# A Role for PPARβ/δ in Tumor Stroma and Tumorigenesis

Authors: Rolf Müller; Martin Kömhoff; Jeffrey M. Peters; Sabine Müller-Brüsselbach
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a transcription factor that is activated by endogenous fatty acid ligands and by synthetic agonists. Its role in the regulation of skeletal muscle fatty acid catabolism, glucose homeostasis, and cellular differentiation has been established in multiple studies. On the contrary, a role for PPARβ/δ in tumorigenesis is less clear because there are contradictory reports in the literature. However, the majority of these studies have not examined the role of PPARβ/δ in the tumor stroma. Recent evidence suggests that stromal PPARβ/δ regulates tumor endothelial cell proliferation and promotes differentiation leading to the properly orchestrated events required for tumor blood vessel formation. This review briefly summarizes the significance of these studies that may provide clues to help explain the reported discrepancies in the literature regarding the role of PPARβ/δ in tumorigenesis.


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## Body

## 1. Introduction

Peroxisome proliferator-activated
receptor-β/δ (PPARβ/δ) is a transcription factor that is activated by lipid-derived ligands [1, 2]. Major functions of PPARβ/δ are associated with the regulation of intermediary metabolism, in
particular energy homeostasis, skeletal muscle lipid catabolism, and glucose
metabolism [3]. PPARβ/δ is also important in the control of inflammatory responses as it
modulates the function, proliferation, differentiation, and survival of immune
cells, notably macrophages and lymphocytes [4]. PPARβ/δ therefore represents a highly relevant drug target for the treatment of
major human diseases such as obesity, metabolic syndrome, inflammatory diseases,
and arteriosclerosis, which has led to the development of several synthetic
drug agonists displaying subtype selectivity and high-affinity binding [5].Mice lacking PPARβ/δ exhibit embryonic lethality due to aberrant development and malfunction
of the placenta, which is, however, modulated by the genetic background [6–8]. In line with these findings, differentiation
and metabolic function of trophoblast giant cells in vitro are dependent on
PPARβ/δ [8]. PPARb null mice also exhibit a defect in wound healing [9], and consistent with this observation, PPARβ/δ is critical for the AKT-mediated survival of keratinocytes during wound healing in skin [10]. However, in contrast to this prosurvival
pathway observed in skin wound healing, PPARβ/δ also stimulates keratinocyte terminal differentiation and inhibits
proliferation [6, 11–14], concomitant with a downregulation of protein
kinase C and MAP kinase signaling [15]. Differentiation of the digestive tract is also
regulated by PPARβ/δ, where it promotes the differentiation of Paneth cells in the
intestinal crypts by downregulating the hedgehog signaling pathway [16].


## 2. PPARβ/δ and Tumorigenesis

Consistent with its functional role
in differentiation and proliferation, PPARβ/δ inhibits chemically induced skin carcinogenesis as enhanced skin cancer is observed
in mice where PPARβ/δ has been deleted globally in all cells [17]. Since no difference in chemically
induced skin carcinogenesis is observed in mice when PPARβ/δ is deleted specifically in basal keratinocytes [18], this suggests that the protective effect of
PPARβ/δ in skin cancer may require functional roles in other cell types found
in skin. Enhanced tumor formation has also been observed in a mouse model of
Raf oncogene-induced lung adenoma formation, but the precise mechanisms and
cell types involved are not known [19]. In the Apc/Min mouse lacking
functional APC protein as well as in azoxymethane-induced intestinal
carcinogenesis, effects of PPARβ/δ have been described for tumor growth with different outcomes. For
example, one study reports that PPARβ/δ is dispensible for intestinal
tumorigenesis [7], while other studies suggest that PPARβ/δ attenuates colon cancer by regulating colonocyte terminal differentiation
[20–24].  Yet others suggest that PPARβ/δ potentiates colon cancer by promoting cell survival pathways [25–27]. The reason for these discrepancies, and thus
the precise function of PPARβ/δ in intestinal tumor cells, remains unclear at present [28]. Importantly, none of these studies addressed the issue as to whether
PPARβ/δ might play a role in cells of the tumor stoma, that is host cells recruited by
the tumor, such as endothelial cells (ECs), fibroblasts and macrophages [29], and would thus add another level of
complexity regarding the interpretation of results obtained with transgenic
tumor mouse models. Indeed, recent work suggests that
PPARβ/δ also has an essential function in the tumor stroma [30, 31], which is discussed in the following section.


