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[Cancer Research 63, 8338-8344, December 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

EWS/ETS Fusions Activate Telomerase in Ewing’s Tumors

Akiko Takahashi1, Fumihiro Higashino1, Mariko Aoyagi1, Koichi Yoshida3, Miyuki Itoh3, Satoru Kyo4, Takatoshi Ohno5, Takahiro Taira6, Hiroyoshi Ariga6, Kohichi Nakajima7, Mitsutoki Hatta1, Masanobu Kobayashi8, Hidehiko Sano2, Takao Kohgo1 and Masanobu Shindoh1

1 Departments of Oral Pathobiological Science and
2 Oral Health Science, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan;
3 Department of Biology, Sapporo Medical University School of Medicine, Sapporo, Japan;
4 Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Kanazawa, Japan;
5 Department of Orthopedic Surgery, Gifu University School of Medicine, Gifu, Japan;
6 Department of Molecular Biology, Hokkaido University Graduate School of Pharmaceutical Sciences, Sapporo, Japan;
7 Department of Immunology, Osaka City University Graduate School of Medicine, Osaka, Japan; and
8 Division of Cancer Pathobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EWS/ETS is a chimeric protein identified in most Ewing’s sarcomas. Although EWS/ETS has been shown to activate transcription as a transcription factor, the detailed targets of EWS/ETS in transformed cells have not been clarified. Herein, we demonstrate that telomerase is a new target of EWS/ETS fusions. Both telomerase activity and the expression level of telomerase reverse transcriptase (TERT) mRNA were up-regulated in NIH3T3 cells transformed by EWS/E1AF and EWS/FLI1 as well as in two Ewing’s sarcoma cell lines. Luciferase assay using the TERT promoter revealed that EWS/E1AF and EWS/FLI1 function as positive regulators of TERT transcription in an ETS binding site-independent manner. EWS/ETS appeared to be included in the initiation complex of TERT transcription and to cooperate with CREB-binding protein (CBP)/p300. When EWS/FLI1 was knocked down in Ewing’s sarcomas cells by RNA interference, the expression level of TERT mRNA and the telomerase activity were significantly decreased. These findings indicate that EWS/ETS fusion proteins activate human telomerase activity in Ewing’s tumors through up-regulation of TERT gene expression, probably as a transcriptional coactivator.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ewing’s sarcoma is an aggressive bone neoplasia that occurs mostly in young adults and adolescents and appears as undifferentiated small round tumor cells that are thought to derive from neural crest progenitors. These tumors are characterized by specific chromosomal translocations wherein the EWS gene on chromosome 22 is fused to one of five members of the ETS gene family (FLI1, ERG, ETV1/ER81, E1AF/PEA3, and FEV). These translocations produce five chimeric proteins that include the NH2-terminal transactivation domain of EWS and the COOH-terminal DNA-binding domain of ETS family transcription factors (1) . In most Ewing’s sarcomas, there is a t(11;22)(q24;q12) chromosome translocation that encodes a chimeric EWS/FLI1 protein (2) . FLI1 was found to be rearranged in 75% of erythroleukemias induced by Friend murine leukemia virus (3) . EWS/FLI1 can bind to DNA with the same consensus sequence as FLI1, and the NH2-terminal EWS region is capable of activating the promoters of the target genes (4, 5, 6, 7, 8) . The fusion protein has an ability to transform NIH3T3 cells, and this transformation requires the COOH-terminal DNA-binding domain (5) . Various attempts to search for the target gene promoter activated by EWS/FLI1 have been carried out, and target genes such as c-fos, platelet-derived growth factor C, and Id2 have been identified (9, 10, 11, 12) . EWS/E1AF was also found to be the gene responsible for tumorigenesis in Ewing’s sarcoma. E1AF was cloned due to its ability to bind to the enhancer element of the adenovirus E1A gene (13) . It has been shown that E1AF positively regulates transcription from matrix metalloproteinase genes associated with cancer cell invasion (14, 15, 16) . However, the transforming activity and the tumorigenic mechanism of EWS/E1AF have remained unclear.

