Loss of Heterozygosity at 2Q37 in Sporadic Wilms' Tumor: Putative Role for Mir-562 Kylie M

Published OnlineFirst September 29, 2009; DOI: 10.1158/1078-0432.CCR-09-1065

Human Cancer Biology

Loss of Heterozygosity at 2q37 in Sporadic Wilms' Tumor: Putative Role for miR-562 Kylie M. Drake,1 E. Cristy Ruteshouser,4 Rachael Natrajan,5 Phyllis Harbor,1 Jenny Wegert,6 Manfred Gessler,6 Kathy Pritchard-Jones,5 Paul Grundy,7 Jeffrey Dome,8 Vicki Huff,4 Chris Jones,5 and Micheala A. Aldred1,2,3

Abstract Purpose: Wilms' tumor is a childhood cancer of the kidney with an incidence of ∼1in 10,000. Cooccurrence of Wilms' tumor with 2q37 deletion syndrome, an uncommon constitutional chromosome abnormality, has been reported previously in three children. Given these are independently rare clinical entities, we hypothesized that 2q37 harbors a tumor suppressor gene important in Wilms' tumor pathogenesis. Experimental Design: To test this, we performed loss of heterozygosity analysis in a panel of 226 sporadic Wilms' tumor samples and mutation analysis of candidate genes. Results: Loss of heterozygosity was present in at least 4% of cases. Two tumors harbored homozygous deletions at 2q37.1, supporting the presence of a tumor suppressor gene that follows a classic two-hit model. However, no other evidence of second mutations was found, suggesting that heterozygous deletion alone may be sufficient to promote tumorigenesis in concert with other genomic abnormalities. We show that miR-562, a microRNA within the candidate region, is expressed only in kidney and colon and regulates EYA1, a critical gene for renal development. miR-562 expression is reduced in Wilms' tumor and may contribute to tumorigenesis by deregulating EYA1. Two other candidate regions were localized at 2q37.3 and 2qter, but available data from patients with constitutional deletions suggest that these probably do not confer a high risk for Wilms' tumor. Conclusions: Our data support the presence of a tumor suppressor gene at 2q37.1 and suggest that, in individuals with constitutional 2q37 deletions, any increased risk for developing Wilms' tumor likely correlates with deletions encompassing 2q37.1. (Clin Cancer Res 2009;15(19):5985–92)

Authors' Affiliations: 1Genomic Medicine Institute, Lerner Research Institute Wilms' tumor (nephroblastoma) is the most common pediat- 2 3 and Taussig Cancer Institute, Cleveland Clinic; and Department of Genetics, ric renal malignancy, affecting ∼1 in 10,000 children (1). Most Case Western Reserve University School of Medicine, Cleveland, Ohio; 4Department of Cancer Genetics, The University of Texas M. D. Anderson cases of Wilms' tumor are sporadic and unilateral, but a minor- Cancer Center, Houston, Texas; 5Pediatric Oncology, Institute of Cancer ity (2%) have a family history (2). Wilms' tumors appear to de- Research/Royal Marsden Hospital NHS Trust, Sutton, United Kingdom; velop from nephrogenic rests, an abnormal structure in the 6 Developmental Biochemistry, Biocenter, University of Wuerzburg, kidney that is formed by a failure of the mesenchymal tissue Wuerzburg, Germany; 7Departments of Pediatrics and Oncology, University of Alberta, Edmonton, Alberta, Canada; and 8Division of Oncology, Children's to differentiate into nephrons (3). Additional genetic events National Medical Center, Washington, District of Columbia are then required to transform these undifferentiated cells, thus Received 4/27/09; revised 6/26/09; accepted 6/26/09; published OnlineFirst causing uncontrolled growth that can lead to the formation of 9/29/09. Wilms' tumor. Grant Support: Association for International Cancer Research grant 07- Wilms' tumor is also a feature in several syndromes, notably 0128 and startup funding from Strategic Investment Funds to the Genomic Medicine Institute (M.A. Aldred). WAGR (Wilms' tumor, aniridia, genitourinary abnormalities, The costs of publication of this article were defrayed in part by the payment of and mental retardation), Simpson-Golabi-Behmel, and Beck- page charges. This article must therefore be hereby marked advertisement with-Wiedemann (4). Analysis of chromosome deletions in pa- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tients with WAGR led to the identification of the Wilms' tumor Note: Supplementary data for this article are available at Clinical Cancer WT1 Research Online (http://clincancerres.aacrjournals.org/). suppressor gene, , on chromosome 11p13 (5, 6). Homozy- Requests for reprints: Micheala A. Aldred, Genomic Medicine Institute gous mutations in WT1 are found in ∼18% of Wilms' tumors (NE-50), Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, (7), whereas point mutations of WT1 in patients with Denys- Cleveland, OH 44195. Phone: 216-445-4336; Fax: 216-636-0009; E-mail: Drash and Frasier syndromes underscore its importance in nor- [email protected]. F 2009 American Association for Cancer Research. mal renal and urogenital development (8). Two other genes, doi:10.1158/1078-0432.CCR-09-1065 WTX (Wilms' tumor on the X) and CTNNB1, have also been