## 3. A Role for PPARβ/δ in Tumor  Vascularization

Two recent studies showed that the growth of syngeneic tumors is impaired in mice lacking PPARβ/δ.
This was seen with two different subcutaneous tumor models, the Lewis lung carcinoma
(LLC1) and the B16F1 melanoma [30, 31]. Tumor growth was initially indistinguishable in Pparb+/+ and Pparb−/− mice, but
halted after approximately 2 weeks selectively in the Pparb−/− mice
(Figure 1), while the inoculated Pparb+/+ mice invariably succumbed to their tumors within 2-3 weeks, the Pparb−/− mice
exhibited a survival rate of >90% after six months. Histological analyses
showed that density of functional microvessels is diminished in LLC1 tumors in Pparb−/− mice [30, 31]. In contrast to tumors examined in Pparb+/+ mice, the majority of tumor microvessels in Pparb−/− mice exhibited a hyperplastic appearance typified by a thickened endothelial
lining and the lack of a lumen (Figure 2(a)). Consistent with this finding,
kinetic DCE-MRI analysis showed an obstructed tumor blood flow in the tumors
developing in the Pparb−/− mice [31]. These alterations were associated with a
striking increase in tumor endothelial cell proliferation in the absence of
PPARβ/δ expression  (Figure 2(b)), and
concomitant with this hyperproliferation, the immature ECs were surrounded by
perivascular cells expressing vast amounts of the myofibroblast marker α-smooth
muscle actin (Figure 2(c)), a picture that is characteristic of endothelial
hyperplasia. These observations strongly suggest that an abnormal organization
caused by a hyperplastic response, rather than a lack of ECs, underlies the
abundance of abnormal microvessels in Pparb−/− mice. This
is consistent with a large body of evidence demonstrating that PPARβ/δ
can inhibit cell proliferation in a number of different cell types [13, 24]. Importantly,
PPARβ/δ-dependent tumor vascularization was not restricted to ectopic tumor models, but was also
seen with intestinal adenomas in APC+/min⁡ mice which showed disorganized microvessels specifically in a Pparb−/− background (Figure 3). Collectively, these observations point to a general role
for PPARβ/δ in the formation or maintenance of tumor blood vessels.Figure 1
Growth of subcutaneous Lewis lung carcinoma (LLC1) in syngeneicPparb+/+
and Pparb−/− mice. Tumor sizes were determined at the times indicated
with a caliper. The calculated volumes are shown as mean ±SD [31].
*All tumor volumes <1000 mm3.(a) Aquaporin-1 immunostaining of endothelial cells and blood vessels in subcutaneous Lewis lung
carcinoma (LLC1) 14 days after inoculation intoPparb+/+ and Pparb−/− mice
(brown stain). Areas of tumor cell necrosis are obvious in the vicinity of the
aberrant vascular structures in Pparb−/− mice. (b)
PCNA (proliferating cell nuclear antigen) staining of an LLC1 tumor section
from a Pparb−/− mouse. The
red stain shows a high fraction of proliferating endothelial cells lining the
tumor microvascular structures (denoted by asterisks; 38.7% in Pparb−/− mice
versus 16.6% in Pparb+/+ mice) [31]. (c) Aquaporin-1/α-smooth
muscle actin double immunofluorescence of LLC1 tumors from Pparb−/− mice,
showing hallmarks of a hyperplastic stroma. Red: aquaporin-1, green: α-smooth
muscle actin.
(a)(b)(c)Figure 3
Analysis of microvessels in intestinal adenomas fromAPC+/min⁡ mice in
a PPARb+/+ or PPARb−/− background (31 ± 3 weeks
old mice) by aquaporin-1 immunostaining of paraffin sections (brown). Arrows point to normal microvessels
in tumors from PPARb+/+ mice, lacking in PPARb−/− mice.
Highly aberrant vascular structures lacking a lumen are seen specifically in Pparb−/− mice.Although a defect in angiogenesis has not been
observed during normal development ofPparb−/− mice [6–9], the findings discussed above are consistent
with previous findings pointing to a role for PPARβ/δ
in terminal differentiation and the control of cell proliferation in different
cell types, including keratinocytes [12, 14, 32, 33], trophoblast giant cells [8], and intestinal epithelial cells [16, 22]. This suggests that PPARβ/δ
is specifically required by tumor ECs to orchestrate their proliferation and
differentiation in an environment providing an abnormally rich source of growth
factors and cytokines. A role for PPARβ/δ
in tumor vascularization is also supported by several pieces of circumstantial
evidence: PPARb is the predominant PPAR subtype expressed in mouse and
human tumor endothelial cells, and it is upregulated by angiogenic growth
factors of the tumor microenvironment [30, 31].


