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Oncogene (2010) 29, 2739–2745 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastoma tumor cells

JJ Molenaar1,2, ME Ebus1, J Koster1, E Santo1, D Geerts1, R Versteeg1 and HN Caron2

1Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 2Department of Pediatric Oncology, Emma Kinderziekenhuis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Almost all neuroblastoma tumors express excess levels of 2000). Several checkpoints normally prevent premature Cyclin D1 (CCND1) compared to normal tissues and cell-cycle progression and cell division. The crucial G1 other tumor types. Only a small percentage of these entry point is controlled by the D-type Cyclins that can neuroblastoma tumors have high-level amplification of the activate CDK4/6 that in turn phosphorylate the pRb Cyclin D1 . The other neuroblastoma tumors have . This results in a release of the transcription equally high Cyclin D1 expression without amplification. factor that causes transcriptional upregulation of Silencing of Cyclin D1 expression was previously found to numerous involved in further progression of the trigger differentiation of neuroblastoma cells. Over- cell cycle (Sherr, 1996). expression of Cyclin D1 is therefore one of the most Neuroblastomas are embryonal tumors that originate frequent mechanisms with a postulated function in neuro- from precursor cells of the sympathetic nervous system. blastoma pathogenesis. The cause for the Cyclin D1 This tumor has a very poor prognosis and despite the overexpression is unknown. Here we show that Cyclin D1 low frequency it is the second cause of cancer-related overexpression results from transcriptional upregulation. deaths in children (van Noesel and Versteeg, 2004; To identify upstream regulators, we searched in mRNA Maris et al., 2007). The G1 checkpoint is deregulated in profiles of neuroblastoma tumor series for transcription neuroblastoma in various ways. Sporadic CDKN2A factors with expression patterns correlating to Cyclin D1. (p16) mutations and CDK4 amplifications have been GATA3 most consistently correlated to Cyclin D1 in four reported but most striking is the extensive overexpres- independent data sets. We identified a highly conserved sion of Cyclin D1 (Van Roy et al., 1995; Molenaar et al., GATA3 binding site 27 bp upstream of the Cyclin D1 2003; Caren et al., 2008). We have previously shown a transcriptional start. immune precipitation very high expression on mRNA and protein level confirmed binding of GATA3 to the Cyclin D1 promoter. compared to normal tissue and even compared to other Overexpression of GATA3 induced Cyclin D1 promoter tumors with known high expression levels of Cyclin D1. activity, which decreased after site-directed mutagenesis of Functional evaluation of Cyclin D1 in neuroblastoma the GATA3 binding site in the Cyclin D1 promoter. Silencing showed a crucial function in maintaining the undiffer- of GATA3 resulted in reduced Cyclin D1 promoter activity entiated of the neuroblasts in vitro and in vivo and reduced Cyclin D1 mRNA and protein levels. Moreover, (Molenaar et al., 2008). A genomic cause for the GATA3 silencing caused differentiation that was similar to extensive Cyclin D1 overexpression could be identified that caused by Cyclin D1 inhibition. These finding implicate in a minority of cases. We have previously reported GATA3 in Cyclin D1 overexpression in neuroblastoma. high-level genomic amplification of the Cyclin D1 gene Oncogene (2010) 29, 2739–2745; doi:10.1038/onc.2010.21; in 5 out of 202 neuroblastoma tumors and cell lines. published online 15 February 2010 Others also reported recurrent high-level genomic amplifications of the 11q13 region encompassing the Keywords: CCND1; ChIP; GATA3; microarray; neuro- Cyclin D1 gene (Molenaar et al., 2003; Michels et al., blastoma 2007). These clonal events clearly pointed to a crucial function for