Oncogene (2009) 28, 2245–2256 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/b- pathway

M Flahaut1, R Meier2, A Coulon1, KA Nardou1, FK Niggli3, D Martinet4, JS Beckmann4, J-M Joseph5,AMu¨ hlethaler-Mottet1 and N Gross1

1Department of Paediatrics, Paediatric Oncology Research, University Hospital CHUV, Lausanne, Switzerland; 2Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, USA; 3Department of Paediatrics, University Children Hospital, Zu¨rich, Switzerland; 4Medical Genetic Service, University Hospital CHUV, Lausanne, Switzerland and 5Department of Paediatrics, Paediatric Surgery, University Hospital CHUV, Lausanne, Switzerland

The development of chemoresistance represents a major Introduction obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood Drug resistance acquired in the course of therapy is a solid tumour. The mechanisms underlyingthe chemore- major obstacle in the successful treatment of many sistant phenotype in NB were addressed by expres- cancers. A promising initial response of the tumour to sion profilingof two doxorubicin (DoxR)-resistant vs chemotherapy by shrinking of the tumour volume is sensitive parental cell lines. Not surprisingly, the MDR1 frequently observed, followed by appearance of multi- gene was included in the identified upregulated , drug resistant variants and chemoresistance (Duhem although the highest overexpressed transcript in both cell et al., 1996; Ludwig et al., 2006). The multiple lines was the -1 Wnt receptor (FZD1) gene, an mechanisms contributing to chemoresistance are only essential component of the Wnt/b-catenin pathway. FZD1 partially elucidated (Gottesman et al., 2002). One of the upregulation in resistant variants was shown to mediate best-studied mechanisms conferring chemoresistance to sustained activation of the Wnt/b-catenin pathway as cancer cells is the increased drug efflux lowering revealed by nuclear b-catenin translocation and target intracellular drug concentration. This drug efflux is genes transactivation. Interestingly, specific micro- partially mediated by cell surface glycoproteins, which adapted short hairpin RNA (shRNAmir)-mediated belong to the family of ATP-binding cassette (ABC) of FZD1 silencinginduced parallel strongdecrease in the multidrug transporters (Gottesman et al., 2002). ABC expression of MDR1, another b-catenin target gene, transporters, such as MDR1 (multidrug resistance gene) revealinga complex, Wnt/ b-catenin-mediated implication and MRP1 (MDR-related ), are highly expressed of FZD1 in chemoresistance. The significant restoration in many human cancers including neuroblastoma (NB) of drugsensitivity in FZD1-silenced cells confirmed the (Haber et al., 1997; Norris et al., 1997; Blanc et al., 2003; FZD1-associated chemoresistance. RNA samples from 21 Munoz et al., 2007), and they are associated with the patient tumours (diagnosis and postchemotherapy), resistance of these tumours to chemotherapeutic drugs showed a highly significant FZD1 and/or MDR1 (Haber et al., 1997; Norris et al., 1997; Gottesman et al., overexpression after treatment, underlininga role for 2002). FZD1-mediated Wnt/b-catenin pathway in clinical che- NB is the most common extracranial solid tumour moresistance. Our data represent the first implication of in childhood that accounts for 8–10% of cancers the Wnt/b-catenin pathway in NB chemoresistance and and 15% of all cancer-related deaths in childhood identify potential new targets to treat aggressive and (Maris et al., 2007). Despite recent advances in resistant NB. combined therapies, recurrent disease in patients with Oncogene (2009) 28, 2245–2256; doi:10.1038/onc.2009.80; high risk NB remains a major clinical problem due to published online 4 May 2009 treatment failure, which is mainly attributed to the development of chemoresistance during treatment. NB Keywords: neuroblastoma; chemoresistance; FZD1; can be regarded as developmental disease as it originates MDR1; Wnt signalling from primitive cells of the sympathetic nervous system (Brodeur, 2003). During development, the Wnt signalling pathway plays a key role by controlling multiple aspects, such as proliferation, fate, specification, polarity and migration Correspondence: Dr M Flahaut, Paediatric Oncology Research, of cells. Sustained activation of this pathway by Paediatric Department, University Hospital CHUV, CH-1011 mutation is a major factor in oncogenesis in many Lausanne, Switzerland. E-mail: [email protected] cancers (Polakis, 2000; Eisenmann, 2005). Upon Wnt Received 16 December 2008; revised 6 March 2009; accepted 14 March ligand binding to cell surface receptors of the 2009; published online 4 May 2009 Frizzled family (FZD) and coreceptors from the Wnt signalling in chemoresistant NB M Flahaut et al 2246 family of low-density lipoprotein receptor-related Results (LRP-5/6), the intrinsic kinase activity of the adenomatous polyposis coli (APC)/Axin/CK1/GSK3b Identification of differentially expressed genes destruction complex is blocked. This leads to the in chemoresistant cells stabilization and accumulation of cytoplasmic b-cate- The profile of IGRN-91-R cells was nin, which is subsequently translocated to the nucleus compared with the IGRN-91 parental cell line. The (Lee et al., 2004) and thereby activated. The interaction comparison revealed 16 significantly upregulated genes of b-catenin with the TCF/LEF transcription (>4-fold) in the IGRN-91-R cell line (Table 1). As factors leads to the transcription of Wnt target expected, the MDR1/ABCB1 transcript was highly genes (Eisenmann, 2005). In a majority of tumours, overexpressed in the resistant cell line (27.5-fold aberrant activation of the Wnt/b-catenin is the conse- increase). Interestingly, FZD1 encoding the frizzled 1 quence of APC, Axin or b-catenin gene mutations receptor, a seven transmembrane receptor member of (Lustig and Behrens, 2003). Moreover, it has been the Wnt/b-catenin signalling pathway was found to be shown that overactivation of the Wnt signalling path- the highest upregulated transcript (34.5-fold stimula- way is due to the overexpression of different FZD tion) in resistant cells. Moreover, 12 out of 16 receptors in a variety of cancers (Milovanovic et al., transcripts in the list of upregulated transcripts corre- 2004; Merle et al., 2005; Ueno et al., 2008). In NB, sponded to genes located on the 7q21 region as b-catenin has been shown to be strongly expressed and published earlier (Flahaut et al., 2006b). As the 7q21 aberrantly localized in the nucleus in highly aggressive amplified region also harbours the FZD1 gene, we now NB cells without MYCN amplification, whereas no investigated whether the two MDR1 and FZD1 genes b-catenin-specific mutations were identified (Liu et al., were coamplified. Fluorescent in situ hybridization was 2008). carried out with MDR1-, FZD1- and 7 To elucidate genes and pathways involved in centromeric-specific probes (labelled green and red for chemoresistance in NB cells, we have further MDR1 and FZD1, respectively) on the resistant IGRN- analysed chemoresistant NB cell lines generated by 91-R and LAN-1-R cells and their corresponding prolonged exposure to doxorubicin (DoxR cells; sensitive counterparts. Figure 1a shows a colocalization Flahaut et al., 2006b). We have recently reported that of strong MDR1 and FZD1 signals detected in some DoxR NB cell lines exhibited overexpression of the resistant IGRN-91-R and LAN-1- of the MDR1 gene due to an amplification at the 7q21 R cells. None of the sensitive parental cell lines revealed locus. As the MDR1/P-gp inhibitor verapamil was not this cytogenetic pattern (data not shown). Interestingly, able to restore 100% of cell sensitivity to DoxR, in the resistant LAN-1-R cells, coamplified MDR1/ etoposide or paclitaxel, we postulated that P-glycopro- FZD1 signals were also detected on , tein-mediated drug efflux was not responsible for implying an underlying general mechanism conferring 100% drug resistance (Flahaut et al., 2006b). In drug resistance. However, only a few coamplified genes this study, microarray expression profile analysis of mapping in this amplicon were also found to be resistant variants revealed 7q21 region-related amplifi- upregulated at the mRNA level (data not shown). Thus, cation and overexpression of several genes, including the high level of FZD1 expression likely to be MDR1 and the Wnt receptor FZD1. These genes biologically relevant was further validated by semi- are shown to mediate chemoresistance in DoxR cell quantitative real-time PCR. MDR1/ABCB1 and FZD1 lines and patients, through Wnt/b-catenin pathway transcripts expression levels were measured in the two activation. DoxR cell lines (IGRN-91-R and LAN-1-R) compared

