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Cancer Letters xxx (2008) xxx–xxx

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Transcription factor AP2alpha (TFAP2a) regulatesdifferentiation and proliferation of neuroblastoma cells

Johannes H. Schulte a,*, Jutta Kirfel b, Soyoung Lim b, Alexander Schramm a,Nicolaus Friedrichs b, Hedwig E. Deubzer c, Olaf Witt c, Angelika Eggert a,

Reinhard Buettner b

a Department of Pediatric Oncology and Hematology, University Children’s Hospital Essen, Hufelandstr. 55, 45122 Essen, Germanyb Institute of Pathology, Bonn Medical School, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany

c CCU Pediatric Oncology, German Cancer Research Center and Department for Pediatric Oncology,

Hematology and Immunology, University of Heidelberg, Germany

Received 9 February 2008; received in revised form 9 February 2008; accepted 23 May 2008

Abstract

Neuroblastoma, the most common extracranial solid tumour of childhood, is derived from neural crest progenitor cells.The TFAP2a transcription factor regulates neural crest patterning. We analysed TFAP2a protein expression in 97 primaryneuroblastic tumors and report that TFAP2a was strongly expressed in poorly differentiated neuroblastomas. TFAP2aexpression in tumor cells of differentiated neuroblastic tumors was below detection. TFAP2a was strongly expressed in4 of 6 neuroblastoma cell lines tested, and TFAP2a siRNA mediated knock down in SH-EP cells reduced proliferationand induced a more differentiated phenotype associated with an increase in the expression of the differentiation markerneurotensin.� 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Neuroblastoma; AP2alpha; TFAP2A; Differentiation; Ganglioneuroma; IHC; Neural crest

1. Introduction

Neuroblastoma is the most common extracranialtumor of childhood and accounts for approximately15% of all childhood cancer deaths [1,2]. Thisembryonal tumor originates from neural crest pro-genitor cells that fail to differentiate along their pre-

0304-3835/$ - see front matter � 2008 Elsevier Ireland Ltd. All rightsdoi:10.1016/j.canlet.2008.05.039

* Corresponding author. Tel.: +49 20172385185; fax: +492017235750.

E-mail address: johannes.schulte@uni-due.de (J.H. Schulte).

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defined route to sympathetic neurons or sympatho-adrenergic adrenal cells. The exact developmentalstage at which cells undergo malignant transforma-tion remains elusive. However, it has been suggestedthat the timepoint of malignant transformation ismirrored by the gene expression pattern of theresulting neuroblastoma [3]. Many genes temporar-ily expressed during normal neural crest develop-ment, e.g. MYCN, CD44, Delta/Notch and theneurotrophin receptors, are indeed expressed in pri-mary neuroblastomas (reviewed in [3]). Differentialexpression of these genes has been shown to be a

reserved.

ion factor AP2alpha (TFAP2a)regulates ..., Cancer Lett.

Table 1Characteristics of neuroblastic tumors used for the tissuemicroarray (TMA)

No. tumors TMA 97NB pd 60NB diff 16GN/GNB 21No. Tumors /w follow-up 68EFS 50Event 18Stage ½ 36Stage 3 11Stage 4 17Stage 4s 4

Abbreviations: NB = neuroblastoma; GN = ganglioneuroma;GNB = ganglioneuroblastoma; pd = poorly differentiated; diff =differentiating; EFS = Event-free survival.

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prognostic factor in neuroblastoma [3] and may wellmark different developmental timepoints of malig-nant transformation.

Presently, the only known mutations inducinghereditary neuroblastoma affect the gene Phox2b,a basal transcriptional regulator of neural crestdevelopment [4,5]. Other important transcrip-tional regulators involved in basic processes ofdevelopment and differentiation include the AP-2 family of basic helix-span-helix transcriptionfactors (TFAP2A-E). In mice, three of the fiveAP-2 family members (AP-2a, AP-2b, AP-2c)are temporarily expressed in neural crest cells.Despite partially overlapping expression patterns,their functions were shown to be non-redundant(reviewed in [6]). TFAP2a was expressed at E8.0(day 8 of embryonic development) in premigrato-ry neural crest cells, and gene disruption resultedin a severe loss of neural crest cells [7–9]. Inparticular, loss of AP-2 transcription factorsimpaired proliferation and induced prematuredifferentiation or apoptosis of neural crest andother cells [6].

