# The Role of PPAR-γ and Its Interaction with COX-2 in Pancreatic Cancer

Authors: Guido Eibl
Journal: PPAR Research
 (2008)

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## Abstract

In recent years, the study of the peroxisome proliferators activated receptor gamma (PPAR-γ) as a potential target for cancer prevention and therapy has gained a strong interest. However, the overall biological significance of PPAR-γ in cancer development and progression is still controversial. While many reports documented antiproliferative effects in human cancer cell and animal models, several studies demonstrating potential tumor promoting actions of PPAR-γ ligands raised considerable concerns about the role of PPAR-γ in human cancers. Controversy also exists about the role of PPAR-γ in human pancreatic cancers. The current review summarizes the data about PPAR-γ in pancreatic cancer and highlights the biologically relevant interactions between the cyclooxygenase and PPAR system.


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## Body

## 1. Introduction

Despite advances in surgical techniques, imaging
modalities, and intensive care, pancreatic cancer is still an almost
universally lethal disease with annual mortality figures virtually equaling
incidence numbers. An estimated number of
37 170 patients hse been diagnosed with pancreatic cancer in
2007, and 33 370
patients have succumbed to that disease in the same year [1]. Absence of specific symptoms, lack
of early detection markers, aggressive tumor growth, and virtual resistance to
conventional chemo- and radiotherapy conspire to culminate in a median overall
survival of less than nine months. Currently, surgical removal of the tumor
offers the only hope of long-term survival with 5-year survival rates
approaching 25–30% in large-volume
centers in the US [2]. Although an adjuvant treatment
regimen after surgical resection seems to prolong survival, the precise
treatment protocol including drug-of-choice is still debated and the focus of
several ongoing clinical trials [3]. Only a disappointing 10–15% of patients
at the time of diagnosis are candidates for surgical resection and even
patients who have undergone “curative” resection often die of recurrent tumor. The
majority of pancreatic cancer patients unfortunately present with locally
advanced or metastatic tumors which render them ineligible for surgical resection. Gem-citabine, an
S-phase nucleoside cytidine analog, has been the standard chemotherapeutic drug
for locally advanced and metastatic pancreatic cancer for more than ten years,
but the improvement of overall survival is unacceptably small, often approaching
only a few weeks [4]. Currently, several trials are underway that
investigate gemcitabine-based combination therapies in patients with advanced pancreatic
cancers. Capecitabine, an oral fluoropyrimidine carbamate and 5-fluorouracil
prodrug, and erlotinib, an inhibitor of the epidermal growth factor receptor, are
two promising agents which seem to improve survival in combination with
gemcitabine compared to gemcitabine monotherapy [4]. The encouraging results from a
large, double-blind, placebo-controlled, international phase III trial led to the
approval of erlotinib for the treatment of locally advanced and metastatic
pancreatic cancer in combination with gemcitabine [5]. Although certainly noteworthy, the
improvement of overall survival with the combination regimen, however, was only
marginal compared to gemcitabine monotherapy [5], strongly emphasizing the need for
the identification of novel targets and the development of more efficacious
therapeutic agents.Although several environmental risk factors for the
development of pancreatic cancers, including tobacco smoking and dietary
factors, have been described, detailed insights into the pathogenetic mechanisms
are virtually lacking [6]. Dietary intake of high-caloric,
high-fat diets with ensuing obesity and metabolic syndrome has been correlated
with an increased risk of pancreatic cancer [7, 8]. An important molecule in fatty acid
sensing and metabolism is the peroxisome proliferator activated receptor gamma
(PPAR-γ), a member of the nuclear receptor superfamily
that functions as a ligand-activated transcription factor [9]. There is now a large body of
evidence demonstrating an important role of PPAR-γ in the metabolic syndrome [10–13]. The thiazolidinedione (TZD) class
of PPAR-γ ligands has been used for the treatment of
hyperglycemia and insulin resistance in type 2 diabetes for the past ten years [14]. In addition, TZDs may also show
beneficial effects on cardiovascular complications associated with type 2
diabetes and the metabolic syndrome [14–17]. More recently, the role of PPAR-γ in various human cancers has been studied. There
is now strong evidence that PPAR-γ is overexpressed in a variety of cancers,
including colon, breast, prostate, stomach, lung, and pancreas [18–20]. However, the biological
significance of PPAR-γ is still controversial [21, 22]. Although several reviews highlight
the antiproliferative actions of PPAR-γ ligands in cell culture and animal models of
human cancers [23, 24], more recent studies illustrating a
tumor-promoting effect of PPAR-γ, in particular in colon and breast cancer
models, raise considerable concern about the significance and safety of PPAR-γ ligands as anticancer drugs [25–29]. This review will summarize and
discuss the data concerning the role of PPAR-γ in pancreatic cancer.


