N-Myc Regulates Expression of the Detoxifying Enzyme Glutathione Transferase GSTP1, a Marker of Poor Outcome in Neuroblastoma

Published OnlineFirst December 27, 2011; DOI: 10.1158/0008-5472.CAN-11-1885

Cancer Clinical Studies Research

Jamie I. Fletcher1, Samuele Gherardi2, Jayne Murray1, Catherine A. Burkhart1, Amanda Russell1, Emanuele Valli2, Janice Smith1, Andre Oberthuer3, Lesley J. Ashton1, Wendy B. London4, Glenn M. Marshall1, Murray D. Norris1, Giovanni Perini2, and Michelle Haber1

Abstract Amplification of the transcription factor MYCN is associated with poor outcome and a multidrug-resistant phenotype in neuroblastoma. N-Myc regulates the expression of several ATP-binding cassette (ABC) transporter genes, thus affecting global drug efflux. Because these transporters do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, we explored the prognostic significance and transcriptional regulation of the phase II detoxifying enzyme, glutathione S-transferase P1 (GSTP1). Using quantitative real-time PCR, GSTP1 gene expression was assessed in a retrospective cohort of 51 patients and subsequently in a cohort of 207 prospectively accrued primary neuroblastomas. These data along with GSTP1 expression data from an independent microarray study of 251 neuroblastoma samples were correlated with established prognostic indicators and disease outcome. High levels of GSTP1 were associated with decreased event-free and overall survival in all three cohorts. Multivariable analyses, including age at diagnosis, tumor stage, and MYCN amplification status, were conducted on the two larger cohorts, independently showing the prognostic significance of GSTP1 expression levels in this setting. Mechanistic investigations revealed that GSTP1 is a direct transcriptional target of N-Myc in neuroblastoma cells. Together, our findings reveal that N-Myc regulates GSTP1 along with ABC transporters that act to control drug metabolism and efflux. Furthermore, they imply that strategies to jointly alter these key multidrug resistance mechanisms may have therapeutic implications to manage neuroblastomas and other malignancies driven by amplified Myc family genes. Cancer Res; 72(4); 845–53. 2011 AACR.

Authors' Affiliations: 1Children's Cancer Institute Australia for Medical Introduction Research, Lowy Cancer Research Centre, Randwick, Sydney, New South Wales, Australia; 2Department of Biology, University of Bologna, Bologna, Italy; 3Children's Hospital, Department of Pediatric Oncology and Hema- Neuroblastoma is a solid tumor of embryonal neural crest tology, Center for Molecular Medicine Cologne, University of Cologne, origin and is the most common extracranial solid tumor of Cologne, Germany; and 4Children's Oncology Group Statistics and Data early childhood, accounting for 15% of all cancer related deaths Center, Dana-Farber Harvard Cancer Care and Children's Hospital Boston, Massachusetts in children (1). Patients diagnosed over one year of age often have disseminated, metastatic disease, often with MYCN Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). ampli cation. While neuroblastomas typically respond to initial therapy regardless of risk group (2), the majority of patients M.D. Norris, G. Perini, and M. Haber contributed equally to this work. with high-risk disease relapse with tumors that are refractory Current address for C.A. Burkhart: Cleveland BioLabs, Inc., Buffalo, New to treatment. There are no salvage regimens known to be York. curative for these patients (3). We have previously shown that the ATP-binding cassette transporter C1 (ABCC1), a key com- Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital, ponent of phase III detoxification, is a powerful independent Randwick, Sydney, Australia. prognostic marker in neuroblastoma (4, 5). Consistent with Corresponding Authors: Michelle Haber, Children's Cancer Institute this observation, ABCC1 is able to efflux a wide range of Australia for Medical Research, Lowy Cancer Research Centre, PO Box chemotherapeutics (6), including many agents of importance 81, Randwick, Sydney, NSW 2031, Australia. Phone: 612-93852170; Fax: 612-93850150; E-mail: [email protected]; and Giovanni Perini, in neuroblastoma therapy. However, several important che- University of Bologna, Department of Biology, via F. Selmi 3, 40126 motherapeutic agents to which resistance is observed, includ- Bologna, Italy. E-mail: [email protected] ing cisplatin and cyclophosphamide, are not substrates of doi: 10.1158/0008-5472.CAN-11-1885 ABCC1. Amongst the well-characterized contributors to mul- 2011 American Association for Cancer Research. tidrug resistance in other types of tumors are cellular