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Human Cancer Biology

kidney and bilateral ovarian dysgenesis in the female transloca- Translational Relevance tion case (23). Features of urogenital anomalies and horseshoe kidney have also been noted in cases of constitutional 2q37 de- 2q37 deletion syndrome is an uncommon constitu- letions without Wilms' tumor (19, 20, 25, 26), suggesting the tional chromosome abnormality. An association with presence of a gene at chromosome 2q37 that, like WT1,isim- Wilms' tumor has been reported in three cases, sug- portant in both normal development and as a tumor suppres- gesting the presence of a tumor suppressor gene. In a sor gene. panel of 226 sporadic Wilms' tumors, we identified We therefore hypothesized that chromosome 2q37 harbors a loss of heterozygosity in at least 4% of tumors. Homo- tumor suppressor gene, the deletion of which predisposes to zygous deletions in two independent tumors strongly Wilms' tumor, and that this gene, or a closely linked gene, is implicate a 360-kb region containing the DIS3L2 gene important in renal/urogenital development. To test this, we and microRNA miR-562. Previously, it has been un- conducted LOH and candidate gene analyses in a large panel clear whether children with 2q37 deletions are at in- of sporadic Wilms' tumors. creased risk for developing Wilms' tumor; ∼100 cases have been described, only 3 of whom had this malignancy. However, the majority of cases without malig- Materials and Methods nancy where the breakpoints have been molecularly defined do not encompass the critical 360-kb region LOH analysis in sporadic Wilms' tumors. Wilms' tumor samples and we identified, suggesting a genotype-phenotype cor- paired normal tissue or blood (where available) were accrued from cen- ters in Europe and North America with appropriate ethics board ap- relation. Overall, our results therefore suggest that proval and written informed consent. The initial panel used for LOH any increased susceptibility to Wilms' tumor in chil- screening comprised 226 randomly selected tumors. Seventy-three of dren with a constitutional 2q37 deletion likely corre- these were subjected to genome-wide screening and aggregate results lates with deletions encompassing 2q37.1. for 2q have been reported previously (14). The remainder were specif- ically analyzed for LOH at 2q37 using a high-density microsatellite panel as described previously (19). Allele ratios were calculated as described, with ratios <0.45 classified as LOH and ratios of 0.45 to 0.66 as possible mosaic LOH or trisomy (14). Additional samples with implicated in the pathogenesis of Wilms' tumor. CTNNB1, β ∼ known copy number loss were subsequently accrued to enrich the which codes for -catenin, is mutated in 15% of Wilms' tu- pool for candidate gene analysis (27). Copy number at 2q37 was de- WT1 mors but rarely occurs without concomitant mutation of termined by multiplex ligation–dependent probe amplification (28) (7, 9), whereas somatic mutations of WTX are present in 11% using custom-designed oligonucleotide probes. Where sufficient DNA to 29% of Wilms' tumors and occur with and without WT1 mu- was available, precise breakpoints were defined by genome-wide single tation (7, 9, 10). Interestingly, germ-line mutations of WTX nucleotide polymorphism analysis using Illumina Hap300 arrays and were recently shown to underlie osteopathia striata congenita Beadstudio software (Illumina). Mutation analysis and promoter meth- with cranial sclerosis, a X-linked sclerosing bone dysplasia, ylation assays were done using standard methods as detailed in Supple- but these patients had no predisposition to Wilms' tumor or mentary Data. miR-562 