# The Role of PPARs in Cancer

Authors: Keisuke Tachibana; Daisuke Yamasaki; Kenji Ishimoto; Takefumi Doi
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated
transcription factors that belong to the nuclear hormone receptor superfamily.
PPARα is mainly
expressed in the liver, where it activates fatty acid catabolism. PPARα activators have been used to treat dyslipidemia, causing a reduction in plasma triglyceride
and elevation of high-density lipoprotein cholesterol. PPARδ is expressed ubiquitously and is
implicated in fatty acid oxidation and keratinocyte differentiation. PPARδ activators
have been proposed for the treatment of metabolic disease. PPARγ2 is expressed
exclusively in adipose tissue and plays a pivotal role in adipocyte differentiation.
PPARγ is involved in glucose metabolism through the improvement of insulin sensitivity
and represents a potential therapeutic target of type 2 diabetes. Thus PPARs are molecular
targets for the development of drugs treating metabolic syndrome. However, PPARs also play
a role in the regulation of cancer cell growth. Here, we review the function of PPARs in tumor
growth.


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## Body

## 1. Introduction

Peroxisome
proliferator-activated receptors (PPARs) areligand-activated transcription
factors that belong to thenuclear hormone receptor superfamily [1]. PPARs bind
to a direct repeat of two hexanucleotides, spaced by one or two nucleotides
(the DR1 or DR2 motif) as heterodimers with the retinoid X receptor (RXR), and
activate several target genes [2–4]. These
peroxisome proliferator responsive elements (PPREs) are found in various genes
that areinvolved in lipid metabolism and energy homeostasis,including lipid
storage or catabolism, and fatty acid transport, uptake, and intracellular
binding [5]. Three subtypes, PPARα, PPARδ (also known as PPARβ), and PPARγ, have been identified and these
subtypes with a high degree of sequence conservation of each subtype across
various species have been characterized. The DNA-binding domains of the three subtypes are 80% identical, while their ligand-binding domains exhibit a lower
degree (approximately 65%) of identity (Figure 1). Consistent with this
relatively high divergence among the subtype-specific ligand-binding domains,
differential activation of PPARs by endogenous and exogenous compounds may
account for the specific biological activity of the three PPAR subtypes [6, 7].The general features of human PPARs. (a) Structure and functional domain of
human PPARs. A/B, C, D, and E/F indicate N-terminal A/B domain containing a
ligand-independent activation function 1, DNA-binding domain (DBD), hinge
region, and C-terminal ligand-binding domain (LBD), respectively. The number
inside each domain corresponds to the percentage of amino acid sequence identity
of human PPARδ and PPARγ relative to PPARα. (b) PPAR/RXR heterodimers bind to a
PPRE located in the promoter of target genes through the DBD. Unliganded PPAR
associates with the corepressor complex. In the presence of ligand, the
ligand-bound LBD associates with the coactivator complex.
(a)(b)PPARα is expressed in the liver, kidney, small
intestine, heart, and muscle, where it activates fatty acid catabolism and is
involved in the control of lipoprotein assembly [8]. PPARα is activated by several molecules, such
as long chain unsaturated fatty acids, eicosanoids, and hypolipidemic dr-ugs
(e.g., fenofibrate) [9–12]. PPARα activators have been used to treat
dyslipidemia, causing a reduction in plasma triglyceride and elevation of high-density
lipoprotein (HDL) cholesterol [13, 14]. PPARδ is expressed ubiquitously and is
implicated in fatty acid oxidation, keratinocyte differentiation, wound healing,
and the response of macrophages for very low-density lipoprotein
[15–19]. PPARδ activators have been proposed for the
treatment of metabolic disease and are under clinical trial [20, 21]. There are
two PPARγ isoforms: PPARγ1 and γ2 [22, 23]. PPARγ2, which is generated by alternative
splicing, contains an additional 28 amino acids at the N-terminal compared to
PPARγ1. PPARγ3 is a splicing variant of PPARγ1 and gives rise to the same protein
[24]. PPARγ2 is expressed exclusively in adipose
tissue and plays a pivotal role in adipocyte differentiation, lipid storage in
the white adipose tissue, and energy dissipation in the brown adipose tissue [22, 25]. On the other hand, PPARγ1 is ex-pressed in the colon, the immune
system (e.g., monocytes and macrophages), and others. Except for the function
of PPARγ2 in adipose tissue, PPARγ also participates in inflammation, cell
cycle regulation, and other functions [26]. PPARγ is involved in glucose metabolism through
the improvement of insulin sensitivity and represents a potential therapeutic
target of type 2 diabetes [26]. Indeed, insulin-sensitizing thiazolidinedione
(TZD) drugs are PPARγ ligands [27]. Thus PPARs are molecular
targets for the development of drugs to treat type 2 diabetes and metabolic
syndrome. On the other hand, PPARs also play a role in the regulation of cancer
cell growth.


## 2. PPARα and Cancer

Fibrates,
which are relatively weak PPARα ligands, are useful for the treatment
of dyslipidemia [7, 9–11]. Fibrates
lower serum triglyceride levels and increase HDL levels through the activation
of PPARα [5]. PPARα induces lipoprotein lipase (LPL)
expression, reduces the expression levels of apolipoprotein C-III (ApoC-III), a
natural LPL inhibitor, and stimulates the uptake of cellular fatty acids and
the conversion of fatty acids to acyl-CoA derivatives [5, 28, 29]. These
catabolism functions are mediated by upregulating the expression of a series of
genes-related carbohydrate and lipid metabolism [5, 30]. In addition, PPARα increases the expressions of ApoA-I and
ApoA-II, resulting in raising HDL cholesterol levels in humans [31, 32]. Thus
PPARα plays a central role in the control of
fatty acid andlipoprotein metabolism, and improves plasma lipid profiles.