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detoxification pathways. In particular, the phase II conjugating When medium palpable tumors (1cm2) were detected, they enzyme glutathione S-transferase P1 (GSTP1) is upregulated in were resected, measured, and snap frozen in liquid nitrogen for a wide range of tumors and high GSTP1 expression is often subsequent analysis. All experimental procedures were associated with multidrug resistance (7–9). GSTP1 catalyses approved by the University of New South Wales Animal Care the conjugation of reduced glutathione to otherwise harmful and Ethics Committee and were conducted under the Animal electrophilic compounds, including cytotoxic drugs, and can Research Act, 1985 (NSW, Australia) and the Australian Code sequester a range of nonsubstrate ligands (10). In addition to of Practice for the Care and Use of Animals for Scientific these roles, GSTP1 has been proposed to inhibit the mitogen- Purposes (1997). activated protein kinase (MAPK) pathway through direct interaction with c-jun-NH2-kinase 1 (JNK1), decreasing sensi- Real-time PCR methods tivity to drug-induced apoptosis (11). We have, therefore, For tumor samples, total cytoplasmic RNA was isolated extended our studies to investigate the prognostic value of from human and mouse tumor material using a guanidinium this gene in neuroblastoma. thiocyanate-phenol-chloroform extraction method (4) and Here, we show that GSTP1 expression is an independent cDNAs were synthesized from 1 mgofRNAusingrandom prognostic indicator of clinical outcome in primary neuroblas- hexanucleotide primers and Moloney murine leukemia virus toma and, like ABCC1, is a direct transcriptional target of the N- (MMLV) reverse transcriptase. Gene expression was deter- Myc oncoprotein. The regulation of multiple components of mined by real-time PCR (RT-PCR) using either a Prism 7700 drug metabolism pathways has implications for understanding or 7900 Sequence Detection System (Applied Biosystems) drug insensitivity in patients with neuroblastoma with MYCN with the following oligonucleotide primers and probes: amplification and potentially in other malignancies with human GSTP1, b-2 microglobulin (B2M)andMYCN and increased expression of Myc family proteins. mouse Gstp1, Gstp2 (the 2 mouse genes corresponding to human GSTP1)andB2m. All sequences are shown in Sup- Materials and Methods plementary Table S4. For cell lines, RNA samples were prepared with Tri-Reagent Patient tumor samples (Sigma-Aldrich) and treated with DNase (DNAfree; Ambion). All patient material used for gene expression analyses has Reverse transcription and PCR were conducted by a Super- been previously described (5, 12). In brief, a small discovery Script reverse transcription-PCR kit (Invitrogen). RT-PCR was cohort of 51 primary tumors from previously untreated carried out with iQ SYBR Green Supermix and the iQCycler patients was obtained from either the Sydney Children's thermocycler (BioRad) with primers and probes for human Hospital, Sydney, Australia or the Neuroblastoma Tumor MYCN, GSTP1, NUC, and GUSB. All sequences are shown in Bank of the Pediatric Oncology Group, USA ("SCH/POG" Supplementary Table S4. cohort; ref. 12) and a large validation cohort of 207 patients Gene expression levels were determined using the DDCt enrolled onto COG Neuroblastoma Biology Study 9047 was method, normalized to the B2M control, and expressed relative obtained from the Tumor Bank of the Children's Oncology to a calibrator (18). Group, USA ("COG" cohort; ref. 5). The study was approved by the individual Institutional Review Boards, and informed Western blot analysis consent was obtained for patients registered on the study. Frozen tissue (50 mg) was pulverized on dry ice with a Publicly available data for GSTP1 expression from a custom- mortar and pestle in 500 mL lysis buffer (2 PBS, 1% NP-40, ized oligonucleotide microarray data set of primary tumors 0.5% sodium deoxycholate, and 0.1% SDS) containing a from 251 patients with neuroblastoma from the German protease inhibitor cocktail (Sigma-Aldrich). Samples were Neuroblastoma Trials NB90-NB2004 were also analyzed as a centrifuged at 13,000 rpm in a benchtop centrifuge and the second independent validation cohort ("Oberthuer cohort"; supernatants retained and quantitated by BCA assay (Pierce). GSTP1 probe ¼ A_23_P202658). The patient cohort and Samples (20 mg) were electrophoresed on a 4% to 20% SDS- microarray study are previously published (13). A summary PAGE gel and transferred to nitrocellulose membrane (GE of patient data for each cohort is provided as Supplementary Healthcare). The membrane was blocked with 5% skim milk Tables S1–S3. powder in Tris-buffered saline with 0.05% Tween 20, then incubated with an anti-GSTP1 antibody (mouse monoclonal Treatment 3F2C2, Abcam) followed by a horseradish peroxidase–conju- Treatments subsequently administered to patients were gated secondary antibody. Membranes were developed with specific to their disease stage, age, and tumor biology and Supersignal reagent (Pierce), visualized on film and quantitat- have been previously described for each of the SCH/POG (4), ed relative to a-tubulin by densitometry with QuantityOne COG (5), and Oberthuer cohorts (14). software (BioRad).

