# PPARγ and MEK Interactions in Cancer

Authors: Elke Burgermeister; Rony Seger
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor-gamma (PPARγ) exerts multiple functions in determination of cell fate, tissue metabolism, and host immunity. Two synthetic PPARγ ligands (rosiglitazone and pioglitazone) were approved for the therapy of type-2 diabetes mellitus and are expected to serve as novel cures for inflammatory diseases and cancer. However, PPARγ and its ligands exhibit a janus-face behaviour as tumor modulators in various systems, resulting in either tumor suppression or tumor promotion. This may be in part due to signaling crosstalk to the mitogen-activated protein kinase (MAPK) cascades. The genomic activity of PPARγ is modulated, in addition to ligand binding, by phosphorylation of a serine residue by MAPKs, such as extracellular signal-regulated protein kinases-1/2 (ERK-1/2), or by nucleocytoplasmic compartmentalization through the ERK activators MAPK kinases-1/2 (MEK-1/2). PPARγ ligands themselves activate the ERK cascade through nongenomic and often PPARγ-independent signaling. In the current review, we discuss the molecular mechanisms and physiological implications of the crosstalk of PPARγ with MEK-ERK signaling and its potential as a novel drug target for cancer therapy in patients.


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## Body

## 1. Introduction

### 1.1. The Janus-Face of PPARγ: Tumor Suppressor versus Tumor Promoter Actions

The metabolic and cell fate regulatory functions of PPARγ place this nuclear receptor (NR) [1, 2] at the cross-road of life style and
diabetic comorbidity risks, which are assumed to result from the diet and/or
chronic inflammation-induced sequence of preneoplastic lesions towards
manifested cancer [3]. Since decades, the association of
aberrant insulin signaling in diabetics and increased cancer risk has been
stated, and recently validated in patient studies with respect to colon,
pancreas, breast, endometrium, prostate, liver, and bladder (see, e.g., [4–7]). Although PPARγ plays an important part in the transmission of insulin responses and
physiological diet, little direct evidence exists relating these factors to
PPARγ activation and the risks of the
development of cancer [6–8]. One of the reasons for the lack of
knowledge on the role of PPARγ is that a bona fide high-affinity natural
ligand(s) for PPARγ has not been identified yet [2].PPARγ can be activated by low-affinity
ligands such as unsaturated long-chain fatty acids derived from nutrient uptake
(e.g., linoleic acid) and/or inflammatory reactions (e.g., 15-deoxy-Δ(12,14)-prostaglandin J2) [9, 10]. However, those do not induce the
full activity of PPARγ in most systems examined [2]. As of today, modulation of PPARγ activity is mediated by synthetics
drugs, and among them the thiazolidinediones (TZDs) rosi- and pioglitazone are
considered to be potent and selective PPARγ agonists [2]. These drugs were approved as
insulin sensitizers for the treatment of type-2 diabetes mellitus [11] and have been proven helpful in
vascular and atherogenic complications [12, 13]. However, TZD drugs can also exert
protumorigenic actions in certain rodent models [14, 15]. In addition, the safety of the
TZDs has been recently evaluated in clinical studies aimed to examine cancer
prevalence in diabetic patients under TZD use [16–18]. One study stated a significant
association of cancer risk in women under any TZD treatment (1003 patients) [17], while the other two stated no
significant associations (126,971 patients [16]; 87,678 patients [18]). On the other hand, patients with
long-term intake of nonsteroidal anti-inflammatory drugs (NSAIDs),
cyclooxygenase (COX) inhibitors that prevent endogenous eicosanoid production
and may act also as low-affinity PPARγ ligands, were reported to profit from a reduced risk for colon cancer
formation [19].These paradoxical
effects resulting from PPARγ activation are derived from a complex balance of anti-versus protumor functions of PPARγ protein and its ligands in a given system. The latter are also related
to the interaction of PPARγ with other oncomodulating proteins (such as MEK1 and βcatenin). In the current review, we
will discuss this janus-faced role of PPARγ and its ligands in cancer with a
major focus on its crosstalk with the ERK signaling cascade, which is a central
signaling pathway deregulated in a majority of tumor types in humans.


### 1.2. The ERK Cascade and Cancer

The MAPK cascades are central signaling pathways that mediate the response of essentially all
cellular processes stimulated by extracellular ligand, including proliferation,
survival, differentiation, apoptosis, stress response, and even oncogenic
transformation. Four main cascades have been identified to date, of which the Ras-Raf-MEK1/MEK2-ERK1/ERK2 cascade (ERK cascade) is the
most prominent one in human cancers [20, 21]. Its multilevel organisation of
kinases guarantees signal amplification and coherence, and its scaffold
proteins [22] organize the pathway into a 3D module that enables
crosstalk and direct interactions with other central signaling pathways such as
the PPARγs.Within the MAPK family,
the ERK cascade constitutes a major signaling pathway, regulating cell
proliferation and survival, as well as cell adhesion and motility,
differentiation, embryonal development, and neuronal regulation [21, 23]. Its deregulation, mainly due to
constitutive upregulation by receptor kinase “gain of function” mutations,
contributes to cancer initiation and progression [24–26]. The majority of human carcinomas
harbour increased expression or activating point mutations for the upstream
components of the ERK cascade (e.g., epidermal growth factor receptor
(EGFR/Her1), Her2/Neu/ErbB2, K-Ras, B-Raf) that culminate in a higher ERK
activity in a large majority of human tumors. The ERK cascade currently
represents the main targeted cascade (next to the angiogenic vascular
endothelial growth factor/receptor (VEGF/R) system) by second-generation low
molecular weight (LMW) kinase inhibitors (e.g., gefitinib, erlotinib) and
monoclonal (humanized) mAbs directed against members of the EGFR family (e.g.,
herceptin), which are in clinical use against cancer (as reviewed in [25, 27, 28]). Therefore, inhibitors of the ERK
cascade are likely to be beneficial in combating most types of cancer.


## 1.1. The Janus-Face of PPARγ: Tumor Suppressor versus Tumor Promoter Actions

The metabolic and cell fate regulatory functions of PPARγ place this nuclear receptor (NR) [1, 2] at the cross-road of life style and
diabetic comorbidity risks, which are assumed to result from the diet and/or
chronic inflammation-induced sequence of preneoplastic lesions towards
manifested cancer [3]. Since decades, the association of
aberrant insulin signaling in diabetics and increased cancer risk has been
stated, and recently validated in patient studies with respect to colon,
pancreas, breast, endometrium, prostate, liver, and bladder (see, e.g., [4–7]). Although PPARγ plays an important part in the transmission of insulin responses and
physiological diet, little direct evidence exists relating these factors to
PPARγ activation and the risks of the
development of cancer [6–8]. One of the reasons for the lack of
knowledge on the role of PPARγ is that a bona fide high-affinity natural
ligand(s) for PPARγ has not been identified yet [2].PPARγ can be activated by low-affinity
ligands such as unsaturated long-chain fatty acids derived from nutrient uptake
(e.g., linoleic acid) and/or inflammatory reactions (e.g., 15-deoxy-Δ(12,14)-prostaglandin J2) [9, 10]. However, those do not induce the
full activity of PPARγ in most systems examined [2]. As of today, modulation of PPARγ activity is mediated by synthetics
drugs, and among them the thiazolidinediones (TZDs) rosi- and pioglitazone are
considered to be potent and selective PPARγ agonists [2]. These drugs were approved as
insulin sensitizers for the treatment of type-2 diabetes mellitus [11] and have been proven helpful in
vascular and atherogenic complications [12, 13]. However, TZD drugs can also exert
protumorigenic actions in certain rodent models [14, 15]. In addition, the safety of the
TZDs has been recently evaluated in clinical studies aimed to examine cancer
prevalence in diabetic patients under TZD use [16–18]. One study stated a significant
association of cancer risk in women under any TZD treatment (1003 patients) [17], while the other two stated no
significant associations (126,971 patients [16]; 87,678 patients [18]). On the other hand, patients with
long-term intake of nonsteroidal anti-inflammatory drugs (NSAIDs),
cyclooxygenase (COX) inhibitors that prevent endogenous eicosanoid production
and may act also as low-affinity PPARγ ligands, were reported to profit from a reduced risk for colon cancer
formation [19].These paradoxical
effects resulting from PPARγ activation are derived from a complex balance of anti-versus protumor functions of PPARγ protein and its ligands in a given system. The latter are also related
to the interaction of PPARγ with other oncomodulating proteins (such as MEK1 and βcatenin). In the current review, we
will discuss this janus-faced role of PPARγ and its ligands in cancer with a
major focus on its crosstalk with the ERK signaling cascade, which is a central
signaling pathway deregulated in a majority of tumor types in humans.


## 1.2. The ERK Cascade and Cancer

The MAPK cascades are central signaling pathways that mediate the response of essentially all
cellular processes stimulated by extracellular ligand, including proliferation,
survival, differentiation, apoptosis, stress response, and even oncogenic
transformation. Four main cascades have been identified to date, of which the Ras-Raf-MEK1/MEK2-ERK1/ERK2 cascade (ERK cascade) is the
most prominent one in human cancers [20, 21]. Its multilevel organisation of
kinases guarantees signal amplification and coherence, and its scaffold
proteins [22] organize the pathway into a 3D module that enables
crosstalk and direct interactions with other central signaling pathways such as
the PPARγs.Within the MAPK family,
the ERK cascade constitutes a major signaling pathway, regulating cell
proliferation and survival, as well as cell adhesion and motility,
differentiation, embryonal development, and neuronal regulation [21, 23]. Its deregulation, mainly due to
constitutive upregulation by receptor kinase “gain of function” mutations,
contributes to cancer initiation and progression [24–26]. The majority of human carcinomas
harbour increased expression or activating point mutations for the upstream
components of the ERK cascade (e.g., epidermal growth factor receptor
(EGFR/Her1), Her2/Neu/ErbB2, K-Ras, B-Raf) that culminate in a higher ERK
activity in a large majority of human tumors. The ERK cascade currently
represents the main targeted cascade (next to the angiogenic vascular
endothelial growth factor/receptor (VEGF/R) system) by second-generation low
molecular weight (LMW) kinase inhibitors (e.g., gefitinib, erlotinib) and
monoclonal (humanized) mAbs directed against members of the EGFR family (e.g.,
herceptin), which are in clinical use against cancer (as reviewed in [25, 27, 28]). Therefore, inhibitors of the ERK
cascade are likely to be beneficial in combating most types of cancer.


## 2. Mechanisms of Crosstalk between PPARγ and the ERK Cascade

The mechanism of action
and the regulation of PPARγ have attracted considerable attention over the years. Although this
protein was initially shown to act as a transcription factor, studies using synthetic
ligands suggested that it may exert its function via activation of signaling as
well [1, 2]. According to the current
knowledge, PPARγ signaling is mediated by several
distinct mechanisms (Figure 1). The best known one is exerted by PPARγ protein itself, which is activated
by ligand binding, heterodimerizes with the retinoic X receptor (RXR) and
requires NR coregulator recruitment, events that lead to binding and
transcriptional activation of PPAR-responsive elements (PPREs) in the DNA [29]
(Figure 1(a)). Simultaneous
activation of the ERK cascade (e.g., by mitogens) therein contributes to
inhibition of this classical genomic action through serine phosphorylation of
PPARγ (Figure 1(a)). Another mechanism is that PPARγ interacts with other transcription
factors at the DNA level, which leads to PPRE-independent genomic actions of
PPARγ protein and its ligands [9, 10] (Figure 1(b)). Activation of
the ERK cascade participates in this mechanism by phosphorylation of the latter
transcription factors that interact with PPARγ (Figure
1(b)). A third
possibility is that nuclear export and cytoplasmic retention of PPARγ by MEK1 [30] results in “off-DNA”-interaction of
PPARγ with distinct protein partners
(e.g., cytoskeleton, lipid droplets, kinases), leading to alternative
cytoplasmic signaling (Figure 1(c)). Finally, PPARγ ligands can function via activation
of intracellular signalling (e.g., the ERK cascade) by a PPARγ-independent mechanism, which is
derived from exogenous application of ligands that bind to plasma
membrane-bound receptors [31]
(Figure 1(d)). The latter
mode of action can be “nongenomic,” that is, involving cytosolic signaling
cascades, or “genomic,” that is, converging on the DNA by activation of
alternative (non-PPAR) transcription factors (Figure 1(d)).Figure 1
Mechanisms of PPARγ-ERK signaling crosstalk:
(a) serine phosphorylation of PPARγ
by the ERK cascade suppresses the
classical genomic action of RXR/PPARγ heterodimers on PPREs in the DNA; (b) ERK cascade phosphorylation
of promitotic and proinflammatory transcription factors (TF) and NR coactivators (NCoA) modulates
their interaction with PPARγ “On-DNA”; (c) nuclear export of PPARγ by MEK1 may result in “Off-DNA”
interactions of PPARγ with alternative protein partners in the cytoplasm; (d) PPARγ-independent
ERK cascade activation by PPARγ ligands through plasma membrane GPCRs, transactivation of the EGFR
(black bars), or calcium signaling.As apparent from the
above description, interaction with the ERK cascade plays an important role in
the regulation and signal transmission of PPARγ and its ligands. Overall, three
main mechanisms of signaling crosstalk between the ERK cascade and PPARγ were described so far as follows: (1)
phosphorylation of PPARγ (and its cofactors) by ERKs and other MAPKs (p38, JNK); (2) nongenomic
activation of the ERK cascade by PPARγ ligands; and (3) compartmentalization of PPARγ by the ERK cascade component MEK1.
Those are described in details in this section.