Mammalian telomeres are composed of the sequence 5'-TTAGGG-3' that caps the ends of linear chromosomes. Telomere shortening causes replicative senescence in most somatic cells and increases genetic instability and tumor formation in mice (17) . Telomeres in most tumor cells maintain a certain length, probably as a result of the function of telomerase (18) . Telomerase is able to add the telomeric repeats to the chromosomes through its enzymatic activity. hTERT,9 in particular, is a catalytic subunit of telomerase capable of inducing in vitro and in vivo telomerase activity and immortalizing cells (19) . Telomerase activity is tightly regulated at the transcriptional level of hTERT, and expression depends on the proximal 181-bp region of the promoter in cancer cells (20) . This promoter region contains E-boxes, GC-boxes, and EBS, putative binding sites of Myc, Sp1, and ETS transcription factors, respectively, which are highly conserved between the human and mouse promoters (21) . Recently, many transcriptional factors have been reported to regulate hTERT expression (22, 23, 24, 25) . For example, Myc has been proved to up-regulate TERT and consequently activate telomerase (26, 27, 28) .

In the present study, we found that both EWS/FLI1 and EWS/E1AF activate telomerase activity by up-regulating TERT transcription in an EBS-independent manner. Additionally, EWS/E1AF interacted with CBP/p300 to activate the hTERT promoter. Knock down of EWS/FLI1 by RNAi resulted in a decrease of hTERT mRNA and telomerase activity. These results strongly suggest that telomerase is the target of EWS/ETS fusions in Ewing’s sarcoma cells, and EWS/ETS functions as a coactivator for TERT transcription.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Construction of Plasmid and Reporter Gene.
The cell lines used in this study were H1299 (human lung carcinoma), NIH3T3 (mouse fibroblast), and HeLa (human cervical carcinoma). Two Ewing’s sarcoma cell lines (SCCH196 and NCR-EW2) were described previously (12) . The cells were grown at 37°C with 5% CO2 in DMEM containing 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD) with penicillin/streptomycin/amphotericin B (Sigma, St. Louis, MO). We subcloned E1AF and EWS/E1AF cDNA into pcDNA3 (Invitrogen, Carlsbad, CA) to produce pcDNA3-E1AF and pcDNA3-FLAG-EWS/E1AF. The c-Myc and EWS/FLI1 expression vectors (pEF-c-Myc and pcDNA3-EWS/FLI1) were constructed as described previously (29 , 30) . A reporter plasmid of hTERT containing a substituted mutant (pGL3-32 MTETS2) was constructed using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA).

Retroviral Vector Construction and Infection.
The entire open reading frame of EWS/E1AF and EWS/FLI1 cDNA was inserted downstream of a cytomegalovirus promoter in the retroviral expression vector pLNCX (CLONTECH, Palo Alto, CA). These constructs were then transfected into a virus packaging cell line (293 10A1) by the calcium phosphate precipitation method. After 48 h, high-titer viral stocks were collected as conditioned media. NIH3T3 cells were infected with each of the replication-deficient viral stocks, and stable transfectants were selected with 400 µg/ml neomycin sulfate (Sigma). Appropriate expression of EWS/E1AF and EWS/FLI1 was assessed by Western blot analysis.

Western Blotting and Antibodies.
Proteins were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were probed with either an anti-PEA3 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-FLI1 polyclonal antibody (Santa Cruz Biotechnology), followed by a horseradish peroxidase-labeled secondary antibody, and visualized with the ECL Detection Kit (Amersham Biosciences, Buckinghamshire, United Kingdom).

TRAP Assay.
The TRAP assay was performed using a TRAPEZE telomerase detection system (Intergen) according to the manufacturer’s instructions.

Luciferase Reporter Gene Assay.
For luciferase reporter gene assays, H1299 cells were grown in 12-well plates and cotransfected with various amounts of effector plasmids (pcDNA3-E1AF, pCMV-EWS/E1AF, pKC-FLI1, pcDNA3-EWS/FLI1, and pEF-c-Myc) together with 200 ng of reporter plasmids (a series of mutant hTERT, pBSB-3XEBS, and pBSB-3XmEBS) and 20 ng of pRL-tk vector using FuGENE 6 transfection reagent (Roche Diagnostics, Tokyo, Japan). After incubation for 24 h, the lysates were assayed in a luminometer (ATTO, Tokyo, Japan) with a dual-luciferase reporter assay system (Promega, Madison, WI). All experiments were performed at least three times, and results represent average relative luciferase activities.