Cyclin D1 in neuroblastoma tumor genesis, but could not explain the overexpression of Cyclin D1 in about 75% of the remaining neuroblastomas, that Introduction showed Cyclin D1 expression levels reaching equal or even higher levels compared to tumors with genomic The cell cycle is a tightly controlled process that is amplification. deregulated in all cancer cells (Hanahan and Weinberg, Identification of the mechanism that drives Cyclin D1 overexpression in neuroblastomas is therefore an Correspondence: Dr JJ Molenaar, Department of Human Genetics, important question. In this paper, we identify by in silico M1-132, Academic Medical Center, University of Amsterdam, analysis the GATA3 as a candidate Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. regulator of Cyclin D1. We show that GATA3 binds to E-mail: [email protected] Received 30 June 2009; revised 9 December 2009; accepted 3 January the Cyclin D1 promoter and that silencing of GATA3 2010; published online 15 February 2010 reduced promoter activity, resulting in decreased Cyclin GATA3 regulates Cyclin D1 in neuroblastoma JJ Molenaar et al 2740 D1 mRNA and protein levels, as well as in differentia- To identify transcription factors that regulate Cyclin tion of neuroblastoma cells. D1 expression in neuroblastoma, we used Affymetrix mRNA expression data. We hypothesized that the mRNA expression level of a transcription factor that Results causes the overexpression of Cyclin D1 might be related to the level of Cyclin D1 mRNA expression. Identification of GATA3 as possible regulator of Cyclin We have built the R2 bioinformatic analysis program D1 using mRNA profiling (http://hgserver1.amc.nl), which allows easy identifica- Cyclin D1 is extremely highly expressed in over 75% of tion of correlations in in tumor the neuroblastoma tumors, whereas CCND1 genetic series analyzed by expression arrays. The R2 application aberrations are much more infrequent. To search for the contains Affymetrix mRNA expression data of four mechanism behind Cyclin D1 overexpression, we first different neuroblastoma tumor sets in the public analyzed whether upregulation was transcriptionally domain. We searched these four data sets for genes in mediated. We cloned a 1882-bp Cyclin D1 promoter the Gene-Ontology category ‘transcription factors’ fragment encompassing the region 1696 bp upstream to (GO 3700). Of the 945 genes in this category, GATA3 186 downstream of the transcription start site of Cyclin showed the highest correlation to the Cyclin D1 D1 (numbered according to NCBI Reference Sequence expression levels (Table 1). This raised the possibility NM_053056.2) in a pGL3 reporter vector. This construct that GATA3 is involved in transcriptional regulation of was transfected together with a Renilla luciferase control Cyclin D1. The correlation in the series of 110 construct into several different neuroblastoma cell lines neuroblastic tumors profiled in our lab is shown in that had a wide range of Cyclin D1 expression levels as detail in Figure 1b. There is a strong correlation between based on Affymetrix (Santa Clara, CA, USA) expression the GATA3 and Cyclin D1 mRNA expression data. The cells were harvested during exponential growth, profiles, with one striking exception (sample with circle). and the relative Cyclin D1 promoter activity determined by This outlier has high Cyclin D1 expression, but only a dual luciferase assay. The relative Cyclin D1 promoter low GATA3 expression. The sample appeared to have activity showed a very clear correlation to the endogenous high-level genomic amplification of the Cyclin D1 gene. Cyclin D1 mRNA expression levels (Figure 1a). This This suggests that high Cyclin D1 expression in suggested that the high expression of Cyclin D1 in neuro- neuroblastoma is either caused by genomic amplifica- blastoma results from transcriptional upregulation. tion of Cyclin D1, or related to high GATA3 expression.

