WT1 Induces Expression of Insulin-Like Growth Factor 2 in Wilms' Tumor Cells'

ICANCER RESEARCH55, 4540-4543. October tS, 19951 Advances in Brief

WT1 Induces Expression of Insulin-like Growth Factor 2 in Wilms' Tumor Cells'

Kim E. Nichols, Gian G. Re, Yu Xin Yan, A. Julian Garvin, and Daniel A. Haber@

Laboratory of Molecular Genetics, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts 02129 (K. E. N., Y. X. Y., D. A. HI, and Department of Pathology and Li.iboratory Medicine, Medical University ofSouth Carolina, Qiarleston, South Carolina 1G. G. R., A. J. G.]

Abstract by the observation that naturally occurring point mutations arising within the transactivation domain of WT1 convert the encoded protein The Wilma' tumor suppressor gene WTJ encodes a zinc finger tran from a transcriptional repressor to a potent activator of the EGR1 scription factor, whose expression inhibits the growth of the RM1 Wilma' promoter in 3T3 cells (7, 16, 17). The nature of the recipient cells tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected themselves is an important factor in these transient transfection stud promoters containing the early growth response gene 1 consensus se ies. We have demonstrated recently that WT1 and p53 proteins are quence. We now show that WT1 has properties of a transcriptional associated in vivo and that transfection of WT1 into cells with a activator in RM1 cells, an effect that may be associated with the presence deleted p53 gene results in transcriptional activation, an effect that is of a mutated p53 gene In these cells. Stable transfection of wild-type WT1 suppressed by the reintroduction of wild-type p53 (18). Thus, the into RM1 cells results in induction of endogenous insulin-like growth transactivational properties of WT1 appear to be complex, involving factor2 (IGF2)butnotof otherpreviouslypostulatedWT1-targetgenes. specific domains within the protein itself, as well as potential inter The induction of IGF2 is dramatically enhanced by WT1 mutants encod actions with other cellular proteins. ing an altered transactivation domain. We conclude that IGF2 is a poten To define further the transactivational properties of WT1 in an tinily physiological target gene for WT1 and that its induction may appropriate cell type, we analyzed the effect of WT1 expression in the contribute to the growth-stimulating effects of WT1 variants. RM1 Wilms' tumor cell line. This cell line, derived from an anaplastic Introduction Wilms' tumor, has the unique property of indefinite passage in vitro, while retaining the ability to produce tumors with characteristic Wilms' tumor, a pediatric kidney cancer, has been linked to the Wilms' histology following inoculation into nude mice (7, 19). The inactivation of a tumor suppressor gene, WTJ, at the chromosome endogenous WT1 transcript in these cells is expressed at very low I lpl3 locus (reviewed in Ref. 1). WT1 encodes a Cys-His zinc finger levels and is comprised ofthe WT1/del2 variant, an aberrantly spliced protein that is normally expressed in cells of the developing genito transcript that is found in a subset of Wilms' tumors and encodes a urinary system (2â€”4).Mutations inactivating WT1 have been detected protein with altered transactivational activity (7). We now demon in â€”10% of sporadic Wilms' tumors (5, 6), and reintroduction of strate that transient transfection of WT1 into RM1 cells, which harbor wild-type WT1 into Wilms' tumor cells expressing an aberrant WT1 a p53 mutation, results in transcriptional activation of a reporter transcript results in growth suppression (7). WT1 has properties of a construct containing the EGR1 consensus site, rather than transcrip transcriptional repressor, based on transient transfection