## 4. PPARβ/δ Target Genes Relevant for  Stroma Cell Function

Microarray and qPCR analysis led to the
identification of a set of genes that are differentially expressed in an in vivo model of growth
factor-induced angiogenesis  (matrigel
plugs) fromPparb+/+ and Pparb−/− mice [31]. Consistent with the observed
hyperproliferative phenotype in Pparb−/− mice, three of these genes have known inhibitory functions in angiogenesis (Cd36, Thbs2) or cell cycle control (Cdkn1c) [34, 35]. Thrombospondins attenuate EC proliferation
and migrationin vitro and inhibit angiogenesis in vivo, which is strictly
dependent on their interaction with the CD36 receptor. In PPARb−/− cells, both ligand (Thbs2) and receptor(Cd36) genes are downregulated,
suggesting that an autocrine or paracrine signaling loop with an essential
function in modulating angiogenesis is impaired in these cells. Very little is
known about the intracellular events that occur after binding of thrombospondin
to CD36, so it is difficult to speculate at present about the CD36-triggered
signal transduction pathway(s) that is/are affected in ECs lacking PPARβ/δ
expression. The third gene identified as a PPARβ/δ
target gene in this context is Cdkn1c [31], which codes for the CIP/KIP family member p57KIP2 that it is likely to function as a
cyclin-dependent kinase inhibitor [34]. Thus, p57KIP2 would have a similar
effect on EC proliferation as CD36 and thrombospondin, suggesting that these
molecules may act in concert. It is likely that additional genes with functions
in growth control and differentiation will be identified as potential PPARβ/δ
target genes in the same experimental system, and it is likely that multiple
PPARβ/δ regulated genes are important in the context of tumor stroma development and
tumor angiogenesis.


## 5. Conclusions

The findings discussed above are consistent with a model where PPARβ/δ
is required to modulate the angiogenic response to growth factors during the
final stages of tumor angiogenesis, which is characterized by an inhibition of
EC proliferation and the acquisition of a fully differentiated phenotype [36]. The lack of PPARβ/δ
with the ensuing decreased expression of negative regulators of proliferation
may result in a deregulation of angiogenesis with the consequence of tumor
endothelial hyperplasia. A similar phenotype of enhanced, but nonproductive,
angiogenesis has very recently been described in mice lacking the Notch ligand
Delta-Like 4 (Dll4) [37, 38]. In contrast to PPARβ/δ,
however, Dll4 is essential not only for tumor angiogenesis but also for
embryonic vascular development and arteriogenesis [39], and there seems to be no cross-talk or
interaction between both the PPARβ/δ
and Notch/Dll4 pathways. This suggests that multiple and presumably mutually
independent regulatory mechanisms are required to prevent the deregulation of
tumor EC proliferation and the occurrence of nonproductive angiogenesis. The
current evidence suggests that PPARβ/δ is such a regulator.Previous studies addressing the role of PPARβ/δ
in tumorigenesis have yielded partly conflicting results leaving it unclear
whether PPARβ/δ has tumor-promoting or suppressing properties, in particular in colon cancer
models (reviewed in [28]). Our
findings provide some insight that may eventually help to resolve this issue.
PPARβ/δ may have different functions in tumor stroma and in certain tumor cells with
opposing effects on tumor growth. Clearly, a detailed understanding of these
complexities will be a prerequisite for the development of PPARβ/δ
directed drugs and their clinical application.



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