TH-MYCN mouse tumor samples Human neuroblastoma cell lines and culture The TH-MYCN transgenic mouse model of neuroblastoma, SH-EP Tet-21N cells (19) were cultured in Dulbecco's Mod- which has the expression of the human MYCN oncogene iﬁed Eagle's medium containing 10% heat-inactivated tetra- targeted to mouse neuroectodermal cells via the rat tyrosine cycline-free FBS, whereas BE(2)-C and LA-N-5 cells were hydroxylase promoter, has been previously described (15–17). cultured in RPMI-1640 medium containing 20% FBS and

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A B 100 Low (n = 25) Low (n = 25) 92% 100 96% 80 80 n 60 High (n = 26) High ( = 26) 51% 60 63% 40 EFS (%)

OS (%) 40 20 P = 0.002 20 0 P = 0.006 0 0 1,000 2,000 3,000 4,000 0 1,000 2,000 3,000 4,000 Analysis time (d) Analysis time (d)

C D E Primary tumors

BE(2)-C 653 905746 mRNA ) t GSTP1

α-Tubulin

110.540.160.30.03 0.68 .4protein

Figure 1. GSTP1 is marker of poor prognosis in neuroblastoma. Kaplan–Meier curves for EFS (A) and overall survival (OS; B) in 51 patients with neuroblastoma from the SCH/COG cohort according to the expression of GSTP1. The level of GSTP1 expression was determined by RT-PCR and tumors dichotomized around the median value. Survival curves were analyzed by log-rank test. C, GSTP1 is expressed at a higher level in MYCN-ampliﬁed tumors in comparison with those without MYCN ampliﬁcation. Graph shows relative expression as determined by DDCt method SD (see Materials and Methods) with P value determined by the Student t test. D, GSTP1 protein levels in 6 representative neuroblastoma tumors. Relative expression at mRNA level is shown above the blots, while protein expression relative to a-tubulin, as determined using densitometry, is shown below. The drug-resistant neuroblastoma cell line BE(2)-C is used as a positive control. E, GSTP1 mRNA levels are closely correlated with GSTP1 protein levels in primary neuroblastoma tumors (r ¼ 0.96, P ¼ 0.002). The relationship was analyzed using linear regression.

50 mg/mL gentamycin. For MYCN repression, SH-EP Tet-21N Statistical analysis cells were treated with 2 mg/mL tetracycline for the indicated The relationship between levels of target gene expression time. Cell lines were obtained from: Manfred Schwab (SH-EP and patient survival was analyzed using the Kaplan–Meier Tet-21N), German Collection of Microorganisms and Cell method with curves analyzed by log-rank test. Standard Cultures BE(2)-C, and Deborah Tweddle (LA-N-5) between errors were calculated according to the method of Peto and 1997 and 2005 and were systematically validated for MYCN Peto (23). Comparisons of outcome between subgroups in copy number and expression of specific markers such as multivariable analyses were conducted using the Cox pro- MYCN, upon receipt and during usage by Dr. Giuliano Della portional hazards regression model. The relationship Valle who runs the cytogenetic facility, Department of Biology, between levels of target gene expression was analyzed University of Bologna, Bologna, Italy. using the Spearman rank correlation. Differences in target gene expression between samples were assessed using a 2- Chromatin immunoprecipitation assays sided Student t test. Statistical analyses were conducted The promoter region of GSTP1 was analyzed using bioin- using either Stata (StataCorp) or SPSS (IBM). Event-free formatics techniques to identify putative N-Myc–binding sites, survival(EFS)timewascalculatedfromthetimeofenrol- including the canonical E-box sequence CACGTG and other ment onto the relevant study, or from diagnosis (SCH noncanonical sequences such as CATGTG and CACGCG. samples), to the time of the first occurrence of an event Promoter regions from 2,000 to þ2,000 base pairs with (relapse, progressive disease, secondary malignancy, or respect to the transcription start site were considered for death) or to the date of last contact if no event occurred. analysis. To test binding of N-Myc, quantitative chromatin Death was the only event considered for the calculation of immunoprecipitation (ChIP) was carried out as previously overall survival time. All samples were deidentified, and described (20, 21) using either SH-EP Tet-21N cells cultured researchers who conducted the gene expression analysis in the absence of tetracycline (presence of N-Myc), BE(2)-C, or were blinded to all clinical characteristics and outcome of LA-N-5 cells. Antibodies used in this study were: IgG (Santa the subjects. Cruz Biotechnology; sc-2027); N-Myc monoclonal antibody Tumors were categorized as having high expression of B8.4.B (22), and Max C-17 (Santa Cruz Biotechnology; sc- GSTP1 based on a range of cutoff points, including the 197). Specific pairs of primers used for quantitative ChIP are median, upper quartile, and upper decile PCR values. The listed in Supplementary Table S5. statistical methodology used to identify the optimal cutoff

