# PPARα Ligands as Antitumorigenic and Antiangiogenic Agents

Authors: Ambra Pozzi; Jorge H. Capdevila
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-activated transcription factors. This subfamily is composed of three members—PPARα, PPARδ, and PPARγ—that differ in their cell and tissue distribution as well as in their target genes. PPARα is abundantly expressed in liver, brown adipose tissue, kidney, intestine, heart, and skeletal muscle; and its ligands have been used to treat diseases such as obesity and diabetes. The recent finding that members of the PPAR family, including the PPARα, are expressed by tumor and endothelial cells together with the observation that PPAR ligands regulate cell growth, survival, migration, and invasion, suggested that PPARs also play a role in cancer. In this review, we focus on the contribution of PPARα to tumor and endothelial cell functions and provide compelling evidence that PPARα can be viewed as a new class of ligand activated tumor “suppressor” gene with antiangiogenic and antitumorigenic activities. Given that PPAR ligands are currently used in medicine as hypolipidemic drugs with excellent tolerance and limited toxicity, PPARα activation might offer a novel and potentially low-toxic approach for the treatment of tumor-associated angiogenesis and cancer.


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## Body

## 1. The Peroxisomal Proliferator-Activated Receptors (PPARs)

PPARs nuclear receptors
that regulate many physiological processes, including lipid and glucose
homeostasis, inflammation, and wound healing [1]. Three PPAR isotypes have
been identified: α, δ (or β), and γ.
Upon ligand binding, PPARs form heterodimers with the retinoic acid receptor
and interact with specific response elements in the promoter region of target
genes [2]. Although PPARs share
extensive structural homology, each isotype appears to possess distinct
functions. PPARγ
is expressed mainly in adipose tissue and at lower levels in intestine
and immune cells [3–5]. It controls adipocyte
differentiation, glucose and lipid homeostasis [5–7] and has been implicated in the
pathophysiology of insulin resistance and atherosclerosis [1, 8]. PPARγ
ligands include long-chain fatty acids, prostaglandins, and other
eicosanoids [4]. Among the synthetic PPARγ
ligands, the thiazolidinediones are currently used as insulin
sensitizers in patients with type-2 diabetes [9]. PPARδ
is ubiquitously expressed and it is most abundant in brain, colon, and
skin [10, 11], and binds molecules such as
fatty acids and prostaglandins [4].PPARα is primarily expressed in liver,
brown adipose tissue, kidney, intestine, heart, and skeletal muscle. This
receptor controls fatty acid metabolism and transport, peroxisomal and
mitochondrial β-oxidation [3, 4]. Moreover, this receptor has
been implicated in the pathophysiology of inflammation and cardiovascular diseases
[12]. Several compounds bind PPARα, including fatty and phytanic acids
[4], as well as the fibric acid
derivatives used in medicine for the treatment of hyperlipidemias [1].


