Transcriptional Regulation of IGF-I Receptor Gene Expression by Novel Isoforms of the EWS-WT1 Fusion Protein

Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS-WT1 fusion protein

Ina Finkeltov1, Scott Kuhn3,4, Tova Glaser1, Gila Idelman1, John J Wright2, Charles T Roberts Jr3 and Haim Werner*,1

1Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Israel; 2Medicine Branch, Division of Clinical Science, National Cancer Institute, NIH, Bethesda, Maryland MD 20889, USA; 3Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon OR 97201, USA

The EWS family of genes is involved in numerous Werner et al., 1994a). In addition to its central role in chromosomal translocations that are characteristic of a normal growth processes, the IGF-I-R plays a pivotal variety of sarcomas. A recently described member of this role in malignant transformation (Baserga et al., 1994; group is desmoplastic small round cell tumor (DSRCT), Grimberg and Cohen, 2000; Werner and LeRoith, which is characterized by a recurrent t(11;22)(p13;q12) 1996). The IGF-I-R is highly expressed in most tumors translocation that fuses the 5' exons of the EWS gene to and cancer cell lines, where it functions as a potent the 3' exons of the WT1 gene. The originally described antiapoptotic agent, conferring enhanced survival to chimera comprises exons 1 ± 7 of EWS and exons 8 ± 10 malignant cells (Resnico et al., 1995; Werner and of WT1. We have previously reported that the WT1 LeRoith, 1997). Transcription of the IGF-I-R gene is protein represses the expression of the IGF-I receptor negatively regulated by a number of tumor suppres- gene, whereas the EWS(1 ± 7)-WT1(8 ± 10) fusion protein sors, including p53, BRCA1 and WT1 (Maor et al., activates IGF-I receptor gene expression. It has recently 2000; Ohlsson et al., 1998; Werner et al., 1995, 1996a). become apparent that EWS-WT1 chimeras produced in Inhibitory control of the IGF-I-R gene, with ensuing DSCRT are heterogeneous as a result of fusions of reduction in the levels of IGF cell-surface binding sites, dierent regions of the EWS gene to the WT1 gene. We has been postulated to keep the receptor in its have recently characterized additional EWS-WT1 trans- `nonmitogenic' mode, thus preventing progression locations that involve the juxtaposition of EWS exons 7 through the cell cycle (Resnico et al., 1995). or 8 to WT1 exon 8, and an EWS-WT1 chimera that The product of the WT1 Wilm's tumor suppressor lacks EWS exon 6. The chimeric transcription factors gene is a nuclear protein of 52 ± 54 kDa that contains encoded by these various translocations dier in their four zinc ®ngers of the C2H2 class in its C terminus DNA-binding characteristics and their ability to trans- (Rauscher, 1993). This domain binds target DNAs that activate the IGF-I receptor promoter. These data suggest contain versions of the consensus sequence that the molecular pathology of DSRCT is more GCGGGGGCG. We have previously shown that the complex than previously appreciated, and that this WT1 protein binds to multiple sites in the IGF-I-R diversity may provide the foundation for predictive promoter and suppresses the activity of cotransfected genotype-phenotype correlations in the future. promoter fragments, as well as the endogenous IGF-I-R Oncogene (2002) 21, 1890 ± 1898. DOI: 10.1038/sj/ gene (Tajinda et al., 1999; Werner et al., 1994b, 1996b). onc/1205042 The ®ndings that the IGF-I-R gene is highly expressed in Wilms' tumors (which in many cases are associated Keywords: DSRCT; IGF-I receptor; EWS; WT1; trans- with mutations in the WT1 gene), and that the growth location of Wilms' tumor heterotransplants in nude mice is inhibited by anti-IGF-I-R antibodies, suggest that the IGF-I-R gene constitutes a bona ®de molecular target of Introduction WT1 action (Gansler et al., 1989; Werner et al., 1993). Tumor-speci®c chromosomal translocations that The insulin-like growth factor-I receptor (IGF-I-R) is a disrupt the molecular architecture of transcription membrane-bound heterotetramer that mediates the factors have emerged as a common theme in oncogen- trophic and dierentiative actions of IGF-I and IGF- esis (Rabbitts, 1994). As a result of these rearrange- II (Cohick and Clemmons, 1993; LeRoith et al., 1995; ments, chimeric proteins are generated that are composed of modules derived from unrelated genes. The EWS gene is a member of the TET gene family (Plougastel et al., 1993). The products of this gene *Correspondence: H Werner; E-mail: [email protected] family are RNA-binding proteins of unknown physio- 4Current address: Agritope, Inc., Beaverton, OR 97008, USA Received 19 March 2001; revised 30 September 2001; accepted 9 logical function that contain potential transcriptional October 2001 activation domains in their N termini (Ohno et al., EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1891 1994). The EWS gene is involved in a number of tion of the IGF-I-R promoter by various EWS-WT1 translocation events that are hallmarks of a variety of isoforms. speci®c cancers (Ladanyi, 1995). Ewing's sarcoma is associated with a t(11 : 22)(q24;q12) translocation that fuses the 5' exons of the EWS gene on chromosome 22 Results to the 3' exons of the FLI-1 gene (Zucman et al., 1993b; Delattre et al., 1992; Jeon et al., 1995). Fusion Reverse transcription-polymerase chain reaction (RT ± of the EWS and ATF-1 genes is characteristic of soft PCR) of DSRCT-derived RNA samples, followed by