Combined RARa- and RXR-specific ligands overcomeN-myc-associated retinoid resistance in neuroblastoma cellsq

Tue Nguyen,a Jayne E. Hocker,a Wayne Thomas,a Stewart A. Smith,a Murray D. Norris,a

Michelle Haber,a Belamy Cheung,a and Glenn M. Marshalla,b,*

a Children�s Cancer Institute Australia for Medical Research, P.O. Box 81, Randwick, NSW 2031, Australiab Centre for Children�s Cancer and Blood Disorders, Level 1 South, Sydney Children�s Hospital, High St., Randwick, NSW 2031, Australia

Received 13 January 2002

Abstract

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through inter-

actions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide

variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid

(aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-

induced RARb levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused amarked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RARasignificantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results

indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more

effective chemo-preventive strategy in the disease.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Retinoic acid; Resistance; Synthetic retinoid; Retinoic acid receptor b; Neuroblastoma

Retinoids induce growth inhibition, differentiation,

and cell death in a wide range of cancer cell types in

vitro, and are used therapeutically in several humancancers [1]. The retinoid signal is mediated by retinoid

ligand binding to nuclear retinoid (RARa; b, and c) andretinoid receptor (RXRa; b, and c) protein dimers,

which then lead to altered transcriptional regulation of

target genes [2]. Many synthetic retinoids have recently

been described with highly specific receptor-subtype

binding, and have served as useful reagents to assess the

relative importance of individual receptor subtypes fordifferent retinoid effects [3–6]. Several lines of evidence

suggest that loss of basal or retinoid-inducible RARb

expression may be a common mechanism for retinoid

resistance in some human cancers [7,8]. However, other

evidence suggests RARb is unlikely to mediate all of theretinoid anti-cancer effects [9].

Neuroblastoma is a common solid tumour of early

childhood, which arises from the embryonic neural

crest. Poor patient prognosis and advanced clinical stage

correlate with amplification and high-level expression of

the proto-oncogene N-myc, as a clonal feature in ma-

lignant neuroblasts [10,11]. A recent clinical trial showed

that 13-cis-retinoic acid (13RA), used as a chemo-pre-ventive agent after conventional chemo-radiotherapy,

increased the survival rates of patients with advanced

stage neuroblastoma [12]. However, 50% of patients

demonstrated resistance to retinoid therapy through

unknown mechanisms. In this study, we sought to define

mechanisms for retinoid resistance of human neuro-

blastoma cells and strategies for overcoming that resis-

tance in vivo.

Biochemical and Biophysical Research Communications 302 (2003) 462–468

www.elsevier.com/locate/ybbrc

BBRC

qAbbreviations: aRA, all-trans retinoic acid; RAR, retinoic acid

receptor; RXR, retinoid X receptor; RARE, retinoic acid responsive

element.* Corresponding author. Fax: +61-2-9382-1789.

E-mail address: g.marshall@unsw.edu.au (G.M. Marshall).

0006-291X/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00177-3

Materials and methods

Chemicals. Synthetic receptor-selective retinoids CD336 (RARa),CD417 (RARb), CD666 (RARc), CD2314 (RARb), CD3640 (RXR),CD2872 (RXR), and 9-cis-retinoic acid (9RA) were supplied by Dr.

Uwe Reichert and Dr. Serge Michel from CIRD-Galderma (Sophia-

Antipolis, France). The natural retinoids, all-trans-retinoic acid (aRA)

and 13RA, were purchased from Sigma (St. Louis, MO, USA).

Cell culture. Neuroblastoma cell lines used in this study were

IMR-32, BE-(2)C, SHEP, and SH-SY5Y. The N-myc transfectant,

SHEP-S1, was a gift from Dr. Susan Cohn (Northwestern University,

Chicago) and Dr. Mary Lou Schmidt (University of Illinios, Chi-

cago). All cell lines were cultured at 37 �C in 5% CO2 in DMEM

supplemented with 10% LL-glutamine and 10% fetal calf serum. The

culture medium, with fresh retinoid, was replaced every 3 days. Viable

cell counts were evaluated using trypan blue. All experiments were

performed in duplicate and repeated 3 times.

RNA preparation and RT-PCR. Total RNA preparation and

mRNA expression level for the RARb2 gene was analysed by com-petitive RT-PCR as previously described [13]. The RARb2 gene wasco-amplified with a control gene (b2-microglobulin, b2M), using gene-specific primers that did not amplify genomic DNA. The RARb2forward primer sequence was 50-CTACACTGCGAGTCCGTCTT-30

and reverse primer sequence was 50-CAGAGCTGGTGCTCTGTG

TT-30.