Although peroxisome proliferators have carcinogenic consequences in the liver
of rodents, epidemiological studies suggest that similar effects are unlikely
to occur in humans [10, 33–36].Several mechanisms have been
proposed to explain the carcinogenesis of peroxisome proliferators in rodents.
Peters et al. reported that
wild-type mice treated with the Wy-14,643 showed increase of replicative DNA
synthesis in hepatic cells and developing liver tumors with 100% incidence,
whereas PPARα-null mice were refractory to this
effect [37]. Peroxisome proliferators increase the peroxisome volume and number
and result in an increase in hydrogen peroxide (H2O2)
levels [38–40]. These
effects may be mediated in part by the increased expression of peroxisomal
enzymes that produce H2O2, such as acyl CoA oxidase (ACO) [39–41]. PPARα upregulates the expression levels of ACO via PPRE in the promoter region [42, 43]. A stably transfected African
green monkey kidney cells (CV-1) overexpressing rat ACO increased H2O2 production, formed transformed foci, and grew efficiently in soft agar when the
cells were treated with linoleic acid [44]. Furthermore, when these cells were
transplanted into nude mice, these cells formed solid tumors [44]. An increase
of intracellular levels of H2O2 could lead to DNA damage
via a variety of mechanisms [45]. Any reduced iron present can catalyze the
cleavage of H2O2, via the Fenton reaction, to produce
hydroxyl radicals (HO•) [46]. The HO• attacks guanine
residues, producing residues of 8-oxo-7,8-dihydroguanine (8-oxoguanine). When
DNA synthesis occurs before the 8-oxoguanine is repaired, this damaged base
will have a chance to pair with adeninenucleotide, re-sulting in a mutation in
the daughter cells [47]. In addition, antioxidants inhibit ciprofibrate-induced
hepatic tumorigenesis by scavenging active oxygen [48]. Thus oxidative stress
by peroxisome proliferators acts as a driving force to malignancy. The
activation of PPARα also leads to increased hepatocellular
proliferation and inhibition of apoptosis. Chron-ic administration of nafenopin,
PPARα agonist, to mice causes significant
increase in the liverweight, hepatic DNA synthesis, and the development of
hepatocellularcarcinomas [49]. Nafenopin treatment of primary cultures of
adult
rat hepatocytes also stimulated DNA synthesis [50]. Indeed,
Peters et al. showed that mRNAs encoding cyclin-dependent kinase (CDK) 1, CDK4,
cyclin D1, and c-myc and their
proteins, which induce cell proliferation, increased in wild-type mice fed by
Wy-14,643 but not in PPARα-null mice [51]. Increase of the average
liver weight and the levels of mRNAs encoding cell cycle regulation, such as
CDK4, proliferating cell nuclear antigen (PCNA) and cyclin B1, were also found
in wild-type mice fed by bezafibrate, the less specific PPARα agonist, and these effects were not
found in PPARα-null mice [52]. Moreover, the treatment
of the primary culture of rat hepatocytes and the rat hepatoma cell line, FaO,
with nafenopin suppressed apoptosis [53, 54]. Thus the activation of PPARα leads to the increase of oxidative
stress, induction of cell proliferation and inhibition of apoptosis, indicating
that PPARα increases hepatocarcinogenesis in mice.A number of experimental
observations suggest that there is a species difference between rodents and
humans in the response to PPARα agonists, although the functional
differences of PPARα derived from species are not clear
(Table 1). One possible explanation for the difference is the expression levels
of PPARα in the liver. The expression levels of
PPARα in human liver are approximately one
order less than that observed in mouse liver [55]. Small expression levels of
PPARα could allow PPREs to be occupied by other
members of the nuclear receptor superfamily,such as RXR, the chicken ovalbumin
upstream promoter transcription factor I (COUP-TFI), COUP-TFII, hepatocyte
nuclear factor-4 (HNF4), retinoic acid receptor (RAR), and thyroid hormone
receptor (TR), and affect responsiveness to peroxisome proliferators
[56–62]. We and
others have shown that elevated expression of PPARα in HepG2 cells dramatically increased
the expression of several target genes, such as 3-hydroxy-3-methylglutaryl-CoA
synthase 2 (mitochondrial) (HMGCS2), carnitine palmitoyltransferase 1A (CPT1A),
and long chain fatty acyl-CoA synthetase (ACS) [30, 63, 64]. In this way, the
lower expression levels of PPARα in human liver might contribute to
holding down peroxisome proliferation and subsequent pathologic effects. Another
explanation is that several PPARα variants, which lack the entire exon 6
or contain mutations, are detected in human cells and these variants act as a
dominant negative regulator of PPAR-mediated gene transcription [55, 65, 66]. But
this has not been found in rodents yet. One PPARα variant containing the mutation
prevents the suppression of hepatocyte apoptosis by nafenopin [55, 65, 66].
