# PPARγ and Agonists against Cancer: Rational Design of Complementation Treatments

Authors: Dorina Veliceasa; Frank Thilo Schulze-Hoëpfner; Olga V. Volpert
Journal: PPAR Research
 (2008)

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## Abstract

PPARγ is a member of the ligand-activated nuclear receptor superfamily: its ligands act as insulin sensitizers and some are approved for the treatment of metabolic disorders in humans. PPARγ has pleiotropic effects on survival and proliferation of multiple cell types, including cancer cells, and is now subject of intensive preclinical cancer research. Studies of the recent decade highlighted PPARγ role as a potential modulator of angiogenesis in vitro and in vivo. These observations provide an additional facet to the PPARγ image as potential anticancer drug. Currently PPARγ is regarded as an important target for the therapies against angiogenesis-dependent pathological states including cancer and vascular complications of diabetes. Some of the studies, however, identify pro-angiogenic and tumor-promoting effects of PPARγ and its ligands pointing out the need for further studies. Below, we summarize current knowledge of PPARγ regulatory mechanisms and molecular targets, and discuss ways to maximize the beneficial activity of the PPARγ agonists.


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## Body

## 1. Introduction

PPARs
are nuclear hormone receptors and targets for the compounds inducing peroxisome proliferation.
The family encompasses three species, PPARα, PPARβ/δ, and PPARγ. PPARγ, the best researched of the three, is
presented by the two isoforms, γ1 and γ2 whereas PPARγ2 contains 30 extra amino acids at the
N-terminus due to initiation from the alternative transcription start (see Figure 1(a)). PPARγ, a key player in adipocyte
differentiation and glucose metabolism, is abundantly expressed in adipose
tissues [1]. On the other hand, it is
expressed in all the cells of the normal and pathological vascular beds, including
endothelial cells (EC), macrophages (MΦ), and vascular smooth muscle cells
(VSMCs), in a variety of tumor cells, and, at lower
levels, in lymphatic tissue, intestinal
epithelium, retina, and skeletal muscle [2]. PPARγ is a potent modulator of the EC and VSMC
function and inflammation: its effects on the tumor cells, tumor-associated MΦs (TAM), and tumor vasculature (EC and
VSMCs) significantly attenuate tumor progression [3, 4], suggesting that PPARγ ligands may become new convenient
therapeutic modifiers targeting simultaneously tumors and their microenvironment
[5]. Unfortunately, recent
studies reveal the tumor-promoting and pro-angiogenic PPARγ activities; while in most cases PPARγ agonists attenuate tumor growth and angiogenesis,
troglitazone (TGZ, a now rejected PPARγ agonist) promotes hepatic
carcinogenesis and liposarcomas. Moreover, some PPARγ agonists promote the differentiation of
the circulating endothelial progenitor cells (EPC) [6] and elicit angiogenesis in
vivo [7]. In some instances, PPARγ ligands increase the production of
angiogenic stimuli, including VEGF or NO, by the EC or tumor cells [8]. Thus, the use of PPARγ modulators to manage tumor progression
is more complex than it
appears at a glance and requires precise knowledge of the molecular events
involved in their pro- and antitumorigenic actions. Below we summarize the current
knowledge of PPARγ effects and molecular mechanisms and delineate
ways to augment PPARγ anti-angiogenic and antitumor effects
while minimizing its pro-angiogenic and tumor-promoting capacities.PPARγ  structure and regulation. (a) Schematic representation of the domain structure of the PPARγ-1 and PPARγ-2. The mutations associated with
metabolic syndrome are indicated. LF: loss of function; GF: gain of function. (b) Positive and negative regulators of the PPARγ gene transcription. (c) The
regulation of PPARγ levels by Rb and E2F. (d) The
mechanism of ligand-dependent PPARγ activation. (e) The regulation
of PPARγ activity by MEK and Erk kinases: MEK1
activates Erk-1/2, which phosphorylates
PPARγ and targets it to proteasomes; in addition, MEK1 binds
PPARγ in the nucleus and exports it to the
cytoplasm. MEK5 can serve as coactivator for the PPARγ.
(a)(b)(c)(d)(e)