Table 1 Affymetrix microarray analysis of significantly upregulated genes in the IGRN-91-R cell line compared with the IGRN-91 cell line Probe set Fold increase Gene Abbreviation Accession no. CHR

204451_at 34.48 Frizzled homologue 1 (Drosophila) FZD1 NM_003505.1 7q21.13 204115_at 31.76 Guanine nucleotide-binding protein 11 GNG11 NM_004126.3 7q21.3 209994_s_at 27.45 ATP-binding cassette, subfamily B, member 1 MDR1/ABCB1 AF016535.1 7q21.12 204688_at 23.88 Sarcoglycan, epsilon SGCE NM_003919.2 7q21.3 202710_at 21.70 BET1 homologue (S. cerevisiae) BET1 BC000899.1 7q21.3 204215_at 16.68 Hypothetical protein MGC4175 MGC4175 NM_024315.1 7q21.12 219342_at 12.75 O-acetyltransferase CASD1 NM_022900.1 7q21.3 209278_s_at 11.58 Tissue factor pathway inhibitor 2 TFPI2 L27624.1 7q21.3 203301_s_at 8.77 Cyclin D-binding myb-like transcription factor 1 DMTF1 NM_021145.1 7q21.12 205694_at 8.09 Tyrosinase-related protein 1 TYRP1 NM_000550.1 9p23 210886_x_at 5.79 TP53 target gene 1 TP53AP1 AB007457.1 7q21.12 208791_at 5.09 Clusterin CLU M25915.1 8p21.1 204604_at 4.90 PFTAIRE protein kinase 1 PFTK1 NM_012395.1 7q21.13 211959_at 4.43 Human insulin-like growth factor-binding protein 5 IGFBP5 L27560.1 2q35 34031_i_at 4.22 Cerebral cavernous malformations 1 Krit1 U90268 7q21.2 220115_s_at 4.16 Cadherin 10, type 2 CDH10 NM_006727.1 5p14.2

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2247 IGRN-91-R LAN-1-R

20 40 ** ** 15 30

10 20 MDR1 5 10 relative expression 0 0 IGRN-91 IGRN-91-R LAN-1 LAN-1-R

7.5 4000 ** *** 3000 5 2000 FZD1 2.5 1000 relative expression 0 0 IGRN-91 IGRN-91-R LAN-1 LAN-1-R IGRN-91 IGRN-91-R LAN-1 LAN-1-R IGRN-91 IGRN-91-R LAN-1 LAN-1-R