Thus the expression levels of PPARα variants might affect the response to
peroxisome proliferators. Next, there appears to be sequence differences in the
PPRE found in the promoter region of ACO. Osumi et al. identified ACO to be
a direct PPARα target gene and a functional PPRE
located in the proximal promoter of the rat ACO gene [42]. In contrast to the
rodent ACO gene promoter, the human ACO gene promoter differs at three bases
within the PPRE from the rat ACO promoter and appears refractory to PPARα [42, 67, 68]. This human PPRE was
unable to drive peroxisome proliferators-induced gene transcription in cell-based
assays [67–69]. Indeed,
human liver cell lines and primary hepatocytes did not induce ACO mRNA by
treatment with fibrates or other PPARα agonists [63, 64]. A similar pattern,
such differences between human and other species, was observed in the
expression of ApoA-I gene [31]. Fibrates influence the ApoA-I gene expression,
raising it in humans, and lowering it in rodents. These differences are due to
a combination of two distinct mechanisms implicating the nuclear receptors PPARα and Rev-erbα, a negative regulator of gene
transcription [31]. The species-distinct regulation is due to sequence
differences in cis-acting elements in
their respective promoters leading to repression by Rev-erbα of rat ApoA-I and activation by PPARα of human ApoA-I. There is a positive
PPRE in the human ApoA-I promoter but not in rats. The expression of Rev-erbα is induced by fibrates [3, 31]. In the
case of rat, this induction leads to the repression of the ApoA-I gene
expression through an Rev-erbα response element. On the other hand,
there is no Rev-erbα response element in the human ApoA-I
gene [31]. Thus the sequence differences in cis-acting
elements cause the species-distinct regulation of target genes expression by
peroxisome proliferators. However, the mechanism of the species differences is
not known in detail.Table 1
Summary
of the species differences of PPARα.
HumanRodentPPARα expression levels+++PPARα variantsYes?Peroxisome proliferation+/−+Fatty acid metabolism++Expression of cell cycle regulator
genes+/−+Expression of miRNA (let-7C)+−Hepatocellular proliferation+/−+Apoptosis+−Liver tumor+/−++To determine the mechanism of species difference in response to peroxisome proliferators, Gonzalez et al.
gen-erated a liver-specific PPARα humanized mouse line (hPPARαTetOff mice) in which the
human PPARα was expr-essed in the liver in a PPARα-null background under the control of
the tetracycline (Tet) responsive regulatory system
[70–72]. The
expression of several target genes encoding peroxisomal and mitochondrial fatty
acid metabolizingenzymes were elevated in hPPARαTetOff mice fed Wy-14,643 or
fenofibrate, resulting in the decrease of serum triglycerides [70, 73].
However, the expressions of various genes involved in cell cycle regulation
(PCNA, c-myc, CDK1, CDK4, and
cyclins) in the liver were unaffected by Wy-14,643. In ad-dition, hPPARαTetOff mice were resistant to
Wy-14,643-induced hepatocarcinogenesis [70, 73].  Recently,
Shah et al. showed that Wy-14,643
regulated mice hepatic MicroRNA (miRNA) expression via a PPARα-dependent pathway
[74].  miRNAs are a
class of nonprotein-coding, endogenous,small RNAs, and regulate gene
expression by translational repression and mRNA cleavage [75].  Some miRNAs
regulate cell proliferation and apoptosis processes that are important in
cancer formation [76].  The activation of PPARα with Wy-14,643 inhibits the expression
of miRNA let-7C, which functions as a tumor suppressor gene [74].  let-7C
degrades c-myc mRNA by binding to 3'
untranslated region (UTR) of the c-myc gene. Treatment of mice with Wy-14,643 showed that let-7C expression was
decreased and a subsequentincrease in c-myc was observed. Following an increase in c-myc,
the levels of the oncogenic mir-17 miRNA cluster were increased [74].  In this
way, inhibition of the let-7C signaling cascade may lead to increased
hepatocellular proliferation andtumorigenesis. In contrast, hPPARαTetOff mice do not exhibit
downregulation of let-7C and induced c-myc and mir-17 expression [74].  Furthermore,
Yang et al. generated another type of PPARα humanized mice, hPPARαPAC mice, that has the
complete human PPARα gene sequence including 5' and 3' flanking
sequences on a P1 phage artificial chromosome (PAC) genomic clone, introduced
onto the mouse PPARα-null background [71].  Upon treatment
with the peroxisome proliferators (Wy-14,643 or fenofibrate), hPPARαPAC mice exhibited peroxisome
proliferation, lowering of serum triglycerides, and induction of PPARα target genes encoding enzymes involved
in fatty acid metabolism. However, let-7C expression was not decreased and the
expression levels of c-myc, cyclin D1
and CDK4 were not increased [71].  Thus these observations suggest that the
species differences in response to peroxisome proliferators could be due in
part to a differential ability of the mouse and human PPARα to suppress let-7C gene expression
[74].  However, the mechanism involved in PPARα-dependent repression of let-7C is
unclear. The differences between the wild-type mice and PPARα humanized mice could be caused by the
structural differences between human and mouse PPARα and differential coactivator
recruitment. However, additional investigation is required to better understand
and clarify the mechanism of action of PPARα in causing hepatocarcinogenesis.