experiments tional repression. Stable transfection of wild-type WT1 in these cells in 3T3 and 293 cells (8). These properties are differentially mediated is associated with an increase in the expression of endogenous IGF2 by alternative splicing variants of wild-type WT1 (9). Whereas the but not of other postulated WT1 target genes with EGR1-containing four zinc finger domains of WT1 bind the 5'-CGCCCCCGC-3' DNA promoters. Transfection of naturally occurring WT1 variants encoding consensus sequence shared by the EGR13 gene product, insertion of an altered transactivation domain leads to a dramatic induction of an alternatively spliced KTS sequence between zinc fingers 3 and 4 endogenous IGF2 mRNA. These results suggest that only a subset of abolishes this DNA-binding activity (10). EGR1-containing, WTI-responsive promoters may be physiologically The EGR1 consensus is found upstream of many transcriptional regulated by WT1. Although we do not detect transcriptional repres start sites, leading to the identification of a number of promoters that sion of endogenous IGF2 by wild-type WT1, the potent induction of bind in vitro-translated WT1 and are repressed in transient trans this growth-inducing gene by naturally occurring WT1 mutants fection assays. WT1 has, therefore, been identified as a potential suggests that these altered proteins may actively contribute to cell transcriptional repressor of genes such as EGR1 (8), IGF2 (1 1), transformation. insulin-like growth factor 1 receptor (12), PDGF-A (13, 14), colony stimulating factor 1 (15), among others. Although transcriptional Materials and Methods suppression of the endogenous genes themselves has not been dem Culture of RM1 Cells. The generation of RM1 cells (also called W4) has onstrated, these experiments have led to the concept that the tumor been described elsewhere (19). RM1 cells were maintained as tumor explants suppressor properties of WT1 might result from its ability to reduce in nude mice by serial s.c. passage. For growth in vitro, tumors were minced expression of growth-inducing gene products. This model is supported and adapted to growth on a collagen matrix and then on standard tissue culture plates and grown in DMEM, 10% FCS, and supplemental glutamine. Sequence Received 7/6/95; accepted 8/30/95. analysis of the WTI transcript has been described previously (7). For p53 The costs of publication of this article were defrayed in part by the payment of page mutational analysis, the p53 transcript was reverse transcribed using random charges. This article must therefore be hereby marked advertisement in accordance with oligonucleotide primers and subjected to PCR amplification using primers 18 U.S.C. Section 1734 solely to indicate this fact. spanning the coding region. The amplified eDNA was analyzed by automated I This grant was supported by NIH Grants CA 37887 (to A. i. G.) and CA 58596 (to D. A. H.). sequencing (ABI), and the mutation observed was confirmed by sequencing 2 To whom requests for reprints should be addressed, at Massachusetts General multiple independent clones. Hospital Cancer Center, CNY 7, Building 149, 13th Street, Charlestown, MA 02129. CAT Assays. To test the transactivational properties of WT1 in RM1 cells, Phone: (617) 726-7805; Fax: (617) 726-5637. cells were transfected by calcium phosphate DNA precipitation with CMV 3 The abbreviations used are: EGRI, early growth response gene 1; IGF2, insulin-like growth factor 2; PDGF, platelet-derived growth factor; CAT, chloramphenicol acetyl driven constructs encoding murine WT1 (with or without the KTS alternative transferase; CMV, cytomegalovirus; RT, reverse transcription. splice), along with the p3X-EGR1-CAT reporter, containing three tandem 4540

