Abstract
Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.
Similar content being viewed by others
Main
Ewing's sarcoma (ES) and primitive neuroectodermal tumours (PNET) belong to the Ewing family of tumours, which is a subgroup of small-round-cell tumours (Ewing, 1984). The clinical prognosis of individuals with these tumours is poor because of the lack of specific therapies and a high incidence of relapse (Kovar et al, 1990; Ambros et al, 1991). Monitoring the disease is also difficult because of the absence of specific phenotypic markers. The majority of ES/PNET have been shown to have a t(11;22)(q24;q12) chromosomal translocation that fuses the amino terminus of the EWS gene to the carboxyl terminus of the FLI-1 gene (Delattre et al, 1992).
The fusion of an Ets DNA-binding domain (DBD) to EWS appears to be an important determinant in the generation of ES/PNET. While 85% of ES/PNET translocations involve FLI-1, another 5% involve ERG, a closely related ETS transcription factor and other rarer translocations in ES have included only closely related members of the ETS family. While human EWS/FLI-1 fusion is capable of transforming, neither the N-terminal EWS nor FLI-1 DBD domain transformed NIH3T3 cells (May et al, 1993a; Lessnick et al, 1995). This suggests that the EWS/FLI-1 fusion protein does not act by blocking the binding of normal cellular FLI-1 and is consistent with the role of EWS/FLI-1 as a novel oncogenic transcription factor. Consistent with this hypothesis, in ES/PNET tumour cell lines or human EWS/FLI-1-transformed NIH3T3 cells many genes associated with tumorigenesis have also been shown to be upregulated including c-myc, stromelysin-1, Manic Fringe, mE2C and EAT-2 (Bailly et al, 1994; Braun et al, 1995; Thompson et al, 1996; May et al, 1997; Arvand et al, 1998; Dauphinot et al, 2001). Since EWS/FLI-1 or EWS/GAL4 can more efficiently activate reporter constructs regulated by HTLV Ets DNA-binding or GAL4 DNA binding elements than similar FLI-1 proteins (May et al, 1993b; Bailly et al, 1994), it has been suggested that EWS/FLI-1 could act by overexpressing genes normally regulated by FLI-1, resulting in tumorigenesis.
Although the EWS/FLI-1 fusion gene is capable of acting as an oncogene, the mechanism that results in the EWS/FLI-1 translocation in ES/PNET and the direct effects of the resultant protein are unknown. Indeed, the EWS/FLI-1 fusion protein may also have alternate activities since mutation of the Ets DBD does not ablate all transforming abilities and EWS/FLI-1 can also affect mRNA splicing (Jaishankar et al, 1999; Knoop and Baker, 2001; Welford et al, 2001). While the use of antisense oligonucleotides or transcripts to inhibit EWS/FLI-1 expression or suppression of a variety of signalling pathways reduced the tumorigenic potential of ES/PNET cells (Ouchida et al, 1995; Kovar et al, 1996; Tanaka et al, 1997; Toretsky et al, 1997), these reports confirm the central role of EWS/FLI-1 in ES/PNET, but do not address the mechanism by which EWS/FLI-1 acts. Since cancer rarely results from a single mutation and, like other cancers, ES/PNET cells contain other mutations (e.g. p53), understanding the precise mechanism by which the EWS/FLI-1 protein results in transformation will be important in the development of effective therapies for this condition.
One approach to inhibit only the transcriptional activity of EWS/FLI-1 is to introduce specific transcriptional suppressors into EWS/FLI-1-transformed cells that can bind and actively suppress EWS/FLI-1 target genes. The Kruppel associated box (KRAB) is a protein domain of approximately 75 amino-acid residues and has been shown to act as a potent DNA-binding-dependent transcriptional repressor (Margolin et al, 1994; Witzgall et al, 1994; Pengue et al, 1995; Vissing et al, 1995) that inhibits the transcriptional machinery and/or alters the chromatin structure (Kingston et al, 1996; Moosmann et al, 1997). Approximately, one-third of the zinc-finger proteins of the Kruppel Cys2His2-type contain KRAB domains at their amino termini (Bellefroid et al, 1991). Recently, constructs linking KRAB to a variety of DBD have been shown to efficiently suppress the activity of promoter–reporter constructs in a DNA-binding specific manner (Beerli et al, 1998; Herchenroder et al, 1999; Ma et al, 1999; de Haan et al, 2000). Furthermore, the introduction of KRAB fused to PAX3 or Myb DBD into rhabdomyosarcoma cells or haemopoietic tumour cell lines, respectively, can inhibit their transformed phenotype (Rossi et al, 1999; Ayyanathan et al, 2000; Fredericks et al, 2000; Nawrath et al, 2000).
