# PPARγ and PPARδ as Modulators of Neoplasia and Cell Fate

Authors: Robert I. Glazer; Hongyan Yuan; Zhihui Xie; Yuzhi Yin
Journal: PPAR Research
 (2008)

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## Abstract

PPARγ and PPARδ agonists represent unique classes of drugs that act through their ability to modulate gene transcription associated with intermediary metabolism, differentiation, tumor suppression, and in some instances proliferation and cell adhesion. PPARγ agonists are used by millions of people each year to treat type 2 diabetes but may also find additional utility as relatively nontoxic potentiators of chemotherapy. PPARδ agonists produce complex actions as shown by their tumor promoting effects in rodents and their cholesterol-lowering action in dyslipidemias. There is now emerging evidence that PPARs regulate tumor suppressor genes and developmental pathways associated with transformation and cell fate determination. This review discusses the role of PPARγ and PPARδ agonists as modulators of these processes.


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## Body

## 1. Introduction

PPARγ and PPARδ are involved in cell cycle regulation,
survival and angiogenesis [1–3], and in inflammation through
ligand-dependent and independent mechanisms [4]. Several recent reviews have
described the role of PPARs in metabolic disease [4–6], cancer treatment [3, 7], and chemoprevention [8]. In addition to their
metabolic actions, an emerging area of investigation for PPARγ and PPARδ agonists is their ability to modulate mammary cell
lineage and genes associated with tumor suppressor function and cell fate
determination. This suggests that PPAR agonists may play a role in
stem/progenitor cell proliferation and differentiation to modify tumor response.


