# Rat Urinary Bladder Carcinogenesis by Dual-Acting  PPARα+γ Agonists

Authors: Martin B. Oleksiewicz; Jennifer Southgate; Lars Iversen; Frederikke L. Egerod
Journal: PPAR Research
 (2008)

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## Abstract

Despite clinical promise, dual-acting activators of PPARα and γ (here termed PPARα+γ agonists) have experienced high attrition rates in preclinical and early clinical development, due to toxicity. In some cases, discontinuation was due to carcinogenic effect in the rat urothelium, the epithelial layer lining the urinary bladder, ureters, and kidney pelvis. Chronic pharmacological activation of PPARα is invariably associated with cancer in rats and mice. Chronic pharmacological activation of PPARγ can in some cases also cause cancer in rats and mice. Urothelial cells coexpress PPARα as well as PPARγ,  making it plausible that the urothelial carcinogenicity of PPARα+γ agonists may be caused by receptor-mediated effects (exaggerated pharmacology). Based on previously published mode of action data for the PPARα+γ agonist ragaglitazar, and the available literature about the role of PPARα and γ in rodent carcinogenesis, we propose a mode of action hypothesis for the carcinogenic effect of PPARα+γ agonists in the rat urothelium, which combines receptor-mediated and off-target cytotoxic effects. The proposed mode of action hypothesis is being explored in our laboratories, towards understanding the human relevance of the rat cancer findings, and developing rapid in vitro or short-term in vivo screening approaches to faciliate development of new dual-acting PPAR agonist compounds.