Western blot. Nuclear proteins were extracted from cells, as pre-

viously described [14]. Protein samples were quantified by BCA protein

assay kit (Pierce, Illinois, USA) according to the manufacturer�s in-structions. Protein samples were separated by 10% SDS–PAGE. The

membrane was blocked with 10% skim milk in PBS overnight. The

membrane was then incubated with polyclonal rabbit anti-RARb(Santa Cruz, CA, USA) diluted 1/400 in PBS/Tween 20 0.05% and then

washed with TBST (20mM Tris, 0.5M NaCl, and 0.3% Tween 20, pH

7.4). Following incubation with anti-rabbit secondary antibody (Santa

Cruz), RARb protein bands were detected with SuperSignal chemilu-minescence (Pierce, Illinois, USA).

Statistic analysis. All graphs and statistic analysis were performed

by Prisms statistical software (CA, USA).

Results

We examined 3 human neuroblastoma cell lines,

originally established from different patient tumours

(SH-SY5Y, BE-(2)C, and IMR-32), for variation in

their sensitivities to the growth inhibitory effects of ret-

inoids in vitro. The N-myc proto-oncogene was ampli-

fied and over-expressed in the BE-(2)C and IMR-32 celllines, while the SH-SY5Y cell line had low expression

and a single copy of N-myc [15]. The 3 cell lines were

treated continuously for 7 days with 0.1, 1.0, and 10 lMaRA. We found a wide variation in the level of retinoid-

induced growth inhibition, when the 3 cell lines were

compared for their sensitivities at the lower aRA con-

centration (0.1 lM) (Fig. 1). The SH-SY5Y cell line wasmarkedly growth inhibited at 0.1 lM aRA, when con-trasted with the relatively resistant BE-(2)C and IMR-32

cells. At the highest concentration of 10 lM aRA, all 3

cell lines were similarly growth inhibited, when com-

pared with their untreated control.

An early and necessary response to the retinoid signal

is induction of RARb2 gene expression and exogenous

expression of RARb enhances aRA sensitivity [2,16]. All3 cell lines demonstrated a similar level of retinoid-in-

duced expression of both RARb2 mRNA and protein at24 h (Figs. 2A and B), respectively. Moreover, the reti-noid-induced RARb2 expression was equally sustainedin all 3 cell lines, following continuous exposure to aRA

for 21 days. There was also concordance between the

level of induction of RARb2 mRNA and protein in all 3cell lines. There was no significant or consistent differ-

ence between the retinoid-induced RARb2 expression

Fig. 1. Sensitivities of neuroblastoma cell lines to various concentra-

tions of aRA. Cells were continuously treated with 0.1, 1, and 10 lMaRA for 7 days. Viable cell counts were performed by trypan blue

staining. Experiments were performed in duplicate and repeated 3

times.

T. Nguyen et al. / Biochemical and Biophysical Research Communications 302 (2003) 462–468 463

level at 0.1 and 10 lM across the 3 cell lines, suggestingthat aRA-induced RARb2 expression is not dependenton retinoid concentration. Thus, differences in the phe-

notypic responses of the 3 neuroblastoma cell lines could

not be explained by differences in the responsiveness of

the endogenous RARb2 gene.We then examined whether over-expressed N-myc

influenced the retinoid sensitivity of neuroblastoma

cells. We evaluated the effect of exogenous over-ex-pression of N-myc in a neuroblastoma cell line without

N-myc expression (SHEP) on the level of retinoid-in-

duced growth inhibition. The SHEP cell line was stably

transfected with an N-myc expression vector (SHEP-S1)

or empty vector control (SHEP-EV). Over-expression of

N-myc increased the growth rate of SHEP cells by 4-fold

(data not shown). SHEP transfectant cells were then

treated continuously for 7 days with 10 lM aRA, andthe level of growth inhibition was expressed as the ratio

of aRA-treated/untreated cell numbers. In SHEP-S1

cells, aRA inhibited cell growth by only 15%, compared

Fig. 3. (A) The effect of N-myc over-expression on the growth inhib-

itory activity of aRA. Neuroblastoma cell lines, SHEP, its empty

vector (SHEP-EV), and N-myc transfectant (SHEP-S1) were treated

with 10 lM aRA for 7 days. Viable cell counts were performed by

trypan blue staining. Experiments were performed in duplicate and

repeated 3 times. The figure represented the means and standard error

of the percentage of growth inhibition by 10 lM aRA in each cell line,

compared to their untreated controls. (B) Analysis of aRA-induced

RARb2 expression in SHEP and SHEP-S1 by RT-PCR. The figurerepresents the means and standard error of mean of the ratio of

RARb2:b2 microglobulin expression from 3-independent RT-PCRs.

