n-myc regulates expression of the detoxifying enzyme ... · 23/12/2011 · 1 n-myc regulates...

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N-Myc regulates expression of the detoxifying enzyme glutathione transferase GSTP1, a

marker of poor outcome in neuroblastoma

Jamie I. Fletcher1, Samuele Gherardi2, Jayne Murray1, Catherine A. Burkhart1,3, Amanda Russell1,

Emanuele Valli2, Janice Smith1, André Oberthuer4, Lesley J. Ashton1, Wendy B. London5, Glenn

M. Marshall1, Murray D. Norris1, Giovanni Perini2, Michelle Haber1.

1Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre

Randwick, NSW, Australia. 2Department of Biology, University of Bologna, Bologna, Italy. 3Current address: Cleveland BioLabs, Inc, Buffalo, New York, USA. 4Children’s Hospital, Department of Pediatric Oncology and Hematology, University of Cologne

and Center for Molecular Medicine Cologne, Cologne, Germany. 5Children’s Oncology Group Statistics and Data Center, Dana-Farber Harvard Cancer Care and

Children’s Hospital Boston, MA, USA.

Running title: Expression and regulation of GSTP1 in neuroblastoma

Key words: Multidrug resistance, tumor, chemotherapy, glutathione S-transferase, MYCN.

Abbreviations: Glutathione S-transferase P1 (GSTP1), ATP-binding cassette (ABC), real-time

polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), peroxisome

proliferator-activated receptor gamma (PPAR-γ), β2-microglobulin (B2M); tetracycline (tet).

Footnotes

Financial support: National Health and Medical Research Council, Australia (JF, MDN, GMM,

MH), Cancer Institute New South Wales (JF, MDN, GMM, MH), Cancer Council New South

Wales (JF, MDN, GMM, MH), the Italian Association for Research on Cancer (GP), the Italian

Ministry of University and Research (GP), the University of Bologna (GP) and the Children’s

Oncology Group (USA).

Corresponding authors: Michelle Haber, Children’s Cancer Institute Australia for Medical

Research, Lowy Cancer Research Centre, PO Box 81, Randwick, New South Wales 2031,

Australia. E-mail: [email protected], or Giovanni Perini, University of Bologna,

Department of Biology, via F. Selmi 3, 40126 Bologna, Italy. E-mail: [email protected].

Conflict of interest: The authors declare no competing financial interests.

Research. on October 4, 2020. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 27, 2011; DOI: 10.1158/0008-5472.CAN-11-1885

http://cancerres.aacrjournals.org/

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Notes: MDN, GP and MH contributed equally to this work. Children’s Cancer Institute Australia

for Medical Research is affiliated with the University of New South Wales and Sydney Children’s

Hospital, Randwick, Sydney, Australia. Word count = 4762; 4 figures; 1 table.




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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 demonstrating 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. Further, 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.




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Introduction

Neuroblastoma is a solid tumor of embryonal neural crest origin and is the most common

extracranial solid tumor of early childhood, accounting for 15% of all cancer related deaths in

children (1). Patients diagnosed over one year of age often have disseminated, metastatic disease,

often with MYCN amplification. While neuroblastomas typically respond to initial therapy

regardless of risk group (2), the majority of patients with high-risk disease relapse with tumors that

are refractory to treatment. There are no salvage regimens known to be curative for these patients

(3). We have previously demonstrated that the ATP binding cassette transporter C1 (ABCC1), a key

component of phase III detoxification, is a powerful independent prognostic marker in

neuroblastoma (4, 5). Consistent with this observation, ABCC1 is able to efflux a wide range of

chemotherapeutics (6), including many agents of importance in neuroblastoma therapy. However,

several important chemotherapeutic agents to which resistance is observed, including cisplatin and

cyclophosphamide, are not substrates of ABCC1. Amongst the well-characterized contributors to

multidrug resistance in other types of tumors are cellular detoxification pathways. In particular, the

Phase II conjugating enzyme glutathione S-transferase P1 (GSTP1) is up-regulated in a wide range

of tumors and high GSTP1 expression is often associated with multidrug resistance (7-9). GSTP1

catalyses the conjugation of reduced glutathione to otherwise harmful electrophilic compounds,

including cytotoxic drugs, and can sequester a range of non-substrate ligands (10). In addition to

these roles, GSTP1 has been proposed to inhibit the mitogen-activated protein kinase (MAPK)

pathway through direct interaction with c-Jun N-terminal kinase 1 (JNK1), decreasing sensitivity to

drug-induced apoptosis (11). We have therefore extended our studies to investigate the prognostic

value of this gene in neuroblastoma.

