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Homeobox genes in neuroblastoma

Revet, I.M.

Publication date2010

Link to publication

Citation for published version (APA):Revet, I. M. (2010). Homeobox genes in neuroblastoma.

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Download date:20 Aug 2021

3MSX1 induces the Wnt pathway antagonist genes DKK1, DKK2, DKK3, and SFRP1 in neuroblastoma

cells, but does not block Wnt3 and Wnt5A signalling to DVL3

Ingrid Revet, Gerda Huizenga, Jan Koster, Richard Volckmann, Peter van Sluis, Rogier Versteeg, and Dirk Geerts

Department of Human Genetics Academic Medical Center – University of AmsterdamMeibergdreef 9 1105 AZ Amsterdam The Netherlands

Published in Cancer Letters (2010) 289:195-207

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Abstract

Neuroblastoma is the most common extra-cranial solid childhood cancer; it arises from neural crest-derived cells of the sympathetic nervous system. The anomalous regulation of embryonic developmental pathways like Delta-Notch and Wnt has been implicated in aberrant cell growth and differentiation in many (childhood) tumours. We have previously found regulation of Delta-Notch pathway genes by the MSX1 neural crest development gene in a neuroblastoma cell line, and significant correlations between these genes in neuroblastic tumours. However, a clear role for the Wnt pathway in neuroblastic tumours has not yet been determined. We now analyze the complete spectrum of genes regulated by inducible expression of MSX1 in the SJNB8 neuroblastoma cell line using Affymetrix expression profiling. We show that MSX1 induces the expression of four different Wnt pathway inhibitor genes: Dickkopf 1-3 (DKK1-3) and secreted frizzled related protein 1 (SFRP1), and provide evidence that high expression of two of these genes correlates with good prognosis. We were able to demonstrate that both the canonical Wnt3 and the alternative Wnt5A ligands are highly expressed in neuroblastic tumours and cell lines, and specifically activate the DVL3 Wnt co-receptor protein in SJNB8 neuroblastoma cells. These results suggest involvement of MSX1 in Wnt signalling and demonstrate activity of the more upstream Wnt pathway in neuroblastic cells.

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Introduction

Neuroblastoma is the most common extra-cranial solid cancer in childhood; it arises from neural crest-derived cells of the sympathetic nervous system. Neuroblastomas account for ~15% of all paediatric cancer deaths. The tumours display a wide spectrum of clinical behaviour; some tumours regress spontaneously whereas others are highly malignant, with very poor prognosis despite intensive therapy [1]. The cause of neuroblastoma tumorigenesis remains largely unknown, since recurrent gene defects were identified in only about one-third of tumours. Amplification of the MYCN oncogene is found about 20% of tumours [1], while CCND1 is amplified in 3% of neuroblastomas [2]. Mutations in the PHOX2B [3,4] and ALK [5] genes are found in 4% and 8% of tumours, respectively.

Neuroblastomas belong to the group of neuroblastic tumours, which also include ganglioneuroblastomas, and ganglioneuromas. Ganglioneuromas are differentiated with an invariably good prognosis, while neuroblastomas have an undifferentiated morphology. Ganglioneuroblastomas are intermixed with a more positive prognosis [6,7]. The histological classification of neuroblastic tumours argues for a biological continuum of differentiation. This hypothesis is supported by the observation that malignant neuroblastoma tumours can occasionally transdifferentiate into ganglioneuroblastomas or ganglioneuromas. Conversely, specific (nodular) cases of ganglioneuroblastoma can progress to neuroblastoma [6-8]. Regulatory pathways that underlie these differentiation processes are therefore of special interest as potential targets for treatment.

The early formation of the neural crest, and the subsequent development, migration and terminal differentiation of neural crest-derived cells is regulated by at least three key pathways: the bone morphogenetic protein (BMP), the fibroblast growth factor (FGF), and Wnt signaling pathways [9-11]. Up-regulation of BMP in non-neural ectoderm triggers the neural crest differentiation program and expression of the MSX1 homeobox transcription factor in neural crest cells. MSX1 on its turn induces expression of BMP and Wnt in neural crest cells (reviewed in [12]). Regulation of Wnt genes by MSX was e.g. shown by loss of Wnt1 expression in Msx1/Msx2 double null-mutant mice, and by induction of Wnt1 in chick brain and lateral ectoderm following ectopic MSX1 expression [13]. While MSX1 induces BMP and Wnt, both pathways in turn up-regulate MSX1 [12,14]. Hence, the MSX genes function as intermediate between the BMP and Wnt signaling pathways and may balance the two pathways to ensure proper differentiation of neural crest cells. In general, the MSX transcription factors fulfill this function by repressing target gene expression [12,15].

Embryonal oncogenesis is thought to occur by the disruption of the normal embryonic developmental program, thereby giving rise to aberrant cell growth and differentiation, and finally to cancer. In line with this, anomalous regulation of developmental pathways such as Delta-Notch and Wnt is involved in malignant growth in different paediatric tumours [16-18]. Both pathways play key roles in normal neural crest development and neuronal differentiation, and are interesting candidate pathways

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for neuroblastoma tumorigenesis [9-12,14]. Several studies have implied the Delta-Notch pathway in neuroblastoma pathogenesis [19-23]. Also the Wnt pathway has been investigated, but the role of Wnt signaling in neuroblastoma has remained elusive [8,24-29].