tissue clear cell sarcoma or malignant melanoma of direct sequencing of PCR products, allowed recently soft parts (Zucman et al., 1993a). EWS-CHN gene one of us to identify a number of novel breakpoint fusions have been reported in human myxoid chon- regions in fusion transcripts (Liu et al., 2000; Figure 1). drosarcoma (Clark et al., 1996; Gill et al., 1995). In addition to the previously described and most Although these fusions exhibit heterogeneity with frequently detected chimeric gene (present in 12 out of respect to the position of the chromosomal breakpoints 14 tumors) resulting from the in-frame fusion of exons (Kuroda et al., 1995; Zucman et al., 1992), a common 1 ± 7 of EWS to exons 8 ± 10 of WT1 (7/8), a novel feature is the fusion of the N-terminal transcription isoform in which EWS exon 8 is fused to WT1 exon 8 activation domain of EWS to either the full-length (8/8) was identi®ed in one tumor. A 6-bp heterologous sequence or the C-terminal DNA-binding domain of sequence inserted at the junction and a 4-bp deletion of any of a number of transcription factors. Antisense the 5' end of WT1 exon 8 forms an in-frame fusion of and antibody inhibition of the chimeric EWS-FLI-1 the EWS and WT1 ORFs. An additional novel variant and EWS-ATF1 products reduces the tumorigenicity resulting from the 7/8 fusion, but lacking EWS exon 6 and viability of Ewing's and clear cell carcinoma cells, (7/8D6), was isolated from four tumors. Since WT1 respectively (Bosilevac et al., 1999; Ouchida et al., exon 9 is subject to alternative splicing, DSRCT 1995), implicating the altered transcriptional activity of samples express isoforms that either include or lack these chimeric proteins in tumor development. the KTS insert. Desmoplastic small round cell tumor (DSRCT), a To verify that the expression constructs to be utilized very aggressive primitive tumor of children and young in functional assays of transcriptional activity and in adults, particularly males (Gerald et al., 1991; DNA-binding experiments encoded proteins of the Gonzalez-Crussi et al., 1990), is a recently described expected size, synthetic RNAs were generated by in malignancy that is also characterized by a recurrent vitro transcription of pcDNA3 constructs and used to translocation involving the EWS gene. The distinctive program rabbit reticulocyte lysates for in vitro hallmark of DSRCT is the t(11;22)(p13;q12) transloca- translation in a coupled system. As shown in Figure tion (Sawyer et al., 1992) that fuses the N-terminal 2, the apparent molecular weight of the 35S-methionine- (activation) domain of EWS to the C-terminal (DNA- labeled EWS-WT1(7/8+KTS) protein was *56 ± binding) domain of WT1, including the three terminal 58 kDa, the size of the EWS-WT1(8/8+KTS) protein zinc ®ngers (Brodie et al., 1995; Gerald et al., 1995; was *60 ± 62 kDa, and the size of the EWS-WT1(7/8 Ladanyi and Gerald, 1994; Rauscher et al., 1994). D67KTS) was *48 ± 50 kDa. Although the sizes of Consistent with the postulate that the EWS-WT1 the in vitro-translated proteins were slightly larger than fusion protein may modulate transcription of target expected, similar discrepancies have been reported by genes containing WT1 binding motifs, we have others (Kim et al., 1998). previously shown that EWS(1 ± 7)-WT1(8 ± 10) can To establish whether WT1 binding sites located in recognize and transactivate the IGF-I-R promoter in the 5'-¯anking region of the IGF-I-R gene are involved transient transfection assays (Karnieli et al., 1996). in EWS-WT1 protein binding, electrophoretic mobility More recently, we and others have shown that the shift assays (EMSA) were performed using a labeled t(11;22)(p13;q12) translocation is also a heterogeneous promoter fragment extending from 7331 to 740. This event that may involve several alternative breakpoints DNA region was previously shown to bind EWS- (Antonescu et al., 1998; Chan et al., 1999; Liu et al., WT1(7/8 7KTS)-derived recombinant proteins con- 2000; Shimizu et al., 1998). Speci®cally, EWS-WT1 taining the 21-amino acid fragment of EWS located N- chimeras have been described in which exons 7, 8, 9 or terminal to the fusion point and most of the C-terminal 10 of the EWS gene are fused to exon 8 of the WT1 domain of WT1, including zinc ®ngers 2 ± 4 (Karnieli et gene. We have also identi®ed an additional EWS exon al., 1996). To assess the potential contribution of the 7-WT1 exon 8 fusion that lacks EWS exon 6 (Liu et dierent N-terminal EWS-derived domains (i.e., exons al., 2000). As a result of the alternative splicing of 1 ± 7, exons 1 ± 7 D6, or exons 1 ± 8) on DNA binding, exon 9 that occurs in the WT1 gene (Haber et al., full-length in vitro-translated proteins were employed. 1991), each of these fusion proteins may include or As shown in Figure 3, incubation of the labeled DNA lack a Lys-Thr-Ser (KTS) insert located between zinc fragment with the full-length EWS-WT1(7/8 D67KTS) ®ngers 3 and 4 of the WT1 portion of the chimera. fusion protein generated one retarded band. Incubation This diversity suggests that the etiology of DSRCT is with the EWS/WT1(7/87KTS) protein resulted in the much more complex than originally suspected. In light formation of three retarded bands, whereas the 7/8 of the important role of the IGF-I-R in tumorigenesis, isoform containing the KTS insert (EWS/WT1(7/ we have investigated the potential dierential regula- 8+KTS)) did not generate any retarded band.

