# Activated PPARγ Targets Surface and Intracellular Signals That Inhibit the Proliferation of Lung Carcinoma Cells

Authors: Shou Wei Han; Jesse Roman
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily.  Their discovery in the 1990s provided insights into the cellular mechanisms involved in the control of energy homeostasis, the regulation of cell differentiation, proliferation, and apoptosis, and the modulation of important biological and pathological processes related to inflammation and cancer biology, among others.  Since then, PPARs have become an exciting target for the development of therapies directed at many disorders including cancer.  PPARs are expressed in many tumors including lung cancer, and their function has been linked to the process of carcinogenesis.  Consequently, intense research is being conducted in this area with the hope of discovering new PPAR-related therapeutic targets for the treatment of lung cancer.  This review summarizes the research being conducted in this area, and focuses on the mechanisms by which a member of this family (PPARγ) is believed to affect lung tumor cell biology.


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## Body

## 1. Introduction

Lung carcinoma is one of the most common malignant tumors in the world, and is the
leading cause of carcinoma death in USA [1]. Primary malignant cancers of the lung are
classified into small cell lung cancer (SCLC) and nonsmall cell lung cancer
(NSCLC). NSCLC accounts for 80% of malignant lung cancer, and SCLC constitutes the remainder [2].
Based on the cellular phenotype, NSCLC is
further subdivided into squamous cell carcinoma, adenocarcinoma, and large cell
carcinomas [3]. Despite advances in understanding the
mechanisms involved in carcinogenesis, the development of new surgical procedures,
and the use of new radio and chemotherapeutic protocols, the five-year survival
rate for lung cancer patients is poor and remains less than 15% [1]. This underscores the desperate need for novel
strategies for early detection, prevention, and treatment of this malignancy.Since their discovery in 1990, peroxisome proliferator-activated receptors (also
known as PPARs) have captured the attention of investigators interested in
learning about the intracellular pathways that control signal transduction and
gene transcription. PPARs were
originally cloned in an attempt to identify the molecular mediators of
peroxisome proliferation in the liver of rodents. Today, PPARs are recognized as versatile
members of the ligand-activated nuclear hormone receptor superfamily of
transcription factors that includes receptors for steroids, thyroid hormone,
retinoic acid, and vitamin D, among others [4]. PPARs are considered to play key roles in
diverse physiological processes ranging from lipid metabolism to inflammation,
and have been implicated in diseases such as cancer, atherosclerosis, and
diabetes [4, 5]. Although information about the function of
PPARs in lung is scarce, data implicating these molecules in key processes in
lung biology are rapidly emerging.Three subtypes of PPARs have been identified and cloned: PPARα, PPARβ/δ,
and PPARγ. These subtypes are
distinguished by their tissue distribution, and to a lesser degree, by their
ligand specificity. PPARα has been implicated in hepatocellular carcinoma in
rodents, whereas activation of PAPRβ/δ promotes human lung carcinoma cell proliferation through
PI3-Kinase/Akt activation [6]. However, of the three
PPARs identified to date, PPARγ
represents the most promising target in view of the many reports implicating
this molecule in lung carcinoma cell growth both in vitro and in vivo. This
review focuses on PPARγ, its role in lung carcinogenesis, and the potential therapeutic role
of PPARγ agonists in lung cancer.