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A COG cohort D Oberthuer cohort All patients All patients 100 100 Low (n = 186) Low (n = 227) 75 75% 75 74%

50 52% 50 n High ( = 21) FS (%) High (n ==24) EFS (%) E 25 E 25 24% P P = 0.002 < 0.001 0 0 0 2,000 4,000 6,000 0 2,000 4,000 6,000 Figure 2. GSTP1 is prognostic for Analysis time (d) Analysis time (d) outcome in 2 large, independent neuroblastoma cohorts. Kaplan– B E Meier curves for patients with No MYCN amplification No MYCN amplification neuroblastoma from the COG and Oberthuer cohorts showing EFS for 100 the overall cohorts (A, D), patients Low (n = 169) 100 80% Low (n = 202) with tumors lacking MYCN 78% 75 75 ampliﬁcation (B, E), and those from 60% n patients of INSS stage 3 or 4 (C, F), 50 High ( = 15) 50 n according to the expression of FS (%)

FS (%) High ( = 15) E E GSTP1. In each case, tumors were 25 25 26% P dichotomized into "low" and "high" = 0.025 P < 0.001 0 0 expression around the upper decile 0 500 1,000 1,500 2,000 2,500 0 2,000 4,000 6,000 value. Survival curves were Analysis time (d) Analysis time (d) analyzed by log-rank test. Five- year EFS rates are indicated. C F Stage 3 and 4 patients Stage 3 and 4 patients 100 100

75 75 Low (n = 74) Low (n = 87) 55% 50 52% 50 EFS (%) 31% EFS (%) n 25 n 25 High ( = 18) High ( = 12) 15% P = 0.022 P = 0.007 0 0 0 500 1,000 1,500 2,000 2,500 0 2,000 4,000 6,000 Analysis time (d) Analysis time (d)

point to maximize the difference in outcome between GSTP1 gene expression accurately reflected expression at the groups with low versus high expression has previously been protein level, we conducted Western blotting on 6 repre- described in detail (24). sentative tumor samples, 3 with high GSTP1 gene expression and 3 with low gene expression (Fig. 1D). Protein levels were Results found to correlate strongly with gene expression in these samples (Fig. 1E; r ¼ 0.96, P ¼ 0.002). Full-length Western Prognostic significance of GSTP1 in primary human blots are presented in Supplementary Fig. S1. neuroblastoma Next, we extended these observations to 2 large patient To assess the prognostic significance of GSTP1 expression cohorts. In the first, we analyzed GSTP1 expression by RT-PCR in neuroblastoma, we initially used RT-PCR to analyze its in 207 primary tumors (COG cohort). In the second, we expression in a small cohort of 51 primary untreated tumors. analyzed publicly available microarray data (13) for GSTP1 When patients were dichotomized around the median RT- expression in 251 primary tumors (Oberthuer cohort). PCR value, high expression of GSTP1 was significantly asso- In the COG cohort, high GSTP1 expression was associated ciated with poorer EFS (P ¼ 0.002, Fig. 1A) with 5-year with worse outcome when patients were dichotomized around survival rates of 92% 5.4% and 51% 11% for tumors with either the median or upper quartile RT-PCR values, although low and high GSTP1 expression, respectively, and also sig- this failed to reach statistical significance at these cutoff points nificantly associated with poorer overall survival (P ¼ (not shown). However, at the upper decile, high GSTP1 expres- 0.006, Fig. 1B), with 5-year survival rates of 96% 3.9% and sion was significantly associated with reduced EFS (P ¼ 63% 10% for tumors with low and high GSTP1 expression, 0.002; Fig. 2A). The 5-year EFS rate for patients with high respectively. Furthermore, GSTP1 expression was signifi- GSTP1 expression was 52% 11% compared with 75% 3.3% cantly higher in tumors with MYCN amplification than in for low GSTP1 expression. The prognostic significance of nonamplified tumors (Fig. 1C, P ¼ 0.024). To verify that GSTP1 was also examined for patients with tumors lacking

