansen H, Niggli F, Gatterer-Menz I, Smith TL, Jurgens H, Gadner H, Kovar H. (1996). J. Clin. Oncol. 14, 1245-1251. MEDLINE

Zoubek A, Pfleiderer C, Salzer-Kuntschik M, Amann G, Windhager R, Fink FM, Koscielniak E, Delattre O, Strehl S, Ambros PF, Gadner H, Kovar H. (1994). Br. J. Cancer 70, 908-913. MEDLINE

Zucman J, Delattre O, Desmaze C, Epstein AL, Stenman G, Speleman F, Fletchers CD, Aurias A, Thomas G. (1993a). Nature Gen. 4, 341-345.

Zucman J, Melot T, Desmaze C, Ghysdael J, Plougastel B, Peter M, Zucker JM, Triche TJ, Sheer D, Turc-Carel C, Ambros P, Combaret V, Lenoir G, Aurias A, Thomas G, Delattre O. (1993b). EMBO J. 12, 4481-4487. MEDLINE

Zwerner JP, May WA. (2001). Oncogene 20, 626-633. MEDLINE

Figures

Figure 1 Schematic depicting molecular consequences of the EFT 11;22 chromosomal translocation. The glutamine-rich N-terminus of EWS is fused to the C-terminus of FLI1 which encodes the ETS DNA-binding domain. The EWS RRM motif that is thought to be involved in RNA binding is not present in the fusion. Differences in chromosome breakpoints lead to variability of sequences juxtaposing the EWS/FLI1 fusion points in EFTs (bracketed region). Legend: clear box - EWS; vertical wave - EWS RRM domain; diagonal lines - FLI1; gray box - ETS DNA-binding domain

Figure 2 Potential EWS/ETS oncogenic mechanisms in EFT cells. ETS proteins involved in EFT fusions cannot interact directly with the RNA polymerase II complex and rely on protein-protein interactions with other transcription factors. EWS, a member of the TET family, appears to act as an adapter molecule tethering RNA processing proteins to the POL II complex. EWS/ETS fusions could act as dominant active ETS transcription factor since the N-terminus of EWS (clear) is able to bind POL II subcomponents. At the same time it is possible though unproven, that EWS/ETS fusions could act as dominant negative TET proteins by denying access of the normal TET-snRNP complex

Tables

Table 1 Listing of cancer-associated gene fusions involving TET family members

10 September 2001, Volume 20, Number 40, Pages 5747-5754
Table of contents    Previous  Article  Next    [PDF]