## 2. Elucidating the Function of PPARγ

PPARγ was discovered based on its similarity to PPARα,
and it is the most intensively studied ligand-dependent transcriptional regulator. By
utilizing three different promoters, a single PPARγ gene encodes three isoforms, namely, PPARγ1,
PPARγ2, and PPARγ3 [7]. Analysis of PPARγ1 and γ3
transcripts revealed that they both translate into the same PPARγ1 protein [8]. PPARγ2
protein contains additional 30 amino acids at its N-terminus compared to PPARγ1. PPARγ
is highly expressed in adipose tissue, and it is a master regulator of
adipocyte differentiation [9, 10]. In addition to its role in adipogenesis, PPARγ
serves as an important transcriptional regulator of glucose and lipid
metabolism, and it has been implicated in the regulation of insulin
sensitivity, atherosclerosis, and inflammation [11, 12]. PPARγ
is also expressed in multiple other tissues such as breast, colon, lung, ovary,
prostate, and thyroid, where it was demonstrated to regulate cellular
proliferation, differentiation, and apoptosis [13, 14]. As will be discussed later, PPARγ
activation inhibits tumor progression in NSCLC [15, 16]. Several leukocyte populations, including
monocytes/macrophages, lymphocytes, and dendritic cells, have also been shown
to express PPARγ suggesting a role for this molecule in the regulation of immune responses [17].
PPARγ has been described as a negative regulator of macrophage function since its
activation suppresses the production of inflammatory cytokines, chemokines,
metalloproteases, and nitric oxide [18, 19]. These PPARγ-mediated anti-inflammatory effects are not
restricted to monocytes, as the treatment with PPARγ agonists results in inhibition of
cytokine/chemokine production in several epithelial and stromal cells [15].Several natural and synthetic compounds have been identified as activators of PPARγ. The insulin-sensitizing antidiabetic drugs
known as thiazolidinediones (TZDs) were the first compounds identified as PPARγ
agonists [20]. The TZDs’ rosiglitazone and pioglitazone are
currently in clinical use for the treatment of type II diabetes, while
troglitazone was withdrawn from clinical use because it was linked to
idiosyncratic liver toxicity [21]. Other non-TZD synthetic ligands include
certain nonsteroidal anti-inflammatory drugs such as isoxazolidinone JTT-501 [22]
and tyrosine-based GW7845 [23]. Naturally occurring compounds that activate
PPARγ include long-chain polyunsaturated fatty acids which are found in fish oil
(e.g., n-3-PUFA and n-6-PUFA), eicosanoids (e.g., 15d-PGJ2), lipid
hydroperoxides (e.g., 9(s)-HODE and 13(s)-HODE), as well as linoleic acid,
15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2), 12/15 lipoxygenase products of 15-hydroxyeicosatetraenoic acid (15-HETE),
and 13-hydroxyoctadecadienoic acid [24–26]. In addition, compounds from several medicinal
plants such as Saurufuran A from Saururus
chinensis [27],
flavonoids like chrysin and kaempferol [28],
phenolic compounds from Glycyrrhiza uralensis [29],
and curcumin from Curcumin longa [30, 31]
are also shown to activate PPARγ.The synthetic ligands and some natural ligands have been used to elucidate the role
of PPARγ in cellular functions both in vitro and in vivo. However, several
caveats should be taken into consideration when interpreting such studies. First, the natural ligands that regulate PPARs in vivohave not been completely
elucidated. Second, not all PPARγ ligands exert their effects through PPARγ since there is strong
evidence for the activation of PPARγ-independent
signals, particularly with the natural ligand 15d-PGJ2. Third, high-affinity ligands for PPARγ
(e.g., TZDs) may exert partial agonist/antagonist activity
[32]. The latter might be
due to the fact that individual TZDs induce different PPARγ conformations that
influence the recruitment of different coactivator/corepressor molecules. Thus, the activity of the PPARγ
transcriptional complex is influenced by the context
of a given gene and its promoter, and by the relative availability of pertinent
coactivator/corepressor molecules in the cell or tissue of interest.