## 2. PPARγ and Angiogenesis

Angiogenesis
is a complex process involving diverse cell types and controled by the pro- and
anti-angiogenic factors produced by the ECs, VSMCs, and in vascular microenvironment
by the stromal, tumor, and inflammatory cells. The balance between positive and
negative angiogenesis regulators determines if the existing capillaries would expand,
regress, or remain quiescent [9]. Active angiogenesis involves
invasion, migration, and proliferation of the EC followed by the morphogenesis
(assembly) of the neovessels. It is aided by the recruitment of the EPCs, which
may constitute up to 50% of the cells in a neovessel  [10]. The newly formed capillaries
recruit vascular smooth muscle cells (VSMCs), which stabilize and render quiescent
the newly formed capillaries: in thus stabilized mature vessels, the
interactions between angiopoietin-1 (Ang-1) on the EC and Tie-2 receptor on the
VSMCs generate signals that dampen EC sensitivity to the pro- and anti-angiogenic
molecules [11]. Brown adipose tissue, a
thermogenic organ in mammals responds to cold by increasing VEGF, thus creating
permissive conditions for the fat expansion. Treatment of brown adipocytes with
PPARγ ligands reduces VEGF-C mRNA pointing to
their anti-angiogenic potential [12]. Moreover, chimeric mice null
for PPARγ show gross defects in placental
vascularization [13]. Natural and synthetic PPARγ ligands block VEGF-driven angiogenesis
in vivo, in matrigel implants, in rodent cornea, and choroid [14–16]. RGZ suppresses the growth
and angiogenesis of the glioblastoma, Lewis lung carcinoma, liposarcoma, and rhabdomyosarcoma in mouse models [17], which is partly due to the
PPARγ-mediated apoptosis of the tumor EC and
the repression of VEGF production by the tumor cells. Below, we elucidate the
PPARγ pleiotropic effects on angiogenesis and
suggest optimization strategies.


## 3. PPARγ Regulatory Mechanisms

PPARγ can be regulated at expression level: PPARγ gene is repressed by the GATA-2 and 3,
TCF4 [18] (see Figure 1(b)), and
transactivated by CAAT enhancer binding proteins (C/EBPs), predominantly C/EBPα, ADD1/SREBP1, and E2F1 (see Figure 1(b))
[19]. E2F proteins have dual
effect on PPARγ expression: during cell cycle progression,
phospho-Rb releases E2F1 to activate PPARγ promoter (see Figure 1(c)), however,
E2F4, if bound to the p103 or p130 Rb, represses PPARγ transcription [2, 18]. Moreover, hypo-phosphorylated
Rb binds PPARγ and recruits histone deacetylase (HDAC)
3 to the complexes, causing transcriptional repression (see Figure 1(c)) [19]. Multiple growth factors including
platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), angiotensin
II, tumor necrosis factor (TNF) α, interleukin (IL) 1β, and  tumor-derived growth factor β(TGF-β) increase PPARγ expression by the vascular smooth
muscle cells (VSMCs), via Egr-1. In contrast, AP-1 aided by Smad3/4 represses
PPARγ promoter activity [20]. Mitotic, stress, and
inflammatory signals cause PPARγ degradation via phosphorylation on Ser84
of the mouse PPARγ
(Ser112 of the human molecule) in a consensus MAPK target motif PXSPP [21] by  ERKs, JNKs, and p38, which leads to ubiquitination
and proteasomal clearance [22]. Ser to Ala PPARγ mutant shows increased transcriptional
activity, similar effect is caused by coexpression of a phosphoprotein
phosphatase [21].  In human PPARγ, substitution of proline to glutamine
at position 115 results in constitutive activation by blocking MAPK
phosphorylation at position 114: patients with such mutation display extreme
obesity [23]. Likewise, increased
phosphorylation on Ser112 in Dok-1 null mice caused lean phenotype, which is
lost in mice expressing phosphorylation-defective PPARγ [24].
The effect of PPARγ on angiogenesis remains to be
determined.The
next regulatory step involves cofactor recruitment: upon ligand binding, PPARγ forms heterodimers with the retinoic
acid X receptor (RXR), and occupies twin PPAR response elements AAGGTCAnAAGGTCA
(PPRE); binding of the RXR ligands further increases transcriptional activity
of the PPARγ/RXR dimers (see Figure 1(d)). Coactivators
including SRC1, CBP/p300, pCAF/GCN, and PGC bind PPARγ/RXR complexes in a ligand-dependent
manner [19]; PGC-1α has recently been linked to HIF-independent
induction of vascular endothelial growth factor (VEGF) and angiogenesis [25].  PPARγ activity can be  suppressed due to phosphorylation, which
results in nuclear export, both executed by MEK-1 (see Figure 1(e)) [26]. In contrast, MEK-5 acts as
PPARγ coactivator (see Figure 1(e)) [27].In
addition to its activator function (see Figure1(d)), PPARγ represses transcription of select genes.
PPARγ transrepression of AP-1, nuclear
factor of the activated T-cells (NFAT), NFκB, and STAT-1 is well documented [19, 28]. Typical PPARγ corepressors SMRT and NCoR corecruit
HDAC3, transducin beta-like protein-1 (TBL-1) and TBL-1-related protein 1
(TBLR1) [29]. The repression can be ligand-independent,
with PPAR/RXR dimers forming repressor complexes in the absence of the ligands (see
Figure 2(a)). Ligand-dependent repression may occur by direct interaction with target
transcription factors (see Figure 2(b)), modulation of the transcriptional
regulators (see Figures 2(c) and 2(d)), by coactivator sequestration (see Figure
2(e)), or the blockade of corepressor clearance (see Figure 2(f)). The latter requires
PPARγ  sumoylation, which keeps HDAC3 associated with
repressor complexes and prevents proteasomal clearance of their components [19]. NCoR complexes interact with
a limited subset of promoters, which explains gene-specific repression by PPARγ.Mechanisms of
transrepression by PPARγ. (a) Ligand-independent
repression: preferential recruitment of corepressors in the absence of
agonists. (b) Direct binding and sequestration of transcription factors
on example of NFκB. (c) Activation of genes
encoding inhibitors of transcription factor (e.g., NFκB inhibitor, IκBα). (d) Direct binding and
inactivation of kinases, which activate transcription factors (e.g., the
blockade of JNK activation of cJun). (e) Competitive binding of the
coactivator complex. (f) The blockade of corepressor clearance:
sumoylated PPARγ stabilizes corepressor complexes (NCoR,
Tab2, and TBL1) on the promoter and facilitates the recruitment of HDAC3. In
the absence of sumoylation, NCoR, Tab2, and TBL1 are subject to ubiquitination
and proteasomal clearance.
(a)(b)(c)(d)(e)(f)