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1892

Figure 1 Schematic representation of EWS, WT1, and EWS-WT1 proteins. The EWS gene is located at 22q12 and includes 17 exons. The EWS gene product is a 656-amino acid protein that includes an N-terminal activation domain (AD, dotted) and an RNA-binding domain (RBD, black). Exon 6, which is missing in the EWS-WT1(7/8 D67KTS) fusion protein, is boxed. The WT1 gene, located at 11p13, includes 10 exons and encodes a 429-amino acid protein. The WT1 protein includes an N-terminal repression domain (RD, dashed) and a DNA-binding domain (DBD) comprised of four zinc-®nger motifs. Alternative splicing of exon 9 results in WT1 molecules containing or lacking a Lys-Thr-Ser (KTS) insert between zinc ®ngers 3 and 4. DSRCT is characterized by a recurrent t(11;22)(p12;q12) translocation that fuses either exons 1 ± 7 or 1 ± 8 of the EWS gene to exons 8 ± 10 of the WT1 gene. As a result of the ®rst rearrangement, EWS-WT1(7/8+KTS) fusion proteins including or lacking the KTS insert are generated. An additional fusion protein that results from the same translocation lacks EWS exon 6 (EWS-WT1(7/8 D67KTS)). As a result of the second event, EWS-WT1(8/8+KTS) fusion proteins are generated. Arrowheads indicate breakpoints in the EWS and WT1 genes. Exon numbers of the EWS and WT1 genes are shown above the protein diagrams

Similarly, no binding was seen with the EWS/WT1(8/ fected with a series of expression vectors encoding 8+KTS) chimeric proteins. Band formation was EWS-WT1(7/8+KTS), EWS-WT1(8/8+KTS), and abrogated when the binding reactions were performed EWS-WT1(7/8 D67KTS), together with a reporter in the presence of a *200-fold molar excess of construct [p(7476/+640)LUC] containing most of the unlabeled probe (data not shown). In addition, proximal region of the IGF-I-R promoter upstream of potential DNA binding of the various chimeric a luciferase reporter gene. This promoter fragment has proteins was assessed using a labeled genomic DNA a high G-C content (475%), and, using DNase I fragment extending from nucleotide 740 to +115 footprinting assays with the expressed zinc-®nger (including the IGF-I-R `initiator' element), and a domain of WT1, we have previously mapped 12 WT1 double-stranded synthetic oligonucleotide extending binding sites in this region (Werner et al., 1994b). from nucleotide 7382 to 7354 (including three Sp1 As shown in Figure 4a, cotransfection of Saos-2 cells binding sites). No speci®c binding was observed to with 2.5 mg of the EWS-WT1(7/8 D67KTS) isoform either of these DNA fragments (data not shown). resulted in the strongest transactivation (617+172% of We then evaluated the ability of the various EWS- control values). Consistent with our previous report WT1 isoforms to regulate the activity of the IGF-I-R (Karnieli et al., 1996), the activity of the EWS-WT1(7/ promoter in transient transfection assays. For this 8+KTS) protein was signi®cantly lower than that of purpose, osteosarcoma-derived Saos-2 cells and malig- the 7KTS isoform (EWS-WT1(7/87KTS)) (172+ nant rhabdoid tumor-derived G401 cells were cotrans- 65% vs 481+147% stimulation). Interestingly, both