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Table 1. Multivariable Cox regression analysis of prognostic factors for neuroblastoma outcome

EFS Overall survival

Factor HR (95% CI) P HR (95% CI) P

COG-209 Tumor stage 4.6 (2.1–10.0) <0.001 9.8 (3.4–28.7) <0.001 Age 2.4 (1.1–5.1) 0.025 2.5 (1.1–6.1) 0.037 MYCN status 1.8 (0.9–3.6) 0.086 2.1 (1.0–4.1) 0.04 GSTP1 expression 2.6 (1.2–5.5) 0.011 2.2 (1.0–4.8) 0.047 Oberthuer Tumor stage 2.7 (1.6–4.8) <0.001 7.7 (2.6–23.1) <0.001 Age 0.8 (0.4–1.4) 0.385 1.5 (0.6–4.1) 0.411 MYCN status 2.3 (1.3–4.1) 0.005 4.3 (2.1–8.9) <0.001 GSTP1 expression 2.0 (1.1–3.8) 0.029 2.3 (1.1–4.9) 0.024

NOTE: Variables adjusted for in the multivariable analysis included tumor stage (1, 2, and 4S vs. 3, 4), age at diagnosis (<12 months vs. 12 months), MYCN status (ampliﬁed vs. nonampliﬁed), and GSTP1 expression (dichotomized at the upper decile, low vs. high).

MYCN amplification. While these patients would be expected retained independent prognostic significance for EFS [HR ¼ to have a relatively favorable outcome compared with patients 2.6; 95% confidence interval (CI) 1.2–5.5; P ¼ 0.011] and overall with MYCN amplification, high levels of GSTP1 expression survival (HR ¼ 2.21; 95% CI 1.0–4.8; P ¼ 0.047). Likewise in the remained predictive of poorer outcome with a 5-year EFS rate Oberthuer cohort, GSTP1 expression retained prognostic sig- of 60% 13% compared with 80% 3% for patients with low nificance for both EFS (relative risk ¼ 2.0; 95% CI 1.1–3.8; P ¼ GSTP1 expression in their tumors (P ¼ 0.025; Fig. 2B). Similarly, 0.029) and overall survival (relative risk ¼ 2.3; 95% CI 1.1–4.9; within poor prognosis patients (INSS stage 3 and 4), high levels P ¼ 0.024). Finally, in a multivariable analysis for stage 4 of GSTP1 expression identified a subset with particularly poor patients only, including GSTP1 expression and age, GSTP1 outcome and a 5-year EFS rate of only 31% 14% compared expression approached significance for overall survival (rela- with 52% 6% for patients with low GSTP1 expression (P ¼ tive risk ¼ 2.1; 95% CI 0.99–4.6; P ¼ 0.054), but was not 0.022; Fig. 2C). In each case, similar effects were observed for significant for EFS (Supplementary Table S6). overall survival (not shown). Within the Oberthuer cohort, GSTP1 was strongly associated Expression of GSTP1 correlates with MYCN in with EFS at the upper decile (P < 0.001; Fig. 2D) and also at the neuroblastoma tumors upper quartile and median (not shown). At the upper decile, As amplification of the transcription factor MYCN is a com- the 5-year EFS rates for patients with high GSTP1 expression mon feature of poor outcome in neuroblastoma, and GSTP1 were 24% 13% compared with 74% 3% for low GSTP1 expression was higher in MYCN-amplified tumors than in those expression. In patients with tumors lacking MYCN amplifica- without MYCN amplification (Fig. 1C), we sought to determine tion, 5-year EFS rates were 26% 20% and 78% 3% for those whether GSTP1 expression was regulated by MYCN. First, we with high and low GSTP1 expression, respectively (P < 0.001, Fig. assessed the correlation between GSTP1 expression and MYCN 2E). For those with poor prognosis (stage 3 and 4), high GSTP1 expression in the COG cohort after conducting RT-PCR analysis expression was associated with significantly worse outcome to determine relative MYCN expression levels. Using the Spear- with 5-year EFS rates of 15% 12% and 55% 6% for high and man rank correlation, GSTP1 levels were found to correlate with low GSTP1 expression, respectively (P ¼ 0.007; Fig. 2F). Within MYCN expression in both tumors with MYCN amplification (r ¼ the stage 4 patients only, EFS was not significantly different for 0.61, P ¼ 0.003; Fig. 3A) and those without (r ¼ 0.73, P < 0.001; Fig. those with low and high GSTP1 expression in their tumors (P ¼ 3B). We also extended this analysis to the TH-MYCN transgenic 0.227), however, overall survival rates retained significance (P mouse model of neuroblastoma (15), where the human MYCN ¼ 0.049) with 5-year survival rates of 51% 8.5% and 37% transgene is expressed in cells of neuroectodermal origin by use 14% for high and low GSTP1 expression, respectively (Supple- of a tyrosine hydroxylase promoter. Using RT-PCR analysis, mentary Fig. S2). MYCN levels were found to correlate particularly closely with Multivariable analysis was conducted to determine whether the expression of mouse Gstp1 (r ¼ 0.91, P < 0.001; Fig. 3C). GSTP1 expression had prognostic significance independent of Similar results were observed for Gstp2 (not shown). Finally, we established prognostic indicators for neuroblastoma. Tumor assessed the expression of GSTP1 using RT-PCR at various time stage (1, 2, or 4S vs. 3 or 4), age at diagnosis (<12 months vs. 12 points after tetracycline-induced suppression of exogenous months), MYCN status (amplified vs. single copy), and dichot- MYCN expression in the neuroblastoma cell line SH-EP Tet- omized GSTP1 expression were tested in a Cox proportional 21N (19). GSTP1 expression was decreased after tetracycline hazards model (Table 1). In the COG cohort, GSTP1 expression exposure, along with MYCN (not shown).