13 13

3000 300 12 12 11

2500 250 2log of GATA3 11 10

2000 200 FFL/RL x 10 10 9

1500 150 8

2log of CCND1 9 E

6 7 1000 100 8 6

Affymetrix CCND1 expression 500 50 7 Samples ordered by CCND1 5 1 survival 2 inss stage 3 4 mycn E 0 6 I 2 1 B 1 B -F -3 2 N B P N JN -N R E K K S J S IM H S S S CCND1 promoter activity Affymetrix CCND1 expression Figure 1 Affymetrix microarray analysis suggests Cyclin D1 transcriptional upregulation by GATA3. (a) Cyclin D1 mRNA expression and Cyclin D1 promoter activity in neuroblastoma cell lines: mRNA was isolated from neuroblastoma cell lines using Trizol (Invitrogen, Breda, Netherlands). mRNA was labeled and hybridized on Affymetrix HG-U133 Plus 2.0 arrays. Expression was normalized using the MAS5.0 algorithm. For Cyclin D1 promoter activity, a Cyclin D1 promoter fragment encompassing nucleotides À1696 to 186 according to NM_053056.2 was taken from a pA3 vector and cloned in the pGL3 Firefly luciferase reporter plasmid (Albanese et al., 1995). Neuroblastoma cell lines were co-transfected with the Cyclin D1 promoter reporter and a Renilla luciferase expression vector as an internal transfection control. Luciferase activity was measured using the Promega (Madison, WI, USA) Dual-Luciferase Reporter Assay System and Cyclin D1 promoter activity was defined as firefly luciferase activity/Renilla luciferase activity. (b) YY plot of Cyclin D1 and GATA3 Affymetrix expression in 110 neuroblastic tumors: Material collection and RNA isolation procedures were described previously (Molenaar et al., 2008). MAS5.0 corrected data are shown. The tumor sample indicated by the circle contains a strong genomic amplification of the 11q13 region containing Cyclin D1. 2log values of Cyclin D1 and GATA3 expression are indicated by red and blue dots, respectively. For each tumor sample several characteristics are given as tracks (color coded boxes). Survival (red, death; light green, survival 0–5 years; dark green, survival 45 years); INSS (red, stage 3 or 4; green, stage 1 or 2; orange, stage 4S); histology (red, neuroblastoma; dark green, ganglioneuroblastoma; light green, ganglioneuroma); MYCN (green, nonamplified; red, amplification410 copies). A full colour version of this figure is available at the Oncogene journal online.

Oncogene GATA3 regulates Cyclin D1 in neuroblastoma JJ Molenaar et al 2741 Table 1 The ten most significant correlating transcription factors with Cyclin D1 in four independent neuroblastoma datasets Versteeg (110, u133plus2) Delatre (64, u133plus2) Seeger (102, u133a) Maris (101, u95a)

HUGO P-value HUGO P-value HUGO P-value HUGO P-value

PKNOX1 4.28EÀ20 GATA3 1.59EÀ12 MYST2 1.29EÀ06 GATA3 1.83EÀ10 SOX4 1.28EÀ17 PCGF2 3.86EÀ10 GATA3 9.89EÀ06 BAZ1B 1.21EÀ06 ZNF3 8.17EÀ17 GATAD2B 5.04EÀ09 PCGF2 5.16EÀ05 MEIS1 3.13EÀ05 GATA3 1.34EÀ16 ZSCAN29 5.34EÀ09 LASS6 0.000267 LASS6 4.51EÀ05 FOXK2 1.38EÀ16 PKNOX1 1.22EÀ08 ZEB1 0.000498 CTCF 5.17EÀ05 ISL1 6.36EÀ16 ZFHX2 1.59EÀ08 PHOX2B 0.000508 PA2G4 6.54EÀ05 MEIS1 7.48EÀ16 POU2F1 1.78EÀ08 BAZ1B 0.000834 TAF4 6.96EÀ05 MAML3 7.88EÀ16 ZNF445 2.00EÀ08 TBX2 0.001724 PHOX2B 7.07EÀ05 PHOX2A 8.66EÀ16 ZKSCAN2 2.24EÀ08 BRD8 0.00173 HCFC1 9.14EÀ05 ZSCAN2 9.22EÀ16 CTCF 3.71EÀ08 TAF4 0.002189 PHF2 0.00012

Correlations were calculated between the Affymetrix expression of Cyclin D1 and all transcription factors defined in the database. The significance of finding a certain correlation (P-value) is calculated by the following formula: t ¼ r/O((1Àr2)/(nÀ2)), where r corresponds to the correlation value and n denotes the number of samples. The text between brackets indicates the number of tumors in the data set and the type of Affymetrix microarray. The Versteeg data set has been previously published (Molenaar et al., 2008). The other data sets were taken from the NCBI GEO database (gse3960, gse3446 and gse12460).

