# PPAR Ligands for Cancer Chemoprevention

Authors: Yumiko Yasui; Mihye Kim; Takuji Tanaka
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferators-activated receptors (PPARs) that are members of the nuclear receptor superfamily have three different isoforms: PPARα, PPARδ, and PPARγ. PPARs are ligand-activated transcription factors, and they are implicated in tumor progression, differentiation, and apoptosis. Activation of PPAR isoforms lead to both anticarcinogenesis and anti-inflammatory effect. It has so far identified many PPAR ligands including chemical composition and natural occurring. PPAR ligands are reported to activate PPAR signaling and exert cancer prevention and treatment in vitro and/or in vivo studies. Although the effects depend on the isoforms and the types of ligands, biological modulatory activities of PPARs in carcinogenesis and disease progression are attracted for control or combat cancer development. This short review summarizes currently available data on the role of PPAR ligands in carcinogenesis.


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## Body

## 1. Introduction

Peroxisome proliferators-activated receptors (PPARs) are
member of the nuclear hormone receptor superfamily that were initially
characterized as molecules that mediated the proliferation of peroxisomes in
rodent liver parenchymal cells  in response to the
hypolipidemic drug clofibrate [1]. Subsequently, PPARs
have been shown to regulate the expression of genes involved in a variety of biological
processes, including lipid metabolism and insulin sensitivity [2, 3]. Three
isotypes of PPAR exist; PPARα, PPABβ/δ or simply δ, and PPARγ which are known and they are encoded
by three separate genes and display distinctly different tissue distributions
and functions. PPARα regulates numerous aspects of fatty acid catabolism, where as PPARγ controls adipocyte
differentiation, systemic glucose levels, and lipid homeostasis [4, 5]. PPARδ is involved in development,
embryo implantation, myelination of the corpus callosum, lipid metabolism, and
epidermal cell proliferation [6]. The PPARs are ligand-dependent transcription factors that regulate target genes
expression by binding to characteristic DNA sequences termed peroxisome proliferators
response element (PPREs) located in the 5′-flanking region of target genes [7, 8]. Each
receptor binds to its PPRE as a heterodimer with the receptor for 9-cis
retinoic acid, the retinoid X receptor (RXR) (Figure 1).
Upon binding a ligand, the conformation of a PPAR is altered and stabilized
such that a binding cleft is created, and recruitment of transcriptional coactivators occurs. The result is an increase in gene
transcription, therefore PPARs are able to regulate such divers effects as cell
proliferation, differentiation, or apoptosis.Figure 1
PPAR activation pathway and its target genes.


## 2. PPARα Ligands and Carcinogenesis

PPARα is the first member of this nuclear receptor subclass to be cloned [9]. PPARα is expressed preferentially in
the liver [10] and tissues with high
fatty acid catabolism, such as the kidney, heart, skeletal muscle, and brown
fat [11–13]. The
PPARα isotype is the cellular target
for leukotriene B4 (LTB4) fibrates such as bezafibrate and fenofibrate, which
are hypolipidemic drugs widely used for reducing triglyceride levels, a risk of
cardiovascular diseases. Several studies
have established a link between PPARα
activation and epidermal differentiation. Fibrates
induce differentiation and inhibit proliferation in normal and
hyperproliferating mouse epidermis and regulate apoptosis, but are inactive in
PPARα-deficient mice [14, 15]. Farnesol
also stimulates PPARα-dependent
differentiation in epidermal keratinocytes [16].
Topical PPARα ligands
have weak preventive effects on tumor promotion in mouse skin, despite
upregulation of PPARα in
untreated tumors compared with normal epidermis [17]. These observations suggest that the use of PPARα activators may have
chemopreventive properties in skin carcinogenesis. PPARα expression is also upregulated
in human prostate adenocarcinomas [18]. In
addition, PPARα
ligands suppress the growth of several cancer lines, including colon [19],
endometrial [20], and
breast [21] in vivo or in vitro.
PPARα ligands are able to suppress
the metastatic potential of melanoma cells in vivo and in vitro [22, 23]. More
recently, a PPARα ligand
WY14643 suppresses both endothelial cell proliferation and tumorigenesis in a
PPARα-dependent manner [24]. These
data suggest that certain PPARα
ligands may act as antitumor agents, although the exact mechanisms remain unclear. PPARα
activation has been associated with both anti and proinflammatory actions in
rodents. PPARα ligands
reduce expression of inflammatory markers [25]. In
contrast, the expression of the inflammatory mediator cyclooxygenase (COX)-2 in
human breast and colon cancer cells is upregulated by PPARα ligands [26]. The increased COX-2
expression is known to link to the risk of
epithelial malignancies [27]. These findings indicate that PPARα ligands may be interesting
candidates for the chemoprevention of several types of cancers, but we should
consider negative face of influence of PPARα
ligands on cancer development.