## 4. Ligands

PPARγ ligands encompass wide range of
structurally diverse compounds, natural and synthetic. Natural ones include
long chain polyunsaturated fatty acids and derivatives (eicosanoids, prostaglandins, like
15-deoxy-Δ12,14-prostaglandin J2 (15D-PGJ2)) and nitrolinoleic acids. Synthetic ones include thiazolinediones
(TZDs, or glitazones), of which rosiglitazone (RGZ) and pioglitazone (PGZ) are
marketed for the treatment of type 2 diabetes and tyrosine-based derivatives (glitazars)
including tesaglitazar
and farglitazar, the dual agonists of PPARα and PPARγ [30].
Although their ability to alleviate insulin resistance, vascular complications,
and angiogenesis is well documented, the adverse effects include hepatotoxicity, renal toxicity, weight gain, and fluid retention [30], all of which complicate the
long-term use. Thus further work is required to develop PPARγ ligands into safe and efficacious
treatment for diabetes, cancer, and angiogenesis-related disease. Selective PPARγ modulators (SPPARMs) represent one way
to overcome this problem: they are designed to retain the desired PPARγ properties, while minimizing adverse
side effects. SPPARMs can be categorized as tightly binding partial agonists
(GW0072) or weakly binding full agonists of PPARγ (MCC-555/netoglitazone, NC-2100) [31].