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## Body

## 1. Introduction

Selective small molecule agonists for the peroxisome
proliferator-activated receptorsα and γ are used to treat metabolic disorders.
PPARα agonists (fibrates) are used for their blood lipid lowering effects, and
PPARγ agonists (thiazolidinediones) for their insulin sensitizing effects [1–3].
Additionally, dual-acting agonists for PPARα and PPARγ, here termed PPARα+γ
agonists, have been shown to have clear therapeutic advantages over selective
PPAR agonists in animals as well as humans [3, 4].
Unfortunately, a high percentage of PPARα+γ agonists exhibited carcinogenic
effect during preclinical safety testing in rats and mice [2–7]. Based
on carcinogenicity findings for 6 PPARα+γ and 5 PPARγ anonymous developmental
compounds in rats and mice, the FDA concluded that “PPAR agonists are
multispecies, multistrain, multisex, multisite carcinogens” (Table 1)
[8]. The
FDA further concluded that “mechanistic data to explain mode of action for
tumour formation is not available. Tumours sites are consistent with the known
distribution of PPAR receptors. Oncogenic potency correlates with PPAR agonist
potency. A receptor-mediated mechanism cannot be ruled out” [8].Table 1
Frequency of cancer findings for PPAR agonists in rats, mice, and hamsters. The table is adapted
from [8–10]
and comprises rodent carcinogenicity data for between 16 and 30 PPARα agonists
(pharmacological as well as industrial compounds) [9, 10],
5 PPARγ agonists (pharmacological compounds only) [8], and 6
dual-acting PPARα+γ agonists (pharmacological compounds only) [8].
Numbers in the cells: number of compounds causing the indicated pathology in
the indicated rodent species; M: male; F: female. The difference
in rodent bladder and liver tumour frequency between PPARα, PPARγ, and PPARα+γ
agonists is significant (P<.0001, Chi-square test). The difference in
rodent bladder cancer frequency betwen PPARγ and dual-acting PPARα+γ agonists
is borderline significant (P=.0357 and .081 by Chi-square and Fischer’s
exact tests, resp.).
(a)PPAR agonist selectivity (n, number of compounds)(b),(f)Hemangio-sarcoma(c),(f)Urinary bladder and renal pelvis(d)Fibrosarcoma(f)Lipoma and sarcoma(e),(f)LiverOtherPPARα agonists  (n=30 for hepatocarcinogenicity, n=16 for extrahepatic tumours)NoneNoneNoneNone30 of 30, in mice or ratsTypically pancreatic acinar cell and Leydig cell tumours. Thyroid and lung tumours and  leukaemia also described.PPARγ agonists (n=5)3 (mice, M and F)1 (rats, M and F)None3 (rats, M and F)2 (rats and mice, F)1 (mice, gallbladder adenoma). 1 (rats, stomach, leiomyosarcoma).Dual acting PPARα+γ agonists(n=6)5 (mice, M and F, hamster, M)5 (rats, M and F)2 (rats, M and F)2 (rats, mice, M and F)3 (rats, mice, M and F)1 (rats, testicular). 1 (rats, mammary). 1 (mice, mammary and stomach).
1 (rats, thyroid). 1 (rats, uterine). 1 (rats, uterine and leukaemia).(a)Comparative
data for PPAR selectivity are lacking. No study has to our knowledge for a
panel of PPAR agonists compared activity on all PPAR isoforms, between rats,
mice, and humans, in the relevant cell type, for example, hepatocyte or
urothelial. However, it is clear that selective PPARγ agonists may have
significant PPARα activity [3, 5, 11].
(b) Mice
appear to be more sensitive to the effect of PPARγ agonists than rats [12].
(c)Rat
urothelium may be more sensitive to the carcinogenic effect of dual-acting
PPARα+γ agonists than mouse urothelium. Bladder cancer was seen in SD, Wistar,
and Fischer rats of both sexes [8]. Renal
proximal tubular carcinoma was also observed with 2 dual agonists
(undifferentiated sarcomatous tumours) [8].
(d)One
dual-acting PPARα+γ agonist for which fibrosarcoma has been described is
tesaglitazar [13].
(e)One
PPARγ agonist for which hepatocarcinogenesis has been described is troglitazone
[14].
(f)PPARα
and PPARγ are typically described as having a tissue-restricted expression,
with PPARβ expression being more ubiquitous [2, 15, 16].
Endothelial as well as urothelial cells coexpress PPARα and PPARγ isoforms [17–20].
White fat expresses mostly PPARγ [2, 15, 16],
but it is increasingly recognized that PPARα may also have function in white
fat [21]. Liver
expresses mostly PPARα [2, 15, 16],
but it is increasingly recognized that PPARγ may also have function in the
liver [22–26].Accordingly, the attrition rate amongst developmental
PPARα+γ agonists has been high, with amongst others tesaglitazar (Galida),
naveglitazar (LY519818), muraglitazar, ragaglitazar, farglitazar, and
imiglitazar (TAK559) recently being discontinued due to clinical cardiac,
kidney or liver toxicity, or preclinical findings [3–5]. These
6 developmental PPARα+γ agonists represent different nonthiazolidinedione
chemical structures, with different balances between PPARα and γ activation [3, 5, 7, 11].
For 4 dual-acting agonists, preclinical carcinogenicity findings have been
published muralitazar, ragalitazar, tesaglitazar, and naveglitazar are
nongenotoxic by standard tests. Muralitazar caused gallbladder adenomas (male
mice), adipocyte neoplasms (male and female rats), and urinary bladder tumours
(male rats) [27]. Ragalitazar
caused urinary bladder and renal pelvis tumours (male and female rats) [28–30].
Naveglitazar caused urinary bladder tumours in female rats, with the evaluation
of carcinogenicity in male rats affected by poor survival [31].
Tesaglitazar caused mesenchymal sarcomas (male and female rats) [13].The involvement of PPARα and PPARγ in cancer pathogenesis
has been reviewed extensively [2, 5, 6, 32–36]. While
PPARα activation is clearly carcinogenic in rodents [9, 32, 33],
the rodent PPARγ data are controversial, and it appears that rodent PPARγ
activation may have oncogenic as well as tumour suppressor activity, likely
depending amongst others on cellular and physiological  contexts [5, 6, 8, 9, 34, 36].
Further, potential interactions between rodent PPARα and PPARγ in coexpressing
cells have, to our knowledge, essentially not been examined at all. Finally,
the human relevance of rodent data is unknown, as there is indication that, for
example, PPARγ agonists may have clinical benefit against certain human cancers
such as lung cancer [37].We base the present manuscript on the observation that in
rats, toxicity of dual-acting PPARα+γ agonists appears to target cells coexpressing
PPARα and PPARγ, resulting in a qualitatively different target organ profile
from that of selective PPARα and PPARγ agonists (Table 1). Then, we review the
literature with the aim of constructing a mode of action hypothesis for the
carcinogenic effect of ragaglitazar in the rat urothelium. Due to the
complexity of the available data, this is by definition speculative, and
involves weighing of probablilities rather than combining facts, but the
presented mode of action hypothesis forms the basis for our own research
regarding the mechanisms by which PPAR agonists induce cancer in the rat
urothelium.