Here we demonstrate that GSTP1 expression is an independent prognostic indicator of

clinical outcome in primary neuroblastoma and, like ABCC1, is a direct transcriptional target of the

N-Myc oncoprotein. The regulation of multiple components of drug metabolism pathways has

implications for understanding drug insensitivity in neuroblastoma patients with MYCN




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amplification, and potentially in other malignancies with increased expression of Myc family

proteins.

Materials and Methods

Patient tumor samples

All patient material used for gene expression analyses has been previously described (5, 12).

In brief, a small discovery cohort of 51 primary tumors from previously untreated patients was

obtained from either the Sydney Children’s Hospital, Sydney, Australia, or the Neuroblastoma

Tumor Bank of the Pediatric Oncology Group, USA (“SCH/POG” cohort) (12) and a large

validation cohort of 207 patients enrolled onto COG Neuroblastoma Biology Study 9047 was

obtained from the Tumor Bank of the Children’s Oncology Group, USA (“COG” cohort) (5). The

study was approved by the individual institutional review boards, and informed consent was

obtained for patients registered on the study. Publicly available data for GSTP1 expression from a

customized oligonucleotide microarray dataset of primary tumors from 251 neuroblastoma patients

from the German Neuroblastoma Trials NB90-NB2004 were also analysed as a second independent

validation cohort (“Oberthuer cohort”; GSTP1 probe = A_23_P202658). The patient cohort and

microarray study are previously published (13). A summary of patient data for each cohort is

provided as Supplementary data Tables 1, 2 and 3.

Treatment

Treatments subsequently administered to patients were specific to their disease stage, age

and tumor biology and have been previously described for each of the SCH/POG (4), COG (5) and

Oberthuer cohorts (14).

TH-MYCN mouse tumor samples




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The TH-MYCN transgenic mouse model of neuroblastoma, which has the expression of the

human MYCN oncogene targeted to mouse neuroectodermal cells via the rat tyrosine hydroxylase

promoter, has been previously described (15-17). When medium palpable tumors (approximately 1

cm2) were detected, they were resected, measured, and snap-frozen in liquid nitrogen for subsequent

analysis. All experimental procedures were approved by the University of New South Wales

Animal Care and Ethics Committee and were conducted under the Animal Research Act, 1985

(New South Wales, Australia) and the Australian Code of Practice for the Care and Use Animals for

Scientific Purposes (1997).

RT-PCR methods

For tumor samples, total cytoplasmic RNA was isolated from human and mouse tumor

material using a guanidinium thiocyanate-phenol-chloroform extraction method (4) and cDNAs

were synthesized from 1 μg of RNA using random hexanucleotide primers and Moloney murine

leukemia virus reverse transcriptase. Gene expression was determined by real-time polymerase

chain reaction (RT-PCR) using either a Prism 7700 or 7900 Sequence Detection System (Applied

Biosystems, Foster City, CA) with the following oligonucleotide primers and probes: human

GSTP1, beta2-microglobulin (B2M) and MYCN, and mouse Gstp1, Gstp2 (the two mouse genes

corresponding to human GSTP1) and B2m. All sequences are shown in Supplementary data Table 4.

For cell lines, RNA samples were prepared using Tri-Reagent (Sigma-Aldrich, St Louis,

MO) and treated with DNase (DNAfree; Ambion, Austin, TX). Reverse transcription and PCR were

performed using a SuperScript reverse transcription-PCR kit (Invitrogen, Carlsbad, CA). RT-PCR

was performed using iQ SYBR Green Supermix and the iQCycler thermocycler (Bio-Rad,

Hercules, CA) with primers and probes for human MYCN, GSTP1, NUC and GUSB. All sequences

are shown in Supplementary data Table 4.

Gene expression levels were determined using the ΔΔCt method, normalized to the B2M

control, and expressed relative to a calibrator (18).