Fig. 2. (A) Analysis of aRA-induced RARb2 mRNA expression levelsby competitive RT-PCR. The figure represents the means and standard

error of mean of the ratio of RARb2:b2 microglobulin expression from3-independent RT-PCRs. (B) Detection of aRA-induced RARb2protein expression in neuroblastoma cell lines. Lane 1 represents un-

treated control. Lanes 2–5 represent cells treated with 10 lM aRA for

1 h, 1 day, 3 days, and 7 days, respectively. Lanes 6 and 7 represent the

untreated BE-(2)C transfected with MEP4 empty-vector, V, or MEP4/

RARb2 full-length cDNA expression vector, used as a negative and

positive control for RARb2 protein expression, respectively. The ex-periment was repeated 3 times.

464 T. Nguyen et al. / Biochemical and Biophysical Research Communications 302 (2003) 462–468

with 52% and 47%, in the parental and SHEP-EV con-trol cells, respectively (Fig. 3A). Retinoid-induced

RARb2 mRNA expression was observed equally in

SHEP-S1 cells and controls (Fig. 3B).

The initiation of the retinoid signal is believed to re-

quire the formation of liganded RAR–RXR protein

heterodimers at cognate retinoic acid response elements

in the promoter regions of retinoid target genes.

Therefore, we examined the hypothesis that combina-tions of receptor-specific retinoids may be more effective

than single, receptor subtype non-specific, retinoids,

such as aRA, 9RA, and 13RA. As single agents, the

receptor non-specific retinoids, aRA and 9RA, were the

most effective inhibitors of cell growth at the end of 7days of continuous retinoid treatment (Fig. 4). The most

effective single, receptor-specific retinoid was the RARa-specific ligand (CD336), while the RXR agonist

(CD3640) enhanced cell growth when compared to un-

treated control.

We next combined RAR-specific retinoids (CD336

[RARa], CD2314 [RARb], CD417 [RARb], and CD666

Fig. 4. Growth inhibitory effects of natural and synthetic receptor-se-

lective retinoids. Neuroblastoma cell lines were treated with 0.1lM of

each of the retinoids as a single agent for 7 days. Viable cell counts

were performed by trypan blue staining. Experiments were performed

in duplicate and repeated 3 times.

Fig. 5. Growth inhibitory effects by natural and synthetic receptor-

selective retinoids used in combination treatments. Neuroblastoma cell

lines were treated with different combinations of retinoids at 0.1 lMconcentration for 7 days. Viable cell counts were performed by trypan

blue staining. Experiments were performed in duplicate and repeated 3

times.

T. Nguyen et al. / Biochemical and Biophysical Research Communications 302 (2003) 462–468 465

[RARc]) with RXR agonists (CD3640, CD2872) in thesame growth inhibition assay at retinoid concentrations

of 0.1 lM. We found that the combination of the

RARa-specific retinoid (CD336) and the RXR agonist

(CD3640) was the most effective growth inhibitory

combination of the synthetic receptor-specific retinoids

(Fig. 5). However, in the N-myc single copy cell line,

SH-SY5Y, aRA, and 9RA remained significantly more

effective than combination therapy with CD336 andCD3640. Combinations of aRA with either CD336 or

CD3640 singly, or in combination, enhanced the growth

inhibitory effect of aRA in the two N-myc amplified celllines, but appeared to block the effect of aRA in SH-

SY5Y cells. The combination of CD336 and CD3640

was a more effective growth inhibitor than 13RA, which

is in current clinical use in neuroblastoma patients. In-

terestingly, the RARb-specific retinoids (CD2314,

CD417), either singly or in combination, appeared to

promote, rather than inhibit, cell growth.

When the level of retinoid-induced RARb2 mRNAexpression and the degree of growth inhibition were

directly compared, the data clustered into two groups of

Fig. 6. (A) Comparison of RARb2 expression levels following treatment with different combinations of natural and synthetic retinoids. The figurerepresents the means and standard error of mean of the ratio of RARb2:b2 microglobulin expression from 3-independent RT-PCRs. (B) Correlation

analysis between the growth inhibitory activity of natural/synthetic retinoids and retinoid-induced RARb2 expression levels. Mean values of RARb2expression (X -axis) from 3-independent experiments are plotted against the means of viable cell counts (Y -axis) from the same experiments. Cor-

relation coefficient values, r, were calculated by Prism statistic software.

466 T. Nguyen et al. / Biochemical and Biophysical Research Communications 302 (2003) 462–468

retinoid therapies (Figs. 6A and B). Those retinoidtherapies which induced RARb2 mRNA expression to alevel represented by an RT-PCR ratio of >0.5 causedsignificant growth inhibition, whereas retinoid therapies

which failed to induce RARb2 mRNA expression be-

yond this level showed little or no growth inhibition.