a) EGRI consensus sites upstream of a minimal promoter (10). The total amount (1) of CMV promoter sequence transfected into the cells was equalized with the Ca) 1@ Cd 0 addition of vector DNA, and transfection efficiency was standardized using a cnm@ ci) cotransfected human growth hormone reporter construct (Nichols Institute). @zzz CAT assays were performed by the method of Gorman et a!. (20), and CAT activity was quantitated by scintillation counting of appropriate sections of the TLC plate. Northern Analysis. Total cellular RNA was prepared by extraction with guanidine isocyanate and electrophoresed on a 1% agarose/formaldehyde gel, wT1-j'. followed by transfer to a Genescreen membrane (Amersham). Northern hy bridizations were performed under standard conditions, using eDNA probes for v5rr1, IGF2, EGR1, PDGF-A, Pax 2, and phosphoglutaraldehyde dehydrogen ase (PGAD). Results IGF2 Transcriptional Activation of a WT1-Target Promoter in RM@ Cells. To examine transcriptionalregulationby WT1 in Wilms' tu mor cells, we transfected CMV-driven expression constructs into RM1 cells, along with a reporter containing a WT1 target site. WT1 lacking the KTS alternative splice, WT1(â€”KTS), has been shown to repress transcription from a promoter containing three tandem EGR1 PGAD â€” consensus sites (p3X-EGR1-CAT) by 2â€”3-fold,following transfection into 3T3 cells (10). In contrast, transient transfection of WT1(â€”KTS) into RM1 cells resulted in 5-fold transcriptional activation of this Fig. 2. Induction ofendogenous IGF2 mRNA in RM1 cells expressing WT1. Northern reporter. Insertion of the KTS splice within the WT1 zinc finger blot of total mRNA from four independent RMI-derived clones, stably transfected with domain, which abrogates binding to the EGR1 consensus, also re antisense WT1, Wi'! lacking the KTS splice (NB! and NB2), and vector alone (Neo), and grown as explants in nude mice. Blots were hybridized with probes for WT1, IGF2, and duced transactivation of the reporter (Fig. 1). PGAD (loading control). The transactivational properties of WT1 appear to be modulated by a number of factors, including promoter structure, cellular context, and the status of p53 (18, 21). To determine the sequence of endog with a role in tumor progression (22, 23).@The absence of wild-type enous p53 in RM1 cells, we amplified the endogenous p53 transcript p53 in RM1 cells may affect the transactivational properties of WT1, by RT-PCR, followed by direct automated sequencing of the ampli as demonstrated for fibroblasts expressing a temperature-sensitive p53 fled product. A homozygous single base deletion of thymidine at allele (18). codon 212 was noted, leading to a frame shift and premature termi Induction of IGF2 by Wild-Type WT1 in RM1 Cells. Although nation at codon 246. p53 mutations in Wilms' tumors are rare (esti WT1 has been reported to repress transcription from a number of mated at <5%), and an association between these mutations and promoters containing the EGR1 consensus site, it has not been shown histological anaplasia has been suggested by recent studies, consistent to alter the expression of endogenous genes containing these promot cr5. Therefore, we examined the expression of putative WT1 target genes in stable RM1 clones, generated by transfection of CM V-driven wild-type WT1, linked to the neomycin resistance (neo) gene (7). These cell lines expressed WT1 mRNA that was detectable by North em blot and did not contain any mutation as determined by RT-PCR and sequencing (7). However, WT1 protein expression was below detection by immunoblot, suggesting that inefficient translation of â€˜1, C WT1 mRNA in these cells made it possible for them to remain viable. â€˜1, as Stable RM1 transfectants showed a decreased cloning efficiency in as .0 soft agar and reduced tumorigenicity in nude mice, consistent with the x tumor suppressor properties of WT1 (7). Two RMI-derived cell lines, NB1 and NB2, expressing .@ WT1(â€”KTS) were compared with cells transfected with the neomy cm resistance vector (Neo) or with an anti-sense WT1 construct. NB1 cells contained a WT1 transcript of increased size, consistent with a read-through transcript. Cell lines were grown both as tumor explants in nude mice (in vivo) and in tissue culture (in vitro), conditions that alter the levels of endogenous growth factors, and Northern blots were used to examine the expression of postulated WTI-target genes with EGR1-containing promoters. WT1 expression in RM1 cells had no Vector WT1 WT1 (-KTS) (+KTS) effect on the expression of EGR1, Pax 2, and PDGF-A. However, endogenous IGF2 expression was increased in RM1 cells expressing Fig. 1. Transcriptional activation by WT1 in RMI cells. CAT activity in RM1 cells wild-type WT1 (Fig. 2). The baseline expression of IGF2 in RM1 transiently transfected with the p3X-EGR1-CAT reporter, containing three tandem EGR1 sites upstream of a minimal promoter, along with constructs encoding CMV-driven cells was low compared with that of most Wilms' tumors, and IGF2 murine W'fl. The WT1 construct lacking the KTh alternative splice, WT1(â€”KTS), mRNA levels were at the limit of detection when cells were grown in encodes a protein that binds to the EGRI consensus site, whereas insertion of KTh between zinc fingers 3 and 4, WT1(+KTS), abrogates DNA binding. Bars, SDs calculated from multiple independent experiments. 4 R. El Bahtimi and A. J. Garvin, unpublished data. 4541