Thus, we have generated a construct that contains KRAB linked to the DBD of FLI-1 and examined its ability to reverse the phenotype of human ES/PNET cell lines or NIH3T3 cells transformed by both human or mouse EWS/FLI-1 oncogenes. We have demonstrated that KRAB/FLI-1 expression reduces the ability of these cells to proliferate in low serum, form colonies in soft agar and tumours in nude mice and reduced the upregulation of c-myc. The initial transformation by EWS/FLI-1 and reversal by KRAB/FLI-1 were similar for both mouse and human genes and demonstrate that the generation of a mouse model for this disease by manipulating the mouse genome is appropriate for elucidating the mechanisms and validating novel therapeutic strategies. Furthermore, these data demonstrate that inhibition of EWS/FLI-1 transcription via sequence-specific DNA binding of the KRAB repressor domain is sufficient to inhibit the transformed phenotype. Therefore, targeted gene repression may be a potential approach for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.
Materials and methods
Plasmids
The murine Ews/Fli-1 fusion gene was designed to recapitulate the human type 1 EWS/FLI-1 fusion sequence. The murine Ews/Fli-1 fusion cDNA construct was generated by first amplifying exons 1–7 on murine Ews cDNA and exons 6–9 of murine Fli-1 cDNA, and then fusing both partial cDNAs together by using an overlap extension PCR technique. The murine Ews/Fli-1 and human type 1 EWS/FLI-1 fusion genes were cloned into the pEF-BOS vector (Mizushima and Nagata, 1990) containing a puromycin resistance gene. Similar constructs were also generated using the cytomegalovirus (CMV) minimal promoter. The KRAB/FLI-1 fusion gene was made by replacing EWS cDNA (amino acids 1–244) in human EWS/FLI-1 type I fusion cDNA with a KRAB domain derived from KOX1 (Figure 2). Mutant KRAB (amino acids 18 and 19 were changed from DV to AA) was used as negative control because the two substitutions abolish binding to the corepressor KAP-1 and thus the KRAB repressor function (Friedman et al, 1996; Moosmann et al, 1997; Figure 2). A KOZAK consensus sequence and initiation codon was added to KRAB and mutant KRAB by PCR amplification using the oligonucleotide; 5′-TCCCCGCGGTTCGCCACCATGGATGCT-3′. A promoterless IRES/Neo/PolyA was cloned behind the KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes to allow selection of clones with high expression.
Cell culture and transfection
NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf serum (FCS; Life Technology), 3.7 g l−1 of sodium bicarbonate (BDH) and 10 mM penicillin/streptomycin. SK-N-MC cell line was obtained from the American Type Tissue Collection (ATCC) and was cultured in Eagle's minimal essential medium (EMEM; GIBCO) supplemented with 10% FCS, 0.1 mM nonessential amino acids (GIBCO), 1.0 mM sodium pyruvate (GIBCO), 1.5 g l−1 sodium bicarbonate (BDH) and 10 mM penicillin/streptomycin. All cell lines were grown in 5% CO2. To examine growth of cell lines in low serum, the cell lines were washed twice in phosphate buffered saline (PBS) and placed in media supplemented with 1% FCS.
To establish cell lines that stably express murine or human EWS/FLI-1, 10 μg of each EWS/FLI-1 expression constructs was electroporated into 1 × 106 NIH3T3 cells in 0.1 ml culture medium at 650 mF and 200 V in a 0.4 ml cuvette with the BIO-RAD Gene Pulser and Pulse Controller Transfection apparatus. Promoter constructs without the fusion protein were similarly transfected as ‘mock’ or normal controls. The transfected cells were selected with 5 μg ml−1 puromycin for 1 week. The puromycin-resistant colonies were cloned, expanded, cryopreserved and evaluated for expression of protein by Western analysis. Clones of EWS/FLI-1-transformed cells were similarly transfected with KRAB/FLI-1 or mutant KRAB/FLI-1, selected with 400 μg ml−1 G418 for 2 weeks and treated as described above.
Antibodies and Western blot
Total cell lysates were prepared from cells at 70% confluence and electrophoresed on an 8% denaturing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were electroblotted onto Hybond C extra membranes (Amersham). The filters were blocked in 10% skim milk powder (Sunshine) dissolved in TBST (20 mM Tris, 137 mM NaCl, 0.1% Tween 20, pH 7.6) for 1h at room temperature. Incubation with primary antibodies were carried out at 4°C overnight. Antibodies used were anti-FLI-1 (Santa Cruz; detects EWS/FLI-1, KRAB/FLI-1 and mutant KRAB/FLI-1), c-myc (Santa Cruz) and α/β tubulin (Boeringer Mannheim). The secondary antibody conjugated to horseradish peroxidase was detected using the ECL Western Blotting Analysis System (Amersham) and exposed to Kodak X-AR film.