GATA3 binding to a newly identified GATA3 binding site Transfac database. This search yielded one highly in the Cyclin D1 promoter conserved match, 27–19 bp upstream of the Cyclin D1 To validate a functional relation between the GATA3 transcriptional start site (Figure 2c). To test whether transcription factor and Cyclin D1, we generated three GATA3 binds this cis element, we performed a different siRNAs targeting various 21 bp sequences in chromatin immunoprecipitation (ChIP) assay. Chroma- the coding region of GATA3. The best siRNA showed tin isolated from IMR-32 cells was used for a pull-down 60% silencing of GATA3 as analyzed by Q-PCR, and experiment with a GATA3 , as well as a FLAG this siRNA was selected for further experiments (data antibody as a negative control. Following DNA not shown). To analyze whether GATA3 silencing isolation, Q-PCR was performed using primers sur- results in a decrease in Cyclin D1 promoter activity, rounding the GATA3 binding site in the Cyclin D1 we used the Cyclin D1 promoter construct as described promoter (PP1), or primers further upstream in the above. The IMR-32 and SK-N-BE neuroblastoma cell promoter (PP2). The results showed a strong enrichment lines were co-transfected with the Cyclin D1 promoter for the DNA fragment containing the postulated reporter construct and a constitutively active Renilla GATA3 binding site, confirming binding of GATA3 luciferase expression vector as internal transfection to the Cyclin D1 promoter in neuroblastoma tumor cells control. In addition, these cells were transfected with (Figure 2d). To show loss of function of this GATA3 GATA3 siRNA or a GFP siRNA control. Figure 2a binding site, we performed site-directed mutagenesis on shows that GATA3 silencing results in a decreased two positions at the core GATA3 binding site Cyclin D1 promoter activity in both neuroblastoma cell (Figure 2c) in the full-length Cyclin D1 promoter lines. Inversely the overexpression of GATA3 in construct. After overexpression of GATA3, the mutated neuroblastoma cell lines with low GATA3 levels should Cyclin D1 promoter construct shows a clear reduction in lead to an induction of Cyclin D1 promoter activity. activity compared to the wild-type construct in both cell Therefore, we selected a neuroblastoma cell line with lines. The reduction in promoter activity is 40% (SHEP- undetectable GATA3 (SHEP-21-N) and a cell line with 21-N) and 35% (SK-N-AS) if we take the activity level low GATA3 (SK-N-AS) expression based on Affyme- without GATA3 overexpression as the basal level trix mRNA profiling data. These cell lines were co- (Figure 2b). The partially reduced activity in the transfected with a GATA3 overexpression construct mutated Cyclin D1 promoter reporter might indicate (Usary et al., 2004) and the Cyclin D1 promoter that there are other less conserved GATA3 binding sites reporter. The Renilla luciferase expression vector was in the Cyclin D1 promoter that also influence the used as internal transfection control. Both cell lines activity. Together these results suggest a functional role show a strong induction of Cyclin D1 promoter activity of GATA3 as transcription factor in activation of the (2.6- and 2.7-fold, respectively) on GATA3 overexpres- Cyclin D1 promoter. sion (Figure 2b). To identify whether the reduction in Cyclin D1 promoter activity resulted from a direct or indirect GATA3 silencing in neuroblastoma cell lines causes regulation by GATA3, we searched for candidate Cyclin D1 downregulation GATA3 binding sites within the Cyclin D1 promoter. Our results predicted that knockdown of GATA3 would We performed a high-stringency search for GATA3 result in reduced levels of Cyclin D1. Therefore, we binding sites conserved among (different) species using transfected the most potent GATA3 siRNA in the three GATA3 position weight matrices provided by the neuroblastoma cell line IMR-32. Control samples were