## 2. PPARγ Signaling

The PPAR nuclear receptor subfamily
consists of the PPARα, PPARγ, and PPARδ/β isotypes that regulate a number of
metabolic pathways controlling fatty acid β-oxidation, glucose utilization,
cholesterol transport, energy balance, and adipocyte differentiation [4–6]. PPARs function as heterodimeric partners with RXR, and
require high-affinity binding of PPAR ligand to engage transcription [7]. PPARs bind to the DR-1 response element (PPRE) consensus
sequence AGG(T/A)CA, which is recognized specifically by the PPAR partner [9]. Like other nuclear receptors, PPARs consist of  a putative N-terminal transactivation domain
(AF-1), a DNA-binding domain (DBD) containing two zinc fingers, a
ligand-binding domain (LBD) containing a large hydrophobic pocket, and a
C-terminal ligand-dependent transactivation region (AF-2) [10].There is >97% homology at the protein
level, 99% homology within the LBD, and minimal functional differences after
ligand-dependent activation between human and mouse PPARγ,  [11]. PPARγ is expressed predominantly in white adipose
tissue, intestine, endothelial cells, smooth muscle and macrophages [12], and is the major isotype expressed in the mammary gland, and
in primary and metastatic breast cancer and breast cancer cell lines [3].Several mutations and polymorphisms have been
identified in PPARγ, such as Lys319X (truncating) and Gln286Pro, in
sporadic colon cancer, which are associated with loss of DNA-binding and
ligand-dependent transcription by the PPARγ agonist, troglitazone [13].  Similar results were found for PPARγ2 polymorphism Pro112Ala [14], but the polymorphism
Ser114Ala resulted in increased transactivation by presumably blocking the
inhibitory effect of Ser114 phosphorylation by ERK [15, 16].  However, in a sampling of approximately 400
breast, prostate, colon, and lung tumors and leukemia's, no mutations of the
PPARγ gene were found, suggesting that if indeed
this does occur, it is a very rare event [17].In follicular thyroid cancer, the t(2;3)(q13;p25)
translocation results in formation of the Pax8-PPARγ fusion protein, which is pathoneumonic for the
majority of cases of this disease [18]. It acts as a
dominant-negative receptor of PPARγ  [18, 19], and reduces expression of the Ras tumor suppressor, NORE1A [20], which inhibits ERK
activation [21]. PPARγ also increases
expression of other tumor suppressor genes, such as PTEN [22] and BRCA1 [23] through their respective PPRE
promoter regions, suggesting that the antitumor effects of PPARγ agonists may be related to their ability to downregulate
multiple tumorigenic signaling pathways. This agrees with the reduction of PTEN
and increased nuclear β-catenin and ERK activity in the mammary gland
and tumors of MMTV-Pax8PPARγ mice [24] (see Figure 1).  Since inactivation of BRCA1 [25] and PTEN [26–28] also increases stem cell
proliferation, Pax8-PPARγ may upregulate specific progenitor cell lineages
that are more susceptible to tumorigenesis.Figure 1
Pax8PPARγ and mammary cell fate determination. Pax8PPARγ acts in a dominant-negative fashion to block PPARγ-dependent transactivation and upregulation of PTEN. MMTV-Pax8PPARγ mice exhibit reduced PTEN and activation of Ras and ERK, presumably through activation of PI3K (p85 and p110).  ERK activates ER transcriptionally and posttranslationally, and Pax8PPARγ may interfere with the ability of PPARγ to inhibit ER transactivation. Mammary epithelial cells isolated from the mammary glands of MMTV-Pax8PPARγ mice contain a higher percentage of CD24+/CD29hi stem/progenitor cells, and present with predominantly ER+ ductal carcinomas following carcinogenesis, suggesting a role of PPARγ in cell fate determination.PPARs interact with the coactivators C/EBP,
SRC-1, and DRIP205, and in the unliganded state with the corepressor SMRT [19, 29–31], and exhibit similar coactivator/corepressor dynamics as  other nuclear receptors, such as estrogen