## 2. PPAR-gamma in Pancreatic Cancer

Reports from several groups have shown that the
thiazolidinedione (TZD) class of PPAR-γ ligands attenuates the growth of pancreatic
cancer cells in vitro by induction of terminal differentiation and G1 phase
cell cycle arrest [30, 31], and by an
increase in apoptotic cell death [32]. Furthermore,
thiazolidinediones attenuated pancreatic cancer cell migration and invasion by
modulation of actin organization and expression of matrix metalloproteinase-2
and plasminogen activator inhibitor-1, respectively [33, 34]. However, many growth-inhibitory
effects of PPAR-γ ligands are independent of PPAR-γ [35]. To date, several
non-PPAR-γ targets have been implicated in the antitumor
activities of certain TZDs ,for example, troglitazone and ciglitazone, including
intracellular Ca2+ stores, mitogen-activated protein kinases, cyclin-dependent
kinase inhibitors p27kip1 and p21WAF/CIP1,
the tumor suppressor protein p53,
and Bcl-2 family members [36]. There is
increasing evidence that TZDs directly affect mitochondrial function which
impairs oxidative respiration leading to increased reactive oxygen species
(ROS) production and ATP depletion, which in turn can activate AMP kinase [37]. An increase in
ROS and activation of AMP kinase can lead to PPAR-γ independent reduction in inflammation and cell
growth [37]. In addition, it
has been shown in pancreatic cancer cells that 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide
(CDDO-Im), a partial PPAR-γ agonist, induces apoptosis directly by
targeting mitochondrial glutathione [38]. Furthermore,
3,3′-diindolylmethane (DIM), another PPAR-γ agonist, induced apoptotic cell death in
pancreatic cancer cells through activation of the endoplasmic stress response [39]. Overall, the
potential to elicit PPAR-γ-independent effects may be ligand- and cell
context-dependent. Our own studies have demonstrated that PPAR-γ is expressed in the nucleus of six human
pancreatic cancer cells and that treatment of these cells in vitro with
15-deoxy-Δ12,14-prostaglandin J2 (15-PGJ2) and
ciglitazone dose- and time-dependently decreases cell growth by induction of
caspase-3-dependent apoptosis [40]. In addition to
their antiproliferative actions, both ligands, 15-PGJ2 and ciglitazone, reduced
the invasive capacity of pancreatic cancer cells in vitro by a PPAR-γ-mediated decrease of urokinase-type
plasminogen activator and elevation of plasminogen activator inhibitor-1 expression
that resulted in an overall reduction in urokinase activity [41]. Taken together,
there is a strong evidence today from cell culture models that PPAR-γ ligands potently reduce the growth of human
pancreatic cancer cells. The discrepancy of the reported underlying mechanisms,
however, may be caused by the use of different cell lines, culture conditions,
and experimental settings. In contrast to the notion of PPAR-γ ligands being potent antitumor drugs in
pancreatic cancers, we have reported that treatment of human pancreatic cancer
cells in vitro with 15-PGJ2 and troglitazone dose-dependently increases the
secretion of the vascular endothelial growth factor (VEGF), which is widely
recognized as a potent stimulus for tumor angiogenesis [42]. In addition, the
culture medium of troglitazone-treated human pancreatic cancer cells enhanced
migration of endothelial cells, another step in the angiogenic cascade (own finding).
These findings are already observed at submicromolar concentrations of the
PPAR-γ ligands, which are usually considerably lower
than the typical ligand concentrations needed for the antiproliferative effects
in pancreatic cancer cells. Our in vitro data suggest that PPAR-γ ligands may have a tumor-promoting effect in
vivo by enhancing tumor angiogenesis. Although the precise role of PPAR-γ in tumor angiogenesis is still debated and controversial,
there is accumulating evidence that activation of PPAR-γ stimulates VEGF production and neoangiogenesis
also in other cell models [43, 44].In addition to the effects of PPAR-γ ligands on the growth of established
pancreatic cancers in preclinical cell culture and xenograft mouse models,
dietary intake of 800 ppm pioglitazone for 22 weeks correlated with an improved
serum lipid profile and a decreased incidence and multiplicity of pancreatic
tumors in the N-nitrosobis(2-oxopropyl)amine (BOP) model of pancreatic
carcinogenesis in Syrian golden hamsters, suggesting a potential
chemopreventive role of TZDs [45].There are very few data concerning the significance of
PPAR-γ in clinical pancreatic cancer specimens. In a
recent study, PPAR-γ was expressed in the majority of human
pancreatic cancer specimens, positively correlated with higher tumor stage and
grade, and interestingly was associated with shorter patient survival,
suggesting a potential role in pancreatic cancer progression [20].