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of GSTP1 was examined bioinformatically for putative N- A Myc–binding sites within 2,000 bp on either side of the Tumors with MYCN amplification transcriptional start site. Two canonical (CACGTG) and 4 r 150 = 0.61 noncanonical (CACGCG, CATGTG) E-box sites were identi- P = 0.003 ﬁed over 5 distinct regions of the promoter (Fig. 4A). Next, n = 21 we used ChIP assays to examine the physical association of 100 N-Myc with each of the identiﬁed regions using the 3

ive expression) independent human neuroblastoma cell lines SH-EP Tet- 50 21N, LA-N-5, and BE(2)-C. In each cell line, the GSTP1 (relati promoter was directly bound by N-Myc, as well as its dimerization partner Max, at a noncanonical E-box proximal 0 totheputativetranscriptionalstartsite(Fig.4A).Thefold GSTP1 0 50,000 100,000 150,000 enrichment for GSTP1 was comparable with that of APEX1, MYCN ΔΔC a well-known N-Myc target gene (25) indicating that the ( t) N-Myc/Max complex is strongly bound to the promoter. B Finally, to determine whether N-Myc directly activates Tumors without MYCN amplification GSTP1 transcription, we cloned the GSTP1 promoter upstream 200 r = 0.73 of a luciferase reporter gene and tested activity in SH-EP Tet- P < 0.001 21N cells as a function of N-Myc expression. Luciferase activity 150 n = 184 was found to be dependent on N-Myc expression for the GSTP1 reporter construct, as for ABCC1, a positive control (26; Fig. expression) 100 4B). Together, these data show that GSTP1 is a direct transcriptional target of N-Myc in neuroblastoma cells.

(relative 50 Discussion 0 GSTP1 0 5,000 10,000 15,000 Using 3 independent patient cohorts, we have shown that high expression of GSTP1 is a marker of poor prognosis in MYCN (ΔΔC ) t primary neuroblastoma. Furthermore, when included in multivariable analyses with the established prognostic indicators C MYCN TH- mouse tumors of age at diagnosis, MYCN ampliﬁcation status and tumor stage as variables, GSTP1 expression retained independent 20 prognostic signiﬁcance. This result raises the possibility that GSTP1 contributes to the clinical behavior of neuroblastoma, 15 consistent with its known roles in drug resistance and tumor expression) 10 biology. Whereas neuroblastomas typically respond to initial therapy regardless of risk group (2), the majority of patients r = 0.91 with high-risk disease relapse with tumors that are refractory