## 5. Anti-Angiogenic Effects of PPARγ in Diverse Cell Types: Endothelial-Specific Events

Human micro- and macrovascular endothelial cells (EC) express PPARγ [32]. PPARγ activation by the natural (15D-PGJ2)
or synthetic ligands (TGZ, RGZ, ciglitazone, and pioglitazone) potently inhibits
in vitro proliferation and morphogenesis by EC of diverse tissue origin [33]. 15D-PGJ2 and
ciglitazone (CGZ) also induce EC apoptosis through PPARγ-dependent pathway. The PPARγ involvement is supported by (1) nuclear
translocation, (2) increased transcriptional activity, (3) attenuation of the EC
apoptosis by the decoy PPRE oligonucleotide, and (4) increased background
apoptosis in PPARγ overexpressing EC, further enhanced by
the ligand exposure [15]. PPARγ activation interferes with EC migration:
TZDs block EC chemotaxis up the VEGF or leptin gradients, by blocking PI3K/Akt and
Erk1/2 signaling
[34–37]. In both cases, PPARγ/SREBP1 complex drives the transcription
of PTEN tumor suppressor, which opposes the induction of Akt [38], see Figure 4(a).PPARγ ligands hamper the response of the
vascular EC to VEGF by lowering VEGFR1 (Flt-1) and VEGFR2 (KDR). The regulation
of VEGFR2 is biphasic: in the absence of the ligands, PPARγ enhances Sp1/Sp3 binding to the
promoter and opposes it if ligands are present [39]. VEGFR2 decrease also reduces
EC survival under stress or in the presence of anti-angiogenic factors, see Figure 4(a).PPARγ induction decreases UPA and increases
PAI-1 expression by the EC, thus lowering their ability to invade surrounding
tissues [14, 16]. In the brain microvasculature,
PPARγ stimulation dampens the activation of RhoA
and  Rac1 GTPases critical for the cell adhesion
and migration [40], see Figure 4(a).Proapoptotic
PPARγ effects in the EC can be mediated by
p53 [41–43] or by the opening of Maxi-K
channel (Ca2+ activated K+ channel) whereas the protective Bcl-2 levels plummet
and apoptotic Bax increases. In addition, increased eNos production causes
elevated NO, which, in contrast with its usual protective effect contributes to
EC death [44]. Downmodulation of the
thioredoxin (Trx-1) by PPARγ via vitamin D3 upregulated protein
(VDUP-1) also contributes to the EC killing, likely via formation of inactive
PTEN/Trx-1 complexes [45]. PPARγ also ameliorates EC activation by
glucose via the induction of diacylglycerol kinase (DGK), the reduction of
diacylglycerol, which attenuates PKC activity and decreases angiogenesis [46]. Importantly, PPARγ activation enhances surface CD36, a
lipid scavenger receptor, which transmits the anti-angiogenic signal of thrombospondin-1
(TSP1) [47] a potent endogenous inhibitor
of angiogenesis, see Figure 4(a).PPARγ produces complex effect on the
endothelial progenitor cells (EPC): RGZ enhances the expression of the endothelial
markers CD31 and VEGFR2 on the circulating EPCs, however VE-cadherin and CD146
remain low; increased uptake of oxidized lipids suggests elevated CD36, which
increases the sensitivity to TSP1. EPCs from the diabetic patients treated with
RGZ display better adherence to fibronectin than those from untreated diabetics
and normal donors [6]. This is consistent with
reduced oxidative stress and improved re-endothelialization by the EPCs from
diabetic patients in RGZ-treated mice [48]. EPCs from the RGZ-treated
diabetics migrate more vigorously than those from untreated subjects, but similarly
to the EPC from untreated normal donors [6] suggesting that RGZ rather
normalizes than increases the EPCs migratory potential. PGZ effect on cultured
EPCs is twofold: it enhances the expression of endothelial markers at a lower
dose (1 μm) and reduces it at higher
(10 μm) concentration. PGZ also stimulates
the expression of TGFβ and TGFβ receptor [49], and thus initiates EPC
conversion to the VSMC phenotype [50]: increased VSMC presence may
stabilize the neovasculature and thus reduce angiogenesis. This may explain why
PPARγ agonists ameliorate glomerulonephritis in
mouse model without increase in EPC homing [51].