### 2.1. The Functions of the PPARγ Protein and  Its Regulation by ERK Phosphorylation

Genetic and pharmacologic studies in cells, rodent models, and human patients corroborated
that the PPARγ protein serves as a master
regulator of adipocyte and macrophage function in normal and pathophysiological
conditions (inflammation, type-2 diabetes, obesity, atherosclerosis) [1]. Its expression in mesenchymal stem
cells also associated this receptor with bone, skin, and muscle differentiation
[2]. This 50-kDa protein consists of
(from N- to C-terminal) the following: a transactivation function-1 (AF1)
harbouring an MAPK-phosphorylation motif PXSP, a zinc-finger-type DNA-binding
domain (DBD), a hinge region, the ligand-binding domain (LBD), and a flexible
AF2 helix. Ligand-binding triggers the formation of the “charge clamp” between
the AF2 and the core LBD, an event that enables the release of NR corepressors
(NCoRs), heterodimerization with RXR, DNA-binding, NR coactivator (NCoA)
recruitment, and transactivation of promoters [29] (Figure 1(a)). The LBD/AF2
interface also constitutes an important docking interface with unusual
coregulators such as kinases and cell-cycle regulators (reviewed
in [32]).PPARγ positively regulates the expression
of a vast spectrum of target genes involved in immunity and inflammation,
differentiation, proliferation, apoptosis, cell survival, and metabolism [10]. However, PPARγ can also repress transcription by
negatively interacting with several proinflammatory [9] and promitotic transcription
factors [33] such as ETS, STAT, AP1, and NFκB (Figure 1(b)). Thereby,
this factor promotes terminal differentiation of various normal and transformed
cells of epithelial and mesenchymal origin. PPARγ(−/+) knockout mice exhibit enhanced
susceptibility to chemically induced tumorigenesis [34, 35], and this enhanced susceptibility
is observed also upon breeding with other strains deficient in tumor
suppressors (such as APC) [36]. In patients, PPARγ protein is expressed (in varying
levels) in leukemias, lipo- and osteosarcomas and in many carcinomas. Gene
polymorphisms within the human population result in several “loss-of-function”
PPARγ variants that are associated with
metabolic diseases (insulin resistance, lipodystrophy) [37] and cancers (e.g., colon, stomach) [4, 5, 38, 39]. These data initially corroborated
PPARγ as a protective transcription factor.In line with the latter
findings, ERK- (and other MAPK-) mediated phosphorylation of PPARγ reduces its genomic activity. A panel
of extracellular/environmental promitotic, stress and inflammatory stimuli
(growth factors, hormones, cytokines, lipid mediators/eicosanoids,
UV-radiation, anisomycin, acetaldehyde, etc.) trigger the activation of the
MAPK-family members: ERK, JNK, and p38 (Figure 1(a)). These MAPKs
phosphorylate (in humans) Ser 84 in the PPARγ1 and Ser 114 in the PPARγ2 isoform, which correspond to Ser
82/112 in mouse and are both located in the AF1 region of the molecules. This
phosphorylation results in suppression of the PPARγ’s ability to transactivate target
gene promoters and thereby its physiological functions (reviewed by [40, 41]). In addition, phosphorylated PPARγ is assumed to be more prone to
other posttranslational modifications (sumoylation, ubiquitination) and
subsequent degradation by the proteasome, an event that promotes its further
downregulation upon MAPK-activation [42, 43]. But these effects are not fully
characterized yet. In any event, the inhibition of PPARγ activity by MAPK phosphorylation is
in accordance with the anti-inflammatory and prodifferentiation action of PPARγ and has been verified for normal
(fibroblasts, adipocytes, macrophages, hepatic stellate cells) as well as
cancer cell lines, various stimulating agents (as reviewed in [31, 44]) and also in vivo [45, 46]. An additional level of crosstalk
is constituted by the fact that PPARγ cofactors, such as steroid receptor coactivator (SRC) family members
(e.g., AIB/SRC3 in breast cancer), are phosphorylated by MAPKs and thereby are
altered in their ability to coactivate transcription [47]
(Figure 1(b)).The effect of PPARγ phosphorylation by MAPKs was also
supported by several in vivo studies. For example, a “knock in” of an
unphosphorylable allele S112A in mice preserved their insulin sensitivity in
absence of lipogenesis (weight gain) in a setting of diet-induced obesity [45]. In addition, a recent study
revealed “downstream of tyrosine kinases-1” (Dok1) as an adapter protein in the
insulin-signaling pathway that inhibits S112 phosphorylation of PPARγ2 in vivo [46]. Dok1 knockout mice on high fat
remain lean and insulin-sensitive, and Dok1 knockout mouse embryonal
fibroblasts (MEFs) show defective adipogenic differentiation, increased ERK
activation and phosphorylation of PPARγ2 on S112. Mutation of S112 of PPARγ2 blocked the lean phenotype in Dok1
knockout mice, indicating that Dok1 promotes adipocyte growth and
differentiation by counteracting the inhibitory effect of ERK on PPARγ. Another current intriguing example
is the identification of parvinβ, a focal adhesion protein (lost in breast cancer patients), that
increases the expression, S84 phosphorylation, and activity of PPARγ1 through cyclin-dependent kinase 9
(CDK) and suppressed breast cancer growth in vivo [48]. These data indicate that
MAPK-mediated S84/S114 phosphorylation alters the activity of PPARγ1/2 in vitro and in vivo.In sum, these studies
initially corroborated the role of PPARγ as a tumor suppressor [2, 14], which may be shut down by
MAPK-phosphorylation [44]. However, more recent evidence was
collected, that PPARγ is a context-specific tumor modulator, whose effector profile is
complemented and modified by PPARγ-independent effects of its ligands (e.g., TZDs and eicosanoids) and by
reciprocal regulation of PPARγ through members of the ERK cascade as
follows [31, 40].


### 2.2. PPARγ Ligands Influence Cellular Processes via  a Nongenomic Activation of the ERK Cascade

A second mechanism of
crosstalk between PPARγ and the ERK cascade comprises the direct activation of ERKs by PPARγ ligands. In the past, ample data
was collected on the effects of chemically distinct classes of PPARγ ligands on cells. Different ligands
induce either cell growth and proliferation or growth arrest and apoptosis in
various human and mouse cancer cell lines and xenografts (as extensively
reviewed in [14, 31]), and also modulate angiogenesis in
vitro and in vivo [49]. These effects are dose-, time-,
and cell type-dependent, and manifest either in a PPARγ receptor-dependent (“genomic”) or
non-PPARγ receptor-mediated (“nongenomic”)
manner or in a combination of both. The mechanisms that underlie these context-dependent
responses are largely unknown. One concept is based on the claim that
nongenomic PPARγ ligand effects manifest at higher
micromolar concentrations (>10 μM) well above the low EC50’s necessary for
classical genomic actions on PPARγ/RXR heterodimers at characterized PPREs in target genes (e.g., 80 nM
for rosiglitazone) [50, 51]. This assumption translated into
the idea that, low doses of PPARγ ligands, for example, that correspond to the pharmacological doses
prescribed for diabetic patients, exert overtly beneficial efficacy, while
supra-pharmacological high doses evoke adverse effects. For example,
troglitazone was retracted from the market due to hepatotoxicity, which was not
a TZD-class effect but due to a drug-specific (possibly “nongenomic”) adverse
action [2]. However, the literature provides
examples for both pro- and antitumor actions of PPARγ ligands at similar dose ranges in
similar cellular systems. Thus, an underlying principle for the separation of
genomic from nongenomic PPARγ ligand effects is currently not available.The PPARγ ligand effects are likely to be
mediated either (i) through so far unknown plasma membrane-bound receptors (Figure 1(d)) or (ii) through cytoplasmatic localized PPARγ protein (Figure 1(c)). Novel
G-protein coupled receptors, such as GPR30 for estradiol [52], TGR5 for bile acids [53], and GPR40 for free fatty acids [54], were identified to function as
alternative signal transducers for NR-ligands. GPR40, a candidate PPAR ligand
receptor, is highly expressed in the pancreas but also in monocytes and in the
lower GI tract (e.g., ileum, colon) [55, 56]. Oleate, a natural PPAR ligand,
increases proliferation of MCF7 human breast adenocarcinoma through binding and
signaling via endogenous GPR40 [57]. TZDs were postulated as bona fide
ligands for ectopic GPR40 in CHO cells and to signal via Gαi/q proteins, cAMP, calcium, and ERK
activation [58]. However, in vivo proof is lacking.
In addition to GPCRs, also plasma membrane-bound classical NRs interact with
specific adapter or scaffold proteins in the cytoplasm and trigger the
initiation of proproliferative and survival signaling [59]. For example, the estrogen receptor
docks to modulator of non-genomic action of estrogen receptor (MNAR) that
recruits Src and leads to activation of the p85 subunit of PI3K [60] and the ERK cascade [61]. If this situation is also relevant
for PPARγ molecules remains to be shown. Many
TZD effects actually target cytoplasmic proteins such as at mitochondria, the
proteasome, or the translational machinery. Thus, it is possible that
cytoplasmic PPARγ molecules are also involved in the
transduction of “nongenomic” TZDs signals.Downstream of the
initial ligand triggering event, nongenomic responses to PPARγ ligands include transient
alterations in mitochondrial functions and activation of stress (production of
reactive oxygen species (ROS)) as well as kinase signaling pathways promoting
proliferation and survival such as PI3K-PKB/AKT, ERK, p38, and JNK [50, 51]. Rapid signaling initiated by
ligands can be mediated by membrane proximal events such as cleavage of
transmembrane proteinases (ADAMs), activation of GPCRs, EGFR transactivation,
calcium influx, and activation of protein tyrosine kinases (Pyk2, Src). Further
downstream effects include PPARγ-independent induction of “early response genes” such as c-Fos and
Egr-1. In this context, it was shown that PPARγ ligands enhance proliferation,
survival and drug resistance in cancer cells, for example, by induction of the
prosurvival and promitotic hormone gastrin [62]. We showed that TZDs enhance drug
resistance in human colon adenocarcinoma HT29 cells in a PPRE-independent but
EGFR-dependent manner, involving Src/MAPK-signaling [63]. In colon carcinoma cells, TZDs
induce matrix metalloproteinase 2 (MMP2) and membrane type 1-MMP (MT1-MMP)
activation and concomitantly increase tumor cell invasion through generation of
ROS and activation of the ERK cascade [64]. On the other hand, ERK cascade
activation by TZDs may also translate into growth inhibition and/or apoptosis [65–69]. It is currently unknown which
mechanism governs the decision for pro-versus antiproliferative responses upon TZD application.In addition to TZD drugs,
also the physiological eicosanoid-type ligands for PPARγ exert tumor-modulating effects
through their ability to trigger ERK cascade activation [70]. Eicosanoids are generated by
cytoplasmic phospholipase A2 and cyclooxygenases (COX1/2). Some of these
arachidonic acid metabolites act as endogenous PPARγ ligands ((e.g., 15-deoxy-Δ(12,14)-PGJ2) [71]), while others, like the
prostaglandins of the E and D series, activate the ERK cascade through
prostanoid GPCRs at the cell membrane [72]. 15-deoxy-Δ(12,14)-PGJ2 directly inhibits
inhibitor-κB kinase (IKK) in an intracellular
fashion and exerts various effects on inflammation, cell growth, and apoptosis
independent of a prostanoid GPCR [71]. For example, in human breast MCF7
adenocarcinoma cells, 15-deoxy-Δ(12,14)-PGJ2 upregulates VEGF synthesis through induction of heme
oxygenase-1, an enzyme that stimulates proliferation and angiogenesis, and
triggers ERK phosphorylation in an PPARγ-independent fashion [73]. In sum, these data point out to
the important role for protumor effects of PPARγ ligands of the TZD- and
eicosanoid-class in the activation of ERK cascade-related proliferation and
survival pathways, which stand in sharp contrast to the otherwise reported
tumor suppressive effects of the latter in similar cellular
systems [65–67].In vivo preclinical and
clinical data of TZDs support the concept of an overlapping profile of PPARγ receptor-dependent and independent
ligand signaling. In contrast to the lessons from PPARγ(+/−) knockout mice [34, 35] and the antineoplastic action of
PPARγ receptor activation in vitro [33], ample in vivo data asserted that
many potent and selective PPARγ ligands actually promote tumorigenesis. Thus, PPARγ ligands induce tumor growth in
rodent xenograft models [14] and enhance in vivo angiogenesis [49]. In addition, TZDs act as
procancerogenic agents in wild-type and APC-deficient mouse models of colon
carcinogenesis [74–77]. Importantly, clinical studies in
humans failed to show a clear benefit of TZD monotherapy in cancer patients [14, 78, 79]. PPARγ ligands are procarcinogenic in
human bladder, as evaluated by the PROactive study [12], and in the rodent bladder [80, 81]. As a reaction towards the
safety-toxicological data collected in preclinical studies and clinical trials
regarding TZD use, the US Food and Drug Administration (FDA) (http://www.fda.gov/cder/present/DIA2004/15) issued a warning of tumor-related adverse effects of novel
potent PPARγ ligands that are currently in clinical trials as novel antidiabetics or
obesity cures (reviewed in [82]) [83, 84]. The FDA classified all PPARγ ligands as multispecies and
multiorgan carcinogens requiring strict dose finding for therapeutical use in
humans. However, the full molecular mechanism of this interplay between tumor
promoting versus suppressing action of PPARγ ligands is so far unknown.