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1893

Figure 3 Dierential binding of EWS-WT1 isoforms to the Figure 2 In vitro transcription/translation analysis of EWS-WT1 IGF-I-R promoter. The promoter region extending from 7331 to expression vectors. T7 RNA polymerase-driven in vitro transcrip- 740 (containing ®ve WT1 binding sites) was end-labeled with tion reactions were performed using the TNT1 T7 Quick Coupled g-32P-ATP in the presence of T4 polynucleotide kinase, and Transcription/Translation System (Promega). Each reaction employed in gel-shift assays with equal amounts of the dierent in included 1 mg of each of the following expression vectors: empty vitro-translated EWS-WT1 isoforms (or with control pcDNA3 pcDNA3, EWS-WT1(7/8 D67KTS), EWS-WT1(7/8+KTS), and reaction product). Binding reactions were performed as indicated EWS-WT1(8/8+KTS). In vitro translation reactions were per- in the Materials and methods section. Arrows denote the formed in the presence of 35S-labeled methionine using rabbit positions of three DNA-protein complexes that result from the reticulocyte lysates. One ml of each translation reaction (out of a binding of the EWS-WT1(7/87KTS) protein to the IGF-I-R total volume of 50 ml) were electrophoresed through 8% SDS ± promoter. The dotted arrow shows the position of a DNA ± EWS- PAGE gels and exposed for 20 h to Kodak X-Omat ®lm. The WT1(7/8 D67KTS) complex sizes of prestained molecular weight markers (Sigma) are indicated

plasmid (199+28% vs 617+172% of controls, respec- EWS-WT1(8/8+KTS) isoforms stimulated promoter tively). No further reduction was seen with the p(740/ activity to a similar extent (326+50% vs 326+84%). +640)LUC construct (221+42% of control) (Figure As expected, cotransfection of a WT1 expression vector 5b). Likewise, the stimulatory eect of EWS-WT1(7/8 consistently suppressed promoter activity (data not D67KTS) in G401 cells was completely abrogated shown). On the other hand, only the EWS-WT1(7/8 when the reporter plasmids p(7188/+640)LUC and D67KTS) isoform exhibited transactivational activity p(740/+640)LUC were transfected (Figure 5c). in G401 cells (329+12% of controls) (Figure 4b). To examine the physiological relevance of the To more precisely map the promoter region transactivation eect of EWS-WT1 chimeras on the responsible for mediating the eect of EWS-WT1, IGF-I-R promoter, we measured the levels of endo- cotransfections were performed using the reporter genous IGF-I-R protein following transient transfec- plasmids p(7188/+640)LUC and p(740/+640)LUC tions using Western blot analysis. Saos-2 cells were (Figure 5a), along with the EWS-WT1(7/8 D67KTS) transfected with 6 mg of the EWS-WT1(7/8 D67KTS) expression vector. Construct p(7188/+640)LUC lacks expression vector, and after 24, 48 and 72 h, cells were four WT1 consensus sites (located at 7262/7254, lysed as described in the Materials and methods 7250/7242, 7220/7212, and 7196/7188) that we section. Cell lysates (50 mg protein) were electrophor- have previously shown in DNaseI footprinting assays esed through 10% SDS ± PAGE gels, blotted onto to bind a recombinant WT1 zinc-®nger domain with nitrocellulose membranes, and incubated with an anti- relatively high anity (Werner et al., 1994b). Construct IGF-I-R b-subunit antibody. Western analysis of p(740/+640)LUC lacks, in addition, a WT1 cis- lysates of transfected cells demonstrated a 1.7-fold element at position 7163/7155 that binds WT1 with increase in the levels of the IGF-I-R at 72 h after medium anity. The transactivation activity of the transfection (Figure 6). EWS-WT1(7/8 D67KTS) protein on the p(7188/ Finally, triple cotransfections were performed using +640)LUC construct in Saos-2 cells was signi®cantly pCB6+-derived expression vectors encoding EWS- reduced in comparison to the p(7476/+640)LUC WT1(7/87KTS) and WT1, together with the