## 2. Comparing Normal Physiology of  PPARs in the Rat and Human Urothelium:  Toward Safety PPARallelograms

Expression of PPAR transcripts by urothelium occurs early
in development and is conserved across species [17, 38],
implying a tissue-specific role. In normal human urothelium, PPARγ is most intensely expressed in the terminally differentiated
superficial cell layer [39, 40], and
expression is decreased in high-grade urothelial cell cancer [41],
further indicating a potential role in urothelial cytodifferentiation.Normal human urothelium isolated from urological
specimens from patients with no history of urothelial cancer can be routinely
established in serum-free primary cell culture and maintained through multiple
serial subcultures (typically 6–10) as cell lines
with a finite lifespan [42, 43]. These cultures show a regenerative phenotype
and do not spontaneously express gene/proteins associated with late/terminal
differentiation [42, 43].Activation of PPARγ  by agonists
(troglitazone or rosiglitazone) in finite cultures of normal human urothelial
cells (NHU cell cultures) has been shown to induce expression of gene/protein
markers associated with late/terminal urothelial differentiation, including
uroplakins, cytokeratins, and tight junction constituents [40, 44–46]. The
proposed differentiation-inducing mechanism is via PPARγ-dependent transduction of intermediary
transcription factors, including HNF3α, IRF-1, and FOXA1, and the induction of
differentiation is specifically blocked by PPARγ antagonists, or siRNAs against PPARγ,  IRF-1,
and FOXA1 [46]. The
induction of differentiation in NHU cell cultures by troglitazone requires
inhibition of PI3K/AKT or MEK1/ERK signalling pathways downstream of EGFR [44], which
in NHU cells is important for driving proliferation [47].
Inhibition of the downstream EGFR pathways resulted in dephosphorylation of
PPARγ  [44]. The
interaction between the signalling pathways that regulate differentiation (PPARγ) and proliferation (EGFR) in urothelium may
lie at the heart of regulating urothelial homeostasis and the switch from
quiescent to regenerative phenotypes.While PPARγ activation in NHU cell cultures induces
differentiation [40, 44–46], some
selective PPARγ and most dual-acting PPARα+γ agonists cause
bladder cancer in rats (Table 1) [8, 48]. Also,
a recent study showed that the specific PPARγ agonist rosiglitazone is a strong
promoter of hydroxybutyl(butyl)nitrosamine-induced bladder cancer in rats [49]. It is
unknown whether this apparent contradiction represents a species difference, or
a difference between in vivo and in
vitro experimental systems. Also, it is well known that the outcome of
PPARγ signalling is highly context specific, that is, diametrally opposite
biological effects can be seen in different situations [5, 6, 34, 36].
Resolution of the different observations in NHU cell cultures in vitro and rat tissue in vivo is of
obvious relevance for elucidating the bladder carcinogenicity mechanisms of
dual-acting PPARα+γ agonists in rats, and elucidating the human relevance of
the rat bladder cancer findings (Table 1). Using a standard “safety
parallelogram” approach for extrapolating the human relevance of rodent
findings, PPAR signalling should be compared between rat urothelial cells in vivo and in vitro, and also
between rat and human urothelial cells in
vitro.In this vein, we have recently compared normal rat and
human urothelia in situ as well
as following culture, and confirmed urothelial expression of all three PPARs and the RXRα and RXRβ isoforms by immunolabelling. Some difference
in relative expression and localisation of the different isoforms was apparent between
species [18]. Also,
rat urothelium exhibited a higher proliferative pool of Ki67 positive
urothelial cells than did human urothelium [18], in
agreement with a high percentage of G2/M cells in rat urothelium [30]. In
contrast, human urothelial cells in
situ appear arrested in G0/G1 [47]. The
relevance of these differences between rat and human urothelia for PPAR
signalling is at present unknown. However, PPAR and RXR expression patterns were
retained by both NHU and cultured normal rat urothelial cells, opening the
possibility that normal urothelial cell culture systems may be used to compare
PPAR signalling between rats and humans [18, 42, 43, 50].In short, most knowledge about PPARγ signalling in urothelium stems from NHU cell
cultures [40, 44–47], and
very little information exists regarding PPARα signalling response in the urothelium [51, 52]. Nevertheless,
based on the observation that bladder cancer appears overrepresented for
dual-acting PPARα+γ agonists (Table 1), and direct experimental indication of
cross-talk between PPARα and PPARγ in urothelium as well as other cell types [19, 28, 53–55], our
current hypothesis is that simultaneous activation of PPARα and PPARγ could in some way modulate the
proliferation/differentiation balance, contributing to carcinogenesis of
dual-acting PPARα+γ agonists in the rat urothelium (Figure 3).