### 2.3. Towards Solving the Tumor Initiation/Suppression Paradox of PPARγ: Interaction of PPARγ with  the ERK Cascade in Cancer

Unlike the impression that is left by many articles to date, PPARγ protein does not always act as a
tumor suppressor, and the PPARγ ligands are not always procancerogenic independently of the receptor.
Notably, the PPARγ itself seems to be important for exacerbating mammary gland tumor
formation in bitransgenic mice expressing a constitutive active PPARγ form independently of application
of an exogenous ligand [85]. An interesting in vitro study
corroborated the functional cooperation of the PPARγ receptor and the ERK cascade in the
promotion of epithelial-mesenchymal transition (EMT) in the mouse small
intestine and rat intestinal epithelial cells, which was dependent on an intact
DNA-binding activity of the PPARγ receptor protein [86]. In this system, PPARγ induced ERK1/2 phosphorylation by
activating PI3K, Cdc42, and p21-activated kinase (PAK), which in turn
phosphorylated S298 of MEK1 that supports its activity [23]. Ectopic expression of dominant negative
MEK1 blocked EMT induced by PPARγ, while constitutively active MEK1
overexpression promoted a mesenchymal morphology. However, as evident in the
latter intriguing example, the exact molecular mechanisms and physiological
relevance of the cooperative interactions between posttranslational regulation
of NRs by kinases and rapid nongenomic kinase activation by
NR-ligands are so far unknown.Ample data supports the
notion that mutual physical/allosterical associations between kinases and NRs
exist that translate into reciprocal regulation of their activities [87, 88]. For example,
3-phosphoinositide-dependent protein kinase-1 (PDK1), that is the upstream
activator of AKT/PKB, binds to and activates PPARγ during adipogenic differentiation [89]. Complexes of cyclins and CDKs are
cofactors for and phosphorylate PPARγ in adipocytes [90, 91]. PPARγ also interacts with and is activated by
ERK5 [92, 93] in order to inhibit (in conjunction
with WNT signaling factors) the proliferation of lung cancer (NSCLC) cells and
inflammation in endothelial cells upon flow (shear stress), indicative of a
protective function of ERK5-PPARγ cooperation. These unusual NR
cofactors [32], that also include retinoblastoma
protein and transcriptional elongation factors, directly interact with
regulatory domains in NRs and considerably add to the pleiotropic effector
profile of a given NR. Several interaction partners for PPARγ protein have been identified
including prominent oncogenic modulators such as βcatenin [94, 95] and MEK1 [30]. Therefore, it is likely that PPARγ interacts with or cooperates with
several signaling pathways and particularly the ERK cascade in order to induce
or prevent oncogenic transformation dependent on the cell type and environment.


#### 2.3.1. Spatial Regulation of PPARγ Activity:  MEKs Export PPARγ to the Cytoplasm

Next to Ser84/114 phosphorylation and the nongenomic ERK activation by PPARγ ligands, the direct interaction of
PPARγ with the ERK cascade component MEK1 constitutes a third mechanism of
crosstalk between PPARγ and the ERK cascade. Subcellular compartmentalization is a major
mechanism in regulating cellular signaling. Interestingly, PPARγ itself can regulate the membrane
translocation of other proteins such as NFκB in gut intestinal epithelial cells
[96] and PKC in macrophages [97]. Several reports have demonstrated
a signal-mediated translocation of PPARγ between the nucleus and the
cytoplasm in vitro (as reviewed in [98]). In addition, it was shown that
PPARγ is expressed predominantly in the nucleus of nonneoplastic tissues,
whereas it is present in both the nucleus and the cytoplasm of tumorous tissues
in squamous cell carcinoma (SCC) of the lung, indicative of a correlation of
malignancy with differential PPARγ compartmentalization [99]. Moreover, a dominant negative PPARγ splice variant was described in
lung SCC patients, an event that leads to the loss of apoptosis sensitivity in
response to oxidative stress and cisplatin [99]. Differential compartmentalization
of PPARγ was also described in gastric cancer patients [100]. The ratio of cytoplasmic/nuclear
PPARγ expression decreased in the progression of intestinal metaplasia to
undifferentiated cancers [100]. In salivary duct carcinoma, an
aggressive tumor type, PPARγ is highly expressed (80%) and topographically located in the cytoplasm [101], indicative of an inactivation of
its genomic activities in the nucleus. Cytoplasmic PPARγ was also detected in the cytoplasm
(58%) of infiltrating breast carcinoma samples and was proposed as an
independent prognostic factor for patients with ductal carcinoma [102]. However, the function of this
subcellular distribution of PPARγ molecules are yet unknown.The mechanism that may
induce the changes in localization of PPARγ upon stimulation, or upon
neoplastic transformation was
only recently elucidated by us [30]. We showed that PPARγ is exported from the nucleus to the
cytoplasm by MEK1/2. This is induced by a reversible interaction of PPARγ with MEK1 through association of
the AF2 of the first with the N-terminal docking domain of MEK1. This export to
the cytoplasm (Figure 1(c)) leads to reduction in its genomic function
in the nucleus [30]. We also elucidated the molecular
mechanisms of the export and the physiological implications, but the question
remained is whether cytoplasmatically located PPARγ is subjected to degradation or
shunted to alternative signaling compartments such as lipid droplets, ER/Golgi,
cytoskeleton, or the plasma membrane. To this regard, we tend to speculate that
alternative locations of PPARγ in the cell may determine the balance between tumor-suppressive and tumor-promoting
functions.


#### 2.3.2. Tumor-Suppressive Functions of PPARγ Related to ERKs and MEKs Interaction

Due to the coexpression of the
ubiquitous proteins PPARγ and MEK1/2 in different organs of the body, it was interesting to
identify their coregulation in various physiological and pathological
processes, as described below.Differentiation
Due to the lethality of MEK1 knockout mice [103] and absence of phenotypes in MEK2
knockout mice [104], the major focus of interest was
directed towards the role of MEK1 overexpression in vivo. Constitutively active
MEK1 (S218E/S222E) has been conditionally overexpressed (among other tissues)
in the skin and bone of mice [105]. All transgenic mice exhibited
increased cell numbers (hyperplasia) and cell size and a defect in terminal
differentiation. Interestingly, both in skin and in bone of mice, PPARγ was shown to be an important player
promoting differentiation [2]. In addition, the constitutively
active MEK1 overexpressing mice show dwarfism and reduced bone size due to
defective ossification and impaired chondrocyte differentiation. In other
systems, it was shown that osteoclast-specific PPARγ knockout mice are characterized by
increased bone mass due to impaired osteoclast differentiation [106], suggesting antagonistic effects of
PPARγ and MEK1 on different bone cell types: with PPARγ promoting osteoclast
differentiation, and MEK1 inhibiting chondrocyte differentiation.
Skin-restricted MEK1
transgenic mice exhibit hyperproliferation, hyperkeratosis and of age
papillomas at sites of wounding [105, 107]. Vice versa, epidermis-specific
knockout of MEK1/2 in mice [108] resulted in hypoproliferation,
apoptosis, skin barrier defects, and death, indicative of a positive role of
MEK1 in skin proliferation and tissue homeostasis. PPARγ knockout mice are characterized by
an increased sensitivity to experimentally-induced skin tumors [35], emphasizing the tumor suppressor
and differentiation promoting activity of PPARγ in the skin. These “mirror-images”
phenotypes in the organs where MEK1/2 and PPARγ are normally coexpressed may give
some indication for the antagonistic regulation of the two proteins, MEK
promoting proliferation and dedifferentiation, PPARγ promoting terminal differentiation.
In line with this idea, it was shown that the kinase activity of MEK1 was
actually dispensable for the hyperproliferative and integrin-inducing effects
of the MEK1 in mouse skin [109]. Instead, a kinase-dead mutant of
MEK1 elicited the same phenotype, indicative of an involvement of other
MEK1-functions such as scaffolding inhibition of differentiation-promoting
cellular factors.
In adipogenic
differentiation systems originating from (mesenchymal) stem cells, synergistic
cooperations between the MEK-ERK cascade and PPARγ have been described. In
fibroblasts, differentiating towards the adipogenic lineage, a positive
cooperation between PPARγ and MEK1 exists that facilitates the adipogenic program by
MEK1-dependent induction of the C/EBPα gene [110]. In bone marrow-derived mesenchymal
stem cells isolated from normal and streptozotocin (STZ)-induced diabetic FVB/N
mice, high glucose enhanced adipogenesis, lipid accumulation, and PPARγ expression via PI3K/AKT and ERK
cascade signaling, events that were all inhibited by the MEK-inhibitor PD98059 [111]. In differentiated C2C12 myocytes,
the free fatty acid palmitate reduces the mRNA levels of PPARγ-coactivator-1α (PGC1α) and activated MEK, while the MEK
inhibitors PD98059 and U0126 prevented such downregulation of PGC1α, indicative of a MEK-mediated
inhibition of an important NR coactivator protein for PPARγ in muscle cells [112]. These findings corroborated that
the MEK-ERK cascade and PPARγ signaling pathways can syn- or antagonistically cooperate to control
the balance of proliferation and differentiation in an organ/cell type-specific
manner.Cell cycle
The ERK cascade participates in
the regulation of cell cycle at (i) G0/G1 and G1/S transitions in response to
mitogenic stimulation (as reviewed in [24]) and (ii) in the process of Golgi
fragmentation [113–115] during mitosis. This is mediated in
part by the nuclear translocation of ERK upon cellular stimulation that
promotes expression of “immediate early” genes such as members of the AP1
family that activate the promoters of the G1 cyclins D and E. However, the
subcellular compartmentalization of ERK signaling by scaffold proteins (KSR,
MP1/p14, Sef) (reviewed in [22]) indicates a novel mode of spatial
separation of substrate specifities and signal translation. For example, MP1
via the adapter protein p14 tethers MEK1 to endosomes [116] and focal adhesions [117]. Sef translocates MEK1 to the Golgi
apparatus, prevents nuclear translocation of ERKs, and, thereby, favours
phosphorylation of cytoplasmic ERK substrates instead of nuclear ones [118]. The latter subcellular
localization-determining systems may thus be as well exploited by the PPARγ-MEK1 nuclear export shuttle to
regulate the cell cycle.
The PPARγ receptor has been involved in the
inhibition of the G0/G1-transition by up-regulation of genes coding for the
CDK-inhibitors p18(INK4C) [119] and p21(WAF1/CIP1) [120, 121], and in the inhibition of G1/S
transition through upregulation of the p27(KIP1) gene [122, 123]. Upregulation of other genes
implicated in cell cycle control such as PTEN or members of the BCL-gene family
contributes to the
growth-arresting and/or apoptosis-inducing action of PPARγ ligands [15]. The cell cycle modulatory actions
of PPARγ are usually not mediated through classical PPRE binding at the DNA but
rather through PPRE-independent “off-DNA” crosstalk to other transcription
factors [15] and through nongenomic effects in
the cytoplasm, such as inhibition of translation initiation [124, 125] and modulation of the proteasomal
machinery [126–128]. The latter processes may be
mediated by ligand-activated cytoplasmic PPARγ molecules or cytoplasmic
alternative signal-transducers for PPARγ ligands. We therefore hypothesize
that, by nuclear export and cytoplasmic retention of PPARγ-MEK1 complexes to other
MEK1-scaffolding locations (e.g., at the Golgi, endosomes, focal adhesions), the
genomic PPARγ functions may as well be redirected in favour of cytoplasmic signaling
events. In sum, the cell cycle modulating effects of PPARγ protein and its ligands may be
caused by its differential subcellular compartmentalization by MEK1.