(relative 5 P < 0.001 ﬂ n = 20 to treatment. It is as yet unclear whether this re ects the 0 acquisition of multidrug resistance following exposure to MYCN 0 5 10 15 20 chemotherapy, as a result of induction of one or more of the Gstp1 (relative expression) mechanisms which have previously been implicated as mediating multidrug resistance in neuroblastoma, such as MRP1 overexpression (5) or p53 mutation (27), or the selection and Figure 3. GSTP1 gene expression correlates with that of MYCN. expansion of preexistent multidrug resistant tumor clones, as Expression of GSTP1 correlates with that of MYCN in tumors from the we have previously described for acute lymphoblastic leukae- MYCN ﬁ COG cohort either with ampli cation (A), or without (B), whereas mia (28). Future studies involving matched pairs of pre- and Gstp1 expression correlates with that of the MYCN transgene in tumors from the TH-MYCN mouse neuroblastoma model (C). The relationships posttreatment tumor samples would be valuable to determine were analyzed using the Spearman rank correlation. whether levels of GSTP1 or other drug resistance–associated genes are elevated at relapse compared with levels at the time Collectively, these data support regulation of GSTP1 expres- of diagnosis. sion by N-Myc in cultured neuroblastoma cell lines, human We also provide evidence that GSTP1 is a direct transcrip- neuroblastoma tumors, and tumors from a biologically rele- tional target of N-Myc. First, expression of GSTP1 correlates vant mouse model of neuroblastoma. with that of MYCN in both human tumors and the tumors derived from the TH-MYCN mouse neuroblastoma model. N-Myc directly regulates GSTP1 expression Second, the N-Myc protein, in complex with its dimerization In light of these results, we assessed whether N-Myc partner Max, directly interacts with a noncanonical E-box directly regulates GSTP1 expression. The promoter region proximal to the putative transcriptional start site of GSTP1.

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Figure 4. GSTP1 is a direct transcriptional target of N-Myc. A, N-Myc associates with the GSTP1 promoter. Quantitative ChIP was applied to the neuroblastoma cell lines BE(2)-C, LA-N-5, and SH-EP Tet21N cells expressing MYCN. Relative enrichment is compared with the preimmune serum. The results represent the mean SE of 5 independent ChIP experiments in which each region was amplified by RT-PCR in triplicate. Bent arrow, transcription start site; gray arrow, canonical E-box; black arrow, noncanonical E-box; and black boxes, amplicons. The chromosome and coordinates (bp) are also given. Quantitative ChIP for APEX1 in BE(2)-C cells is also shown. B, N-Myc regulates GSTP1 in a luciferase reporter assay. Firefly luciferase activity was determined following transient transfection of either a GSTP1 or ABCC1 reporter construct into SH-EP Tet21N cells. Cells were either not treated (NT; MYCN ON) or treated with tetracycline for 24 or 48 hours (TET 24 h or TET 48 h; MYCN OFF). Cloned regions are shown at left, with bent arrows denoting the transcription start site, the cloned DNA region (bp) indicated below the promoter map and fragment coordinates expressed relative to the transcription start point. The results are the means of the relative fluorescence units (RFU) SE of 3 independent transfections.

Finally, repression of MYCN expression reduces activity in a conjugated chemotherapeutic agents (29) and ABCC4, which luminescent reporter assay using the GSTP1 promoter. Nota- can efflux both irinotecan and topotecan (12), are also inde- bly, both the multidrug transporter ABCC1, a phase III detox- pendently prognostic of neuroblastoma outcome (4, 5, 12, 30) ification component that can efflux a number of glutathione- as well as directly regulated by N-Myc (26). MYCN amplification