The PHOX2B homeobox transcription factor is exclusively expressed in the nervous system, and is crucial for differentiation of neural crest cells into sympathetic neurons and chromaffin cells [30, 31]. PHOX2B can be mutated in both sporadic and familial neuroblastoma [3,4]. We recently showed that expression of a PHOX2B transgene silenced MSX1 expression in a neuroblastoma cell line. Accordingly, we observed an inverse correlation between PHOX2B and MSX1 expression in a panel of 110 neuroblastic tumours, with PHOX2B expression being highest in neuroblastomas and MSX1 expression peaking in ganglioneuroma and ganglioneuroblastoma [22].

We set out to identify MSX1 downstream target genes in neuroblastoma by Affymetrix

Figure 1: MSX1 induces expression of the DKK1-3 and SFRP1 Wnt inhibitor genes in SJNB8. Expression micro-array and Northern blot analysis of MSX1 expression in SJNB8-MSX1 (clones K2 and K5) cells. MSX1 expression was induced with doxycycline (50 ng/ml), and total RNA was isolated during a time course experiment at the time points indicated after addition of doxycycline (+ dox). All micro-array data in this study were produced using the Affymetrix HG-U133 Plus 2.0 platform. (Panel A): Expression micro-array analysis. Visual representation of the Affymetrix expression values for the DKK1-3 and SFRP1 Wnt inhibitor genes. Time points analyzed were 0 and 48 h after addition of doxycycline for clone K2, and 0, 24, 48, and 192 h for clone K5. In Affymetrix analyses of parallel time-course experiments with the SJNB8-TetR parental cell line, no changes in expression could be detected for DKK1-3 or SFRP1 with or without doxycycline (results not shown). (Panel B): Northern blot analysis. Northern blot autoradiographs of 20 μg of total RNA from the SJNB8-MSX1 clone K5 time series. The blots were hybridized with radio-active probes specific for DKK1-3 and SFRP1 mRNA. Time points analyzed were 0, 2, 4, and 8 h, and 1, 2, 4, 6, and 8 days after the addition of doxycycline (+ dox). Ethidium bromide staining of the 28S rRNA band of the agarose gel used for the blot is provided as a loading control.

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expression analysis using time-course experiments of SJNB8 neuroblastoma cell line clones with inducible expression of an MSX1 transgene. We have previously reported activation of the Delta-Notch signalling pathway, inhibition of cell growth, and decreased anchorage-independence as a result of MSX1 expression [22]. Here we report that the MSX1 transcription factor induces expression of several inhibitors of the Wnt pathway. We also observed significant prognostic values for these Wnt inhibitors in neuroblastic tumours. We confirm earlier data on the low activity of the canonical β-catenin/TCF Wnt signalling pathway in neuroblastoma cell lines. However, many upstream Wnt and DVL genes were well expressed in neuroblastic tumours and cell lines, and we could show activation of DVL3 by the canonical Wnt3 and non-canonical Wnt5A ligands. These data for the first time show that upstream Wnt signalling is active in neuroblastoma cells, and that key genes in the pathway are regulated by the MSX1 neural crest differentiation gene.

Materials and Methods

Generation of cell linesThe SJNB8 NB cell line was a gift from the St. Jude’s Research Hospital (Memphis,

Figure 2: DKK2 and DKK3 are correlated to MSX1 and prognostic in neuroblastic tumours. (Panel A): Correlations of MSX1-DKK2 and MSX1-DKK3 expression in the NB110 series. Affymetrix micro-array analysis of MSX1, DKK2 or DKK3 expression in NB110. Visual representation of expression values in all 110 tumours. Tumours are ranked on the horizontal axis from left to right according to their MSX1 expression. MSX1 and DKK2 or DKK3 expression values for each tumour are visualized with black circles and red rectangles, respectively. Tumour histology is depicted below the graph (12 ganglioneuromas in green, 10 ganglioneuroblastomas in yellow and 88 NBs in red). (Panel B): Prognostic value of DKK2 and DKK3 in the NB110 series. Kaplan–Meier curve for overall survival based on mRNA expression of DKK2 or DKK3 in NB110. Tumours were grouped according to high (red) or low (blue) expression for each gene (median expression cut-off for DKK2 and DKK3 was 17.4 and 734, respectively).

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TN, USA). It was derived from a metastatic NB tumour (N1108L) before treatment, and has MYCN amplification [32,33]. SJNB8 was chosen for MSX1 expression manipulation since it showed median MSX1 expression in a panel of 24 NB cell lines tested (results not shown), allowing both MSX1 over-expression and shRNA-mediated knock-down to levels comparable to those in other NB cell lines. Cell lines in this study were maintained in high-glucose DMEM without sodium-pyruvate with pyridoxine-HCl (Gibco 41965-039, Invitrogen, Breda, The Netherlands), supplemented with 10 % heat-inactivated fetal calf serum (Gibco 10106), 2 mM L-Glutamine (ICN 1680149, Cleveland, OH), 1 x Minimal Non-Essential Amino Acids (Gibco 11140-035) and 10 U penicillin/10 μg streptomycin (Sigma P0781, St. Louis, MO) per ml. All cells were maintained at 37 °C, in a humidified atmosphere containing 5% CO2. Generation of the SJNB8-TetR-MSX1 cell line clones capable of tetracycline/doxycycline-inducible MSX1 transgene expression (called SJNB8-MSX1 in this study) using the SJNB8-TetR parental cell line has been described previously [22].