## 2. PPARs and Cancer

The observation that members of
the PPAR family are expressed by tumor and endothelial cells [13, 14] together with the finding
that PPAR ligands regulate cell growth, survival, migration, and invasion [15, 16] prompted investigators to
determine whether these receptors play a role in the pathophysiology of
tumorigenesis and angiogenesis [17, 18].The
anticancer effects of PPARγ agonists have been extensively studied because of their antiproliferative, proapoptotic, antiapoptotic, and
differentiation-promoting activities [19]. In this context, activation
of PPARγ has been reported to reduce tumor cell
proliferation and invasion [20] and to enhance apoptosis
[21]. PPARγ ligands also regulate endothelial cell
growth, migration, and angiogenesis [22–25], and influence the
progression of vascular inflammation and tumorigenesis [26, 27]. Moreover, disruption of the PPARγ gene in the intestine enhances tumorigenesis in ApcMin/+ mice
[28].  Although these studies suggest that PPARγ functions as a tumor suppressor factor
and its activation might be beneficial
for patients with tumors, PPARγ agonists have been shown also to
increase the frequency of colon tumors [29] and to promote edema
[30].In contrast to PPARγ,
PPARδ
has been described as protumorigenic as its ligand-mediated activation increases
tumor-associated angiogenesis [31].  Moreover, treatment of ApcMin/+ mice with PPARδ antagonists or crossing these mice with
PPARδ-null mice prevents tumor growth and
angiogenesis [31]. However, a recent study showed
that activation of this receptor attenuates chemically-induced colon
carcinogenesis, and that PPARδ-null
mice exhibit increased colon polyp multiplicity, suggesting that ligand
activation of this receptor can also inhibit carcinogenesis [32].The analysis of the antitumorigenic
properties of PPARα ligands has been less studied mostly due
to the observation that long-term administration of certain PPARα agonists (Clofibrateand
WY14643) induces hepatocarcinogenesis in rodents [33–35], despite the fact that PPARα ligands are widely used in medicine as
antilipidemic drugs with excellent tolerance and little or no reported side
effects. The finding that fenofibrate decreases VEGF levels in
patients with hyperlipidemiaand atherosclerosis [36] provided a rationale for analyzing
PPARα and its ligands as a molecular target
for cancer therapy. In this review, we highlight some of the key functions
attributed to PPARα in the context of endothelial and tumor
cell biology.


