# NO-Donating NSAIDs, PPARδ, and Cancer: Does PPARδ Contribute to Colon Carcinogenesis?

Authors: Gerardo G. Mackenzie; Shaheen Rasheed; William Wertheim; Basil Rigas
Journal: PPAR Research
 (2008)

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## Abstract

The chemopreventive NO-donating NSAIDs (NO-NSAIDs; NSAIDs with an NO-releasing moiety) modulate PPARδ and offer the opportunity to revisit the controversial role of PPARδ in carcinogenesis (several papers report that PPARδ either promotes or inhibits cancer). This review summarizes the pharmacology of NO-NSAIDs, PPARδ cancer biology, and the relationship between the two. In particular, a study of the chemopreventive effect of two isomers of NO-aspirin on intestinal neoplasia in Min mice showed that, compared to wild-type controls, PPARδ is overexpressed in the intestinal mucosa of Min mice; PPARδ responds to
m- and p-NO-ASA proportionally to their antitumor effect (p- >m-). This effect is accompanied by the induction of epithelial cell death, which correlates with the antineoplastic effect of NO-aspirin; and NO-aspirin's effect on PPARδ is specific (no changes in PPARα or PPARγ). Although these data support the notion that PPARδ promotes intestinal carcinogenesis and its inhibition could be therapeutically useful, more work is needed before a firm conclusion is reached.


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## Body

## 1. Introduction

Cancer represents a major health
challenge of our time. In the last decade, biomedical science has pursued with
unusual vigor the molecular understanding of cancer.  Cell signaling cascades
have, in particular, been examined or even recognized in the context of cancer
research. The implicit assumption (as well as the expectation) has been that
understanding the mechanisms of carcinogenesis will facilitate the development
of rational, mechanism-driven interventions for either the treatment or even
better the prevention of cancer. The ultimate “deliverable" of such systematic
efforts will be successful cancer therapeutic or preventive agents.As is, however, sometimes the case
in science, mechanistic progress can also be made while trying to understand
the mode of action of agents already developed. Such appears to be the case
with the opportunity that presented itself while we were exploring the mode of
action of a novel chemopreventive agent, nitric oxide-donating aspirin (NO-ASA),
and its relationship to peroxisome proliferator-activated receptorδ (PPARδ).
Here, we discuss our findings, and to provide an appropriate perspective, we
summarize relevant aspects of the pharmacology of nitric oxide-donating nonsteroidal
anti-inflammatory drugs (NO-NSAIDs), PPARδ cancer biology, and the relationship
between the two.


## 2. NO-NSAIDs and Cancer

NO-ASA, initially intended for rheumatologic and cardiovascular
applications [1, 2], is a member of a large
family of pharmacologically active compounds known as NO-donating NSAIDs
(NO-NSAIDs). NO-NSAIDs consist of a conventional NSAID to which the
NO-releasing moiety–ONO2 has been attached via a chemical linker [3, Figure 1]. In the case of NO-ASA, the spacer
can vary in its chemical structure, generating a great number of derivatives.
There are three positional isomers of the NO-ASA molecule (ortho, meta, and para), generated by varying the position of the –CH2ONO2 group with respect to the ester bond linking the two benzenes [4]. NO-ASA is the
best studied NO-NSAID to date.The impetus for the development of NO-NSAIDs for cancer prevention has
been provided by extensive epidemiological data and interventional studies which over fifteen years have established conclusively conventional NSAIDs as
chemopreventive agents against colon and other cancers [5, 6]. The limited efficacy (less than 50%) and side
effects that accompany NSAIDs have prompted the search for better performing
agents. NO-NSAIDs, especially NO-ASA, promise to be such an alternative, and their
anticancer properties are now under intense study by our group and others. Even
though significant progress has been made, the mechanism by which NO-ASA exerts
its chemopreventive effect against colon cancer is still not completely
understood [2].
Our data indicate that NO-ASA could exert its colon chemopreventive
effect, at least in part, by modulating PPARδ function [7].Extensive preclinical results have
established that NO-ASA, which is now FDA approved for clinical trials,
displays properties consistent with a chemopreventive effect [8]. These findings can be grouped into
those documenting a favorablein
vitro cytokinetic effect and those demonstrating chemopreventive efficacy
in animal models of cancer. Compared to their corresponding parent compounds, several
NO-NSAIDs (including NO-ASA, NO-sulindac and NO-ibuprofen, NO-salicylic acid,
NO-indomethacin, and NO-flurbiprofen) have greater potency in inhibiting the
growth of cancer cell lines, for example, colon, prostate, lung, pancreas,
tonsil, breast cancer, and leukemia [9–12]. For example, in the case of colon cancer cell lines, the IC50 values
of NO-NSAIDs were enhanced between 1.7- and 1083-fold. The growth-inhibitory
effect of NO-NSAIDs is due to a profound cytokinetic effect, consisting of
reduced cell proliferation, enhanced cell death, and inhibition of
cell-cycle-phase transitions. Beyond classical apoptosis, NO-ASA induced another
form of cell death, termed as atypical cell death [4]. Likely, a form
of cell necrosis, atypical cell death, was initially described in vitro, but may actually occur in vivo [7].The in vivo studies used orthotopic animal
models of cancer as well as xenotransplants of human cancer cell lines in
appropriate murine hosts. For colon cancer, the results from the various models
are congruent and demonstrate a clear-cut chemopreventive effect. InMin mice, 3 weeks of treatment with NO-ASA decreased the number of tumors by 55% [13]. In F344 rats treated with the carcinogen
azoxymethane, NO-indomethacin and meta NO-ASA significantly suppressed
both tumor incidence and multiplicity (NO-indomethacin was more effective than
NO-ASA). Of the two NO-ASA isomers, the para was more efficacious than
the meta in Min mice [4]. When combined
with 5-fluorouracil or oxaliplatin, para NO-ASA showed additive effects [14].
Sequential NO-ASA and oxaliplatin treatment reduced tumor growth more effectively than single-drug treatments,  perhaps by sensitizing
colon cancer cells to the effect of antitumor drugs. Studies using a hamster
model of pancreatic cancer generated impressive results [15]. Compared with
the control group, NO-ASA reduced the incidence and multiplicity of pancreatic
cancer by 88.9% and 94%, respectively, whereas conventional ASA had no
significant effect.An exciting aspect
of NO-ASA is its extraordinarily enhanced potency. We and others have attempted
to understand this through studies assessing their effects on potentially
informative pathways (summarized in [8]). It appears
that NO-ASA has a pleiotropic effect involving several pathways, as depicted
in Figure 1. PPARδ is a significant component of this array of signaling
molecules [7, 16–19]. Below, after an overview of the role of PPARδ in
cancer, we discuss the relationship between NO-ASA and PPARδ.Figure 1
Effect of NO-ASA on PPARδ and other signaling
pathways. NO-ASA consists of
a traditional ASA molecule (shaded), the spacer, and –ONO2, which
releases NO, with the molecule being considered responsible for much of its
pharmacological properties. There are three positional isomers of NO-ASA (ortho, meta, and para), depending
on the position of –ONO2 in the benzene ring with respect to the
ester bond linking the ASA and spacer moieties. NO-ASA affects several cell
signaling pathways, all relevant to carcinogenesis. The modulation of these
often cross-talking pathways culminates in a net inhibitory effect on cell
growth, one of the crucial determinants of the fate of a tumor. It is likely
that such mechanistic pleiotropism by NO-ASA is central to its efficacy against
cancer.