receptor-α (ER) [32].  PPARγ can interfere with ER transactivation through
its binding to the ERE [33, 34], and preferentially partitions with ER for its canonical
response elements [35]; conversely, ER can block PPRE-dependent transcription [36] (see Figure 1). PPARγ also modifies ER signaling by promoting its
ubiquitination and degradation [37] as well as by upregulating CYP19A1 (aromatase) activity [38, 39], which can blunt the activity of aromatase inhibitors used
to treat patients with ER+ breast cancer.  PPARγ agonists block the ER-dependent growth of
leiomyoma cells, further suggesting crosstalk between the ER and PPARγ signaling pathways. PPARγ and ER pathways have opposite effects on
PI3K/AKT signaling that may also account for the inhibitory action of PPARγ ligands on ER-dependent breast cancer cells [36] (see Figure 1).
These findings imply that PPARγ antagonism should upregulate ER expression in
responsive tissues, which is precisely the phenotype observed in mammary tumors
induced in transgenic mice expressing Pax8PPARγ  [24].Studies using transgenic and knockout mouse models of PPARγ have led to disparate conclusions regarding
the role of PPARγ in tumorigenesis. Mice expressing
constitutively active VP16-PPARγ in the mammary gland did not exhibit a
tumorigenic phenotype but accelerated tumorigenesis when crossed with
MMTV-polyoma middle-T antigen mice [40], intimating that the
unliganded receptor may have interfered with tumor suppressor transactivation
by endogenous PPARγ through corepressor recruitment.  Alternatively, the VP16 fusion protein is
known to induce many genes that are not indicative of PPARγ activation [41]. In the probasin-SV40
T-antigen prostate tumor model, tumorigenesis was unaffected by a PPARγ null background [42], indicating that oncogenic
signaling was already maximally activated. However, in the ApcMin mouse colon tumor model, “glitazone” PPARγ agonists increased the number of colon, but
not small intestine polyps [43, 44], as well as colon adenomas [45].  Since the small intestine, and not the colon,
is the predominant site of neoplasia in this mouse model, the significance of
this observation is unclear. It should also be stressed that PPARγ agonists did not induce malignant changes in
wild type mice, indicating their lack of carcinogenicity. Contrary to these
results, PPARγ haploinsufficiency produced a greater rate and
number of colon tumors following azoxymethane-induced carcinogenesis [46], implying that PPARγ acts as a tumor suppressor rather than as an
oncogene.  APC+/1638N mice
heterozygous for PPARγ did not exhibit changes in polyp formation [46]. This result indicates that
the induction of β-catenin in the colonic crypt cells of PPARγ haplosufficient mice, a protumorigenic factor
that is constitutively activated in APC mice, is the target of tumor
suppression in wild-type mice [47]. A tumor suppressor role for
PPARγ is also supported by the inhibitory effect of PPARγ agonists on colon tumor growth [48, 49], and mammary carcinogenesis [50–52]. This effect may be mediated
in breast tumors through induction of apoptosis due to reduction of Bcl-2 [53], and in pancreatic and liver
tumors through a reduction of cyclin D1 and HB-EGF [54] and an increase of p27Kip1 [55–57]. PPARγ agonists may also find utility as modifiers of
the response to chemotherapy. CS-7017, a potent thiazolidinedione agonist,
synergized with paclitaxel to inhibit the growth of anaplastic thyroid tumors through
induction of p21Cip1 [58].  Notwithstanding possible “off-target” effects
[59, 60], most studies indicate that PPARγ agonists as a class have antitumor activity, and
thus may have efficacy as a relatively nontoxic adjunct to chemotherapy and
possibly to radiation therapy through their ability to act as “tumor suppressor
enhancers.”