## 3. PPARδ Ligands and Carcinogenesis

A number of reports have described a variety of biological functions of the PPARα and γ isotypes. These two isotypes also have
clinical significance in the treatment of dyslipideamia and type II diabetes mellitus [28]. In contrast, less is
known about the physiological role of the PPARδ isoform, although there is
some evidence supporting its involvement in embryo implantation and development
[6, 29],
epidermal maturation and wound healing [30], and
regulation of fatty acid metabolism [31].  Recently, the effect of  PPARδ function on colon carcinogenesis has been reported. However, the role of PPARδ in
colon cancer is still unclear, as there are data suggesting that it either inhibits
or promotes colon carcinogenesis. PPARδ
expression is increased in colon tumor cells with a mutant Apc (adenomatous polyposis coli) allele (min) [32]. The number of polyps
was the same among the multiple
intestinal neoplasia (Min) mice that were PPARd−/−, PPARd+/−, or PPARd+/+. These findings
suggest that PPARδ  is not essential for colon
carcinogenesis, but PPARδ may
affect size and/or growth of polyps [29]. The
most striking results were provided by a study demonstrating that in PPARδ deficient (PPARd−/−) mice, both Min mutants and those with chemically induced cancers, colon polyp formation
was significantly greater in those nullizygous for PPARδ [33]. These results suggest
that PPARδ attenuates colon carcinogenesis. On the other hand, the following observations strongly
suggest that PPARδ enhances colon cancer formation. PPARδ was elevated
in colon cancer cells and was repressed by APC gene via the β-catenin/Tcf-4
response elements in its promoter [32]. Genetic disruption of
PPARδ decreases the tumorigenicity
of human colon cancer cells [34]. Nitric oxide donating
aspirin is reported to suppress intestinal tumors in Min mice and downregulates the expression of PPARδ and enhance apoptosis and perhaps
atypical cell death [35]. This suggests that
PPARδ contributes to intestinal carcinogenesis.GW501516 was shown to be a PPARδ subtype-selective ligand using
combinatorial chemistry and structure-based drug design [36]. There
are some reports describing the effects of PPARδ ligand on colon
carcinogenesis. Exposure of APCmin⁡/+ mice to the GW501516 resulted in activation of PPARδ and significant acceleration of intestinal adenoma growth [37]. Furthermore, PPARδ activation by PPARδ ligand promotes tumor growth
by inhibiting epithelial tumor cell apoptosis through activation of a VEGF autocrine
signaling loop in APCmin⁡/+ mice [38]. GW501516
stimulates proliferation of human breast, prostate, and hepatocellular
carcinoma cells [39, 40]. In a mouse
mammary tumorigenesis model, GW501516 activates 3-phospholinositide-dependent
protein kinase-1 that is oncogenic when expressed in mammary ductal cells, and
leads to accelerated tumor formation [41]. From
these findings, PPARδ
selective-ligand tends to exert enhancing effects on carcinogenesis, while its antagonists
are expected to prevention and/or treatment of cancer.