## 3. PPARδ and Cancer

PPARs, having their first member cloned in 1990 [20], are
ligand-activated transcription factors belonging to the superfamily
of nuclear receptors. They facilitate the response of cells to
extracellular stimuli by transcriptionally regulating gene
expression [21, 22]. Three distinct PPAR subclasses have been
identified: PPARα, PPARδ (also referred to as PPARβ/δ), and PPARγ. These isoforms
are encoded by separate genes and differ in their tissue distribution and
function. PPARδ is the more ubiquitously expressed isoform. Each of the PPAR
isoforms heterodimerizes with the 9-cis-retinoic receptor,
their obligate partner. PPARs regulate diverse physiological processes ranging
from lipogenesis to inflammation, and have been implicated in several disorders
including the metabolic syndrome, diabetes, and atherosclerosis, as well as cancer.
More recently, PPARγ was shown to play a significant role
in cell growth, inflammation, apoptosis, and angiogenesis [23–27].The study of PPARδ lags behind our fairly advanced understanding of
PPARα and PPARγ; the development of high-affinity PPARδ agonists has recently expedited progress [28, 29]. PGI2 and cPGI  are naturally occurring PPARδ
agonists [30]. PPARδ is involved in a wide range
of phenomena affecting several functions, and some of them are critical to the
life of an organism. PPARδ stimulates fatty acid oxidation in heart and
skeletal muscle [31, 32], and  plays a role in cell differentiation [33–35], placental
development [36], cancer, wound repair [37], and atherosclerosis [38–41]. PPARδ-null mouse
models revealed that PPARδ deficiency is associated with multiple developmental
and metabolic abnormalities, including
frequent embryonic
lethality [36].


## 4. The Pros and Cons for a Role of PPARδ in Cancer

There have been both significant work on and
significant excitement about a potential role of PPARδ in cancer. As with any
evolving field, some controversy is almost inevitable. This controversy arises
mainly from the varying results from animal studies (summarized in [42]). Available data can be divided into
two: those which support the notion that PPARδ plays a crucial role in carcinogenesis,
and those which indicate that PPARδ is devoid of any such role. Below, we
present the main points supporting each one of these antithetic conclusions
(Table 1).Table 1
Thepros and cons for a role of PPARδ in cancer.
EvidenceReferenceProsPPARδ expression is enhanced in colon cancer cells[43]PPARδ expression is repressed by the APC gene[44]PPARδ expression increases as tumor progresses[45]PPARδ genetic disruption decreases tumorigenicity of colorectal cancer cells[46]PPARδ activation accelerates intestinal adenoma growth in Min mice[47]Ppard−/− HCT116 cells exhibit decreased ability to form xenograft tumors[46]Dietary fish oil/pectin protects against radiation-enhanced colon cancer by upregulating apoptosis, in part,[48]through PPARδ suppressionPPARδ expression levels are correlated with advanced pathological tumor stage in tumor patients[49]PPARδ-targeted removal of a hub node of the angiogenic network markedly impairs angiogenesis[49]and tumor growth in miceInhibition of PPARδ function reduces growth of epithelial ovarian cancer[50]Activation of PPARδ upregulates VEGF in colon cancer cells[51]PPARδ activation stimulates the proliferation of human breast and prostate cancer cell lines[52]PGE2 indirectly transactivates PPARδ promoting cell survival and intestinal adenoma formation[53]ConsPPARδ-null Min mice exhibit increased predisposition to intestinal tumorigenesis[54]PPARδ-deficient mice show higher polyp formation[55]PPARδ agonists do not increase cell growth in human cancer cell lines[56]PPARδ is dispensable for polyp formation in the intestine and colon of Min mice[36]RNA interference againstPpard promotes proliferation of HCT116 cells[57]Lung tumorigenesis is attenuated in mice
with disruptedPpard[58]PPARδ does not modify impaired mismatch repair-induced neoplasia[59]PPARδ promotes differentiation, inhibiting cell proliferation in keratinocytes[60]PPARδ ligands inhibit TNFα-induced expression of the vascular cell adhesion molecule-1 and[61]E-selectin in HUVEC, preventing inflammationInhibition of colon carcinogenesis by a PPARδ agonist in an  azoxymethane mouse model[62]PPARδ activators inhibit TNFα-induced endothelial inflammation, in part by interfering with[63]the NF-κB signaling pathwayPPARδ activation by a PPARδ agonist produces no change in colon cancer cell growth[52]PPARδ activation by GW0742 inhibits colon polyp multiplicity in Ppard+/+ mice,[64]but not inPpard−/− mice