## 6. In Vascular Smooth Muscle Cells

Genetic
variations associated with atherosclerosis point to PPARγ role in associated metabolic and
vascular events [52]. In
atherosclerotic lesions, PPARγ promotes vascular repair and
re-endothelialization, while suppressing neointima formation. PPARγ attenuates vasoconstrictive remodeling
by blocking NADPH oxidases [53] and inhibits VSMCs
proliferative and migratory responses to multiple cytokines and growth factors
including PDGF-BB, bFGF, thrombin, insulin, and angiotensin II (AngII). PPARγ interferes with VSMC proliferation and
survival by blocking the downstream targets of ERK1/2 and PI3K/Akt, SHIP2 and
two important regulators
of mRNA translation, p70S6 kinase and 4-EBP translation initiation inhibitor [54]. In
addition, PPARγ activation enhances the expression of Shp-2
phosphatase, which dephosphorylates/inactivates Vav, a guanidine exchange
factor for RhoA, impairs the activation of Rho-associated kinase (ROCK), and suppresses
VSMC proliferation and migration [55]. PPARγ
inhibits VSMC migration but not the attachment and motility components
of the migratory response: the inhibition of PDGF-BB driven VSMC migration is
due to the transcriptional repression of Ets-1, which, in turn, drives MMP-9
and invasion [56], see Figure 4(b).PPARγ
activation causes VSMC growth arrest via multiple pathways: (1) by suppressing
proteasomal degradation of the p27/Kip; (2) via transrepression of the E2F target, minichromosome maintenance
protein, MCM7, which blocks replication [2]; (3)
by blocking Ets-1 dependent transactivation of telomerase promoter [57]. PPARγ
and its agonists potently induce VSMC apoptosis (1) through direct upregulation of GADD45
and p53 via an Oct-1 dependent mechanism (PPRE are identified in GADD45 and p53
promoters) [58, 59]; (2) by inducing the TFG-β/ALK/Smad pathway, subsequent Bcl-2 repression,
and Smad-dependent induction of GADD45 [60]; (3) through transcriptional upregulation
of the interferon regulatory factor-1 (IRF-1), a proapoptotic,
antiproliferative transcription factor [61], see Figure 4(b).All
PPARγ-dependent
changes in VSMC behavior can contribute to its anti-angiogenic function:
decreased VSMC migration, and proliferation, plus increased apoptosis restrict VSMC
incorporation in the vasculature and therefore the stability of neovessels.
Moreover, ECs of the immature, VSMC-poor vessels are vulnerable to the
apoptotic signals by angiogenesis inhibitors, see Figure 4(b).


## 7. Anti-Inflammatory Effects

PPARγ affects inflammation directly, by
driving CD36-dependent apoptosis in MΦs  [62, 63], or indirectly, by reducing VCAM-1
expression by the ECs and thus blocking transendothelial migration (TEM) of
monocytes and MΦs during chronic inflammation typical
for diabetes and cancer. In contrast, E-selectin, a mediator of the acute
immune response, is not altered by PPARγ [64]. Statins increase
anti-inflammatory Cox-2 in MΦs, which, in turn, increases endogenous 15D-PGJ2,
activates PPARγ, and upregulates its downstream target,
CD36 [65]. In addition, PPARγ ligands cause NFκB transrepression, thus reducing the production
of inflammatory cytokines (IL-8, IL-6, MCP-1, and CX3CL1-1) by MΦs, and thus disrupting paracrine loop
that attracts tumor-associated MΦs (TAM) and thus stimulates angiogenesis
and tumor growth [66], see Figure 4(c).