MDR1/Pgp FZD1

β- β-actin

Figure 1 MDR1 and FZD1 are co-amplified and co-overexpressed in the two doxorubicin (DoxR) neuroblastoma (NB) cell lines. (a) Metaphase fluorescent in situ hybridisation (FISH) analysis (magnification  100) showing strong MDR1 signals (CTB 137N13 bacterial artificial chromosome (BAC) clone specific for MDR1, green dots, red arrows) and FZD1 signals (RP11 20K20 BAC clone specific for FZD1, red dots, red arrows) on distinct chromosomes in IGRN-91-R and LAN-1-R cells. Chromosome 7 was localized using a specific centromeric probe (chromosome 7 a-satellite probe D7Z1, red signal, white arrows). (b) MDR1 and FZD1 mRNA expression levels in the IGRN-91-R and in the LAN-1-R cells were compared to the respective parental cells by semiquantitative real- time PCR. Plots represents means±s.d. (c) Western blot analysis of MDR1/PgP (130 kDa) and FZD1 receptor (67 kDa) in total protein extracts (one representative experiment is shown). Equal loading was controlled with b-actin hybridization. with their respective counterparts (IGRN-91 and LAN- the resistant IGRN-91-R and LAN-1-R cells. Higher 1). The expression of MDR1/ABCB1 was significantly levels of the FZD1 protein were also detected in IGRN- increased 13.04±0.0009-and 30.88 ±5.3-fold in the 91-R cells compared with the IGRN-91 cells, confirming IGRN-91-R and LAN-1-R, respectively (Figure 1b). the earlier gene expression data. Despite the increase of The FZD1 transcript was upregulated with a mRNA level (4.7-fold) in the LAN-1-R cells, FZD1 2003±605.8-and 4.71 ±0.5-fold increase in the resistant receptor protein expression could not be detected in this IGRN-91-R and LAN-1-R, respectively (Figure 1b). cell line. However, the undetectable expression of the The MDR1/P-gp and FZD1 protein expression was FZD1 protein in the parental and resistant LAN-1 cells examined by western blot. As shown in Figure 1c, the is likely to be due to the low sensitivity of the antibody MDR1/P-gp glycoprotein level was highly increased in rather than the absence of the receptor in these cell lines.

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2248 The Wnt/b-catenin pathway is activated in the DoxR cells constitutive accumulation of the stabilized form of b- Deregulation of b-catenin signalling is an important catenin was detected in both DoxR cell lines, confirming event in the genesis of several human malignancies an activation of the Wnt/b-catenin pathway in these (Clevers, 2006). We, therefore, investigated whether the cells. To further investigate whether the Wnt/b-catenin overexpression of the FZD1 receptor resulted in the pathway was activated in these cells, the translocation of activation of the Wnt/b-catenin pathway in the che- active b-catenin into the nucleus was measured by moresistant DoxR cell lines. Western blot analyses were confocal immunofluorescence analysis. As shown in carried out to detect the non-phosphorylated, active Figure 2b, IGRN-91-R cells showed higher b-catenin form of b-catenin, using as positive control the colon expression with a distinct nuclear localization compared cancer SW480 cell line. This cell line displays a to their sensitive counterparts. The level and localization constitutive activation of b-catenin due to an APC gene of the staining was comparable to that of the colon mutation (Smith et al., 1993). As shown in Figure 2a, cancer cell line, SW480, harbouring a constitutional IGRN-91 IGRN-91+ LiCl IGRN-91-R LAN-1 LAN-1+LiCl LAN-1-R SW480 Active β-catenin β-actin

IGRN-91 IGRN-91-R LAN-1 LAN-1-R SW480

non specific

Active β-catenin

2 * *** 3 1.5 2 1 IGF2 0.5 1

relative expression 0 0 IGRN-91 IGRN-91-R LAN-1 LAN-1-R

3 4 ** *** 3 2 2 1 Cyclin D1 1

relative expression 0 0 IGRN-91 IGRN-91-R LAN-1 LAN-1-R Figure 2 b-Catenin is translocated into the nucleus of the doxorubicin (DoxR) cell lines. (a) Western blot analysis of the activated non-phosphorylated form of b-catenin. Sensitive cells were treated for 24 h with 20 mM of LiCl to activate b-catenin. The SW480 colon cancer cell line was used as a positive control. (b) Immunofluorescence staining for active b-catenin using a Cy3-conjugated secondary antibody (magnification  400). Nuclei are visualized with DAPI counterstain. Non-specific labelling was controlled by staining the cells with the secondary antibody alone. (c) IGF2 and Cyclin-D1 mRNA expressions were quantified by semiquantitative real-time PCR in the IGRN-91-R and LAN-1-R cell lines compared to the sensitive counterparts. Plots are means±s.d. of three experiments.

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2249 activation of the pathway. To a lesser extent, b-catenin the acquisition of a functionally activated Wnt/b-catenin nuclear staining was also observed in LAN-1-R cells. pathway in the resistant cells. Interestingly, it was mainly localized at the periphery of the nucleus. Altogether, these observations indicate that Wnt signalling is constitutively activated by transloca- Inhibition of the FZD1 receptor restores tion of active b-catenin into the nucleus of chemoresis- chemotherapeutic drug sensitivity of the resistant cells tant cells. To discern whether the increased drug resistance is the To further show a functional activation of the Wnt/b- consequence of b-catenin activation in the DoxR cells, catenin pathway in the resistant cell lines, we measured FZD1 was silenced in chemoresistant cells by lentiviral transcriptional activity of b-catenin by evaluating the micro-adapted short hairpin RNA (shRNAmir, green expression of known b-catenin target genes as a result of fluorescent protein-tagged) strategy. The extent of an increased b-catenin-mediated transcriptional activity FZD1 interference was first evaluated by semiquantita- in DoxR cells. Activation of selected target genes, such tive real-time PCR. A significant decrease in FZD1 as Cyclin-D1 (Shtutman et al., 1999; Tetsu and mRNA level was achieved, with 71 and 64% inhibition McCormick, 1999), IGF2 (Longo et al., 2002) and of its expression in the LAN-1-R-shFZD1 and IGRN- TCF4 (Kolligs et al., 2002) was measured by real-time 91-R-shFZD1, respectively, compared with the corre- semi quantitative PCR (Figure 2c). Significant increases sponding control (LAN-1-R-shC and IGRN-91-R-shC; of Cyclin-D1 (2.45±0.32-and 1.86 ±0.25-fold increase) Figure 3a). We functionally evaluated the consequence and IGF2 expression (2.9±0.07-and 1.38 ±0.09-fold of FZD1 interference on cell sensitivity to doxorubicin, increase) were detected in LAN-1-R and IGRN-91-R, etoposide, paclitaxel or cisplatin. For this purpose, the respectively. Surprisingly, decreased TCF4 expression FZD1-silenced DoxR cells were treated with the was measured in LAN-1-R cells (0.723±0.063-fold different drugs, and cell viability was measured using decrease), although it was not changed in IGRN-91-R MTS/PMS assay. As shown in Figure 3b, FZD1 cells (data not shown). These results strongly support silencing significantly restored LAN-1-R cell sensitivity