RNA isolation, Affymetrix micro-array analysis and Northern blot analysis RNA isolation, Affymetrix micro-array analysis, and Northern blot were performed as described previously [22]. All expression micro-array analysis in this study was performed on a genome-wide mRNA expression platform (Affymetrix HG-U133 Plus 2.0, Santa Clara, CA). The Affymetrix probe-sets used were selected using the TranscriptView application (http://bioinfo.amc.uva.nl/human-genetics/transcriptview), and are listed in Table 1-3. Probes for Northern blot analysis

Figure 3: β-catenin activity and localization in SJNB8-MSX1 cells. (Panel A): β-catenin activity: Reporter plasmids TOP- or FOP-flash firefly luciferase, which contain several TCF4-binding elements (TOP) or mutant sequences (FOP), respectively, were transfected into each cell line together with the phRG-TK renilla luciferase standard plasmid. Colon carcinoma cell lines HCT-116 and CaCo-2A were used as a positive control. The neuroblastoma cell line SJNB8-MSX1 was treated with or without doxycycline (+ dox) for 48 h before transfection, and cell lysates were harvested 24 h after transfection. Similar low TOP-flash/FOP-flash ratios were found in the SJNB8-TetR parental cell line (not shown). Luciferase data are reported in relative light units (RLU) of relative TOP over relative FOP luciferase activity. The data shown are the means of 2 experiments. (Panel B): β-catenin localization: Western blot analysis of total lysates (Total), nuclear (N), and cytoplasmic (C) fractions from SJNB8-MSX1 cells. Total protein was isolated, and 10 μg protein used to prepare Western blots. Since the cytoplasmic fraction usually contained 3-4 times more protein than the nuclear fraction, nuclear protein bands are actually underrepresented. The blots were incubated with antisera specific to β-catenin or MSX1. In addition, the relative purity of the nuclear and cytoplasmic fractions was confirmed by probing the blots with antisera specific for the cytoplasmic marker tubulin, and the nuclear marker TBP (TATA-binding protein), respectively.

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Table 1: Genes regulated by MSX1 in SJNB8 cells. Gene: NCBI Entrez Gene name (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene). Probe-set: Affymetrix probe-set for the gene listed, selected using TranscriptView (see Materials and Methods). The following cut-offs were used for the gene selection: Expression; present call ≥ 100, change in expression during the time course; 2log fold ≥ 0.8, correlation of regulation between clones K2 and K5; cut-off: 2logPearson correlation ≥ 1.0. Clone K2, K5: expression regulation (in 2log) in SJNB8-MSX1 clones K2 and K5, respectively. P value: lowest P value during the time-course (cut-off in this test is P < 0.0025, see also [22]). KEGG Pathway: Gene assignment to KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathways in “Cellular Processes” and “Environmental Information Processing”. Information about KEGG is found on http://www.genome.jp/kegg/

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3were generated using RT-PCR on neuroblastoma cDNA with primers specific for MSX1 (5’-ccaaaaagtggctggaagag-3’ and 5’-cgatttctctgcgcttttct-3’, producing a probe encoding nucleotides 1316-1523 of the MSX1 RefSeq NM_002448), DKK1 (5’-gacaactaccagccgtaccc-3’ and 5’-tgcatttggatagctggttt-3’, 386-973 of NM_012242), DKK2 (5’-agggcctgtcttgcaaagta-3’ and 5’-cactgcatttgtcacccatt-3’, 1409-1675 of NM_014421), DKK3 (5’-agcatgtactgccagtttgc-3’ and 5’-tcatactcatcggggacctc-3’, 705-1165 of NM_015881) and SFRP1 (5’-tccaacagcaacacagccac-3’ and 5’-ttctcagcccgaaaatcgcc-3’, 1536-2054 of NM_003012). Probes were verified by sequencing before hybridisation. The SJNB8-MSX1 time-course micro-array data have been deposited for public access in a MIAME-compliant format through the Gene Expression Omnibus (GEO) database at the NCBI website under number GSE9339. Other Affymetrix data sets were retrieved from public GEO datasets on the NCBI website [34]: The EXPO dataset of 1,908 samples representing 41 different human tumor types (https://expo.intgen.org/expo/public), the Roth set of 353 samples representing 65 different normal human tissues, and three different GEO datasets for 13 samples of normal adrenal gland tissue. CEL data from the Affymetrix GeneChip Human Genome U133 Plus 2.0 array data sets were downloaded, and analyzed as described in [22]. Annotations and clinical data for the tissue samples analyzed are available from http://www.ncbi.nlm.nih.gov/geo/query/ thru their GEO ID’s: GSE2109 (EXPO), GSE3526 (Roth), and GSE3526/7307/8514 (adrenal gland).

Wnt stimulation, protein isolation, CIP treatment, and Western blot analysisPrior to Wnt stimulation, cells were grown to ~70% confluence, upon which Wnt1 (Peprotech 120-17, Neuilly-Sur-Seine, France), Wnt3, or Wnt5A (R&D Systems 1324-WN or 645-WN, respectively, Abingdon, UK) was added to the medium at a final concentration of 100 ng/ml for the time period indicated. Cells were washed with PBS and lysed using Radio-immunoprecipitation (RIPA) buffer (150 mM NaCl, 1% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, in 50 mM Tris-HCl (pH

Figure 4: Wnt gene expression in neuroblastic tumours and cell lines.(Panel A): Wnt gene expression in expression in the NB110 tumour series (NB110), 24 neuroblastoma cell lines (NBC24), and the SJNB8 neuroblastoma cell line (SJNB8). Visual representation of expression of all 18 Wnt genes analyzed using Affymetrix profiling. The graphs represent the average expression; see Table 3 for the complete data. (Panel B): Correlation of MSX1 and Wnt5A expression in the NB110 series: Visual representation of MSX1 and Wnt5A expression in all 110 tumours, ranked on the horizontal axis from left to right according to their MSX1 expression. MSX1 and Wnt5A expression values for each tumour are visualized with black circles and red rectangles, respectively. Other details are as in Figure 2A.