Total RNA extraction
Total RNA from each of the cell lines was prepared by harvesting the cells when they were at 70% confluency. Approximately, 1 × 106 cells were washed twice with PBS and total RNA extracted using the High Pure RNA Isolation kit (Roche) as described by the manufacturer and then quantified by absorbance at OD260 using a PE spectrophotometer (Perkin-Elmer).
Cell proliferation assay
The growth rate of each cell line was examined in media containing 1% FCS. NIH3T3 (5 × 103 cells) or SK-N-MC (1 × 103 cells) were seeded in triplicate in 3 cm wells in low serum media and cell proliferation measured by direct counting of trypan-blue negative cells.
Soft agar assays
In all, 5000 cells of each cell line were trypsinized and suspended in 2 ml of complete medium plus 0.3% agarose (Promega). The agar–cell mixtures were plated on top of a bottom layer with 1% complete medium agar mixture in triplicate. After 2–3 weeks, the agar assays were scored for viable colonies.
Mouse tumour formation assay
The tumorigenic potential of each cell line was evaluated by injecting of cell suspensions into 2- to 3-week-old BALB/c nu/nu female mice. For each clone tested, five mice received subcutaneous injections of 2 × 106 cells (NIH3T3 cell lines) or 5 × 106 cells (SK-N-MC cell lines) in a volume of 0.2 ml of PBS into both dorsal midline sides. The mice were housed in microisolator cages, given food and water ad libitum, and handled in a sterile laminar-flow hood. Tumour sizes were measured every 3 days using Vernier calipers along two perpendicular axes. The volume of the tumours was calculated by using the following formula: (mean diameter)3 × π/6. All animal experimentation was performed with approval from the Monash University Ethics Committee and conformed to UKCCCR guidelines for the Welfare of Animals in Experimental Neoplasia (UKCCR, 1998).
Results
Transformation of NIH3T3 cells by murine or human EWS/FLI-1 fusion genes
To determine whether murine Ews/Fli-1 is capable of transforming cells in a similar manner to that of the human EWS/FLI-1 fusion gene, we generated stable NIH3T3 cell lines transfected with mouse or human EWS/FLI-1. Clones were selected with puromycin and testing for mRNA expression by RT–PCR (data not shown). Positive clones were demonstrated to express the expected 68 kDa EWS/FLI-1 band on Western blot analysis using a polyclonal antibody to the C-terminal of the Fli-1 protein (Figure 1A). Cells transfected with the empty vector were used as controls, which displayed a weak nonspecific band that has been observed with this commercial antibody in other studies (May et al, 1997). Both human and mouse EWS/FLI-1 transfectant clones displayed a variety of levels of EWS/FLI-1 fusion protein expression.
One of the hallmarks of the transformed phenotype is the ability of cells to grow in semisolid medium (Rizzino, 1987). At least three cell lines from each genotype were tested in triplicate with data from representative clones shown (Figure 1B). The control cell lines grew poorly in soft agar with only 15±10 small colonies (<20 cells) per 5000 cells plated observed. In contrast, all human EWS/FLI-1 and murine Ews/Fli-1-expressing clones grew efficiently in soft agar, forming large macroscopic colonies of greater than 200 cells, indicating anchorage-independent growth (Figure 1B). For example, human EWS/FLI-1-transformed clone HuEF#1 formed 403±26 colonies/5000 cells and mouse Ews/Fli-1 clone mEF#14 formed 317±33 colonies/5000 cells. The efficiency of colony formation was similar for cell lines expressing mouse or human EWS/FLI-1. Moreover, the level of expression of the fusion protein appeared to correspond to the efficiency of colony formation (Figure 1A and 1B). For example, HuEF#16 formed 623±27 colonies/5000 cells, whereas HuEF#1, which expressed less EWS/FLI-1, only formed 403±27 colonies/5000 cells. These data demonstrate that murine EWS/Fli-1 is capable of transformation similar to human EWS/FLI-1 and may indicate that the amount of EWS/FLI-1 oncoprotein is proportional to the proliferation rate and potency of transformation of NIH3T3 cells.
To determine whether c-myc, which is one of the genes known to be activated in ES/PNET tumour cells, was also altered in our human and murine EWS/FLI-1-transformed NIH3T3 cells, we examined the expression levels of c-myc by Western blot (Figure 1A). Levels of c-myc were elevated in both mouse and human EWS/FLI-1-transfected cells compared with controls, demonstrating that human and mouse EWS/FLI-1 activate a similar downstream signalling cascade.