### 4.1. Pros

PPARδ was ascribed as an oncogenic function after being
identified as a direct transcriptional target of β-catenin, and as a repression
target of the NSAID sulindac, a potent suppressor of colorectal tumors [44]. A close association between PPARδ
and colon carcinogenesis was suggested by immunohistochemical analyses showing
that the expression of PPARδ increases progressively as the colonic epithelium advances
from normal to malignant [45].A series of observations inMin mice support a procarcinogenic role of PPARδ. When Min mice were treated with azoxymethane,
PPARδ levels were increased in flat dysplastic aberrant crypt foci [65], although the same authors indicate
that PPARδ expression in adenomas from Min mice does not differ compared to normal epithelium [65]. Deletion of PPARδ decreased
intestinal adenoma growth and inhibited the tumor-promoting effects of a PPARδ
agonist [51]. Interestingly,
the same group also showed that prostaglandin E2(PGE2),
the predominant prostanoid found in most colorectal cancers, indirectly
transactivates PPARδ promoting cell survival and intestinal adenoma formation [53]. PGE2 treatment did not increase intestinal adenoma burden in Min mice lacking PPARδ, concluding that PPARδ is a focal point of
cross-talk between the prostaglandin and Wnt signaling pathways, which results
in a shift from cell death to cell survival, leading to increased tumor growth [53]. Treatment of Min mice with a synthetic agonist of
PPARδ increased significantly the number and size of intestinal polyps. The
most prominent effect was on polyp size; the PPARδ activator increased the
number of polyps by >2 mm five-fold [47]. The same group also showed that compared with
control ApcMin/+ mice (Ppard+/+/ApcMin/+), small intestinal polyps in PPARδ-deficient ApcMin/+ mice (Ppard−/−/ApcMin/+) were reduced three-fold; the number of large polyps (>1 mm) was reduced
about ten-fold. Heterozygous deletion of PPARδ(Ppard+/−/ApcMin/+) did not significantly reduce the total number of small and large intestinal
polyps in malemice, but this disruption significantly diminished
the number of small intestinal polyps that were >1 mm [51].In cultured colon cancer cells, PPARδ inhibited
differentiation, conferred apoptotic resistance, and promoted cell migration [43], whereas
prostacyclin, a metabolic product of COX-2 which modulates intestinal
tumorigenesis [66], increased PPARδ
activity [43]. PPARδ expression
was elevated in colon cancer cells and was repressed by apc via the β-catenin/TCF-4 response elements in its promoter [44]. Genetic disruption of PPARd decreased the tumorigenicity of human
coloncancer cells [46]. HCT116 Ppard−/− cells, inoculated as xenografts onto nude mice, exhibited decreased ability to
form tumors compared to Ppard+/− and wild-type
controls [46]. Dietary fish oil/pectin protected rats
against radiation-enhanced colon cancer by upregulating apoptosis in colonic
mucosa, in part, by suppressing PPARδ [48].Data from noncolonic cell lines and tissues also
support a role for PPARδ in cancer. Activation of PPARδ results in increased
growth in sex hormone-responsive breast (T47D, MCF7) and prostate (LNCaP,
PNT1A) cell lines [52]. Epithelial ovarian
cancer cells express high levels of PPARδ, and inhibition of PPARδ reduced
tumor growth [50]. In epithelial
ovarian cancer cells, aspirin suppressed PPARδ function and cell growth by
inhibiting ERK1/2 [50]. Activation of PPARδ
by its pharmacologic ligand GW501516 enhanced the growth of human hepatoma cell
lines, whereas PPARδ knockdown by siRNA prevented cell growth [67]. In murine
knockout experiments, targeted removal of a hub node (PPARδ) of the angiogenic
network markedly impaired angiogenesis and tumor growth [49]. In human
cholangiocarcinoma, a positive feedback loop between PPARδ and PGE2 was
recognized; this interaction plays an important role in cell growth [68]. In patients with pancreatic
cancer, PPARδ levels were correlated with advanced pathological tumor stage,
increasing the risk of tumor recurrence and distant metastases [49].