## 3. PPARδ and Cancer

The role of PPARδ in oncogenesis is controversial,
especially in colon cancer. Some reports show that PPARδ promotes tumorigenesis by increasing
cell proliferation. Indeed, the levels of PPARδ mRNA are increased in both human and
rodent colorectal carcinomas [77, 78]. PPARδ is a potential downstream target gene
of the adenomatous polyposis coli (APC)/β-catenin/T cell factor-4 (TCF-4) pathway
[77]. APC is a tumor suppressor gene and is mutated in familial adenomatous
polyposis (FAP) and most sporadic colorectal tumors [79–83]. β-catenin, which binds to APC and axin in
a large protein complex, can be phosphorylated by glycogen synthase kinase-3β (GSK3β) and is followed by ubiquitination and
degradation. Mutation of APC results in the accumulation of β-catenin, which in turn translocates to
the nucleus and associates with the transcription factor TCF-4 [84]. The β-catenin-TCF-4 transcription complex
increases the transcription of growth-promoting genes, such as c-myc and cyclin D1 [85, 86]. The β-catenin-TCF-4 transcription complex
also activates the human PPARδ promoter activity via TCF-4 binding
sites, namely, APC suppresses the PPARδ expression through the degradation of β-catenin [77]. K-Ras mutation is found
in colorectal cancer [80, 87]. Activation mutations in Ras result in the
activation of the mitogen-activated protein kinase (MAPK) pathway and induce
tumor growth and progression [88]. The expression levels and activity of PPARδ were increased by the induction of
mutated K-Ras in conditionally K-Ras-transformed rat intestinal epithelial
cells [89]. Thus PPARδ is also a downstream target gene of Ras/Raf/MAPK and
extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway [89]. In
this way, PPARδ may play a role in colon cancer.Epidemiological studies have
shown that nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin,indomethacin, and sulindac, reduce the overall number and size of adenomas in
patients with FAP. Healthy individuals using NSAIDs regularly can lead to a 40–50% reduction in
the relative risk of developing colon cancer [90]. NSAIDs inhibit cyclooxygenase
(COX) activity and thereby reduce prostaglandin synthesis [91]. COX is a key
enzyme inarachidonic acid metabolism and prostaglandin production. COX catalyzes a two-step reaction that converts arachidonic acid to prostaglandin H2⁢(PGH2), which in turn serves as the precursor for the synthesis of
all biologically active prostaglandins, including PGD2, PGE2,
PGF2α, prostacyclin (PGI2), and
thromboxane A2⁢(TXA2)  [92]. COX exists in two isoforms
that are encoded by two separate genes. COX-1 is constitutively expressed in
most tissues, on the other hand, the expression of COX-2 is normally low or
absent in most tissues but is rapidly upregulated by proinflammatory cytokines
[93]. Expression of COX-2 is also elevated in colorectal cancer and in a subset
of adenomas [94].Moreover, since both the introduction of the knockout
mutation of the COX-2 gene into ApcΔ716 mice, amodel of human
FAP, and treating ApcΔ716 mice with NSAIDs reduce
the development of intestinal tumors, COX-2inhibitors have been considered as
therapeutic agents for colorectal polyposis and cancer [95].
He et al. reported that NSAIDs inhibited
the transcriptional activity of PPARδ by disruption of the DNA binding
ability of PPARδ/RXR heterodimers, and ectopic
expression of PPARδ in the human colorectal cancer cell
line, HCT116, protected the cells from sulindac-induced apoptosis [77]. PPARδ and COX-2 mRNA are expressed in similar
regions in human colon cancer, and the stable PGI2 analog,
carbaprostacyclin (cPGI), acts as a PPARδ ligand [11, 78]. Indeed, ectopic
expression of COX-2 and PGI synthase (PGIS) in the human osteosarcoma cell
line, U2OS, produced high levels of endogenous PGI2 and
transactivation of PPARδ [78]. PGE2 levels are also
elevated in human colorectal cancers and adenomas, and PGE2 increases the growth and motility of colorectal carcinoma cells [96, 97]. D. Wang et al. showed that PGE2 promoted resistance to serum starvation-induced apoptosis of cultured human
colon carcinoma cells, LS-174T, through indirectly upregulation PPARδ transcriptional activity via a phosphotidylinositol-3-kinase
(PI3K)-Akt pathway [98]. Furthermore, PGE2 accelerates intestinal
adenoma growth of Apcmin⁡ mice, a model of human FAP that harbors a mutation in the
apc gene, via PPARδ [98].
Xu et al. showed that PGE2 activated cytosolic
phospholipase A2α(cPLA2α) through PI3K or MAPK pathway, and
subsequently cPLA2α enhanced PPARδ activity in the human
cholangiocarcinoma cells [99]. They also showed that PPARδ enhanced COX2 expression and PGE2 production. This positive feedback loop may play an important role in
cholangiocarcinoma cell growth, although it is not known whether this kind of
positive feedback loop exists in the colorectal cancer cells [99]. Thus PPARδ induces the cell proliferation through
the inhibition of apoptosis. However, sulindac sulfide induces apoptosis not
only in wild-type HCT116, but also in HCT116 PPARδ-null cell lines [100]. On the basis of
these observations, although NSAIDs may reduce tumorigenesis through the
inhibition of PPARδ activity, PPARδ is not a major mediator of
sulindac-mediated apoptosis.Recent evidence supports the
hypothesis that PPARδ promotes tumor progression. HCT116 PPARδ-null cell lines grew slightly more
slowly than wild-type HCT116 cells, and exhibited a decreased ability to form
tumors compared with wild-type mice when inoculated as xenografts in nude mice
[100]. Gupta et al. showed that
exposure of Apcmin⁡ mice to
10 mg/kg of GW501516, a high-affinity PPARδ-selective agonist, led to a two-fold
increase in polyp number in the small intestine [101].