ns 2500 ns *** 10 2000 7.5 *** 1500 5

FZD1 1000 500 2.5

relative expression 0 0 nt nt shC shFZD1 nt nt shC shFZD1 IGRN-91 IGRN-91-R LAN-1 LAN-1-R

IGRN-91 IGRN-91-R-shC LAN-1 LAN-1-R-shC IGRN-91-R IGRN-91-R-shFZD1 LAN-1-R LAN-1-R-shFZD1 110 * 100 *** ** *** 100 *** *** 90 *** 90 80 80 *** 70 70

Viability, % 60 60

50 50

40 40

Cisplatin Cisplatin Etoposide Paclitaxel Etoposide Paclitaxel Doxorubicin Doxorubicin Figure 3 FZD1 silencing partially restores sensitivity of neuroblastoma (NB) cells to different chemotherapeutic drugs. (a) FZD1 mRNA expression was measured in IGRN-91-R and LAN-1-R cells infected with non-specific lentiviral micro-adapted short hairpin RNA (shRNAmir) control particles (shC) or specific shRNAmir particles against FZD1 (shFZD1). Expression was related to that of the sensitive non-transfected corresponding cell lines. Bars are means±s.d. of 2–6 experiments. (b) Cells were plated in a 96-wells dish. Cell viability was measured and compared with the non-treated cells (100% viability) after 48-h drug treatment (1 mM doxorubicin (Sigma), 100 mg/ml etoposide (Calbiochem, Nottingham, UK), 100 nM paclitaxel (Sigma) or 16 mg/ml cisplatin (Sigma)). Bars are means±s.d. of 2–3 experiments.

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2250 to the different drugs in a dose-dependent manner MDR1 inhibitor verapamil on the drug sensitivity in (Figure 3b, only one dose of each drug is shown). FZD1 these cells was measured by caspase-3 activity. As shown silencing in IGRN-91-R cells was less effective in in Figure 4a, this had no apparent additive effect on restoring doxorubicin, etoposide and cisplatin sensitiv- LAN-1-R-shFZD1 cell death. The above-described ity, whereas the IGRN-91-R-shFZD1 cells remained results reveal that related mechanisms are used by fully resistant to paclitaxel (Figure 3b). MDR1 and FZD1 to induce chemoresistance in NB We next explored the mechanisms involved in drug cells. sensitization after FZD1 silencing. The induction of a In addition, as illustrated in Figure 4b, the expression caspase-dependent cell death was measured by caspase- of MDR1/ABCB1 was significantly decreased in the 3-like activity assay. As shown in Figure 4a, shRNAmir- LAN-1-R-shFZD1 group (75% inhibition in the LAN- mediated FZD1 silencing in the LAN-1-R group (LAN- 1-R-shFZD1 and 18% in the IGRN-91-R-shFZD1 1-R-shFZD1) resulted in a higher relative caspase-3-like compared with the shRNAC-transfected cells). activity in response to etoposide, paclitaxel or cisplatin, compared with the control cells (LAN-1-R-shC). In Expression of FZD1 receptor in patient tumours addition, the effect of simultaneous inhibition of FZD1 To investigate whether FZD1 was associated with and MDR1/P-gp-mediated drug efflux by adding the clinical chemoresistance in NB patients, tumour samples

LAN-1 LAN-1-R-shC LAN-1-R LAN-1-R-shFZD1 17.5 10 15 8 12.5

6 10 7.5 * 4 **

relative values 5 * Caspase-3 activity 2 2.5 0 0 no ttt doxorubicin doxorubicin no ttt etoposide etoposide verapamil verapamil 20 15

15 10 10 *** ** ** 5

relative values 5 Caspase-3 activity

0 0 no ttt paclitaxel paclitaxel no ttt cisplatin cisplatin verapamil verapamil ns 30 50

40 *** 20 30 20 MDR1 MDR1 10 10 relative expression relative expression 0 0 ntnt shC shFZD1 ntnt shC shFZD1 IGRN-91 IGRN-91-R LAN-1 LAN-1-R Figure 4 FZD1 silencing induces caspase-dependent apoptosis in the LAN-1-R in response to the different drugs through MDR1 downregulation. (a) LAN-1, LAN-1-R, LAN-1-R-shC and LAN-1-R-shFZD1 were non-treated (no ttt) or treated for 48 h with the different drugs (1 mM doxorubicin, 100 mg/ml etoposide, 100 nM paclitaxel or 32 mg/ml cisplatin) in presence or absence of 10 mM verapamil (Sigma). Caspase-3-like activities relative to non-treated cells were measured for each condition. The caspase-3/7 inhibitor DEVD-fmk was used as control to inhibit hydrolysis of DEVD-pNA (data not shown). Bars are means±s.d. of duplicates in one representative experiment. (b) MDR1 mRNA expression was measured in IGRN-91-R and LAN-1-R cells infected or not with non- specific lentiviral micro-adapted short hairpin RNA (shRNAmir) control particles (shC) or specific shRNAmir particles against FZD1 (shFZD1) compared to the parental cells. Bars are means±s.d. of 2–6 experiments.