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1894

Figure 5 Mapping of EWS-WT1 target regions of the IGF-I-R promoter. (A) Schematic representation of reporter constructs. Plasmids p(7476/+640)LUC, p(7188/+640)LUC, and p(740/ +640)LUC contain, respectively, 476, 188, or 40 bp of 5'-¯anking region (open bar), and 640 bp of 5'-untranslated region (closed bar), linked to a luciferase cDNA (LUC). The arrow indicates the transcription initiation site. Triangles denote the location of WT1 binding sites (7262/7254, 7250/7242, 7220/7212, and 7196/ 7188) that were footprinted by the puri®ed zinc-®nger domain of WT1 with relatively high anity. The site at 7163/7155 (denoted by a circle) was shown to bind WT1 with medium anity. The luciferase cDNA is not shown to scale. Saos-2 (B) and G401 (C) cells were cotransfected with the indicated reporter plasmid, along with the EWS-WT1(7/8 D67KTS) and b- galactosidase expression plasmids, as described in the legend to Figure 3 and in the Materials and methods section. The luciferase Figure 4 Stimulation of IGF-I-R promoter activity by EWS- values, normalized for b-galactosidase (closed bars), are expressed WT1 isoforms. Five micrograms of the p(7476/+640)LUC as a percentage of the activity seen in the absence of expression reporter plasmid (shown in Figure 5A) were cotransfected into vector (open bars). Results of transfections in Saos-2 (B) are Saos-2 cells (A) with 2.5 mg of each of the EWS-WT1 expression mean+s.e.m. (n=3 experiments, performed each one in dupli- vectors (or empty pcDNA3) and 2.5 mg of the pCMVb plasmid cate). (C) (G401) shows the results of a representative experiment, using the calcium phosphate method. G401 cells (B) were performed in duplicate. Note the dierences in scale in the y axis cotransfected with 0.6 mg of reporter plasmid, 0.2 mgof between panels B and C expression vector, and 0.2 mg of the pCMVb plasmid using the Fugene-6 Reagent (Roche Molecular Biochemicals). After 40 h, cells were harvested and the levels of luciferase and b- galactosidase activities were measured. Luciferase values, normal- p(7476/+640)LUC reporter plasmid. The rationale ized for b-galactosidase, are expressed as a percentage of the for these experiments was the fact that tumor cells luciferase activity of the empty pcDNA3 expression vector. containing the t(11;22)(p13;q12) translocation still Experiments were performed between three and ®ve times retain one normal allele encoding wild-type WT1. (Saos-2), and two times (G401), each time in duplicate. Bars are mean+s.e.m. Where not shown, the s.e.m. bars are smaller than Because WT1 was previously shown to bind to the symbol size consensus cis elements in the IGF-I-R promoter with