## 3. Ranking the Possible Mechanisms for the Carcinogenic Effects of Ragaglitazar  and Naveglitazar in the Rat Urothelium: What Is PPARt of the PPARcel?

Ragaglitazar is a phenyl propanoic acid derivative with
dual PPARα+γ agonist activity [56, 57]. In 2-year
rat carcinogenicity assays, papillomas and carcinomas originating from the
transitional epithelial (urothelial) lining of the urogenital tract were
observed for all groups receiving ragaglitazar, for both male and female
animals [29]. The
urothelial papillomas and carcinomas were observed in the urinary bladder, ureters,
and renal pelvis [29]. In
mouse 2-year studies, one urinary bladder tumour was observed in a high-dose
male mouse [29]. The
higher sensitivity of rats than mice to urothelial tumours induced by
ragaglitazar may be shared by other dual-acting PPARα+γ agonists (Table 1).Four nonexclusive mechanisms were initially considered
for the urothelial tumours in ragaglitazar-treated rats (Figure1): (i) a receptor-mediated effect of the
parent compound, with carcinogenesis caused by activation of PPARα and γ
transcription factors in the urothelium, that is, an exaggerated
pharmacological effect, (ii) a genotoxic
effect of metabolites of the parent compound (the parent compound itself is not
genotoxic), (iii) a cytotoxic
effect of parent compound or metabolites on urothelium, causing cancer due to a
proliferation-driven chronic wound healing response, (iv) formation of urinary solids (urolithiasis) due to urinary
changes induced by parent compound or metabolites, leading to cancer due to
chronic irritation of the urothelium.Possible mechanisms for the carcinogenic effect of dual-acting PPARα+γ
agonists in the rat urothelium. (a) Simplified view of
the rat urinary tract, showing the urothelial lining of urinary bladder,
ureters, and kidney pelvis. The star in the bladder lumen indicates the
expected predilection site for urolith residence, the ventral part of the
bladder. In the shown part of the urogenital tract, there are no gross
anatomical differences between male and female rats. The epithelial lining is
contiguous but exhibits differentiation differences through the urogenital
tract [58, 59]. The
drawing is not to scale. (b) Four possible
mechanisms for carcinogenic effect in rat urothelium by dual-acting
PPARα+γ agonist.
(a)(b)It is well known that certain agents cause urinary
bladder cancer in rodents secondary to urolith formation [48], and
that such carcinogenic effect is not relevant for humans because in humans
uroliths do not predispose for bladder cancer [60]. A
urolithiasis-mediated mechanism would be expected to affect primarily (albeit
not exclusively) male rats due to the lower efficacy with which males void
uroliths and act primarily in the ventral part of the urinary bladder (Figure 1) [48].
Therefore, a urolithiasis-mediated mechanism was ruled out primarily by the
observation that ragalitazar caused tumours also in the ureters and renal
pelvis, a conclusion supported by the occurrence of bladder tumours in females [29]. In
detailed follow-up examinations in ragaglitazar-treated animals, urinary
calculi were not detected during necropsy, no microcrystals were found to
adhere to the urothelium by scanning electron microscopy, sediments were not
increased in the urine by light microscopy, no significant changes were
observed in urinary composition [29].
Likewise, and naveglitazar did not cause changes in urinary composition [31].To explore mechanism (ii), profiling of urinary metabolites by mass spectroscopy and
examination of DNA damage in urothelium isolated from ragaglitazar-treated rats
by single-cell gel electrophoresis assay (COMET) were performed. Ragaglitazar
exhibited multiple metabolites in rat urine (>10), but there was low overall
urinary excretion, and DNA damage was not observed in the urinary bladder of
ragaglitazar-treated rats [29].In summary, neither urinary calculi nor genotoxic damage
by urinary metabolites could explain the carcinogenic effect of ragaglitazar in
the rat urothelium. As a working hypothesis, we, therefore, assumed that
ragaglitazar caused urothelial cancers in rats by a receptor-mediated effect of
the parent compound (mechanism (i)
above), which may be exacerbated by a cytotoxic effect of parent compound or
metabolites on urothelium, promoting a
chronic wound healing response (mechanism (iii) above). This mode of action hypothesis (Figure3),
comprising 2 nonexclusive mechanisms (Figure 1(b), (i) and (iii))
was in agreement with coexpression of PPARα and γ by the urothelium [17, 18, 38],
with the known propensity of various PPAR agonists to exhibit cytotoxic effects
[36, 61, 62],
and with the known positive correlation between cytotoxic and carcinogenic
effects for some small molecule drugs [63, 64].
Similarly, it was concluded for naveglitazar-induced bladder cancer in rats
that a mechanism involving a direct effect of the compound on PPARs in the
urothelium should be considered [31].