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8.0)) supplemented with protease (Roche RE 1836153, Woerden, the Netherlands) and phosphatase inhibitors (Calbiochem CALB524625, Merck, Nottingham, UK) according to the manufacturer’s protocol. For the nuclear and cytoplasmic protein fractions the CelLyticTM NuCLEARTM Extraction Kit was used (Promega NXTRACT, Leiden, the Netherlands). Protein concentrations were determined using the DC Protein assay kit I (Biorad 500-0116, Veenendaal, the Netherlands). CIP treatment of DVL3 protein was performed on 100 μg protein from Wnt-stimulated SJNB8 cells with 20 U of CIP (calf intestine alkaline phosphatase; New England Biolabs M0290S, Ipswich, MA) according to the manufacturer’s protocol (http://www.neb.com/nebecomm/products/protocol18asp). Total protein (10 µg) in 1x Laemmli buffer with 10% 2-mercaptoethanol was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (Millipore IPVH 00010, Amsterdam, The Netherlands) by electro-blotting with 20% (v/v) methanol, and blocked for 1 h. Membranes were incubated overnight at 4 °C with primary antiserum following

Figure 5: DVL gene expression and prognostic value in neuroblastic tumours and cell lines. (Panel A): DVL gene expression in the NB110 tumour series (NB110), 24 neuroblastoma cell lines (NBC24) and the SJNB8 neuroblastoma cell line (SJNB8). Visual representation of expression of DVL1-3 analyzed using Affymetrix profiling. The graphs represent the average expression; see Table 3 for the complete data. (Panel B): Prognostic value of DVL1-3 in the NB110 series. Kaplan–Meier curve for overall survival based on mRNA expression of DVL2 or DVL3 in NB110. Tumours were grouped according to high or low expression for each gene (median expression cut-offs for DVL2 and DVL3 were 158.6 and 149.8, respectively). DVL1 expression was not significantly prognostic at any cut-off. DVL3 expression also correlated with poor prognosis in the 88 neuroblastoma-only tumour set (median expression cut-off: 150.1, P value: 4.2 • 10-4, not shown). Other details are as in Figure 2B.

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incubation with a horseradish peroxidase conjugated secondary antiserum for 1 h and developed using enhanced chemiluminescence (GE Healthcare RPN2132, Eindhoven, The Netherlands). Blocking solution were 5% (w/v) dry milk/TBS/ 0.2% (v/v) Tween-20 for MSX1 and 5% (w/v) dry milk/TBS/ 0.1% (v/v) Tween-20 for all other antibodies. The antibodies used were: MSX1: goat anti-MSX1, sc-17727; DVL1: mouse anti-DVL1, sc-8025; DVL2 mouse anti-DVL2, sc-8027; DVL3: mouse anti-DVL3, sc-8027 all from Santa Cruz (Santa Cruz, CA); β-catenin: mouse anti-β-catenin (BD Transduction Laboratories 610153, Erembodegem, Belgium); β-actin: mouse anti-β-catenin (Abcam ab6267, Cambridge, UK); TATA-binding protein: mouse anti-TATA-binding protein (Genetex II GTX20818, Irvine, CA); Tubuline: mouse anti-tubuline (Roche 1111876).

TCF-responsive TOP-flash/FOP-flash luciferase reporter experimentsSJNB8-MSX1 cells were seeded in 24-well plates and subsequently treated with or without 100 ng/ml doxycycline to ensure induction of both MSX1 and the Wnt inhibitors 48 h prior to transfection. The plasmids phRG-TK Renilla Luciferase (Promega E6291) and FOP- or TOP-flash Firefly Luciferase (Upstate Biotechnology, Lake Placid, NY) were used for transfection with a ratio 1:8. The transfection reagent Lipofectamine 2000 (Invitrogen 11668) was used according to the standard protocol. After 24 h cell lysates were harvested. The Dual-Luciferase® Reporter Assay System (Promega E1910) was used to detect luciferase activity according to the manufacturer’s protocol.

Table 2: Wnt inhibitor gene expressions and correlations in neuroblastic tumours and cell lines. Expression of Wnt inhibitor genes in the NB110 tumour series (NB110), 24 neuroblastoma cell lines (NBC24), and the SJNB8 neuroblastoma cell line as determined by Affymetrix analysis. Average expression values are represented for each set with the number of present calls for each set represented between brackets (n). Absent means no significant expression was detected. Prognosis of each gene was determined via Kaplan Meier analysis (n.a. = not applicable, n.s. = not significant). Prognostic values were also found for DKK3 (median expression cut-off: 625.0, P value: 0.012), SFRP1 (427.3 and 3.3 • 10-5), and FRZB (362.5 and 3.3 • 10-3) in the 88 neuroblastoma-only tumor set (not shown). In all cases, higher gene expression corresponded to better survival. Other details on this analysis are as in Figure 2B. See also Figure 2.

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StatisticsAffymetrix analysis: Intensity values and their p-values were assigned to probe-sets with GeneChip® Operating Software (GCOS) using the MASS5.0 algorithm (Affymetrix Inc., Santa Barbara, CA, USA). Used in Figures 1A, 2, 4, 5, Tables 1-3, and Supplemental Figure 2. Significant changes in gene expression in time-course experiments were assessed using the p-values generated by the ‘comparison analysis’ provided by the Affymetrix GCOS software. GCOS software uses a Wilcoxon Rank sum test to calculate p-values. Within a time-course experiment, all the time points are compared to time point zero. Used in Figure 1A and Table 1. Gene expression correlations (r and P) were calculated with a 2log Pearson test. The significance of a correlation is determined by t=r/sqrt((1-r^2)/(n-2)), where r is the correlation value and n is the number of samples. Distribution measure is approximately as t with n-2 degrees of freedom. Used in Figures 2A and 4B and Tables 1 and 2. Kaplan Meier analysis was performed using a Wilcoxon log rank test [54]. The graphs depicts the logrank significance. Used in Figures 2B and 5B and Table 2. Standard error was calculated using Microsoft Excel (Microsoft, Redmond, WA). Used in Figure 3A.