Expression of the KRAB/FLI-1 repressor in EWS/FLI-1-transformed NIH3T3 cells
Murine and human EWS/FLI-1-transformed clones were used to test the effects of the KRAB/FLI-1 hybrid protein on the transformed phenotype. KRAB/FLI-1 and mutant KRAB/FLI-1 vectors were generated such that they contained the equivalent region of the FLI-1 DBD as that found in human type I EWS/FLI-1 translocations and our human and mouse EWS/FLI-1 constructs. The mutant KRAB domain, which was used as a control, contains two amino-acid substitutions which abolishes KRAB binding to the corepressor KAP-1 and thus repressor function (Margolin et al, 1994; Friedman et al, 1996). Both KRAB/FLI-1 and mutant KRAB/FLI-1 fusion genes were placed under the control of the human EF-1α promoter (Figure 2). These genes were followed by a promoterless IRES/neomycin cassette to ensure that all G418-resistant clones expressed the KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes. Protein expression in G418-resistant clones was demonstrated by Western blot with the Fli-1 polyclonal antibody that detected both the 68 kDa EWS/FLI-1 protein band and the 45 kDa KRAB/FLI-1 or mutant KRAB/FLI-1 protein bands. Three murine and human EWS/FLI-1-transformed clones were each transfected with KRAB/FLI-1 and data from two representative clones, mEF#1 and HuEF#16, are shown in Figure 3A and 3B. Clones K1, K22 and mK12 are KRAB/FLI-1 and mutant KRAB/FLI-1 cotransfectants, respectively, of the mouse EWS/Fli-1-transformed clone mEF#1 (Figure 3A). These clones had similar EWS/Fli-1 protein levels to the parental mEF#1 relative to β-tubulin controls. Clone mK12 displayed a slightly higher expression of mutant KRAB/FLI-1 protein levels compared with K1 and K22 cotransfectants. Similar expression of human EWS/FLI-1, KRAB/FLI-1 and mutant KRAB/FLI-1 was observed in cotransfectants of the human EWS/FLI-1-transformed clone HuEF#16 (Figure 3B). These data indicate KRAB/FLI-1 and mutant KRAB/FLI-1 proteins are expressed at similar levels in these clones, and thus differences in the transformed phenotypes of these clones is likely to be a protein function.
KRAB/FLI-1 reduces the proliferation rate of human or murine EWS/FLI-1 transformed NIH3T3 cells in low serum media
Under normal culture conditions with 10% FCS there was no difference in the growth rate of clones expressing EWS/Fli-1 and/or KRAB/FLI-1 or mutant KRAB/FLI-1 fusion genes (data not shown). KRAB/FLI-1 also had no effect on the growth of wild-type NIH3T3 cells. However, under low serum culture conditions (1% FCS), the EWS/FLI-1-transformed cell lines and clones coexpressing mutant KRAB/FLI-1 grew rapidly (Figure 4A and 4B), whereas EWS/FLI-1-transformed cells coexpressing KRAB/FLI-1 had significantly slower or no growth (Figure 4A and 4B). The growth rate of these KRAB/FLI-1 cotransfectants was similar to the control cell lines that did not express either fusion construct (Figure 4A and 4B). Thus, KRAB/FLI-1 was able to inhibit the growth of both human or murine EWS/FLI-1-transformed cells.
KRAB/FLI-1 reduces the colony formation of murine and human EWS/FLI-1-transformed NIH3T3 cells in soft agar
The ability of KRAB/FLI-1 to reverse the transformation phenotype of EWS/FLI-1-expressing cells was also tested using the soft agar assay. Human or murine EWS/FLI-1-transformed NIH3T3 cell clones were tested in triplicate in three separated experiments. Data from two representative EWS/FLI-1 expressing clones (one human, HuEF#16, and one murine, mEF#1) are shown in Figure 5A and 5B. After 2 weeks, the parental human and mouse EWS/FLI-1-transformed NIH3T3 cells formed ∼650 and ∼200 colonies of >20 cells, respectively (Figure 5A and 5B). Clones cotransfected with mutant KRAB/FLI-1 (mEF#1mK12 and HuEF#16mK11), showed no significant inhibition of soft-agar growth, however, clones cotransfected with KRAB/FLI-1 (mEF#1K1 and mEF#1K7, and HuEF#16K13 and HuEF#16K19) showed a significant reduction in the number of colonies formed (Figure 5A and 5B). These data indicate that the KRAB/FLI-1 repressor impairs anchorage-dependent growth of EWS/FLI-1-transformed cells.