## 8. In Tumor Cells and Stroma

PPARγ
is expressed in human carcinomas of the breast, colon, esophagus, liver, lung, pancreas
prostate, stomach, and thyroid, also in neuroblastoma, astrocytoma, and glioma:
in all of these PPARγ ligands repress or delay xenograft growth in mouse models
[67].PPARγ ligands affect tumor cells in several
ways: they reduce proliferation, enhance apoptosis, and modulate angiogenic phenotype
of the tumor cells. PPARγ targets cyclin D1 via the inhibitors of
cyclin-dependent kinases (Cdk), p18, p21, and p27, causing a decline in Rb phosphorylation
[1] and arresting cells in G1
phase: PPARγ acts via p21 and p27 in pancreatic
cancer and via p18 in hepatoma (see Figure 3(a)). On the other hand, glitazones
repress the production of Cdk2, 4 and 6 in carcinomas of the bladder, breast, lung,
and pancreas via GADD45 [67] (see Figure 3(b)). PPARγ activation also restores PTEN expression
in tumor cells and thus blocks PI3K/Akt axis [38], it can also initiate a negative
feedback loop, which consists of calcineurin phosphatase, nuclear factor of the
activated T-cells (NFAT), and down syndrome critical region 1 (DSCR1), which
inhibits calcineurin and blocks NFAT activity necessary for proliferation and
survival (see Figure 3(c)) [68], see Figure 4(c).PPARγ effects in cancer cells. (a) The induction of Cdk inhibitor, p27 causes growth arrest due to reduced MCM7 activity and subsequent blockade of replication. (b) The induction of GADD45 impairs Cyclin B and causes
G2M growth arrest. In addition, the activation of JNK and p38 kinases via MEKK4
initiates cell death by apoptosis. (c) PPARγ activation by hormones and nutrition in
normal cells and by agonists in cancer cells may activate the differentiation programs.
(a)(b)(c)PPARγ effects on the endothelial, pericytic,
tumor and immune cells in the tumor microenvironment: the consequences of angiogenesis and
possible ways to augment antitumor actions. Pro-angiogenic and tumor-promoting
events are shown in red. The opposing effects are in blue. The proposed drugs
are shown in black. (a) Summary of the PPARγ molecular effects in the endothelial
cells. TEM, transendothelial migration. (b) PPARγ molecular effects on the VSMCs. (c) The
effects on macrophages and tumor cells.
(a)(b)(c)PPARγ induction also causes tumor cell
apoptosis by downmodulating prosurvival proteins cFLIP and Bcl-2, while
increasing proapoptotic Bax and BAD, as occurs in glioblastoma [69] or by the interference with the
PI3K/Akt signaling [38]. Conversely, PPARγ often augments the expression of
TNF-related apoptosis inducing ligand (TRAIL), which selectively eliminates
cancer cells [70], see Figure 4(c).In some cases, PPARγ activation induces tumor cell
differentiation (e.g., liposarcoma, breast and pancreatic cancer,
neuroblastoma, glioma, bladder carcinoma, and lung carcinoma). The differentiation
is evidenced by the increase of the general markers of differentiated state,
such as E-cadherin, and downregulation of the specific markers of progenitor
lineages, also by morphology changes consistent with differentiated state (see Figure 3(d)) [1, 67].Finally, treatment with the PPARγ ligands frequently downregulates the
expression of pro-angiogenic
factors VEGF [17], IL-8 [71], Ang-1 [72], and Cox-2 [73] and thus suspends tumor
angiogenesis. Moreover, mice
null for PPARγ show impaired tumorgenesis, due to the dramatic increase in TSP-1
[5], see Figure 4(c).


## 9. PPARγ Pro-Angiogenic/Tumorigenic Effects

In
contrast to the majority of findings, a recent study suggests that PPARγ
ligands may have pro-angiogenic properties both in vitro [74], in an
endothelial/interstitial cell coculture assay, and in a murine corneal
angiogenesis model in vivo [74]. The magnitude of the
angiogenic response caused by PPARγ ligands has not been compared to the angiogenesis
elicited by typical stimuli (VEGF, bFGF); also, the contradiction between these
results and previous studies has not yet been addressed.PPARγ pro-angiogenic effects are associated
with the induction of VEGF and increased phosphorylation of eNOS and AKT [7, 75], which cause elevated VEGF
production in human and rodent VSMCs, MΦs and tumor cells [76–79], VEGF and VEGFR levels in the
ECs and myofibroblasts [80]. Although PPARγ ligands inhibit
xenografted human tumors [1, 33], in one study using mouse
model of colon cancer (APC/Min) PPARγ ligands increased the number of
precancerous polyps, tumor frequency and size [81]. However, in two other
models, APC-deficient HT-29 xenografts and azoxymetane-induced tumors PPARγ ligands suppress tumor growth and
angiogenesis [82, 83]. Of the multiple small-scale
clinical trials using PPARγ ligands for cancer treatment, only two showed
promising results: in an early study TGZ caused prolonged PSA stabilization in
prostate cancer patients [84], while PGZ combined with
low-dose chemotherapy and rofexoxib produced moderate improvement in the
patients with high-grade glioma [85]. In contrast, patients with
breast, colon, and thyroid cancers showed no significant response [86–88]. Thus, the use of PPARγ
ligands in clinical practice obviously requires optimization, and the answers
may come from the use of combination or complementation treatments.