Oncogene GATA3 regulates Cyclin D1 in neuroblastoma JJ Molenaar et al 2742 120 SHEP21N SKNAS 300 300

100 250 250

80 200 200

GFP siRNA 150 150 60 GATA3 siRNA 100 100 40 promoter activity 50 50 Relative full length CCND1 20 0 0 Cyclin D1 promoter reporter +mut + mut +mut + mut

Relative full length CCND1 promoter activity 0 GATA3 over-expression -+- + -+- + IMR-32 SK-N-BE 8 CyclinD1 promoter region at 11q13 7 69163500 69164000 69164500 69165000 69165500 Chr11 position 6 5 Promoter construct -1696 186 4 Input PP2 PP1 3 ChIP primers GATA3 ab -1164 -1032-31 89 2 Ct = input - FLAG ab Δ GATA3 binding site o 1 -27/-19 0 Exon1 Q-PCR CCND1 Gene 5’UTR CCND1 -1 1 210 407 -2 -3 PP1 PP2 Figure 2 GATA3 binding to the Cyclin D1 promoter. (a) Cyclin D1 promoter activity after GATA3 siRNA knockdown in IMR-32 and SK-N-BE: both cell lines were co-transfected with the full-length Cyclin D1 promoter reporter construct and a constitutive active Renilla luciferase expression vector as an internal transfection control as described above. In addition these cells were transfected with GATA3 siRNA or a GFP siRNA control (see Supplementary Table 1 for sequences). Luciferase activity was measured and Cyclin D1 promoter activity defined as described above. The relative Cyclin D1 promoter activity in the control sample was set to 100. (b) Activity of the Cyclin D1 promoter and Cyclin D1 promoter with mutated GATA3 binding site after GATA3 overexpression in cell lines with low endogenous GATA3 expression. The Cyclin D1 promoter construct was mutated in the core GATA3 binding site at two positions using the Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Primers are given in the supplementary data. SHEP-21-N and SK-N-AS cell lines were co-transfected with the full-length Cyclin D1 promoter reporter construct or a full- length Cyclin D1 promoter construct with mutated GATA3 binding site together with a constitutively active Renilla luciferase expression vector as an internal transfection control. In addition, these cells were transfected with a pBabe plasmid containing the GATA3 coding sequence or an empty vector (Usary et al., 2004). After 48 h the Luciferase activities were measured as described above. The relative Cyclin D1 promoter activity of the cells transfected with the nonmutated construct without GATA3 overexpression was set to 100. Assays were performed in triplicate and the error bars give the standard deviation. (c) Overview of the Cyclin D1 promoter region with the location of the GATA3 binding site, the ChIP primer pairs and the full-length promoter construct. The CCND1 promoter sequence was extracted from assembly version 18 (Hg18) as provided by the UCSC Genome Browser. The conserved GATA3 binding site was detected using three available matrices for GATA3 from Transfac 11.1 (V$GATA3_01, V$GATA3_02 and V$GATA3_03). These were searched against the CCND1 promoter region using the Storm program from the CREAD package. A P-value of 0.0001 was used as a match threshold against whole-genome intergenic sequence hit-tables constructed for each genome searched. This search of the CCND1 promoter region was first carried out in the Hg18 sequence and subsequently in the orthologous regions of 19 other genomes. Requiring that a site be conserved in at least seven species, a single GATA3 binding site was identified that most closely matched the V$GATA3_03 matrix. (d) ChIP analysis using GATA3 antibody and Cyclin D1 promoter primers. A modified protocol of the Upstate ChIP kit was used (Upstate, Charlottesville, VA, USA). ChIP was performed with either 1 mg of GATA3 antibody or 1 mg of anti-FLAG-antibody (as a negative control) and protein-A agarose. For each ChIP sample, the precipitated chromatin was finally dissolved in 30 ml water. These samples were amplified using a ligation-mediated PCR. DNA (50 ng) of all ChIP samples and of the input DNA was analyzed with SYBR Green based quantitative PCR. Primer pairs were checked for specificity by melting curve analysis and gel electrophoresis. Primer pair efficiencies were determined by the slope of PCR curves, and were comparable at different template concentrations. Each ChIP and following PCR was performed in fourfold. A complete list of all the primers and used for ChIP analysis is listed in the Supplementary Table 1. The Ct was calculated using iCycler (Bio-Rad, Veenendaal, Netherlands) software and the figure shows the DCt, which is the Ct of the input sampleÀthe Ct of the calculated sample. Error bars show the standard error.