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can, therefore, contribute to multidrug resistance in neuro- While the most familiar role for GSTP1 is the conjugation blastoma through the upregulation of a suite of drug resistance of reduced glutathione to cytotoxic drugs, GSTP1 may genes covering a range of chemotherapeutic drugs. While possess other functions that contribute to chemoresistance, previous in vitro studies suggest cooperativity between ABCC1 including inhibition of JNK1 (34) and activation of extracel- and GSTs (31, 32), GSTP1 has a relatively weak affinity for many lular signal–regulated kinase (ERK; ref. 35). In addition to anticancer drugs (7), implying that these proteins do not potential roles in mediating drug resistance in neuroblas- necessarily form sequential steps for the detoxification of a toma, GSTP1 could potentially impact other tumor proper- particular drug. ties through its ability to conjugate the cyclopentenone 12,14 It is notable that of the 2 cohorts analyzed by RT-PCR, GSTP1 prostaglandins 15d-D -PGJ2 and PGA2 (36), leading to expression was significantly associated with poor outcome their export through ABCC1 and ABCC3 (37) and its ability 12,14 when dichotomized around the median RT-PCR value in the to sequester 15d-D -PGJ2 in the cytosol independently of initial SCH/POG 51 sample cohort, whereas in the larger COG conjugation (38). Both mechanisms may prevent cyclopen- cohort, the association between high GSTP1 expression and tenone prostaglandins from binding to the nuclear localized poor outcome was evident but not significant at this cutoff point gamma isoform of the peroxisome proliferator–activated and in fact did not achieve prognostic significance until the data receptor (PPAR-g), activation of which is antiproliferative, were dichotomized around the upper decile RT-PCR value. One prodifferentiation, and proapoptotic (39) in cancer cells. In 12,14 contributing factor for this difference is most likely due to the neuroblastoma, activation of PPAR-g by 15d-D -PGJ2 has differences between the patient characteristics of the 2 cohorts. been shown to induce apoptosis (40) and differentiation (41). Specifically, in the retrospectively accrued SCH/POG cohort of Whilewecannotexcludetheseadditionalfunctionsinour 51 tumors, 23.5% of tumors were MYCN amplified and 55% were experiments, analysis of microarray data available for the INSS stage 3 or 4 (12), whilst in the prospectively accrued COG Oberthuer cohort did not reveal any evidence of an associ- cohort (5), only 10.1% of tumors were MYCN amplified and 42% ation between GSTP1 expression and that of JNK1 (MAPK8), were INSS stage 3 or 4. The underrepresentation of MYCN ERK1 (MAPK1) or PPARg (PPARG; Supplementary Fig. S4), or amplified, advanced stage tumors in the COG cohort can be with pathways associated with these genes (data not shown). related to the frequent diagnosis of stage 4 disease based on GSTP1 is upregulated in numerous human tumors and high bone marrow sampling rather than tumor biopsy in that cohort, GSTP1 expression is associated with chemoresistance (7–9). To as we have previously described (5). As GSTP1 is transcription- our knowledge, this is the first study to reveal the prognostic ally regulated by N-Myc, and given the strong correlation significance of GSTP1 expression in neuroblastoma and the between MYCN amplification and GSTP1 overexpression, it is, first to show that GSTP1 is a direct transcriptional target of the therefore, not surprising that GSTP1 expression is more predic- MYCN oncogene, which is frequently amplified in poor out- tive of outcome in a cohort with a higher proportion of MYCN- come neuroblastoma. These observations have significance amplified tumors and less predictive in a cohort where MYCN- both for MYCN-amplified neuroblastoma and for other dis- amplified tumors are underrepresented. Indeed, those MYCN eases with amplification of Myc family oncogenes. amplified patients lost to the COG cohort would most likely only further strengthen the association between high GSTP1 expres- Disclosure of Potential Conflicts of Interest sion and poor patient outcome evident in that cohort. The distributions of RT-PCR values for each cohort are shown in No potential conflicts of interest were disclosed. Supplementary Fig. S3. The identification of GSTP1 as an N-Myc target gene also has Grant Support importance for malignancies with high expression or ampli- fi The study was supported by National Health and Medical Research Council, cation of c-Myc. N-Myc and c-Myc share a common set of Australia (to J.I. Fletcher, M.D. Norris, G.M. Marshall, M. Haber), Cancer Institute target genes and are functionally interchangeable in the mouse New South Wales (to J.I. Fletcher, M.D. Norris, G.M. Marshall, M. Haber), Cancer (33). Furthermore, GSTP1 was one of a large number of genes Council New South Wales (to J.I. Fletcher, M.D. Norris, G.M. Marshall, M. Haber), fi the Italian Association for Research on Cancer (to G. Perini), the Italian Ministry identi ed as potential c-Myc transcriptional targets in a high- of University and Research (to G. Perini), the University of Bologna (to G. Perini), throughput ChIP study (25) and GSTP1 is transcriptionally and the Children's Oncology Group (Philadelphia, PA). regulated by c-Myc in osteosarcoma cell lines (G. Perini, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in manuscript in preparation). Finally, as Gstp1 is tightly coupled accordance with 18 U.S.C. Section 1734 solely to indicate this fact. to MYCN expression in neuroblastomas arising in TH-MYCN mice, this is likely to be a suitable preclinical model in which to Received June 8, 2011; revised December 11, 2011; accepted December 12, 2011; assess the efficacy of GSTP1 inhibitors. published OnlineFirst December 27, 2011.

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www.aacrjournals.org Cancer Res; 72(4) February 15, 2012 853

N-Myc Regulates Expression of the Detoxifying Enzyme Glutathione Transferase GSTP1, a Marker of Poor Outcome in Neuroblastoma

Jamie I. Fletcher, Samuele Gherardi, Jayne Murray, et al.

Cancer Res 2012;72:845-853. Published OnlineFirst December 27, 2011.

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