The most prominent effect was on polyp size, mice treated with the PPARδ activator had a five-fold increase in
the number of polyps larger than 2 mm, suggesting that PPARδ activation primarily aff-ected the rate
of polyp growth rather than initiating polyp formation. Pretreatment of
wild-type HCT116 cells with GW501516 significantly suppressed serum
starvation-induced apoptosis in a dose-dependent manner,  but not HCT116 PPARδ-null cells [101]. Furthermore,
D. Wang et al. showed that PPARd−/−/Apcmin⁡
mice decreased intestinal adenoma growth and
inhibited the tumor-promoting effect of GW501516 [102]. They also showed that PPARδ activation with GW501516 upregulated vascular
endothelial growth factor (VEGF) transcription, expression, and peptide release
in intestinal epithelial tumor cells, and subsequently activated PI3K-Akt
signaling [102]. Similar results were obtained in the human endothelial cells
[103, 104]. Piqueras et al. showed that GW501516 induced VEGF mRNA and peptide release, and thus PPARδ induced endothelial cell proliferation
and angiogenesis [103]. Stephen et al.
showed that the activation of PPARδ resulted in increased expression of
VEGF and its receptor fms-related tyrosine kinase 1 (FLT-1), and they suggested
that PPARδ might initiate an autocrine loop for
cellular proliferation and possibly angiogenesis [104]. These results
demonstrate that VEGF mediates the antiapoptotic effects of PPARδ in intestinal epithelial tumor cells by
activating the PI3K-Akt cell survival pathway, and the VEGF autocrine loop
plays an important role in cell survival. Diminished apoptosis is also linked
to downregulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer
cells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primary
product of 15-LOX-1 metabolism of linoleic acid, inhibits cell proliferation
and induces cell cycle arrest and apoptosis in transformed colonic epithelial
cells [105]. 15-LOX-1 protein expression and 13-S-HODE intracellular levels are
decreased in human colonic tumors [105]. Shureiqi et al. showed that 13-S-HODE bound to PPARδ and then downregulated PPARδ expression and activation in colorectal
cancer cells, DLD-1 and RKO, and that the loss of PPARδ expression in HCT116 markedly
suppressed 13-S-HODE-mediated apoptosis [106]. 15-LOX-1 expression also
downregulated PPARδ expression and transcriptional activity
in these colorectal cancer cells [106]. Furthermore, NSAIDs increase 15-LOX-1
protein expression and its product 13-S-HODE levels and downregulate PPARδ expression in association with
subsequent growth inhibition and apoptosis [106, 107]. Thus it is considered
possible that PPARδ promotes the growth of colon cancers.On the contrary, other reports
suggest that ligandactivation of PPARδ promotes the induction of terminal
differentiation and inhibition of cell growth. PPARδ was found in intestinal epithelial
cells in both the normal intestine and adenomas of Apcmin⁡ mice [101].
Reed et al. reported that targeted deletion of the APC alleles in
mouse intestines decreased the expression levels of PPARδ mRNA and protein, although β-catenin and c-myc were increased
[108]. Marin et al. showed that PPARδ expression was
reduced in both the Apcmin⁡ mouse colon polyps and
azoxymethane (AOM)-treated wild-type mouse polyps, though the expression lev-els
of PPARδ mRNA in colonic epithelium were notdifferent between Apcmin⁡  mice and wild-type mice with or without AOM-treatment
[109]. Several reports
identified that the transcription factor binding sites for AP-1,
CCAAT/enhancer-binding proteins, vitamin D receptor, and others were found in
human or mouse PPARδ promoter, and these transcription
factors regulated PPARδ expression [16, 110, 111]. However,
further investigation is required to certify the regulation of PPARδ expression in cancer.Hollingshead et al. reported that GW501516 and
GW0742, highly specific PPARδ ligands, did not increase the growth of
human colon cancer cell lines (HT-29, HCT116, and LS-174T) and liver cancer
cell lines (HepG2 and HuH7) cultured in the presence or absence of serum [112].
In addition, treatment of these cell lines with either GW501516 or GW0742
did not change the phosphorylation of Akt, and no increase in the expression levels
of COX2 or VEGF were detected [112]. Similar results were observed in the colon or liver of Apcmin⁡ mice treated with
GW501516 or GW0742 [109, 112]. Barak et
al. showed that theaverage number of intestinal polyps was not
significantly different between PPARd+/+/Apcmin⁡, PPARd+/−/Apcmin⁡, and PPARd−/−/Apcmin⁡  mice, although this
study was limited to a small number [113]. On the other hand, several studies
showed that colon polyp formation was enhanced in the absence of PPARδ expression in both PPARd−/−/Apcmin⁡  and AOM-treated PPARd−/− mice [108, 109, 114]. Moreover, Marin et
al. showed that the administration of GW0742 had no effect on colon or
small intestinal tumorigenesis in either PPARd−/−/Apcmin⁡ or PPARd+/+/Apcmin⁡ mice as compared with controls [109]. In
addition, decrease of colon polyp multiplicity was observed in PPARd+/+ AOM-treated mice
administrated with GW0742 compared with control wild-type mice. This effect was
likely due in part to PPARδ-dependent induction of colonocyte
differentiation and enhancement of apoptosis [109]. Indeed, PPARδ induces keratinocyte terminal
differentiation, which normally opposes cell proliferation [115, 116].