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Western blot analysis

Frozen tissue (approximately 50 mg) was pulverized on dry ice with a mortar and pestle in

500 μL lysis buffer (2 x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a

protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO). Samples were centrifuged at 13,000

RPM in a benchtop centrifuge and the supernatants retained and quantitated by BCA assay (Pierce,

Rockford, IL). Samples (20 μg) were electrophoresed on a 4-20% SDS-PAGE gel and transferred to

nitrocellulose membrane (GE Healthcare, Rydalmere, NSW, Australia). The membrane was

blocked with 5% skim milk powder in Tris buffered saline with 0.05% Tween 20, then incubated

with an anti-GSTP1 antibody (mouse monoclonal 3F2C2, Abcam, Cambridge, UK) followed by a

horseradish peroxidase conjugated secondary antibody. Membranes were developed using

Supersignal reagent (Pierce, Rockford, IL), visualized on film and quantitated relative to α-tubulin

by densitometry using QuantityOne software (BioRad, Hercules, CA).

Human neuroblastoma cell lines and culture

SH-EP Tet-21N cells (19) were cultured in Dulbecco’s modified Eagle’s medium containing

10% heat-inactivated tetracycline-free fetal bovine serum, while BE(2)-C and LA-N-5 cells were

cultured in RPMI medium 1640 containing 20% fetal bovine serum and 50 mg/mL gentamycin. For

MYCN repression, SH-EP Tet-21N cells were treated with 2μg/mL tetracycline for the indicated

time. Cell lines were obtained from: Manfred Schwab (SH-EP Tet-21N), German Collection of

Microorganisms and Cell Cultures (BE(2)-C) and Deborah Tweddle (LA-N-5) between 1997 and

2005, and were systematically validated for MYCN copy number and expression of specific markers

such as MYCN, upon receipt and during usage by dr. Giuliano Della Valle who runs the cytogenetic

facility, Department of Biology, University of Bologna, Italy.

Chromatin immunoprecipitation (ChIP) assays




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The promoter region of GSTP1 was analysed using bioinformatics techniques to identify

putative N-Myc binding sites, including the canonical E-box sequence CACGTG and other non-

canonical sequences such as CATGTG and CACGCG. Promoter regions from -2000 to +2000 base

pairs with respect to the transcription start site were considered for analysis. To test binding of N-

Myc, quantitative ChIP was performed as previously described (20, 21) using either SH-EP Tet-

21N cells cultured in the absence of tetracycline (presence of N-Myc), BE(2)-C or LA-N-5 cells.

Antibodies employed in this study were: IgG (Santa Cruz Biotechnology, Santa Cruz, CA, sc-

2027); N-Myc monoclonal antibody B8.4.B (22) and Max C-17 (Santa Cruz Biotechnology, sc-

197). Specific pairs of primers used for quantitative ChIP are listed in Supplementary data Table 5.

Statistical analysis

The relationship between levels of target gene expression and patient survival was analyzed

using the Kaplan-Meier method with curves analyzed by log-rank test. Standard errors were

calculated according to the method of Peto and Peto (23). Comparisons of outcome between

subgroups in multivariable analyses were performed using Cox’s proportional hazards regression

model. The relationship between levels of target gene expression was analysed using Spearman’s

rank correlation. Differences in target gene expression between samples were assessed using a two-

sided Student’s t test.�Statistical analyses were conducted using either Stata (StataCorp, College

Station, TX) or SPSS (IBM, Chicago, IL). Event Free Survival (EFS) time was calculated from the

time of enrolment onto the relevant study, or from diagnosis (SCH samples), to the time of the first

occurrence of an event (relapse, progressive disease, secondary malignancy, or death) or to the date

of last contact if no event occurred. Death was the only event considered for the calculation of

overall survival time. All samples were de-identified, and researchers who performed the gene

expression analysis were blinded to all clinical characteristics and outcome of the subjects.

Tumors were categorized as having high expression of GSTP1 based on a range of cut

points, including the median, upper quartile, and upper decile PCR values. The statistical




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methodology used to identify the optimal cut point to maximize the difference in outcome between

groups with low versus high expression has previously been described in detail (24).