## 4. Early Biomarkers for Ragaglitazar and Naveglitazar Actions in the Rat Urothelium

The mode of action hypothesis detailed above (Figure3)
predicted that very early (precancerous) changes should occur in the urothelium
of ragaglitazar-dosed rats, reflecting exaggerated pharmacology and/or
cytotoxicity of the compound.To test whether this was the case, a method was developed
where a lysing guanidine buffer is injected in situ in the bladder of anaesthetised treated animals,
providing selective lysis of the urothelial layer and minimizing the risk of
preparation artefacts (Figure2).Lysis of the rat bladder urothelial cell layer in situ. (a), (b) On a fully
anesthetized rat, the bladder is exposed through an abdominal incision, a thin
needle or catheter (27G) is introduced into the bladder at the bladder neck,
and ligated in place with a silk suture. (c) The bladder is
emptied for urine, and filled with approximately 0.5 mL lysis solution (4 M
guanidine isothiocynate, 0.5% sarcosine, 25 mM citrate, pH 5.5), which is left
in place for 2 minutes and withdrawn. The resulting urothelial lysates can be
used for RNA isolation or protein analysis by Western, as described in [28]. By
infusing a trypsin solution into the bladder lumen, suspensions of urothelial
cells for flow cytometric analysis can be made [30].
(a)(b)(c)Figure 3
Current mode-of-action hypothesis for the carcinogenic
effect of dual-acting PPARα+γ agonists  in the rat urothelium. To explain the carcinogenicity of dual-acting PPARα+γ
agonists in the rat urothelium, we favor a multifactorial mode-of-action (MOA)
hypothesis, compatible with the observation that PPAR agonists can cause
diametrally opposite biological effects (mitogenesis as well as cytotoxicity in
vitro, carcinogenicity as well as tumour inhibition in vivo) depending on
context (species, PPAR activation profile of agonist, agonist dose, cell type
as well as PPAR expression, etc.) [2, 4–6, 8, 33–36, 62, 106, 107]. (The shown MOA hypothesis is based on previously published
ragaglitazar data [18, 28–30, 61], but
may be applicable to other dual-acting agonists (Table 1) [8, 31]. The shown MOA hypothesis is applicable to rats only due
to the known profound species differences in PPAR function [26, 32].
Bladder cancer was seen in SD, Wistar, and Fischer rats of both sexes [8, 29], but
the shown MOA hypothesis may nevertheless be rat strain dependent due to rodent
strain differences in PPAR function [10]. The shown MOA hypothesis is compatible with gender
differences, due to gender differences in PPAR expression and function [108–113], and
does not assume urinary excretion of the PPAR agonist [39].)Such urothelial lysates from male rats dosed orally for 2-3 weeks were
examined by a combination of microarray, RT-PCR, and Western blotting methods [28]. We
found that within 4 days of ragaglitazar treatment, the transcription factor
Egr-1 was strongly upregulated in the bladder urothelium of animals treated
with 50 mg/kg/day ragaglitazar [28].
Interestingly, Egr-1 was not upregulated in the bladder urothelium of rats
daily receiving either 8 mg/kg/day rosiglitazone (a selective PPARγ agonist) or
200 mg/kg/day fenofibrate (a selective PPARα agonist), but appeared upregulated
in the bladder urothelium of rats receiving a combination of rosiglitazone  and fenofibrate [28]. The
significance of these findings is being confirmed, but the data support that in
rats orally dosed with ragaglitazar,
expression of Egr-1 was acutely induced in the bladder urothelium, and coactivation
of PPARγ and PPARα was required for
this. Other early changes observed in the bladder urothelium involved
phosphorylation of the S6 ribosomal protein, and the c-jun transcription factor
[28].Microscopically, hypertrophy (increased cell size),
hyperplasia (increased number of cells), and increased proliferation (increased
DNA synthesis, measured by BrdU incorporation) were observed in the bladder and
kidney pelvis urothelium of ragaglitazar-dosed rats, within 3 weeks of daily
oral dosing [28–30].
Because urothelial hypertrophy is difficult to quantitate by light microscopy,
we utilized flow cytometry as well as DNA/protein measurements to show that
within 2-3 weeks of oral
dosing with 5–50 mg/kg/day
ragaglitazar, the bladder urothelium underwent diffuse, generalized
hypertrophy; that is, the hypertrophy affected the whole urothelial cell
population [30].
Urothelial hypertrophy was also observed in the kidney pelvis [29, 30].
Finally, hypertrophy and hyperplasia were likewise observed in the urothelium
of ragaglitazar-dosed dogs and monkeys [29].
Interestingly, in naveglitazar-dosed rats, urothelial hypertrophy was the
earliest change, seen at 27 weeks, followed by urothelial hyperplasia at 53–79 weeks [31].