## 3. Interaction between the PPAR-gamma and COX-2 Pathways

Besides the
TZD class of antidiabetic drugs, various intracellular lipids and lipid
mediators are capable of activating PPAR-γ. Among those, polyunsaturated fatty
acids (e.g., arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and
eicosanoids (e.g., 15-deoxy-Δ12,14-prostaglandin J2 (15-PGJ2))
are also substrates and products, respectively, of intracellular cyclooxygenase
(COX) enzymes strongly suggesting relevant interactions between the PPAR and
COX pathways (see Figure 1).Figure 1
Possible
interactions between the COX-2 and PPAR-γ pathways: polyunsaturated fatty acids (PUFAs)
are substrates for cyclooxygenase-2 (COX-2) enzymes leading to the formation of
various prostaglandins (PGs). Certain PUFAs and PGs can also activate PPAR-γ. Selective and nonselective COX-2 inhibitors
(COX Inh) block PG formation by COX-2 but can also at higher concentrations
activate PPAR-γ. Solid arrows indicate activation; dashed
arrow indicates metabolic pathway; blocked arrow indicates inhibition.


### 3.1. COX Products as PPAR-γ Activators

COX activity
leads to the formation of an unstable hydroxy-endoperoxide, prostaglandin H2,
which can be further converted to various prostanoid species by tissue specific
isomerases [46]. While parent prostaglandins (e.g.,
PGE2, PGF2α, and PGD2) transduce their signals
through binding to G-protein coupled cell surface receptors [47], cyclopentenone prostanoids (e.g.,
PGJ2) are known ligands of PPAR-γ [48]. In fact, there is evidence
suggesting that COX-2 is preferentially located on the nuclear membrane
allowing cyclopentenone prostaglandins to directly enter the nucleus and bind
to ligand-activated transcription factors [49]. In this regard, human pancreatic
cancer cells seem to express COX-2 preferentially in a perinuclear
localization [50]. 15-deoxy-Δ12,14-prostaglandin J2 (15-PGJ2),
a nonenzymatically formed dehydration product of PGD2, is detectable
in COX-2 expressing human pancreatic cancer cells (own observation) and able to
activate PPAR-γ in these cells [42]. Furthermore, a selective COX-2 inhibitor at a concentration that
inhibits COX-2 activity and consequently prostanoid production reduces PPAR-γ activity, presumably by decreasing the levels
of cyclopentenone prostaglandins (own observation).