Hatae et al. also showed that intracellular
PGI2, an endogenous PPARδ ligand, formed by expressing PGIS in
human embryonic kidney 293 (HEK293) cells, promoted apoptosis by activating
PPARδ [117]. In this way, PPARδ inhibits tumor growth by inducing apoptosis or differentiation.Thus the conflicting reports in
the literature suggest that PPARδ either potentiates or attenuates colon
cancer. Similar discrepancies were observed in other tissues.
Di-Poï et al. showed that the activation of
PPARδ inhibited apoptosis in keratinocyte
[118]. The activation of PPARδ by L-165041, one type of PPARδ ligand, upregulates
3-phosphoinositide-dependent kinase-1 (PDK1) and integrin-linked kinase (ILK)
gene expression via PPRE and downregulates phosphatase and tensin homolog
(PTEN) protein expression, and subsequently leads to the activation of Akt1 in
a PI3K- dependent manner in mouse primary keratinocytes and human keratinocyte
HaCaT cells [118]. Yin et al.
showed that PPARδ ligand GW501516 accelerated progestin-
and carcinogen-induced mouse mammary carcinogenesis [119]. Stephen
et al. reported that PPARδ selective agonists stimulated the
proliferation of human breast and prostate cancer cell lines and primary endothelial cells
[104]. On the
other hand, Burdick et al.
reported that ligand activation of    PPARδ with GW0742 inhibited the cell growth
of either human keratinocyte cell line N/TERT1 or mouse primary keratinocytes
[120]. In these cells, ligand activation of PPARδ by GW0742 did not alter expression
and/or modulation of the PTEN/PDK1/ILK1/Akt pathway [120]. Girroir
et al. reported that both GW0742 and
GW501516 inhibited the growth of the human breast cancer cell line, MCF7, and
human melanoma cell line, UACC903 [121].To date, however, the reason for
the contradiction in these observations is unclear. One explanation for these conflicting
results may be the ability of PPARδ to repress the transcription of target
genes. We and others observed that unliganded PPARδ repressed target gene expression,
though ligand-activated PPARδ induced these genes [30, 122–124]. It has been
reported that unliganded PPARδ bound to PPRE and recruited
corepressors, such as B-cell lymphoma 6 (BCL-6), silencing mediator for
retinoid and thyroid hormone receptor (SMRT), nuclear receptor corepressor
(NCoR), and others. On the other hand, liganded PPARδ is thought to release the corepressor
and form a complex with coactivators [122–124].
Furthermore, binding of ligand to the PPARδ or deletion of PPARδ expression may lead to the release of
BCL-6. Subsequently, BCL-6 represses the transcription of a number of
inflammatory cytokine genes [124]. Thus the PPARδ activity may be influenced by the
cellular environment, such as the existence of PPARδ ligands, cofactors, and others. From
this viewpoint, the conflicting resultsmay be due to differences in the
condition of cell cultures or the genetic background of animal models. Secondly,prostaglandins, some of which act as PPAR ligands, have a variety of biological
activities. Prostaglandins, synthesized via the COX pathway from arachidonic
acid, are released outside the cells and lead to changes in the cellular levels
of cyclic AMP and Ca2+ through binding to G-protein-coupled
receptors on the plasma membrane [90]. Indeed,
Hatae et al. suggested that cAMP produced by the PGI2-PGI
receptor (IP)-cAMP pathway might protect vascular endothelial cells from
intracellular PGI2-PPARδ-mediated apoptosis
[117]. On the other
hand, Fauti et al. showed that
the ectopic expression of COX-2 and PGIS in HEK293 cells results in a dramatic
induction of PGI2 synthesis, but no increase in PPARδ transcriptional activity is observed
[125]. Thus they suggest that PGI2 lacks agonistic activity for PPARδ. Since PGI2 is
unstable and
rapidly hydrolyzed to 6-keto-PGF1α within minutes and increases the
production of intracellular cAMP via stimulation of adenylyl cyclase through
the cell surface IP receptor, further investigation is necessary to certify the
mechanism of the effect of the PGI2 on PPARδ activity in detail. Therefore, additional
analyses are necessary to define the PPARδ functions in cancer
(Figure 2).Figure 2
Does PPARδ progress or suppress tumor growth?


## 4. PPARγ and Cancer

Cancer cells
represent dysregulaton of the cell cycle and lead to cell proliferation. In
this viewpoint, modulators of the cell cycle and/or apoptosis are useful as
chemotherapeutic agents for cancer [126, 127]. A number of investigators have
shown that PPARγ was expressed in a variety of tumor
cells, and the activation of PPARγ by ligands led to either inhibition of
cell proliferation or induction of apoptosis (Table 2) [128, 129]. PPARγ is expressed in colonic tumors, normal
colonic mucosa, and colon cancer cell lines
[130–135]. Kitamura et al. showed that TZDs, such as
troglitazone and rosiglitazone, inhibited the cell growth and induced G1 cell
cycle arrest of rat intestinal epithelial cells [136]. The cell growth
inhibition by TZDs was caused by the decrease of the expression of cyclin D1,
critical for entering the S phase of the cell cycle. TZDs suppressed the cyclin
D1 promoter activity through inhibition of the transcriptional activities of AP-1
and Ets [136]. Shao et al.