Reverse Transcription-PCR Analysis.
The expression of TERT and EWS/FLI1 mRNA was analyzed by real-time reverse transcription-PCR. Total RNA was isolated from cells using TRI REAGENT (Sigma). cDNA was synthesized from 1 µg of RNA using Rever Tra Ace (Toyobo, Osaka, Japan) with random primers (Takara, Otsu, Japan). PCR was performed on the reverse-transcribed cDNA with each of the specific primers using a DNA master SYBR Green I kit (Roche Diagnostics) and DNA Engine Opticon Real-Time system (MJ Research, Waltham, MA). For PCR reactions, sense and antisense oligonucleotide primers for hTERT (5'-CGGAAGAGTGTCTGGAGCAA-3' and 5'-GGATGAAGCGGAGTCTGGA-3'), EWS/FLI1 (5'-TATGGACAGCAGAGTAGCTATG-3' and 5'-CCGTTGCTCTGTATTCTTACTG-3'), and mouse TERT (5'-CAGACATTTCCTTTACTC-3' and 5'-ACCATATACCTGCCAGGG-3') were used as described previously (22 , 31 , 32) . The amount of cDNA from each sample was estimated by PCR with GAPDH-specific primers for human (5'-CAACTACATGGTTTACATGTTC-3' and 5'-GCCAGTGGACTCCACGAC-3') and mouse (5'-CAACTACATGGTCTACATGTTC-3' and 5'-CGCCAGTAGACTCCACGAC-3').

DNA IP Assay.
H1299 cells were cotransfected with pGL3-32 and pCMV-FLAG-EWS/FLI1 or pcDNA3-FLAG-EWS/E1AF. Forty-eight h after transfection, DNA and proteins were cross-linked by the addition of 1% formaldehyde to the medium for 15 min at 37°C, and the reaction was quenched by addition of 100 mM glycine for 15 min at room temperature. Cells were collected in ice-cold PBS, and DNA IP assays were performed as described previously (33) .10 Target sequences were amplified by using RV3 and GLP2 primers from Promega (25 PCR cycles) and resolved in 1.2% agarose gel.

RNAi.
The targeted sequence of the EWS/FLI1 dsRNA was 5'-ACGGGCAGCAGAACCCTTCTT-3'. RNA oligomers in water were annealed according to the procedure used by the Tuschl laboratory.11 This construct was transfected into SCCH196 cells by using GeneSilencer siRNA transfection reagent (Gene Therapy Systems, San Diego, CA). The knock-down level of EWS/FLI1 mRNA was analyzed by real-time quantitative reverse transcription-PCR methods as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EWS/ETS Activates Telomerase Activity.
To examine the effect of EWS/ETS on telomerase activity, NIH3T3 cells expressing EWS/E1AF and EWS/FLI1 (Fig. 1A)Citation , which showed anchorage-independent growth in soft agar,12 were established by retroviral (pLCNX) infection. Telomerase activity was analyzed by the PCR-based telomerase activity detection method TRAP (34) . Cells expressing EWS/E1AF and EWS/FLI1 showed higher telomerase activity than control cells (pLCNX), although the internal control bands showed the same intensities (Fig. 1B)Citation . H1299 cells used as the positive control for the TRAP assay showed high TRAP activity; however the band of the internal control was hardly seen (Fig. 1B)Citation . This seemed to be due to excessive telomerase activity because amplification of the TRAP products and the internal control band are semicompetitive. Semiquantitative reverse transcription-PCR assays were performed to examine the expression of the TERT mRNA in EWS/ETS-expressing cells. NIH3T3 cells expressing EWS/E1AF and EWS/FLI1 showed increased levels of TERT mRNA compared with control cells (Fig. 1C)Citation . Additionally, in two cell lines of Ewing’s sarcoma (SCCH196 and NCR-EW2), the telomerase activities were high (Fig. 1D)Citation , and expression of hTERT mRNA was also detected (Fig. 1E)Citation . From these results, it was considered that transformation by EWS/ETS transfection led to up-regulation of telomerase activity.



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  		Fig. 1. EWS/ETS activates telomerase activity. A, EWS/E1AF and EWS/FLI1 expression in NIH3T3 cells. B, to measure telomerase activity, TRAP assays were performed on lysate equivalent to 1 x 103 cells for all samples. H1299 cells were used as positive controls. IC, internal control to verify the amplification efficiency in each reaction. C, quantity of TERT mRNA of NIH3T3 cells was evaluated by reverse transcription-PCR. GAPDH mRNA was amplified from the same samples. D, cell extracts from the two different Ewing’s cells (SCCH196 and NCR-EW2) were analyzed for telomerase activity. HI, heat inactivation of the enzyme activity of TERT was used as a negative control. E, total RNA was extracted from SCCH196 and NCR-EW2 cells, and reverse transcription-PCR was performed to examine the expression of hTERT mRNA.