transfected with a GFP siRNA. RNA was harvested analysis showed a good knockdown of GATA3 48 h after transfection, and GATA3 and Cyclin D1 followed by a decrease in Cyclin D1 protein levels determined using Q-PCR analysis. Figure 3a (Figure 3b). Phenotypic analysis of the IMR-32 cells shows a 60% knock down of GATA3 followed by a using light microscopy clearly showed reduced cell 40% reduction of Cyclin D1 mRNA. Also western blot proliferation as well as the formation of neurite

Oncogene GATA3 regulates Cyclin D1 in neuroblastoma JJ Molenaar et al 2743 1.20

1.00

0.80 IMR-32 SK-N-BE

GATA3 shRNA 0.60 none Sh002 GATA3 none Sh002 GATA3 CCND1 GATA3 0.40 Cyclin D1 Relative gene expression Actin 0.20

0.00 GFP GATA3

IMR-32 IMR-32

siRNA GFP GATA3 GATA3

Cyclin D1

Actin GFP siRNA GATA3 siRNA

Figure 3 Silencing of GATA3 in neuroblastoma cell lines causes Cyclin D1 downregulation. (a) Q-PCR analysis of GATA3 and Cyclin D1 mRNA expression after GATA3 knockdown. IMR-32 cells were transfected with double-stranded 21 bp siRNA targeting GATA3 and with an siRNA targeting GFP as control, following previous described procedures (target sequences are listed in Supplementary Table 1) (Molenaar et al., 2008). Cells were harvested after 48 h. Trizol RNA isolation and cDNA production were performed using standard procedures. Q-PCR was carried out on cDNA with primers described in supplementary information using the real-time iCycler PCR platform (Bio-Rad) in combination with the intercalating fluorescent dye SYBR Green I. Expression was normalized to a control (PSMB1) gene and calculated using iCycler software. The expression of GATA3 and Cyclin D1 in the GFP siRNA control samples was set to 1. Error bars show the standard error. (b) Western blot of IMR32 after GATA3 knockdown. Cells used for the experiments described in Figure 3a were harvested for protein isolation using Laemmli extraction buffer. Western blot analysis was performed as described previously (Molenaar et al., 2003). Antibody descriptions can be found in the supplementary data. (c) Light microscopy pictures of cells after GATA3 or GFP control siRNA treatment after 72 h. (d) Western blot analysis of GATA3 and Cyclin D1 after lentiviral GATA3 shRNA knockdown in IMR-32 and SK-N-BE. Lentivirus expressing shRNA constructs targeting GATA3 or a control nontargeting shRNA were produced in the HEK-293T packaging cell line following standard procedures. Virus was concentrated by ultracentrifugation at 32 000 r.p.m. for 1 h, and virus titers determined by a p24 ELISA assay. A MOI (multiplicity of infection) of 1 was used for the experiments. At 24 h after virus addition, medium was refreshed, and protein isolated after an additional 72 h. Western blot analysis was as described above. extensions (Figure 3c). To exclude off-target effects, we transcription factor target genes by comparative geno- used a second RNA interference technique and another mics based on expression profiling has been shown target sequence. Lentiviral transduction was used to before (Vallania et al., 2009). We now used the same infect IMR-32 and SK-N-BE cells with a GATA3 method in reverse. Identification of correlating tran- shRNA expressing vector. Again, western blot analysis scription factors made it possible to identify a likely showed that GATA3 was efficiently silenced and that upstream regulator of an important oncogene in Cyclin D1 expression also decreased (Figure 3d), con- neuroblastic tumors. The exception formed by the firming the findings in the transient GATA3 siRNA tumor with strong genomic Cyclin D1 amplification in experiments. the GATA3/Cyclin D1 correlation analysis is striking (Figure 1b). One could consider including this outlier approach to predict upstream regulators even more accurate. Discussion The transcriptional regulation of Cyclin D1 suggests oncogenic potential for the GATA3 transcription factor. Our results identify GATA3 as a gene involved in GATA3 has been shown to be crucial in cell transcriptional regulation of Cyclin D1. Identification of differentiation by transcriptional activation of the TH