#### 2.3.3. Tumor-Promoting Functions of PPARγ,  Related to Crosstalk with the ERK Cascade

Metastasis
In contrast to the initial
assumption of PPARγ mainly acting as a tumor suppressor whose activity and/or expression is
lost in cancers, PPARγ expression and activity can also be a negative predictor of cancer
aggressiveness; and positive cooperation between PPARγ and components of the ERK cascade
in malignant phenotypes takes place. For example, strong nuclear PPARγ expression was detected in thyroid
carcinomas compared to normal tissue, and patient samples of thyroid
carcinoma-associated lymph node metastasis also showed a higher percentage of
PPARγ-positive staining than other case categories [129]. PPARγ expression was also elevated in
human prostate cancer compared to normal prostate [130]. In patients with invasive breast
carcinoma, cytoplasmic MT1-MMP and MMP9 expression positively correlated with
PPARγ levels [131]. These data corroborated a positive
relationship between PPARγ expression and malignancy state in certain tumor entities, a fact that
was shown to be therapeutically exploitable by the use of PPARγ antagonists or siRNA. This was
described in primary esophageal tumor specimen and in esophageal cancer cell
lines [132], in human primary squamous cell
carcinoma (SCC) and lymph node metastases [133] and in hepatocellular carcinoma
(HCC) samples [134], where PPARγ expression is elevated compared to
matched normal tissue. In all three cell systems, PPARγ antagonists (T0070907,GW9662) and
RNAi-mediated knock-down of PPARγ levels reduced the invasiveness and
adherence of cells to the extracellular matrix, triggered anoikis, or inhibited
proliferation by decreasing the phosphorylation status of focal adhesion kinase
(FAK), MEK, and ERK. Therefore, in tumors where elevated PPARγ and activated ERK and MEK levels
contribute to the malignant phenotype, inhibition of PPARγ may be beneficial as a therapeutic
strategy (see also Section 3).Angiogenesis
The overall vascular protective and
antiatherogenic effects of PPARγ ligands provide essential add-ons for the clinical application as
insulin sensitizers (reviewed in [13]). However, the proangiogenic
effects of PPARγ ligands via modulation of the VEGF/VEGF-receptor system (that signals
via the ERK cascade) have gained recognition (reviewed by [49]), which may be beneficial for
therapy of vascular diseases (e.g., infarction) [135, 136] but detrimental in cancer tissue.
For example, in rat myofibroblasts, rosiglitazone and 15-deoxy-Δ(12,14)-PGJ2 induce expression of
VEGF and its receptors (Flt1 and KDR, that signal via the ERK cascade), and
augment tubule formation on a matrigel, indicative of a promoting function of
PPARγ and ERKs in angiogenesis [137]. In osteoblast-like MC3T3E1 cells,
pioglitazone and ciglitazone augmented FGF2-induced VEGF release in a PPARγ-dependent manner and enhanced the
phosphorylation of JNK [138]. In human RT4 bladder cancer cells,
VEGF mRNA and protein are upregulated by PPARγ via activation of the VEGF
promoter. Interestingly, the MEK inhibitor PD98059 reduced PPARγ ligand-induced expression of VEGF [139], indicative of a positive
cooperation of PPARγ-ERK pathways in angiogenesis. These positive effects on angiogenesis
were examined also in two clinical studies with rosiglitazone [140] and pioglitazone [136], in which it was demonstrated that
chronic addition of the TZDs increased endothelial cell precursor counts and
migration in diabetic patients, raising concern on the proangiogenic potential
of TZDs.
Taken together, the data
which revealed an antagonistic cooperation of PPARγ and ERK signaling in several cell
or tissue-specific differentiation systems (skin, bone, muscle, fat) is now
challenged by the findings of positive cooperation of the same components  in tumor progression (metastasis,
angiogenesis). Thus, the role of PPARγ as a MEK/ERK-regulated tumor
suppressor seems to be of importance in normal tissue or in prevention of tumor
initiation, while in advanced stages of certain tumors a synergistic
cooperation between PPARγ and the ERK cascade may contribute to the malignancy of the disease.
Future studies have to clarify whether PPARγ agonists, PPARγ antagonists, or PPARγ modulators/partial agonists
(SPPARMs) with a selective effector profile [141] may be of interest for the therapy
of certain tumor entities.


## 2.1. The Functions of the PPARγ Protein and  Its Regulation by ERK Phosphorylation

Genetic and pharmacologic studies in cells, rodent models, and human patients corroborated
that the PPARγ protein serves as a master
regulator of adipocyte and macrophage function in normal and pathophysiological
conditions (inflammation, type-2 diabetes, obesity, atherosclerosis) [1]. Its expression in mesenchymal stem
cells also associated this receptor with bone, skin, and muscle differentiation
[2]. This 50-kDa protein consists of
(from N- to C-terminal) the following: a transactivation function-1 (AF1)
harbouring an MAPK-phosphorylation motif PXSP, a zinc-finger-type DNA-binding
domain (DBD), a hinge region, the ligand-binding domain (LBD), and a flexible
AF2 helix. Ligand-binding triggers the formation of the “charge clamp” between
the AF2 and the core LBD, an event that enables the release of NR corepressors
(NCoRs), heterodimerization with RXR, DNA-binding, NR coactivator (NCoA)
recruitment, and transactivation of promoters [29] (Figure 1(a)). The LBD/AF2
interface also constitutes an important docking interface with unusual
coregulators such as kinases and cell-cycle regulators (reviewed
in [32]).PPARγ positively regulates the expression
of a vast spectrum of target genes involved in immunity and inflammation,
differentiation, proliferation, apoptosis, cell survival, and metabolism [10]. However, PPARγ can also repress transcription by
negatively interacting with several proinflammatory [9] and promitotic transcription
factors [33] such as ETS, STAT, AP1, and NFκB (Figure 1(b)). Thereby,
this factor promotes terminal differentiation of various normal and transformed
cells of epithelial and mesenchymal origin. PPARγ(−/+) knockout mice exhibit enhanced
susceptibility to chemically induced tumorigenesis [34, 35], and this enhanced susceptibility
is observed also upon breeding with other strains deficient in tumor
suppressors (such as APC) [36]. In patients, PPARγ protein is expressed (in varying
levels) in leukemias, lipo- and osteosarcomas and in many carcinomas. Gene
polymorphisms within the human population result in several “loss-of-function”
PPARγ variants that are associated with
metabolic diseases (insulin resistance, lipodystrophy) [37] and cancers (e.g., colon, stomach) [4, 5, 38, 39]. These data initially corroborated
PPARγ as a protective transcription factor.In line with the latter
findings, ERK- (and other MAPK-) mediated phosphorylation of PPARγ reduces its genomic activity. A panel
of extracellular/environmental promitotic, stress and inflammatory stimuli
(growth factors, hormones, cytokines, lipid mediators/eicosanoids,
UV-radiation, anisomycin, acetaldehyde, etc.) trigger the activation of the
MAPK-family members: ERK, JNK, and p38 (Figure 1(a)). These MAPKs
phosphorylate (in humans) Ser 84 in the PPARγ1 and Ser 114 in the PPARγ2 isoform, which correspond to Ser
82/112 in mouse and are both located in the AF1 region of the molecules. This
phosphorylation results in suppression of the PPARγ’s ability to transactivate target
gene promoters and thereby its physiological functions (reviewed by [40, 41]). In addition, phosphorylated PPARγ is assumed to be more prone to
other posttranslational modifications (sumoylation, ubiquitination) and
subsequent degradation by the proteasome, an event that promotes its further
downregulation upon MAPK-activation [42, 43]. But these effects are not fully
characterized yet. In any event, the inhibition of PPARγ activity by MAPK phosphorylation is
in accordance with the anti-inflammatory and prodifferentiation action of PPARγ and has been verified for normal
(fibroblasts, adipocytes, macrophages, hepatic stellate cells) as well as
cancer cell lines, various stimulating agents (as reviewed in [31, 44]) and also in vivo [45, 46]. An additional level of crosstalk
is constituted by the fact that PPARγ cofactors, such as steroid receptor coactivator (SRC) family members
(e.g., AIB/SRC3 in breast cancer), are phosphorylated by MAPKs and thereby are
altered in their ability to coactivate transcription [47]
(Figure 1(b)).The effect of PPARγ phosphorylation by MAPKs was also
supported by several in vivo studies. For example, a “knock in” of an
unphosphorylable allele S112A in mice preserved their insulin sensitivity in
absence of lipogenesis (weight gain) in a setting of diet-induced obesity [45]. In addition, a recent study
revealed “downstream of tyrosine kinases-1” (Dok1) as an adapter protein in the
insulin-signaling pathway that inhibits S112 phosphorylation of PPARγ2 in vivo [46]. Dok1 knockout mice on high fat
remain lean and insulin-sensitive, and Dok1 knockout mouse embryonal
fibroblasts (MEFs) show defective adipogenic differentiation, increased ERK
activation and phosphorylation of PPARγ2 on S112. Mutation of S112 of PPARγ2 blocked the lean phenotype in Dok1
knockout mice, indicating that Dok1 promotes adipocyte growth and
differentiation by counteracting the inhibitory effect of ERK on PPARγ. Another current intriguing example
is the identification of parvinβ, a focal adhesion protein (lost in breast cancer patients), that
increases the expression, S84 phosphorylation, and activity of PPARγ1 through cyclin-dependent kinase 9
(CDK) and suppressed breast cancer growth in vivo [48]. These data indicate that
MAPK-mediated S84/S114 phosphorylation alters the activity of PPARγ1/2 in vitro and in vivo.In sum, these studies
initially corroborated the role of PPARγ as a tumor suppressor [2, 14], which may be shut down by
MAPK-phosphorylation [44]. However, more recent evidence was
collected, that PPARγ is a context-specific tumor modulator, whose effector profile is
complemented and modified by PPARγ-independent effects of its ligands (e.g., TZDs and eicosanoids) and by
reciprocal regulation of PPARγ through members of the ERK cascade as
follows [31, 40].