Results

Prognostic significance of GSTP1 in primary human neuroblastoma

In order to assess the prognostic significance of GSTP1 expression in neuroblastoma, we

initially used RT-PCR to analyse its expression in a small cohort of 51 primary untreated tumors.

When patients were dichotomized around the median real-time PCR value, high expression of

GSTP1 was significantly associated with poorer event free survival (P = 0.002, Figure 1A) with 5-

year survival rates of 92% ± 5.4% and 51% ± 11% for tumors with low and high GSTP1 expression,

respectively, and also significantly associated with poorer overall survival (P = 0.006, Figure 1B),

with 5-year survival rates of 96% ± 3.9% and 63% ± 10% for tumors with low and high GSTP1

expression, respectively. Furthermore, GSTP1 expression was significantly higher in tumors with

MYCN amplification than in non-amplified tumors (Figure 1C, P = 0.024). To verify that GSTP1

gene expression accurately reflected expression at the protein level, we conducted Western blotting

on six representative tumor samples, three with high GSTP1 gene expression and three with low

gene expression (Figure 1D). Protein levels were found to correlate strongly with gene expression

in these samples (Figure 1E; r = 0.96, P = 0.002). Full-length Western blots are presented in

Supplementary Figure S1.

Next, we extended these observations to two large patient cohorts. In the first, we analysed

GSTP1 expression by RT-PCR in 207 primary tumors (COG cohort). In the second, we analysed

publicly available microarray data (13) for GSTP1 expression in 251 primary tumors (Oberthuer

cohort).

In the COG cohort, high GSTP1 expression was associated with worse outcome when

patients were dichotomized around either the median or upper quartile real-time PCR values,

although this failed to reach statistical significance at these cut-points (not shown). However at the




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upper decile, high GSTP1 expression was significantly associated with reduced EFS (P = 0.002;

Figure 2A). The 5-year EFS rate for patients with high GSTP1 expression was 52% ± 11%,

compared with 75% ± 3.3% for low GSTP1 expression. The prognostic significance of GSTP1 was

also examined for patients with tumors lacking MYCN amplification. While these patients would be

expected to have a relatively favourable outcome compared to patients with MYCN amplification,

high levels of GSTP1 expression remained predictive of poorer outcome with a 5-year EFS rate of

60% ± 13% compared to 80% ± 3% for patients with low GSTP1 expression in their tumors (P =

0.025; Figure 2B). Similarly, within poor prognosis patients (INSS stage 3 and 4), high levels of

GSTP1 expression identified a subset with particularly poor outcome and a 5-year EFS rate of only

31% ± 14% compared to 52% ± 6% for patients compared to patients with low GSTP1 expression

(P = 0.022; Figure 2C). In each case, similar effects were observed for overall survival (not shown).

Within the Oberthuer cohort, GSTP1 was strongly associated with EFS at the upper decile

(P < 0.001; Figure 2D), and also at the upper quartile and median (not shown). At the upper decile,

the 5-year EFS rates for patients with high GSTP1 expression were 24% ± 13% compared with 74%

± 3% for low GSTP1 expression. In patients with tumors lacking MYCN amplification, 5-year EFS

rates were 26% ± 20% and 78% ± 3% for those with high and low GSTP1 expression respectively

(P < 0.001, Figure 2E). For those with poor prognosis (stage 3,4), high GSTP1 expression was

associated with significantly worse outcome with 5-year EFS rates of 15% ± 12% and 55% ± 6%

for high and low GSTP1 expression respectively (P = 0.007; Figure 2F). Within the stage 4 patients

only, EFS was not significantly different for those with low and high GSTP1 expression in their

tumors (P = 0.227), however overall survival rates retained significance (P = 0.049) with 5 year

survival rates of 51% ± 8.5% and 37% ± 14% for high and low GSTP1 expression respectively

(Supplementary Figure S2).