### 4.2. Cons

The strongest evidence that PPARδ plays no appreciable
role in carcinogenesis comes from a series of animal studies, cell culture data,
and from studies evaluating the role of PPARδ in inflammation, the
latter being considered as a contributor to carcinogenesis.Barak et al. evaluated the hypothesis that
if PPARδ is a critical transducer of the
tumorigenic signal, then its loss should substantially reduce, if not eliminate, intestinal polyps in Min mice [36]. Min mice that were Ppard-null
harbored intestinal and colonic polyps. Histologically, all of the
12 intestinal polyps from Ppard+/+ mice and the 9 from Ppard−/− mice were low-grade tubular adenomas. The number of intestinal polyps
was not significantly different between Ppard+/+,Ppard+/−, and Ppard−/−Min mice. Loss of PPARδ
did not significantly change the median size of intestinal polyps, although
polyps > 1 mm were decreased upon PPARδ dosage reduction, which was further pronounced for polyps > 2 mm. The number of polyps < 1 mm was essentially identical in all PPARδ
genotype groups. Their conclusion was that PPARδ is qualitatively dispensable for the tumorigenic
process, although they could not rule out the possibility that it
influences the pace of polyp growth. In agreement with these
findings, Marin et al. showed
that PPARδ activation by GW0742 inhibits colon polyp multiplicity in Ppard+/+ but not in Ppard−/− mice, suggesting that
ligand activation of PPARδ attenuates azoxymethane-induced colon carcinogenesis
[64].The most striking result is provided by a
study demonstrating that inMin mice differing in their Ppard genotype (Ppard−/−, which did
not express PPARδ protein; Ppard+/−; and Ppard+/+),
the incidence of polyp formation was not significantly different between groups
[54]. In fact, Ppard−/−Min mice had about 3–6 times as many
colon polyps as those of Ppard+/+ or Ppard+/− mice. No significant differences in polyp size were found between any of the
genotypes. Congruent results were obtained when they examined colon
carcinogenesis with a more colon-specific, azoxymethane-induced model. The data
from these two different colorectal cancer models suggest that PPARδ attenuates colon carcinogenesis.Finally, Reed et
al. reported that PPARδ-null Min mice exhibited increased predisposition to intestinal tumorigenesis [55]. Another report from the same
group, evaluating the incidence and severity of intestinal neoplasia in mice
deficient in both PPARδ and the mismatch repair gene Mlh1, showed that deficiency of PPARδ in mice with compromised
mismatch DNA repair failed to affect intestinal neoplasia [59], with the implication being that
PPARδ is not required for intestinal adenoma formation.Similar results have
been obtained for noncolonic tumors. For example, mice lacking one or both
alleles ofPpard had enhanced growth
of lung tumors [58]. In another
example, the onset of tumor formation, tumor size, and tumor multiplicity of
the skin was
significantly enhanced in PPARδ-null mice compared with wild-type mice [69].There are also data from cell culture models
contradicting the notion that PPARδ plays a role in carcinogenesis. For
example, in several human cancer cell lines, two PPARδ ligands failed to
increase cell growth, Akt phosphorylation, or the expression of VEGF or COX-2 [56]. PPARδ activation by a PPARδ
agonist does not induce cell growth in HT29, SW480, and HCA-7 colon cancer
cells [64]. Furthermore, Raf oncogenes can contribute to tumorigenesis by augmenting the secretion
of tumor growth promoting prostaglandins, such as PGI2. However, using
several cell lines, Fauti et al.
showed that the increase in PGI2 synthesis did not induce the
transcriptional activity of PPARδ, suggesting that the
oncogenic effect of PGE2 does not involve PPARδ [70]. Another PPARδ function is the modulation of
cell cycle. Knockdown of the PPARδ gene by siRNA promoted proliferation of
HCT116 cells, suggesting that PPARδ may, in fact, inhibit their proliferation
by arresting them in the G1 phase of the cell cycle [57].The chemopreventive
action of PPARδ is also suggested by studies showing that in many cell types PPARδ
promotes differentiation and inhibits proliferation [33, 60, 69, 71]. For example, Hollingshead et al. examined in azoxymethane-treated
PPARδ-null mice whether PPARδ activation and COX2 inhibition attenuate colon
cancer independently. Inhibition of COX2 by nimesulide attenuated colon cancer,
and activation of PPARδ by GW0742 had inhibitory effects. The effects of these
compounds occurred through independent mechanisms as increased levels of
differentiation markers resulting from ligand activation of PPARδ were not
found with COX-2 inhibition, and reduced PGE2 levels resulting from
COX-2 inhibition were not observed in response to ligand activation of PPARδ [62]. In another study by
the same group, wild-type (Ppard+/+) and Ppard−/− mice were treated
with azoxymethane, together with GW0742, a specific PPARδ ligand, to test if Ppard−/− mice exhibit increased colon
polyp multiplicity [64]. Ligand activation
of PPARδ in Ppard+/+ mice
increased the expression of mRNA encoding the adipocyte differentiation-related
protein, fatty acid-binding protein, and cathepsin E, all being indicative of
colonocyte differentiation [64]. Thus, the induction
of differentiation and the inhibition of proliferation in response to PPARδ
activation support the hypothesis that PPARδ attenuates colon carcinogenesis [62].Another contrarian point of view concerns the role of
PPARδ in inflammation, with studies suggesting that activation of PPARδ has
anti-inflammatory effects. In hepatocytes, the PPARδ agonist suppressed
IL-6-mediated acute phase reaction, prompting the speculation that PPARδ
agonists may be used  to suppress
systemic inflammatory reactions in which IL-6 plays a central role [72]. Two synthetic PPARδ ligands
inhibited TNFα-induced expression of the vascular cell adhesion molecule-1 and
E-selectin in human umbilical vein endothelial cells, suggesting that PPARδ
activation has a potent anti-inflammatory effect [61].Relevant to cancer is the presumed role of PPARδ in
inflammation and NF-κB regulation [63, 73, 74]. Such a role is
exemplified by studies on the skin, where activation of PPARδ by IFN-γ and TNFα
accelerated keratinocyte differentiation [75]. Studies with
PPARδ agonists have shown anti-inflammatory
properties of PPARδ attributed to inhibition of NF-κB DNA-binding activity [74, 76]. Inflammation induced by TPA (O-tetradecanoylphorbol-13-acetate)
in the skin was lower in wild-type mice fed sulindac than in similarly treated
PPARδ-null mice [77]. In human
endothelial cells, PPARδ activators inhibited TNFα-induced endothelial
inflammation (VCAM-1 expression, monocyte adhesion, and MCP-1 secretion), in
part by interfering with the NF-κB signaling pathway [63].
Lipopolysaccharide-induced TNFα production in cultured cardiomyocytes through
NF-κB activation was inhibited by overexpression of PPARδ or the PPARδ
synthetic ligand GW0742 [73].The foregoing arguments and counterarguments make it
clear that this controversy remains unresolved. This is the reason why we
attempted to obtain an insight into the role of PPARδ in carcinogenesis by
exploiting the unique opportunity offered by studying the effect of NO-ASA on PPARδ.
Our work is presented in the following section.