Members of the AP-2 family have been foundto be dysregulated in various cancer types.Whether AP-2 transcription factors act as onco-genes or tumor suppressors appear to dependon the cellular context. Loss of TFAP2a in mel-anoma cells resulted in a highly metastatic phe-notype [10], but reduced proliferation ofpancreatic tumor cells [11]. TFAP2c has beenshown to induce ErbB family oncogenes in breastcancer, to be highly expressed in germ celltumors and to increase the malignant potentialof various other cancer cells [12–14]. Gershonand colleagues analysed expression of neural cresttranscription factors in a panel of various neuro-ectodermal tumors, and indeed report expressionof TFAP2a in 2 out of 11 analysed neuroblas-toma [15]. In addition, they report expression ofTFAP2a in the schwannian stroma of 3 out of3 ganglioneuroma, while TFAP2a was absent inthe neuroblastic tumor cells (i.e. ganglionic cells)of this tumors. Nevertheless, the functional roleof TFAP2a in neuroblastoma cells was notaddressed in their study [15]. Considering themajor importance of developmental neural crestgenes in neuroblastoma biology, as well as theclinical and biological relevance of differentiationand its regulation in this tumor, we asked ifTFAP2a plays a role in neuroblastomapathogenesis.

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2. Materials and methods

2.1. Tissue Microarrays (TMAs)

A TMA was prepared from formalin-fixed, par-affin-embedded tissue specimens of 97 primaryneuroblastic tumors (76 neuroblastomas, 21 gan-glioneuroma/ganglioneuroblastoma) selected fromthe archival files of the Institute of Pathology,University of Kiel Medical School, the Instituteof Pathology, University Hospital Essen, and theInstitute of Pathology, University of Bonn. Alltumors were surgically obtained from patients atthe time of diagnosis before treatment initiation.Three different tissue cores representative of therespective tumor were arrayed from formalin-fixed, paraffin-embedded tissue blocks using amanual device (Beecher Instruments, Sun Prairie,WI). Four micrometer paraffin sections were cutfrom every tissue microarray, and used for subse-quent immunohistochemical analysis within oneweek. The German Neuroblastoma Study Centreprovided complete clinical and diagnosticallyimportant molecular data for neuroblastoma sam-ples. Clinical follow-up was available for only 5 of21 ganglioneuromas/ganglioneuroblastomas, whichall were treated by initial surgery alone and notincluded into a clinical study. For 13 of 76 neu-roblastomas, no follow-up >5 yrs was available.Informed consent was obtained from all patientsor parents within the German NeuroblastomaTrail for the use of neuroblastoma tumor samplesfor research purposes (Table 1).

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2.2. Immunohistochemistry

Immunohistochemical staining was performed aspreviously described [16] using an a-TFAP2a anti-body (Active Motif, Rixenart, Belgium) diluted1:250. TFAP2a nuclear immunostaining was evalu-ated using a semi-quantitative scoring system [17].Briefly, the number and intensity of TFAP2a posi-tive neuroblastic cells (in neuroblastoma) and gan-glionic cells (in ganglioneuromas) were countedand scaled 0–4 (0 = no positive nuclei, 1 = 1–25%positive nuclei, 2 = 26–50% positive nuclei,3 = 51–75% positive nuclei, 4 = 76–100% positivenuclei). Note that no stroma cells, in particular, sch-wannian stroma cells, were included in scoringTFAP2a expression. These scores were multipliedby a staining intensity scale (0 = negative, 1 = weak,2 = moderate, 3 = intense). All slides were reviewedindependently by two pathologists in a blindedfashion.

2.3. Statistics

Statistical analysis was performed using R statis-tical language (www.r-project.org). A Kruskal–Wal-lis test was used to compare different groups(differentiation, MYCN status, event-free survival(EFS) vs. event, tumor stage). The survival packageof R was used for Kaplan–Meier analysis. Eventwas defined as either local or distant relapse or asdisease progression.