## 3. PPARα Targets in Angiogenesis

PPARα controls the transcription of many
genes involved in cell functions such as lipid metabolisms, inflammation, cell
cycle progression, and angiogenesis. Among the angiogenic targets, PPARα has been shown to regulate the
expression of the vascular endothelial growth factor (VEGF), fibroblast growth
factors (FGFs), members of the arachidonic acid P450 monooxygenases,
thrombospondin and endostatin to name few (see also Figure 1 and Table
1). Biscetti
et al. have recently shown that the selective PPARα agonist WY14643 promotes cornea
angiogenesis in vivo and
enhances endothelial tubulogenesis in
vitro [37]. Interestingly, WY14643 can
enhance endothelial cell tubulogenesis in
vitro only when endothelial cells are cocultured with interstitial cells
and this effect is accompanied by upregulation of interstitial-derived VEGF
synthesis [37].  However, WY14643 does not directly promote endothelial
cell migration or proliferation, and when used at 10–20 μM range it reduces both endothelial cell
proliferation and migration [37]. Thus, this study indicates
that while WY14643 might directly prevent endothelial cell functions, it might
also promote angiogenesis by stimulating the production of nonendothelial VEGF.
The observation that activation of PPARα prevents endothelial cell
proliferation/migration parallels our findings that WY14643 prevents—in a PPARα-dependent fashion—endothelial cell
proliferation in vitro and
tumorigenesis in vivo [38]. The antiangiogenic
properties of WY14643 are associated   with a PPARα-dependent downregulation of the epoxygenase
branch of the cytochrome P450 arachidonic acid monooxygenases
[38]. The arachidonic acid epoxygenases
are expressed by endothelial cells both in
vitro and in vivo [39–41] and catalyze the oxidation of
arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs)
[42, 43]. EETs have been shown to
possess proangiogenic activities [39, 44–47] and we have demonstrated that
WY14643-mediated PPARα activation directly prevents
endothelial cell migration and proliferation by downregulating endothelial
arachidonate epoxygenase expression and EET biosynthesis [38]. Most importantly, in vivo treatment with WY14643
prevents primary tumor growth and tumor-associated angiogenesis by
downregulating the levels of circulating EETs [38].Table 1
Effect of PPARα activation on angiogenesis and
tumorigenesis.
LigandCell typeEffectTargetReferenceWY14643Endothelial cellsInhibition of cell proliferation
and tubulogenesis in vitro  Antiangiogenic activity in vivoDownregulation of arachidonate epoxygenase synthesis[38]WY14643Endothelial cellsEnhanced endothelial tube formation in vitro
Proangiogenic activity in vivoUpregulation of VEGF production[37]Fenofibrate
WY14643 ETYAEndothelial cellsInhibition of VEGF- or FGF2-mediated cell proliferation in vitro
Antiangiogenic activity in
vivoDownregulation of VEGF production
Upregulation of thrombospondin and endostatin production[48]Fenofibrate WY14643Endothelial cellsReduced cell migrationInhibition of Akt activation[24]FenofibrateEndothelial cellsReduced cell proliferationInhibition of cyclooxygenase-2 expression[25]FenofibrateEndothelial cellsReduced cell proliferationInhibition of VEGFR2 expression[23]ClofibrateOvarian cancer cellsReduced cell proliferation in
vitro Antitumorigenic activity in
vivoReduced prostanoid and VEGF levels via upregulation of carbonyl
reductase expression[49]MethylclofenapateColonic adenocarcinomaReduced cell proliferationNot investigated[50]MethylclofenapateApcMin/+ miceReduced number of intestinal polypsNot investigated[50]BezafibrateAPC1309mice  ApcMin/+ miceReduced number of intestinal polypsReduced serum level of triglycerides and increased
lipoprotein lipase synthesis[27, 51]WY14643Wild-type miceEnhanced hepatocellular proliferation and tumorigenesis in vivoDownregulation of the miRNA let-7C with increased c-myc expression[52]Figure 1
Schematic representation of the antiangiogenic and antitumorigenic
properties of PPARα. PPARα ligands reduce tumor growth by direct
inhibition of tumor cell functions (black pathway). In addition, they prevent
tumor-associated angiogenesis via direct (red pathway) as well as indirect
(green pathway) inhibition of endothelial cell functions.Consistent
with the observation that PPARα ligands might act as potent direct
and/or indirect antiangiogenic factors, Panigrahy et al. have recently shown
that fenofibrate suppresses VEGF-mediated endothelial cell proliferation as
well as tumor cell-derived VEGF and FGF2 synthesis with concomitant stimulation
of tumor-cells derived thrombospondin and endostatin [48]. Moreover, fenofibrate  and
WY14643 prevent
VEGF-mediated endothelial cell migration by inhibiting Akt phosphorylation
[24] and fenofibrate prevents
endothelial cell proliferation by inhibiting cyclooxygenase-2 expression
[25]. Finally, PPARα agonists were found to inhibit
endothelial VEGFR2 expression by preventing Sp1-dependent promoter binding and
transactivation [23]. Some of the major PPARα targets known to control endothelial
cell functions and the effects of PPARα ligands on angiogenesis are summarized
in Figure 1 and Table 1.In
conclusions these studies strongly suggest that by preventing endothelial cell
functions PPARα ligands may protect the vasculature
from pathological alterations associated with either metabolic disorders (i.e.,
atherosclerosis, diabetes) or cancer. Thus, PPARα can be considered as a new class of “antiangiogenic”
gene, and suggest that its ligands may function as effective antiangiogenic drugs.