Results

Characterization of the MSX1 downstream pathways in SJNB8-MSX1 cellsTo investigate the downstream signalling network of the MSX1 transcription factor, we have generated SJNB8 neuroblastoma cell line clones capable of doxycycline-inducible MSX1 transgene expression, called SJNB8-MSX1 (see Materials and Methods, and [22]). Genome-wide expression profiling of RNA isolated from time course experiments of two SJNB8-MSX1 clones, K2 and K5 were performed with Affymetrix HG-U133 Plus 2.0 arrays. The time points chosen were 0 and 48 h after doxycycline addition for K2, and 0, 24, 48, and 192 h for K5. The R2 bio-informatics platform developed in our lab (Koster et al., manuscript submitted), was used for identification of the genes regulated as a result of MSX1 induction.

We selected genes that had a 2log fold regulation of ≥ 0.8 in both clones and with correlating expression in the time-courses of both clones (2logPearson correlation ≥ 1.0). We identified 71 genes that showed a significant up- or down-regulation upon induction of the MSX1 transgene (see Table 1). They comprehend 23 down-regulated and 48 up-regulated targets suggesting that in the SJNB8-MSX1 cells, MSX1 functions as a transcriptional activator rather than as a repressor (see Introduction). Analysis of these genes using the KEGG Pathway annotation tool in R2 revealed that the Wnt pathway was among the most heavily represented signalling routes: Three Wnt pathway genes, Dickkopf 1 (DKK1), Dickkopf 3 (DKK3) and Soluble Frizzled-Related Protein 1 (SFRP1), that are all capable of blocking the Wnt signalling pathway, were significantly up-regulated by MSX1 in both clone K2 and K5. This strongly suggested that MSX1 can regulate the Wnt pathway in neuroblastoma cells. This result, together with the clear involvement of the Wnt pathway in (paediatric) tumours as well as neural crest development urged us to focus on the MSX1 target genes from this pathway.

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MSX1 induces expression of DKK1-3 and SFRP1 in SJNB8-MSX1 cells Figure 1A shows the strong up-regulation of DKK1, DKK3, and SFRP1 mRNA during an MSX1 induction time-course experiment (Figure 1A). Additionally, we identified increased expression levels of the Wnt inhibitor Dickkopf 2 (DKK2) in clone K5. DKK2 was not identified in our initial screen due to the absence of induction between 0 and 48 h in clone K2. The up-regulation of the three DKK genes and SFRP1 in clone K5 was confirmed by Northern blot analysis (Figure 1B). Expression of DKK1 and DKK2 was up-regulated within 4 h and of SFRP1 within 8 h after MSX1 induction. DKK3 up-regulation was more delayed: DKK3 mRNA was first observed after 24 h.

We asked whether the up-regulation of the Wnt inhibitors in vitro, in SJNB8-MSX1 cells, represented a regulatory principle active in vivo, in the neuroblastic tumours. We therefore analysed the expression profiles of MSX1, DKK1-3, and SFRP1 in an Affymetrix series of 110 neuroblastic tumours, called “NB110” (see Table 2). In agreement with the results obtained in the SJNB8-MSX1 cell line, MSX1 expression showed significant, positive correlation with DKK2 and DKK3 in neuroblastic tumours (Figure 2A, r = 0.313, P = 8.8 • 10-4, and r = 0.336, P = 3.4 • 10-4, respectively). No

Figure 6: Wnt stimulation of SJNB8 cells results in DVL3 phosphorylation. (Panel A): Western blot analysis of DVL3 levels in the SJNB8-MSX1 neuroblastoma cell line. MSX1 expression was induced for 48 h with 100 ng/ml doxycycline (+ dox), and stimulated with 100 ng/ml Wnt5A for respectively 90 and 180 min. Total protein was isolated, and 10 μg protein used to prepare Western blots. The blots were probed with DVL3, MSX1 and β-actin antiserum. (Panel B): As in (Panel A), but stimulation with Wnt3. A titration experiment with Wnt3 and Wnt5A showed that 100 ng/ml was necessary for DVL3 phosphorylation (Supplemental Figure 1), so this amount was used for the experiments. (Panel C): Phosphatase treatment of DVL3 protein. To prove that the DVL3 protein mobility change was due to phosphorylation, protein extracts stimulated with Wnt5A for 180 min (see Figure 6A) were treated with CIP (calf intestine alkaline phosphatase) in the presence or absence of phosphatase inhibitors as described in the Materials and Methods. The upward mobility shift was completely abolished after CIP treatment, but only in the absence of phosphatase inhibitors.

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significant correlations could be found for MSX1 and DKK1 or SFRP1 (not shown). Interestingly, high DKK2 and DKK3 expression were both indicative of good prognosis in Kaplan-Meier analysis of the neuroblastic tumour series: High expression (above average) correlated with a significant better prognosis than low expression (below average) for both genes (~ 85% versus ~ 58% survival for both genes, P = 4.7 • 10-3 and 2.3 • 10-3 for DKK2 and DKK3, respectively). These results suggested that MSX1 might be involved in neuroblastoma cell survival by interaction with the Wnt signalling pathway.

