# The Critical Role of PPARγ in Human Malignant Melanoma

Authors: Christian Freudlsperger; Udo Schumacher; Siegmar Reinert; Jürgen Hoffmann
Journal: PPAR Research
 (2008)

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## Abstract

The past 30 years have only seen slight improvement in melanoma therapy. Despite a wide variety of therapeutic options, current survival for patients with metastatic disease is only 6–8 months. Part of the reason for this treatment failure is the broad chemoresistance of melanoma, which is due to an altered survival capacity and an inactivation of apoptotic pathways. Several targetable pathways, responsible for this survival/apoptosis resistance in melanoma, have been described and current research has focused on mechanism inactivating these pathways. As PPARγ was shown to be constitutively active in several tumour entities and PPARγ agonists extent strong anticancer effects, the role of PPARγ as a possible target for specific anticancer strategy was investigated in numerous studies. However, only a few studies have focused on the effects of PPARγ agonists in melanoma, showing conflicting results. The use of PPARγ agonists in melanoma therapy has to be
carefully weighted against considerable,
undesirable side effects, as their mode of action is not fully
understood and even pro-proliferative effects have
been described. In the current review, we discuss the role of
PPARs, in particular PPARγ in melanoma and their potential role as a molecular target for melanoma therapy.


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## Body

## 1. Malignant Melanoma and Molecular Targets in Melanoma Therapy

Cutaneous malignant melanoma is the most aggressive form of skin cancer. Despite attempts
to treat melanoma using a large variety of therapies, including immuno-, radio,
and chemotherapies, survival remains very poor once the disease has spread to
distant sites (median survival: 6–8 months) [1]. Systemic therapy,
immunotherapy, or even biochemotherapy have failed to improve the survival of
these patients. Until now, the only drug approved by the FDA for treatment of
metastatic melanoma is the alkylating agent dacrabazine (DTIC), which results
in clinical response only of 5–10% of cases when
given as a single agent [2]. This treatment failure is mainly due to the
notorious chemoresistance of melanoma cells. In contrast to other cancer cells,
this chemoresistance of melanoma cells seems not to be acquired selectively
following drug therapy, but to be more intrinsic in melanoma cells. Alteration
of survival capacity and inactivation of apoptotic pathways are the molecular
mechanisms responsible of conventional drug resistance in melanoma cells (see Figure 1). One example for a targetable pathway is the mitogen-activated protein
kinase (MAP-kinase) pathway, which plays a crucial role in cell proliferation,
invasion, and enhanced survival in diverse cancers [3]. A key player in the
MAP-kinase pathway is B-RAF, a serin/threonine protein kinase acting as an
oncogene [4]. The recent identification of activating mutations in B-RAF in
over 60% cases of melanoma has offered the first opportunity for a rationale
treatment program [3] and early clinical trials using the RAF kinase inhibitor
BAY 43-9006 have been encouraging, being the first positive example of how
targeted therapy can work in malignant melanoma. Other examples of targetable
pathways in melanoma are the phosphoinositide-3-kinase (PI3K)/Akt pathway,
which can be activated either through growth factors or loss of negative
regulators of this pathway [5]. One of the most critical regulators of Akt (also
known as protein kinase B) is the phophatase and tensin homologue (PTEN), which
degrades the products of PI3K, preventing the activation of Akt [6]. In several
studies it has been shown that up to 30% of melanoma, cell lines have lost PTEN
expression [7]. Finally, Huang et al.
investigated that the NFκ-B signaling pathway, acting as a key regulator of
survival in cancer cells, is constitutively activated in melanoma cells [8]. In
addition, a recent study has demonstrated that inhibiting NFκ-B activity, using
the proteasome inhibitor bortezomib, reduced melanoma cell growth in vitro [9].
Although these targets seem to be attractive ones for melanoma therapy in the
future, most of the findings in this area do not give a comprehensive picture
which would warrant a review. As several studies have shown an antiproliferative effect of PPARγ
agonists on several tumour entities including melanoma, this review focuses on
the role of the PPARγ as a possible target in melanoma therapy.Figure 1
Activating pathways known to be constitutively active and contributing to the
chemoresistance of melanoma cells. Over 60% of melanomas have activating
mutations of B-RAF, 30% have lost PTEN expression, majority with NF-κB
activation.