vitro. However, growth of these cells in vivo led to an increase in c'Ja)0:@ baseline expression, and WT1-transfected cells had a 5-fold increase

in IGF2 mRNA, compared with vector-transfected cells. RM1 cells @- 5- L@ 0 0 expressing high levels of an antisense WT1 transcript had no detect Iâ€” 1@c'J @) able expression of IGF2 mRNA. Of the two major IGF2 mRNA @:t@a:@ @:< zâ€˜-z species, the increased expression following WT1 transfection was observed with the 6-kb IGF2 transcript originating primarily from @ the P3 promoter, which has been shown to have WT1-responsive â€” .4â€” 28S sites(11). IGF2 _ Enhanced Induction of IGF2 by WT1 Transactivation Mutants. We have described previously naturally occurring WT1 proteins with . @4-18S an altered transactivation domain that display potent transcriptional activation of reporter constructs in transient transfection assays. The aberrantly spliced WT1/del2, lacking WT1 exon 2, is observed in a subset of Wilms' tumor specimens (7), and mutant WT1/201, encod ing a gly to asp substitution in WT1 exon 3, was found in a tumor specimen from a patient with WAGR, a Wilms' tumor predisposition syndrome (17). Stable RM1 transfectants were generated with CMV driven WT1/del2 and WT1/201 . Expression of the transfected gene in these cells was only detectable by RT-PCR, suggesting selection against high levels of expression of these transactivating WT1 van ants. WT1/del2 and WT1/201 transfectants were grown in vitro and compared with RM1 cells transfected with the different wild-type isoforms of WT1 : WTA (lacking both the KTh splice and the alter natively spliced exon 5); WTB (containing exon S and lacking KTS); and WTD (containing both exon 5 and KTS). IGF2 mRNA expression in RMI cells grown in vitro was low, and no significant change was detected in cells transfected with wild-type WT1 isoforms. However, Fig. 3 Induction of endogenous IGF2 transcript by WT1 variants. Northern blot of total WT1/del2 and WT1/201-transfected cells expressed dramatically in mRNA from RM1 cells grown in vitro and stably transfected with vector (Neo 1 and Neo creased levels of IGF2 mRNA (Fig. 3). The 6-kb IGF2 transcript 2), wild-type Wfl lacking both alternative splices (WTA), WT1 containing exon 5 but lacking KTh (WJ'B), WT1 containing both exon 5 and KTh (YvTD), or two WT1 variants: induced was that derived primarily from the P3, WT1-responsive a gly to asp mutation at codon 201 within exon 3 (Wfl/201; Ref. 17) and an aberrantly promoter. No other previously postulated WT1-target genes with spliced product with an in-frame deletion of exon 2 (WT!/del2; Ref. 7). Blots were hybridized with a probe for IGF2. An ethidium bromide-stained gel is shown to demon EGR1-containing promoters were found to be induced in RM1 cells strate equal loading and RNA integrity. transfected with the WT1/del2 and WT1/201 variants.