## 4.1. Pros

PPARδ was ascribed as an oncogenic function after being
identified as a direct transcriptional target of β-catenin, and as a repression
target of the NSAID sulindac, a potent suppressor of colorectal tumors [44]. A close association between PPARδ
and colon carcinogenesis was suggested by immunohistochemical analyses showing
that the expression of PPARδ increases progressively as the colonic epithelium advances
from normal to malignant [45].A series of observations inMin mice support a procarcinogenic role of PPARδ. When Min mice were treated with azoxymethane,
PPARδ levels were increased in flat dysplastic aberrant crypt foci [65], although the same authors indicate
that PPARδ expression in adenomas from Min mice does not differ compared to normal epithelium [65]. Deletion of PPARδ decreased
intestinal adenoma growth and inhibited the tumor-promoting effects of a PPARδ
agonist [51]. Interestingly,
the same group also showed that prostaglandin E2(PGE2),
the predominant prostanoid found in most colorectal cancers, indirectly
transactivates PPARδ promoting cell survival and intestinal adenoma formation [53]. PGE2 treatment did not increase intestinal adenoma burden in Min mice lacking PPARδ, concluding that PPARδ is a focal point of
cross-talk between the prostaglandin and Wnt signaling pathways, which results
in a shift from cell death to cell survival, leading to increased tumor growth [53]. Treatment of Min mice with a synthetic agonist of
PPARδ increased significantly the number and size of intestinal polyps. The
most prominent effect was on polyp size; the PPARδ activator increased the
number of polyps by >2 mm five-fold [47]. The same group also showed that compared with
control ApcMin/+ mice (Ppard+/+/ApcMin/+), small intestinal polyps in PPARδ-deficient ApcMin/+ mice (Ppard−/−/ApcMin/+) were reduced three-fold; the number of large polyps (>1 mm) was reduced
about ten-fold. Heterozygous deletion of PPARδ(Ppard+/−/ApcMin/+) did not significantly reduce the total number of small and large intestinal
polyps in malemice, but this disruption significantly diminished
the number of small intestinal polyps that were >1 mm [51].In cultured colon cancer cells, PPARδ inhibited
differentiation, conferred apoptotic resistance, and promoted cell migration [43], whereas
prostacyclin, a metabolic product of COX-2 which modulates intestinal
tumorigenesis [66], increased PPARδ
activity [43]. PPARδ expression
was elevated in colon cancer cells and was repressed by apc via the β-catenin/TCF-4 response elements in its promoter [44]. Genetic disruption of PPARd decreased the tumorigenicity of human
coloncancer cells [46]. HCT116 Ppard−/− cells, inoculated as xenografts onto nude mice, exhibited decreased ability to
form tumors compared to Ppard+/− and wild-type
controls [46]. Dietary fish oil/pectin protected rats
against radiation-enhanced colon cancer by upregulating apoptosis in colonic
mucosa, in part, by suppressing PPARδ [48].Data from noncolonic cell lines and tissues also
support a role for PPARδ in cancer. Activation of PPARδ results in increased
growth in sex hormone-responsive breast (T47D, MCF7) and prostate (LNCaP,
PNT1A) cell lines [52]. Epithelial ovarian
cancer cells express high levels of PPARδ, and inhibition of PPARδ reduced
tumor growth [50]. In epithelial
ovarian cancer cells, aspirin suppressed PPARδ function and cell growth by
inhibiting ERK1/2 [50]. Activation of PPARδ
by its pharmacologic ligand GW501516 enhanced the growth of human hepatoma cell
lines, whereas PPARδ knockdown by siRNA prevented cell growth [67]. In murine
knockout experiments, targeted removal of a hub node (PPARδ) of the angiogenic
network markedly impaired angiogenesis and tumor growth [49]. In human
cholangiocarcinoma, a positive feedback loop between PPARδ and PGE2 was
recognized; this interaction plays an important role in cell growth [68]. In patients with pancreatic
cancer, PPARδ levels were correlated with advanced pathological tumor stage,
increasing the risk of tumor recurrence and distant metastases [49].