β-catenin activation and localisation in SJNB8 cells is not affected by MSX1DKK as well as SFRP proteins inhibit Wnt signalling by extracellular inhibition of the Wnt pathway. DKK proteins act by direct binding to the Wnt co-receptor LRP6. SFRPs act as a soluble frizzled receptor and can bind to either Wnt proteins or Frizzled receptors. Even though DKKs and SFRPs interact with different proteins in the Wnt pathway, both function as antagonists of normal Wnt activation (reviewed in [35-37]).We hypothesized that MSX1-induced up-regulation of these Wnt inhibitors could inhibit the downstream activity of the pathway. Wnt signalling can act on at least three downstream pathways: the well-studied canonical Wnt/β-catenin cascade and the less well characterized non-canonical pathways like planar cell polarity (PCP), and Wnt/Ca2+ signalling.

Activation of the canonical Wnt pathway ultimately leads to nuclear accumulation of β-catenin and activation of TCF-mediated transcription of target genes [16]. We tested the ability of MSX1 to inhibit the Wnt pathway using the TCF4-responsive TOP-flash/FOP-flash luciferase reporters [38]. SJNB8-MSX1 cells were transfected with TOP-flash or FOP-flash plasmid DNA 48 h after the induction of MSX1 expression with doxycycline. SJNB8-MSX1 cells that were not induced with doxycycline were taken along as a negative control, and the human colon cancer cell lines HCT-116

Table 3: Wnt and DVL expression and correlations in neuroblastic tumours and cell lines. Expression of Wnt and DVL genes in the NB110 tumour series (NB110), 24 neuroblastoma cell lines (NBC24), and the SJNB8 neuroblastoma cell line as determined by Affymetrix analysis. Other details are as in Table 2. See also Figures 4A, and 5A.

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Figure 7: Overview of the Wnt pathways discussed in this manuscript. Secreted Wnt binds to the LRP5/6 and/or Fzd receptor membrane proteins. This results in activation of DVL protein by phosphorylation. Activated DVL in its turn activates further downstream pathways. In the canonical pathway, DVL activation inhibits the phosphorylation (by e.g. GSK3B) and degradation of β-catenin. Cytoplasmic β-catenin can then accumulate, and transfer to the nucleus where it activates TCF/β-catenin- dependent downstream target genes. The Ca2+ pathway inhibits the canonical Wnt pathway, and controls cell migration, The PCP pathway is involved in cell polarity regulation and also in cell migration. Proteins discussed in this manuscript are depicted in grey. The diagram shows a hugely over-simplified scheme of the different Wnt pathways. Only the core proteins are depicted, most molecular interactions are not described, and not more than 2 of at least 9 different non-canonical Wnt pathways are shown. Adapted from [37], see also [53]).

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and CaCo-2A, which contain a constitutively activated Wnt pathway, as positive controls [39,40]. The luciferase activities showed that both colon cell lines displayed high ratios of TOP-flash/FOP-flash activity (Figure 3A). Activation of wild type TCF-binding sequences was therefore much higher than that of the mutant TCF-binding sites. In contrast, a very low TOP-flash/FOP-flash ratio was measured in the neuroblastoma cell line SJNB8-MSX1, as well as in the parental SJNB8-TetR cells (not shown), suggesting that the TOP-flash reporter construct was not significantly activated in SJNB8 cells. The TOP-flash/FOP-flash ratio was not affected by the induction of MSX1. These results suggest no or low activity of the Wnt/β-catenin cascade in SJNB8-MSX1 cells and no effect of the MSX1-regulated Wnt inhibitors on this Wnt reporter activity. This low TOP-flash/FOP-flash ratio, indicative of low Wnt/β-catenin activity, has been reported previously for both MYCN-single copy and MYCN-amplified neuroblastoma cell lines [8,25,26]. β-catenin can also regulate its downstream target genes through less well-characterized interactions with non-TCF partners such as SOX or FOXO families [41,42], to which the TOP-flash/FLOP-flash reporter constructs do not respond. We therefore also investigated the stabilization and translocation of β-catenin to the nucleus, which should precede the transcriptional activation of all β-catenin target genes in the Wnt pathway. SJNB8-MSX1 cells with or without induction with doxycycline were harvested after 48h. Total, nuclear, and cytoplasmic fractions were isolated (see Materials and Methods), and analyzed by Western blot (Figure 3B). MSX1 induction as a result of doxycycline addition is clearly visible (upper section), and is located exclusively in the nuclear fraction. The nuclear fraction was properly isolated, as shown by the enrichment for the nuclear TATA-binding protein (third section from the top). In addition, incubation of the Western blot with an anti-tubulin antiserum (fourth section) showed only very low presence of the cytoplasmic protein tubulin, indicating that the nuclear fraction is almost free of contaminating cytoplasm. However, incubation with a β-catenin-specific antiserum (second section) showed that β-catenin was present in both the nuclear and cytoplasmic fractions of SJNB8-MSX1 cells, and that this localization was not affected by MSX1 induction.

These results strongly indicate that up-regulation of Wnt inhibitor genes by MSX1 does not act on the canonical Wnt/β-catenin signalling pathway. Our results in SJNB8-MSX1 corroborate with the aberrant nuclear localization of β-catenin and low TCF-β-catenin complex activities observed in studies in other neuroblastoma cell lines [8,25,26].