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2251 removed at diagnosis, and after chemotherapy in a or MDR1 expression after chemotherapy, whereas no cohort of 21 patients with stage 3 or 4 tumours were significant increase was observed in the non-relapsed analysed. The patients were divided into two groups group of patients. Despite the difficulty to obtain a large according to their clinical outcome: Group 1 includes number of matched patient samples, these results patients who relapsed after chemotherapy; Group 2 obtained on a cohort of 21 paired patients nevertheless comprises patients who did not relapse and whose strongly indicate that Wnt/b-catenin activation is tumours were removed after one or several courses of involved in chemoresistance in patients. chemotherapy (Table 2). Matched pre-and post- treatment samples (after one or several cycles of chemotherapy) were compared for FZD1 and MDR1 mRNA expression by semiquantitative real-time PCR. Discussion Among the group of relapsed patients, 70% of patient samples (7/10, P ¼ 0.0098) showed a significant increase Constitutive or acquired resistance of cancer cells to of FZD1 expression, and 60% of patient samples (6/10, multiple drugs remains a major clinical challenge in a P ¼ 0.002) showed an increase of MDR1 expression in majority of cancers, and most deaths result from their tumours after chemotherapy (Figure 5 and resistant, progressive disease. One of the major, Table 2). In two patients, the tumours after chemother- extensively studied mechanisms of chemoresistance, is apy showed an increased FZD1 expression alone with- the overexpression of several ATP-dependent drug out an increase of MDR1, and in another patient, an efflux pumps (Gillet et al., 2007). In NB as well, high MDR1 increase alone was observed. Moreover in five levels of MDR1 mRNA are often associated with (50%) samples, a simultaneous increase of FZD1 and acquired resistance to chemotherapy (Goldstein et al., MDR1 was measured. In parallel, in the non-relapsed 1990) and thus poor survival of the patients. group of patients, only one patient showed a significant We recently reported that two chemoresistant NB cell increase of FZD1 expression (1/11, 9%, P>0.05) and lines, generated by prolonged exposure to doxorubicin, five patients showed an increase in MDR1 expression (5/ acquired a similar amplification on chromosome 7 (7q21 11, 45%, P>0.05). Overall, the group of relapsed amplicon), harbouring the region of the MDR1 gene patients showed a significant increase of either FZD1 (Flahaut et al., 2006b). A strong increase of MDR1

Table 2 Clinical characterisitcs of the patients and their respective FZD1 and MDR1 expressions Patient Sex Date of Stagea MYCN Sampleb Date of FZD1 expression MDR1 expression diagnosis status death (year) Fold changec P-value Fold change P-value

Group 1: relapse E-1 M 1987 4 NA BM/PT 1988 2.968±0.572 0.028 1.401±0.078 0.0082 Q-2 M 2002 4 NA HM/nodule 51.23±10.37 0.0084 1.623±0.114 0.0058 GGL R-3 M 2003 4 NA PT/PT 25.97±1.484 o0.0001 17.63±0.671 o0.0001 X-4 F 1991 4 A GGLM/BM 1993 1.026±0.061 NS 1.23±0.457 NS Y-5 M 1994 4 NA GGLM/PT 1997 4.105±0.675 0.0451 1.956±0.241 NS A-6 M 1985 3 A PT/PT 1986 1.627±0.451 NS 2.754±0.124 0.0001 G-7 M 1991 3 NA PT/PT 8.201±0.123 o0.0001 57.56±7.039 0.0013 J-8 F 1992 3 A PT/PT 1993 3.607±0.324 0.0013 1.621±0.115 0.0065 F-9 M 2006 4 NA PT/PT 2006 decreased 5.881±1.399 NS B-10 M 2004 4 NA BM/BM 2009 4.543±0.436 0.0157 2.771±0.763 NS

Group 2: no relapse C-11 F 1986 4 ND PT/PT 1.724±0.261 NS 0.663±0.067 0.0069 H-12 M 1991 4 NA PT/SKM 0.497±0.022 o0.0001 1.818±0.079 0.0005 I-13 M 1992 4 NA PT/PT 0.337±0.076 0.0008 1.388±0.218 NS N-14 M 1997 4 A PT/PT 0.907±0.191 NS 0.324±0.163 0.0137 O-15 F 1998 4 A PT/PT 0.767±0.028 0.0014 13.28±2.856 0.0129 P-16 F 1999 4 NA GGLM/PT 1.555±0.355 NS 0.371±0.062 0.0005 D-17 M 1987 4 A BM/BM 1.187±0.133 NS 14.39±2.755 0.0083 K-18 M 1992 4 NA GGLM/PT 43.56±1.642 o0.0001 2.476±0.394 0.0204 L-19 M 1992 4 NA BM/PT 15.61±3.506 NS 3.895±0.430 0.0026 S-20 F 2003 3 NA PT/PT 0.553±0.351 NS 0.766±0.514 NS T-21 F 2008 3 NA PT/PT 0.891±0.154 NS 0.885±0.179 NS

Abbreviations: A, amplified; BM, bone marrow; GGL, ganglion; GGLM, ganglion metastasis; HM, hepatic metastasis; NA, non amplified; ND, not determined; NS, non-statistically significant; PT, primary tumour; SKM, skin metastasis. aInternational Neuroblastoma Staging System (INSS). bSample at diagnosis/sample after treatment. cFold change is the mean of three different experiments±s.d.