## 4. PPARα Targets in Cancer

The
observation that PPARα is expressed by tumor cells [59–61] started studies of the role of this
nuclear receptor and its
ligands on the prevention of tumor cell proliferation in vitro and in vivo.  In this context it has been shown that PPARα ligands suppress the growth of
several cancer lines—including colon, liver,
breast, endometrial, and skin—in vitro [62–66], as we
all inhibit the metastatic potential of melanoma cells in vitro and in vivo [67, 68].
Furthermore, PPARα  ligands decrease colon carcinogenesis [62] and the growth of human
ovarian cancer in mice [49].  Although the mechanisms whereby PPARα directly prevents tumor cell functions have
not been investigated in details, potential targets have been identified. Clofibrate,
a PPARα ligand, significantly suppressed the
growth of OVCAR-3 xenotransplanted tumors and inhibited ovarian tumor cell
proliferation by increasing the expression of carbonyl reductase, an enzyme that
promotes the conversion of protumorigenic prostaglandin E2 to inactive PGF2α [49]. Moreover, clofibrate reduced
the levels of circulating VEGF in tumor-bearing mice [49], while bezafibrate, another
PPARα   ligand, decreased the number of
intestinal polyps in ApcMin/+ mice possibly by lowering serum level
of triglycerides and upregulating lipoprotein lipase synthesis
[27, 51]. Finally, PPARα activation has been shown to inhibit
vascular smooth muscle cell proliferation underlying intimal hyperplasia by
inducing the expression of the tumor suppressor p16INK4a [56].Whereas
these studies clearly suggest that PPARα activation might be beneficial in reducing
cancer growth, studies from the Gonzales laboratory demonstrate that long-term administration
of certain PPARα agonists (clofibrate and
WY14643) induces liver adenoma and carcinomas in rats and mice [35, 52, 69, 70]. The ability of PPARα ligands to induce hepatocarcinoma is
PPARα-dependent and mediated by the novel microRNA
let-7C/c-myc axis [52]. Let-7C is a micro RNA that
controls cell growth by directly downregulating c-myc expression [52]. Upon treatment of mice with
WY14643, the hepatic expression of let-7C decreases with the concomitant
induction of c-myc and the increased expression of the oncogenic mir-17-92
cluster [52]. Thus, this novel rodent
specific PPARα-regulated pathway might be responsible
for increased hepatocellular proliferation and tumorigenesis.All
together, these findings
indicate that, with
few exceptions, PPARα ligands can be viewed as
antitumorigenic agents either by directly preventing tumor cell functions or by
preventing tumor-derived production of proangiogenic molecules. Some of the
potential PPARα targets that control tumor cell
functions and the effects of PPARα ligands on tumorigenesis are summarized
in Figure 1 and Table 1.


## 5. PPARα Ligands and Tumorigenesis: Lessons from Mice

The
generation of PPARα null mice has provided an excellent
tool not only to determine whether the effects exerted by PPARα ligand are indeed PPARα-dependent, but also for discerning
between host versus tumor-mediated PPARα responses (see Table 2 for details). In
this regard, we have shown that wild-type mice injected with isogenic PPARα expressing tumor cells respond to WY14643
treatment and develop fewer and smaller tumors than untreated wild-type mice
[38]. In contrast, the growth of
the same tumor cells is not prevented in WY14643-treated PPARα null mice
[38]. In agreement with our finding,
absence of PPARα in the host animals abrogated the
potent antitumor effect of fenofibrate [48]. Finally whereas in vivo activation of PPARα prevents vascular smooth muscle cell
proliferation underlying intimal hyperplasia, PPARα deficiency leads to hyperplasia
[56]. Taken together, these
results strongly suggest that activation of PPARα in the host is a key element in preventing
unwanted pathological cell growth.Table 2
PPARα and tumorigenesis: lessons from the
PPARα-null mice.
LigandHostChallengeEffectTargetReferenceWY14643PPARα-null miceResistant to the development of spontaneous hepatocarcinomaInability to downregulate the miRNA let-7C[52]WY14643  FenofibratePPARα-humanized transgenic mouseResistant to the development of spontaneous hepatocarcinomaInability to downregulate the microRNA let-7C[53, 54]WY14643PPARα-null miceInjection of isogenic tumor cellsResistant to the Wyeth-mediated antiangiogenic and
antitumorigenic activitiesInability to downregulate arachidonate epoxygenase
expression[38]DEHPPPARα-null miceDevelopment of hepatocarcinomaIncreased PPARα-independent oxidative stress[55]WY14643
FenofibratePPARα-null miceCarotid
arterial injuryIntimal hyperplasiaInability to induce the expression of the tumor suppressor
p16INK4a[56]PPARα-null miceInjection of isogenic tumor cellsResistant to the development of primary and metastatic
tumor growthIncreased recruitment of granulocyte responsible for
thrombospondin production[57]PPARα-null miceIncreased susceptibility to spontaneous adenomas and hepatocellular
carcinomasNot explored[58]Although
rodents are the only species in which activation of PPARα promotes liver cancer, for a long time
it was thought that Di(2-ethylhexyl)phthalate (DEHP), a commonly used
industrial plasticizer, might cause liver tumorigenesis presumably via
activation of PPARα [55, 71]. The use of PPARα null mice has disproved this idea, as
this plasticizer is able to induce tumorigenesis in both wild-type and PPARα-null mice
[55, 71]. These results suggest the
existence of pathways for DEHP-induced hepatic tumorigenesis that are
independent of PPARα, but most likely dependent on DEHP-mediated
oxidative stress [55].PPARα null mice have been also instrumental
to determine the role of rodent versus human PPARα in the promotion of liver
carcinogenesis. Morimura et al. have generated a PPARα-humanized mouse in which the human PPARα is expressed in liver under control of
the Tet-OFF system. Interestingly, prolonged exposure to WY14643 in these mice
only led to a 5% incidence of liver tumors—including
hepatocellular carcinoma—compared to the
71% observed in mice expressing the mouse PPARα [53]. More recently, Yanget al. generated a PPARα-humanized transgenic mouse where the
complete human PPARα gene was introduced onto a PPARα-null background [54]. These PPARα-humanized mice express the human PPARα in liver as well as other tissues and
respond to fenofibrate treatment by lowering serum triglycerides and by
inducing the expression of enzymes involved in fatty acid metabolism [54]. However, in contrast to wild-type
mice, treatment with fenofibrate did not cause significant hepatomegaly, hepatocyte
proliferation, and most importantly hepatocarcinoma [54]. Thus, this study shows that
the protumorigenic let-7C/c-myc pathway is activated only by the rodent, but
not the human PPARα receptor. Most importantly, this work highlights
the possibility that PPARα ligands might be used as safe drugs for
the treatment of cancer in humans.Although
activation of PPARα in either endothelial or tumor cells has
been proven to be beneficial in inhibiting cancer growth, it has also been
shown that loss of host-derived PPARα can be advantageous as it prevents
tumor growth and development [57]. The host cells responsible
for this protection, however, are granulocytes rather than endothelial cells. Loss
of PPARα leads to an increased infiltration to
the side of injury of granulocytes that suppress tumor-associated angiogenesis
via excess production of the endogenous angiogenesis inhibitor thrombospondin [57].  This study clearly indicates that both
activation of PPARα in specific host cells (i.e., endothelia
cells) and concomitant inhibition of PPARα in immuno cells (i.e., granulocytes) might
lead to the same effects, namely protection from tumor growth.