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1895

Figure 6 Eect of EWS-WT1 expression on endogenous IGF-I- R gene expression. Saos-2 cells were transfected with 6 mg of the EWS-WT1 (7/8 D67KTS) expression vector (or empty pcDNA3) using the Fugene 6 Reagent. Cells were collected after 24, 48, and 72 h as indicated in the Materials and methods section. Equal amounts of protein (50 mg) were separated by 10% SDS- polyacrylamide gel electrophoresis, transferred to nitrocellulose ®lters and blotted with an anti-human IGF-I-R antibody an anity similar to that displayed by the fusion protein, it was important to assess potential functional interactions between WT1 and EWS-WT1 in transcriptional control of the IGF-I-R gene. As shown in Figure 7, cotransfection of EWS-WT1(7/87KTS) attenuated the repression of the IGF-I-R promoter seen with WT1 Figure 7 Functional interactions between EWS-WT1 and WT1 proteins in regulation of IGF-I-R promoter activity. Cotransfec- alone. tions were performed in Saos-2 cells using 5 mg of the p(7476/ +640)LUC plasmid and 0.2 mg pCMVb, together with 2.1 mgof pCB6+DNA, 0.1 mg of the pCB6+(WT1-KTS), 2.0 mgof Discussion pCB6+(EWS-WT1(7/87KTS)), or both expression vectors. Total DNA amount transfected was kept constant with pCB6+. The values of luciferase normalized for b-galactosidase DSRCT is a recently described primitive tumor that are expressed as a percentage of the activity seen with the pCB6+ occurs most frequently in adolescent males and is control DNA. The results of a typical experiment repeated at least mainly localized in the abdomen. As is the case with four times are presented Wilms' tumors, pathological analyses of DSRCT samples revealed the presence of epithelial, mesenchy- mal, and neural-derived cells. The distinctive hallmark negatively controlled by a number of tumor suppres- of DSRCT is a recurring t(11;22)(p13;q12) chromoso- sors, including WT1. In the present study, we have mal translocation that fuses the N-terminal domain of expanded our previous observations showing that the the EWS gene in frame to the C-terminal, DNA- IGF-I-R gene is a molecular target for the EWS- binding domain of WT1. The chimeric protein that WT1(7/8) protein, and that the transactivating eect of results from this rearrangement, EWS-WT1, is an the fusion protein involves binding to WT1-related example of a growing family of aberrant transcription sites in the IGF-I-R promoter (Karnieli et al., 1996). factors harboring domains encoded by unrelated genes. Speci®cally, the recent ®nding that alternative break- In general, these disrupted transcription factors display points in the EWS and WT1 genes are involved in the gain-of-function activity that abrogates the biological t(11;22)(p13;q12) translocation suggested that the role of each one of the parental genes, resulting in an molecular basis underlying DSRCT pathology may, oncogenic role for the chimeric protein. In the in fact, be more complex than originally appreciated, in particular case of EWS-WT1, the translocation event that variant translocation products may display seems to abolish the RNA-binding activity of EWS as dierent transactivating activities. The results of this well as the transcriptional repression activity of WT1. study are in accord with this concept by demonstrating Furthermore, the EWS-WT1(7/87KTS) protein ex- that EWS-WT1 isoforms stimulated IGF-I-R promoter hibits oncogenic activity as determined by formation of activity to dierent extents and, additionally, exhibited transformed foci in cell monolayers, anchorage-inde- distinct DNA-binding properties. pendent growth, and tumor formation in nude mice. One of us has recently reported that EWS- The EWS-WT1(7/8+KTS) protein, on the other hand, WT1(7KTS) isoforms signi®cantly activated an arti®- exhibited no transforming potential (Kim et al., 1998). cial promoter containing three tandem copies of the The involvement of the IGF system in the etiology early growth response (EGR) binding site (EBS), as of a number of solid tumors, including Wilms' tumor, well as the activity of the EGR-1 promoter (Liu et al., rhabdomyosarcoma, and others, has been well estab- 2000). These activities correlated with the speci®c lished. The main ligand in most of these malignancies binding of the 7KTS forms of EWS-WT1 to labeled appears to be IGF-II, whose mitogenic and prolif- EBS oligonucleotides. The results of the present study erative activity is mediated by the IGF-I-R (El-Badry extend the spectrum of potential targets of EWS-WT1 et al., 1990). The expression of the IGF-I-R gene is isoforms to a gene encoding a growth factor receptor