## 3. PPARγ in Lung Cancer

Among the three subtypes, the role of PPARγ
has been investigated the most in lung carcinogenesis. PPARγ
is expressed in many cancers including colon, breast, and prostate cancers, and
with few exceptions, PPARγ ligands are antiproliferative in these cancers. Similarly, PPARγ
is expressed in SCLC and NSCLC [33]. Furthermore, PPARγ ligands induce growth arrest and promote
changes associated with differentiation as well as apoptosis in a variety of
lung carcinoma cell lines although most of the knowledge available in this area
has been generated in NSCLC [34, 35]. The
exact mechanisms linking modulation of PPARγ with cancer growth inhibition remain unclear. However, current evidence suggests that PPARγ ligands affect a number of mechanisms
including regulation of the intracellular machinery involved in cell signaling
and cell cycle control, inhibition of mitogenic factors and tumor promoters, prevention
of tumor cell recognition of extracellular mitogenic signals, breakdown of nicotine-induced
cell survival, and modulation of the expression of angiogenic factors needed
for the development of the vascular networks that supply tumor cell (see Figure 1). These mechanisms are discussed below
as they relate to the action of PPARγ
ligands in lung cancer.Figure 1
Anticancer actions of PPARγ ligands. In addition to genetic abnormalities, lung
carcinoma cells receive mitogenic and antiapoptotic signals that promote their progression and
metastasis through the activation of key intracellular pathways (e.g., COX-2,
Akt, and mTOR). Lung carcinoma cells
recognize these signals via diverse receptors for growth factors (e.g., EGFR),
prostanoids (e.g., EP2 and EP4), and extracellular matrices (e.g., integrins),
among others. In addition, angiogenic
factors assist in the vascularization of tumors, while inflammatory signals
further promote tumor progression. PPARγ
ligands inhibit tumor growth in animal models, but the mechanisms responsible
for these effects appear to be multidimensional. In vitro studies reveal that PPARγ
ligands affect tumors by inhibiting the expression of key prostanoid and integrin receptors, by
reducing the expression of fibronectin, a matrix glycoprotein that stimulates
tumor cell proliferation, and by inhibiting the production of angiogenic and
inflammatory signals. In addition, PPARγ
ligands increase the expression and/or activity of tumor suppressors like PTEN
and p21. Although many of these
anticancer effects are mediated through PPARγ, others appear to be independent of this
nuclear transcription factor (e.g., via targeting TSC2, AMPK, and ROS
production and ERK activation, and interacting with CRE, AP-1).Several studies demonstrate that PPARγ ligands affect cell cycle control in tumor cells. For
example, PPARγ ligands have been found to inhibit the growth
of A549 adenocarcinoma cells due to G0/G1 cell cycle arrest through the
upregulation of mitogen-activated protein kinases ERK1/2 and the downregulation
of G1 cyclins D and E [15]. Troglitazone inhibits NSCLC proliferation in
part by stimulating the expression of the GADD 153 (for growth arrest and DNA damage-inducible gene-153) [36]. Also, troglitazone was found to induce
apoptosis in NCI-H23 cells via a mitochondrial pathway through the activation
of ERK1/2 [37]. Others have shown similar results using
CRL-202 cells, and further demonstrated that troglitazone downregulated the
expression of the antiapoptotic molecules Bcl-w and Bcl-2, as well as decreasing
the activity of SAPK/JNK [38]. PPARγ
ligands also induce the expression of death receptor 5 (DR5) and increase DR5
distribution at the cell surface in addition to reducing c-FLIP levels in human
lung cancer cells. These agents cooperated with TRAIL to enhance apoptosis in human lung carcinoma
cells [39]. One recent report found that PPARγ
ligands 1-[(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1H-indene-2-carboxylic
acid ethyl ester (KR-62980)] and rosiglitazone induce NSCLC apoptotic cell
death mainly through PPARγ-dependent
reactive oxygen species formation via increased expression of proline oxidase,
a redox enzyme expressed in mitochondria [35].Tumor suppressor genes are also affected by PPARγ ligands. For example, PGJ2 and ciglitazone stimulated the expression
of p21 mRNA and protein expression in NSCLC, and this coincided with a
reduction in cyclin D1 mRNA expression [40]. Of note, p21 antisense oligonucleotides