## 2.2. PPARγ Ligands Influence Cellular Processes via  a Nongenomic Activation of the ERK Cascade

A second mechanism of
crosstalk between PPARγ and the ERK cascade comprises the direct activation of ERKs by PPARγ ligands. In the past, ample data
was collected on the effects of chemically distinct classes of PPARγ ligands on cells. Different ligands
induce either cell growth and proliferation or growth arrest and apoptosis in
various human and mouse cancer cell lines and xenografts (as extensively
reviewed in [14, 31]), and also modulate angiogenesis in
vitro and in vivo [49]. These effects are dose-, time-,
and cell type-dependent, and manifest either in a PPARγ receptor-dependent (“genomic”) or
non-PPARγ receptor-mediated (“nongenomic”)
manner or in a combination of both. The mechanisms that underlie these context-dependent
responses are largely unknown. One concept is based on the claim that
nongenomic PPARγ ligand effects manifest at higher
micromolar concentrations (>10 μM) well above the low EC50’s necessary for
classical genomic actions on PPARγ/RXR heterodimers at characterized PPREs in target genes (e.g., 80 nM
for rosiglitazone) [50, 51]. This assumption translated into
the idea that, low doses of PPARγ ligands, for example, that correspond to the pharmacological doses
prescribed for diabetic patients, exert overtly beneficial efficacy, while
supra-pharmacological high doses evoke adverse effects. For example,
troglitazone was retracted from the market due to hepatotoxicity, which was not
a TZD-class effect but due to a drug-specific (possibly “nongenomic”) adverse
action [2]. However, the literature provides
examples for both pro- and antitumor actions of PPARγ ligands at similar dose ranges in
similar cellular systems. Thus, an underlying principle for the separation of
genomic from nongenomic PPARγ ligand effects is currently not available.The PPARγ ligand effects are likely to be
mediated either (i) through so far unknown plasma membrane-bound receptors (Figure 1(d)) or (ii) through cytoplasmatic localized PPARγ protein (Figure 1(c)). Novel
G-protein coupled receptors, such as GPR30 for estradiol [52], TGR5 for bile acids [53], and GPR40 for free fatty acids [54], were identified to function as
alternative signal transducers for NR-ligands. GPR40, a candidate PPAR ligand
receptor, is highly expressed in the pancreas but also in monocytes and in the
lower GI tract (e.g., ileum, colon) [55, 56]. Oleate, a natural PPAR ligand,
increases proliferation of MCF7 human breast adenocarcinoma through binding and
signaling via endogenous GPR40 [57]. TZDs were postulated as bona fide
ligands for ectopic GPR40 in CHO cells and to signal via Gαi/q proteins, cAMP, calcium, and ERK
activation [58]. However, in vivo proof is lacking.
In addition to GPCRs, also plasma membrane-bound classical NRs interact with
specific adapter or scaffold proteins in the cytoplasm and trigger the
initiation of proproliferative and survival signaling [59]. For example, the estrogen receptor
docks to modulator of non-genomic action of estrogen receptor (MNAR) that
recruits Src and leads to activation of the p85 subunit of PI3K [60] and the ERK cascade [61]. If this situation is also relevant
for PPARγ molecules remains to be shown. Many
TZD effects actually target cytoplasmic proteins such as at mitochondria, the
proteasome, or the translational machinery. Thus, it is possible that
cytoplasmic PPARγ molecules are also involved in the
transduction of “nongenomic” TZDs signals.Downstream of the
initial ligand triggering event, nongenomic responses to PPARγ ligands include transient
alterations in mitochondrial functions and activation of stress (production of
reactive oxygen species (ROS)) as well as kinase signaling pathways promoting
proliferation and survival such as PI3K-PKB/AKT, ERK, p38, and JNK [50, 51]. Rapid signaling initiated by
ligands can be mediated by membrane proximal events such as cleavage of
transmembrane proteinases (ADAMs), activation of GPCRs, EGFR transactivation,
calcium influx, and activation of protein tyrosine kinases (Pyk2, Src). Further
downstream effects include PPARγ-independent induction of “early response genes” such as c-Fos and
Egr-1. In this context, it was shown that PPARγ ligands enhance proliferation,
survival and drug resistance in cancer cells, for example, by induction of the
prosurvival and promitotic hormone gastrin [62]. We showed that TZDs enhance drug
resistance in human colon adenocarcinoma HT29 cells in a PPRE-independent but
EGFR-dependent manner, involving Src/MAPK-signaling [63]. In colon carcinoma cells, TZDs
induce matrix metalloproteinase 2 (MMP2) and membrane type 1-MMP (MT1-MMP)
activation and concomitantly increase tumor cell invasion through generation of
ROS and activation of the ERK cascade [64]. On the other hand, ERK cascade
activation by TZDs may also translate into growth inhibition and/or apoptosis [65–69]. It is currently unknown which
mechanism governs the decision for pro-versus antiproliferative responses upon TZD application.In addition to TZD drugs,
also the physiological eicosanoid-type ligands for PPARγ exert tumor-modulating effects
through their ability to trigger ERK cascade activation [70]. Eicosanoids are generated by
cytoplasmic phospholipase A2 and cyclooxygenases (COX1/2). Some of these
arachidonic acid metabolites act as endogenous PPARγ ligands ((e.g., 15-deoxy-Δ(12,14)-PGJ2) [71]), while others, like the
prostaglandins of the E and D series, activate the ERK cascade through
prostanoid GPCRs at the cell membrane [72]. 15-deoxy-Δ(12,14)-PGJ2 directly inhibits
inhibitor-κB kinase (IKK) in an intracellular
fashion and exerts various effects on inflammation, cell growth, and apoptosis
independent of a prostanoid GPCR [71]. For example, in human breast MCF7
adenocarcinoma cells, 15-deoxy-Δ(12,14)-PGJ2 upregulates VEGF synthesis through induction of heme
oxygenase-1, an enzyme that stimulates proliferation and angiogenesis, and
triggers ERK phosphorylation in an PPARγ-independent fashion [73]. In sum, these data point out to
the important role for protumor effects of PPARγ ligands of the TZD- and
eicosanoid-class in the activation of ERK cascade-related proliferation and
survival pathways, which stand in sharp contrast to the otherwise reported
tumor suppressive effects of the latter in similar cellular
systems [65–67].In vivo preclinical and
clinical data of TZDs support the concept of an overlapping profile of PPARγ receptor-dependent and independent
ligand signaling. In contrast to the lessons from PPARγ(+/−) knockout mice [34, 35] and the antineoplastic action of
PPARγ receptor activation in vitro [33], ample in vivo data asserted that
many potent and selective PPARγ ligands actually promote tumorigenesis. Thus, PPARγ ligands induce tumor growth in
rodent xenograft models [14] and enhance in vivo angiogenesis [49]. In addition, TZDs act as
procancerogenic agents in wild-type and APC-deficient mouse models of colon
carcinogenesis [74–77]. Importantly, clinical studies in
humans failed to show a clear benefit of TZD monotherapy in cancer patients [14, 78, 79]. PPARγ ligands are procarcinogenic in
human bladder, as evaluated by the PROactive study [12], and in the rodent bladder [80, 81]. As a reaction towards the
safety-toxicological data collected in preclinical studies and clinical trials
regarding TZD use, the US Food and Drug Administration (FDA) (http://www.fda.gov/cder/present/DIA2004/15) issued a warning of tumor-related adverse effects of novel
potent PPARγ ligands that are currently in clinical trials as novel antidiabetics or
obesity cures (reviewed in [82]) [83, 84]. The FDA classified all PPARγ ligands as multispecies and
multiorgan carcinogens requiring strict dose finding for therapeutical use in
humans. However, the full molecular mechanism of this interplay between tumor
promoting versus suppressing action of PPARγ ligands is so far unknown.


## 2.3. Towards Solving the Tumor Initiation/Suppression Paradox of PPARγ: Interaction of PPARγ with  the ERK Cascade in Cancer

Unlike the impression that is left by many articles to date, PPARγ protein does not always act as a
tumor suppressor, and the PPARγ ligands are not always procancerogenic independently of the receptor.
Notably, the PPARγ itself seems to be important for exacerbating mammary gland tumor
formation in bitransgenic mice expressing a constitutive active PPARγ form independently of application
of an exogenous ligand [85]. An interesting in vitro study
corroborated the functional cooperation of the PPARγ receptor and the ERK cascade in the
promotion of epithelial-mesenchymal transition (EMT) in the mouse small
intestine and rat intestinal epithelial cells, which was dependent on an intact
DNA-binding activity of the PPARγ receptor protein [86]. In this system, PPARγ induced ERK1/2 phosphorylation by
activating PI3K, Cdc42, and p21-activated kinase (PAK), which in turn
phosphorylated S298 of MEK1 that supports its activity [23]. Ectopic expression of dominant negative
MEK1 blocked EMT induced by PPARγ, while constitutively active MEK1
overexpression promoted a mesenchymal morphology. However, as evident in the
latter intriguing example, the exact molecular mechanisms and physiological
relevance of the cooperative interactions between posttranslational regulation
of NRs by kinases and rapid nongenomic kinase activation by
NR-ligands are so far unknown.Ample data supports the
notion that mutual physical/allosterical associations between kinases and NRs
exist that translate into reciprocal regulation of their activities [87, 88]. For example,
3-phosphoinositide-dependent protein kinase-1 (PDK1), that is the upstream
activator of AKT/PKB, binds to and activates PPARγ during adipogenic differentiation [89]. Complexes of cyclins and CDKs are
cofactors for and phosphorylate PPARγ in adipocytes [90, 91]. PPARγ also interacts with and is activated by
ERK5 [92, 93] in order to inhibit (in conjunction
with WNT signaling factors) the proliferation of lung cancer (NSCLC) cells and
inflammation in endothelial cells upon flow (shear stress), indicative of a
protective function of ERK5-PPARγ cooperation. These unusual NR
cofactors [32], that also include retinoblastoma
protein and transcriptional elongation factors, directly interact with
regulatory domains in NRs and considerably add to the pleiotropic effector
profile of a given NR. Several interaction partners for PPARγ protein have been identified
including prominent oncogenic modulators such as βcatenin [94, 95] and MEK1 [30]. Therefore, it is likely that PPARγ interacts with or cooperates with
several signaling pathways and particularly the ERK cascade in order to induce
or prevent oncogenic transformation dependent on the cell type and environment.


### 2.3.1. Spatial Regulation of PPARγ Activity:  MEKs Export PPARγ to the Cytoplasm

Next to Ser84/114 phosphorylation and the nongenomic ERK activation by PPARγ ligands, the direct interaction of
PPARγ with the ERK cascade component MEK1 constitutes a third mechanism of
crosstalk between PPARγ and the ERK cascade. Subcellular compartmentalization is a major
mechanism in regulating cellular signaling. Interestingly, PPARγ itself can regulate the membrane
translocation of other proteins such as NFκB in gut intestinal epithelial cells
[96] and PKC in macrophages [97]. Several reports have demonstrated
a signal-mediated translocation of PPARγ between the nucleus and the
cytoplasm in vitro (as reviewed in [98]). In addition, it was shown that
PPARγ is expressed predominantly in the nucleus of nonneoplastic tissues,
whereas it is present in both the nucleus and the cytoplasm of tumorous tissues
in squamous cell carcinoma (SCC) of the lung, indicative of a correlation of
malignancy with differential PPARγ compartmentalization [99]. Moreover, a dominant negative PPARγ splice variant was described in
lung SCC patients, an event that leads to the loss of apoptosis sensitivity in
response to oxidative stress and cisplatin [99]. Differential compartmentalization
of PPARγ was also described in gastric cancer patients [100]. The ratio of cytoplasmic/nuclear
PPARγ expression decreased in the progression of intestinal metaplasia to
undifferentiated cancers [100]. In salivary duct carcinoma, an
aggressive tumor type, PPARγ is highly expressed (80%) and topographically located in the cytoplasm [101], indicative of an inactivation of
its genomic activities in the nucleus. Cytoplasmic PPARγ was also detected in the cytoplasm
(58%) of infiltrating breast carcinoma samples and was proposed as an
independent prognostic factor for patients with ductal carcinoma [102]. However, the function of this
subcellular distribution of PPARγ molecules are yet unknown.The mechanism that may
induce the changes in localization of PPARγ upon stimulation, or upon
neoplastic transformation was
only recently elucidated by us [30]. We showed that PPARγ is exported from the nucleus to the
cytoplasm by MEK1/2. This is induced by a reversible interaction of PPARγ with MEK1 through association of
the AF2 of the first with the N-terminal docking domain of MEK1. This export to
the cytoplasm (Figure 1(c)) leads to reduction in its genomic function
in the nucleus [30]. We also elucidated the molecular
mechanisms of the export and the physiological implications, but the question
remained is whether cytoplasmatically located PPARγ is subjected to degradation or
shunted to alternative signaling compartments such as lipid droplets, ER/Golgi,
cytoskeleton, or the plasma membrane. To this regard, we tend to speculate that
alternative locations of PPARγ in the cell may determine the balance between tumor-suppressive and tumor-promoting
functions.


### 2.3.2. Tumor-Suppressive Functions of PPARγ Related to ERKs and MEKs Interaction

Due to the coexpression of the
ubiquitous proteins PPARγ and MEK1/2 in different organs of the body, it was interesting to
identify their coregulation in various physiological and pathological
processes, as described below.Differentiation
Due to the lethality of MEK1 knockout mice [103] and absence of phenotypes in MEK2
knockout mice [104], the major focus of interest was
directed towards the role of MEK1 overexpression in vivo. Constitutively active
MEK1 (S218E/S222E) has been conditionally overexpressed (among other tissues)
in the skin and bone of mice [105]. All transgenic mice exhibited
increased cell numbers (hyperplasia) and cell size and a defect in terminal
differentiation. Interestingly, both in skin and in bone of mice, PPARγ was shown to be an important player
promoting differentiation [2]. In addition, the constitutively
active MEK1 overexpressing mice show dwarfism and reduced bone size due to
defective ossification and impaired chondrocyte differentiation. In other
systems, it was shown that osteoclast-specific PPARγ knockout mice are characterized by
increased bone mass due to impaired osteoclast differentiation [106], suggesting antagonistic effects of
PPARγ and MEK1 on different bone cell types: with PPARγ promoting osteoclast
differentiation, and MEK1 inhibiting chondrocyte differentiation.
Skin-restricted MEK1
transgenic mice exhibit hyperproliferation, hyperkeratosis and of age
papillomas at sites of wounding [105, 107]. Vice versa, epidermis-specific
knockout of MEK1/2 in mice [108] resulted in hypoproliferation,
apoptosis, skin barrier defects, and death, indicative of a positive role of
MEK1 in skin proliferation and tissue homeostasis. PPARγ knockout mice are characterized by
an increased sensitivity to experimentally-induced skin tumors [35], emphasizing the tumor suppressor
and differentiation promoting activity of PPARγ in the skin. These “mirror-images”
phenotypes in the organs where MEK1/2 and PPARγ are normally coexpressed may give
some indication for the antagonistic regulation of the two proteins, MEK
promoting proliferation and dedifferentiation, PPARγ promoting terminal differentiation.
In line with this idea, it was shown that the kinase activity of MEK1 was
actually dispensable for the hyperproliferative and integrin-inducing effects
of the MEK1 in mouse skin [109]. Instead, a kinase-dead mutant of
MEK1 elicited the same phenotype, indicative of an involvement of other
MEK1-functions such as scaffolding inhibition of differentiation-promoting
cellular factors.
In adipogenic
differentiation systems originating from (mesenchymal) stem cells, synergistic
cooperations between the MEK-ERK cascade and PPARγ have been described. In
fibroblasts, differentiating towards the adipogenic lineage, a positive
cooperation between PPARγ and MEK1 exists that facilitates the adipogenic program by
MEK1-dependent induction of the C/EBPα gene [110]. In bone marrow-derived mesenchymal
stem cells isolated from normal and streptozotocin (STZ)-induced diabetic FVB/N
mice, high glucose enhanced adipogenesis, lipid accumulation, and PPARγ expression via PI3K/AKT and ERK
cascade signaling, events that were all inhibited by the MEK-inhibitor PD98059 [111]. In differentiated C2C12 myocytes,
the free fatty acid palmitate reduces the mRNA levels of PPARγ-coactivator-1α (PGC1α) and activated MEK, while the MEK
inhibitors PD98059 and U0126 prevented such downregulation of PGC1α, indicative of a MEK-mediated
inhibition of an important NR coactivator protein for PPARγ in muscle cells [112]. These findings corroborated that
the MEK-ERK cascade and PPARγ signaling pathways can syn- or antagonistically cooperate to control
the balance of proliferation and differentiation in an organ/cell type-specific
manner.Cell cycle
The ERK cascade participates in
the regulation of cell cycle at (i) G0/G1 and G1/S transitions in response to
mitogenic stimulation (as reviewed in [24]) and (ii) in the process of Golgi
fragmentation [113–115] during mitosis. This is mediated in
part by the nuclear translocation of ERK upon cellular stimulation that
promotes expression of “immediate early” genes such as members of the AP1
family that activate the promoters of the G1 cyclins D and E. However, the
subcellular compartmentalization of ERK signaling by scaffold proteins (KSR,
MP1/p14, Sef) (reviewed in [22]) indicates a novel mode of spatial
separation of substrate specifities and signal translation. For example, MP1
via the adapter protein p14 tethers MEK1 to endosomes [116] and focal adhesions [117]. Sef translocates MEK1 to the Golgi
apparatus, prevents nuclear translocation of ERKs, and, thereby, favours
phosphorylation of cytoplasmic ERK substrates instead of nuclear ones [118]. The latter subcellular
localization-determining systems may thus be as well exploited by the PPARγ-MEK1 nuclear export shuttle to
regulate the cell cycle.
The PPARγ receptor has been involved in the
inhibition of the G0/G1-transition by up-regulation of genes coding for the
CDK-inhibitors p18(INK4C) [119] and p21(WAF1/CIP1) [120, 121], and in the inhibition of G1/S
transition through upregulation of the p27(KIP1) gene [122, 123]. Upregulation of other genes
implicated in cell cycle control such as PTEN or members of the BCL-gene family
contributes to the
growth-arresting and/or apoptosis-inducing action of PPARγ ligands [15]. The cell cycle modulatory actions
of PPARγ are usually not mediated through classical PPRE binding at the DNA but
rather through PPRE-independent “off-DNA” crosstalk to other transcription
factors [15] and through nongenomic effects in
the cytoplasm, such as inhibition of translation initiation [124, 125] and modulation of the proteasomal
machinery [126–128]. The latter processes may be
mediated by ligand-activated cytoplasmic PPARγ molecules or cytoplasmic
alternative signal-transducers for PPARγ ligands. We therefore hypothesize
that, by nuclear export and cytoplasmic retention of PPARγ-MEK1 complexes to other
MEK1-scaffolding locations (e.g., at the Golgi, endosomes, focal adhesions), the
genomic PPARγ functions may as well be redirected in favour of cytoplasmic signaling
events. In sum, the cell cycle modulating effects of PPARγ protein and its ligands may be
caused by its differential subcellular compartmentalization by MEK1.