2.4. Cell culture

All neuroblastoma cell lines were grown in RPMImedium supplemented with 10% FCS, L-glutamine,penicillin, streptomycin and amphotericin B asdescribed [18].

2.5. siRNA transfection

Human SH-EP neuroblastoma cells were seededat 4000 cells/well into 96-well plates in 6 replicates(3 for microscopy readout and 3 for qRT-PCR anal-ysis) and cultured for 24 h in RPMI medium con-taining 10% FCS. TFAP2A siRNAs (Ambion, (1)#4618, (2) #4522, (3) #4710) were transfected using0.25 ll/well Dharmafect I (Dharmacon, Lafayette,CO, USA) at a final concentration of 100 nM.Scrambled siRNAs (Ambion, negative control #2,#AM4637) were used as controls (2 � 6 Replicates,separately shown as Control 1 and Control 2). Cells

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were either fixed and stained for microscopy orlysed for qRT-PCR analysis 56 h post-transfection.

2.6. Quantitative real-time PCR

RNA was extracted from six neuroblastoma celllines using the RNeasy Mini kit (Qiagen, Hilden,Germany) according to the manufacturer’s proto-col. Alternatively, 56 h after siRNA transfection,total RNA was extracted from the cells using theInvisorb 96-well kit (Invitek, Munich, Germany)following the manufacturer’s instructions. cDNAwas synthesised using TaqMan RT reagents(Applied Biosystems, Foster City, CA) followingthe protocol provided by the manufacturer. Real-time qPCR with gene-specific primers was per-formed in an 11 ll reaction mix, comprising 5.5 ll2� SybrGreen PCR mix (ABgene, Surrey, UK),3.0 ll cDNA and 2.5 ll 2 lM primers. The follow-ing program was run on an ABI-7900-HT real-timePCR machine (Applied Biosystems): 50 �C 2 min–95 �C 10 min – 45 cycles (95 �C 15 sec–60 �C1 min)–95 �C 15 sec–60 �C 15 sec–95 �C 15 sec(melting curve). Expression was normalised to 18 srRNA expression to account for inter-sample vari-ability. After normalisation for 18s rRNA, thedegree of knock down yielded by a gene-specificsiRNA was presented as percentage of the mRNAexpression of the respective gene in negative controlsiRNA-treated samples.

2.7. Fixation and staining of cells

Cells were fixed using 3% paraformaldehyde 56 hpost-transfection, permeabilised with 0.5% TritonX-100, and nuclei were stained with Hoechst33342. Apoptotic cells were detected using TUNELstaining (In situ cell death detection kit, Roche,Mannheim, Germany) for 1 h at 37 �C, at a 1:10ratio for enzyme/TUNEL reagent, diluted in50 mM Tris buffer containing 1 mg/ml BSA.

2.8. Determination of cell numbers and apoptosis

Each siRNA was tested in triplicate using threeseparate 96-well plates. An MDC ImageXPressMicro automated microscope was used to acquire9 images per well through the 10� objective. Imageanalysis was performed using Definiens eCognitionsoftware (Definiens, Munich, Germany). Numbersof Hoechst- and TUNEL-stained nuclei were aver-aged from different sites in each well, and the mean

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and standard deviation of these averages were calcu-lated for triplicates. Gene-specific siRNA-treatedsamples were then normalised to control-treatedsamples. Numbers of Hoechst-stained and apopto-tic nuclei were then presented as percentages ofthe negative control-treated samples, which wereset to 100%.

3. Results

3.1. TFAP2a expression inversely correlates with

differentiation

To assess a potential role of TFAP2a in neuroblas-toma, we first analysed TFAP2a protein expression in acohort of primary neuroblastic tumors. These includedneuroblastoma as well as its more benign and differenti-ated derivates, ganglioneuroblastoma and ganglioneu-roma. A tissue microarray was established incorporating97 primary, untreated neuroblastic tumors, including 21ganglioneuromas/ganglioneuroblastomas and 76 neurobl-astomas. Immunohistochemistry detected variable levelsof TFAP2a protein expression (Score 2–12) in the nucleiof 69 neuroblastic tumors and very weak to no expressionin 28 tumors (Score 0–1) (Fig. 1b). TFAP2a proteinexpression was significantly higher in the nuclei of neurob-lastic cells and ganglionic cells in undifferentiated orpoorly differentiated neuroblastomas compared with dif-ferentiating neuroblastomas or ganglioneuromas/ganglio-neuroblastomas (Table 2). Consistent with Gershon et al.[15], TFAP2a was also detectable in cells of the schwan-nian stroma, which is per definition predominant in gan-glioneuroma and ganglioneuroblastomas. However, asschwannian stroma cells are most often considered to beattracted non-transformed cells not originating from thetransformed original tumor cells [19,20], ganglioneuromasor ganglioneuronblastomas with negative ganglionic cellsbut positive schwann cells were considered TFAP2a neg-ative. The TFAP2a protein was not differentiallyexpressed between neuroblastic tumors of different stage(Table 2), and TFAP2a expression did not correlate withpatient age at diagnosis (Table 2).

Fig. 1. (a) Representative immunohistochemical staining of TFAPdifferentiated neuroblastomas, nuclear staining of almost all nuclei istumor cells (i.e. neuroblasts or ganglionic cells) in differentiating neuroabsent in most cases. Note the weak cytoplasmic staining occurring inpredominant schwann cell stroma. Note that the nuclei are TFAP2a neNeuroblastic tumor cells in ganglioneuroblastoma, intermixed. Greenintermixed. (b) Expression profiling of TFAP2a in neuroblastoma usiTFAP2a expression: While a large proportion of tumors displayed littlecharacterised by moderate to high TFAP2a expression. Box-plot of Tdifferentiation: TFAP2a expression in poorly differentiated neuroblasneuroblastomas and ganglioneuromas/ganglioneuroblastomas. In addsignificantly higher (p< = 0.05) than in ganglioneuromas/ganglioneuro

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3.2. TFAP2a is expressed in neuroblastoma cell lines

derived from high-stage tumors

TFAP2a expression was analysed in 6 neuroblastomacell lines, all derived from aggressive, high-stage primaryneuroblastomas and demonstrating an undifferentiatedmorphology in cell culture. Real-time quantitative PCRrevealed high TFAP2a expression in 4 of 6 cell lines(Fig. 2a). No correlation was observed between TFAP2aexpression and MYCN status. The SH-EP cell linestrongly expressed TFAP2a and was selected for furtherfunctional analyses.

3.3. TFAP2a siRNA mediated knock down resulted in

reduced cell numbers

To analyse the functional role of TFAP2a in neuro-blastoma, we used 3 different siRNAs to knock downTFAP2a mRNA expression in the SH-EP cell line. Whileno significant knock down was observed using siRNA#3,siRNA#1 and #2 reduced TFAP2a mRNA levels to42.12% and 42.51% of control cells, respectively (Fig.2b). Knock down of TFAP2a mRNA resulted in a 40%reduction in SH-EP cell numbers compared to controlcells, as measured by counting cell nuclei 56 h afterTFAP2a knock down (Fig. 3a).

3.4. Decreased proliferation and enhanced differentiation

cause reduced cell numbers following TFAP2 knock down

Although cell numbers were significantly reduced fol-lowing TFAP2a knock down, no increase in apoptosiswas detected. Apoptosis was assessed using the TUNELassay 56 h post-transfection (Fig. 3b). CDKN1A mRNAexpression was induced 3-fold at 56 h post-transfection(Fig. 3c), suggesting that the rate of proliferation wasreduced in TFAP2a knock down cells. As a decrease inproliferation is often associated with differentiation, weassessed the expression of the neuronal differentiationmarker, neurotensin. Indeed, neurotensin mRNA levelswere induced 2-fold following TFAP2a knock down, indi-cating a significant step towards a neuronal phenotype(Fig. 3 d).This is consistent with the morphological shift

2a in neuroblastic tumors (magnification, 400�). In poorlyobserved. In contrast, nuclear TFAP2a staining of neuroblastic

blastomas, ganglioneuromas or ganglioneuroblastomas is mild orsome cases. Black arrows: ganglionic tumor cells-embedded in

gative, while a weak cytoplasmic stating is observed. Red arrows:Arrow: TFAP2a positive schwann cell in ganglioneuroblastoma,ng immunohistochemistry on a tissue microarray. Histogram ofor no TFAP2a expression (score < = 3), a significant subgroup isFAP2a expression in neuroblastic tumors of different levels of

tomas is significantly higher (p< = 0.05) than in differentiatingition, TFAP2a expression in differentiating neuroblastomas is

blastomas.

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of SH-EP cells upon TFAP2a knock down from an epi-thelial-like, round shape to a more differentiated pheno-type of rectangular shape with significant neuriteoutgrowth (Fig. 2c). These data support decreased prolif-eration and enhanced differentiation rather than theinduction of apoptosis as the underlying mechanism forthe reduction in cell numbers.

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4. Discussion

Here we provide evidence that TFAP2a is highlyexpressed in a subset of neuroblastomas, and thatTFAP2a expression inversely correlates with differ-entiation of neuroblastic tumors. Based upon data

ion factor AP2alpha (TFAP2a)regulates ..., Cancer Lett.

Table 2Results of Kruskal–Wallis analysis of TFAP2a protein expressionin 97 neuroblastic tumors

Feature Strata p-value

Stage (1,2,3,4,4s) 0.06736Differentiation (NB pd, NB diff, GNB/GN) 4.61e–08Clinical Course (EFS, Event) 0.9775

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derived from functional experiments using siRNAto knock down TFAP2a expression in SH-EP neu-roblastoma cells, we suggest that TFAP2a isinvolved in maintaining an undifferentiated, highlyproliferative phenotype of neuroblastoma cells.

Several AP-2 transcription factors are expressedin the neural crest, with TFAP2a being the first fam-ily member to be expressed in undifferentiated,migrating neural crest cells. We show here thatTFAP2a protein is expressed in a subset of primaryneuroblastomas and neuroblastoma cell lines, whichare mainly characterised by an undifferentiated orpoorly differentiated morphology. This is in linewith a long-discussed model for the origin of neu-roblastomas from neural crest cells of differentdevelopmental stages and different degrees of differ-entiation [3]. Each tumor inherits the specific set ofdevelopmentally expressed transcription factors ofthe neural crest cell of origin according to thismodel. TFAP2a has been described as a marker ofundifferentiated neural crest cells, it seems plausiblethat, in particular, the undifferentiated neuroblasto-

Fig. 2. (a) Real-time quantitative PCR analysis of TFAP2aexpression in 6 neuroblastoma cell lines (MYCN non-amplified:NB69, SH-EP; MYCN amplified: NGP, LA-N-1, IMR-5, CHP-134). TFAP2a expression was normalised to expression levels of18s RNA. (b) Quantitative real-time PCR analysis of TFAP2aexpression after transfection of SH-EP cells with TFAP2a-directed siRNA (Ambion #4618 (1), Ambion #4522 (2), Ambion#4710 (3)). Expression levels were normalised to TFAP2aexpression in scrambled control siRNA-transfected SH-EP cells(marked by dotted line). Ribosomal 18s RNA served as aninternal control for expression. Note that while treatment withsiRNA (1) and (2) led to a significant decrease of TFAP2aexpression (42,12% and 42,51% of control cell expression,respectively) no decrease was detected after transfection withsiRNA (3). (c) Phase-contrast microscopy of SH-EP cellstransfected with either siRNA against TFAP2a (Ambion#4618) or control siRNA. SH-EP cells transfected with controlsiRNA displayed an undifferentiated, round-cell phenotypeidentical to the phenotype of untreated SH-EP cells. In contrast,SH-EP cells transfected with siRNA against TFAP2a (Ambion#4618) displayed a more differentiated phenotype of morerectangular cells possessing neurite-like outgrowths.

"

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mas arising from these cells are characterised byhigh expression levels of TFAP2a. It is also likelythat the more differentiated neuroblastomas arisingfrom later stages of neural crest development havereduced or lost TFAP2a expression similar to sym-pathetic ganglion cells [7].

Notably, schwannian stroma cells also expressedTFAP2a. As schwann cells are considered to benon-tumoral stroma cells of reactive nature whichare attracted within the course of differentiation[19,20], these cells cannot be included into the anal-ysis of neuroblastic tumor cells.

To discriminate whether TFAP2a expression inneuroblastic tumor cells is only a marker for thedevelopmental stage of the originator neural crestcells or whether TFAP2a has a functional role ofits own in neuroblastoma, we used an siRNA knock

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Fig. 3. Analysis of SH-EP neuroblastoma cells 56h post-siRNA (Ambion #4618) mediated TFAP2a knock down in comparison to SH-EPneuroblastoma cells treated with control siRNA. Student’s t-test was used to calculate significance levels. For proliferation and apoptosis,a two sided t-test was applied, while a single sided test was used for p21 and neurotension expression. For calculation of significance levels,measures obtained in both mock-treated control panels were pooled and compared to the siRNA-treated controls. Therefore, only one p-value is displayed per figure. (a) Cellular growth, as indicated by the number of nuclei. (b) Number of apoptotic nuclei in TUNEL staining.(c) CDKN1A expression analysed by quantitative real-time PCR. (d) Neurotensin (NTS) expression analysed by quantitative real-timePCR. Neurotensin is a well established marker of differentiation in neuroblastoma.

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down approach. Indeed, TFAP2a knock down ledto differentiation and reduced proliferation of neu-roblastoma cells. Thus, the functional implicationof TFAP2a in neuroblastoma tumor biology paral-lels its function in neural crest cells, where knock-

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out of AP-2 transcription factors also resulted inimpaired proliferation and premature differentiation[7].

Here we report the TFAP2a expression pattern ina large series of neuroblastic tumors. We show that

ion factor AP2alpha (TFAP2a)regulates ..., Cancer Lett.

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strong TFAP2a expression in neuroblastic tumorcells correlates with an undifferentiated phenotypeand a trend towards poor prognosis. Our resultssupport that TFAP2a contributes to the malignantphenotype, since down-regulation led to a more dif-ferentiated and less proliferative phenotype in vitro.This work contributes to the understanding of thecomplex network of transcription factors constitut-ing the malignant phenotype of neuroblastoma.

Acknowledgements

We thank Francoise Halley, Theresia Walter andBirte Sonnichsen (Cenix BioScience GmbH, Dres-den, Germany) for performing the siRNA experi-ments; Sabine Dreesmann for excellent technicalassistance; Kathy Astrahantseff for critical readingof the manuscript as well as Barbara Hero and theGerman Neuroblastoma Study Group for providingclinical data. A.E., O.W. and A.S. were supportedby grants from the National Genome Research Net-work (NGFN), R.B. was supported by a grant fromthe DFG.

References

[1] J.M. Maris, M.D. Hogarty, R. Bagatell, S.L. Cohn, Neuro-blastoma, Lancet 369 (2007) 2106–2120.

[2] J.M. Maris, K.K. Matthay, Molecular biology of neuro-blastoma, J. Clin. Oncol. 17 (1999) 2264–2279.

[3] G.M. Brodeur, Neuroblastoma: biological insights into aclinical enigma, Nat. Rev. Cancer 3 (2003) 203–216.

[4] Y.P. Mosse, M. Laudenslager, D. Khazi, A.J. Carlisle, C.L.Winter, E. Rappaport, J.M. Maris, Germline PHOX2Bmutation in hereditary neuroblastoma, Am. J. Hum. Genet.75 (2004) 727–730.

[5] V. van Limpt, A. Schramm, A. van Lakeman, P. Sluis,A. Chan, M. van Noesel, F. Baas, H. Caron, A.Eggert, R. Versteeg, The Phox2B homeobox gene ismutated in sporadic neuroblastomas, Oncogene 23(2004) 9280–9288.

[6] D. Eckert, S. Buhl, S. Weber, R. Jager, H. Schorle, The AP-2family of transcription factors, Genome Biol. 6 (2005) 246.

[7] P.J. Mitchell, P.M. Timmons, J.M. Hebert, P.W. Rigby, R.Tjian, Transcription factor AP-2 is expressed in neural crestcell lineages during mouse embryogenesis, Genes Dev. 5(1991) 105–119.