Multivariable analysis was performed to determine whether GSTP1 expression had

prognostic significance independent of established prognostic indicators for neuroblastoma. Tumor

stage (1, 2, or 4S v 3 or 4), age at diagnosis (<12 mo v �12 mo), MYCN status (amplified v single




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copy) and dichotomized GSTP1 expression were tested in a Cox’s proportional hazards model

(Table 1). In the COG cohort, GSTP1 expression retained independent prognostic significance for

EFS (hazard ratio = 2.6; 95% CI 1.2 to 5.5; P = 0.011) and overall survival (hazard ratio = 2.21;

95% CI 1.0 to 4.8; P = 0.047). Likewise in the Oberthuer cohort, GSTP1 expression retained

prognostic significance for both EFS (relative risk = 2.0; 95% CI 1.1 to 3.8; P = 0.029) and overall

survival (relative risk = 2.3; 95% CI 1.1 to 4.9; P = 0.024). Finally, in a multivariable analysis for

stage 4 patients only, including GSTP1 expression and age, GSTP1 expression approached

significance for overall survival (relative risk = 2.1; 95% CI 0.99 to 4.6; P = 0.054), but was not

significant for event-free survival (Supplementary data Table 6).

Expression of GSTP1 correlates with MYCN in neuroblastoma tumors

As amplification of the transcription factor MYCN is a common feature of poor outcome in

neuroblastoma, and GSTP1 expression was higher in MYCN-amplified tumors than in those without

MYCN amplification (Figure 1C), we sought to determine whether GSTP1 expression was regulated

by MYCN. First, we assessed the correlation between GSTP1 expression and MYCN expression in

the COG cohort after conducting RT-PCR analysis to determine relative MYCN expression levels.

Using Spearman’s rank correlation, GSTP1 levels were found to correlate with MYCN expression in

both tumors with MYCN-amplification (r = 0.61, P = 0.003; Figure 3A) and those without (r =

0.73, P < 0.001; Figure 3B). We also extended this analysis to the TH-MYCN transgenic mouse

model of neuroblastoma (15), where the human MYCN transgene is expressed in cells of

neuroectodermal origin by use of a tyrosine hydroxylase promoter. Using RT-PCR analysis, MYCN

levels were found to correlate particularly closely with the expression of mouse Gstp1 (r = 0.91, P <

0.001; Figure 3C). Similar results were observed for Gstp2 (not shown). Finally, we assessed the

expression of GSTP1 using RT-PCR at various time points after tetracycline-induced suppression of

exogenous MYCN expression in the neuroblastoma cell line SH-EP Tet-21N (19). GSTP1

expression was decreased after tetracycline exposure, along with MYCN (not shown).




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Collectively, these data support regulation of GSTP1 expression by N-Myc in cultured

neuroblastoma cell lines, human neuroblastoma tumors, and tumors from a biologically relevant

mouse model of neuroblastoma.

N-Myc directly regulates GSTP1 expression

In light of these results, we assessed whether N-Myc directly regulates GSTP1 expression.

The promoter region of GSTP1 was examined bioinformatically for putative N-Myc-binding sites

within 2000bp on either side of the transcriptional start site. Two canonical (CACGTG) and four

non-canonical (CACGCG, CATGTG) E-box sites were identified over five distinct regions of the

promoter (Figure 4A). Next, we used ChIP assays to examine the physical association of N-Myc

with each of the identified regions using the three independent human neuroblastoma cell lines SH-

EP Tet-21N, LA-N-5 and BE(2)-C. In each cell line, the GSTP1 promoter was directly bound by N-

Myc, as well as its dimerization partner Max, at a non-canonical E-box proximal to the putative

transcriptional start site (Fig. 4A). The fold enrichment for GSTP1 was comparable to that of

APEX1, a well-known N-Myc target gene (25) indicating that the N-Myc/Max complex is strongly

bound to the promoter.

Finally, to determine whether N-Myc directly activates GSTP1 transcription, we cloned the

GSTP1 promoter upstream of a luciferase reporter gene and tested activity in SH-EP Tet-21N cells

as a function of N-Myc expression. Luciferase activity was found to be dependent on N-Myc

expression for the GSTP1 reporter construct, as for ABCC1, a positive control (26) (Figure 4B).

Together, these data demonstrate that GSTP1 is a direct transcriptional target of N-Myc in

neuroblastoma cells.