expression analysis. Total RNA from Wilms' tumor and other malignancies, suggesting temporal or spatial constraints WTX normal adjacent kidney tissue was extracted using Trizol (Invitrogen). on the action of during tumorigenesis (11). RNA from other human organs was purchased commercially (Agilent Due to an improved combination of surgery, chemotherapy, Technologies). Cell lines used included the fetal kidney-derived HEK- and radiotherapy, there has been a dramatic improvement 293 and 293T lines, the breast cancer–derived MCF-7 cell line, and in Wilms' tumor survival over the past 40 years, with the cure WiT-49 cells derived from a Wilms' tumor. RNA from these lines was rate now approaching 90% (12). Despite this, the molecular extracted using the miRNeasy mini kit (Qiagen). RNase protection assay pathogenesis of Wilms' tumor and factors determining the of miR-562 was done using 2 μg RNA with the mirVana miRNA detec- subset that relapse remain largely unknown. Identification of tion kit (Ambion) according to the manufacturer's protocol. Probes for 32 other genes involved in the etiology of sporadic Wilms' tumor microRNA detection were end-labeled with γ- P by using the mirVana therefore remains an important priority. Studies of loss of probe and marker kit (Ambion). For quantitative real-time PCR, two commercially available quantita- heterozygosity (LOH), loss of imprinting, and constitutional tive PCR assays for mature miR-562 failed completely even on control chromosomal defects have implicated several recurrent RNA samples that showed high expression in our RNase protection changes in Wilms' tumor at chromosomes 11p15, 1p, 1q, assay. We therefore opted to amplify the primary miR-562 transcript, – 7p, 9q, 14q, 16q, and 22 (13 16). designing primers within the precursor stem loop. Validation on the 2q37 deletion syndrome is a chromosomal disorder charac- RNA panel used for RNase protection assays gave concordant results terized by developmental delay, dysmorphic facies, skeletal ab- (data not shown). Total RNA was DNase I treated (Invitrogen) and re- normalities, and an increased risk of congenital heart defects verse transcribed using SuperScript III (Invitrogen) and oligo(dT) primer. (17–20). Although most cases have no associated malignancies, Samples were amplified on an Eppendorf Realplex MasterCycler (Ep- three children with constitutional 2q37 monosomy and Wilms' pendorf) with QuantiTect SYBR Green PCR master mix (Qiagen). The miR-562 tumor have been reported (21–24). Two of these were de novo relative abundance of was determined by using a standard curve generated from 5-fold serial dilutions of fetal kidney cDNA and deletions and the third case resulted from unbalanced segrega- normalized to GAPDH mRNA. To analyze changes in microRNA expres- tion of a reciprocal translocation from an unaffected parent sion, ratios of the geometric means between control (fetal kidney) and with a balanced karyotype. All three cases showed additional experimental (Wilms' tumor and adjacent normal kidney) samples urogenital anomalies: hypospadias and a small penis in a male were calculated. Significance was determined by testing the difference patient (21); gonadal dysgenesis, bifid uterus, and dysplasia of of two means. One advantage of analyzing the primary miR-562 tran- the contralateral kidney in one female (24); and a horseshoe script is that the same aliquots of cDNA could also be used for analysis

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2q37 Deletions in Wilms' Tumor

Table 1. Wilms' tumors showing LOH at 2q37

Tumor Copy no. Proximal Distal Size Genes WT1 Additional breakpoint* breakpoint* (Mb) affected mutation abnormalities