demonstrated that treatment with rosiglitazone inhibited the K-Ras-induced
elevation of the expression levels of cyclin D1 by inhibition of the  K-Ras-induced phosphorylation of Akt, resulting in the G1 cell cycle arrest
[89]. Furthermore, J.-A. Kim et al.
showed that treatment of the human colorectal cell line, HCT15, with
troglitazone induced the expression of p21Cip1/Waf1, that is, a CDK
inhibitor (CKI) and negatively regulates the cell cycle progression, through
the ERK pathway, and inhibited HCT15 cell growth [155]. PPARγ ligands also induceapoptosis in human
colon cancer cells [156]. Chen et al.
sho-wed that PPARγ ligands, 15-Deoxy-Δ12,14-prostaglandin
J2⁢(15dPGJ2), or ciglitazone, induced apoptosis in HT-29
by inhibiting nuclear factor kappa B (NF-κB) activity, which upregulates various
antiapoptotic genes, and suppressing the expression of BCL-2, which protects
cells against apoptosis [133]. Furthermore, using the in vivo mouse model, the administration of TZD to mice reduced AOM
and/or dextran sodium sulfate-induced formation of aberrant crypts foci and
colon carcinogenesis [131, 157]. In addition, PPARγ ligands also inhibit the cell growth of
several breast cancer cell lines and mammary gland tumor development
[137, 158–162]. Elstner
et al. showed that PPARγ ligands, troglitazone, 15dPGJ2, and indomethacin, caused inhibition
of proliferation in several human breast cancer cell lines, such as MCF7,
MDA-MB-231, BT474, and T47D [162]. Troglitazone also inhibited MCF7 tumor
growth in triple-immunodeficient BNX nude mice [162].
Clay et al. reported that 15dPGJ2 and troglitzaone attenuated cellular proliferation of MDA-MB-231 by blocking
cell cycle progression and inducing apoptosis [160]. Pretreatment of MDA-MB-231
cells with 15dPGJ2 attenuated the capacity of these cells to induce
tumors in nude mice [160]. Yin et al. showed that treatment of MCF7
with troglitazone also decreased the expression of several regulators of pRb
phosphorylation, such as cyclin D1, CDK4, CDK6, and CDK2 [158]. pRB is a
retinoblastoma tumor suppressor gene product, and phosphorylated pRB induces
cell cycle progression [163]. Troglitazone induced the G1 cell cycle arrest by
attenuation of pRb phosphorylation, resulting in inhibition of cell
proliferation [158]. Suh et al.
showed that GW7845, synthetic PPARγ ligand, prevented mammary
carcinogenesis in the rat model that used nitrosomethylurea as the carcinogen
[159]. Mehta et al. also
reported that troglitazone prevented the induction of preneoplastic lesions by
7, 12-dimethylbenz[a]anthracene in a
mouse mammary gland organ culture model [161]. Moreover, PPARγ ligands inhibit the cell proliferation
in other types of cancer. PPARγ ligands inhibited the growth of
esophageal squamous carcinoma cell lines by inducing G1 arrest associated with
an increased level of several CKIs, such as p27Kip1, p21Cip1/Waf1, and p18Ink4c [138]. PPARγ ligands also induced apoptosis and G1
cell cycle arrest in human gastric cancer cells, and that inhibited cell
proliferation [139, 164]. In human pancreatic cancer cells, PPARγ  ligands induced apoptosis and growth
inhibition associated with G1 cell cycle arrest through increasing p27Kip1 protein expression
[140, 165–167]. In human
hepatocellular carcinoma cell lines, PPARγ ligands induced cell cycle arrest
through increased expression of p21Cip1/Waf1, p27Kip1, and
p18Ink4c protein levels [141, 168]. Troglitazone also induced the
activation of the cell death protease, caspase 3, and that induced apoptosis of
human liver cancer cell lines [169]. PPARγ is abundantly expressed in human
adrenal tumors including adrenocortical carcinomas and normal adrenal tissues.
PPARγ agonists suppress adrenocorticaltumor
cell proliferation, increase apoptosis, and induce adrenal differentiation
[142, 170]. Moreover, PPARγ ligand showed antitumor effect against
human prostate cancer cells and human lung cancer cells [
171,
143, 172,
173, 144]. Thus PPARγ ligands could suppress the
tumorigenesis. Therefore, PPARγ ligands could be used as antineoplastic
drugs.Table 2
The
expression of PPARγ in cancer.
ReferencesColonic tumor[135]Breast tumor[137]Esophageal tumor[138]Gastric cancer[139]Pancreatic cancer[140]Hepatocellular carcinoma[141]Adrenocortical carcinoma[142]Lung tumor[143]Prostate cancer[144]Liposarcoma[145]Thyroid carcinoma[146]Bladder cancer[147]Renal cell carcinoma[148]Melanoma[149]Squamous cell carcinoma[150]Cervical
carcinoma[151]Testicular cancer[152]Neuroblastoma[153]Pituitary tumor[154]In contrast, both troglitazone
and rosiglitazone treatment increased the frequency and size of colon tumors inApcmin⁡ mice [174, 175].