## 5. Potential Relevance of Early Urothelial Changes for Later Cancer Development

The c-jun transcription factor is a recognized oncogene [65] and
has been implicated in human bladder cancer development [66, 67].
Futhermore, increased c-jun activity has been linked to bladder cancer
development in mice exposed to the model bladder carcinogen arsenic [66, 67].Egr-1 (Zif268) is a zinc finger transcription factor
mediating a broad range of cellular responses such as proliferation,
differentiation, apoptosis, neuronal plasticity, and neovascularization [68–71]. Egr-1
is closely related to the WT1 Wilms’ tumour suppressor, with these two zinc
finger transcription factors being able to  be bound to the same DNA sequence, but exerting
opposite effects on transcription [72–74]. Given
the importance of the WT1 transcription factor for kidney development [58], it is
perhaps unsurprising that Egr-1 also has functional roles through the length of
the urogenital tract. Egr-1 expression is regulated during kidney development [75], and
postnatally, Egr-1 is involved in control of kidney function [76], and
bladder urothelium function [77–79]. Egr-1
overexpression and interaction with the WT1 Wilms’ tumour suppressor may be
involved in the pathogenesis of nephroblastoma [72–74].
Further, the bladder and prostate epithelia are contiguous and have common
embryological origin [58, 80, 81],
and interestingly, Egr-1 is absolutely required for the development of prostate
cancer in a mouse model [82, 83]. Egr-1
has also been implicated in human prostate cancer development [84]. In vitro, Egr-1 is induced in human
urothelial cancer cells treated with the model bladder carcinogen arsenic [67], and
Egr-1 physically associates with BLCA-4, a recognized marker of bladder cancer,
in human urothelial tumour cells [85, 86]. c-jun
and Egr-1 have also been shown to physically interact in rat spontaneous
pheochromocytoma PC12 cells [70].
Importantly, it is currently unknown whether the phosphorylation of c-jun and
induction of Egr-1 in the urothelium of ragaglitazar-treated rats  correspond to increased
activity of these transcription factors.Hypertrophy (increased cell size) is a surrogate
parameter for increased protein synthesis (translation) [87].
Intriguingly, phosphorylation of the ribosomal S6 protein is known to stimulate
protein translation, and S6 phosphorylation is also linked to cellular size [88–93]. Thus,
the increased S6 phosphorylation and hypertrophy observed in the urothelium of
ragaglitazar-treated rats may be causally linked [28, 30]. As
mentioned, hypertrophy was also the earliest change in the urothelium of
naveglitazar-dosed rats [31].Urothelial hypertrophy can also be induced by noncarcinogenic
agents [94].
Nevertheless, both hypertrophy and increased protein synthesis have been
reported as  precancerous changes
following exposure to model bladder carcinogens [95], and
translational deregulation is increasingly being recognized as playing a key
role in cancer development [88–90, 93, 96].In summary, while completely speculative, a causal link
between early urothelial changes (hypertrophy, S6 phosphorylation, c-jun
phosphorylation, Egr-1 induction) and later urothelial cancer development in
ragaglitazar-treated rats appears possible (Figure3). As mentioned above,
early urothelial hypertrophy was also
observed in naveglitazar-dosed rats [31].