## 3. PPARδ Signaling

As with PPARγ, PPARδ is involved in adipocyte
differentiation by promoting clonal expansion of preadipocyte progenitor cells [61], possibly through activation
of PPARγ expression [62]. The PPARδ agonist GW501516
has been tested clinically as a cholesterol lowering drug in dyslipidemic
patients, but the results have been mixed [63]. In animal models, homozygous
disruption of PPARδ resulted in a runted phenotype [64] and in  90% embryonic lethality with runted survivors [65], indicating its importance in
embryonic development.  PPARδ null
macrophages exhibited loss of the dominant inhibitory effect by unliganded
PPARδ [60], which was previously identified
by its ability to block PPARα and PPARγ transactivation through corepressor
recruitment [60, 66, 67]. In breast cancer cells, PPARδ
expression was greater in ER− MDA-MB-231 breast cancer cells than in
ER+ MCF-7 cells [68], also suggesting a correlation  with a more aggressive form of this disease. Indeed,
tissue microarray analysis of invasive
breast cancers indicated that PPARδ is strongly expressed (see Figure 2,
“+3”) in 52% of 164 samples, and thus may have value as a prognostic
marker and therapeutic target. There are no examples of the development of PPARδantagonists as anticancer therapeutics.PPARδ expression in invasive breast cancer. Representative samples from a tissue microarray analysis of invasive breast cancers are shown. PPARδ staining intensity is indicated as low (+1), medium (+2) or high (+3). The magnified image shows examples of +1 and +3 staining. The bar graph depicts the percentage of samples expressing PPARδ in DCIS, node (+), node (−) and node distant tumors.
(a)(b)GW501516 accelerated the onset of tumor formation during mammary
carcinogenesis, in contrast to the delay of tumor formation by PPARγ agonist GW7845 [52]. PPARδ expression increased
in K-Ras-transformed intestinal epithelial cells [69] and PDGF-stimulated vascular
smooth muscle cells [70]. Similar findings were
reported for conditional expression of PPARδ, where GW501516 increased
proliferation of hormone-dependent breast and prostate cancer cells and
endothelial cells, and increased expression of genes associated with proliferation
and angiogenesis [71]. PPARδ can suppress the
antiproliferative effects of PPARα and PPARγ [7] and directly associate with
PDK1 [52] to affect its localization
and activation [72, 73], which implicate it as a
protumorigenic factor, and therefore raise a caution for the general use of
this class of agonists [74].Colon cancer presents an interesting model to
exam the role of PPARδ in tumorigenesis since ApcMin  mice exhibit constitutive
activation of β-catenin/TCF signaling, the pathway believed to
activate PPARδ  [75]. PPARδ is highly expressed in
colorectal cancer cells [75], and somatic cell knockout of
PPARδ reduced tumorigenicity in nude mice [76]. Crossing PPARδ null or
heterozygous mice with ApcMin  mice showed a gene dosage dependent
reduction in large intestinal polyps [65], and treatment of ApcMin  mice with GW501516 produced an increase in both polyp number and size [77], all suggesting that PPARδ is protumorigenic.  However, a study using a different targeting
scheme to delete PPARδ reported no change in polyp number or size in the small
intestine of ApcMin  mice, and a  greater number but not size of carcinogen-induced
colon tumors in mice with this background [78]. Since the PPARδ knockout
mice generated by Barak contained a deletion of exon 4 encoding the hinge
region [65], whereas, that generated by Peters et al.
[64] contained a deletion of the
last exon encoding the AF2 domain, it is possible that the truncated PPARδ may not
be as susceptible to corepression as the wild-type receptor. This would explain
why their results [79, 80] differ from studies showing
that keratinocytes from mice heterozygous or null for PPARδ exhibit less
proliferation [81] and those in ApcMin  mice in a
PPAR null background exhibit increased tumorigenesis [65]. From a mechanistic
standpoint, PPARδ is activated in colon cancer cells by prostacyclin (PGI2)
[82] and inhibited by the NSAID
indomethacin [75], suggesting that its tumor
promoting action is related to inflammation, a condition that increases the
risk of colon cancer [83]. NSAIDs downregulate PPARδ and
reduce eicosanoid-mediated inflammation [84], and induce apoptosis in
colon cancer cells [85], in contradistinction to the anti-inflammatory
effects elicited by PPARγ agonists in colitis [86]. Increased expression of PPARδ
in tumors may also inhibit PPARγ transcription [60, 66, 67], and reduce its tumor
suppressor activity, as mentioned above in colon tumorigenesis.  In addition, the tumor promoting effects of
PPARδ in the mammary gland relate to activation of β-catenin/TCF signaling [76, 87]  (see Figure 3), which is increased in cells
transformed by PDK1 [88, 89].  PDK1 is a key regulator downstream of PI3K
that is increased by PPARδ in keratinocytes [72, 73].  Mammary tumors formed after administration of
GW501516 exhibit an association between PDK1 and PPARδ [52], which further suggests that PPARδ
may function as an integrator of proliferative and prosurvival pathways
downstream of oncogenic signaling and inflammation [90, 91], which are likely to account
for its tumor promoting effects.Figure 3