## 4.2. Cons

The strongest evidence that PPARδ plays no appreciable
role in carcinogenesis comes from a series of animal studies, cell culture data,
and from studies evaluating the role of PPARδ in inflammation, the
latter being considered as a contributor to carcinogenesis.Barak et al. evaluated the hypothesis that
if PPARδ is a critical transducer of the
tumorigenic signal, then its loss should substantially reduce, if not eliminate, intestinal polyps in Min mice [36]. Min mice that were Ppard-null
harbored intestinal and colonic polyps. Histologically, all of the
12 intestinal polyps from Ppard+/+ mice and the 9 from Ppard−/− mice were low-grade tubular adenomas. The number of intestinal polyps
was not significantly different between Ppard+/+,Ppard+/−, and Ppard−/−Min mice. Loss of PPARδ
did not significantly change the median size of intestinal polyps, although
polyps > 1 mm were decreased upon PPARδ dosage reduction, which was further pronounced for polyps > 2 mm. The number of polyps < 1 mm was essentially identical in all PPARδ
genotype groups. Their conclusion was that PPARδ is qualitatively dispensable for the tumorigenic
process, although they could not rule out the possibility that it
influences the pace of polyp growth. In agreement with these
findings, Marin et al. showed
that PPARδ activation by GW0742 inhibits colon polyp multiplicity in Ppard+/+ but not in Ppard−/− mice, suggesting that
ligand activation of PPARδ attenuates azoxymethane-induced colon carcinogenesis
[64].The most striking result is provided by a
study demonstrating that inMin mice differing in their Ppard genotype (Ppard−/−, which did
not express PPARδ protein; Ppard+/−; and Ppard+/+),
the incidence of polyp formation was not significantly different between groups
[54]. In fact, Ppard−/−Min mice had about 3–6 times as many
colon polyps as those of Ppard+/+ or Ppard+/− mice. No significant differences in polyp size were found between any of the
genotypes. Congruent results were obtained when they examined colon
carcinogenesis with a more colon-specific, azoxymethane-induced model. The data
from these two different colorectal cancer models suggest that PPARδ attenuates colon carcinogenesis.Finally, Reed et
al. reported that PPARδ-null Min mice exhibited increased predisposition to intestinal tumorigenesis [55]. Another report from the same
group, evaluating the incidence and severity of intestinal neoplasia in mice
deficient in both PPARδ and the mismatch repair gene Mlh1, showed that deficiency of PPARδ in mice with compromised
mismatch DNA repair failed to affect intestinal neoplasia [59], with the implication being that
PPARδ is not required for intestinal adenoma formation.Similar results have
been obtained for noncolonic tumors. For example, mice lacking one or both
alleles ofPpard had enhanced growth
of lung tumors [58]. In another
example, the onset of tumor formation, tumor size, and tumor multiplicity of
the skin was
significantly enhanced in PPARδ-null mice compared with wild-type mice [69].There are also data from cell culture models
contradicting the notion that PPARδ plays a role in carcinogenesis. For
example, in several human cancer cell lines, two PPARδ ligands failed to
increase cell growth, Akt phosphorylation, or the expression of VEGF or COX-2 [56]. PPARδ activation by a PPARδ
agonist does not induce cell growth in HT29, SW480, and HCA-7 colon cancer
cells [64]. Furthermore, Raf oncogenes can contribute to tumorigenesis by augmenting the secretion
of tumor growth promoting prostaglandins, such as PGI2. However, using
several cell lines, Fauti et al.
showed that the increase in PGI2 synthesis did not induce the
transcriptional activity of PPARδ, suggesting that the
oncogenic effect of PGE2 does not involve PPARδ [70]. Another PPARδ function is the modulation of
cell cycle. Knockdown of the PPARδ gene by siRNA promoted proliferation of
HCT116 cells, suggesting that PPARδ may, in fact, inhibit their proliferation
by arresting them in the G1 phase of the cell cycle [57].The chemopreventive
action of PPARδ is also suggested by studies showing that in many cell types PPARδ
promotes differentiation and inhibits proliferation [33, 60, 69, 71]. For example, Hollingshead et al. examined in azoxymethane-treated
PPARδ-null mice whether PPARδ activation and COX2 inhibition attenuate colon
cancer independently. Inhibition of COX2 by nimesulide attenuated colon cancer,
and activation of PPARδ by GW0742 had inhibitory effects. The effects of these
compounds occurred through independent mechanisms as increased levels of
differentiation markers resulting from ligand activation of PPARδ were not
found with COX-2 inhibition, and reduced PGE2 levels resulting from
COX-2 inhibition were not observed in response to ligand activation of PPARδ [62]. In another study by
the same group, wild-type (Ppard+/+) and Ppard−/− mice were treated
with azoxymethane, together with GW0742, a specific PPARδ ligand, to test if Ppard−/− mice exhibit increased colon
polyp multiplicity [64]. Ligand activation
of PPARδ in Ppard+/+ mice
increased the expression of mRNA encoding the adipocyte differentiation-related
protein, fatty acid-binding protein, and cathepsin E, all being indicative of
colonocyte differentiation [64]. Thus, the induction
of differentiation and the inhibition of proliferation in response to PPARδ
activation support the hypothesis that PPARδ attenuates colon carcinogenesis [62].Another contrarian point of view concerns the role of
PPARδ in inflammation, with studies suggesting that activation of PPARδ has
anti-inflammatory effects. In hepatocytes, the PPARδ agonist suppressed
IL-6-mediated acute phase reaction, prompting the speculation that PPARδ
agonists may be used  to suppress
systemic inflammatory reactions in which IL-6 plays a central role [72]. Two synthetic PPARδ ligands
inhibited TNFα-induced expression of the vascular cell adhesion molecule-1 and
E-selectin in human umbilical vein endothelial cells, suggesting that PPARδ
activation has a potent anti-inflammatory effect [61].Relevant to cancer is the presumed role of PPARδ in
inflammation and NF-κB regulation [63, 73, 74]. Such a role is
exemplified by studies on the skin, where activation of PPARδ by IFN-γ and TNFα
accelerated keratinocyte differentiation [75]. Studies with
PPARδ agonists have shown anti-inflammatory
properties of PPARδ attributed to inhibition of NF-κB DNA-binding activity [74, 76]. Inflammation induced by TPA (O-tetradecanoylphorbol-13-acetate)
in the skin was lower in wild-type mice fed sulindac than in similarly treated
PPARδ-null mice [77]. In human
endothelial cells, PPARδ activators inhibited TNFα-induced endothelial
inflammation (VCAM-1 expression, monocyte adhesion, and MCP-1 secretion), in
part by interfering with the NF-κB signaling pathway [63].
Lipopolysaccharide-induced TNFα production in cultured cardiomyocytes through
NF-κB activation was inhibited by overexpression of PPARδ or the PPARδ
synthetic ligand GW0742 [73].The foregoing arguments and counterarguments make it
clear that this controversy remains unresolved. This is the reason why we
attempted to obtain an insight into the role of PPARδ in carcinogenesis by
exploiting the unique opportunity offered by studying the effect of NO-ASA on PPARδ.
Our work is presented in the following section.