KRAB/FLI-1 impairs tumour development of murine and human EWS/FLI-1-transformed NIH3T3 cells in nude mice
For each murine or human EWS/FLI-1-transformed clone, one KRAB/FLI-1 cotransfectant and one mutant KRAB/Fli-1 cotransfectant were inoculated into BALB/c nu/nu mice. Control cells were not observed to form any tumours (data not shown). The HuEF#16-transformed clone formed tumours of 222±53 mm3 by 30 days after inoculation, whereas the mEF#1 transformed clone had formed tumours of only 128±26 mm3 at the same stage (Figure 6A and 6B). The faster tumour growth rate of this clone HuEF#16 was consistent with its more rapid growth rate observed in vitro and higher expression of EWS/FLI-1. Clones that expressed both mutant KRAB/FLI-1 and EWS/FLI-1 displayed a similar tumour growth rate in nude mice to that of the parental EWS/FLI-1-expressing cells; however, clones that expressed KRAB/FLI-1 showed significantly reduced tumour size (Figure 6A and 6B). For example, mouse Ews/Fli-1-transformed cells coexpressing mutant KRAB/FLI-1 formed tumours in nine out of 10 mice inoculated with a mean size of 96±26 mm3 by day 30; however, at the same stage no tumours were observed in cells coexpressing KRAB/FLI-1. Similarly, in mice inoculated with human EWS/FLI-1 clone #16 coexpressing KRAB/FLI-1, no tumours were observed at day 27; however, tumours formed in nine out of 10 mice inoculated with the same clone expressing mutant KRAB/FLI-1 with a mean volume of 147±32 mm3. In all 50% of the mice inoculated with clones expressing both KRAB/FLI-1 and EWS/FLI-1 did develop small palpable tumours (7±2 mm3) in the latter part of the experiment (Figure 6A and 6B); however, these tumours continued to grow very slowly (data not shown).
KRAB/FLI-1 suppresses the transformed phenotype of SK-N-MC, a PNET cell line
Our data demonstrate that human and mouse EWS/FLI-1 transforms NIH3T3 cells and this transformation could be repressed by KRAB/FLI-1. Since the precise aetiology of ES/PNET is not defined and, like other cancers, these tumours contain other mutations, we were interested to determine whether KRAB/FLI-1 could also suppress the transformed phenotype of a human PNET cell line. Thus, we transfected KRAB/FLI-1 (or mutant KRAB/FLI-1 as a control) into the human PNET cell line SK-N-MC. This cell line has a type I EWS/FLI-1 translocation similar to our constructs generated above, but also has a defined mutation in the tumour suppressor p53 (Beerli et al, 1998). Clones were generated and expression of both EWS/FLI-1 (68 kDa) and KRAB/FLI-1 or mutant KRAB/FLI-1 (45 kDa) proteins was examined by Western blot (Figure 7A). Unlike our NIH3T3 transfectants, the amount of EWS/Fli-1 fusion protein was higher than that of the KRAB/FLI-1/mutant KRAB/FLI-1 chimaeric proteins.
To assess the ability of KRAB/FLI-1 to inhibit transformation of SK-N-MC cells in vitro, we determined their growth rates in media with low serum. KRAB/FLI-1-expressing clones proliferated at a slower rate compared to both mutant KRAB/FLI-1-expressing clones and control SK-N-MC cells (Figure 7B). Unlike NIH3T3 cells coexpressing KRAB/FLI-1 and EWS/FLI, KRAB/FLI-1-expressing SK-N-MC cells continued to proliferate suggesting that the KRAB/FLI-1 repressor could not completely abolish the transformed phenotype of SK-N-MC cells, perhaps because of the relative levels of KRAB/FLI-1 and EWS/FLI-1 proteins.
To determine the ability of the KRAB/FLI-1 repressor to influence tumour development of SK-N-MC cells, we inoculated clones of SK-N-MC cells expressing KRAB/FLI-1 or mutant KRAB/FLI-1 and control cells into nude mice as described above. The mean tumour volume was significantly reduced in KRAB/FLI-1 expressing SK-N-MC cells compared with both mock-transfected and mutant KRAB/FLI-1-expressing cells (Figure 7C). For example, at day 15 100% of mice had developed tumours in the parental and mutant KRAB/FLI-1-expressing cells (mean tumour volume 113±23 and 111±22 mm3, respectively), whereas only 50% of mice inoculated with KRAB/FLI-1-expressing cells had palpable tumours (mean volume 34±8 mm3). These data indicate that the KRAB/FLI-1 repressor could significantly reduce tumour development, similar to the in vitro data.
Discussion
The majority of Ewing's sarcoma and primitive neuroectodermal tumours are associated with a t(11;22)(q24;q12) chromosomal translocation, which results in the production of an EWS/FLI-1 fusion protein that comprises the amino terminus of the EWS gene and the carboxyl terminus of the FLI-1 gene. The continued expression of human EWS/FLI-1 protein has been shown to be sufficient and necessary for in vitro transformation of cell lines (May et al, 1993a, 1993b). Although EWS/FLI-1 has been shown to be a potent transcription factor (Delattre et al, 1992; May et al, 1993a, 1993b; Bailly et al, 1994 and references therein), whether its ability to transform is due only to its transcriptional activation activity has been unclear. Furthermore, its importance in the aetiology of human disease is also unclear as these human tumours are likely to contain multiple mutations. To address these issues, we have demonstrated herein that both human and murine EWS/FLI-1 can transform NIH3T3 cells and that the transformation phenotype in these cells and a human ES/PNET cell line can be inhibited by a specific transcriptional repressor KRAB/FLI-1. The latter also sheds light on the mechanism of EWS/FLI-1 transformation since the FLI-1 DBD is necessary to target an active repressor domain to the effector site.