 
EWS/ETS Is a Positive Regulator of hTERT Transcription.
A 181-bp fragment upstream from the transcription start site has been defined as a core functional promoter essential for the expression of hTERT mRNA in cancer cells (20) . Some consensus sequences for multiple transcription factors such as c-Myc, Sp1, and ETS are in the core promoter (Fig. 2A)Citation . To determine whether EWS/ETS activates the hTERT promoter, activity of the full-length hTERT promoter (pGL3-3328) was examined by luciferase assay. We used p53-null H1299 cells for luciferase assay to eliminate the effect of p53, which has been established to be a transcriptional repressor of the hTERT core promoter through interaction with Sp1 (23) . Although EWS/ETS did not affect the transcription of the empty luciferase vector (pGL3-basic), the full-length promoter of hTERT was activated by EWS/ETS (Fig. 2B)Citation . Furthermore, EWS/E1AF activated the hTERT core promoter (pGL3-181) more than 10-fold versus the control, and EWS/FLI1 activated the same promoter as well as the full-length promoter (Fig. 2C)Citation . However, E1AF and FLI1 showed lower activities compared with the fusion proteins. In this system, the expression of c-Myc activated transcription of the hTERT core promoter approximately 2-fold in H1299 cells. These data indicated that EWS/ETS had the potential to activate the hTERT promoter.



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  		Fig. 2. EWS/ETS is a positive regulator of the hTERT promoter. A, schematic diagram of transcription factor binding sites in hTERT core promoter and luciferase reporters. The , {bullet}, and indicate c-Myc (E-box), Sp1 (GC-box), and EBS, respectively. B, the full-length promoter region was used for luciferase reporter assay. H1299 cells were cotransfected with pGL3-basic or pGL3-3328 and EWS/ETS expression vectors. The parent vectors of EWS/ETS were used as negative controls (mock). Twenty-four h later, luciferase assays were performed. C, various amounts of EWS/E1AF or EWS/FLI1 expression vector (10, 50, 100, and 200 ng, respectively), or 200 ng of E1AF or FLI1 expression vectors were transfected with a reporter plasmid including hTERT core promoter (pGL3-181) into H1299 cells. After 24 h, transcriptional activity was measured by luciferase assay. The same experiment design was applied to c-Myc. All assays were repeated individually at least three times.

 
EBS and E-Box in hTERT Promoter Are Not Direct Targets of EWS/ETS.
To identify the region responsible for EWS/ETS, various reporters, which have point mutations in the binding motifs of transcription factors in the hTERT core promoter, were used (as illustrated in Fig. 3ACitation ). Two EBSs are included in the 181-bp core promoter, one of which was determined to be the minimal responsive element for epidermal growth factor (25) . Substitution EBS mutants did not affect the responsiveness to either EWS/E1AF or EWS/FLI1 (Fig. 3B)Citation . Abrogation of the E-box by substituted mutations in pGL3-181 did not change the responsiveness to EWS/ETS (Fig. 3C)Citation either. These data indicated that EBS and E-box were not the targets of EWS/ETS.



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  		Fig. 3. EBS and E-box in the hTERT promoter are not direct targets of EWS/ETS. A, schematic diagram of hTERT core promoter with substituted mutations in transcription factor binding sites. Two of the E-boxes, consensus motif of c-Myc binding site, are located at -165 and +44, and two EBSs are located at -23 and +48 in the hTERT promoter. Black-shaded areas indicate the mutated sequences of each binding motif. Mutant reporter plasmids containing mutations in two E-boxes were constructed from pGL3-181; i.e., CACGT at -165 was altered to TTTGT (pGL3 Myc MT1), and CACGTG at +44 was altered to CACAAG (pGL3 Myc MT2). Two putative EBSs at -23 and +48 were mutated in pGL3-32; i.e., TTCCTTTCCG at -23 was altered to TTAATTTAAG (pGL3-32 MTETS1), and GGAAG at +48 was altered to GCTAG (pGL3-32 MTETS2). B, pGL3-32 or its mutants of EBS (pGL3-32 MTETS1 and pGL3-32 MTETS2) were cotransfected with 200 ng of EWS/E1AF or EWS/FLI1 expression vectors. C, expression vector of EWS/E1AF or EWS/FLI1 (200 ng) was transfected with the hTERT reporter plasmid mutated at either of the c-Myc binding sites (pGL3 Myc MT1 and pGL3 Myc MT2). All cells were harvested after 24 h, and luciferase activities were analyzed. All assays were repeated individually at least three times.