Discussion Wilms' tumors are heterozygous, and WT1/del2 is an aberrant splic ing variant that is often coexpressed with the wild-type WT1 tran We have used a Wilms' tumor-derived cell line to study the script in these tumors (5â€”7).Thus, the altered transactivation domain transactivational properties of WT1 and to confirm the identity of of these mutant WT1 proteins may have dysfunctional, potentially IGF2 as a potentially physiological target gene for WT1. The presence dominant properties. The induction of growth-inducing genes, such as of the EGR1 consensus sequence in a promoter, together with tran IGF2, provides an attractive mechanism for their function in tumon scriptional suppression of that promoter in transient transfection as genesis. An analogous mechanism may be inferred from the fusion of says, has been taken as evidence that a gene is a potential target for the Ewing sarcoma gene EWS to zinc fingers 2â€”4ofWT1 that defines WT1 . However, the relative loss of binding specificity associated with the mesothelial cancer desmoplastic small round cell tumor (24). overexpression of WT1 in transient transfection assays may lead to However, the altered WT1 DNA binding domain in the EWS-WT1 the identification of putative target genes that are not physiologically fusion appears to result in distinct DNA target specificity.5 regulated by WT1. Furthermore, the cell type-specific expression of The characterization of WT1 as a repressor of transcription is WT1 may affect the identity of putative target genes. Thus, the supported by transient transfection experiments using a number of observation that endogenous IGF2 expression is altered in Wilms' EGR1-containing promoters, recipient cell lines, and chimenic tumor cells stably expressing WT1 confirms that IGF2 is a genuine Ga14 constructs (8, 10â€”15). However, wild-type WT1 has also target gene for WT1. Our results, however, do not exclude the been shown to act as a potent transcriptional activator, depending possibility that other genes with WT1-responsive promoters may be upon promoter and cellular contexts (18, 21). It is possible that affected by WT1 expression in other appropriate cell types. IGF2 induction by WT1 in RM1 cells results from the absence of The induction of endogenous IGF2 by WT1 was dramatic in cells wild-type p53 in these cells. We have shown previously that WT1 transfected with transactivating variants, WT1/del2 and WT1/201. activates an EGR1-containing reporter when transfected into These mutant proteins may prove particularly useful in identifying Saos-2 cells lacking p53 or into rat embryo fibroblasts expressing bona fide WT1 targets, since their effect does not appear to be a temperature-sensitive p53 in the mutant conformation (18). Al dependent on the baseline expression level of the target gene. In ternatively, WT1-mediated transcriptional activation could be af contrast, the induction of IGF2 by wild-type WT1 was more modest fected by the low baseline expression level of IGF2 in RM1 cells, and only evident when the baseline IGF2 expression was enhanced by with transcriptional repression by WT1 dependent upon competi growth in vivo. Our observations also suggest a potential physiolog tion with other transcription factors that normally regulate IGF2 ical consequence of the potent IGF2 induction by naturally occurring WT1 mutants. A substantial fraction of WT1 mutations observed in 5 K. E. Nichols and D. A. Haber, unpublished data. 4542