### 3.2. COX Substrates as PPAR-γ Activators

Certain polyunsaturated
fatty acids (PUFAs) (e.g., arachidonic acid (AA; 20 :4⁢n−6) and eicosapentaenoic
acid (EPA; 20 : 5⁢n−3)) are substrates for COX enzymes and also known PPAR-γ ligands [51]. Both PUFAs are released from the sn-2 position of major membrane
phospholipids by phospholipase A2 (PLA2) enzymes,
particularly by the cytoplasmic PLA2, which upon activation seems to
preferentially locate to the nuclear membrane [52, 53]. Once released, the PUFAs can be
metabolized by COX enzymes or enter the nucleus to activate PPAR-γ. Our own studies demonstrated that EPA
decreased the growth of human pancreatic cancer cells through COX-2 dependent
and independent mechanisms (manuscript in press). The COX-2 independent
mechanism involved activation of PPAR-γ by EPA as the growth-inhibitory effect of EPA
was abolished by a pharmacological PPAR-γ antagonist. Furthermore, EPA and to a lesser
extent AA can activate PPAR-γ transcriptional activity in human pancreatic
cancer cells (own observation). This effect is less pronounced in pancreatic
cancer cells that express COX-2 presumably because EPA is rapidly metabolized
by COX-2 in these cells. The overall efficacy of PUFAs to activate PPAR-γ may therefore be dependent on the cellular
expression and activity of COX-2.


### 3.3. COX Inhibitors as PPAR-γ Activators

In addition to
COX-2 substrates and products, certain nonselective and selective COX-2
inhibitors have also been shown to activate PPAR-γ independent of their ability to inhibit COX-2
enzymatic activity [54], although the precise molecular
mechanisms are still unknown. There is a compelling evidence today that the inducible
COX-2 isoform plays an important role in pancreatic cancer development and
growth and that selective COX-2 inhibitors may be efficacious for pancreatic
cancer prevention and therapy [50]. Our own studies demonstrated
that dietary intake of a selective COX-2 inhibitor delayed the progression of
recognized pancreatic cancer precursor lesions in a genetically engineered
mouse model of pancreatic cancer development [55]. Furthermore, a selective COX-2
inhibitor decreased the growth of COX-2 positive human pancreatic cancers in a
xenograft mouse model by induction of apoptosis in cancer cells and by
inhibition of tumor angiogenesis [42]. In contrast, the selective COX-2
inhibitor enhanced the growth of xenografted human pancreatic cancers that
lacked or had very little COX-2 protein expression. This tumor-promoting effect
was associated with an increase in intratumoral VEGF levels and tumor
angiogenesis [42]. Additional studies showed that
the tumor-enhancing effect of the selective COX-2 inhibitor in COX-2 negative
or weakly COX-2 expressing human pancreatic cancers was abolished by GW9662, an
irreversible pharmacological PPAR-γ antagonist, suggesting biologically important
interactions between the COX-2 inhibitor and PPAR-γ [42]. Further studies demonstrating
enhanced PPAR-γ binding activity in tumors that were treated
with a selective COX-2 inhibitor confirmed that interaction [42]. The findings obtained in vivo were corroborated by in vitro experiments. Human
pancreatic cancer cells treated with relatively high concentrations of selective
COX-2 inhibitors showed an increased production and secretion of VEGF, which
was inhibited by a pharmacological PPAR-γ antagonist and a dominant-negative PPAR-γ receptor [42]. Additionally, the selective
COX-2 inhibitor at that concentration stimulated PPAR-γ transcriptional and DNA-binding activities [42]. These data clearly indicated
that a biologically significant interaction between selective COX-2 inhibitors
and PPAR-γ exists and that activation of PPAR-γ by these drugs may have detrimental, that is
tumor-promoting, effects on pancreatic cancer growth. It is important to note
that the tumor-promoting effects of selective COX-2 inhibitors were only
observed at relatively high concentrations (much higher than needed to inhibit
COX-2 enzymatic activity) in tumors that had no or only very little COX-2 expression [42]. Although the selective COX-2
inhibitor stimulated VEGF production by pancreatic cancer cells through a PPAR-γ mediated mechanism also in COX-2 expressing
pancreatic cancers, the potential proangiogenic and tumor-promoting effect in
COX-2 positive cancers was masked by a significant reduction of COX-2 generated
proangiogenic and protumorigenic prostanoids [42].