In order to facilitate mouse model studies, we engineered a murine Ews/Fli-1 fusion gene to mimic the human fusion gene formed by the most common chromosomal translocation, t(11;22)(q24;q12), observed in ES/PNET. This murine fusion gene showed 98% nucleotide identity to the human fusion gene and fibroblast transfectants showed similar transformed phenotypes to human EWS/FLI-1-transfected cells. This included anchorage-independent growth in soft agar, increased proliferation rate in low serum media and tumour development in BALB/c nu/nu mice. Variations in the efficiency of clones to display a transformed phenotype appeared to correlate with the level of the EWS/FLI-1 fusion proteins rather than the species from which the sequences were derived. These findings validate at a protein level previous reports that suggested a correlation between the mRNA levels of human EWS/FLI-1 and the proliferation rate of ES/PNET cells (Tanaka et al, 1997). These data indicate the feasibility and validity of a murine model for ES/PNET, which would provide a valuable resource for elucidating the mechanisms of EWS/FLI-1 transformation and the development of a model in which to test novel therapeutic strategies.
To inhibit EWS/FLI-1 transactivation activity in both human and mouse EWS/FLI-1-transformed cells and a human PNET cell line, we generated a KRAB/FLI-1 chimaeric protein. When bound to DNA by a specific DBD, the KRAB domain binds to the corepressor KAP-1, which suppresses transcription by either directly inhibiting the transcriptional machinery and/or by altering the chromatin structure (Friedman et al, 1996; Le Douarin et al, 1996; Moosmann et al, 1996; Darnell and Richardson, 1999). Indeed, KRAB/FLI-1 inhibited EWS/FLI-1 transformation in our study. Since KRAB/FLI-1 will only inhibit transcription of genes with FLI-1 DNA-binding elements and will not have any effect on the nontransactivation activity of EWS/FLI-1, these data demonstrate that transactivation of FLI-1 target genes by EWS/FLI-1 is essential for its ability to transform. This is in contrast with previous data, which indicated that mutation of the EWS/FLI-1 DBD did not eliminate its ability to transform (Jaishankar et al, 1999; Welford et al, 2001). We suggest that the remaining transformation activity reported for the mutant EWS/FLI-1 could be explained by the ability of the fusion protein to still complex with the transcription initiation complex independent of DNA-binding and/or residual-DNA-binding activity not detected in in vitro assays.
Our observation that suppression of EWS/FLI-1-activated genes inhibits transformation in vitro and in vivo is consistent with previous studies, which demonstrated that expression of a FLI-1 DBD can suppress Ewing's sarcoma and EWS/FLI-1-transformed cell growth in vitro (Kovar et al, 1996; Welford et al, 2001), presumably by competing with EWS/FLI-1 DNA binding. However, in Welford et al, (2001) expression of this FLI-1 DBD did not affect tumour growth of EWS/FLI-1-transformed NIH3T3 cells in vivo. This was suggested to be a result of DBD-independent effects of the EWS/FLI-1 protein, although inhibition of an ES cell line was observed (Welford et al, 2001). It is possible that the levels of the human FLI-1 DBD protein used were not sufficient to inhibit EWS/FLI-1 transactivation in murine NIH3T3 cells in vivo. This is supported by our study where the active repressor KRAB/FLI-1 efficiently inhibited transformed cell growth in vitro and in vivo, whereas similar levels of mutant KRAB/FLI-1 had no significant effect on the transformed phenotype. It is logical to conclude that levels of an active repressor required to inhibit transcription would be much lower than those of a DNA-binding competitor.
The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. Many members of this large family of transcription factors have also been implicated as potential oncogenes in other malignancies including acute lymphoblastic leukaemia, myelomonocytic leukaemias and breast cancer (Santoro et al, 1992; Ichikawa et al, 1994; Golub et al, 1995; Chen et al, 1996; Tymms et al, 1997). Thus, similar use of DNA sequence-specific transcriptional repressors will be an important avenue for the development of broader therapeutic strategies.
The use of a targeted and potent repressor domain increases the efficiency of transcriptional repression of cancer-causing genes. Thus, an active transcriptional repressor is more efficient for the inhibition of tumour growth than overexpression of a DBD. Effective therapies will thus be possible with much lower amounts of protein.