## 6. Conclusions

The
studies summarized in this review identify PPARα as a potential host-based target for
the development of new antiangiogenic approaches to inhibit and/or prevent
tumor growth. As an established modulator of gene transcription, PPARα regulates the expression of genes known
to be involved in energy metabolism, cellular proliferation, and angiogenesis
and to have positive effects on the control of dyslipidemia, inflammation, and
cardiovascular diseases. Furthermore, several fibric acid derivatives bind to
and activate human PPARα with limited or no documented unwanted
consequences and have proven to be safe and effective hypolipidemic drugs. In
this context, gemfibrozil safely reduced the risk of death from coronary heart
disease, nonfatal myocardial infarction, or stroke by raising HDL
cholesterol levels and lowering levels of triglycerides [72, 73].The
effects of PPARα ligands in animal models of tumor
angiogenesis should help not only to stimulate further research of their
usefulness as antitumorigenic agents, but also to facilitate their evaluation
as valid tools for the treatment and/or prevention of human cancers. In this
context, it is our hope that these studies will serve to encourage epidemiological
studies of cancer incidence in patients using hypolipidemic drugs, and help to
identify their potential beneficial effects as agents for tumor prevention
and/or treatment. The urgency of new approaches for cancer treatment are
indicated by the fact that most current antitumorigenic therapies are oriented
towards a general inhibition of tumor cell growth and, as such, they suffer
from lacking target selectivity and, in most cases, causing severe side effects
and overall systemic toxicity. Thus, targeting PPARα may prove to be a potential therapeutic
strategy—either alone or
in combination with conventional chemotherapy—to inhibit and
ideally prevent cancer with excellent tolerance and limited toxicity.



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