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2252 Group 1 Group 2

60 ** 60 ns 50 50 40 40 25

20 20 15

10 10 FZD1 mRNA level (relative expression) 5 0 0 before ttt after ttt before ttt after ttt

** ns 60 60 50 50 40 40 20 20

15 15

10 10 MDR1 mRNA level (relative expression) 5 5

0 0 before ttt after ttt before ttt after ttt Figure 5 FZD1 and MDR1 mRNA expression was measured by real-time semiquantitative PCR from patients’ paired tumour samples in (a) relapsed patients (Group 1) and (b) in non-relapsed patients (Group 2), before and after chemotherapy (ttt). The expression value at diagnosis was used as the reference. Scatter plots are means±s.d. of three experiments in triplicates.

expression was in consequence measured in both 2008). However, the Wnt/b-catenin pathway has not yet resistant variants. However, acquired chemoresistance been implicated in chemoresistance in any cancer and is not restricted to MDR1/P-gp overexpression as this study is the first to unravel a potential role of the sensitivity could only partially be restored by the Wnt/b-catenin pathway in mediating chemotherapy addition of the P-gp inhibitor verapamil, and resistance resistance. to cisplatin is not mediated by P-gp-mediated efflux Here, we explored the mechanisms of chemoresistance (Flahaut et al., 2006b). acquisition in NB cells and determined whether resis- Comparison of gene expression profiles of sensitive vs tance could be mediated by overexpression of the FZD1 DoxR cell lines revealed FZD1, a major player of Wnt/ receptor and subsequent activation of the Wnt/b-catenin b-catenin signalling, as the highest overexpressed gene in pathway. both DoxR cell lines, in addition to MDR1 (Table 1). Comparison of resistant and sensitive cell lines by Sustained activation of the canonical Wnt pathway due gene expression profile analysis revealed a strong over- to mutational deregulation of Wnt cascade components, expression of several genes, including FZD1 and MDR1. such as APC, axin or b-catenin, is associated with several The latter is already known for its role in conferring cancers (Lustig and Behrens, 2003; Gregorieff et al., chemoresistance in cancers (Gottesman et al., 2002; 2005). Moreover, abnormal activation of the Wnt Modok et al., 2006). The strong expression of FZD1 signalling pathway could be independent of Wnt suggested a potential role of the Wnt/b-catenin pathway component mutations (Bafico et al., 2004; Merle et al., in the development of doxorubicin resistance. Hence, we 2005). Recently Liu et al. (2008) described activation of subsequently investigated whether the Wnt/b-catenin the pathway in the absence of activating mutations in pathway was constitutively activated. Western blot NB cell lines and primary NBs. Therefore, upstream analyses revealed that, even in the absence of the elements, such as the aberrantly signalling frizzled ligand, both resistant cell lines expressed significantly receptors and their corresponding ligands, are potential enhanced levels of the active, non-phosphorylated form candidates for activation of the canonical Wnt pathway. of b-catenin, indicating a constitutive activation of Mutations or altered expression of several compo- Wnt/b-catenin signalling in these cells. The overall nents of the Wnt/b-catenin pathway leading to its accumulation and preferred nuclear localization of pathological activation have been described to promote active b-catenin as detected by confocal microscopy in tumour progression and survival (Holcombe et al., 2002; both resistant cell lines, but particularly pronounced in Milovanovic et al., 2004; Merle et al., 2005; Ueno et al., IGRN-91-R cells, which also express extremely high