Discussion

Using three independent patient cohorts, we have shown that high expression of GSTP1 is a

marker of poor prognosis in primary neuroblastoma. Furthermore, when included in multivariable




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analyses with the established prognostic indicators of age at diagnosis, MYCN amplification status

and tumor stage as variables, GSTP1 expression retained independent prognostic significance. This

result raises the possibility that GSTP1 contributes to the clinical behaviour of neuroblastoma,

consistent with its known roles in drug resistance and tumor biology. While neuroblastomas

typically respond to initial therapy regardless of risk group (2), the majority of patients with high-

risk disease relapse with tumors that are refractory to treatment. It is as yet unclear whether this

reflects the acquisition of multidrug resistance following exposure to chemotherapy, as a result of

induction of one or more of the mechanisms which have previously been implicated as mediating

multidrug resistance in neuroblastoma, such as MRP1 over-expression (5) or p53 mutation (27), or

the selection and expansion of pre-existent multidrug resistant tumor clones, as we have previously

described for acute lymphoblastic leukaemia (28). Future studies involving matched pairs of pre-

and post-treatment tumor samples would be valuable to determine whether levels of GSTP1 or other

drug-resistance associated genes are elevated at relapse compared to levels at the time of diagnosis.

We also provide evidence that GSTP1 is a direct transcriptional target of N-Myc. Firstly,

expression of GSTP1 correlates with that of MYCN in both human tumors and the tumors derived

from the TH-MYCN mouse neuroblastoma model. Secondly, the N-Myc protein, in complex with

its dimerization partner Max, directly interacts with a non-canonical E-box proximal to the putative

transcriptional start site of GSTP1. Finally, repression of MYCN expression reduces activity in a

luminescent reporter assay using the GSTP1 promoter. Notably, both the multidrug transporter

ABCC1, a Phase III detoxification component that can efflux a number of glutathione conjugated

chemotherapeutic agents (29) and ABCC4, which can efflux both irinotecan and topotecan (12), are

also independently prognostic of neuroblastoma outcome (4, 5, 12, 30) as well as directly regulated

by N-Myc (26). MYCN amplification can therefore contribute to multidrug resistance in

neuroblastoma through the upregulation of a suite of drug resistance genes covering a range of

chemotherapeutic drugs. While previous in vitro studies suggest co-operativity between ABCC1

and GSTs (31, 32), GSTP1 has a relatively weak affinity for many anti-cancer drugs (7), implying




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that these proteins do not necessarily form sequential steps for the detoxification of a particular

drug.

It is notable that of the two cohorts analyzed by RT-PCR, GSTP1 expression was

significantly associated with poor outcome when dichotomized around the median RT-PCR value in

the initial SCH/POG 51 sample cohort, while in the larger COG cohort, the association between

high GSTP1 expression and poor outcome was evident but not significant at this cut-point and in

fact did not achieve prognostic significance until the data was dichotomized around the upper decile

RT-PCR value. One contributing factor for this difference is most likely due to the differences

between the patient characteristics of the two cohorts. Specifically, in the retrospectively accrued

SCH/POG cohort of 51 tumors, 23.5% of tumors were MYCN-amplified and 55% were INSS stage

3 or 4 (12), whilst in the prospectively accrued COG cohort (5), only 10.1% of tumors were MYCN-

amplified and 42% were INSS stage 3 or 4. The under-representation of MYCN amplified, advanced

stage tumors in the COG cohort can be related to the frequent diagnosis of stage 4 disease based on

bone marrow sampling rather than tumor biopsy in that cohort, as we have previously described (5).

As GSTP1 is transcriptionally regulated by MYCN, and given the strong correlation between MYCN

amplification and GSTP1 overexpression, it is therefore not surprising that GSTP1 expression is

more predictive of outcome in a cohort with a higher proportion of MYCN-amplified tumors and

less predictive in a cohort where MYCN-amplified tumors are under-represented. Indeed, those

MYCN amplified patients lost to the COG cohort would most likely only further strengthen the

association between high GSTP1 expression and poor patient outcome evident in that cohort. The

distributions of RT-PCR values for each cohort are shown in Supplementary Figure S3.