Oncogene GATA3 regulates Cyclin D1 in neuroblastoma JJ Molenaar et al 2744 and DBH noradrenaline synthesis genes, but also by GATA3 by Southern blot analysis of 105 primary controlling cell survival and early sympathetic neuronal neuroblastoma tumor samples (results not shown). development (Lim et al., 2000; Tsarovina et al., 2004; Alternatively, the overexpression of GATA3 could Hong et al., 2006, 2008). The direct transcriptional result by other upstream events that maintain a high control of Cyclin D1 could be in line with the function GATA3 expression. PHOX2B is known to regulate of GATA3 in development of a fast dividing population GATA3 and PHOX2B activating mutations have been of neural crest cells. Moreover, we have previously identified in neuroblastoma tumors (van Limpt et al., shown that Cyclin D1 is crucial in maintaining the 2004; Tsarovina et al., 2004). In addition, GATA3 undifferentiated phenotype of neuroblastoma cells regulating miRNAs could be downregulated in neuro- (Molenaar et al., 2008). This raises the possibility that blastoma. the function of GATA3 in maintaining sympathetic We have shown direct involvement of the GATA3 neuronal cells in a certain differentiation state could be transcription factor in activation of the driving mediated through its activation of Cyclin D1. Sustained oncogene Cyclin D1 that is overexpressed in 75% of activity of GATA3 in neuroblastoma cells could then be the neuroblastomas, implicating GATA3 in the patho- considered oncogenic, because it maintains the high genesis of neuroblastoma. Cyclin D1 expression with a consequent increased proliferation rate and block in further differentiation. We show an overexpression of GATA3 in neuroblas- Conflict of interest toma tumors but also in breast cancer GATA3 is known to be highly expressed in a subset of tumors (estrogen The authors declare no conflict of interest. -positive cells) (Hoch et al., 1999; Kouros-Mehr et al., 2006, 2008). Within the group of -positive cells the GATA3 transcription factor is crucial for cell-cycle progression (Eeckhoute et al., Acknowledgements 2007; Wilson and Giguere, 2008) possibly through the We kindly thank Dr RG Pestell for the full-length Cyclin D1 inhibition of CDKN2C (Pei et al., 2009). It might be pA3 promoter construct that we used to generate our pGL3 also of interest to study the activity of GATA3 on the Cyclin D1 promoter construct. The work was supported by Cyclin D1 promoter in breast cancer cells. Stichting Koningin Wilhelmina Fonds (KWF), Stichting The high GATA3 expression suggests clonal events Kindergeneeskundig Kankeronderzoek (SKK) and Kinderen but we could not detect any amplifications or gain of Kankervrij (KiKa).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene