Transcription factor AP2alpha (TFAP2a) regulates differentiation and proliferation of neuroblastoma cells

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Cancer Letters 271 (2008) 56–63 www.elsevier.com/locate/canlet

Transcription factor AP2alpha (TFAP2a) regulates differentiation 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, Germany b 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 primary neuroblastic tumors and report that TFAP2a was strongly expressed in poorly differentiated neuroblastomas. TFAP2a expression in tumor cells of differentiated neuroblastic tumors was below detection. TFAP2a was strongly expressed in 4 of 6 neuroblastoma cell lines tested, and TFAP2a siRNA mediated knock down in SH-EP cells reduced proliferation and induced a more differentiated phenotype associated with an increase in the expression of the differentiation marker neurotensin. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; AP2alpha; TFAP2A; Differentiation; Ganglioneuroma; IHC; Neural crest

1. Introduction Neuroblastoma is the most common extracranial tumor of childhood and accounts for approximately 15% of all childhood cancer deaths [1,2]. This embryonal tumor originates from neural crest progenitor cells that fail to differentiate along their pre-

* Corresponding author. Tel.: +49 20172385185; fax: +49 2017235750. E-mail address: [email protected] (J.H. Schulte).

defined route to sympathetic neurons or sympathoadrenergic adrenal cells. The exact developmental stage at which cells undergo malignant transformation remains elusive. However, it has been suggested that the timepoint of malignant transformation is mirrored by the gene expression pattern of the resulting neuroblastoma [3]. Many genes temporarily expressed during normal neural crest development, e.g. MYCN, CD44, Delta/Notch and the neurotrophin receptors, are indeed expressed in primary neuroblastomas (reviewed in [3]). Differential expression of these genes has been shown to be a

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.05.039

J.H. Schulte et al. / Cancer Letters 271 (2008) 56–63

prognostic factor in neuroblastoma [3] and may well mark different developmental timepoints of malignant transformation. Presently, the only known mutations inducing hereditary neuroblastoma affect the gene Phox2b, a basal transcriptional regulator of neural crest development [4,5]. Other important transcriptional regulators involved in basic processes of development and differentiation include the AP2 family of basic helix-span-helix transcription factors (TFAP2A-E). In mice, three of the five AP-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 premigratory neural crest cells, and gene disruption resulted in a severe loss of neural crest cells [7–9]. In particular, loss of AP-2 transcription factors impaired proliferation and induced premature differentiation or apoptosis of neural crest and other cells [6]. Members of the AP-2 family have been found to be dysregulated in various cancer types. Whether AP-2 transcription factors act as oncogenes or tumor suppressors appear to depend on the cellular context. Loss of TFAP2a in melanoma cells resulted in a highly metastatic phenotype [10], but reduced proliferation of pancreatic tumor cells [11]. TFAP2c has been shown to induce ErbB family oncogenes in breast cancer, to be highly expressed in germ cell tumors and to increase the malignant potential of various other cancer cells [12–14]. Gershon and colleagues analysed expression of neural crest transcription factors in a panel of various neuroectodermal tumors, and indeed report expression of TFAP2a in 2 out of 11 analysed neuroblastoma [15]. In addition, they report expression of TFAP2a in the schwannian stroma of 3 out of 3 ganglioneuroma, while TFAP2a was absent in the neuroblastic tumor cells (i.e. ganglionic cells) of this tumors. Nevertheless, the functional role of TFAP2a in neuroblastoma cells was not addressed in their study [15]. Considering the major importance of developmental neural crest genes in neuroblastoma biology, as well as the clinical and biological relevance of differentiation and its regulation in this tumor, we asked if TFAP2a plays a role in neuroblastoma pathogenesis.

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2. Materials and methods 2.1. Tissue Microarrays (TMAs) A TMA was prepared from formalin-fixed, paraffin-embedded tissue specimens of 97 primary neuroblastic tumors (76 neuroblastomas, 21 ganglioneuroma/ganglioneuroblastoma) selected from the archival files of the Institute of Pathology, University of Kiel Medical School, the Institute of Pathology, University Hospital Essen, and the Institute of Pathology, University of Bonn. All tumors were surgically obtained from patients at the time of diagnosis before treatment initiation. Three different tissue cores representative of the respective tumor were arrayed from formalinfixed, paraffin-embedded tissue blocks using a manual device (Beecher Instruments, Sun Prairie, WI). Four micrometer paraffin sections were cut from every tissue microarray, and used for subsequent immunohistochemical analysis within one week. The German Neuroblastoma Study Centre provided complete clinical and diagnostically important molecular data for neuroblastoma samples. Clinical follow-up was available for only 5 of 21 ganglioneuromas/ganglioneuroblastomas, which all were treated by initial surgery alone and not included into a clinical study. For 13 of 76 neuroblastomas, no follow-up >5 yrs was available. Informed consent was obtained from all patients or parents within the German Neuroblastoma Trail for the use of neuroblastoma tumor samples for research purposes (Table 1).

Table 1 Characteristics of neuroblastic tumors used for the tissue microarray (TMA) No. tumors TMA NB pd NB diff GN/GNB No. Tumors /w follow-up EFS Event Stage ½ Stage 3 Stage 4 Stage 4s

97 60 16 21 68 50 18 36 11 17 4

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

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2.2. Immunohistochemistry Immunohistochemical staining was performed as previously described [16] using an a-TFAP2a antibody (Active Motif, Rixenart, Belgium) diluted 1:250. TFAP2a nuclear immunostaining was evaluated using asemi-quantitativescoringsystem[17].Briefly,thenumber and intensity of TFAP2a positive neuroblastic cells (in neuroblastoma) and ganglionic cells (in ganglioneuromas) were counted and 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% positive nuclei). Note that no stroma cells, in particular, schwannian stroma cells, were included in scoring TFAP2a expression. These scores were multiplied by a staining intensity scale (0 = negative, 1 = weak, 2 = moderate, 3 = intense). All slides were reviewed independently by two pathologists in a blinded fashion. 2.3. Statistics Statistical analysis was performed using R statistical language (www.r-project.org). A Kruskal–Wallis test was used to compare different groups (differentiation, MYCN status, event-free survival (EFS) vs. event, tumor stage). The survival package of R was used for Kaplan–Meier analysis. Event was defined as either local or distant relapse or as disease progression. 2.4. Cell culture All neuroblastoma cell lines were grown in RPMI medium supplemented with 10% FCS, L-glutamine, penicillin, streptomycin and amphotericin B as described [18]. 2.5. siRNA transfection Human SH-EP neuroblastoma cells were seeded at 4000 cells/well into 96-well plates in 6 replicates

(3 for microscopy readout and 3 for qRT-PCR analysis) and cultured for 24 h in RPMI medium containing 10% FCS. TFAP2A siRNAs (Ambion, (1) #4618, (2) #4522, (3) #4710) were transfected using 0.25 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 were either fixed and stained for microscopy or lysed for qRT-PCR analysis 56 h posttransfection. 2.6. Quantitative real-time PCR RNA was extracted from six neuroblastoma cell lines using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Alternatively, 56 h after siRNA transfection, total RNA was extracted from the cells using the Invisorb 96-well kit (Invitek, Munich, Germany) following the manufacturer’s instructions. cDNA was synthesised using TaqMan RT reagents (Applied Biosystems, Foster City, CA) following the protocol provided by the manufacturer. Realtime qPCR with gene-specific primers was performed in an 11 ll reaction mix, comprising 5.5 ll 2 SybrGreen PCR mix (ABgene, Surrey, UK), 3.0 ll cDNA and 2.5 ll 2 lM primers. The following program was run on an ABI-7900-HT real-time PCR machine (Applied Biosystems): 50 °C 2 min– 95 °C 10 min – 45 cycles (95 °C 15 sec–60 °C 1 min)–95 °C 15 sec–60 °C 15 sec–95 °C 15 sec (melting curve). Expression was normalised to 18 s rRNA expression to account for inter-sample variability. After normalisation for 18s rRNA, the degree of knock down yielded by a gene-specific siRNA was presented as percentage of the mRNA expression of the respective gene in negative control siRNA-treated samples.

" Fig. 1. (a) Representative immunohistochemical staining of TFAP2a in neuroblastic tumors (magnification, 400). In poorly differentiated neuroblastomas, nuclear staining of almost all nuclei is observed. In contrast, nuclear TFAP2a staining of neuroblastic tumor cells (i.e. neuroblasts or ganglionic cells) in differentiating neuroblastomas, ganglioneuromas or ganglioneuroblastomas is mild or absent in most cases. Note the weak cytoplasmic staining occurring in some cases. Black arrows: ganglionic tumor cells-embedded in predominant schwann cell stroma. Note that the nuclei are TFAP2a negative, while a weak cytoplasmic stating is observed. Red arrows: Neuroblastic tumor cells in ganglioneuroblastoma, intermixed. Green Arrow: TFAP2a positive schwann cell in ganglioneuroblastoma, intermixed. (b) Expression profiling of TFAP2a in neuroblastoma using immunohistochemistry on a tissue microarray. Histogram of TFAP2a expression: While a large proportion of tumors displayed little or no TFAP2a expression (score < = 3), a significant subgroup is characterised by moderate to high TFAP2a expression. Box-plot of TFAP2a expression in neuroblastic tumors of different levels of differentiation: TFAP2a expression in poorly differentiated neuroblastomas is significantly higher (p< = 0.05) than in differentiating neuroblastomas and ganglioneuromas/ganglioneuroblastomas. In addition, TFAP2a expression in differentiating neuroblastomas is significantly higher (p< = 0.05) than in ganglioneuromas/ganglioneuroblastomas.

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2.7. Fixation and staining of cells Cells were fixed using 3% paraformaldehyde 56 h post-transfection, permeabilised with 0.5% Triton X-100, and nuclei were stained with Hoechst

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33342. Apoptotic cells were detected using TUNEL staining (In situ cell death detection kit, Roche, Mannheim, Germany) for 1 h at 37 °C, at a 1:10 ratio for enzyme/TUNEL reagent, diluted in 50 mM Tris buffer containing 1 mg/ml BSA.

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2.8. Determination of cell numbers and apoptosis Each siRNA was tested in triplicate using three separate 96-well plates. An MDC ImageXPress Micro automated microscope was used to acquire 9 images per well through the 10 objective. Image analysis was performed using Definiens eCognition software (Definiens, Munich, Germany). Numbers of Hoechst- and TUNEL-stained nuclei were averaged from different sites in each well, and the mean and standard deviation of these averages were calculated for triplicates. Gene-specific siRNA-treated samples were then normalised to control-treated samples. Numbers of Hoechst-stained and apoptotic nuclei were then presented as percentages of the negative control-treated samples, which were set to 100%.

transformed original tumor cells [19,20], ganglioneuromas or ganglioneuronblastomas with negative ganglionic cells but positive schwann cells were considered TFAP2a negative. The TFAP2a protein was not differentially expressed between neuroblastic tumors of different stage (Table 2), and TFAP2a expression did not correlate with patient age at diagnosis (Table 2). 3.2. TFAP2a is expressed in neuroblastoma cell lines derived from high-stage tumors TFAP2a expression was analysed in 6 neuroblastoma cell lines, all derived from aggressive, high-stage primary neuroblastomas and demonstrating an undifferentiated morphology in cell culture. Real-time quantitative PCR revealed high TFAP2a expression in 4 of 6 cell lines (Fig. 2a). No correlation was observed between TFAP2a expression and MYCN status. The SH-EP cell line strongly expressed TFAP2a and was selected for further functional analyses.

3. Results 3.1. TFAP2a expression inversely correlates with differentiation

3.3. TFAP2a siRNA mediated knock down resulted in reduced cell numbers

To assess a potential role of TFAP2a in neuroblastoma, we first analysed TFAP2a protein expression in a cohort of primary neuroblastic tumors. These included neuroblastoma as well as its more benign and differentiated derivates, ganglioneuroblastoma and ganglioneuroma. A tissue microarray was established incorporating 97 primary, untreated neuroblastic tumors, including 21 ganglioneuromas/ganglioneuroblastomas and 76 neuroblastomas. Immunohistochemistry detected variable levels of TFAP2a protein expression (Score 2–12) in the nuclei of 69 neuroblastic tumors and very weak to no expression in 28 tumors (Score 0–1) (Fig. 1b). TFAP2a protein expression was significantly higher in the nuclei of neuroblastic cells and ganglionic cells in undifferentiated or poorly differentiated neuroblastomas compared with differentiating neuroblastomas or ganglioneuromas/ganglioneuroblastomas (Table 2). Consistent with Gershon et al. [15], TFAP2a was also detectable in cells of the schwannian stroma, which is per definition predominant in ganglioneuroma and ganglioneuroblastomas. However, as schwannian stroma cells are most often considered to be attracted non-transformed cells not originating from the

To analyse the functional role of TFAP2a in neuroblastoma, we used 3 different siRNAs to knock down TFAP2a mRNA expression in the SH-EP cell line. While no significant knock down was observed using siRNA#3, siRNA#1 and #2 reduced TFAP2a mRNA levels to 42.12% 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 control cells, as measured by counting cell nuclei 56 h after TFAP2a knock down (Fig. 3a).

Table 2 Results of Kruskal–Wallis analysis of TFAP2a protein expression in 97 neuroblastic tumors Feature

Strata

p-value

Stage Differentiation Clinical Course

(1,2,3,4,4s) (NB pd, NB diff, GNB/GN) (EFS, Event)

0.06736 4.61e–08 0.9775

3.4. Decreased proliferation and enhanced differentiation cause reduced cell numbers following TFAP2 knock down Although cell numbers were significantly reduced following TFAP2a knock down, no increase in apoptosis was detected. Apoptosis was assessed using the TUNEL assay 56 h post-transfection (Fig. 3b). CDKN1A mRNA expression was induced 3-fold at 56 h post-transfection (Fig. 3c), suggesting that the rate of proliferation was reduced in TFAP2a knock down cells. As a decrease in proliferation is often associated with differentiation, we assessed the expression of the neuronal differentiation marker, neurotensin. Indeed, neurotensin mRNA levels were induced 2-fold following TFAP2a knock down, indicating a significant step towards a neuronal phenotype (Fig. 3 d).This is consistent with the morphological shift of SH-EP cells upon TFAP2a knock down from an epithelial-like, round shape to a more differentiated phenotype of rectangular shape with significant neurite outgrowth (Fig. 2c). These data support decreased prolif-

J.H. Schulte et al. / Cancer Letters 271 (2008) 56–63

Here we provide evidence that TFAP2a is highly expressed in a subset of neuroblastomas, and that TFAP2a expression inversely correlates with differentiation of neuroblastic tumors. Based upon data derived from functional experiments using siRNA to knock down TFAP2a expression in SH-EP neuroblastoma cells, we suggest that TFAP2a is involved in maintaining an undifferentiated, highly proliferative phenotype of neuroblastoma cells. Several AP-2 transcription factors are expressed in the neural crest, with TFAP2a being the first family member to be expressed in undifferentiated, migrating neural crest cells. We show here that TFAP2a protein is expressed in a subset of primary neuroblastomas and neuroblastoma cell lines, which are mainly characterised by an undifferentiated or poorly differentiated morphology. This is in line with a long-discussed model for the origin of neuroblastomas from neural crest cells of different developmental stages and different degrees of differentiation [3]. Each tumor inherits the specific set of developmentally expressed transcription factors of the neural crest cell of origin according to this model. TFAP2a has been described as a marker of undifferentiated neural crest cells, it seems plausible

a

0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 LAN1

b

IMR5

NGP

CHP134 NB69

SHEP

140.00 120.00

" Fig. 2. (a) Real-time quantitative PCR analysis of TFAP2a expression in 6 neuroblastoma cell lines (MYCN non-amplified: NB69, SH-EP; MYCN amplified: NGP, LA-N-1, IMR-5, CHP134). TFAP2a expression was normalised to expression levels of 18s RNA. (b) Quantitative real-time PCR analysis of TFAP2a expression after transfection of SH-EP cells with TFAP2adirected siRNA (Ambion #4618 (1), Ambion #4522 (2), Ambion #4710 (3)). Expression levels were normalised to TFAP2a expression in scrambled control siRNA-transfected SH-EP cells (marked by dotted line). Ribosomal 18s RNA served as an internal control for expression. Note that while treatment with siRNA (1) and (2) led to a significant decrease of TFAP2a expression (42,12% and 42,51% of control cell expression, respectively) no decrease was detected after transfection with siRNA (3). (c) Phase-contrast microscopy of SH-EP cells transfected with either siRNA against TFAP2a (Ambion #4618) or control siRNA. SH-EP cells transfected with control siRNA displayed an undifferentiated, round-cell phenotype identical 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 more rectangular cells possessing neurite-like outgrowths.

0.90 0.80

TFAP2a Expression

4. Discussion

that, in particular, the undifferentiated neuroblastomas arising from these cells are characterised by high expression levels of TFAP2a. It is also likely that the more differentiated neuroblastomas arising from later stages of neural crest development have reduced or lost TFAP2a expression similar to sympathetic ganglion cells [7]. Notably, schwannian stroma cells also expressed TFAP2a. As schwann cells are considered to be non-tumoral stroma cells of reactive nature which are attracted within the course of differentiation [19,20], these cells cannot be included into the analysis of neuroblastic tumor cells. To discriminate whether TFAP2a expression in neuroblastic tumor cells is only a marker for the developmental stage of the originator neural crest

% Expression

eration and enhanced differentiation rather than the induction of apoptosis as the underlying mechanism for the reduction in cell numbers.

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100.00 80.00 60.00 40.00 20.00 0.00 1

2

Anti TFAP2a siRNA

c

3

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J.H. Schulte et al. / Cancer Letters 271 (2008) 56–63 160

160

P=0,0034

140

140

120

120

100

80

60

40

100

80

60

40

20

20

0

0

Control1

c

b

P=1,3x10-9

Percentage of Apoptotic Nuclei

Relative Number of Nuclei

a

Control2

siRNA

Control1

d

400

siRNA

Control2

siRNA

400

P=0,027

P=0,026

350

350

300

300

Relative NTS Expression

Relative CDKN1A Expression

Control2

250

200

150

100

50

250

200

150

100

50

0

0

Control1

Control2

siRNA

Control1

Fig. 3. Analysis of SH-EP neuroblastoma cells 56h post-siRNA (Ambion #4618) mediated TFAP2a knock down in comparison to SH-EP neuroblastoma 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 pvalue 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-time PCR. Neurotensin is a well established marker of differentiation in neuroblastoma.

cells or whether TFAP2a has a functional role of its own in neuroblastoma, we used an siRNA knock down approach. Indeed, TFAP2a knock down led to differentiation and reduced proliferation of neuroblastoma cells. Thus, the functional implication

of TFAP2a in neuroblastoma tumor biology parallels its function in neural crest cells, where knockout of AP-2 transcription factors also resulted in impaired proliferation and premature differentiation [7].

J.H. Schulte et al. / Cancer Letters 271 (2008) 56–63

Here we report the TFAP2a expression pattern in a large series of neuroblastic tumors. We show that strong TFAP2a expression in neuroblastic tumor cells correlates with an undifferentiated phenotype and a trend towards poor prognosis. Our results support that TFAP2a contributes to the malignant phenotype, since down-regulation led to a more differentiated and less proliferative phenotype in vitro. This work contributes to the understanding of the complex network of transcription factors constituting the malignant phenotype of neuroblastoma.

[9]

[10]

[11]

Acknowledgements We thank Francoise Halley, Theresia Walter and Birte So¨nnichsen (Cenix BioScience GmbH, Dresden, Germany) for performing the siRNA experiments; Sabine Dreesmann for excellent technical assistance; Kathy Astrahantseff for critical reading of the manuscript as well as Barbara Hero and the German Neuroblastoma Study Group for providing clinical data. A.E., O.W. and A.S. were supported by grants from the National Genome Research Network (NGFN), R.B. was supported by a grant from the DFG.

[12]

[13]

[14]

[15]

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