NWTS-99 1 232.24 234.37 2.2 PTMA-UTG1A cluster No LOH 11pter-p15.5 0 232.48 233.88 1.4 NPPC-ATG16L1 1 239.39 240.55 1.2 TWIST2, HDAC4 06-0116 2 218.54 Telomere 25 TNS1-telomere 0 232.58 233.60 1.1 DIS3L2-NEU2 MDA-31T 2 134.98 232.85 98 TMEM163-DIS3L2 No dup 3pter-p14.3; del 11q12.1-qter MDA-28T 1 195.32 Telomere 48 No MDA-30T 2 221.72 Telomere 22 No MDA-1T 1 230.39 233.74 3.4 TRIP12-INPP5D Germ-line del 7p; dup 7q; del 11pter-p11 Rassekh et al. (16) 1 230.83 233.29 2.5 SP140-GIGYF2 Robinow syndrome (caseA7) 08-0144 1 231.98 239.79 7.8 ARMC9-HDAC4 None RMH562 2 Not defined Telomere >13 Includes DIS3L2 No RMH781 2 Not defined Telomere >13 Includes DIS3L2 No NWTS-7T 2 Not defined Telomere >13 Includes DIS3L2 No NWTS-11T 2 Not defined Telomere >13 Includes DIS3L2 No Germ-line 2q37 deletions Conrad et al. (21) 1 Not defined Telomere t(10;11)(q?;p13) Viot-Szoboszlai et al. (24) 1 Not defined Telomere No MDA-74T (also refs. 22, 23) 1 242.29 Telomere 0.6 ING5- telomere Somatic der15, t(2;15) (q37.3;q24.1); +8

*Approximate breakpoints are shown in Mb on chromosome 2, hg18 assembly. Tumors with homozygous deletions are shown first followed by new and published cases in order of proximal breakpoint.

miR-562 of target genes (see below) and direct comparison could be Results made using a common housekeeping gene. miR-562 Bioinformatic analysis of miR-562 targets. targets were LOH and homozygous deletions at 2q37. In the initial panel identified using the Targetscan,9 miRBase,10 miRGator,11 and miRNA of 226 Wilms' tumors, 9 (4%) showed LOH with clonal loss of map12 databases. The targets were then analyzed using the KEGG gPro- filer13 and Metacore14 pathway tools. Three target genes, EYA1, MET, one allele, indicating that this was an early event in tumorigen- and PSEN1, were chosen for further study based on their previously es- esis. Six of these were copy neutral LOH and three harbored de- tablished expression in Wilms' tumor or their roles in renal and urolog- letions. A further ∼6% showed ratios in the range 0.45 and 0.66 ic system development and function (Supplementary Table S1). and may represent later mosaic LOH events or trisomy. A mo- Luciferase assays. The putative miR-562 binding sites in the saic deletion at 2q36.3-q37.1 has recently been documented 3′-untranslated region from genes of interest were cloned into the (16). We subsequently ascertained an additional three tumors pMIR-REPORT miRNA expression reporter vector system (Ambion). with known deletions, making a total of 12 samples for detailed 5 293T cells (1 × 10 per well on a 12-well dish) were transfected with analysis (Table 1). Two had a WT1 mutation, one was somatic, β 200 ng of the reporter vector along with 200 ng -gal-REPORT (Am- and the other was heterozygous in blood DNA and reduced to bion) or an empty vector and pre-microRNA miR-562 precursor mole- miR-562 homozygosity in the tumor. In most of these tumors, the region cule (Ambion) and/or anti-microRNA inhibitor (Ambion). ≥ The ratio of firefly luciferase to β-galactosidase activity was measured of LOH is extensive, encompassing the terminal 13 Mb of after 48 h using the Dual-Light combined reporter gene assay system chromosome 2q. Crucially, however, two tumors (NWTS-99 (Applied Biosystems) following the manufacturer's protocol. Changes and 06-0116) were identified with homozygous deletion at in luciferase activity were determined by taking the ratios of the geomet- 2q37.1 (Figs. 1 and 2). Additional cases showing interstitial ric means for reporters cotransfected with pMIR-REPORT and β-gal- LOH in this region define a minimal 360-kb interval bounded REPORT. Variability of mean ratios for each reporter was determined by the polymorphisms rs2679184 and rs13386477 (region A, by calculating the limits of a 95% confidence interval. Significance Fig. 1). The only known gene within this region is DIS3L2,a t was determined by using Student's test. homologue of the yeast mitotic control gene DIS3. Within intron 9 of DIS3L2 is miR-562, a previously uncharacterized mi- croRNA. Case NWTS-99 also showed a heterozygous deletion of ∼1 Mb at 2q37.3 (region B), encompassing histone deacetylase 4(HDAC4) and TWIST2 (Figs. 1 and 2). The t(2;15) transloca- 9 www.targetscan.org tion case (MDA-74T) was the only tumor available from a pa- 10 www.mirbase.org 11 tient with a known constitutional 2q37 rearrangement. Only genome.ewha.ac.kr/miRGator ∼ 12 http://mirnamap.mbc.nctu.edu.tw the terminal 500 kb of 2q37.3 was deleted in this patient, in- 13 ING5 http://biit.cs.ut.ee/gprofiler cluding the gene (region C, Fig. 1). Thus, a total of three 14 www.genego.com/metacore.php distinct candidate regions were identified within 2q37.