The identification of GSTP1 as an N-Myc target gene also has importance for malignancies

with high expression or amplification of c-Myc. N-Myc and c-Myc share a common set of target

genes and are functionally interchangeable in the mouse (33). Furthermore, GSTP1 was one of a

large number of genes identified as potential c-Myc transcriptional targets in a high-throughput

ChIP study (25) and GSTP1 is transcriptionally regulated by c-Myc in osteosarcoma cell lines (GP,




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manuscript in preparation). Finally, as Gstp1 is tightly coupled to MYCN expression in

neuroblastomas arising in TH-MYCN mice, this is likely to be a suitable pre-clinical model in which

to assess the efficacy of GSTP1 inhibitors.

While the most familiar role for GSTP1 is the conjugation of reduced glutathione to

cytotoxic drugs, GSTP1 may possess other functions that contribute to chemoresistance, including

inhibition of JNK1 (34), and activation of extracellular signal regulated kinase (ERK) (35). In

addition to potential roles in mediating drug resistance in neuroblastoma, GSTP1 could potentially

impact other tumor properties through its ability to conjugate the cyclopentenone prostaglandins

15d-Δ12,14-PGJ2 and PGA2 (36), leading to their export through ABCC1 and ABCC3 (37), and its

ability to sequester 15d-Δ12,14-PGJ2 in the cytosol independently of conjugation (38). Both

mechanisms may prevent cyclopentenone prostaglandins from binding to the nuclear localized

gamma isoform of the peroxisome proliferator-activated receptor (PPAR-γ), activation of which is

anti-proliferative, pro-differentiation and pro-apoptotic (39) in cancer cells. In neuroblastoma,

activation of PPAR-γ by 15d-Δ12,14-PGJ2 has been shown to induce apoptosis (40) and

differentiation (41). While we cannot exclude these additional functions in our experiments,

analysis of microarray data available for the Oberthuer cohort did not reveal any evidence of an

association between GSTP1 expression and that of JNK1 (MAPK8), ERK1 (MAPK1) or PPARγ

(PPARG; Supplementary Figure S4), or with pathways associated with these genes (data not

shown).

GSTP1 is up-regulated in numerous human tumors and high GSTP1 expression is associated

with chemoresistance (7-9). To our knowledge, this is the first study to reveal the prognostic

significance of GSTP1 expression in neuroblastoma and the first to demonstrate that GSTP1 is a

direct transcriptional target of the MYCN oncogene, which is frequently amplified in poor-outcome

neuroblastoma. These observations have significance both for MYCN-amplified neuroblastoma and

for other diseases with amplification of Myc family oncogenes.




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19

Table 1

Event-free survival Overall survival

Factor Hazard

ratio 95% CI P Hazard

ratio 95% CI P COG-209 Tumor stage 4.6 2.1 to 10.0 <0.001 9.8 3.4 to 28.7 <0.001 Age 2.4 1.1 to 5.1 0.025 2.5 1.1 to 6.1 0.037 MYCN status 1.8 0.9 to 3.6 0.086 2.1 1.0 to 4.1 0.04 GSTP1 expression 2.6 1.2 to 5.5 0.011 2.2 1.0 to 4.8 0.047 Oberthuer Tumor stage 2.7 1.6 to 4.8 <0.001 7.7 2.6 to 23.1 <0.001 Age 0.8 0.4 to 1.4 0.385 1.5 0.6 to 4.1 0.411 MYCN status 2.3 1.3 to 4.1 0.005 4.3 2.1 to 8.9 <0.001 GSTP1 expression 2.0 1.1 to 3.8 0.029 2.3 1.1 to 4.9 0.024

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




20

Figure and table legends

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

Figure 1: GSTP1 is marker of poor-prognosis in neuroblastoma.

Kaplan-Meier curves for event free survival (A) and overall survival (B) in 51 NB patients 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

analysed by log-rank test. (C) GSTP1 is expressed at a higher level in MYCN-amplified tumors by

comparison with those without MYCN amplification. Graph shows relative expression as

determined by ΔΔCT method ± SD (see Materials and Methods) with P value determined by

Student’s t-test. (D) GSTP1 protein levels in six representative neuroblastoma tumors. Relative

expression at mRNA level is shown above the blots, while protein expression relative to α-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 = 0.96, P = 0.002). The relationship was analysed

using linear regression.

Figure 2: GSTP1 is prognostic for outcome in two large, independent neuroblastoma cohorts.