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1896 whose involvement in the etiology of a large number of appears to bind to at least three sites). On the other pediatric tumors is well established. In addition, our hand, the presence of EWS exon 8 had a negative ®ndings with the complex IGF-I-R promoter (which impact on the DNA-binding activity and transactivat- contains 12 WT1/EBS binding sites in both the 5'- ing eect of 8/8 isoforms, probably as a result of steric ¯anking and 5'-untranslated regions, in comparison to interference of exon 8-encoded sequences with promo- only one site in the EGR-1 promoter) dier from those ter binding. In addition, the ®nding that the EWS- obtained by Liu et al. (2000) and therefore suggest that WT1(7/8 D67KTS) isoform induced an increase in EWS-WT1 isoforms activate target promoters of levels of endogenous IGF-I-R protein strongly suggests dierent complexities via distinct mechanisms. that the IGF-IR gene is a physiological target of EWS- The EWS-WT1(7/8 D67KTS) chimera exhibited the WT1 chimeric proteins. strongest transactivation activity (*6.2-fold stimula- In the pathophysiological context of DSRCT, EWS- tion above control), and this eect was associated with WT1 fusion proteins are presumably functioning in the its speci®c binding to a promoter fragment extending presence of wild-type WT1 encoded by the untranslo- from nucleotides 7331 to 740 that includes four high- cated allele. The results of triple cotransfection anity WT1 sites (7262/7254, 7250/7242, 7220/ experiments suggest that EWS-WT1 chimeras can 7212, and 7196/7188) and one medium-anity site attenuate the repression of the IGF-I-R gene exerted (7163/7155). Because the stimulatory eect of EWS- by intact WT1. This interference with normal WT1 WT1 (7/8 D67KTS) was abrogated when a promoter function, along with WT1 haploinsuciency and fragment lacking the four high-anity sites was potential gain-of-function eects of the EWS-WT1 employed, and since EMSA data demonstrated the chimeras, may all contribute to the etiology of formation of a single retarded band, we infer that only DSRCT. one site in the cluster of high-anity WT1 elements In conclusion, our results demonstrate that the IGF- was responsible for this eect. Furthermore, the fact I-R gene is a target for a novel family of EWS-WT1 that no further reduction in transcriptional activity was fusion proteins, and that the binding activities of EWS- seen with the p(740/+640) LUC construct in WT1 isoforms are not only determined by the WT1 comparison to p(7188/+640)LUC suggests that the component of the chimera, as usually believed, but medium-anity WT1 site at position 7163/7155 had may also be aected to a large extent by the particular no major eect. EWS-derived exons that are retained in the transloca- A strong stimulatory eect was also displayed by the tion product. The contribution of this variability in EWS-WT1 (7/87KTS) chimera (*4.8-fold stimulation EWS-WT1 translocation products to heterogeneity in above control), but not by the 7/8 isoform that DSRCT remains to be established. includes the KTS insert. These dierences in the results of functional assays correlated with marked dierences in DNA-binding patterns. Thus, whereas the 7KTS isoform generated three retarded bands in EMSA, no Materials and methods discernible binding was displayed by the+KTS isoform. Furthermore, we have previously shown that, Construction of EWS-WT1 expression vectors unlike the exon 6-deleted isoform, the exon 6-contain- EWS-WT1/pcDNA3 expression plasmids were constructed by ing chimera retained its stimulatory eect towards the excising EcoRI ± XbaI fragments from previously described p(7188/+640)LUC, but not towards the p(740/ pAMP vectors (Liu et al., 2000) and ligating these fragments +640)LUC region (Karnieli et al., 1996). These results into the corresponding EcoRI ± XbaI sites in the multiple suggest that the medium-anity site at position 7163/ cloning site of pcDNA3 (Invitrogen). With the exception of 7155 is important for the action of the EWS-WT1(7/ the 8/8+KTS fusion, this positioned the gene in the sense 87KTS) protein. Finally, both EWS-WT1(8/8) var- orientation under the in¯uence of the CMV promoter. The iants had a relatively lower transactivating eect 8/8+KTS cDNA was originally cloned in the opposite orientation in the pAMP vector, so a HindIII ± SmaI (*3.3-fold stimulation), regardless of the presence or fragment was isolated and ligated into the HindIII and absence of the KTS insert. Accordingly, we were EcoRV sites of pcDNA3. All pcDNA3 constructs were unable to detect any speci®c DNA binding by either sequenced with both T7 and Sp6 primers to ensure their 8/8 isoform. It is possible that the mechanism of action proper orientation. underlying the transactivating eect of particular EWS- WT1 isoforms involves protein ± protein interactions Cell cultures and transfections with cell-type speci®c DNA-binding or non-binding transcription factors. This possibility is underscored by The human osteogenic sarcoma-derived cell line Saos-2 and the dierent transactivating activities displayed by the the human malignant rhabdoid tumor cell line G401 were various isoforms in Saos-2 and G401 cells. An obtained from the American Type Culture Collection (Rock- ville, MD, USA). Saos-2 cells were grown in Dulbecco's alternative mechanism of action may involve binding modi®ed Eagle's medium and G401 cells were cultured in to another region of the IGF-IR promoter. McCoy's 5A medium. Media were supplemented with 10% Taken together, the results of this study show that fetal bovine serum, 2 mM glutamine, and 50 mg/ml gentami- the lack of the EWS exon 6-encoded sequence results cin sulfate. Both cell lines were previously shown to express in speci®c binding to a unique high-anity site (in signi®cant amounts of IGF-I-R mRNA and to support comparison to the exon 6-containing isoform that transcription of IGF-I-R promoter-driven reporter constructs