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Mutation analysis of candidate genes. The presence of homo- therefore screened a panel of 96 Wilms' tumor samples with zygous deletions in two tumors strongly suggested that any tu- no LOH at 2q37, looking for heterozygous mutations of genes mor suppressor gene at 2q37.1 follows the classic two-hit within regions A and B: DIS3L2, miR-562, HDAC4,and hypothesis. We therefore analyzed the subset of Wilms' tumor TWIST2. A 18-bp deletion of miR-562 was identified in one tu- samples that showed LOH in region A for evidence of a second mor (Fig. 3A), the follow-up of which is detailed below. No mutation, screening candidate genes within the homozygously pathogenic mutations were identified in DIS3L2, HDAC4,or deleted region: DIS3L2, GIGYF2 (GRB10-interacting GYF pro- TWIST2. We also screened for microdeletions in region A across tein 2), NPPC (natriuretic peptide precursor C), and miR-562. thesamepanelusingsevenpolymorphisms,∼60 kb apart No additional genetic changes were identified. Bisulfite se- (rs2679184, rs12988522, rs4973500, rs3100586, rs3116179, quencing of the CpG island at the DIS3L2 promoter was also rs923333, and rs2633254). No additional microdeletions were done, but there was no evidence for abnormal methylation in detected. these tumors (data not shown). Similarly, there was no evi- Mutation and expression analysis of miR-562. To further dence of a second mutation in TWIST2 or HDAC4 among the characterize the 18-bp deletion of miR-562, we extended se- tumors with LOH in region B. quence analysis to a total of 176 Wilms' tumor samples and As no second mutations were identified in the Wilms' tumors 210 controls. The heterozygote frequency was 3 of 176 Wilms' exhibiting LOH, it is possible that hemizygous deletion or mu- tumors (0.017) and 5 of 210 controls (0.024), suggesting that tation might be sufficient to contribute to tumorigenesis. We it is an uncommon polymorphism. miR-562 expression has

Fig. 1. 2q37 deletions identified in Wilms' tumors. A, ideogram of chromosome 2 enlarged to show regions of LOH at 2q37. Pale gray columns, copy neutral LOH; dark gray columns, heterozygous deletion; black columns, homozygous deletion (06-0116 and NWTS-99T); thin black lines, minimum candidate regions A, B, and C. For simplicity, six additional tumors that show LOH across the entire region have been omitted. B, enlargement of region A showing the position of DIS3L2 and miR-562.

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2q37 Deletions in Wilms' Tumor

(P ≤ 0.005), indicating that it is a genuine target of miR-562 (Fig. 4). Consistent with this, EYA1 was highly expressed in Wilms' tumors but not in normal adjacent kidney tissue (Fig. 5), a result that is consistent with previous microarray expression analysis (30).