However, the correlation coefficient values for RARb2expression level and the level of growth inhibition were

not strong for any of the three cell lines: IMR-32,r ¼ 0:688; BE-(2)C, r ¼ 0:486; and SH-SY5Y,

r ¼ 0:184. When used singly, none of the receptor-spe-cific retinoids-induced RARb2 expression (data not

shown).

Discussion

Our study suggests that N-myc gene amplification,

and consequent N-myc over-expression, may play a role

in mediating retinoid resistance in vivo. Combination

retinoid therapies with synthetic RARa- and RXR-

specific agonists were more effective growth inhibitors in

N-myc-amplified cell lines than the retinoid currently in

clinical use, 13RA. Differences in the retinoid-induced

endogenous RARb2 expression did not explain thevariation in the phenotypic response of neuroblastoma

cells to exogenous retinoids, or the N-myc-mediated

retinoid resistance. However, induction of RARb2 geneexpression broadly correlated with effective growth in-

hibition in experiments using combination retinoid

treatments. These results have general and specific im-

plications for the action of retinoids as a chemo-pre-

ventive strategy in malignant diseases.The mechanism whereby high-level N-myc expression

blocked the retinoid anti-cancer effect is unclear, but

may involve several possibilities. First, N-myc and aRA

may have competing effects on the activation of cell

cycle regulatory proteins. Deregulated expression of N-

myc in neuroblastoma cell lines promotes rapid cell cycle

progression from G1 to S phase by activating cyclin/

cyclin-dependent kinase (Cdks) complexes, and seques-tering p27 expression and activity [17–20]. In contrast,

aRA induces G1 arrest by modulating the activity of G1

cyclin/Cdks and the Cdk inhibitors, p21/Waf1 and p27/

Kip1, in a cell-type-specific manner [21–27]. In neuro-

blastoma cell lines, aRA-inducible growth inhibition

and differentiation are associated with increased p27/

Kip1 expression [28,29]. Furthermore, cell cycle arrest

imposed by p27/Kip1 can be overcome by high expres-sion of c-myc or N-myc [27,30]. Collectively, these data

suggest that N-myc may block the retinoid-induced

growth arrest through effects on Cdk inhibitor proteins.

Second, repression of N-myc expression in neuroblas-

toma cells may be necessary for retinoid-induced growth

arrest to proceed. Repression of N-myc transcription

coincides with the retinoid anti-cancer effect in neuro-

blastoma cells [31,32]. However, N-myc transcriptionfrom an amplicon, or from the constitutive promoter of

a transfected expression vector, may be relatively resis-

tant to repression by retinoids. Thus, retinoid resistance

of N-myc-amplified neuroblastoma cells may relate to

unregulated transcription from the N-myc amplicon.

We have shown that combination synthetic RXR-

and RARa-specific ligands enhanced aRA-induced

growth inhibition in N-myc-amplified neuroblastomacell lines. Moreover, our results show that the retinoid

anti-cancer effect is mediated by both RARb-dependentand -independent mechanisms. Targeting RXR/RARadimers efficiently activated not only RARb-dependentgrowth inhibition, but also signalling pathways that

were independent of RARb expression [5,9]. The in-

creased efficacy of these synthetic ligands may have been

due to enhanced induction of apoptosis or reducedsusceptibility to enzymatic catabolism by pathways in-

volving retinoic acid hydroxylase [33–37]. More detailed

studies of the comparative phenotypic effects of the

combination RXR/RARa treatment and elucidation ofpathways controlled by the liganded RXR/RARa dimerwill address these different possibilities. More impor-

tantly, the effectiveness of combination retinoid therapy

will need to be tested in vivo.In summary, our study has provided evidence for the

general importance of RARb2 and N-myc expression inneuroblastoma cells as determinants of the retinoid anti-

cancer action in the disease. Moreover, we have defined

a novel therapeutic strategy to enhance retinoid sensi-

tivity of neuroblastoma cells by specifically targeting the

RXR/RARa heterodimer.

Acknowledgments

We thank Dr. U. Reichert and Dr. S. Michel from CIRD-Gal-

derma (France) for all of the synthetic retinoids. We also thank Dr. S.

Cohn and Dr. M.L. Schmidt (USA) for the N-myc transfectant cell

line, SHEP-S1. Tue Nguyen is a PhD candidate at Children�s CancerInstitute Australia for Medical Research, which is affiliated with the

University of New South Wales and Sydney Children�s Hospital. Thiswork was supported by National Health and Medical Research

Council grant (G.M.M., M.H., M.D.N.) and New South Wales State

Cancer Council (G.M.M., M.H., M.D.N.).

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