PDK1 and PPARδ autoregulatory cascade. Growth factor receptor (GFR) activation activates PDK1 leading to PKCα and β-catenin/TCF activation [88]. TCF target genes include cyclin D1, c-Myc, and PPARδ [75]. PPARδ transactivates PDK1 [72], which in turn perpetuates the oncogenic signaling cascade. Preliminary data suggests that PDK1 maintains the expression of the murine stem/progenitor cell marker, stem cell antigen-1 (Sca-1), which is under the control of AP1 and Stat1 [92].PPARs and stem cells
There is evidence
that PPARs can modulate stem and progenitor cell expansion and the differentiated
or malignant phenotype.  PPARγ agonists enhance adipocyte differentiation [5, 6], and its ability to
upregulate this process has a negative effect on osteoblast proliferation and
bone development from mesenchymal stem cells [93]. To counteract this
inhibitory effect in bone stem cells, PPARγ must be transrepressed through corepressor
recruitment by the NFκB and Wnt-5a pathways [94].  It is therefore likely that PPARs influence
the fate of other stem and progenitor cell populations in normal and malignant
tissues. PPARγ agonists have been used as chemopreventive
agents [8] to delay mammary
carcinogenesis [51, 52].  One aspect to their chemopreventive action
may relate to their influence on specific cell lineages, as in mesenchymal stem
cells.  Carcinogens target stem cells
rather than terminally differentiated cells [95, 96] as well as hormone-responsive
lineages [97] during mammary carcinogenesis.
Carcinogenesis is markedly attenuated in PR-null mice[98], and is accelerated by
progestin treatment of wild-type mice  [52, 99–101], where progestins are
believed to stimulate the proliferation of stem or early progenitor cells that are
intrinsically more susceptible to tumor initiation [102]. The ability of PPARγ and PPARδ agonists to modulate distinct cell lineages
during mammary tumorigenesis [52] also suggests that they
modulate a complex transcriptional network linked to cell fate [3, 5]. PPARδ agonist GW501516 promoted the development of
adenosquamous carcinomas with high expression of the stem cell markers CK19 and
Notch1, as well as Proliferin, a growth factor that mediates many of the
effects of the stem cell marker, Musashi1, in mammary cells [103]. PPARδ is expressed in the crypt cells of the small
intestine and negatively regulates Hedgehog signaling to block differentiation [104], a process that would be
expected to promote transformation. PPARδ expression lies downstream of
β-catenin/TCF [75], and activation of this
pathway increases expression of luminal epithelial and myoepithelial cells [102] as well as mammary tumor
cells expressing the stem cell marker Sca-1 [105].  Thus, PPARδ activation may promote expansion of a less
differentiated lineage or stem cells that is intrinsically more susceptible to
tumorigenesis. The association of Wnt activation with stem cell expansion,
activation of β-catenin/TCF signaling by PDK1, the identification of PPARδ as a
β-catenin/TCF target gene and PDK1 as a PPARδ responsive gene, as well as the
modulation of Sca-1+ stem/progenitor cells by the Wnt pathway, all
suggest a common mechanism for the tumor promoting action of PPARδ agonists
that may involve stem and progenitor cell proliferation (see Figure 3). This
mechanism also suggests that the development of PPARδ antagonists may have utility
as cancer therapeutics
PPARγ increases
expression of the PPRE-dependent tumor suppressor genes PTEN [22] and BRCA1 [23], suggesting that their
chemopreventive effects may be related to the ability of these suppressor genes
to promote a more differentiated lineage. On the contrary, inactivation of BRCA1 [25]  and PTEN [26–28] should increase stem cell
proliferation, which is precisely the case. This effect is similar to what has
been described for PPARδ agonists in preventing differentiation and
increasing stem cell abundance, and would be expected to complement their tumor
promoting activity. Although studies examining the influence of PPARs on cell
fate determination are just in their infancy, many of the studies cited imply
that their opposing roles in tumorigenesis may be related to their ability to
control the programming of specific cell lineages.


## 4. Conclusions

The ability of
PPAR agonists to modulate the transcriptional activity of this class of nuclear
receptors has generated an enormous interest in being able to pharmacologically
manipulate entire sets of genes that can modulate metabolism, inflammation,
transformation, differentiation and thus, tumorigenesis. Both genetic and pharmacological
approaches to determining the function of PPARγ and PPARδ have yielded some inconsistencies, but that
may be explained by the inherent deficiency of either approach. Gene targeting
resulting in a truncated gene product may not necessarily recapitulate gene
inactivation, and homozygous loss of gene expression can affect the
developmental programming of various tissues that can impact directly or
indirectly on the outcome of tumorigenesis in a particular organ. By the same
token, pharmacological approaches are fraught with the structure-specific and class-specific
side effects inherent in most drugs, which may be unrelated to their specific
actions on the drug target. Nevertheless, the majority of studies in this field
implicate PPARγ activation as an antitumorigenic and
prodifferentiation factor, in contrast to the protumorigenic and less
differentiated phenotype resulting from PPARδ activation. Although the latter characteristic
will likely preclude the clinical development of PPARδ agonists, it will be interesting to see the
outcome of current clinical trials utilizing PPARγ agonists as antitumor and chemotherapy
modulating therapy.



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