Discussion

The discovery of cytogenetic abnormalities in syndromes predisposing to Wilms' tumor, such as 11p deletions in WAGR and 11p trisomies and translocations in Beckwith-Wiedemann syndrome, has been proven critical for the identification of Wilms' tumor genes (31, 32). This combination of LOH data and rare constitutional chromosome abnormalities, used to Fig. 2. Homozygous deletion at 2q37.1. Illumina single nucleotide pinpoint WT1, has also identified up to three loci on chromo- polymorphism microarray data are shown as the difference in log intensity ratio between tumor NWTS-99T and matched normal tissue. A some 7p that have a possible role in the etiology of Wilms' homozygous deletion is evident at 2q37.1, flanked by small regions of tumor (13, 33–35). 2q37 deletion syndrome is a rare consti- heterozygous deletion. An additional heterozygous deletion is present tutional chromosome abnormality, and although most cases at 2q37.3, with normal copy number and heterozygosity in the intervening DNA. have no associated malignancies, reports of Wilms' tumor in three cases (21–24) suggested the presence of a tumor suppressor gene. Our analysis of a large panel of sporadic Wilms' tumors iden- not been characterized previously, except for a single report of tified clonal LOH at 2q37 in 4% of cases and allelic imbalance expression in colorectal cancer cells (29). We therefore char- in another 6%. Remarkably, two of these tumors harbor homo- acterized the expression pattern of miR-562 in a range of zygous deletions, strongly supporting our hypothesis of a tu- human tissues using RNase protection assays. Human fetal mor suppressor gene present in this region and suggesting kidney and colon tissue, as well as WiT-49, 293T, and HEK- that it follows a classic Knudson two-hit model (36). Further- 293 cells, all showed miR-562 expression (Fig. 3B). In con- more, genome-wide single nucleotide polymorphism array trast, no expression was detectable in human fetal heart analysis of these two tumors showed few additional cytoge- and liver samples, adult heart, brain, ovary, testis, and lung netic abnormalities (Table 1). In contrast, the tumors that tissue or MCF-7 cells. miR-562 expression was also undetect- showed only a heterozygous loss at 2q37 harbored multiple able in the mouse kidney, confirming genome sequence data additional chromosomal abnormalities, such as 11p LOH and that suggest miR-562 is only present in primate genomes isochromosome-7q (Table 1). We propose that homozygous (data not shown). deletion of one or more key genes at 2q37.1 is sufficient to This very tissue-restricted expression pattern made miR-562 initiate Wilms' tumor development, whereas, in the absence an attractive candidate gene for a role in kidney development of a second mutation, heterozygous loss can contribute to and Wilms' tumor pathogenesis. Expression of miR-562 in the pathogenesis in concert with other abnormalities else- Wilms' tumors ranged from 0.11 to 1.15 times that observed where in the genome. in fetal kidney (Fig. 3C). The difference in mean miR-562 ex- The significance of two additional regions of localized LOH pression levels between Wilms' tumor and normal adjacent kid- (Fig. 1) is less clear-cut. Region B, encompassing HDAC4 and ney samples was significant (0.35 ± 0.075; P = 0.046). Closer TWIST2, is defined by a 1-Mb deletion in sample NWTS-99. inspection of the data suggests that there are two distinct group- This same sample also harbors one of the homozygous dele- ings: three tumors show normal expression, whereas the re- tions in region A, yet the intervening DNA shows normal copy maining nine, which include two tumors with copy neutral number and no LOH. These deletions were all confirmed to be LOH and one with the heterozygous miR-562 deletion poly- de novo in the tumor. One explanation is that they represent a morphism, show at least a 2-fold decrease in expression. This more complex rearrangement, such as an inversion/deletion suggests that miR-562 expression is significantly downregulated event that masquerades as discontiguous deletions at the in a subset of Wilms' tumor cases. DNA microarray level. Dividing tumor cells are not available Identification of miR-562 target genes. To elucidate the role for the metaphase analysis needed to investigate this further, of miR-562, we performed functional analysis to determine its but it is possible that the region B deletion is a bystander and target genes. Bioinformatic analyses of putative miR-562 targets does not contribute to the pathogenesis of Wilms' tumor. Sim- revealed 37 genes with established roles in kidney or urogenital ilarly, the significance of the ∼500-kb terminal deletion in the development and/or disease (Supplementary Table S1). Lucifer- translocation case (region C) is somewhat unclear. It encom- ase reporter assays were undertaken for the top three candi- passes five genes, including ING5, a putative tumor suppressor dates: EYA1, MET,andPSEN1 to determine whether they are gene that may modulate p53 function (37). The translocation genuinely regulated by miR-562. Because microRNAs negatively is unbalanced and duplicates a 28-Mb region of distal regulate their target genes, a positive interaction in this assay 15q, a rearrangement that has also been implicated in the results in a decrease in luciferase activity. MET and PSEN1 lucif- pathogenesis of Wilms' tumor (15, 38). Thus, although this erase levels were not significantly altered in this system (data isoneofthethreegerm-linerearrangementsthatinspired not shown), but EYA1 expression was significantly decreased the study, the partial trisomy 15q likely also contributed