## 6. Cytotoxic and Nongenomic Effects of  PPAR Agonists In Vitro

Surprisingly, structurally different agonists for PPARα
and PPARγ show a common propensity for PPAR-independent (off-target) effects,
particularly relating to growth inhibition (cytostasis) and cell death in a
variety of cell types [36, 62, 97, 98].
The mechanisms for the nongenomic actions of PPAR agonists are unknown, but may
have parallels in, for example, the nongenomic actions of steroid hormones [99].We found that exposure of NHU cultures to ciglitazone or troglitazone
(PPARγ) or ragaglitazar (PPARα+γ) rapidly induced apoptosis in NHU cells [61]. These
effects were independent of p38 or pERK activation and were not seen with fenofibrate
(PPARα), L165041 (PPARβ), or rosiglitazone (PPARγ). Proapoptotic agonists induced rapid,
sustained increases in intracellular calcium that were attenuated by removal of
extracellular calcium, indicating the involvement of store-operated calcium
entry. Proapoptotic agonists also induced cell membrane disruption, loss of
mitochondrial membrane potential, and activation of caspases-9 and -3. PPAR agonist-induced apoptosis was partially
attenuated by store-operated calcium channel inhibitors, but was unaffected by
PPARγ antagonists. This demonstrates that
structurally different PPAR agonists activate intrinsic apoptotic pathways in normal
human urothelial cells in a PPAR-independent manner. Interestingly, PPAR
agonists associated with hepatotoxicity and carcinogenicity in vivo also exhibited the most severe
cytotoxicity profile in vitro, comprising
apoptosis and sustained increases in intracellular calcium [61].
Recently, sustained increases in
intracellular calcium were linked to transactivation of the EGF receptor by
nongenomic actions of PPARγ agonists [98].Because the nongenomic cytotoxic actions of PPAR agonists
appear to be relatively cell type independent [62], and
because it is well known that cytotoxic effect in vitro may positively correlate with a carcinogenic effect in vivo [63, 64], we
currently favor incorporating the NHU cytotoxicity findings [61] into a
mode of action hypothesis for the carcinogenic effect of dual-acting PPARα+γ agonist in the rat urothelium (Figure 3).
Interestingly, normal urothelial cells are more sensitive to the nongenomic cytotoxic actions of PPAR agonists
than are transformed urothelial cells [41]. Thus,
nongenomic cytotoxic actions could hypothetically contribute not only to
initiating the carcinogenic process (detailed in Figure 3) but also to
selecting transformed urothelial cells.Intriguingly, and further complicating matters, some
studies suggest that PPARγ agonists previously associated with nongenomic
cytotoxicity at high concentration can at lower concentrations stimulate
proliferation and prevent apoptosis [6, 34, 100–103]. This
stimulatory effect does not occur for PPARγ agonists in NHU cultures [104], but
weak stimulatory effects and bell-shaped responses have been observed with
unsaturated fatty acids in NHU cultures [105]. In
short, because bell-shaped responses from activation of a specific PPAR may be
related to agonist characteristics, and because detection of weak mitogenic
responses in cell cultures (at low drug concentrations) may technically be more
difficult than detection of cytotoxicity in cell cultures (at high drug
concentrations), the phenomenon of bell-shaped response curves encompassing mitogenic as well as cytotoxic
effects may be underreported in studies of PPAR agonist effects in vitro.