Wnt5A stimulation of SJNB8 cells results in activation of DVL3As we did not detect differences in the downstream canonical Wnt pathway, we wondered whether alternative Wnt signalling might occur in neuroblastoma. We therefore studied the more upstream, plasma-membrane located, events in the Wnt signal transduction by stimulating SJNB8 cells with purified Wnt protein. We chose to look at phosphorylation of DVL by different Wnt proteins. For instance, Wnt1 and Wnt3 are known to activate DVL and the canonical Wnt pathway, while Wnt5A can activate DVL resulting in non-canonical signalling [35-37]. We first determined which

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Wnt protein genes were expressed in neuroblastic tumours and cell lines using Affymetrix profiles. Figure 4A shows that mRNA expression of 13 of the 18 Wnt family genes (all except Wnt1, -7A/B, -8B and -9B) could be detected in the NB110 tumour series, as well as in a panel of 24 neuroblastoma cell lines (NBC24). The Wnt3 and Wnt5A genes were most commonly highly expressed in NB110 and NBC24, and were present in the SJNB8 cell line (see also Table 3). Interestingly, Wnt5A expression also showed a significant positive correlation with MSX1 expression in the NB110 series (Figure 4B, r = 0.363, P = 9.5 • 10-5). A similar analysis showed that all three DVL genes were well expressed in neuroblastoma tumours and cell lines, including SJNB8 (Figure 5A). High expression of DVL2 and DVL3, but not of DVL1, showed a significant positive correlation with poor prognosis in the NB110 series (Figure 5B and not shown). Since DVL genes are activating members of the Wnt pathway, this further suggests a role for the Wnt pathway as an oncogenic route in neuroblastic tumours.

SJNB8-MSX1 cells were treated with purified Wnt5A protein, after 48 h prior incubation with doxycycline to allow accumulation of the Wnt inhibitor proteins induced by MSX1. Western blot analysis of these experiments using DVL antisera showed that DVL1 and DVL2 were not phosphorylated by Wnt5A stimulation. In contrast, DVL3 protein was shifted to a higher molecular weight, indicating complete phosphorylation, after 90 or 180 min incubation (Figure 6A). To prove that this mobility change was due to phosphorylation, protein extracts stimulated with Wnt5A for 180 min from the same time course were treated with CIP (calf intestine alkaline phosphatase) in the presence or absence of phosphatase inhibitors. The upward mobility shift was completely abolished after CIP treatment, but only in the absence of phosphatase inhibitors (Figure 6C). In similar experiments, DVL3 was also found to be phosphorylated by Wnt3 with comparable kinetics (Figure 6B). When we tested whether induction of the Wnt pathway inhibitors DKK1-3 and SFRP1 by MSX1 was able to interfere with DVL3 phosphorylation by Wnt3 or Wnt5A, we observed no effect of MSX1 induction (Figure 6A, 6B). In addition to DVL1-3, the phosphorylation of JNK, and PKC-θ, two proteins further downstream in the Wnt signalling cascades ([24, 43], see also Figure 7) were analyzed on Western blots, but no changes were detected after stimulation with Wnt5A in MSX1-induced or non-treated SJNB8-MSX1 cells (results not shown).

These experiments showed that the Wnt pathway can be activated at the plasma membrane in SJNB8 neuroblastoma cells, and that Wnt pathway genes have prognostic value in neuroblastic tumours. However, the influence of MSX1 on this Wnt signalling, as well as the exact (alternative) Wnt downstream pathways involved in neuroblastic tumours yet remain to be elucidated.

Discussion

Studies in various model organisms and experimental settings have linked MSX1 to the Wnt pathway. MSX1 and MSX2 expression in the neural crest is strongly connected

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to the BMP, Wnt and FGF signalling pathways [12-15]. Wnt signalling plays a crucial role in the induction and specification of the neural crest, but is also required for the maintenance of neural crest (-derived) cells: Mouse embryo’s lacking both Wnt1 and Wnt3 display severe loss of neural crest cells [44]. In addition, exposing naive chicken neural plates to Wnt suffices to causes the induction of neural crest cells [45]. Finally, β-catenin activation is necessary for the development of sensory neurons at the expense of other neural crest derivatives [45]. Hence, the entire process of neurulation, from development of pre-migratory neural crest [12], subsequent expansion of the neural crest cell population [46] to the survival, differentiation and proliferative control of neural progenitors [47] are all controlled by the Wnt pathway. Disturbance in any of these processes may contribute to oncogenesis in neural crest-derived cells. However, so far only few reports have described a connection between the Wnt pathway and neuroblastoma pathogenesis, and no functioning of the canonical Wnt pathway has been observed in neuroblastoma cells [8,24-29].

The finding that the MSX1 transcription factor up-regulates the DKK1-3 and SFRP1 inhibitors of the Wnt pathway provides the first direct link between MSX1 and the Wnt pathway in neuroblastoma. Although MSX1 is characterized as a transcriptional repressor [12,15], we observed that 48 of 71 MSX1 downstream genes were up-regulated, including the four Wnt inhibitor genes. The strong induction of these Wnt pathway inhibitors suggested that MSX1 may inhibit Wnt pathway signalling.

Inhibitors of Wnt signalling can function as tumour suppressor genes. In fact, the DKK1-3 and SFRP1-5 Wnt inhibitor genes have all been reported to act as tumour suppressor genes in several different tumours (examples in [8,25,48-50]). Wnt inhibitors diminish anchorage-independent growth in some cancers: Expression of DKK1 in cervical or breast cancer cell lines caused decreased colony formation and expression of SFRP1 in breast or renal cell carcinoma cell lines had similar effects [51,52]. This is in line with the reduced proliferation and anchorage-independent growth we previously observed in NB cells upon MSX1 induction [22]. Finally, we recently found that the MYCN oncogene can strongly down-regulate expression of DKK3 in neuroblastoma. DKK3 also showed an inverse correlation to MYCN expression in a neuroblastoma series, and correlated with a good prognosis, further corroborating an attenuating effect of DKK3 on tumour aggressiveness [8].