## 3.1. COX Products as PPAR-γ Activators

COX activity
leads to the formation of an unstable hydroxy-endoperoxide, prostaglandin H2,
which can be further converted to various prostanoid species by tissue specific
isomerases [46]. While parent prostaglandins (e.g.,
PGE2, PGF2α, and PGD2) transduce their signals
through binding to G-protein coupled cell surface receptors [47], cyclopentenone prostanoids (e.g.,
PGJ2) are known ligands of PPAR-γ [48]. In fact, there is evidence
suggesting that COX-2 is preferentially located on the nuclear membrane
allowing cyclopentenone prostaglandins to directly enter the nucleus and bind
to ligand-activated transcription factors [49]. In this regard, human pancreatic
cancer cells seem to express COX-2 preferentially in a perinuclear
localization [50]. 15-deoxy-Δ12,14-prostaglandin J2 (15-PGJ2),
a nonenzymatically formed dehydration product of PGD2, is detectable
in COX-2 expressing human pancreatic cancer cells (own observation) and able to
activate PPAR-γ in these cells [42]. Furthermore, a selective COX-2 inhibitor at a concentration that
inhibits COX-2 activity and consequently prostanoid production reduces PPAR-γ activity, presumably by decreasing the levels
of cyclopentenone prostaglandins (own observation).


## 3.2. COX Substrates as PPAR-γ Activators

Certain polyunsaturated
fatty acids (PUFAs) (e.g., arachidonic acid (AA; 20 :4⁢n−6) and eicosapentaenoic
acid (EPA; 20 : 5⁢n−3)) are substrates for COX enzymes and also known PPAR-γ ligands [51]. Both PUFAs are released from the sn-2 position of major membrane
phospholipids by phospholipase A2 (PLA2) enzymes,
particularly by the cytoplasmic PLA2, which upon activation seems to
preferentially locate to the nuclear membrane [52, 53]. Once released, the PUFAs can be
metabolized by COX enzymes or enter the nucleus to activate PPAR-γ. Our own studies demonstrated that EPA
decreased the growth of human pancreatic cancer cells through COX-2 dependent
and independent mechanisms (manuscript in press). The COX-2 independent
mechanism involved activation of PPAR-γ by EPA as the growth-inhibitory effect of EPA
was abolished by a pharmacological PPAR-γ antagonist. Furthermore, EPA and to a lesser
extent AA can activate PPAR-γ transcriptional activity in human pancreatic
cancer cells (own observation). This effect is less pronounced in pancreatic
cancer cells that express COX-2 presumably because EPA is rapidly metabolized
by COX-2 in these cells. The overall efficacy of PUFAs to activate PPAR-γ may therefore be dependent on the cellular
expression and activity of COX-2.