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. WTI-INDUCED IGF2 IN wILMS' TUMOR CELLS expression. Nonetheless, our observations suggest that wild-type splicing and genomic structure of the Wilms tumor gene WT!. Proc. NatI. Acad. Sci. USA, 88:9618â€”9622,1991. WT1inducesexpressionofIGF2in RM1Wilms'tumorcellsin 10. Rauscher, F., III, Morris, J., Tournay, 0., Cook, D., and Curran, T. Binding of the which it exerts a tumor-suppressor effect (7). Thus, it is unlikely Wilms' tumor locus zinc finger protein to the EGR-l consensus sequence. Science that WT1 suppresses the growth of RM1 cells by inhibiting an (Washington DC), 250: 1259â€”1262, 1990. autocnine growth factor-signaling pathway involving IGF2. How 11. Drummond, I., Badden, S., Rohwer-Nutter, P., Bell, G., Sukhatme, V., and Rauscher, F., III. Repression of the insulin-like growth factor II gene by the Wilms tumor ever, in addition to IGF2, WT1 may either activate or repress suppressor WI!. Science (Washington DC), 257: 674â€”678, 1992. transcription of additional target genes. Identifying these genuine 12. Werner, H., Re, G., Drummond, I., Sukhatme, V., Rauscher, F., III, Sens, D., Garvin, A., LeRoith, D., and Roberts, C., Jr. Increased expression of the insulin-like growth target genes of WT1 and demonstrating whether their expression is factor I receptor gene IGF1R in Wilma tumor is correlated with modulation of IGFI R induced on repressed in appropriate cell types will be required to promoter activity by the WT1 Wilms tumor gene product. Proc. Nail. Acad. Sci. USA, understand the tumor suppressor properties of WT1. 90: 5828â€”5832,1993. 13. Gashler, A., Bonthron, D., Madden, S., Rauscher. F., III, Collins, T., and Sukhatme, V. Human platelet-derived growth factor A chain is transcriptionally repressed by the Acknowledgments Wilma tumor suppressor WT1. Proc. Natl. Acad. Sci. USA, 89: 10984â€”10988,1992. 14. Wang, Z., Madden, S., Deuel, T., and Rauscher, F., III. The Wilms' tumor gene We thank Dr. V. Sukhatme for providing the p3X-EGR1-CAT reporter product, WT1, represses transcription of the platelet-derived growth factor A-chain construct, Dns. S. Friend and P. Romero for their assistance in p53 sequencing, gene. J. Biol. Chem., 267: 21999â€”22002, 1992. S. Park for expert technical assistance, and Dr. J. Settleman for helpful 15. Harrington, M., Konicke, B., Song, A., Xia, X., Fredericks, W., and Rauscher, F., Ill. Inhibition of colony-stimulating factor-i promoter activity by the product of the criticism. Wilms' tumor locus. J. Biol. Chem., 268: 21271â€”21275.1993. 16. 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Maheswaran, S., Park, S., Bernard, A., Morris, J., Rauscher, F., III, Hill, D., and gous deletion in Wilms tumours of a zinc-finger gene identified by chromosome Haber, D. Physical and functional interaction between WT1 and p53 proteins. Proc. jumping.Nature(Land.),343:774â€”778,1990. NatI. Acad. Sci. USA, 90: 5100-5104, 1993. 4. Pritchard-Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous, D., Gosden, 19. Hazen-Martin, D., Garvin, A., Gansler, T., Tarnowski, B., and Sens, D. Morphology C., Bard,J., Buckler,A., Pelletier,J., Housman,D., van Heyningen,V., and Hastie, and growth characteristics of epithelial cells from classic Wilma' tumors. Am. J. N., The candidate Wilma' tumour gene is involved in genitourinary development. Pathol., 142: 893â€”905,1993. Nature (Land.), 346: 194â€”197,1990. 20. Gorman, C., Moffat, L., and Howard, B. Recombinant genomes which express 5. Haber, D., Buckler, A., Glaser, T., Call, K., Pelletier, J., Sohn, R., Douglass, E., and chloramphenicol acetyl transferase in mammalian cells. Mol. Cell. Biol., 2: 1044â€” Housman, D. An internal deletion within an 1lpl3 zinc finger gene contributes to the 1051,1982. development of Wilms' tumor. Cell, 6!: 1257â€”1269,1990. 21. Wang, Z-Y., Qiu, 0-0., and Deuel, T. The Wilms' tumor gene product WTI activates 6. Utile, M., Prosser, J., Condie, A., Smith, P., van Heyningen, V., and Hastie, N. Zinc or suppresses transcription through separate functional domains. J. Biol. Chem., 268: finger point mutations within the WI! gene in Wilma tumor patients. Proc. NatI. 9172â€”9175,1993. Acad.Sci.USA,89: 4791â€”4795,1992. 22. Bardeesy, N., Falkoff, D., Petruzzi, M., Nowak, N., Zabel, B., Adam, M., Aguiar, M., 7. Haber, D., Park, S., Maheswaran, S., Englert, C., Re, G., Hazen-Martin, D., Sens, D., Grundy, P., Shows, T., and Pelletier, J. Anaplastic Wilma' tumour, a subtype dis and Garvin, A. 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Kim E. Nichols, Gian G. Re, Yu Xin Yan, et al.

Cancer Res 1995;55:4540-4543.

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