A major issue in understanding the aetiology of ES/PNET is understanding how EWS/FLI-1 activates the many genes shown to be increased. Since the promoters of some of the genes activated in ES/PNET are believed to not bind FLI-1, including c-myc, it has been suggested that the EWS/FLI-1 protein may have additional properties such as RNA processing (Jaishankar et al, 1999) or the binding and modulation of genes not normally regulated by FLI-1 (May et al, 1993a). Significantly, c-myc was increased in each of our cell lines expressing human or mouse EWS/FLI-1 and decreased in clones coexpressing KRAB/FLI-1. Since the KRAB domain shows no DNA-binding activity (Margolin et al, 1994) the regulation of this gene together with transformed phenotype appears to be a result of FLI-1-specific DNA binding. This suggests that the many genes that are activated by EWS/FLI-1, but not by direct FLI-1 DNA binding, may be indirectly activated. The mechanism by which indirect activation occurs is unclear; however, the analysis of KRAB/FLI-1 expressing ES/PNET cell lines will enable the identification of genes specifically regulated through FLI-1-specific DNA binding in ES/PNET. The identification of the primary target genes of EWS/FLI-1 will be important in understanding the oncogenic processes in ES/PNET and will further assist in the development of new therapeutic strategies.
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Ambros IM, Ambros PF, Strehl S, Kovar H, Gadner H, Salzer-Kuntschik M (1991) MIC2 is a specific marker for Ewing's sarcoma and peripheral primitive neuroectodermal tumors. Cancer 67: 1886–1893
Arvand A, Bastians H, Welford SM, Thompson AD, Ruderman JV, Denny CT (1998) EWS/FLI1 up regulates mE2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction. Oncogene 17: 2039–2045
Ayyanathan K, Fredericks WJ, Berking C, Herlyn M, Balakrishnan C, Gunther E, Rauscher III FJ (2000) Hormone-dependent tumor regression in vivo by an inducible transcriptional repressor directed at the PAX3-FKHR oncogene. Cancer Res 60: 5803–5814
Bailly RA, Bossclut R, Zucman J, Cormier F, Delattre O, Roussel M, Thomas G, Ghysdael J (1994) DNA-binding and transcriptional activation properties of the EWS/FLI1 fusion protein resulting from the t(11;22) translocation in Ewing's sarcoma. Mol Cell Biol 14: 3230–3241
Beerli RR, Segal DJ, Dreier B, Barbas III CF (1998) Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci USA. 95: 14628–14633
Bellefroid EJ, Poncelet DA, Lecocq PJ, Revelant O, Martial JA (1991) The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc Natl Acad Sci USA 88: 3608–3612
Braun BS, Frieden R, Lessnick SL, May WA, Denny CT (1995) Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis. Mol Cell Biol 15: 4623–4630
Chen JH, Vercamer C, Li Z, Paulin D, Vandenbunder B, Stehelin D (1996) PEA3 transactivates vimentin promoter in mammary epithelial and tumor cells. Oncogene 13: 1667–1675
Darnell G, Richardson DR (1999) The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents III: the effect of the ligands on molecular targets involved in proliferation. Blood 94: 781–792
Dauphinot L, De Oliveira C, Melot T, Sevenet N, Thomas V, Weissman BE, Delattre O (2001) Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2 and c-Myc expression. Oncogene 20: 3258–3265
de Haan G, Chusacultanachai S, Mao C, Katzenellenbogen BS, Shapiro DJ (2000) Estrogen receptor-KRAB chimeras are potent ligand-dependent repressors of estrogen-regulated gene expression. J Biol Chem 275: 13493–13501
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) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359: 162–165
Ewing J (1984) Diffuse endothelioma of bone. Reprinted in Clin Orthop Rel Res 185: 2 Proc New York Path Soc 21: 17(1921)
Fredericks WJ, Ayyanathan K, Herlyn M, Friedman JR, Rauscher III FJ (2000) An engineered PAX3-KRAB transcriptional repressor inhibits the malignant phenotype of alveolar rhabdomyosarcoma cells harboring the endogenous PAX3-FKHR oncogene. Mol Cell Biol 20: 5019–5031
Friedman JR, Fredericks WJ, Jensen DE, Speicher DW, Huang X-P, Neilson EG, Rauscher FJ (1996) KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev 10: 2067–2078
Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P, Morgan E, Raimondi SC, Rowley JD, Gilliland DG (1995) Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci USA 92: 4917–4921
Herchenroder O, Hahne JC, Meyer WK, Thiesen HJ, Schneider J (1999) Repression of the human immunodeficiency virus type 1 promoter by the human KRAB domain results in inhibition of virus production. Biochim Biophys Acta 1445: 216–223
Ichikawa H, Shimizu K, Hayashi Y, Ohki M (1994) An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16;21) chromosomal translocation. Cancer Res 54: 2865–2868
Jaishankar S, Zhang J, Roussel MF, Baker SJ (1999) Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity. Oncogene 18: 5592–5597
Kingston RE, Bunker CA, Imbalzano AN (1996) Repression and activation by multiprotein complexes that alter chromatin structure. Genes Dev 10: 905–920
Knoop LL, Baker SJ (2001) EWS/FLI alters 5′-splice site selection. J Biol Chem 276: 22317–22322