significantly blocked lung carcinoma cell growth inhibition observed with PPARγ
ligands, thereby establishing an important role for p21 in this process. These findings are consistent with those of
others showing that the proliferation of A549 cells injected subcutaneously
into nude mice was inhibited significantly by treatment with ciglitazone, and
this coincided with increased expression of PPARγ and p21 and with downregulation of cyclin
D1 [41]. A connection between another tumor suppressor
gene (p53) and PPARγ ligands has also been demonstrated by showing that 15-deoxy-PGJ2, together
with docetaxel, stimulates apoptosis in NSCLC through inhibition of Bcl2 and
cyclin D1 and overexpression of caspases and p53 [34].Other reports implicate alterations in the mammalian target of rapamycin (mTOR)
signaling pathway in the antitumor effects of PPARγ ligands. Rosiglitazone, for example, was reported to reduce the phosphorylation
of Akt, an upstream positive modulator of mTOR, and to increase PTEN, a
negative modulator of mTOR, in NSCLC H1792 and H1838 cells; this resulted in
inhibition of cell proliferation [42]. Although the effects of rosiglitazone on Akt
and PTEN were blocked by the selective PPARγ antagonist GW9662 and restored by transient
overexpression of PPARγ, cell growth was not entirely restored suggesting the involvement of additional
PPARγ-independent mechanisms of action. Further work
revealed that rosiglitazone increased the phosphorylation of AMPKα, a target of LKB1, and TSC2, another potential
tumor suppressor and upstream downregulator of mTOR. The latter pathway was independent of PPARγ
since GW9662 and PPARγ siRNA did not affect it [42, 43];
others have shown similar increases in PTEN expression induced by rosiglitazone
[44].More recently, we found that rosiglitazone and dietary compounds such as fish oil
(which contain certain kinds of fatty acids likeω3 and ω6
polyunsaturated fatty acids known to work as PPARγ ligands) inhibit integrin-linked kinase (ILK) expression through PPARγ signaling and the recruitment of a PPARγ
coactivator, PGC-1α (Han et al., unpublished data). ILK is a unique intracellular adaptor and
kinase that links cell-adhesion receptors, integrins, and growth factors to the
actin cytoskeleton and to a range of signaling pathways that are implicated in
the regulation of anchorage-dependent tumor cell growth/survival, cell cycle
progression, invasion and migration, and tumor angiogenesis [45]. This effect was associated with activation of
p38 MAPK followed by induction of transcription factor AP-2α. In
turn, this resulted in inhibition of NSCLC cell proliferation (Han et al., unpublished
data).Several studies suggest that PPARγ ligands also exert antitumor effects by blocking access to mitogenic agents
such as PGE2, a major cyclooxygenase metabolite that plays important
roles in tumor biology. The functions of
PGE2 are mediated through one or more of its receptors: EP1, EP2,
EP3, and EP4 [46]. Human NSCLC cell lines express EP2 receptors,
among other EP receptors, and the inhibition of cell growth by PPARγ
ligands like GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone
is associated with a significant decrease in EP2 mRNA and protein
expression. Notably, the inhibitory effects of rosiglitazone and ciglitazone, but not PGJ2,
were reversed by a specific PPARγ antagonist GW9662, suggesting the involvement of PPARγ-dependent
and PPARγ-independent mechanisms [46]. Also, a recent study showed that ciglitazone
suppressed cyclooxygenase-2 (COX-2) mRNA expression and COX-2 promoter activity,
while upregulating peroxisome proliferators’ response element (PPRE) promoter
activity in NSCLC cells, further suggesting a negative modulator role for PPARγ ligands in
the COX-2/PGE2 pathway in NSCLC [47].Nicotine, a major component of tobacco, stimulates NSCLC cell proliferation through
nicotinic acetylcholine receptor- (nAChR-) mediated signals. A recent case-control study of 500 incident
lung cancer cases and 517 age and sex frequency-matched cancer-free controls suggested
that PPARγ polymorphisms in Chinese smokers may contribute to the etiology of lung cancer [48]. Monocytes and monocyte-derived macrophages from
healthy smokers showed increased PPARγ
expression as compared to those from healthy nonsmokers, which were reproduced
by nicotine in vitro [49]. Interestingly, concomitant administration of
PPARγ agonists can effectively attenuate the effects of nicotine on alveolar type II