In the present study, like in previous studies by others [8,25,26], no canonical Wnt signalling could be detected in SJNB8 neuroblastoma cells, with or without MSX1 induction. This lack of activity is not due to low β-catenin/TCF4 expression, since both CTNNB1 and TCF4 mRNA are well expressed in neuroblastoma tumours and cell lines (including SJNB8), compared with other normal or cancer tissues (See Supplemental Figure 2). In addition, β-catenin protein shows nuclear localization in SJNB8 cells (see Figure 3B), which was also reported for a number of other neuroblastoma cell lines [26]. These results again define the prominent question whether Wnt signalling is at all active in neuroblastoma, and if so, which of the different downstream Wnt pathways are activated (See Figure 7 for an overview). Our results show for the first time that two Wnt proteins, Wnt3 and Wnt5A, can induce

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phosphorylation of one of the DVL co-receptors. This process was very specific, as Wnt1 did not induce phosphorylation of any DVL protein, while Wnt3 and Wnt5A only phosphorylated DVL3, not DVL1 or DVL2. Although DVL stimulation can lead to TCF/β-catenin canonical Wnt signalling in most cell types [16] earlier studies have shown uncoupling of DVL and canonical Wnt activity in N2A neuroblastoma cells [27,28]. Stimulation with Wnt3 or Wnt5A did not induce phosphorylation of JNK or PKC-θ, targets of the planar cell polarity and Wnt/Ca2+ alternative Wnt signalling [43,53], in our experiments (data not shown). This is in apparent contrast with a study by Bénard and others [24], showing that Wnt5A could phosphorylate PKC-θ in the neuroblastoma cell line IGR-N-91. However, the Wnt5A ligand has recently been identified in several additional alternative Wnt signalling pathways (for an overview see [53]). In addition, interactions have been reported between the different Wnt proteins, including between Wnt3 and Wnt5A, which influence the downstream Wnt signalling events [36,37]. This suggests that the compendium of Wnt proteins expressed at the plasma membrane by a specific tumour cell (Figure 4A) can influence the integration of Wnt signals further downstream. Additional studies into the signalling route of Wnt3/Wnt5A via DVL3 will be very important to understand Wnt signalling in neuroblastoma. Further insight in this route might also lead to an explanation of our paradoxical finding that while Wnt3/Wnt5A can activate DVL3; this was not inhibited by the four Wnt-inhibitors induced by MSX1.

In summary, induction of MSX1 up-regulates several inhibitors of the Wnt pathway in neuroblastoma cells. Both the canonical Wnt3 and the non-canonical Wnt5A can active upstream Wnt signalling in neuroblastoma cells. Expression profiles and correlation studies provided further evidence that Wnt signalling is an important signalling network for neuroblastic tumour pathogenesis, but further experiments will be needed to define the downstream Wnt signalling in neuroblastic tumours.

Acknowledgments

This work was supported by grants from the Dutch Cancer Society “KWF Kankerbestrijding” (Grants UVA 2003-2849 to D.G. and R.V., and UVA 2005-3665 to D.G.), and the Foundation for Children with Cancer “Stichting Kinderen met Kanker” (SKK, to R.V.).

Role of the Funding Source:The authors declare that the KWF and SKK organizations had no role in the design of the study, the collection, analysis or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication.

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Supplemental figures

Supplemental Figure 1: Wnt-stimulation of SJNB8 cells – titration. Titration of DVL3-phosphorylation by the addition of Wnt3 and Wnt5A. Western blot analysis of DVL3 levels in SJNB8 neuroblastoma cells after stimulation with different concentrations of Wnt3 or Wnt5A for 90 min. Total protein was isolated, and 10 μg was used to prepare a Western blot. The blot was probed with DVL3 and β-actin antiserum.

Supplemental Figure 2: Expression of β-catenin and TCF4 expression in NB, other tumours, and normal tissue. (Panel A): β-catenin (CTNNB1) mRNA expression profiling in human tumours by Affymetrix profiling. Affymetrix expression profiling in three sets of expression data in the public domain: a set of 41 different human tumor types (EXPO, 1,908 samples total), a set of 65 different normal human tissue types (Roth, 353 samples total), normal adrenal gland tissue (13 samples), together with new sets of neuroblastic tumours (total of 110 tumour samples), and 24 neuroblastoma cell lines. β-catenin was found to be well expressed in all 110 neuroblastoma tumours, and 24 neuroblastoma cell lines, with average expressions of 728 ± 22, and 825 ± 62, respectively (for comparison: average CTNNB1 expression in this set was 774, with GAPDH and ACTB expression at 9,183, and 12,867, respectively). CTNNB1 expression in the SJNB8 cell line was 752, Set annotation is below the graph. Neuroblastoma tumors and cell lines are shown in white, normal adrenal gland in black, other tumors in light grey and normal tissues (Roth) in dark grey. For reasons of representation, only Roth sets containing 4 or more samples are shown, the results remain similar to an analysis of the complete Roth set. In brackets behind the set name is the number of samples for that set (Panel B): TCF4 mRNA expression profiling. β-catenin was found to be moderately expressed in all 110 neuroblastoma tumours, and 24 neuroblastoma cell lines, with average expressions of 360 ± 23, and 469 ± 68, respectively (average TCF4 expression in this set was 872.6). TCF4 expression in the SJNB8 cell line was 770. Other details are as in (Panel A).