 
Potential of Aberrant ETS Proteins on Transcriptional Activation Is Higher Than That of Normal ETS Proteins.
To determine the transcriptional activation properties of EWS/ETS, we used a luciferase reporter containing three copies of EBS (CAGGAAGT), which is responsible for the PEA3 family of the protein (35) . Expression of E1AF activated the transcription from 3xEBS luciferase reporter to 27-fold over the basal level of the reporter (Fig. 4A)Citation . EWS/E1AF also activated transcription in a dose-dependent manner. Likewise, FLI1 and EWS/FLI1 showed transcriptional activation abilities for the same reporter; however, lower levels of luciferase activities were detected (Fig. 4B)Citation . EWS/E1AF and EWS/FLI1 showed significantly high transcriptional activation compared with the activities of E1AF and FLI1. These results corresponded to those of previous reports using chloramphenicol acetyl transferase analysis (4, 5, 6, 7) . Surprisingly, EWS/ETS was able to active the 3xmEBS promoter, which includes mutant EBS, although normal ETS proteins failed to activate it (Fig. 4C)Citation . These data suggested that EWS/ETS proteins acted as a transcriptional activator.



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  		Fig. 4. Comparison of transcriptional activation properties between normal and aberrant ETS proteins. H1299 cells were transiently transfected with a reporter plasmid (pBSB-3XETS binding site) and increasing amounts of E1AF or EWS/E1AF expression plasmid (A) or FLI1 or EWS/FLI1 (B; 10, 20, 50, 100, and 200 ng, respectively). C, reporter mutant of EBS (pBSB-3XmEBS) was transfected with 200 ng of each effector plasmid. After 24 h of incubation, a luciferase assay was performed, and the activity in control samples (mock) was normalized to 1.0.

 
EWS/ETS Acts on hTERT Promoter as a Transcriptional Activator.
To confirm that EWS/ETS was included in the transcriptional initiation complex, we performed DNA IP assay (33) . H1299 cells were cotransfected with pGL3-32 and FLAG-tagged expression plasmids of EWS/ETS (EWS/E1AF and EWS/FLI1). After 48 h, each potential component of the EWS/ETS complexes was precipitated using the FLAG antibody, and the promoter region was detected by PCR (Fig. 5A)Citation . The data indicated that the complexes containing EWS/E1AF or EWS/FLI1 bound to the hTERT promoter.



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  		Fig. 5. Complexes containing EWS/E1AF or EWS/FLI1 bound to the hTERT promoter as a transcriptional activator. A, H1299 cells were transfected with the hTERT luciferase reporter gene together with FLAG-EWS/E1AF or FLAG-EWS/FLI1 expression vectors. DNA IP assays were performed with FLAG antibody. As a negative control, the same amount of antimouse IgG was used for IP for each sample. Immunoprecipitated DNA was analyzed by PCR (top panel). As an input of each assay, the lysate before IP was subjected to PCR using the same primers (bottom panel). B, EWS/E1AF interacts with p300 for up-regulation of hTERT transcription. H1299 cells were transfected with pGL3-181 and EWS/E1AF expression vectors. Wild-type E1A or its CR2 mutant (E1AmCR2) or NH2-terminal deletion mutant (E1AdlN) was cotransfected. After 24 h of incubation, cells were harvested, and luciferase activity was performed.

 
EWS forms a complex with CBP and its homologue p300 protein (36 , 37) . In oncogenic chimera proteins, EWS/ATF-1 has been demonstrated to associate with CBP and interfere with p53-mediated transcriptional activation (38) . To investigate whether the activities of EWS/E1AF and EWS/FLI1 involved an interaction with endogenous p300, we analyzed the effect of E1A gene product on their activities. Overexpression of E1A protein sequesters endogenous CBP/p300 (39) . Indeed, E1A repressed the transcriptional activity of EWS/E1AF almost to the basal level (Fig. 5B)Citation . An E1A mutant that was deficient in retinoblastoma binding (E1A mCR2) did not abolish the repression ability. However, an NH2-terminal deletion mutant of E1A that was deficient in CBP/p300 binding never repressed EWS/E1AF-dependent transcription. These data strongly suggest that EWS/E1AF cooperated with CBP/p300 to activate the hTERT promoter.