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Fig. 3. Characterization of miR-562. A, sequence analysis identified a heterozygous 18-bp deletion of miR-562 in 1.7% of Wilms' tumors and 2.4% of controls. The miR-562 hairpin is shown with the mature microRNA in bold text. Large arrows, terminal ends of miR-562 in the reference sequence; black box, 18-bp deletion. RNA folding analysis using the program Mfold (http://mfold.bioinfo.rpi.edu/) indicates that the 18-bp deletion will abolish hairpin formation. B, RNase protection assay detected miR-562 expression in human colon, fetal kidney, and several kidney-derived cell lines but not in other major organs. The miR-562 probe is 21 nucleotides in length. C, primary miR-562 expression in Wilms' tumor samples was determined by quantitative real-time PCR and normalized to GAPDH. NK, normal adjacent kidney from Wilms' tumor patients; 08-03XX samples are Wilms' tumors; *, a Wilms' tumor heterozygous for the miR-562 deletion polymorphism; #, Wilms' tumor samples with 2q copy neutral LOH. Bars, SE. Nine of the 12 tumor samples show at least a 2-fold decrease in expression compared with normal kidney.

to the pathogenesis of Wilms' tumor in this patient and the tion of homozygous somatic deletions in two tumors, these data 2q37 deletion alone may not have been causal. In support strongly suggest that region A in 2q37.1 is the primary Wilms' of this, among the constitutional 2q37 deletions that have tumor susceptibility locus on 2q. been well characterized at the DNA level, almost all harbor DIS3L2 is the only gene in the minimum 360-kb interval at HDAC4/ terminal deletions that extend proximally to 2q37.1. Because members of the yeast DIS3 family are essential TWIST2 and are therefore deleted for both regions B and in mitotic control and spindle formation (43, 44), we hypoth- – C, yet these patients do not have Wilms' tumor (19, 39 esized that deletion of DIS3L2 may contribute to the pathogen- 42). In contrast, most of the proximal breakpoints localize esis of Wilms' tumor by disrupting normal cell division and distal of region A; in our panel of 30 deletion patients with- predisposing to aneuploidy. However, no second mutations out malignancy, only one has a deletion of region A, with a breakpoint between NPPC and DIS3L2.15 Additionally, both of the constitutional deletion cases with Wilms' tumor had breakpoints in 2q37.1 (21–24). Combined with our identifica- 15 M.A. Aldred, unpublished data.

