# PPARγ in Neuroblastoma

Authors: Alessandro Peri; Ilaria Cellai; Susanna Benvenuti; Paola Luciani; Silvana Baglioni; Mario Serio
Journal: PPAR Research
 (2008)

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## Abstract

Neuroblastoma (NB) is the most common extracranial tumor in children and accounts for around 15% of all paediatric oncology deaths. The treatment of NB includes surgery, chemotherapy, and radiotherapy. Unfortunately, most children with NB present with advanced disease, and more than 60% of patients with high-risk features will have a poor prognosis despite intensive therapy. Agonists of the nuclear receptor peroxisome proliferator-activated receptorγ (PPARγ) have been shown to have pleiotropic effects, including antineoplastic effects. The studies that addressed the role and the possible mechanism(s) of action of PPARγ in NB cells are reviewed.


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## Body

## 1. Introduction

Neuroblastoma (NB), the
most common extracranial solid tumor in children, accounts for more than 7% of
malignancies in patients younger than 15 years and around 15% of all paediatric
oncology deaths [1]. The disease has a heterogeneous clinical presentation and
course [2]. First of all, NB is a disease of the sympaticoadrenal lineage of
the neural crest, and therefore tumors can develop anywhere in the sympathetic
nervous system. The majority of NB is developed within the abdomen and at least
50% of these tumors arise in the adrenal medulla [2]. Other frequent
localizations include the neck, chest, and pelvis
[3]. The
clinical presentation of the disease may be also highly variable and depends on
the site of the primary tumor as well as on the presence or absence of
metastatic disease (mostly haematogeneous dissemination to cortical bone, bone
marrow, liver, and noncontiguous lymph nodes) or paraneoplastic syndromes. The
diagnosis of NB is based on histopathological assessment of tumor tissue or on
the detection of cancer cells in a bone marrow aspirate/biopsy, together with
the presence of increased levels of urinary catecholamines [2]. Imaging studies
for the localization of the disease include computed tomography, magnetic resonance, 99mTc-diphosphonate, or metaiodobenzylguanidine (using 123I)
scintigraphy for the detection of bone metastases.The treatment of NB
includes surgery, chemotherapy (i.e., cisplatin, etoposide, doxorubicin,
cyclophosphamide, vincristine) [4], and radiotheraphy. Unfortunately, although
substantial improvement in outcome of certain subsets of patients has been
observed during the past few decades [2], most children with NB present with
advanced disease and more than 60% of patients with high-risk features will
have a poor prognosis despite intensive therapy [5, 6]. Thus, research efforts
to understand the biological basis of NB and to identify new and more effective
therapies are essential to improve the outcome for these children. In the last
years an expanding number of new agents have been developed for use in
high-risk patients affected by recurrent disease. Cytotoxic agents, such as the
topoisomerase 1 inhibitors topotecan and irinotecan, have an acceptable
toxicity profile and are effectively used in early relapsing NB [7–10]. The
delivery of radioactive molecules that are selectively concentrated in NB
cells, such as metaiodobenzylguanidine, somatostatin analogues, anti-GD2 (a
disialoganglioside) antibodies, has been used in clinical trials [11–22]. GD2-targeted
therapies using monoclonal antibodies are under investigation in phase III
trials [19, 23, 24], and other immunotherapeutic strategies (i.e., vaccination
or cellular immunotherapy using engineered cytolityc T lymphocytes) are currently
investigated [25, 26]. Similarly, angiogenesis [27–33] and tyrosine kinase [34–38]
inhibitors appear as an attractive therapeutic option and clinical trials are ongoing. Retinoids
have been shown to interfere with cell growth and to induce apoptosis in NB
cells [39, 40] and preliminary clinical trials with retinoids in NB resulted in
improved event-free survival in high-risk patients, with limited toxic effects [41, 42]. Thiazolidinediones (TZDs) are a class of molecules that activate the
nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) [43] and
promote association with the 9-cis retinoic X receptor (RXR) to form functional heterodimers that recognize its
cognate DNA response element within target genes [44, 45]. TZDs have been shown to have antineoplastic effects, as
extensively discussed in this issue of the journal, in agreement with the
demonstration that PPARγ/RXR signalling exerts an important role in
inhibiting cell proliferation and/or in inducing apoptosis [46]. It has been
also shown that PPARγ and RXR ligands may have a synergistic effect in inducing
cell differentiation [47, 48] and in inhibiting cell growth in different
tumors, such as colon, lung, and breast cancer [49–51]. There is evidence
that also PPARα and PPARβ ligands may play a role in counteracting tumoral cell
growth and in promoting cell differentiation, including neuroblastoma cells [52, 53]. However, most of the reports covering this issue, that have been published
in the literature so far, deal with PPARγ agonists. Therefore, the role of
PPARγ ligands as a possible therapeutic option in NB is reviewed and discussed here.