## 2. PPARγ and PPARγ Agonists

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription
factors, belonging to the nuclear receptor superfamily [10]. Activated
PPAR forms complexes with the retinoid receptor, which bind as a heterodimer to
peroxisome proliferator response elements (PPREs) on the DNA, initiating
transcription of downstream genes. The PPAR subfamily comprises three isoforms, PPARα, PPARβ/δ,
and PPARγ, each showing a distinct distribution and ligand preference. While PPARα is predominantly expressed in metabolically
active tissue, like liver, kidney, skeletal muscle and brown fat [11], PPARδ is expressed ubiquitously. PPARγ is highly expressed in adipocytes, where it functions as a key regulator of adipocyte
differentiation [12] and insulin-dependent glucose utilization [13].
Prostaglandin15-deoxy-Δ12,14-prostaglandin
J2 (15d-PGJ2) is the most
potent naturally occurring ligand for PPARγ and the thiazolindinedione (TZD), also called
glitazones, a class of antidiabetic, insulin-sensitizing drugs, are specific exogenous ligands
for PPARγ. The
TZD family of PPARγ
agonists includes rosiglitazone, pioglitazone, ciglitazone, and troglitazone,
rosiglitazone being the most potent agonist (Kd = 40 nM). In general, TZDs are
selective for PPARγ in
concentrations of 10 μM or less [14]. Recently, expression of PPARγ has been demonstrated in tumor cells
originating from various malignancies, including breast, colon, lung, gastric,
pancreatic, prostate, and bladder cancer and its activation through PPARγ agonists led to a significant decrease in
proliferation of tumor cells in vitro [15–21], however, the
exact mecahnisms underlying this effect are still being explored. As a
consequence, PPARγ has
become a molecular target for potential anticancer drug development.


## 3. PPARγ and Melanocytes

Until now, there is little information on the PPAR
subtypes and relative levels of PPAR protein expressed in human skin. The three
PPAR subtypes have been investigated in human keratinocytes [22], and PPARγ ligands have been shown to induce the
expression of genes associated with keratinocyte differentiation in vitro [23]. In addition, Kang et al. showed the expression of all
three PPAR subtypes in human melanocytes [24]. Immunocytochemistry showed that
PPAR staining was mostly confined to the cytoplasm. Furthermore, proliferation
of melanocytes was inhibited through administration of PPARα (WY-14643) and PPARγ (ciglitazone) agonists but not through PPARβ/δ
(bezafibrate) agonists in a dose dependent manner at concentrations ranging
between 0 and 100 μM. The inhibitory effect of ciglitazone seemed to occur
through induction of apoptosis, which was observed by the TUNEL method and flow
cytometry [25]. Moreover, Lee et al.
showed that pigmentation in melanocytes was accelerated with PPARα and PPARγ agonists, suggesting a possible role for PPARα
and PPARγ in modulating melanocyte proliferation and differentiation (pigmentation) [26].
Eastham et al. investigated the expression of mRNA for PPARα, PPARβ/δ, and PPARγ in human
melanocytes [27]. In addition, the natural PPARγ
agonist 15d-PGJ2 and the synthetic PPARγ agonists ciglitazone and troglitazone inhibited
the cell growth of human melanocytes, whereas the PPARα agonists WY14643 and
Leukotriene B4 had no effect on the proliferation of human
melanocytes.