## 3.3. COX Inhibitors as PPAR-γ Activators

In addition to
COX-2 substrates and products, certain nonselective and selective COX-2
inhibitors have also been shown to activate PPAR-γ independent of their ability to inhibit COX-2
enzymatic activity [54], although the precise molecular
mechanisms are still unknown. There is a compelling evidence today that the inducible
COX-2 isoform plays an important role in pancreatic cancer development and
growth and that selective COX-2 inhibitors may be efficacious for pancreatic
cancer prevention and therapy [50]. Our own studies demonstrated
that dietary intake of a selective COX-2 inhibitor delayed the progression of
recognized pancreatic cancer precursor lesions in a genetically engineered
mouse model of pancreatic cancer development [55]. Furthermore, a selective COX-2
inhibitor decreased the growth of COX-2 positive human pancreatic cancers in a
xenograft mouse model by induction of apoptosis in cancer cells and by
inhibition of tumor angiogenesis [42]. In contrast, the selective COX-2
inhibitor enhanced the growth of xenografted human pancreatic cancers that
lacked or had very little COX-2 protein expression. This tumor-promoting effect
was associated with an increase in intratumoral VEGF levels and tumor
angiogenesis [42]. Additional studies showed that
the tumor-enhancing effect of the selective COX-2 inhibitor in COX-2 negative
or weakly COX-2 expressing human pancreatic cancers was abolished by GW9662, an
irreversible pharmacological PPAR-γ antagonist, suggesting biologically important
interactions between the COX-2 inhibitor and PPAR-γ [42]. Further studies demonstrating
enhanced PPAR-γ binding activity in tumors that were treated
with a selective COX-2 inhibitor confirmed that interaction [42]. The findings obtained in vivo were corroborated by in vitro experiments. Human
pancreatic cancer cells treated with relatively high concentrations of selective
COX-2 inhibitors showed an increased production and secretion of VEGF, which
was inhibited by a pharmacological PPAR-γ antagonist and a dominant-negative PPAR-γ receptor [42]. Additionally, the selective
COX-2 inhibitor at that concentration stimulated PPAR-γ transcriptional and DNA-binding activities [42]. These data clearly indicated
that a biologically significant interaction between selective COX-2 inhibitors
and PPAR-γ exists and that activation of PPAR-γ by these drugs may have detrimental, that is
tumor-promoting, effects on pancreatic cancer growth. It is important to note
that the tumor-promoting effects of selective COX-2 inhibitors were only
observed at relatively high concentrations (much higher than needed to inhibit
COX-2 enzymatic activity) in tumors that had no or only very little COX-2 expression [42]. Although the selective COX-2
inhibitor stimulated VEGF production by pancreatic cancer cells through a PPAR-γ mediated mechanism also in COX-2 expressing
pancreatic cancers, the potential proangiogenic and tumor-promoting effect in
COX-2 positive cancers was masked by a significant reduction of COX-2 generated
proangiogenic and protumorigenic prostanoids [42].


## 4. Conclusion

While several in vitro studies demonstrate that
PPAR-γ activation decreases pancreatic cancer cell
growth, the finding that PPAR-γ ligands can stimulate VEGF production by
pancreatic cancer cells raises serious concerns that PPAR-γ activation in vivo may lead to enhanced angiogenesis and tumor growth.
Further detailed studies using pancreatic cancer animal models and specific
PPAR-γ ligands are necessary to evaluate possible
proangiogenic and protumorigenic properties of PPAR-γ activation in vivo. Unfortunately, information about the role of PPAR-γ in pancreatic carcinogenesis is almost
nonexistent. The use of the recently developed genetically engineered mouse
models of pancreatic cancer development that closely recapitulate our current
knowledge of pancreatic cancer development on a histological and genetic level
should shed some needed insights into the role of PPAR-γ in pancreatic carcinogenesis.There is now
clear evidence of a biologically relevant interaction between the COX and PPAR-γ pathways. Our data suggest that activation of PPAR-γ by selective and nonselective COX-2 inhibitors
may have tumor-promoting effects in
vivo by enhancing tumor angiogenesis. The effect of COX-2 inhibitors on PPAR-γ activation seems to be observed only at
relatively high concentrations of the inhibitors and the overall biological
phenotype of that interaction is dependent on the cellular expression and
activity of the COX-2 protein. Although the role of PPAR-γ in pancreatic cancer development and growth
has begun to be elucidated in recent years, a precise knowledge of molecular
targets downstream of PPAR-γ, a more comprehensive elucidation of PPAR-γ-independent actions of PPAR-γ ligands, and a detailed understanding of
crosstalks between PPAR-γ and other intracellular signaling pathways
seem to be absolutely necessary and needed to eventually clarify the role of
PPAR-γ in human cancer development and progression.



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*Source: 326915-2008-07-02.xml*