Knock Down of EWS/FLI1 Leads to the Reduction of hTERT mRNA and Telomerase Activity.
To further understand the relationship between EWS/ETS and hTERT expression, EWS/FLI1 was knocked down by RNAi. The dsRNA molecule of EWS/FLI1, which is the target region of EWS/FLI1 (Fig. 6A)Citation , was applied to SCCH196 Ewing’s tumor cells. Real-time quantitative reverse transcription-PCR analysis revealed that EWS/FLI1 mRNA was reduced to 24% of the corresponding value for mock cells after treatment with dsRNA (Fig. 6BCitation , top panel). In these knock-down cells, the expression level of hTERT mRNA was reduced to 28% (Fig. 6BCitation , bottom panel), and the telomerase activity also decreased compared with that of mock cells (Fig. 6C)Citation .



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  		Fig. 6. Knock down of EWS/FLI1 by dsRNA reduced the expression of hTERT mRNA and telomerase activity. A, schematic structure of the dsRNA that cleaves the target mRNA. After sense and antisense RNA were annealed to dsRNA, either the construct or water (mock) was transfected into SCCH196 cells. B, the expression level of EWS/FLI1 and hTERT mRNA was analyzed by real-time quantitative reverse transcription-PCR methods 48 h after transfection. The quantities of EWS/FLI1 and hTERT mRNA were normalized by the amounts of GAPDH mRNA and expressed as a percentage of expression. C, TRAP analysis revealed that telomerase activity of dsRNA sample was decreased compared with the water-transfected mock-treated cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that EWS/ETS fusion activated telomerase in Ewing’s sarcoma cells. EWS/ETS appeared to activate the transcription of hTERT as a transactivator probably interacting with CBP/p300. RNAi of EWS/FLI1 using Ewing’s tumor cells reduced the TERT mRNA level and the consequent telomerase activity. These results strongly suggested that TERT was one of the targets of EWS/ETS fusions for its oncogenic activity.

Although most cancer cells express TERT and have telomerase activity, the relation between telomerase and Ewing’s family tumors is still unknown. EWS/ETS fusions are recognized to be important for the genesis of these tumors. Various genes were identified as the targets of EWS/ETS in transformed cells. On the EBS in the c-fos promoter, EWS/FLI1 is able to form a ternary complex, although normal FLI1 is unable to do so (9) . Platelet-derived growth factor C is a biologically potent secreted growth factor and is significantly induced in EWS/FLI1-driven transformation and oncogenesis (10 , 11) . On the other hand, it has been reported that EWS/ETS also functions as a transcriptional repressor. EWS/FLI1, EWS/ERG, and EWS/ETV1 fusions have reduced sensitivity to transforming growth factor ß type II receptor, which is a putative tumor suppressor, through suppression of transforming growth factor ß type II expression (40 , 41) . Our finding is the first evidence showing EWS/ETS is the major activator of TERT in tumor cells.

Many regulators of hTERT expression such as hormones, differentiation-inducing agents, growth factors, cell cycle regulators, and oncogenes have been identified previously (42) . Among these, c-Myc is well known to activate telomerase (21 , 27) . In this study, overexpression of c-Myc activated the hTERT core promoter in H1299 cells. Interestingly, EWS/E1AF and EWS/FLI1 showed higher levels of hTERT expression than normal ETS proteins. These results suggested that EWS/ETS might be a more potent activator of human telomerase.

Recent studies have suggested a new aspect of the tumorigenic mechanism of EWS/ETS; i.e., the DNA-binding domain is not essential for its transforming activity (43 , 44) . Indeed, several gene promoters are activated by EWS/ETS regardless of lacking direct binding to DNA (7) . Our findings also indicated that transcriptional activation by EWS/E1AF and EWS/FLI1 did not require direct binding to the hTERT promoter. First, we speculated that EWS/ETS activated the TERT promoter through the E-boxes by up-regulating c-Myc expression level, because c-Myc is consistently expressed in Ewing’s tumor cells at a high level (45) , and EWS/FLI1 is a transactivator of the c-Myc promoter (7) . However, the E-box mutants did not affect the transcriptional activity of EWS/ETS fusion (Fig. 3C)Citation . Thus, the indirect pathway through c-Myc up-regulation was denied in this study.