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2253 levels of the Wnt receptor FZD1, provided additional contribute to Dox-mediated cell death cannot be confirmation of the functional Wnt/b-catenin canonical excluded and are currently under investigation. Inter- pathway in chemoresistant cells. Like others, we were estingly, the addition of the MDR1-efflux pump unable to convincingly show b-catenin transactivation inhibitor verapamil to the shRNAmirFZD1 cells did using the pTOP/pFOP-flash luciferase reporter assay not further sensitize cells to drugs, indicating that the (Liu et al., 2008). The inability to measure TCF/LEF two mechanisms are interrelated. transcriptional activity in these cells may be a common Hence, this study shows for the first time the phenomenon in NB. It was proposed that in NB cells, b- involvement of FZD1 in chemoresistance. Our results catenin may function through interactions with non- indicate that FZD1 activates the Wnt/b-catenin path- TCF/LEF heterodimeric partners, such as those of the way and thereby leads to an upregulation of the MDR1 SOX or FOXO families (Sinner et al., 2004; Essers et al., gene, which is known to mediate chemoresistance. In 2005). Alternatively, mutations in TCF/LEF could also addition to the FZD1-mediated increase of MDR1 explain absence of TCF-dependent transcriptional expression through Wnt/b-catenin activation, the tran- activity in these cells (Wielenga et al., 1999). We, scription of several genes involved in cell proliferation therefore, showed the b-catenin transactivation in and survival, such as Cyclin-D1 and IGF2 was also chemoresistant cells through expression of b-catenin enhanced. This indicates that FZD1 not only confers target genes. The confirmation that the Wnt/b-catenin chemoresistance to cancer cells, but may also enhance canonical pathway is activated was given by the their growth advantage through the upregulation of observation of an increased transcription of several genes increasing cell proliferation and survival. well-recognized Wnt/b-catenin target genes, including Chromosomic alterations in chromosome 7q, in Cyclin-D1 and IGF2 (Figure 2c). In contrast, TCF4, particular, gains and amplifications (Lastowska et al., another known b-catenin target, revealed no transcrip- 2001; Bedrnicek et al., 2005; Johnsson et al., 2005), have tional changes in chemoresistant variants compared been frequently described in NB and other tumours. It is with sensitive cells. thus essential to exclude that MDR1 and FZD1 MDR1, another Wnt pathway target gene (Yamada amplification and overexpression are exclusively related et al., 2000), was also strongly upregulated in the to cell culture. Overexpression of MDR1 in patient resistant cell lines compared to their respective sensitive tumour tissue following chemotherapy is well documen- counterparts (Figures 1b and c). The MDR1 upregula- ted, whereas increased expression of FZD1 has not been tion in resistant cells was initially attributed to the reported (Haber et al., 1997; Norris et al., 1997; Blanc chromosomal amplification of the MDR1 gene locus et al., 2003; Munoz et al., 2007). Moreover, attempts to resulting from the 7q21 amplicon (Flahaut et al., 2006b). overcome P-gp-mediated drug resistance in patients Interestingly, specific silencing by shRNAmirFZD1 using specific inhibitors of P-gp has had limited success revealed a concomitant decrease in MDR1 expression (Duhem et al., 1996; Ludwig et al., 2006). Hence, we in the LAN-1-R-shFZD1 cells (Figure 4b), suggesting measured the mRNA expression of FZD1 and MDR1 that FZD1 mediates MDR1 transcription through in 21 pre-(at diagnosis) and post-treatment(after one or b-catenin activation (Yamada et al., 2000). Altogether, more cycles of chemotherapy)-matched patient samples, our data indicate that FZD1 may mediate chemoresis- including 10 relapsed and 11 non-relapsed patients. All tance by regulating the transcription of Wnt/b-catenin samples were derived from patients diagnosed with targets, thereby leading to increased MDR1 transcription. advanced stage 3 and 4 disease, associated to poor 5- Consequently, we investigated the specific influence of year survival (Brodeur, 2003). Given the low frequency FZD1 on chemoresistance. A significant restoration of of this disease and the difficulty to obtain matched chemosensitivity was obtained by FZD1 downregula- patient samples, this cohort represents an important tion in LAN-1-R and IGRN-91-R cells (Figure 3b). A population size. The cohort of 21 patients was divided in higher remaining chemoresistance in the IGRN-91-R- a group of 10 relapsed patients, representing a failure in shFZD1 cells compared with that of LAN-1-R-shFZD1 the treatment likely due to chemoresistance, and 11 cells is due to the inability to fully knock down the patients who did not relapse. This latter group includes considerable level of FZD1 in IGRN-91-R. patients in partial or complete remission after treatment, Interestingly, significant sensitivity to cisplatin could therefore harbouring drug-sensitive tumours. The ana- be recovered after FZD1 silencing, showing that FZD1 lysis showed that enhanced FZD1 was indeed measured can mediate cisplatin resistance independently of in patient tumours, and assessment of the relapsed MDR1/P-gp, as cisplatin is not an MDR1 target patients showed that FZD1 and/or MDR1 expression (Gottesman et al., 2002). Restoration of caspase- was significantly enhanced in samples after chemother- dependent sensitivity to etoposide, paclitaxel and apy (Figure 5a). On the contrary, no significant increase cisplatin was partially obtained by specific down of FZD1 and/or MDR1 expression was measured in the modulation of FZD1 in the LAN-1-R cells group of non-relapsed patients (Figure 5b). These (Figure 4a). Surprisingly, shRNAmir-mediated FZD1 observations, that need to be confirmed and extended knockdown expression did not significantly improve the on a larger cohort of patients, nevertheless strongly response to doxorubicin. This might be explained by our support the in vitro observation that FZD1 and MDR1 earlier observations that doxorubicin induces a caspase- are both implicated in chemoresistance and cell survival. independent cell death in N-type NB cells (Hopkins- Altogether, our data suggest that amplification and Donaldson et al., 2002). Further mechanisms that upregulation of FZD1 in chemoresistant cancer cells

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2254 leads to Wnt/b-catenin pathway activation and thereby cDNA expression profile and data analysis to Wnt target gene activation. As MDR1 is one of the Expression profiling experiments were carried out on the b-catenin target genes, activation of the Wnt/b-catenin IGRN-91 and IGRN-91-R cell lines using the pathway leads to an amplification of the resistance U133plus 2.0 Affymetrix GeneChip oligonucleotides contain- through further MDR1 upregulation in addition to ing 38 000 probe sets. Each GeneChip was hybridized using targets synthesized from 100 to 250 ng total RNA. Target increased proliferative and survival characteristics synthesis, hybridization, staining and washing were carried out mediated by the upregulation of other targets, such as using standard protocols as recommended by the manufac- Cyclin-D1 and IGF2. turer (Affymetrix). The data analysis was carried out using a This is the first report describing the involvement of Remote Analysis System developed by the DAFL (DNA FZD1 and the subsequent activation of the Wnt/b- Array Facility Lausanne, Lausanne, Switzerland) bioinfor- catenin pathway in chemoresistance in cell lines as well matics group (Psarros et al., 2005). as in patient samples. Further investigation is needed to elucidate the precise mechanisms of FZD1 activation. FZD1 receptor knockdown by shRNAmir Additional studies may lead to the identification of For shRNA knockdown of FZD1 receptor in the 2 DoxR cell potential therapeutic targets to improve survival of lines, one specific shRNAmir cloned into the bicistronic children and adults suffering from devastating chemore- pGIPZ lentiviral vector was obtained from Open Biosystems sistant cancers. (Surrey, UK) (clone Id: V2LHS_172585 (shFZD1)). The non- silencing shRNAmir construct (scrambled shRNA) in pGIPZ was used as the negative control (shC). Lentiviruses were produced by co-transducing the lentiviral vector plasmid Materials and methods containing the shRNAmir (21 mg) and the second generation packaging plasmids (10.5 mg pMD2-VSVG, 21 mg Cell culture pCMVDR8.91), (Naldini et al., 1996) into 293 T cells by the Neuroblastoma cell lines, such as IGRN-91 (Valent et al., CaCl2 method. At 24 h post-transfection, the lentivirus 1999) and LAN-1 (Seeger et al., 1977), as well as the IGRN-91- containing supernatants were collected, supplemented with R and LAN-1-R cells (Flahaut et al., 2006b) were grown in 8 mg/ml of polybrene (hexadimethrindibromide, Fluka, Buchs, Dulbecco’s Modified Eagle’s Medium (Gibco, Paisley, UK) Switzerland) and filtered through a 0.45-mm filter unit. Target containing 10% fetal calf serum (Sigma, Taufkirchen, cells (IGRN-91-R and LAN-1-R cells) were infected by a 24-h Germany), 100 U/ml penicillin and 100 mg/ml streptomycin incubation at 37 1C in virus-containing media. The cells were (Invitrogen, Grand Island, NY, USA). The colon cancer cell then kept under standard cell culture conditions for 2 weeks, line SW480 (Smith et al., 1993) and the 293T virus packaging sorted by fluorescence-activated cell sorting, and the highest cell line were cultured in the same conditions. green fluorescent protein-expressing cell population was selected for each cell line.