### 2.3.3. Tumor-Promoting Functions of PPARγ,  Related to Crosstalk with the ERK Cascade

Metastasis
In contrast to the initial
assumption of PPARγ mainly acting as a tumor suppressor whose activity and/or expression is
lost in cancers, PPARγ expression and activity can also be a negative predictor of cancer
aggressiveness; and positive cooperation between PPARγ and components of the ERK cascade
in malignant phenotypes takes place. For example, strong nuclear PPARγ expression was detected in thyroid
carcinomas compared to normal tissue, and patient samples of thyroid
carcinoma-associated lymph node metastasis also showed a higher percentage of
PPARγ-positive staining than other case categories [129]. PPARγ expression was also elevated in
human prostate cancer compared to normal prostate [130]. In patients with invasive breast
carcinoma, cytoplasmic MT1-MMP and MMP9 expression positively correlated with
PPARγ levels [131]. These data corroborated a positive
relationship between PPARγ expression and malignancy state in certain tumor entities, a fact that
was shown to be therapeutically exploitable by the use of PPARγ antagonists or siRNA. This was
described in primary esophageal tumor specimen and in esophageal cancer cell
lines [132], in human primary squamous cell
carcinoma (SCC) and lymph node metastases [133] and in hepatocellular carcinoma
(HCC) samples [134], where PPARγ expression is elevated compared to
matched normal tissue. In all three cell systems, PPARγ antagonists (T0070907,GW9662) and
RNAi-mediated knock-down of PPARγ levels reduced the invasiveness and
adherence of cells to the extracellular matrix, triggered anoikis, or inhibited
proliferation by decreasing the phosphorylation status of focal adhesion kinase
(FAK), MEK, and ERK. Therefore, in tumors where elevated PPARγ and activated ERK and MEK levels
contribute to the malignant phenotype, inhibition of PPARγ may be beneficial as a therapeutic
strategy (see also Section 3).Angiogenesis
The overall vascular protective and
antiatherogenic effects of PPARγ ligands provide essential add-ons for the clinical application as
insulin sensitizers (reviewed in [13]). However, the proangiogenic
effects of PPARγ ligands via modulation of the VEGF/VEGF-receptor system (that signals
via the ERK cascade) have gained recognition (reviewed by [49]), which may be beneficial for
therapy of vascular diseases (e.g., infarction) [135, 136] but detrimental in cancer tissue.
For example, in rat myofibroblasts, rosiglitazone and 15-deoxy-Δ(12,14)-PGJ2 induce expression of
VEGF and its receptors (Flt1 and KDR, that signal via the ERK cascade), and
augment tubule formation on a matrigel, indicative of a promoting function of
PPARγ and ERKs in angiogenesis [137]. In osteoblast-like MC3T3E1 cells,
pioglitazone and ciglitazone augmented FGF2-induced VEGF release in a PPARγ-dependent manner and enhanced the
phosphorylation of JNK [138]. In human RT4 bladder cancer cells,
VEGF mRNA and protein are upregulated by PPARγ via activation of the VEGF
promoter. Interestingly, the MEK inhibitor PD98059 reduced PPARγ ligand-induced expression of VEGF [139], indicative of a positive
cooperation of PPARγ-ERK pathways in angiogenesis. These positive effects on angiogenesis
were examined also in two clinical studies with rosiglitazone [140] and pioglitazone [136], in which it was demonstrated that
chronic addition of the TZDs increased endothelial cell precursor counts and
migration in diabetic patients, raising concern on the proangiogenic potential
of TZDs.
Taken together, the data
which revealed an antagonistic cooperation of PPARγ and ERK signaling in several cell
or tissue-specific differentiation systems (skin, bone, muscle, fat) is now
challenged by the findings of positive cooperation of the same components  in tumor progression (metastasis,
angiogenesis). Thus, the role of PPARγ as a MEK/ERK-regulated tumor
suppressor seems to be of importance in normal tissue or in prevention of tumor
initiation, while in advanced stages of certain tumors a synergistic
cooperation between PPARγ and the ERK cascade may contribute to the malignancy of the disease.
Future studies have to clarify whether PPARγ agonists, PPARγ antagonists, or PPARγ modulators/partial agonists
(SPPARMs) with a selective effector profile [141] may be of interest for the therapy
of certain tumor entities.


## 2.3.1. Spatial Regulation of PPARγ Activity:  MEKs Export PPARγ to the Cytoplasm

Next to Ser84/114 phosphorylation and the nongenomic ERK activation by PPARγ ligands, the direct interaction of
PPARγ with the ERK cascade component MEK1 constitutes a third mechanism of
crosstalk between PPARγ and the ERK cascade. Subcellular compartmentalization is a major
mechanism in regulating cellular signaling. Interestingly, PPARγ itself can regulate the membrane
translocation of other proteins such as NFκB in gut intestinal epithelial cells
[96] and PKC in macrophages [97]. Several reports have demonstrated
a signal-mediated translocation of PPARγ between the nucleus and the
cytoplasm in vitro (as reviewed in [98]). In addition, it was shown that
PPARγ is expressed predominantly in the nucleus of nonneoplastic tissues,
whereas it is present in both the nucleus and the cytoplasm of tumorous tissues
in squamous cell carcinoma (SCC) of the lung, indicative of a correlation of
malignancy with differential PPARγ compartmentalization [99]. Moreover, a dominant negative PPARγ splice variant was described in
lung SCC patients, an event that leads to the loss of apoptosis sensitivity in
response to oxidative stress and cisplatin [99]. Differential compartmentalization
of PPARγ was also described in gastric cancer patients [100]. The ratio of cytoplasmic/nuclear
PPARγ expression decreased in the progression of intestinal metaplasia to
undifferentiated cancers [100]. In salivary duct carcinoma, an
aggressive tumor type, PPARγ is highly expressed (80%) and topographically located in the cytoplasm [101], indicative of an inactivation of
its genomic activities in the nucleus. Cytoplasmic PPARγ was also detected in the cytoplasm
(58%) of infiltrating breast carcinoma samples and was proposed as an
independent prognostic factor for patients with ductal carcinoma [102]. However, the function of this
subcellular distribution of PPARγ molecules are yet unknown.The mechanism that may
induce the changes in localization of PPARγ upon stimulation, or upon
neoplastic transformation was
only recently elucidated by us [30]. We showed that PPARγ is exported from the nucleus to the
cytoplasm by MEK1/2. This is induced by a reversible interaction of PPARγ with MEK1 through association of
the AF2 of the first with the N-terminal docking domain of MEK1. This export to
the cytoplasm (Figure 1(c)) leads to reduction in its genomic function
in the nucleus [30]. We also elucidated the molecular
mechanisms of the export and the physiological implications, but the question
remained is whether cytoplasmatically located PPARγ is subjected to degradation or
shunted to alternative signaling compartments such as lipid droplets, ER/Golgi,
cytoskeleton, or the plasma membrane. To this regard, we tend to speculate that
alternative locations of PPARγ in the cell may determine the balance between tumor-suppressive and tumor-promoting
functions.