We next assumed that EWS/ETS acts as a coactivator. EWS as well as its close homologue, hTAFII68, binds to some subunits of the basal transcription factor TFIID and to RNA polymerase II (46 , 47) . Additionally, EWS physically and functionally interacts with two homologues of coactivators, CBP and p300 (38) . In particular, EWS forms a complex with CBP and HNF4 and cooperatively enhances HNF4-dependent transactivation, suggesting that EWS is a coactivator of transcription (37) . In this study, we found that EWS/E1AF and EWS/FLI1 were able to activate transcription in an EBS-independent manner (Figs. 3BCitation and 4CCitation ), and the activities were stronger than those of normal ETS proteins (Figs. 2CCitation and 4, A and BCitation ). Furthermore, to confirm whether EWS/ETS was contained in the protein-DNA complex of the hTERT promoter, we performed DNA IP assay. The results indicated that EWS/E1AF and EWS/FLI1 were surely recruited to the promoter region of hTERT (Fig. 5A)Citation . As far as we know, no biochemical evidence of EWS/ETS complexes bound to the hTERT promoter region in Ewing’s tumor has been reported. Experiments with E1A and its mutant revealed that p300 was essential for EWS/E1AF to activate transcription of hTERT. Taken together, we concluded that Ewing’s fusion proteins activate the TERT promoter as a coactivator and recruit other coactivators to the complex of basal transcription factors. The relationship between the hTERT promoter and CBP/p300 has never been reported before. These findings demonstrate a new mechanism regulating transcription of the hTERT promoter. On the other hand, activation of the hTERT promoter by EWS/FLI1 was not affected by the cotransfection of wild-type E1A (data not shown). Thus EWS/FLI1 would activate the transcription of the hTERT promoter by a different mechanism from that of EWS/E1AF.

In SCCH196 cells, two different EWS/FLI1 fusion transcripts were identified, EWS exon 7 to FLI1 exon 6 and EWS exon 8 to FLI1 exon 6 (31) . The former, called type I, is found to be in-frame and most abundantly seen in Ewing’s sarcoma cell lines. Therefore, the type I translocation region in EWS/FLI1 mRNA was chosen for the target of RNAi. Knock down of EWS/FLI1 mRNA using RNAi led to decreases of hTERT mRNA and telomerase activity in SCCH196 cells. These results indicate that chimeric EWS/FLI1 actually activates telomerase, and it may be possible that immortalization of Ewing’s tumor cells is controlled by the application of this system. A recent report revealed that the level of hTERT mRNA expression is proportional to the clinical aggressiveness in soft tissue tumors (48) . The application of this system may make the control of the life of the cancer cell possible, and it may be useful for gene-specific therapeutic treatment of patients with genetic diseases.

Recently, multifunction of transcription factors has been reported. FKHR, a forkhead family member, was demonstrated to regulate transcription as a cofactor in a DNA binding-independent manner in addition to the original function as a DNA binding-dependent transcription factor (49 , 50) . In the present study, we report for the first time a new function of EWS/ETS, which up-regulates the expression of hTERT mRNA and telomerase activity of Ewing’s tumor cells, as a DNA binding-independent transcriptional coactivator.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grant-in-Aid from Akiyama Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Masanobu Shindoh, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13 West 7, Kita-ku, Sapporo 060-8586, Japan. Phone and fax: 81-11-706-4239; E-mail: mshindoh{at}den.hokudai.ac.jp

9 The abbreviations used are: hTERT, human telomerase reverse transcriptase; TERT, telomerase reverse transcriptase; EBS, ETS binding site; RNAi, RNA interference; dsRNA, double-stranded RNA; TRAP, telomeric repeat amplification protocol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CBP, cAMP-responsive element binding protein-binding protein; IP, immunoprecipitation. Back

10 http://www.iam.u-tokyo.ac.jp/bnsikato/index.html. Back

11 http://www.mpibpc.gwdg.de/abteilungen/100/105/siRNAuserguide.pdf. Back

12 K. Yoshida, unpublished data. Back

Received 7/17/03. Accepted 9/ 9/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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