## 4. PPARγ and Melanoma Cells

Only a few studies have focussed on PPARγ expression and effects of PPARγ agonists in melanoma cell lines (summarized in
Table 1). Mössner et al. investigated
the expression of PPARγ in four human melanoma cell lines MM-358, MM-201, MM-254 (established from lymph
node metastasis of cutaneous malignant melanoma), and KAII (derived from a
cutaneous nodular melanoma) [29]. In accordance with the immunocytochemistry of
the melanocytes, staining was predominantly localized in the cytoplasm. In addition, the
PPARγ agonists 15d-PGJ2, troglitazone,
and rosiglitazone dose-dependently inhibited the cell proliferation of all
melanoma cells at concentrations between 0 and 50 μM. As shown by flow
cytometry, this antiproliferative effect was not mediated through induction of
apoptosis, but rather by induction of G1 phase cell cycle arrest. Eastham et al. investigated the
expressions of PPARα, PPARβ/δ, and PPARγ in human melanoma cells SK-mel28 and A375 [27].
Both melanoma cell lines express PPARα protein levels 20–47% higher and
PPARγ protein levels 40–50% higher,
respectively, than the normal human melanocytes. However, mRNA levels and
protein levels for these receptors did not match. In addition, the natural PPARγ agonist 15d-PGJ2
and the synthetic PPARγ agonists ciglitazone and troglitazone inhibited the cell growth of the human
melanoma cell line A375 in concentrations of 0–10 μM, whereas
the SK-mel28 cells were not affected in this concentration range. The PPARα
agonists WY14643 and leukotriene B4 had no effect on the cell proliferation of
both cell lines. Placha et al. investigated PPARγ
expression in the melanoma cell lines WM35, derived from a primary tumour site,
and A375, derived from a solid metastatic tumour. Furthermore, an
antiproliferative effect of the PPARγ
agonist ciglitazone and 15d-PGJ2 in both melanoma
cell lines was observed in concentrations of 10–15 μM [30]. The antiproliferative
effect of ciglitazone was mediated through induction of apoptosis, as evidenced
by fluorescence microscopy. Núñez et al. showed an antiproliferative effect of 15d-PGJ2 on
the melanoma cell line A375 at concentration of
16 μM or higher, which was mediated through induction of apoptosis, while
ciglitazone showed no growth inhibitory effect [31]. Our own results showed
expression of PPARγ in six different human melanoma cells MV3, Lox, MeWo, G361, FemX-1, and UISO-Mel6,
which were established from primary malignant melanoma or metastatic melanoma
lymph node [28]. Similar to the findings of Mössner et al., immunocytochemical staining of PPARγ was mostly confined to the cytoplasm. The PPARγ agonists rosiglitazone, pioglitazone,
troglitazone, and ciglitazone all showed a dose-dependent antiproliferative
effect on all melanoma cell lines tested at concentrations of 30 μM or higher.
This antiproliferative effect was due to a mechanism independent from
apoptosis, which was shown by assessment of the nuclear morphology or by
molecular analysis of DNA fragmentation. Interestingly, all four PPARγ agonists showed an increase in cell
proliferation of all six melanoma cell lines at concentrations of 3 μM.Table 1
Effects of PPARγ agonists on melanoma cell growth.
Cell linePPARγ agonistConcentrationResultsMechanism of actionReferenceUISO-Mel6,Rosiglitazone,0.3–300μM– Growth inhibition of all cell lines at– Independent from apoptosisFreudlsperger et al. [28]MV3, MeWo,pioglitazone,30–300μM,G361,ciglitazone,– increase in cell
proliferation atLoxtroglitazone3μMMM-358,15d-PGJ2,0.1–50μM– Growth inhibtion of all cell lines at 20–50μM– Independent from apoptosis,Mössner et al. [29]MM-201,troglitazone,– induction of G1 phaseMM-254, KAIIrosiglitazonecell cycle arrestSK-mel28, A375Ciglitazone,0–10μM– Growth inhbition only of A375 at 10μM– Not investigatedEastham et al. [27]troglitazone,15d-PGJ2WM35, A375Ciglitazone,10–15μM– Growth inhibition of all cell lines at– Induction of apoptosisPlacha et al. [30]15d-PGJ210–15μMA375Ciglitazone,0–32μM– Growth inhibtion of A375 at 16μM of 15d-PGJ2– Induction of apoptosisNúñez et al. [31]15d-PGJ2,– no growth inhibition by ciglitazone