## 5. NO-NSAIDs and PPARδ

Our limited
understanding of the mechanism by which NO-ASA exerts its colon chemopreventive
effect combined with the possibility that PPARδ plays a role in colon
carcinogenesis prompted us to assess the expression of PPARδ during intestinal
carcinogenesis, and also whether NO-ASA modulates it [7].We studiedMin mice and their congenic (wild-type) mice, C57BL/6J+/+.
Three groups of each type of mice were treated for 21 days with vehicle or m-NO-ASA or p-NO-ASA, each at 100 mg/kg/day. As
expected from their relative in vitro potency, after 21 days m-NO-ASA
suppressed the number of intestinal tumors in Min mice (wt mice had no tumors) by 38%, and p-NO-ASA by 59%.Most of the PPARδ
positive cells (staining being always nuclear) were in the
intestinal villi, with only few in the crypts. PPARδ was minimally expressed among the three groups of wild-typemice. In contrast, the
expression of PPARδ in Min mice, similar in tumors and histologically normal mucosa, was more than ten-fold increased compared to
wild-type mice. The two NO-ASA positional isomers inhibited the expression of PPARδ in both normal and neoplastic cells of Min mice. m-NO-ASA suppressed PPARδ expression in histologically normal mucosa by
23% and in neoplastic tissue by 41%; p-NO-ASA suppressed
PPARδ expression in histologically normal mucosa by 27% and by 55% in
neoplastic tissue. The reduction in the number of tumors by each
NO-ASA isomer and the respective suppression of PPARδ expression in
neoplastic cells are strikingly similar; the meta isomer
reduced tumor incidence by 38% and PPARδ expressionby 42%, whereas
the corresponding reduction for the para isomer was 59% and
55%. Of note, the expression of PPARα and PPARγ was sparse, and treatment with NO-ASA had no
appreciable effect on either of them.The changes in PPARδ expression induced by NO-ASA
seemed to have a significant impact on the cell kinetics of the intestinal
mucosa, rendering such an effect mechanistically important. The induction of
apoptosis by NO-ASA, more prominent in neoplastic epithelial cells,
followed closely the pattern of PPARδ reduction. Thus, in the neoplastic tissues, m-NO-ASA
increased apoptosis by 22% and p-NO-ASA by 70%. The percentage of changes in PPARδ expression and
apoptosis is significantly correlated (P<.03), suggesting a potential etiological association between the two
events.We have previously
reported that NO-ASA induces two types of cell death, classical apoptosis as
well as atypical cell death, which based on a variety of criteria appears to be
a variant of necrosis [4]. Documentation of atypical cell
death in vivo had been elusive. This study, however, provided a glimpse into
this phenomenon in vivo. As shown in Figure 2, we were able to record the
evolution of necrotic areas in NO-ASA-treated intestinal tumors. Initially,
TUNEL positive cells coalesce and, as the necrotic area develops, they populate
its margins (being extremely rare in the surrounding tissue). As the necrotic
area increases in size, the TUNEL positive cells persist at the margins. We
have identified multiple TUNEL positive spots within the necrotic areas,
suggesting their cellular origin. We believe that these TUNEL positive cells
are necrotic cells [78]. The relationship of PPARδ and cell
death induced by NO-ASA was ascertained by studying successive sections of
intestinal tumors from both treated and untreated animals (Figure 2). Untreated
tumors show strong PPARδ expression and few apoptotic cells. After treatment
with meta or para NO-ASA, tumors show decreased PPARδ expression
and increased apoptosis. If the apoptosis index of tumors from NO-ASA-treated
mice is plotted against the expression of PPARδ, the association between the
two is statistically significant; Figure 2
makes this correlation obvious. It should, however, be pointed out
that these data have two methodological limitations. First, the specificity of
the antibody is not considered by experts in the field ideal for
immunohistochemistry, as nonspecific binding is possible. Second, no
corroborating methodology was employed such as determination of PPARδ protein
levels in these tissues by immunoblotting.Effect of NO-ASA on PPARδ and apoptosis in colon tissue of Min mice. Upper panel: the evolution of necrotic
areas in intestinal tumors treated withp-NO-ASA; sections are
stained by the TUNEL method. (I) Coalescence of TUNEL positive cells (arrow),
representing the earliest stage; (II) abundant apoptotic cells at the
margins of the developing area with contrast to their rarity in the surrounding
area; (III) and (IV) the necrotic area is increasing in size, but
TUNEL positive cells persist at its margins; TUNEL positive areas within the
necrotic areas (arrows) suggest their cellular origin. Lower panel: the relationship of PPARδ and apoptosis in NO-ASA
treated intestinal tumors. Successive sections of intestinal tumors were
stained for PPARδ expression and apoptosis. The untreated tumor shows strong
PPARδ expression (A) and rare apoptotic cells (B). After treatment
with meta or para NO-ASA, tumors show decreased PPARδ expression
(C) and (E) and
increased apoptosis (D) and (F). Magnification is x400, adapted
from Ouyang et al. [7].Other NSAIDs such
as aspirin (of which NO-ASA is a derivative) have been reported to have PPARδ
as one of their molecular targets. In epithelial ovarian cancer cells, aspirin
suppressed PPARδ function and cell growth by inhibiting ERK1/2 [50]. Sulindac sulfide
and indomethacin inhibit both 14-3-3 proteins and PPARδ levels in HT29 cells,
suggesting that this could be the mechanism by which NSAIDs induce apoptosis
in colorectal cancer [79]. Furthermore, in
SW480 cells, sulindac sulfone significantly decreased PPARδ expression more
potently than the sulfide metabolite [80]. A case-control study in a large
population showed that a polymorphism
in the promoter of PPARδ
modified the protective effect
of NSAIDs on colorectal adenomas [81]. However, the opposite was observed
by another group, which found that regular NSAIDs use reduced the risk of
colorectal cancer, but none of the polymorphic genes studied, including PPARδ,
modified their protective effect [82].Several reports
present evidence that NSAIDs induce apoptosis independently of PPARδ. For
example, sulindac significantly inhibited chemically induced skin carcinogenesis
in both wild-type and PPARδ-null mice [83]. In addition, aspirin-induced
apoptosis in Jurkat cells was not mediated by PPARδ [84]. Aspirin at a concentration which
induces apoptosis did not affect the DNA binding of PPARδ, whereas neither
addition of a specific PPARδ ligand nor transient transfection of PPARδ
expression vectors protected Jurkat cells from aspirin-induced apoptosis. Finally,
as the work of Hollingshead et al.
presented above suggested, COX-2 inhibition by the NSAID nimesulide and PPARδ activation
during colon carcinogenesis occurred through independent mechanisms [62].