cells [50]. We recently found that rosiglitazone reduced
nicotine-induced NSCLC cell growth through downregulation of α7 nAChR-dependent signals including ERK
and p38 MAPK; this effect appeared to be PPARγ-independent (Han et al., unpublished data). If confirmed, this may unveil a novel mechanism
by which rosiglitazone inhibits human lung carcinoma cell growth.Other studies suggest that PPARγ
ligands might prevent the interaction of tumor cells with their surrounding stromata, thereby
interfering with host-derived and tumor-derived factors and mitogenic and prosurvival effects. An example of this is fibronectin, a matrix
glycoprotein residing in the lung stroma that is increased in most, if not all,
chronic forms of lung disease [51]. This is true for tobacco-related lung
disorders and fibrotic disorders—all associated
with increased incidence of lung cancer [52]. Several studies suggest that fibronectin
serves as a mitogen and survival factor for NSCLC [53],
and fibronectin has been recently shown to stimulate tumor cell expression of
matrix metalloproteinases, proteases implicated in metastatic disease [54]. These observations support the idea that
tumor cell interactions with fibronectin through surface integrin receptors are
advantageous for tumors since they stimulate proliferation, survival, and
metastases [53]. This idea remains to be proven in vivo, but if found to be true,
this might unveil a new target for anticancer strategies. In this regard, PPARγ ligands were shown to inhibit fibronectin
expression in NSCLC cells by inhibiting transcription factors involved in
regulation of fibronectin gene expression [55]. PPARγ
ligands (rosiglitazone and GW1929, but not PGJ2) have been also
recently reported to inhibit the expression of the gene encoding for the α5
integrin subunit resulting in reduced expression of the integrin α5β1,
a fibronectin receptor that mediates fibronectin’s mitogenic effects in NSCLC
cells and nontumor lung cells [56]. Thus, by inhibiting the expression of
fibronectin and its integrin α5β1, PPARγ ligands might reduce tumor cell recognition of fibronectin
with consequent changes in cell proliferation and apoptosis.PPARγ might also regulate the generation of the
complex vascular network that supplies tumor cells. This idea is supported by studies showing a reduction
in blood vessel density in the lung tumors generated by the injection of A549
cells into the flanks of SCID mice treated with
PPARγ ligands [57]. In in
vitro studies, the treatment of A549 cells with troglitazone or their
transient transfection with a constitutively active PPARγ construct blocked the production of angiogenic
molecules such as ELR+CXC chemokines IL-8 (CXC-8), ENA-78 (CXCL5), and
Gro-alpha (CXCL1) [57]. Furthermore, PPARγ activation inhibited NF-κB, a transcription factor known to regulate the
expression of many of the proangiogenic factors mentioned above. Similarly, rosiglitazone was shown to inhibit
mouse lung tumor cell growth and metastasis in vivo through direct and indirect antiangiogenic effects [16]. It is important to note that PPARγ
signaling has also been associated with tumor promoter activities in some tumor cells such as colon
and breast, and that this effect was linked to increased beta-catenin and c-Myc
expression [58, 59] (Table 1). These findings need to be
confirmed and tested in other tumors. However, these data suggest that activation of specific
PPARγ-related pathways may differ depending upon the cells and tumors examined. More than one
pathway was involved in the effect of PPARγ ligands in one cell line which was not observed in
others. Internal genetic variations and other factors may be responsible for these outcomes, and
these need to be explored further followed by confirmation in in vivo models of cancer.Table 1
PPARγ-dependent signals in mediating the effects of PPARγ ligands.
(1) PPARγ ligands inhibit cancer cell growth and induce apoptosis via:↓ PGE2receptors (e.g., EP2 and EP4)↑ Tumor suppressors (e.g., PTEN, p21)↓ Inflammatory factors (e.g., NF-κB, MCP-1, COX-2)↓ Angiogenic factors (e.g., VEGF)↓ Survival factors (e.g., PI3-K/Akt, mTOR)↑↓ Other kinase signals (e.g., ERK, p38 MAPK)↓ Growth factor receptors (e.g., EGF-R, PDGF-R)↓ Extracellular matrices (e.g., fibronectin,MMP-9)↓ Integrin receptors (e.g., α5β1)↑↓
Others (e.g., cytokines (e.g., IL-13, IL-21, TGF-β1) and chemokines (e.g., MIP-1β))