Patients RNA preparation and semiquantitative real-time PCR MDR1 and FZD1 expressions were analysed in the tumours of Total RNAs from NB cell lines (1–2 Â 106 cells), from frozen patients with NB at diagnosis and after treatment. The cohort tumour biopsies and from paraffin-embedded tissues were of patients includes 21 patients (from our local tumour obtained using the RNeasy Mini kit according to the collection): 5 patients with stage 3 NB and 16 patients with manufacturer’s instructions (Qiagen, Hilden, Germany). stage 4 NB, diagnosed from 1986 to 2008. All patients were Semiquantitative real-time PCR details are given in supple- treated according to local and international protocols (SIOP, mentary information. POG), including doxorubicin, in the chemotherapeutic treat- ment. Tumour biopsies were removed at diagnosis, and after at Cell viability assays least one course of chemotherapy. Tumour material was Cell viability was measured in quadruplicates using the MTS/ collected after informed consent and in agreement with local PMS cell proliferation kit (Promega, Du¨ bendorf, Switzerland) ethical regulations. according to the manufacturer’s instructions as previously described (Flahaut et al., 2006b).

Fluorescent in situ hybridization Fluorescent in situ hybridization analysis was carried out on Caspase-3 activity assay metaphase chromosome spreads. Two-colour fluorescent in Caspase-3-like protease activity was measured using the situ hybridization analysis was carried using a-satellite probe caspase-3 colorimetric protease assay kit from MBL as specific for the chromosome 7 centromere (D7Z1, Cytocell, described earlier (Muhlethaler-Mottet et al., 2004). Amplitech, Compie` gne, France) labelled with Texas red and bacterial artificial chromosome clones of chromosome 7, Protein extracts and immunoblotting provided by the Wellcome Trust Sanger Institute and selected Whole cell extracts were prepared as previously described from the Ensembl Human Genome Browser build 35 (http:// (Hopkins-Donaldson et al., 2000). We used rabbit polyclonal www.ensembl.org). Clone CTB 137N13 located on 7q21.12 antibody against MDR1/P-gp (1/100, Oncogene Research, (genomic localization 86,740,029–86,838,500 bp, MDR1/ Boston, MA, USA), mouse monoclonal antibodies to detect ABCB1 gene) was labelled with spectrum green (Vysis Inc., non-phosphorylated b-catenin (1/5000, Upstate, Temecula, Downers Grove, IL, USA) and clone RP11-20K20 on 7q21.13 CA, USA) and b-actin (Sigma) and rat monoclonal antibody (genomic localization 90,459,980–90,589,706 bp, FZD1 gene) against FZD1 receptor (1/500, R&D Systems, Minneapolis, with spectrum Red (Vysis Inc.). The probes were labelled and MN, USA). Binding of the first antibody was revealed by used for hybridization as described earlier (Flahaut et al., incubation with either goat anti-rabbit/anti-mouse or anti-rat 2006b). immunoglobulin G (Jackson ImmunoResearch, West Grove,

Oncogene Wnt signalling in chemoresistant NB M Flahaut et al 2255 PA, USA). Bound antibodies were detected using the ECL represent significance, **Po0.01 and ***Po0.001 were advance western blotting detection kit (Amersham Pharmacia, interpreted to be highly significant. Otelfingen, Switzerland).

Immunofluorescence analysis Conflict of interest Immunofluorcence analysis was carried out on cells (105 cells) grown on glass slides using a Lab-Tek chamber slide system The authors declare no conflict of interest. (Milian, Gene` ve, Switzerland; see experimental details in the Supplementary information). Acknowledgements Statistical analyses Statistical analyses were carried out using GraphPad We thank M Wicht and N Besuchet Schmutz (Medical Genetic Prism 4.0 (La Jolla, CA, USA). Student’s t-test was carried Service, CHUV) for their skillful help. This study was out for each quantitative experiment to compare two different supported by the Schweizer Forschungsstiftung Kind und conditions. Wilcoxon ranked test was chosen to evaluate the Krebs, the Swiss National Science foundation and the FORCE results of the patient samples; *Po0.05 was considered to foundation.

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

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