## 6. Conclusions and Future Directions

The contrast in
data that were reviewed here on the potential role of PPARδ in cancer, with
colon cancer being most extensively evaluated, could not be starker. Excellent
studies from fine laboratories led “conclusively" to diametrically opposite
results. As no grey zone seems to exist, the reader is left in bewilderment.Our data indicate that,
compared to wild-type mice, the nuclear receptor PPARδ is
overexpressed in the intestinal mucosa of Min mice, and that
two isomers of NO-ASA, which suppress their intestinal neoplasia,
inhibit to a commensurate degree the expression of PPARδ as well.
This effect is accompanied by the induction of epithelial cell death,
which correlates well with the antineoplastic effect of NO-ASA. As
discussed earlier, these findings are, however, limited by the fact that PPARδ was
detected using an antibody whose specificity may not be perfect and also by the
lack of any corroborating methodology (e.g., immunoblot detection of PPARδ levels).One could,
nevertheless, consider that these findings support the notion that PPARδ promotes
colon carcinogenesis. The key elements of support come from three findings.
First, PPARδ is overexpressed in the intestinal mucosa of the Min mice but not in the wild-type
control mice; being the same in histologically normal and neoplastic mucosas further suggests
that it has a role in early events of carcinogenesis. There is also specificity in the induction of PPARδ, as neither PPARα nor PPARγ was
induced. Second, PPARδ responds to two NO-ASA molecules that are structurally
identical except for their positional isomerism, proportionally to their
antitumor effect. And, third, changes in tumor response, PPARδ, and cytokinetic
parameters (apoptosis and necrosis) are closely correlated and mechanistically
congruent.Clarifying the role
of PPARδ in colon carcinogenesis and the response to medications is of
substantial interest. The mechanistic significance
of this question is apparent. The implications for the rational design of
therapeutic and/or preventive approaches are also clear. Finally, the fact that
PPARδ agonists may be used for other indications
raises the concern of unintended consequences of such modulation of PPARδ, which
may have a direct effect on the patient’s risk of colon and perhaps other cancers.At this stage, the
jury should be considered out on the role of PPARδ in cancer. As with any evolving field, the mundane but accurate
conclusion is that more work is needed to clarify such an important question.



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*Source: 919572-2008-05-29.xml*