## 10. PPARγ Ligands in Combination Treatments: Can We Augment the Beneficial Effects?

The
information above narrows down the list of PPARγ targets critical for its anti-angiogenic
and antitumor effects (see Figure 4(a)). PPARγ reverses angiogenic functions in the
ECs by blocking the expression of VEGF-A and its receptor, VEGFR2 by blocking
Ets-1 transcription factor, and by dampening the prosurvival PI3K/Akt cascade,
likely via PTEN induction. It also deactivates RhoA/Rac1 small GTPases which
enable EC migration. NFAT deactivation lowers the levels of the apoptosis
inhibitors, cFLIP and Bcl-2, and critical invasion molecules UPA and MMP 9. In
addition, PPARγ promotes the following proapoptotic
events: it elevates expression of the proapoptotic CD36 and TSP1 receptor-ligand
duo; increases p53 stability; opens of the Maxi-K channel to upregulate nitric
oxide (NO), which, paradoxically, causes apoptosis. In addition, PPARγ suppresses Trx-1 and ROS levels by
upregulating VDUP-1, a vitamin D3 target. Finally, PPARγ ligands block protein synthesis via
4-eBP and p70S6 kinase, both the targets of mTOR pathway.In
the VSMC, PPARγ represses the activation of prosurvival
Erk-1 and PI3K/Akt and SHIP thus sustaining the unphosphorylated, active state
of 4-EPB, a negative regulator of translation. It also enhances the activity of
Shp-2 phosphatase, which blocks Vav, the trigger of RhoA/ROCK pathway necessary
for survival and migration; PPARγ also interferes with VSMC Bcl-2
expression by enhancing TGFβ/Smad2 and disrupts MMP-9 production by
blocking Ets-1 (see Figure 4(b)).In
MΦs and tumor cells, PPARγ through transrepression of NFκB and NFAT lowers the production of
multiple growth factors and inflammatory cytokines including VEGF, Ang-1, cyclo-oxygenase
(Cox) 2, IL-6, IL-8, MCP-1, and CX3CL-1. PPARg also enhances the production of thrombospondin (TSP) 1:
therefore angiogenic balance tips in favor of vascular quiescence. In addition,
PPARγ lowers the resistance of tumor cells
and tumor-associated MΦs (TAM) to stress and apoptotic stimuli
by blocking cyclin D1 via cdk inhibitors p18, p21, p27, by repressing
antiapoptotic Bcl-2 and FLIP, by upregulating proapoptotic CD36 in MΦs, and Bax and BAD in tumor cells (see Figure 4).This
comprehensive list of PPARγ targets and interacting proteins can be
used for intelligent design of the optimal combination therapies based on PPARγ  ligands to achieve the best anti-angiogenic and
anticancer activity. For example, it stands to reason to expect that EC apoptosis caused by PPARγ can be augmented by supplying CD36
ligand, TSP1 or its peptide mimics, such as ABT-510 [89]. Indeed, PPARγ ligands 15PG-E2, TGZ and RGZ, and TSP1
anti-angiogenic peptide ABT-510 synergistically block angiogenesis and curtail
the growth of lung and bladder carcinoma xenografts, by initiating CD36-dependent
apoptotic events in remodeling tumor endothelium [47]. Furthermore, TSP1 expression
is enhanced by the low-dose metronomic chemotherapy, including cytoxan, docetaxel,
and 5-fluorouracil [90–92]. Thus cytoxan, docetaxel, and
5-fluorouracil are likely to potentiate the PPARγ anti-angiogenic effects in EC and to reduce
tumor-associated inflammation responses by killing TAMs. This is supported by
the fact that 15D-PGE2 enhances antitumor activity of docetaxel
against lung carcinoma cell lines [93]. In addition, metronomic
chemotherapy enhances the expression of Fas, a critical apoptosis mediator
induced by the TSP1/CD36 interaction and thus potentiates the activity of TSP1
derivatives, such as ABT-510 [94, 95]. Hence, combined use of PPARγ ligands and metronomic regimens of
chemotherapy agents is likely to be more effective than individual treatments.PPARγ blockade of the EC and VSMC migration
involves the inhibition of RhoA/ROCK signaling [40, 65], which makes ROCK inhibitors
likely candidates for the use in combination with PPARγ ligands. This is doubly important,
since ROCK activates Myc pathway and thus abolishes TSP1 expression by the
tumor cells [96]. ROCK inhibitors show strong toxic effects at therapeutic
doses, thus their clinical use is problematic. However, combined use with PPARγ ligands may allow to lower their effective concentration and therefore limit drug-induced toxicity.Since
PPARα strongly increases TSP1 production,
combined use of PPARα and PPARγ agonists or the use of dual PPARα/γ ligands may present an advantage. Interestingly,