## 4. Implications for Therapy

The studies mentioned above suggest that PPARs are involved in lung cancer cell
biology. However, their roles remain uncertain and much needs to be learned before they are targeted for therapeutic
intervention, especially when considering PPARγ. Nevertheless,
activation of PPARγ is strongly associated with decreased lung carcinoma cell proliferation both
in vitro and in vivo. Furthermore, in primary NSCLC, the expression of PPARγ
has been correlated with tumor histological type and grade, and decreased PPARγ
expression was correlated with poor prognosis [60]. Because of this and the fact that synthetic
agonists of PPARγ with good safety profiles are currently in use in the clinical arena, PPARγ
has emerged as a reasonable target for the development of novel antilung cancer
therapies. Synthetic and natural PPARγ activators might be useful as well. For
example, arachidonic acid inhibits the growth of A549 cells, and this effect is
blocked by the synthetic PPARγ inhibitor GW9662 [61]. MK886, a 5-lipoxygenase activating
protein-directed inhibitor, stimulates apoptosis and reduces the growth of A549
cells through activation of PPARγ
[62]. These and related drugs can be used alone or
in combination with other drugs for synergistic effects. This was observed when using low doses of
MK886 in combination with ciglitazone and 13-cis-retinoic acid on A549 and
H1299 cells [62]. Also, dramatic synergistic anticancer effects
have been reported for lovastatin (an HMG-CoA reductase inhibitor) and the PPARγ
ligand troglitazone in several cell lines including lung cancer cells [63].
An enhancement of the antitumor effects of
gefitinib by rosiglitazone on A549 cell growth has been recently noted,
suggesting that combination strategies using selective nuclear receptor
activators in conjunction with epidermal growth factor receptor inhibitors
might be effective [64].A recent study demonstrated that combining the PPARγ ligand rosiglitazone with carboplatin
dramatically reduced lung tumor growth in
vivo [65]. More tantalizing data were derived from
retrospective analysis demonstrating that thiazolidinedione (TZD) use was
associated with reduced risk of lung cancer. This study revealed 33% reduction in lung cancer risk among thiazolidinedione
users as compared to the nonusers after adjusting other variables [66]. Interestingly, similar risk
reduction was not observed for colorectal and prostate cancers [66].Despite the above, enthusiasm for the use of PPARγ ligands as anticancer agents should be tempered by the fact that PPARγ
ligands stimulated PPARγ transactivation in lung adenocarcinoma cell lines, while few to no effects were
noted in squamous cell or large cell carcinomas. Also, it is important that we
better define PPARγ-independent
pathway to avoid unforeseen events and to identify new targets for intervention
[64, 67]
(Table 2). Furthermore, a novel splice
variant of human PPARγ1
which is expressed strongly in tumor tissues of primary human lung SCC has been
recently identified. This splice variant
exhibits dominant-negative properties in human lung tumor cells, and its
overexpression renders transfected cells more resistant to chemotherapeutic
drug- and chemical-induced cell death [68]. This suggests that the decreased drug
sensitivity of PPARγ1-expressing
cells may be associated with increased tumor aggressiveness and poor clinical
prognosis in patients. Thus, a better
understanding of the mechanisms of action of activated PPARs in tumors (and
host cells) is required since the dissection of these pathways might unveil
better targets for therapy. Nevertheless,
the data available to date regarding PPARγ are promising and justify engaging in clinical
studies to determine the true role of PPARγ ligands in lung cancer, while further work should
be performed to identify more selective and effective strategies.Table 2
PPARγ-independent signals triggered by PPARγ ligands.
(2) PPARγ ligands stimulate cancer cell growth and reduce apoptosis via:↑ Wnt signaling and oncogenes (e.g., cyclin D1, β-catenin, c-myc)↑ Tumor suppressors (e.g., LKB1, AMPK, TSC2)↑ ROS production and ERK activation (note that this also occurs in PPARγ-dependent pathways)↓ Effects on transcription factors (e.g., AP-1, NF-κB, Smads, Sp1, CRE)


## 5. Conclusion

In summary, although its exact role in controlling lung tumor growth and apoptosis
remains undefined, PPARγ has been implicated both as a tumor suppressor (in
most cases) and as a tumor promoter (in rare cases). Hence, targeting this receptor for
therapeutic purposes while minimizing side effects represents a great
challenge. Nevertheless, it is clear
that selective PPARγ modulation of desired gene sets can be achieved by
targeting corepressor interactions, separating transactivation from
transrepression, and favoring specific subsets of coactivators. Although the exact mechanisms mediating this
effect remain incompletely elucidated, data available to date regarding this
member of the PPAR family are promising and justify engaging in prospective,
randomized clinical studies to determine the true role of PPARγ ligands in lung
cancer biology.



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