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2q37 Deletions in Wilms' Tumor or promoter methylation were identified in tumors with LOH of this region and no mutations or microdeletions were identified in our wider tumor panel. We therefore focused on miR-562, a microRNA that lies within intron 9 of DIS3L2. micro- RNAs are a group of noncoding ∼22-nucleotide RNA molecules that post-transcriptionally regulate the expression of target mRNAs (45). These small RNAs are evolutionarily conserved and regulate processes as fundamental as cellular proliferation, differentiation, and apoptosis. It is increasingly recognized that dysregulation of microRNAs plays an important role in cancer (46, 47). miR-562 showed a tissue-restricted expression pattern, stron- gest in fetal kidney. Real-time PCR analysis showed a significant reduction in Wilms' tumors compared with normal kidney and suggested that tumors may stratify into two groups based on miR-562 expression level. Clinical data do not suggest a correlation with therapy response, histology, or survival between Fig. 5. EYA1 is overexpressed in Wilms' tumor. Expression of EYA1 was determined by quantitative real-time PCR and normalized to GAPDH. NK, these two groups, but complete data were only available for 6 normal adjacent kidney from Wilms' tumor patients; 08-03XX samples are of the 12 cases where expression analysis was done, so no firm Wilms' tumors; *, a Wilms' tumor heterozygous for the miR-562 deletion conclusions can be drawn. A polymorphic deletion of miR-562 polymorphism; #, Wilms' tumor samples with 2q copy neutral LOH. Bars, SE. EYA1 is significantly overexpressed in all tumors compared with was identified, which probably does not represent a major pre- normal kidney. However, there was no clear inverse correlation with disposing factor in the etiology of Wilms' tumor but could po- mir-562 expression, suggesting that multiple factors contribute to the tentially increase susceptibility to Wilms' tumor in the presence regulation of EYA1. of additional mutations. Decreased miR-562 expression was also observed in 8 of 11 tumors with no deletion. This suggests that miR-562 is frequently downregulated at the transcriptional EYA1, a gene essential for cell survival and proliferation in ear- level perhaps due to mutations of its promoter. In general, mi- ly metanephric development (50, 51), was validated as a target croRNAs residing in introns are coexpressed with their “parent” of miR-562. We confirmed that EYA1 was significantly overex- gene, presumably directed by that gene's promoter (48), where- pressed in Wilms' tumors (30), suggesting that haploinsuffi- as miR-562 expression is apparently independent of the DIS3L2 ciency of miR-562 is likely one factor that contributes to promoter (data not shown). miR-562 is believed to be derived increased EYA1 expression in Wilms' tumors. Given that we from a transposable element (49) and its expression may there- did not see a strong inverse correlation between these two tran- fore be regulated by its own transposon-derived transcription scripts, it is clear that other genetic events also influence EYA1 machinery or by another locus. Defining the promoter for expression. Notably, the gene is located on chromosome 8, miR-562 will be important in further examining its regulation the gain of which is observed in up to 30% of Wilms' tumors and its role in normal kidney development and Wilms' tumor. (27). Downregulation of miR-562 in conjunction with gain of chromosome 8 would therefore be predicted to result in syner- gistic overexpression of EYA1. In summary, we have shown LOH at 2q37 in at least 4% of sporadic Wilms' tumors. Identification of two tumors with homozygous deletions strongly suggests the presence of a Wilms' tumor suppressor gene at 2q37.1. Expression of miR-562,ami- croRNA within this region, is significantly reduced in Wilms' tumors even in the absence of LOH or other detectable abnormality of the microRNA sequence. We showed that EYA1, which is overexpressed in Wilms' tumors, is a target of miR- 562, suggesting that haploinsufficiency of miR-562 contributes to the etiology of Wilms' tumor by promoting deregulation of EYA1. Further study of the role of miR-562 in normal renal development and Wilms' tumor is hampered by the fact it is primate-specific and no orthologue is present in model organ- isms such as mouse or zebrafish. Clinically though, our data may be helpful in clarifying the risk of Wilms' tumor in children diagnosed with a constitutional 2q37 deletion. Our re- Fig. 4. miR-562 regulates EYA1 expression in vitro. A luciferase reporter sults from sporadic Wilms' tumors are broadly concordant construct containing the putative EYA1 3′-untranslated region binding site with published data on constitutional breakpoints: deletion pa- for miR-562 was transfected into 293T cells. Relative luciferase activity was measured after 48 h. Luciferase activity in the presence of three different tients who developed Wilms' tumor had breakpoints in 2q37.1, concentrations of miR-562 was significantly reduced compared with empty whereas the majority of patients with no malignancy have vector (pMIR-REPORT), indicating transcriptional downregulation of EYA1 smaller deletions encompassing only 2q37.2-q37.3. Some cau- by miR-562. This was reversed by addition of anti-miR-562 competitor, confirming specificity of the response. Bars, SD from three independent tion is still required, because we identified two regions of un- experiments. certain significance at 2q37.3 in sporadic Wilms' tumors, but

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Human Cancer Biology overall our data suggest that any increased risk for developing Acknowledgments Wilms' tumor likely correlates with deletions encompassing 2q37.1. We thank the National Wilms' Tumor Study Group, the Children's Oncol- ogy Group, and the Cooperative Human Tissue Network, funded by the National Cancer Institute, for access to samples; the Genomics and Integra- Disclosure of Potential Conflicts of Interest tive Genomic Analysis Cores at the Lerner Research Institute for genotyping services and bioinformatics support; and Dr. Bryan Williams for kindly No potential conflicts of interest were disclosed. providing the WiT-49 cells.

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Loss of Heterozygosity at 2q37 in Sporadic Wilms' Tumor: Putative Role for miR-562

Kylie M. Drake, E. Cristy Ruteshouser, Rachael Natrajan, et al.

Clin Cancer Res 2009;15:5985-5992. Published OnlineFirst September 29, 2009.

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