## 2. PPARγ and PPARγ Agonists in Neuroblastoma

The
first demonstration that PPARγ is expressed in NB cells was provided by Han et al. in 2001 [54]. Using RT-PCR the
authors showed that LA-N-5 NB cells express also PPARβ, but not PPARα. Similarly,
in sections from human primary NB immunostaining for PPARγ was detected in the
nucleus and occasionally in the cytoplasm of cells, particularly in those showing
ganglionic differentiation. Sato et al.
[55] addressed the possibility that the amount of expression of PPARγ in NB might
be correlated to patients' outcome. To this purpose, the level of mRNA was
measured by semiquantitative RT-PCR in NB samples from 17 patients under the
age of one year. In this subset of patients, spontaneous differentiation and
regression are often observed [56], and some investigators suggested to observe
these patients without surgery until there is an increase of vanilmandelic acid
(VMA) or tumoral growth occurs [57, 58]. PPARγ mRNA was present in 12 samples. No
difference between the expression of PPARγ and histology, age, staging, DNA
ploidy was observed, yet a correlation with the
change in urinary VMA was found. In fact, in samples resected from patients,
who showed a reduction of VMA in the period of time preceding surgery (2–7
months), higher PPARγ expression was detected compared to those patients in
which VMA increased. The authors hypothesized that PPARγ might play a role in
the decrease of VMA and hence in the regression of early-onset NB. Thereafter,
several studies addressed the potential role of endogenous or synthetic PPARγ
ligands in counteracting NB cell growth.5-Deoxy-Δ12,14-prostaglandin J2 (15-deoxy-PGJ2) is a naturally
occurring downstream metabolite of PGD2, that is produced by
degradationof PGD2 [59]. In contrast to classic
prostaglandins, which act after binding to cell surface G-protein
coupled receptors (GPCRs), 15-deoxy-PGJ2 is a
high-affinity endogenous ligand of PPARγ. A pro-apoptotic effect of 15-deoxy-PGJ2 in SH-SY5Y NB cells, that was reverted by the caspase inhibitor Z-VAD, was
reported by Rohn et al. [60]. A
subsequent study confirmed that 15-deoxy-PGJ2 was able to inhibit
cell growth and to induce apoptosis via the activation of ERK2 in two
additional NB cell lines (i.e., SK-N-SH and SK-N-MC). An increase of the
expression of the proapoptotic proteins caspase-3, caspase-9, and Bax, together
with the decrease of the anti-apoptotic protein Bcl-2, was also observed [61]. The
PPARγ antagonist GW9662 reverted the effects of 15-deoxy-PGJ2,
including the activation of ERK2. The authors concluded that 15-deoxy-PGJ2 induced apoptosis in a PPARγ-dependent manner through the activation of
ERK pathway. Another study showed that the mechanism by which 15-deoxy-PGJ2 arrests cell growth may vary depending on the content of lipids in the
culture medium [62]. In particular, the delipidation of fetal calf serum, which
removes known serum lipid mitogens including lysophosphatidic
acid [63] and sphingosine 1-phosphate [64], potentiated the degree of
15-deoxy-PGJ2-induced growth inhibition via PPARγ-dependent apoptosis in the NB cell
line IMR-32. Conversely, growth inhibition in the presence of complete medium occurred
through programmed cell death typeII (autophagy).PPARγ-independent
effects of 15-deoxy-PGJ2 have been also described. Jung et al. reported that this PPARγ ligand was
able to increase NGF-induced differentiation of PC-12 NB cells, as assessed by
neurite extension and expression of neurofilament [65]. Pretreatment with the
PPARγ antagonist bisphenol A diglycidyl either did not alter the differentiating
activity of 15-deoxy-PGJ2. The fact that PC-12 cells do not express
PPARγ further supported the hypothesis that the biological effects elicited by 15-deoxy-PGJ2 were not