Kovar H, Aryee DN, Jug G, Henockl C, Schemper M, Delattre O, Thomas G, Gadner H (1996) EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. Cell Growth Differ 7: 429–437
Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF, Gadner H (1990) Overexpression of pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor. Oncogene 5: 1067–1070
Le Douarin B, Nielsen AL, Garnier JM, Ichinose H, Jeanmougin F, Losson R, Chambon P (1996) A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. EMBO J 15: 6701–6715
Lessnick SL, Braun BS, Denny CT, May WA (1995) Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 10: 423–431
Ma ZQ, Tsai MJ, Tsai SY (1999) Suppression of gene expression by tethering KRAB domain to promoter of ER target genes. J Steroid Biochem Mol Biol 69: 155–163
Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher III FJ (1994) Kruppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 91: 4509–4513
May WA, Arvand A, Thompson AD, Braun BS, Wright M, Denny CT (1997) EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic. Nat Genet 17: 495–497
May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, Zucman J, Thomas G, Denny CT (1993a) Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci USA 90: 5752–5756
May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB, Hromas R, Denny CT (1993b) The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 13: 7393–7398
Mizushima S, Nagata S (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18: 5322
Moosmann P, Georgiev O, Le Douarin B, Bourquin JP, Schaffner W (1996) Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1. Nucleic Acids Res 24: 4859–4867
Moosmann P, Georgiev O, Thiesen HJ, Hagmann M, Schaffner W (1997) Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Kruppel-type zinc finger factor. Biol Chem 378: 669–677
Nawrath M, Pavlovic J, Moelling K (2000) Inhibition of human hematopoietic tumor formation by targeting a repressor Myb-KRAB to DNA. Cancer Gene Ther 7: 963–972
Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy ES (1995) Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 11: 1049–1054
Pengue G, Caputo A, Rossi C, Barbanti-Brodano G, Lania L (1995) Transcriptional silencing of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by the Kruppel-associated box repressor domain targeted to the transactivating response element. J Virol. 69: 6577–6580
Rizzino A (1987) Soft agar growth assays for transforming growth factors and mitogenic peptides. Methods Enzymol 146: 341–352
Rossi C, Gibellini D, Barbanti-Brodano G, Betti M, Boarini C, Pengue G, Lania L, Caputo A (1999) Transiently transfected and stably integrated HIV-1 LTR responds differentially to the silencing activity of the Kruppel-associated box (KRAB) transcriptional repressor domain. J Med Virol 58: 264–272
Santoro A, Maggio A, Carbone P, Mirto S, Caronia F, Acuto S (1992) Amplification of ETS2 oncogene in acute nonlymphoblastic leukemia with t(6;21;18). Cancer Genet Cytogenet 58: 71–75
Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y (1997) EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99: 239–247
Thompson AD, Braun BS, Arvand A, Stewart SD, May WA, Chen E, Korenberg J, Denny C (1996) EAT-2 is a novel SH2 domain containing protein that is up regulated by Ewing's sarcoma EWS/FLI1 fusion gene. Oncogene 13: 2649–2658
Toretsky JA, Connell Y, Neckers L, Bhat NK (1997) Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides. J Neurooncol 31: 9–16
Tymms MJ, Ng AY, Thomas RS, Schutte BC, Zhou J, Eyre HJ, Sutherland GR, Seth A, Rosenberg M, Papas T, Debouck C, Kola I (1997) A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer. Oncogene 15: 2449–2462
UKCCCR (1998) United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (2nd edn.). Brit J Cancer 77: 1–10
Vissing H, Meyer WK, Aagaard L, Tommerup N, Thiesen HJ (1995) Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins. FEBS Lett 369: 153–157
Welford SM, Hebert SP, Deneen B, Arvand A, Denny CT (2001) DNA binding domain independent pathways are involved in EWS/FLI1 mediated oncogenesis. J Biol Chem 276: 41977–41984
Witzgall R, O'Leary E, Leaf A, Onaldi D, Bonventre JV (1994) The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc Natl Acad Sci USA 91: 4514–4518
Acknowledgements
We would like to thank Drs J Margolin and FJ Rauscher III for the KRAB and mutant KRAB cDNA, Dr O Delattre for human type I EWS/FLI-1 fusion cDNA, and Dr P Mountford for the IRES/Neo cassette. We also express our thanks to Drs B Scott, C Owczarek and M Pritchard for their critical comments on this manuscript. This work was supported by the Anti-Cancer Council of Victoria (ACCV).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Chan, D., Wilson, T., Xu, D. et al. Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1. Br J Cancer 88, 137–145 (2003). https://doi.org/10.1038/sj.bjc.6600669
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.bjc.6600669
Keywords
This article is cited by
-
Patterns of gene amplification in gastrointestinal stromal tumors (GIST)
Laboratory Investigation (2005)
-
Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody
Modern Pathology (2004)