Kaplan-Meier curves for neuroblastoma patients from the COG and Oberthuer cohorts showing

EFS for the overall cohorts (A, D), patients with tumors lacking MYCN amplification (B, E), and

those from patients of INSS stage 3 or 4 (C, F), according to the expression of GSTP1. In each case,

tumors were dichotomized into “low” and “high” expression around the upper decile value.

Survival curves were analysed by log-rank test. Five year EFS rates are indicated.

Figure 3: GSTP1 gene expression correlates with that of MYCN.




21

Expression of GSTP1 correlates with that of MYCN in tumors from the COG cohort either with

MYCN-amplification (A), or without (B), while Gstp1 expression correlates with that of the MYCN

transgene in tumors from the TH-MYCN mouse neuroblastoma model (C). The relationships were

analysed using Spearman’s rank correlation.

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 Tet21/N cells expressing MYCN. Relative

enrichment is compared to the pre-immune serum. The results represent the mean ±S.E. of five

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; 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 Tet21/N cells. Cells were either not treated (NT; MYCN

ON), or treated with tetracycline for 24 or 48 h (TET 24h, or TET 48h; 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) ±S.E. of

three independent transfections.




Figure 1: GSTP1 is a marker of poor prognosis in neuroblastoma

A BA

2040

6080

100

0

EFS

(%)

Low (n=25)

High (n=26)

92%

51%

P = 0.002 2040

6080

100

OS

(%)

Low (n=25)

High (n=26)63%

96%

P = 0 006

C

0

0 1000 2000 3000 4000Analysis time (d)

00 1000 2000 3000 4000

P 0.006

Analysis time (d)

D EBE

(2)-

C

primary tumors

3 5 6 46 57 90 mRNA

�-tubulin

GSTP1

B 3 5 6 46 57 90 mRNA

1 .03 .3 .16 .54 .68 protein1.4




Figure 2: GSTP1 is prognostic for outcome in two, large independent neuroblastoma cohorts

COG cohort Oberthuer cohort

A D

50

75

100

High (n=21)

Low (n=186)

High (n=24)

Low (n=227)

52%

75%

All patientsAll patients

74%

EFS

(%)

FS (%

)

50

75

100

B E

0

25

0 2000 4000 6000Analysis time (d)

0 2000 4000 6000

Analysis time (d)

High (n 24)

P = 0.002 P < 0.00124%E E

0

25

B ENo MYCN amplification

High (n=15)

Low (n=169)

No MYCN amplification

60%

80%

EFS

(%)

High (n=15)

Low (n=202)78%

EFS

(%)

50

75

100

50

75

100

C F

0 500 1000 1500 2000 2500

Analysis time (d)

P = 0.025

E

0 2000 4000 6000Analysis time (d)

P < 0.001

26%E

0

25

0

25

C F

Low (n=74)

Stage 3 and 4 patientsStage 3 and 4 patients

31%

52%

EFS

(%)

Hi h ( 18)

Low (n=87)55%

EFS

(%)

50

75

100

50

75

100

0 500 1000 1500 2000 2500

Analysis time (d)

High (n=12)P = 0.022

31%

0 2000 4000 6000

Analysis time (d)

P = 0.007

High (n=18)15%

0

25

0

25




Figure 3: GSTP1 expression correlates strongly with that of MYCN in tumours.

A

100

150iv

e ex

pres

sion

)r = 0.61P = 0.003n = 21

ATumors with MYCN amplification

0

50

0 50000 100000 150000

GSTP1

(rel

ati

MYCN (ΔΔCT)

150

200

expr

essi

on)

r = 0.73P < 0.001n = 184

BTumors without MYCN amplification

0

50

100

0 5000 10000 15000GSTP1

(rel

ativ

e

MYCN (ΔΔCT)

exp

ress

ion)

15

20

C TH-MYCN mouse tumors

Gstp1 (relative expression)

MYCN

(rel

ativ

e

0

5

10

0 5 10 15 20

r = 0.91P < 0.001n = 20




Figure 4: GSTP1 is a direct transcriptional target of N-Myc




Published OnlineFirst December 27, 2011.Cancer Res Jamie I Fletcher, Samuele Gherardi, Jayne Murray, et al. neuroblastomaglutathione transferase GSTP1, a marker of poor outcome in N-Myc regulates expression of the detoxifying enzyme

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