mediated by this receptor. Conversely, 15-deoxy-PGJ2 enhanced NGF-induced
p38 MAP kinase expression and phosphorylation as well as the activation of
transcription factor AP-1, that on turn were counteracted by a specific
inhibitor of p38 MAP kinase (SB203580). Altogether, these data suggested that
the promoting effect of 15-deoxy-PGJ2 on cell differentiation may be
mediated by the activation of p38 MAP kinase in conjunction with the AP-1
signalling pathway.Other studies addressed
the role ofsynthetic PPARγligands in
counteracting cell growth in NB. In the already mentioned work by Han et al., in which the presence of PPARγ
in NB cells was described for the first time, the authors also demonstrated
that the synthetic PPARγ agonist GW1929 induced the differentiation of LA-N-5
cells and inhibited cell proliferation [54]. A subsequent study of the same
group showed that the prodifferentiating effect of GW1929 is mediated by PPARγ,
because it was inhibited by the cotreatment with specific antagonists [66].   The
antiproliferative effects of the TZDs ciglitazone, pioglitazone, troglitazone,
and rosiglitazone in different NB cell lines (i.e., LAN-1, LAN-5, LS, IMR-32,
SK-N-SH, SH-SY5Y) were determined by Valentiner et al. [67]. In these cell lines, which express PPARγ, the four
ligands were able to markedly inhibit cell growth at the highest doses that
were used (10 and 100μM). Ciglitazone determined the strongest inhibitory
effect (more than 90% inhibition). The potency of the different PPARγ ligands was
not related to the amount of expression of PPARγ in NB cell lines. Thus, the
authors hypothesized that the effects of the molecules that were used seem to
be independent of the amount of PPARγ protein in one particular cell line.
Conversely, they concluded that the response to PPARγ ligands may rather depend
on various cellular conditions, which are associated with the function of the
receptor, such as its activation, translocation to the nucleus and binding to PPAR
response elements (PPRE). The role played by PPARγ transactivation was confirmed
by the finding that growth inhibition determined by 15-deoxy-PGJ2 and
ciglitazone in NB cells was counteracted by the repression of PPARγ
transactivation via retinoblastoma protein overexpression [68]. Further studies
investigated whether the inhibitory effect of TZDs on cell growth was mediated,
at least partially, by a stimulatory effect on apoptosis. Kato et al. found that in NB-1 cells
troglitazone induced PPARγ-dependent apoptosis [69]. Similar data were reported
later on by Schultze et al. [70], who
showed that in SHEP NB
cells the pro-apoptotic effect of the
death ligand TRAIL is reinforced by troglitazone. However, troglitazone-induced
sensitization to TRAIL appeared to be PPARγ-independent, because it was achieved
at concentrations that failed to activate PPARγ. Conversely, the authors highlighted
the fact that troglitazone may induce apoptotic death by various
PPARγ-independent mechanisms. In particular, troglitazone led to a marked
downregulation of the antiapoptotic protein Survivin, as well as to an upregulation
of the agonistic TRAIL receptor TRAIL-R2.Overall, these data strongly
indicate that PPARγ ligands are able to effectively counteract cell growth and
to induce apoptosis in NB cells. Undoubtedly, the role of PPARγ in eliciting
these responses would be further clarified by studies designed for instance to
manipulate gene expression (i.e., by small interfering RNA or dominant negative
strategies). To our knowledge, there are only two reports from one Korean group
showing, in contrast to the current opinion, that a PPARγ agonist (i.e.,
rosiglitazone) protects NB (SH-SY5Y) cells against the neurotoxins acetaldehyde
and 1-methyl-4-phenylpyridinium ion, through inhibition of apoptosis [71, 72].