TZD18, a novel PPARα/γ dual agonist induces apoptosis of
glioma cells with high efficiency [97]. Unfortunately, glitazars
have carcinogenic activity of their own [98].PPARγ ligands sensitize leukemic, lung and
endothelial cells to the TRAIL-induced apoptosis by enhancing DR5 expression [99, 100] pointing to possible synergy between
PPARγ agonists and TRAIL therapies.The
inhibition of VEGFR2 expression by vascular endothelium, which contributes to
the antiangiogenesis by the PPARγ, could be assisted by VEGF sequestering
agents, such as Avastin, or by the inhibitors VEGF RTK activity, such as sunitinib,
sorafenib or VEGF decoy receptor. This hypothesis is yet to be tested.The
downstream target of the PI3K/Akt pathway, which is blocked by PPARγ via PTEN activation, is tuberous
sclerosis tumor suppressor complex, which, when phosphorylated by Akt, allows
the activation of mammalian target of rapamycin (mTOR) kinase, protein
synthesis, and cell survival [101]. On the other hand, PPARγ ligands interfere with translation by augmenting
the activity of 4-EBP and blocking S6 kinase [102]. Thus PPARγ disrupts mTOR regulation of protein
synthesis at two distinct steps. Moreover, the blockade of mTOR pathway is
likely to suppress VEGF in all cell types in the tumor microenvironment [103]. Hence, mTOR inhibitors such
as tacrolimus are likely to complement the anti-angiogenic and antitumor
activity of PPARγ agonists. Cyclic AMP analogs, which
block mTOR activity via AMPK1 pathway [101], may also contribute to the
PPARγ beneficial effects: this is
particularly important, since cAMP analogs are capable of increasing PPARγ activity (Schulze-Hoepfner and Volpert,
unpublished observations). The fact that amino acid deprivation, the main off
switch for the mTOR, enhances PPARγ proapoptotic effects in tumor cells [104] lends further support to this
hypothesis.PPARγ transrepression of NFκB and NFAT signaling leads to the
inhibition of multiple angiogenic stimuli, including interleukins 6 and 8,
MCP-1 and CX3CL-1, as well as protective Ang-1 and proinflammatory Cox-2. This
PPARγ function suggests a wide range of
possible treatment combinations with NFκB inhibitors, including synthetic inhibitors
of IKK kinases [105] or naturally occurring plant
substances, like curcumin [106]. On the other hand, the
inhibition of Cox-2 with highly selective agents, like celexoxib, has direct
anti-angiogenic tumor-preventing effects [107] and is quite likely to
contribute to the PPARγ antitumor and anti-angiogenic
activities, especially in the light of potentiating effect of celexoxib on
docetaxel treatment [108] and beneficial effects of PGZ
combined with rofexoxib and low-dose chemotherapy [85].PPARγ activity is opposed by MEK kinases:
thus MEK inhibitors are likely to improve the efficacy of PPARγ ligands: indeed, MEK-1 inhibitor,
PD98059, improves CGZ antitumor effect in colon cancer xenografts [109]. PPARγ activity is also augmented by RXR
ligands: 9-cis retinoic acid (RA) enhances PPARγ-induced differentiation and gene
expression. In colon cancer, PPARγ and RXR ligands induce differentiation
and apoptosis more potently than each individual compound [110, 111]. Nine-cis retinoic acid partially
overcomes RXR phosphorylation, which reduces PPARγ/RXR dimerization and opposes PPARγ activity: MEK-1 inhibitors improve the
combined effect of CGZ and 9-cis RA [109]. Finally, HDAC inhibitor,
trichostatin A, potentiates the effects of phenolfibrate on the differentiation
and attenuation of stemness of the lung adenocarcinoma cells [112]. While combining PPARγ agonists with other drugs, particular
attention should be paid to the agonist dosage: studies of PPARγ effects metabolic syndrome demonstrate
that overactive and hypoactive mutants cause similar metabolic consequences and
suggest the use of SPPARMs versus full agonists [113].The list of agents with the
potential to enhance the antitumor and anti-angiogenic effects of PPARγ ligands is not limited by the examples
above, however we hope that it provides a convincing example of rational design
of the complementation therapies, based on the knowledge of molecular mediators
of a given agent. The examples, which demonstrate the improved efficacy of
predicted combinations, provide an impetus for the evaluation of the
combinations, which have not yet been tested.



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*Source: 945275-2008-11-18.xml*