[8] J. Zhang, S. Hagopian-Donaldson, G. Serbedzija, J. Else-more, D. Plehn-Dujowich, A.P. McMahon, R.A. Flavell, T.Williams, Neural tube, skeletal and body wall defects in mice

Please cite this article in press as: J.H. Schulte et al., Transcrip(2008), doi:10.1016/j.canlet.2008.05.039

lacking transcription factor AP-2, Nature 381 (1996) 238–241.

[9] H. Schorle, P. Meier, M. Buchert, R. Jaenisch, P.J. Mitchell,Transcription factor AP-2 essential for cranial closure andcraniofacial development, Nature 381 (1996) 235–238.

[10] L. Zhuang, C.S. Lee, R.A. Scolyer, S.W. McCarthy, X.D.Zhang, J.F. Thompson, P. Hersey, Mcl-1, Bcl-XL and Stat3expression are associated with progression of melanomawhereas Bcl-2, AP-2 and MITF levels decrease duringprogression of melanoma, Mod. Pathol. 20 (2007) 416–426.

[11] V. Fauquette, S. Aubert, S. Groux-Degroote, B. Hemon, N.Porchet, I. Van Seuningen, P. Pigny, Transcription FactorAP-2{alpha} Represses Both the Mucin MUC4 Expressionand Pancreatic Cancer Cell Proliferation, Carcinogenesis(2007).

[12] N. Friedrichs, R. Jager, E. Paggen, C. Rudlowski, S.Merkelbach-Bruse, H. Schorle, R. Buettner, Distinct spatialexpression patterns of AP-2alpha and AP-2gamma in non-neoplastic human breast and breast cancer, Mod. Pathol. 18(2005) 431–438.

[13] R. Jager, U. Werling, S. Rimpf, A. Jacob, H. Schorle,Transcription factor AP-2gamma stimulates proliferationand apoptosis and impairs differentiation in a transgenicmodel, Mol. Cancer Res. 1 (2003) 921–929.

[14] K. Pauls, R. Jager, S. Weber, E. Wardelmann, A. Koch, R.Buttner, H. Schorle, Transcription factor AP-2gamma, anovel marker of gonocytes and seminomatous germ celltumors, Int. J. Cancer 115 (2005) 470–477.

[15] T.R. Gershon, O. Oppenheimer, S.S. Chin, W.L. Gerald,Temporally regulated neural crest transcription factorsdistinguish neuroectodermal tumors of varying malignancyand differentiation, Neoplasia 7 (2005) 575–584.

[16] P. Kahl, L. Gullotti, L.C. Heukamp, S. Wolf, N. Friedrichs,R. Vorreuther, G. Solleder, P.J. Bastian, J. Ellinger, E.Metzger, R. Schule, R. Buettner, Androgen receptor coac-tivators lysine-specific histone demethylase 1 and four and ahalf LIM domain protein 2 predict risk of prostate cancerrecurrence, Cancer Res. 66 (2006) 11341–11347.

[17] W. Remmele, H.E. Stegner, [Recommendation for uniformdefinition of an immunoreactive score (IRS) for immuno-histochemical estrogen receptor detection (ER-ICA) inbreast cancer tissue], Pathologe 8 (1987) 138–140.

[18] J.H. Schulte, S. Horn, T. Otto, B. Samans, L.C. Heukamp,U.C. Eilers, M. Krause, K. Astrahantseff, L. Klein-Hitpass,R. Buettner, A. Schramm, H. Christiansen, M. Eilers, A.Eggert, B. Berwanger, MYCN regulates oncogenic MicroR-NAs in neuroblastoma, Int. J. Cancer 122 (2008) 699–704.

[19] I.M. Ambros, A. Zellner, B. Roald, G. Amann, R. Laden-stein, D. Printz, H. Gadner, P.F. Ambros, Role of ploidy,chromosome 1p, and Schwann cells in the maturation ofneuroblastoma, N. Engl. J. Med. 334 (1996) 1505–1511.

[20] I.M. Ambros, A. Zellner, C. Stock, G. Amann, H. Gadner,P.F. Ambros, Proof of the reactive nature of the Schwanncell in neuroblastoma and its clinical implications, Prog.Clin. Biol. Res. 385 (1994) 331–337.

tion factor AP2alpha (TFAP2a)regulates ..., Cancer Lett.