## 3. Differential PPARγ Transactivation in Neuroblastoma Cell Lines with a Different Phenotype: Relationship with the Response to Rosiglitazone

NB
is a phenotypically heterogeneous tumor, displaying cells of neuronal,
melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also
present in vitro, where cells
of neuroblastic (N) or stromal (S) type may be identified. It has been
hypothesized that the sensitivity to PPARγ ligands may be, at least partially,
dependent on the different cell phenotype. To this purpose, Servidei et al. examined the response of 8
different NB cell lines with N (SH-SY5Y, LA-N-5, SMS-KCNR, SK-N-DZ), mixed
(SK-N-FI, LA-N-1), or S (SH-EP1, SK-N-AS) phenotype to PPARγ agonists [73]. All
the cell lines investigated expressed a functionally active PPARγ. 15-deoxy-PGJ2 and rosiglitazone inhibited cell growth in all cell lines, and the sensitivity
appeared to be more related to the cell phenotype than to PPARγ expression. In
particular, the N type cells appeared the most sensitive to treatment. In this
experimental setting, the cotreatment with PPARγ ligands and the RXR ligand 9-cis
retinoic acid did not determine any synergistic effect on growth inhibition. The
more evident response of N type cells to PPARγ ligands was in part related to
their higher capability to undergo apoptosis, although only 15-deoxy-PGJ2 appeared
to effectively induce the apoptotic cascade in these cells. It has to be said
that in this study some experimental observations (i.e., apoptosis and cell
viability) were not performed in all the investigated NB cell lines.In order to further clarify the
mechanisms underlying the response of NB cells to PPARγ agonists, we compared
the response of two cell lines (SH-SY5Y, N type, and SK-N-AS, S type) to
rosiglitazone. In contrast to the above-mentioned findings, we observed that micromolar
concentrations of rosiglitazone inhibited cell proliferation and reduced cell
viability more effectively in SK-N-AS than in SH-SY5Y [74]. The PPARγ
antagonist BADGE reverted the effect of rosiglitazone, thus suggesting a direct
role of PPARγ in mediating the effects of this agonist on cell proliferation
and viability. In addition, we found that SK-N-AS cells were more sensitive to
rosiglitazone in terms of reduction of cell adhesion and invasiveness. The
latter effect was in agreement with rosiglitazone-dependent reduced expression
of matrix metalloproteinase-9 (MMP-9). In addition, rosiglitazone determined a trend
toward increased expression levels of tissue inhibitor of matrix metalloproteinase-1
(TIMP-1). MMPs, which promote the invasion of extracellular matrix by tumoral
cells, have been related to the progression of different tumors, including NB [75, 76]. In our study, we also addressed the possible role of rosiglitazone in inducing
apoptosis. We demonstrated that micromolar concentrations of this molecule were
able to induce caspase-3 activation in SK-N-AS, but not in SH-SY5Y (up to 50μM). Therefore, all our data indicated that rosiglitazone played an effective
antitumoral role in the S type SK-N-AS, yet not in the N type SH-SY5Y NB cells.
Although our study was limited to two cell lines, this apparent prevalent
effect on a particular cell phenotype may have clinical resonance. In fact, it
is known that in NB, following cytotoxic therapy, the residual tumor often
shows a reduction of the neuroblastic elements and the persistence of stromal
components [77].   Hence, a molecule that appears to have S type NB cells as a
preferential target might be of interest in the setting of residual disease.A
further aim of our study was to determine the reason underlying the peculiar
sensitivity to rosiglitazone displayed by SK-N-AS cells. Both SK-N-AS and
SH-SY5Y expressed a similar amount of PPARγ. However, in transient transfection
experiments, in which a PPRE-thimidine kinase luciferase reporter plasmid was
inserted, we observed that in SK-N-AS 20μM rosiglitazone induced a near
three-fold increase of the reporter activity compared to untreated cells.
Conversely,  no effect was elicited in SH-SY5Y.
Only when these cells were co-transfected with a human PPARγ expression
plasmid, the response to rosiglitazone was present. These data indicated that
the original lack of response showed by SH-SY5Y was due to a very low or absent
transactivation potential of the endogenous PPARγ (Figure 1). The different
efficacy of PPARγ as a transcriptional activator in the two cell lines might be
hypothetically due to the presence of a PPARγ gene mutation. However, no
mutation was found in the entire coding region of the gene. Conversely, we
found that the amount of phosphorylated PPARγ was markedly lower in SK-N-AS
than in SH-SY5Y cells (Figure 2). There is evidence that phosphorylation reduces
the activity of the receptor [78]. Therefore, our conclusion was that the
higher efficacy of rosiglitazone in SK-N-AS cells was due to a reduced
phosphorylation status, hence to increased activity, of PPARγ. To our
knowledge, this was the first demonstration that the response of NB cells to TZDs
may be dependent on PPARγ transactivation.Figure 1
PPARγ transcriptional activity in control untreated
NB cells (C), in cells treated with rosiglitazone (RGZ) (20μM), and in cells
transfected with PPARγ and treated with RGZ. L/C: peroxisome proliferator response element-n73-tk-luciferase
reporter activity, normalized for CAT
activity. *=P<0.05versus C.**=P<0.05versus C, and versus RGZ-treated
cells, in the absence of PPARγ transfection (from [74], modified).Figure 2
Detection of total (anti-PPARγ antibody) and phosphorylated (anti-P-Ser antibody) PPARγ, by Western
blot analysis after PPARγ immunoprecipitation (from  [74], modified).