## 2.3.2. Tumor-Suppressive Functions of PPARγ Related to ERKs and MEKs Interaction

Due to the coexpression of the
ubiquitous proteins PPARγ and MEK1/2 in different organs of the body, it was interesting to
identify their coregulation in various physiological and pathological
processes, as described below.Differentiation
Due to the lethality of MEK1 knockout mice [103] and absence of phenotypes in MEK2
knockout mice [104], the major focus of interest was
directed towards the role of MEK1 overexpression in vivo. Constitutively active
MEK1 (S218E/S222E) has been conditionally overexpressed (among other tissues)
in the skin and bone of mice [105]. All transgenic mice exhibited
increased cell numbers (hyperplasia) and cell size and a defect in terminal
differentiation. Interestingly, both in skin and in bone of mice, PPARγ was shown to be an important player
promoting differentiation [2]. In addition, the constitutively
active MEK1 overexpressing mice show dwarfism and reduced bone size due to
defective ossification and impaired chondrocyte differentiation. In other
systems, it was shown that osteoclast-specific PPARγ knockout mice are characterized by
increased bone mass due to impaired osteoclast differentiation [106], suggesting antagonistic effects of
PPARγ and MEK1 on different bone cell types: with PPARγ promoting osteoclast
differentiation, and MEK1 inhibiting chondrocyte differentiation.
Skin-restricted MEK1
transgenic mice exhibit hyperproliferation, hyperkeratosis and of age
papillomas at sites of wounding [105, 107]. Vice versa, epidermis-specific
knockout of MEK1/2 in mice [108] resulted in hypoproliferation,
apoptosis, skin barrier defects, and death, indicative of a positive role of
MEK1 in skin proliferation and tissue homeostasis. PPARγ knockout mice are characterized by
an increased sensitivity to experimentally-induced skin tumors [35], emphasizing the tumor suppressor
and differentiation promoting activity of PPARγ in the skin. These “mirror-images”
phenotypes in the organs where MEK1/2 and PPARγ are normally coexpressed may give
some indication for the antagonistic regulation of the two proteins, MEK
promoting proliferation and dedifferentiation, PPARγ promoting terminal differentiation.
In line with this idea, it was shown that the kinase activity of MEK1 was
actually dispensable for the hyperproliferative and integrin-inducing effects
of the MEK1 in mouse skin [109]. Instead, a kinase-dead mutant of
MEK1 elicited the same phenotype, indicative of an involvement of other
MEK1-functions such as scaffolding inhibition of differentiation-promoting
cellular factors.
In adipogenic
differentiation systems originating from (mesenchymal) stem cells, synergistic
cooperations between the MEK-ERK cascade and PPARγ have been described. In
fibroblasts, differentiating towards the adipogenic lineage, a positive
cooperation between PPARγ and MEK1 exists that facilitates the adipogenic program by
MEK1-dependent induction of the C/EBPα gene [110]. In bone marrow-derived mesenchymal
stem cells isolated from normal and streptozotocin (STZ)-induced diabetic FVB/N
mice, high glucose enhanced adipogenesis, lipid accumulation, and PPARγ expression via PI3K/AKT and ERK
cascade signaling, events that were all inhibited by the MEK-inhibitor PD98059 [111]. In differentiated C2C12 myocytes,
the free fatty acid palmitate reduces the mRNA levels of PPARγ-coactivator-1α (PGC1α) and activated MEK, while the MEK
inhibitors PD98059 and U0126 prevented such downregulation of PGC1α, indicative of a MEK-mediated
inhibition of an important NR coactivator protein for PPARγ in muscle cells [112]. These findings corroborated that
the MEK-ERK cascade and PPARγ signaling pathways can syn- or antagonistically cooperate to control
the balance of proliferation and differentiation in an organ/cell type-specific
manner.Cell cycle
The ERK cascade participates in
the regulation of cell cycle at (i) G0/G1 and G1/S transitions in response to
mitogenic stimulation (as reviewed in [24]) and (ii) in the process of Golgi
fragmentation [113–115] during mitosis. This is mediated in
part by the nuclear translocation of ERK upon cellular stimulation that
promotes expression of “immediate early” genes such as members of the AP1
family that activate the promoters of the G1 cyclins D and E. However, the
subcellular compartmentalization of ERK signaling by scaffold proteins (KSR,
MP1/p14, Sef) (reviewed in [22]) indicates a novel mode of spatial
separation of substrate specifities and signal translation. For example, MP1
via the adapter protein p14 tethers MEK1 to endosomes [116] and focal adhesions [117]. Sef translocates MEK1 to the Golgi
apparatus, prevents nuclear translocation of ERKs, and, thereby, favours
phosphorylation of cytoplasmic ERK substrates instead of nuclear ones [118]. The latter subcellular
localization-determining systems may thus be as well exploited by the PPARγ-MEK1 nuclear export shuttle to
regulate the cell cycle.
The PPARγ receptor has been involved in the
inhibition of the G0/G1-transition by up-regulation of genes coding for the
CDK-inhibitors p18(INK4C) [119] and p21(WAF1/CIP1) [120, 121], and in the inhibition of G1/S
transition through upregulation of the p27(KIP1) gene [122, 123]. Upregulation of other genes
implicated in cell cycle control such as PTEN or members of the BCL-gene family
contributes to the
growth-arresting and/or apoptosis-inducing action of PPARγ ligands [15]. The cell cycle modulatory actions
of PPARγ are usually not mediated through classical PPRE binding at the DNA but
rather through PPRE-independent “off-DNA” crosstalk to other transcription
factors [15] and through nongenomic effects in
the cytoplasm, such as inhibition of translation initiation [124, 125] and modulation of the proteasomal
machinery [126–128]. The latter processes may be
mediated by ligand-activated cytoplasmic PPARγ molecules or cytoplasmic
alternative signal-transducers for PPARγ ligands. We therefore hypothesize
that, by nuclear export and cytoplasmic retention of PPARγ-MEK1 complexes to other
MEK1-scaffolding locations (e.g., at the Golgi, endosomes, focal adhesions), the
genomic PPARγ functions may as well be redirected in favour of cytoplasmic signaling
events. In sum, the cell cycle modulating effects of PPARγ protein and its ligands may be
caused by its differential subcellular compartmentalization by MEK1.


## 2.3.3. Tumor-Promoting Functions of PPARγ,  Related to Crosstalk with the ERK Cascade

Metastasis
In contrast to the initial
assumption of PPARγ mainly acting as a tumor suppressor whose activity and/or expression is
lost in cancers, PPARγ expression and activity can also be a negative predictor of cancer
aggressiveness; and positive cooperation between PPARγ and components of the ERK cascade
in malignant phenotypes takes place. For example, strong nuclear PPARγ expression was detected in thyroid
carcinomas compared to normal tissue, and patient samples of thyroid
carcinoma-associated lymph node metastasis also showed a higher percentage of
PPARγ-positive staining than other case categories [129]. PPARγ expression was also elevated in
human prostate cancer compared to normal prostate [130]. In patients with invasive breast
carcinoma, cytoplasmic MT1-MMP and MMP9 expression positively correlated with
PPARγ levels [131]. These data corroborated a positive
relationship between PPARγ expression and malignancy state in certain tumor entities, a fact that
was shown to be therapeutically exploitable by the use of PPARγ antagonists or siRNA. This was
described in primary esophageal tumor specimen and in esophageal cancer cell
lines [132], in human primary squamous cell
carcinoma (SCC) and lymph node metastases [133] and in hepatocellular carcinoma
(HCC) samples [134], where PPARγ expression is elevated compared to
matched normal tissue. In all three cell systems, PPARγ antagonists (T0070907,GW9662) and
RNAi-mediated knock-down of PPARγ levels reduced the invasiveness and
adherence of cells to the extracellular matrix, triggered anoikis, or inhibited
proliferation by decreasing the phosphorylation status of focal adhesion kinase
(FAK), MEK, and ERK. Therefore, in tumors where elevated PPARγ and activated ERK and MEK levels
contribute to the malignant phenotype, inhibition of PPARγ may be beneficial as a therapeutic
strategy (see also Section 3).Angiogenesis
The overall vascular protective and
antiatherogenic effects of PPARγ ligands provide essential add-ons for the clinical application as
insulin sensitizers (reviewed in [13]). However, the proangiogenic
effects of PPARγ ligands via modulation of the VEGF/VEGF-receptor system (that signals
via the ERK cascade) have gained recognition (reviewed by [49]), which may be beneficial for
therapy of vascular diseases (e.g., infarction) [135, 136] but detrimental in cancer tissue.
For example, in rat myofibroblasts, rosiglitazone and 15-deoxy-Δ(12,14)-PGJ2 induce expression of
VEGF and its receptors (Flt1 and KDR, that signal via the ERK cascade), and
augment tubule formation on a matrigel, indicative of a promoting function of
PPARγ and ERKs in angiogenesis [137]. In osteoblast-like MC3T3E1 cells,
pioglitazone and ciglitazone augmented FGF2-induced VEGF release in a PPARγ-dependent manner and enhanced the
phosphorylation of JNK [138]. In human RT4 bladder cancer cells,
VEGF mRNA and protein are upregulated by PPARγ via activation of the VEGF
promoter. Interestingly, the MEK inhibitor PD98059 reduced PPARγ ligand-induced expression of VEGF [139], indicative of a positive
cooperation of PPARγ-ERK pathways in angiogenesis. These positive effects on angiogenesis
were examined also in two clinical studies with rosiglitazone [140] and pioglitazone [136], in which it was demonstrated that
chronic addition of the TZDs increased endothelial cell precursor counts and
migration in diabetic patients, raising concern on the proangiogenic potential
of TZDs.
Taken together, the data
which revealed an antagonistic cooperation of PPARγ and ERK signaling in several cell
or tissue-specific differentiation systems (skin, bone, muscle, fat) is now
challenged by the findings of positive cooperation of the same components  in tumor progression (metastasis,
angiogenesis). Thus, the role of PPARγ as a MEK/ERK-regulated tumor
suppressor seems to be of importance in normal tissue or in prevention of tumor
initiation, while in advanced stages of certain tumors a synergistic
cooperation between PPARγ and the ERK cascade may contribute to the malignancy of the disease.
Future studies have to clarify whether PPARγ agonists, PPARγ antagonists, or PPARγ modulators/partial agonists
(SPPARMs) with a selective effector profile [141] may be of interest for the therapy
of certain tumor entities.


## 3. Clinical Use of PPARγ Interaction with the ERK Cascade as a Drug Target

Reactivation (“differentiation”) therapy targeting functional PPARγ protein in cancer cells/tissues by
exogenous application of TZD-class PPARγ ligands was lately expected to
represent a novel approach to fight cancer [142]. However, differentiation-inducing
monotherapy with TZDs did not show the expected clinical benefit [11]. Instead, evidence accumulated that
alternative (“nongenomic”) PPARγ signaling pathways, crosstalk with the ERK cascade and elevated PPARγ expression levels in certain tumor
types (where PPARγ is postulated to act as a prosurvival factor, e.g., in hepatocellular
carcinoma, squamous cell carcinoma), are the cause for the observed tumor
promoting effects of PPARγ ligands, and may explain the absence of clear therapeutical benefit of
TZDs in cancer patients [78, 79, 143]. Therefore combination therapy of
PPARγ ligands with kinase inhibitors may represent a novel strategy to
circumvent the crosstalk of PPARγ and ERK cascade signaling and limit
PPARγ protein activation to its classical differentiation-inducing feature (Figure 2). This dual approach is expected to avoid (a) ERK cascade-mediated
downregulation of PPARγ, (b) MEK-driven nuclear export and cytoplasmic retention of PPARγ and (c) nongenomic amplification
loops of PPARγ ligands towards the ERK cascade, but to promote (d) the
growth-arresting and proapoptotic genomic functions of PPARγ and its ligands, and (e) the
negative crosstalk of PPARγ with promitotic and proinflammatory transcription factors in the
nucleus. This concept may not be suitable for tumor types with elevated
“malignant” PPARγ expression/activities. However, due to the lack of clinically approved
PPARγ antagonists, no statement can be currently made on the potential
therapeutical benefit of PPARγ and kinase coinhibition.Figure 2
Model of the combination therapy using PPARγ ligand and ERK cascade
inhibitors. The simultaneous inhibition of EGF
receptor-initiated ERK cascade activation by specific kinase inhibitors (-ibs)
or antibodies (-MABs) and supply of PPARγ ligands (in tumors that have a need
for restored PPARγ activity) will avoid: (a) ERK-mediated downregulation of PPARγ through Ser84/114 phosphorylation,
(b) MEK1-driven nuclear export and cytoplasmic retention of PPARγ, (c) activation of prosurvival and
proproliferative ERK cascade signaling by exogenous PPARγ ligands (e.g., by TZD drugs) or
endogenous eicosanoid type of PPARγ ligands (e.g., generated by
COX1/2), but is expected to (d) restore the differentiation-inducing and
proapoptotic functions of PPARγ and its ligands, and (e) promote the transrepressive activity of PPARγ on other promitotic and
proinflammatory transcription factors (e.g., AP1, ETS, STAT, NFκB). Legend: Yellow circles =
PPARγ-ligand; TF = transcription factors; ROS = reactive oxygen species; GPCR
= G protein coupled receptor; RPTK = receptor protein tyrosine kinase; crm1 =
exportin1; NSAID = nonsteroidal anti-inflammatory drug; COX = cyclooxygenase;
-Ibs = LMW tyrosine kinase inhibitors; MABs = monoclonal tyrosine kinase
antibodies.


### 3.1. In Vitro Studies

The combination of PPARγ ligands and inhibitors against
receptor tyrosine kinases of the EGFR-family or cytoplasmic tyrosine kinases
(e.g., Abl) revealed some promising results in leukemia and carcinoma cells.
Gefitinib, an inhibitor of the EGFR/Her1 kinase, exhibits antitumor activity in
only a fraction of 10–20% of patients
with nonsmall cell lung cancer (NSCLC) [144]. The mechanisms underlying this
resistance to gefitinib are not known. However, application of rosiglitazone
reduced the growth of the NSCLC A549 cells and potentiated the
antiproliferative effects of gefitinib and increased PPARγ and PTEN expression in these cells,
indicative of a potential benefit of this drug combination also in cancer
patients. MCF7 breast cancer cells stably transfected with ErbB2/Her2 displayed
reduced differentiation and enhanced resistance to TZD-driven inhibition of
anchorage-independent growth [145]. Herceptin, a monoclonal antibody
against Her2 kinase, sensitized cells for the differentiation-promoting and
growth-inhibitory effects of troglitazone. This concept also held true for
chronic myeloid leukemia (CML) cell lines, where TZD18 (a dual PPARα/γ ligand) enhanced CDK-inhibitor
p27(KIP1) expression and inhibited cyclin E, cyclin D2 and CDK2 [122]. TZD18 synergistically enhanced the
antiproliferative and proapoptotic effect of imatinib, a clinically used kinase
inhibitor of the Bcr-Abl fusion protein. Collectively, this work demonstrated
that the targeting of receptor tyrosine kinase signaling with LMW inhibitors or
monoclonal antibodies can improve the sensitivity of cancer cells to PPARγ ligand-mediated
growth inhibition.