Treatment with rosiglitazone also increased the expression levels of β-catenin, a protein involved in Wnt
signaling and correlating with enhanced cell proliferation, in the colon of Apcmin⁡ mice and HT-29 cells
[174]. To investigate the basis for this contradiction, Girnun
et al. used mice heterozygous for PPARγ with both chemical and genetic models
of human colon cancer [176]. Heterozygous loss of PPARγ caused a greater incidence of colon
cancer when these mice were treated with AOM. Although there was no difference
in β-catenin expression levels in colorectal
tumors between AOM-treated PPARg+/-
and wild-type mice, β-catenin expression levels in the colonic
epithelium of untreated PPARg+/-
mice were greater than that of untreated wild-type mice. When crossing to Apc1638N⁡
mice, the mouse
model for FAP, there were also no difference in β-catenin levels between PPARg+/-/Apc1638N⁡ and  PPARg+/+/Apc1638N⁡ mice before polyp
formation. Survival and the number of tumors formed in the colon also showed no
difference in both mice. Thus although PPARγ has the potential to suppress β-catenin levels and colon
carcinogenesis, PPARγ has no effect on β-catenin levels or tumorigenesis in the
presence of APC signaling dysfunction [176]. Furthermore, PPARγ mutations, some of which show the loss
of the transactivation ability, are found in colon cancers in humans, and that
PPARγ may be considered as a tumor suppressor
gene [134]. On the other hand, to evaluate the contribution of PPARγ to breast cancer,
Saez et al. generated transgenic mice,
MMTV-VpPPARγ mice, that express a constitutively
active form of PPARγ in mam-mary gland [177]. MMTV-VpPPARγ mice showed normal development of
mammary gland and no increased tendency to develop tumors. To assess the
influence of in-creased PPARγ signaling on mammary gland neoplasia,
MMTV-VpPPARγ mice were crossed to mice that express
a polyoma virus middle T antigen (PyV-MT) in mammary tissue, MMTV-PyV mice,
which rapidly develop tumors. These mice that expressed both activated PPARγ and PyV-MT showed accelerated
development of mammary tumors. Therefore, although increased PPARγ activation does not initiate tumor
formation in normal mammary tissue, once a tumor-initiating event occurs, PPARγ signaling serves as a tumor promoter in
the mammary gland. Furthermore, there is no difference in tumor development
between MMTV-PyV mice and the mice, generated by crossing PPARg+/-
mice to MMTV-PyV mice [177]. Thus in this
model, PPARγ does not act as a tumor suppressor
gene.Furthermore, PPARγ ligands exert their biological effects
through a PPARγ-independent pathway. Palakurthi et al. reported that troglitazone and
ciglitazone induced G1 arrest by inhibiting translation initiation in both PPARg−/-
and PPARg+/+
mouse embryonic stem
cells. Thus TZDs inhibit cell proliferation and tumor growth in a PPARγ-independent manner
[178]. Therefore, although
PPARγ ligands are used as insulin
sensitizers, further investigation is needed to clarify whether PPARγ ligands are effective chemotherapeutic
agents for cancer in humans.


## 5. Summary

PPARs are
linked to metabolic disorders and are interesting pharmaceutical targets. Among
the synthetic ligands, fibrates are hypolipidemic compounds that activate PPARα, and TZDs, which selectively activate
PPARγ, are hypoglycaemic molecules that are
used to treat type 2 diabetes. PPARδ agonists might form effective drugs for
obesity, diabetes, and cardiovascular disease. Moreover, recent evidence
suggests that PPAR modulators may have beneficial effects as chemopreventive
agents [179]. However, as mentioned above, it remains unclear whether PPARs act
as oncogenes or as tumor suppressors. From this viewpoint, current strategies
are aimed at reducing the side effects and improving the efficacy and safety
profile of PPAR agonists, termed selective PPAR modulators (SPPARMs) [180, 181]. This model proposes that SPPARMs bind in distinct manners to the ligand
binding pocket of PPAR and induce distinct conformational changes of the
receptor, resulting in differential interactions with cofactors according to
the combination of their expression levels in different organs. Thus each
SPPARM leads to differential gene expression and biological response. However,
what kinds of cofactors are recruited to PPAR by each SPPARM is still unknown.
Thus it is important to identify the cofactor complex for PPAR with each SPPARM
and the expression patterns of cofactors in various tissues. Furthermore,
recent evidence suggests that the ligand binding protein in the cytosol that
transports ligands into the nucleus is important to modulate the action of
nuclear receptors. Long-chain fatty acids, endogenous PPAR ligands, are highly
hydrophobic and fatty acids are bound to fatty acid binding proteins (FABPs) in
the aqueous intracellular compartment [182]. FABPs also bind to PPAR ligands
and transport them from the cytosol into the nucleus
[183–191]. In the
nucleus, FABPs interact directly with PPARs and deliver ligands to PPARs, and
the activity of PPARs is modulated [186, 187, 190–192]. Recently,
Schug et al. showed that when
the cellular retinoic acid binding protein-II (CRABP-II) expression levels were
higher than FABP5 in the cells, retinoic acid (RA) bound to CRABP-II.
Subsequently, CRABP-II relocated to the nucleus and delivered RA to RAR,
resulting in inhibition of cell proliferation and induction of apoptosis
[187, 193]. On the contrary, when the FABP5 to CRABP-II ratio is high, RA serves as a
physiological ligand for PPARδ, which induces cell survival and
proliferation [187, 194]. Therefore, it is important to identify the cytosolic
ligand binding proteins and the expression levels of the proteins for defining
the physiological effects of ligands. Furthermore, several ligands exert their
biological effects through a PPAR-independent pathway [195]. Thus further
studies are required to elucidate the role of PPARs for developing new
efficiently and safety chemotherapeutic agents for cancer.



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