## 4. PPARγ Ligands and Carcinogenesis

PPARγ plays an important role in the regulation of proliferation and differentiation of
several cell types. PPARγ is known to be expressed in various organs, including adipose tissue [42], mammary glands [43], small
intestine [44], lung [45], colon
[44], and stomach [46], and
is also upregulated in various types of cancer cells.This receptor has the ability to bind a variety of small
lipophilic compounds derived from both metabolism and nutrition. These ligands,
in turn, direct cofactor recruitment to PPARγ,
regulating the transcription of genes in a variety of complex metabolic
pathways. Several specific ligands (Figure2) have
been identified, such as the thiazolidinediones (including pioglitazone,
rosiglitazone, and troglitazone), naturally occurring lipid, polyunsaturated
fatty acids (PUFA) (including arachidonic, oleic, and linoleic acid) and the
cyclopentenone prostaglandin (PG) 15-deoxy Delta12,14-PGJ2,
a metabolite of PGD2. PPARγ
ligands have been reported to induce cell differentiation and apoptosis in
several types of cancer [47–51],
suggesting potential application as anticancer agents. Furthermore, some
reports recently suggested that PPARγ
ligands can be used as chemopreventive agents for colon, breast, and tongue
carcinogenesis [52–54].Figure 2
Synthetic and naturally occurring ligands for PPARγ.The most widely used synthetic agents belong to the
thiazolidinedione class of antidiabetic drugs (also referred to as glitazones).
These include ciglitazone, troglitazone, pioglitazone, rosiglitazone, and
LY171.833. Pioglitazone, rosiglitazone, and troglitazone have already been used
clinically to treat type 2 diabetes, making use of the ability of synthetic
PPARγ ligands to sensitize insulin and to lower blood glucose
concentration. Recent evidence indicates that certain thiazolidinedione
members, especially troglitazone and ciglitazone, exhibit moderate anitproliferative
activities against epithelial-derived human cancer cell lines, including those
of prostate [55],
breast [56], colon
[57],
thyroid [51], lung [58], and
pituitary carcinoma [50]. PPARγ is known to be expressed in a
variety of cancer, and the treatment of these cancer cells with PPARγ ligands often induces cell
differentiation and apoptosis [47–51], and
exerts antiproliferative effects on human colon cancer [59],
breast cancer [47],
pituitary adenomas [50],
gastric cancer [60], and bladder cancer [61]. Furthermore,
postulated mechanisms by which PPARγ
ligands exert their effects include modulation of the oncogenic Wnt pathway,
inhibition of nuclear factor kappaB (NF-κB), and
modulation of cell cycle pro and antiapoptotic proteins (Figure 3). Wnt signaling
is a complex pathway in which β-catenin binds to transcription factors in the nucleus and plays a role as a central
mediator in regulating cell proliferation and differentiation [62]. PPARγ activation causes a decrease
in β-catenin expression in
adipocytes in vitro and in normal intestinal mucosa in mice [63]. In the
cultured human monocytes, PPARγ inhibits
NF-κB activation thus influencing
the transcription of both survival- and apoptosis-related genes [64]. PPARγ activation also induces the
activation of the proapoptotic caspase-3 protein in human liver cancer cell
lines and a reduction in antiapoptotic Bcl-2 and Bcl-XL protein level in human
colon and gastric cancer cell lines, respectively [65–67]. Furthermore,
colon cancer development is related to hyperlipidemia [68], with clear links to
high level of serum triglycerides (TGs) [69]. A PPARγ ligand, pioglitazone, suppresses
both hyperlipidemia and intestinal polyp formation in the APC-deficient mice in
conjunction with elevation of lipoprotein lipase (LPL), which catalyzes TG
hydrolysis [70].Figure 3
Molecular mechanisms for anticarcinogenic and/or chemopreventive effects of
PPARγ ligands.We previously investigated the modifying effects of PPARγ or α ligands (troglitazone, pioglitazone, or
bezafibrate) on early phase of colon carcinogenesis with or without colitis in
male F344 rats [19, 71]. The role of PPARγ in
AOM-induced colon tumorigenesis was directly demonstrated by the study showing
that the incidence of colonic tumors increased in the hemizygous knockout of