## 5. PPARγ-Dependent or -Independent Effects of PPARγ Agonists in Melanoma Cells

Several studies have documented various mechanisms for the antiproliferate effect of PPARγ
agonists, both being dependent or independent of PPARγ
activation, which holds also true for melanoma cells. Using a reporter gene
assay, Eastham et al. showed that the PPARγ agonists 15d-PGJ2 and ciglitazone
stimulated PPRE reporter gene activity in a dose-dependent manner in B16
melanoma cells. This activity correlated with their ability to inhibit cell
proliferation, hence a PPARγ-dependent
mechanism was postulated [27]. Simularely, Placha et al. investigated, that the apoptosis inducing effect of
ciglitazone in human melanoma cells was clearly associated with the strong
induction of transcription by endogenous PPARγ through PPRE target sequences, as shown in the
reporter gene assay system [30]. On the other hand, PPAR agonists have been
reported to have nonreceptor mediated effects too. In our own studies,
quantitative analyses of PPARγ protein showed no correlation between the amount of the PPARγ protein
and the respective susceptibility of the melanoma cell lines towards PPARγ
agonists. Therefore, a PPARγ-independent effect of PPAR-γ
agonists was assumed [28]. In other cancer cells, resistance pathways which are
constitutively activated in melanoma cells (Figure 1) were affected through
PPARγ agonist independent from PPARγ activation. For example, 15d-PGJ2 has been shown to
alter NF-κB activity in hepatocellular carcinoma cells where 15d-PGJ2 induces apoptosis via
caspase-dependent and -independent pathways [32]. In addition, Straus et al. showed a PPARγ-independent repression of NF-κB by 15d-PGJ2 through two mechanisms,
inhibition of IκB kinase (IKK) and inhibition of NF-κB DNA binding [33]. The
inhibition of NF-κB by PPARγ agonists through PPARγ-independent
mechanisms could be a possible way for the antiproliferative effect in melanoma
cells, especially for the combination of the PPARγ agonist rosiglitazone with bortzezomib, a
potent inhibitor of NF-κB, has led to an augmented antiproliferative effect on
melanoma cells [34]. In addition, Han and Roman investigated that rosiglitazone inhibited the cell growth of human
lung carcinoma cells through inactivation of PI3K/Akt pathway and increase of
PTEN expression [35]. These changes were inhbited by GW9662, a potent
antagonist of PPARγ, suggesting that they depend upon PPARγ activation. If this inactivation of the PI3K/Akt pathway by rosiglitazone also contributes to the antiproliferative effect of PPARγ agonists in melanoma needs to be further elucidated.


## 6. Conclusion

The rapid increase in incidence of malignant melanoma has not been
accompanied by better therapeutic options [36]. The past few years have seen
great leaps in our understanding of the mechanism of drug resistance and cell
survival in melanoma. Many reports have indicated the central role of PPARγ in the control of malignant cell growth in
various tumour entities including melanoma. In addition, evidence has been accumulated that PPARγ is
expressed in human melanoma cells and that PPARγ specific agonists dose-dependently inhibited
proliferation of melanoma cells [27–31]. However, these studies are
inconsistent regarding the concentration of PPARγ agonists initiating an antiproliferative
effect and the mechanism underlying these growth inhibitory effects. In contrast to PPARγ,
PPARα agonists were not shown to have antiproliferative effects on melanoma
cells. Significant inhibition of melanoma cell proliferation did not occur
until 20 μM or higher concentrations of PPARγ ligands were used. However, many natural and
synthetic PPARγ
ligands loose their receptor selectivity at these concentrations [37].
Furthermore, conflicting evidence exists on the ability of PPARγ agonists to promote tumour growth, depending
on the cell model. For example, the PPAR-γ agonist troglitazone increased cell
proliferation of breast cancer cells in low concentrations (<5 μM), while
higher concentrations of troglitazone (100 μM) inhibited cell growth [38].
These investigations corroborate our published data in melanoma cells. The PPARγ agonists rosiglitazone, troglitazone,
pioglitazone, and ciglitazone showed an increase in cell proliferation of all
six melanoma cell lines tested in low concentrations (3 μM), however, in higher
concentrations (>30 μM) a significant growth-inhibitory effect was
observerd [28]. In addition, Lucarelli et al. reported that troglitazone
promoted the survival of osteosarcoma cells at concentrations of 5 μM, through
the activation of the PI3-kinase/Akt survival pathway (see Figure 1) [39].
Therefore, the administered dose of PPAR-γ ligands in clinical trials for melanoma therapy
needs to be carefully defined and monitored closely.In conclusion, the current studies concerning the role of PPARγ in melanoma proliferation and progression
report conflicting results. The concentrations inducing growth inhibitory
effects in melanoma cells seem to be different depending on the PPARγ agonist used and the melanoma cell employed.
In particular, it remains to be further explored whether activation of PPARγ itself or PPARγ-independent effects of PPARγ agonists contribute to the inhibition of
melanoma cell growth. Although the antiproliferative effect of PPARγ agonists in certain concentration ranges in
melanoma is undisputable, more detailed information concerning the exact
mechanisms seems to be necessary. However, PPARγ might be a promising approach for target
specific anticancer strategy in the treatment of melanoma.



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