## 4. PPARγ Agonists in Neuroblastoma Xenograft Models

To our knowledge, no
study on the in vivo effect of TZDs in neuroblastoma has
been published so far. However, our very recent preliminary in vivo observations on CD-1 athymic nude mice, in which SK-N-AS cells
were subcutaneously inoculated, appear to confirm our previous in vitro observations [74].
Rosiglitazone (150 mg/kg/day, in agreement with the average dose used in other in vivo studies addressing different
tumors) was administered by gavage for 4 weeks. Tumoral
growth was markedly reduced compared to control mice, treated with the vehicle
alone. At the end of treatment, the weight of the tumor in
rosiglitazone-treated animals was about 60% less than in control animals [Cellai et al; unpublished data]. An
extensive molecular characterization of tumor specimens is currently ongoing, in
order to elucidate the mechanisms underlying the growth inhibitory effect of
rosiglitazone observed in vivo
in our xenograft model.


## 5. Conclusions

In
the last few years in vitro studies have shown that PPARγ agonists
may inhibit NB cell growth by stimulating cell differentiation and/or by inducing
apoptosis. The different molecules that have been tested have generally
produced similar results. However, the mode of action may change depending on
the agonist and/or on the different cell line used. In addition, both
PPARγ-dependent as well as PPARγ-independent effects have been described. Our
recent data suggest that PPARγ transactivation, determined at least in part by
the phosphorylation status of the receptor, may play an important role in
determining the response of NB cells to PPARγ agonists. However, the exact
mechanisms of action and the possibility to predict the success or failure of
the treatment of NB with these molecules are, at this time, matter of further in vitro as well as in vivo research.



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