PPARγ that received AOM [72]. Although
thiazolidinediones inhibit AOM-induced colon carcinogenesis in the wild type
mice, the observation using APC-deficient mouse models showed conflicting
results regarding the effects of PPARγ ligand
treatment [73–76]. This
may be caused by use of different PPARγ
agonists (troglitazone versus pioglitazone), and different doses (100–2000 ppm in diet) examined. Colonic
inflammation is associated with a high risk of colorectal cancer (CRC) [77]. CRC is thus one of the
most serious complications of inflammatory bowel disease, such as ulcerative
colitis and Crohn’s disease [77]. In the experiments,
dietary administration of PPARα or γ ligands effectively suppressed azoxymethane (AOM)-induced
or dextran sodium sulfate (DSS)/AOM-induced aberrant crypt foci, which are
precursor lesions for colon carcinoma (Table 1).
Our findings suggested that synthetic PPARγ and PPARα ligands are able to inhibit
the early stages of colon tumorigenesis with or without colitis, and the
findings were confirmed by the study conducted by Osawa et al. [78]. Furthermore,
we demonstrated ligands for PPARγ and PPARα inhibit colitis-related colon
carcinogenesis [79] using
our AOM/DSS mouse model [80]. In the experiment,
dietary administration (0.05% in diet for 14 weeks) with troglitazone and
bezafibrate significantly inhibited both the incidence and multiplicity of
colonic adenocarcinoma induced by the treatment with AOM/DSS, although
bezafibrate feeding did not significantly lower the multiplicity (Table 2). Dietary exposure of troglitazone and bezafibrate
suppressed cell proliferation and induced apoptosis and lowered immnoreactivity
of COX-2, inducible nitric oxide, and nitrotyrosine in the colonic
malignancies.Table 1
Effects of PPAR ligands on ACF formation in rats.
Treatment (No. of mice)ACF/colon (% inhibition)ACs/colon (% inhibition)AOM alone (12)83 ± 6(a)2.0±0.24AOM + 0.01%
troglitazone(8)68 ± 16 (18%)1.7±0.21
(15%)AOM + 0.05%
troglitazone(8)55 ± 13(b) (34%)1.5±0.13(c) (25%)AOM + 0.01% bezafibrate
(8)75±8
(10%)2.0±0.20 (0%)AOM + 0.05% bezafibrate
(8)53± 9(d) (36%)1.9±0.10 (5%)None001% DSS + AOM (10)115 ± 222.4±0.291% DSS + AOM + 0.01%
pioglitazone (7)71 ± 24(e) (38%)1.8±0.17(f) (25%)1% DSS + AOM + 0.01% troglitazone(7)57 ± 14(g) (50%)1.6±0.14(g) (33%)1% DSS + AOM + 0.01%
bezafibrate (7)59 ± 18(h) (49%)1.7±0.16(i) (29%)None00(a)Mean ±SD.(b–d)Significantly different
from the AOM alone group: (b)P < .01; (c)P < .005; and (d)P < .001.(e–i)Significantly different
from the DSS/AOM group: (e)P < .05; (f)P < .01; (g)P < .001; (h)P < .005; and (i)P < .002.Table 2
Effects of PPAR ligands on colon carcinogenesis in mice.
Treatment (no. of mice)Incidence/Multiplicity (% inhibition)TotalAdenomaAdenocarcinomaAOM/DSS100%/5.2±3.0(a)100%/2.1±1.8100%/3.0±1.8AOM/DSS/0.05%
Troglitazone90%/2.5±1.8(b)(52%,)90%/1.6±1.1 (24%)40%(c)/1.2±2.5(b) (60%)AOM/DSS/0.05%
Bezafibrate80%/2.6±2.5(b)(50%)70%/1.1±1.0(b) (48%)60%(b)/1.8±2.6 (40%)None0%/00%/00%/0(a)Mean ±SD.(b,c)Significantly different from the AOM/DSS group: (b)P < .05; and (c)P < .01.PPARγ receptors are activated by certain lipophilic ligands, such as PUFAs and
eicosanoid derivatives. They bind to the PPARγ receptor at micromolar
concentrations. The essential fatty acids (arachidonic acid, docosahexanoic acid,
and eicosapentaenoic acid) as well as modified oxidized lipids (9-hydroxyoctadecanoic acid and
13-hydroxy-octadecanoic acid) bind to and activate PPARγ [5]. Recently, conjugated
linoleic acid (CLA) was shown to act as a high affinity ligand and an activator
of PPARγ [81]. Anticarcinogenic
activity of CLA is mediated by PPARγ
activation in susceptible tumors [81]. When treated with CLA,
PPARγ expression is increased, and APC
and c-myc proteins are downregulated in the human colon cancer cells, and
finally proliferation of cancer cells is inhibited by CLA [82–85]. In
fact, feeding with seed oils containing 9c, 11t, 13t-, 9c, 11t, 13c-, and 9t, 11t, 13c-conjugated linolenic acid, which are converted
to 9c, 11t- and 9t, 11t-CLA within colonic and liver cells,
suppresses AOM-induced colon carcinogenesis by increased expression of PPARγ protein in the colon mucosa [86–89].