Oncogene EWS-WT1 regulation of the IGF-I receptor gene I Finkeltov et al 1897 (Ohlsson et al., 1998; Werner et al., 1995). Furthermore, the 1mM DTT. Protein content of the lysates was determined activity of IGF-I-R promoter constructs in G401 cells was using the Bradford reagent (Bio-Rad, Hercules, CA, USA) shown to be strongly suppressed by cotransfection of a WT1 using BSA as a standard. Samples were subjected to 10% expression plasmid (Werner et al., 1995). SDS ± PAGE, followed by electrophoretic transfer of the The p(7476/+640)LUC, p(7188/+640)LUC, and p(740/ proteins to nitrocellulose membranes. Membranes were +640)LUC IGF-I-R promoter-pOLUC constructs used in blocked with 3% BSA in T-TBS (20 mM Tris-HCl, pH 7.5, transient transfection assays have been described previously 135 mM NaCl and 0.1% Tween-20). Blots were incubated (Karnieli et al., 1996). Cells were seeded in 6-well plates and with a rabbit polyclonal anti-human IGF-I-R b-subunit transfected using the calcium phosphate method (Saos-2) or antibody (Santa Cruz Biotechnology, Santa Cruz, CA, the Fugene-6 Reagent (Roche Molecular Biochemicals, USA; 0.2 mg/ml), washed extensively with T-TBS, and Indianapolis, IN, USA) (G401). Saos-2 cells received 5 mg incubated with an HRP-conjugated secondary antibody. of reporter plasmid and 2.5 mg of expression vector (or Proteins were detected using the SuperSignal1 West Pico empty pcDNA3), along with 2.5 mgofab-galactosidase Chemiluminescent Substrate (Pierce, Rockford, IL, USA). expression plasmid (pCMVb, Clontech, Palo Alto, CA, USA). G401 cells were transfected with 0.6 mg of reporter Electrophoretic mobility shift assays plasmid, 0.2 mg of expression vector, and 0.2 mg of pCMVb. Cells were harvested 40 h after transfection, and luciferase A DNA fragment extending from nucleotides 7331 to 740 and b-galactosidase activities were measured as previously of the IGF-I-R 5'-¯anking region was isolated by digestion of described (Werner et al., 1992). Promoter activities were an IGF-I-R genomic clone with XmaI and PmlI. Following expressed as luciferase values normalized for b-galactosidase digestion, the 7331/740 fragment was gel puri®ed and end- activity. labeled with g-32P-ATP using T4 polynucleotide kinase. Triple cotransfections were performed using the p(7476/ Binding reactions were performed in a total volume of +640)LUC reporter vector together with EWS-WT1 (7/ 10 ml for 30 min at 48Cin20mM HEPES, pH 7.5, 70 mM 87KTS) and WT1(7KTS) (pCMVhWT) expression vectors KCl, 12% glycerol, 0.05% Nonidet P-40, 100 mM ZnSO4, in the pCB6+plasmid (Werner et al., 1993). The total DNA 50 mM dithiothreitol, 5 mM MgCl2, 1 mg/ml bovine serum amount transfected was kept constant with empty pCB6+ albumin, 0.1 mg/ml poly(dI:dC), 75 000 d.p.m. (0.15 ± plasmid. The pCB6+ derived expression vectors were 0.75 ng) of the labeled fragment, and 5 ml of the in vitro provided by Dr Frank J Rauscher III (The Wistar Institute, translated EWS-WT1 proteins. Changes in mobility were PA, USA). assessed by electrophoresis through native 5% polyacrylamide gels that were run at 120 V for 2 h in 0.56TBE. After ®xation in 10% acetic acid, gels were autoradiographed at In vitro transcription and translation reactions 7708C. Coupled in vitro transcription/translation of the various EWS-WT1 fusion proteins was performed using the TNT1 T7 Quick-Coupled Transcription/Translation System (Pro- Abbreviations mega, Madison, WI, USA). Brie¯y, T7 RNA polymerase- IGF-I-R, insulin-like growth factor-I receptor; DSRCT, driven in vitro transcription reactions were followed by in desmoplastic small round cell tumor; EWS, Ewing's 35 vitro translations in the presence of S methionine (or Sarcoma; WT, Wilms' tumor; RT ± PCR, reverse transcrip- unlabeled methionine for EMSA) using rabbit reticulocyte tion polymerase chain reaction. lysates. In vitro translation products were electrophoresed through 8% SDS ± PAGE and exposed to Kodak X-Omat ®lm. Acknowledgments This work was performed in partial ful®llment of the Western immunoblotting requirements for the MSc degree by Ina Finkeltov, in the Transiently transfected cells were harvested with ice-cold PBS Sackler Faculty of Medicine, Tel Aviv University. HW is containing 5 mM EDTA and lysed in a buer composed of the recipient of a Guastalla Fellowship from the Rashi 150 mM NaCl, 20 mM HEPES, pH 7.5, 1% Triton X-100, Foundation, Israel. This work was supported by a grant 2mM EDTA, 2 mM EGTA, 1 mM PMSF, 2 mg/ml aprotinin, from the Israel Cancer Association to H Werner and by 1mM leupeptin, 1 mM pyrophosphate, 1 mM vanadate and NIH grant DK50810 to CT Roberts.

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Oncogene