### 3.2. In Vivo Rodent and Clinical Studies

The clinical outcome of
selective MEK inhibitors in patients studies was disappointing (CI-1040,
PD0325901, AZD-6244) (reviewed in [146, 147]). On the other hand, a Raf
inhibitor, sorafenib, was recently approved for clinical use; and novel
selective Raf inhibitors are under development [148]. So far no clinical studies were
performed using MEK or Raf inhibitors in combination with PPARγ ligands. However, successful
treatment data in mouse models or patients are available for combinations of
PPARγ ligands and three other types of inhibitory drugs: classical
chemotherapeutics, COX-inhibitors (NSAIDs), and established tyrosine kinase
inhibitors (imatinib, gefitinib, herceptin).NSAID/COX-inhibitors
have been shown to reduce the risk for colon carcinoma formation, however at
the expense of gastric ulcer and cardiovascular complications [19]. Several NSAIDs are also low-affinity
PPARγ ligands, a fact that led to the speculation that a part of the clinical
profile of these compounds is related to low-level activation of PPARγ [19]. Therefore, clinical trials with
combination therapies were initiated to exploit PPARγ activation and simultaneous
blockage of the promitotic and proinflammatory COX1/2-mediated eicosanoid
production, which contributes to nongenomic signaling in cancer tissues (Figure 2). Pilot clinical studies with an angiostatic triple combination of
pioglitazone, rofecoxib (a selective COX2 inhibitor), and trofosfamide showed
benefit in patients with angiosarcoma and hemangioendothelioma [151, 152] and advanced sarcoma [153]. A phase-II trial with the same
triple combination in patients with metastatic melanoma or soft-tissue sarcoma
evinced disease stabilization [152], indicative of a beneficial effect
of COX2 inhibition (whose eicosanoid metabolites activate the ERK cascade) and
simultaneous PPARγ activation in sensitization of tumor cells to differentiation and/or
apoptosis. A recently published outcome of a phase-II trial in high-grade
glioma patients (glioblastoma or anaplastic glioma) under pioglitazone and
rofecoxib combined with chemotherapy (capecitabine or temozolomide) also stated
some disease stabilization [157]. However, due to the severe side
effects of selective COX2-inhibitors this therapeutic regimen may raise concerns.Preclinical studies in
rodents provided evidence for a therapeutic potential of combination therapy
with other inhibitory agents. In mice xenografted with NSCLC A549 cells, the
PI3K inhibitor PX-866 potentiated the antitumor activity of gefitinib [149]. The glucose intolerance related to
PX-866 in mice was reversed by insulin and pioglitazone. PX-866 in combination
with insulin sensitizers may thus be useful in facilitating the response to
EGFR inhibition. The antitumoral action of rosiglitazone on experimentally
induced mammary tumors induced by N-nitroso-N-methylurea (NMU) in
Sprague-Dawley rats was potentiated by the selective estrogen-receptor
modulator (SERM) tamoxifen with respect to the extent of tumor cell apoptosis
and necrosis [150]. The PPARγ ligand RS5444 in combination with
paclitaxel had additive antiproliferative effect in vitro and minimized tumor
growth in nude mice xenografts of anaplastic thyroid carcinoma (ATC) cells [120]. These preclinical studies
underline that the combination of PPARγ ligands and established anticancer
drugs may be of clinical benefit also in cancer patients.Interestingly, two
studies provided already first-line evidence for the potential of an in vivo
reactivation of PPARγ protein function by simultaneous inhibition of the COX pathway-mediated activation of the ERK cascade: LY293111, an oral PPARγ ligand, leukotriene B4 receptor
antagonist and 5-lipoxygenase inhibitor, was validated for its antineoplastic
efficacy in combination with chemotherapy (irinotecan, gemcitabine) in
preclinical models [154] and evoked disease stabilization in
patients with advanced solid tumors [155, 156]. The NSAID R-etodolac inhibits
growth of prostate cancer (CWRSA6, LuCaP35) xenografts in mice by
downregulation cyclin D1. However, the combination of R-etodolac with herceptin
elicited an additive antitumor effect, reduced ERK phosphorylation and
stabilized PPARγ protein levels [158]. These therapeutic regimens
inhibited the eicosanoid-mediated activation of the ERK cascade, and in conjunction
with PPARγ activation, may provide a basis for differentiation-inducing therapy in
combination with classical chemotherapeutics or biologicals.So far no clinical
evidence was published on the combined use of ERK cascade inhibition and PPARγ activation (in tumors with low PPARγ expression/activity) or PPARγ inhibition (in tumors with high PPARγ expression/activity). In the
future, the combination of PPARγ ligands with kinase inhibition selectively targeted by MABs against the
EGFR tyrosine receptor kinase family or LMW selective inhibitors of the
downstream ERK cascade, such as Raf and MEK, may constitute a possible new
approach to treat cancer.


## 3.1. In Vitro Studies

The combination of PPARγ ligands and inhibitors against
receptor tyrosine kinases of the EGFR-family or cytoplasmic tyrosine kinases
(e.g., Abl) revealed some promising results in leukemia and carcinoma cells.
Gefitinib, an inhibitor of the EGFR/Her1 kinase, exhibits antitumor activity in
only a fraction of 10–20% of patients
with nonsmall cell lung cancer (NSCLC) [144]. The mechanisms underlying this
resistance to gefitinib are not known. However, application of rosiglitazone
reduced the growth of the NSCLC A549 cells and potentiated the
antiproliferative effects of gefitinib and increased PPARγ and PTEN expression in these cells,
indicative of a potential benefit of this drug combination also in cancer
patients. MCF7 breast cancer cells stably transfected with ErbB2/Her2 displayed
reduced differentiation and enhanced resistance to TZD-driven inhibition of
anchorage-independent growth [145]. Herceptin, a monoclonal antibody
against Her2 kinase, sensitized cells for the differentiation-promoting and
growth-inhibitory effects of troglitazone. This concept also held true for
chronic myeloid leukemia (CML) cell lines, where TZD18 (a dual PPARα/γ ligand) enhanced CDK-inhibitor
p27(KIP1) expression and inhibited cyclin E, cyclin D2 and CDK2 [122]. TZD18 synergistically enhanced the
antiproliferative and proapoptotic effect of imatinib, a clinically used kinase
inhibitor of the Bcr-Abl fusion protein. Collectively, this work demonstrated
that the targeting of receptor tyrosine kinase signaling with LMW inhibitors or
monoclonal antibodies can improve the sensitivity of cancer cells to PPARγ ligand-mediated
growth inhibition.


## 3.2. In Vivo Rodent and Clinical Studies

The clinical outcome of
selective MEK inhibitors in patients studies was disappointing (CI-1040,
PD0325901, AZD-6244) (reviewed in [146, 147]). On the other hand, a Raf
inhibitor, sorafenib, was recently approved for clinical use; and novel
selective Raf inhibitors are under development [148]. So far no clinical studies were
performed using MEK or Raf inhibitors in combination with PPARγ ligands. However, successful
treatment data in mouse models or patients are available for combinations of
PPARγ ligands and three other types of inhibitory drugs: classical
chemotherapeutics, COX-inhibitors (NSAIDs), and established tyrosine kinase
inhibitors (imatinib, gefitinib, herceptin).NSAID/COX-inhibitors
have been shown to reduce the risk for colon carcinoma formation, however at
the expense of gastric ulcer and cardiovascular complications [19]. Several NSAIDs are also low-affinity
PPARγ ligands, a fact that led to the speculation that a part of the clinical
profile of these compounds is related to low-level activation of PPARγ [19]. Therefore, clinical trials with
combination therapies were initiated to exploit PPARγ activation and simultaneous
blockage of the promitotic and proinflammatory COX1/2-mediated eicosanoid
production, which contributes to nongenomic signaling in cancer tissues (Figure 2). Pilot clinical studies with an angiostatic triple combination of
pioglitazone, rofecoxib (a selective COX2 inhibitor), and trofosfamide showed
benefit in patients with angiosarcoma and hemangioendothelioma [151, 152] and advanced sarcoma [153]. A phase-II trial with the same
triple combination in patients with metastatic melanoma or soft-tissue sarcoma
evinced disease stabilization [152], indicative of a beneficial effect
of COX2 inhibition (whose eicosanoid metabolites activate the ERK cascade) and
simultaneous PPARγ activation in sensitization of tumor cells to differentiation and/or
apoptosis. A recently published outcome of a phase-II trial in high-grade
glioma patients (glioblastoma or anaplastic glioma) under pioglitazone and
rofecoxib combined with chemotherapy (capecitabine or temozolomide) also stated
some disease stabilization [157]. However, due to the severe side
effects of selective COX2-inhibitors this therapeutic regimen may raise concerns.Preclinical studies in
rodents provided evidence for a therapeutic potential of combination therapy
with other inhibitory agents. In mice xenografted with NSCLC A549 cells, the
PI3K inhibitor PX-866 potentiated the antitumor activity of gefitinib [149]. The glucose intolerance related to
PX-866 in mice was reversed by insulin and pioglitazone. PX-866 in combination
with insulin sensitizers may thus be useful in facilitating the response to
EGFR inhibition. The antitumoral action of rosiglitazone on experimentally
induced mammary tumors induced by N-nitroso-N-methylurea (NMU) in
Sprague-Dawley rats was potentiated by the selective estrogen-receptor
modulator (SERM) tamoxifen with respect to the extent of tumor cell apoptosis
and necrosis [150]. The PPARγ ligand RS5444 in combination with
paclitaxel had additive antiproliferative effect in vitro and minimized tumor
growth in nude mice xenografts of anaplastic thyroid carcinoma (ATC) cells [120]. These preclinical studies
underline that the combination of PPARγ ligands and established anticancer
drugs may be of clinical benefit also in cancer patients.Interestingly, two
studies provided already first-line evidence for the potential of an in vivo
reactivation of PPARγ protein function by simultaneous inhibition of the COX pathway-mediated activation of the ERK cascade: LY293111, an oral PPARγ ligand, leukotriene B4 receptor
antagonist and 5-lipoxygenase inhibitor, was validated for its antineoplastic
efficacy in combination with chemotherapy (irinotecan, gemcitabine) in
preclinical models [154] and evoked disease stabilization in
patients with advanced solid tumors [155, 156]. The NSAID R-etodolac inhibits
growth of prostate cancer (CWRSA6, LuCaP35) xenografts in mice by
downregulation cyclin D1. However, the combination of R-etodolac with herceptin
elicited an additive antitumor effect, reduced ERK phosphorylation and
stabilized PPARγ protein levels [158]. These therapeutic regimens
inhibited the eicosanoid-mediated activation of the ERK cascade, and in conjunction
with PPARγ activation, may provide a basis for differentiation-inducing therapy in
combination with classical chemotherapeutics or biologicals.So far no clinical
evidence was published on the combined use of ERK cascade inhibition and PPARγ activation (in tumors with low PPARγ expression/activity) or PPARγ inhibition (in tumors with high PPARγ expression/activity). In the
future, the combination of PPARγ ligands with kinase inhibition selectively targeted by MABs against the
EGFR tyrosine receptor kinase family or LMW selective inhibitors of the
downstream ERK cascade, such as Raf and MEK, may constitute a possible new
approach to treat cancer.


## 4. Conclusion and Perspectives

In conclusion, PPARγ emerges as a tumor-type and
tumor-stage-specific modulator that is regulated by at least three mechanisms
through the ERK cascade. Downregulation is carried out through (1)
MAPK-mediated Ser84/114 phosphorylation, (2) ERK cascade
activation through PPARγ ligands, and (3) cooperation of PPARγ with tumor modulating proteins
(such as MEK1). The overlay of these 3 mechanisms of crosstalk is likely to
determine the physiological outcome of PPARγ effector functions. Consequently,
interference with these interactions by LMW inhibitors, antibodies, or
peptidomimetic drugs against protein docking interfaces may constitute a novel
approach to redirect PPARγ effector functions from a protumorigenic towards an antitumorigenic
profile. Simultaneous inhibition of ERK cascade-mediated signaling is expected
to prevent adverse promitotic and prosurvival pathways triggered by PPARγ and
its ligands. This
therapeutic approach is assumed to be reasonable in tumors where the tumor-suppressor
activities of PPARγ  are lost/reduced/dysfunctional
and should be restored. However, it may not be applicable for tumors where high
PPARγ expression/activity levels positively correlate with the state of
malignancy. Since no PPARγ antagonist or PPARγ modulator is in clinical use so far, future studies have to evaluate
whether (depending on the tumor type and stage) the combination of the latter
drugs with kinase inhibitors may be of therapeutical benefit in tumor entities
with high PPARγ expression.Table 1
Combination therapy with PPARγ ligands.
Cancer typePPARγ ligandCombinationInhibitor typeReferenceIn vitroCMLTZD18ImatinibAbl, other RPTKs[122]NSCLC A549RosiglitazoneGefitinibEGFR/Her1[144]Breast
MCF7TroglitazoneHerceptinMab-Her2/ErbB2[145]In vivo (human xenografts or
chemically-induced tumors in rodents)NSCLC A549PioglitazonePX-866
GefitinibPI3K-p110α
Her1/EGFR[149]Breast (by NMU)RosiglitazoneTamoxifenSERM[150]Thyroid ATCRS5444PaclitaxelChemotherapeutic[120]Clinical studiesMelanoma
SarcomaPioglitazoneRofecoxib TrofosfamideCOX2 Chemotherapeutic[151–153]Advanced Solid tumorsLY293111Irinotecan GemcitabineChemotherapeutic Chemotherapeutic[154–156]Glioblastoma Anaplastic GliomaPioglitazoneRofecoxib
Capecitabine TemozolomideCOX2 Chemotherapeutic
Chemotherapeutic[157]



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