## 5. Clinical Trial for PPARγ Ligands against Tumors

There are several clinical studies on the
effects of PPARγ ligands
on malignancies (Table3). The beneficial
effects of glitazones on liposarcomas have been demonstrated in a small
clinical trial [90]. Three
patients with intermediate to high-grade liposarcomas were given troglitazone (800 mg/day orally). In the patients, differentiation of the neoplasms occurred as revealed
by histological and biochemical analysis. The clinical outcome of these patients
was not reported, but the therapy was well tolarated [90].
However, a phase II study on 12 patients with liposarcoma showed that the PPARγ ligand rosiglitazone did not
significantly improve clinical outcome [94]. In prostate,
PPARγ immunoreactivity was
significantly higher in prostate cancer and prostatic intraepithelial neoplasia
than in those with benign prostate hyperplasia and with healthy prostate [98]. A high incidence of
prolonged stabilization of serum prostate-specific antigen (PSA) was observed
in phase II clinical study, where patients with advances prostate cancer who
had no symptoms of metastasis were treated with troglitazone (800 mg/day
orally). Moreover, one patient had a striking decrease in PSA concentration to
almost undetectable amounts [91]. In a
75-year-old man with occult recurrent prostate cancer showed a decrease in PSA
after oral treatment with toroglitazone (600–800 mg/day for
1.5 years) [92]. Thus,
PPARγ is expressed in prostate
cancer and activation of PPARγ might
offer an additional therapeutic option for treatment of prostate cancer in the
near future. At present, most of the available data suggest that PPARγ has antineoplastic effect on malignant
neoplasms [99], including colonic
malignancies. However, in a clinical phase II study on CRC, orally
administrated troglitazone did not lengthen median progression-free survival or
median survival in 25 patients with chemotherapy-resistant metastatic colon
carcinoma [93]. In a
phase II study [95] for the use of
troglitazone to treat patients with advanced refractory breast cancer, no
objective tumor response was observed. However, the study was incomplete
because troglitazone was withdrawn from commercial availability after a warning
by the US Food and Drug Administration about hepatic toxic effects. On the
other hand, it is important to note that neither hormone status of the tumors
nor the amount of PPARγ
protein is assessed before patients were included in the study. In an open
labeled phase II study where ten patients with thyroglobulin-positive and
radioiodine-negative differentiated thyroid cancer were enrolled and they were
given oral rosiglitazone treatment (4 mg/day for 1 week, then 8 mg per day for
7 weeks), rosiglitazone treatment resulted in a 40% partial response rate, but
no complete responses, and the expression level of PPARγ mRNA and protein in the
neoplasm appeared unrelated to rosiglitazone treatment response [96]. The
findings also suggest that higher doses and longer duration of rosiglitazone
therapy may be useful to better define the role of rosiglitazone as a
redifferentiation agent in differentiated thyroid cancer. There is a phase I
clinical study of a PPARγ ligand
(LY293111) that is not thiazolidinedione members [97]. LY293111
is a novel diaryl ether carboxylic acid derivative and is known as PPARγ agonist and LTB4 antagonist. The
study suggested the dose (600 mg) of LY293111 in combination with irinotecan
(200 mg/m2 IV every 21 days for phase II clinical study against
solid tumors.Table 3
Clinical trials on the anticancer effects of PPARγ ligands.
Clinical trialsDrugResultsReference no.Patients with intermediate to high-grade liposarcomas (case reports)TroglitazoneHistlogical and biochemical differentiation[90]Phase II study  on patients with histologically-confirmed
prostate cancer and no symptomatic metastatic diseaseTroglitazoneLengthened stabilization of prostate-specific antigen[91]75-year-old patient with an occult recurrent prostate cancer (case reports)TroglitazoneReduced prostate-specific antigen[92]Phase II study  on patients with metastatic colon cancerTroglitazoneNo significant effect[93]Phase II study on patients with liposarcomaRosiglitazoneLengthened mean time of progression[94]Phase II study  on patients with refractory breast cancerTroglitazoneNo significant effect[95]Phase II study on patients with thyroglobulin-positive and radioiodine-negative differentiated
thyroid cancerRosiglitazoneInduced radioiodine
uptake[96]Phase I study on patients with solid tumorsLY293111The recommended oral dose (600 mg/day) for phase II trial[97]


## 6. Conclusions

PPARs were originally recognized
to be genetic regulators of complex pathways of mammalian metabolism, including
fatty acid oxidation and lipogenesis. However, the receptors have been shown to
be implicated in carcinogenesis and inflammation. PPARs are involved in cell
proliferation and differentiation of a variety of cancer. Numerous reports
indicate that PPARs ligands could play an important role in prevention and inhibition
of cancer development. Synthetic PPAR ligands used for drugs or those of naturally
occurring lipids are promising
cancer chemopreventive agents with slight side effects against several types of
cancer. We should characterize expression patterns of different isoforms of
PPAR in cancerous and precancerous tissues and determine their precise roles in
the carcinogenic process for development of PPARs ligands as a novel class of
cancer preventive/theraputic drugs. Based on current data from preclinical and
clinical studies, we believe that thiazolidinediones, especially PPARγ
agonists, have important role in short-term prophylactic therapy designed to
reduce the number of putative preneoplasia, ACF, in patients who are at high risk for
CRC development.



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