## 7. Receptor-Mediated Carcinogenesis  in Rat Urothelium by Dual-Actingα+γ  Agonists Such As Ragaglitazar: PPARadigm or PPARadox

The dual-acting PPARα+γ agonist muraglitazar also caused
urothelial tumours in rats, but in this case it was concluded that a
urolithiasis-mediated mechanism was responsible [27, 29, 114, 115].
In contrast, uroliths were not involved in the urothelial cancers seen in
ragaglitazar or naveglitazar-treated rats [29, 31].
Furthermore, tesaglitazar did not induce bladder cancers in rats [13]. The
reasons for the difference in carcinogenic potential in the rat urothelium for
the four dual-acting PPARα+γ agonists muraglitazar, ragaglitazar, naveglitazar,
and tesaglitazar are unknown. It is tempting to speculate that the differences
in PPAR affinity and selectivity between these three PPARα+γ agonists influence
carcinogenic potential in the rat urothelium [3, 5, 11]. PPARα
and PPARγ activation profiles can be compiled for muraglitazar, ragaglitazar,
naveglitazar, and tesaglitazar from different studies [3, 5].
However, in order to evaluate whether PPAR affinity and selectivity correlates
with carcinogenicity, we believe it would be required to compare these
dual-acting PPARα+γ  agonists
in the same study, preferably using urothelial cells and monitoring the
activation of endogenous PPARα and PPARγ which are coexpressed in this cell
type. Technically, this could, for example, be done by, in urothelial cells,
separately monitoring expression of genes known to be activated by PPARα on one
hand, and genes known to be activated by PPARγ, on the other (PPAR-regulated
genes listed in [15]). Such
data unfortunately do not exist. Nevertheless, the findings in Table 1 suggest
that while agonists with a high degree of PPARγ selectivity (i.e., specific PPARγ agonists) can cause bladder
cancer in rats, they may be less prone to do so than are agonists with a lower
degree of PPARγ selectivity (i.e., dual-acting PPARα+γ agonists). This
interpretation of the data in Table 1 is speculative (disregards, e.g., dose
level or PPAR agonist efficacy differences between the animal trials with the
listed agents), but is plausible given the relatively unique coexpression of
PPARα and PPARγ by urothelial cells. Thus, the hypothesis deserves further
exploration, that combined PPARα+γ activation, by agents with low PPARγ
selectivity or high doses of agents with high PPARγ selectivity, may predispose
to urothelial cancer in rats by receptor-mediated mechanisms (Figure 3).It has been
proposed that it is unlikely that any of the urothelial cancers observed in
rats treated with dual-acting PPARα+γ agonists are due to receptor-mediated
effects (exaggerated pharmacology) [48]. We
have a different interpretation of the available data: it is clear that
activation of PPARα can cause tumours in rats and mice (Table 1) [9], and
while more controversial, activation of PPARγ can at least in some cases cause
cancer in rats and mice (Table 1) [5, 6, 8, 34, 36].
Of special interest is the recent finding that selective PPARγ agonists such as
rosiglitazone can promote hydroxybutyl(butyl)nitrosamine-induced bladder cancer
in rats [49] (Table 1).
Thus, it is plausible that the carcinogenic effect of dual-acting PPARα+γ
agonists in cells coexpressing PPARα and PPARγ (Table 1) may be due to
receptor-mediated mechanisms (exaggerated pharmacology). Further, it has been
described that PPARα and PPARγ agonists may exhibit synergistic effects in
cells coexpressing PPARα and PPARγ [19, 28, 53–55]. Thus,
the hypothesis of receptor-mediated carcinogenicity (carcinogenicity due to
exaggerated pharmacology) would predict that in rat tissue coexpressing PPARα
and PPARγ, dual-acting PPARα+γ agonists
may have a higher propensity for carcinogenic effect than selective PPARα and
PPARγ agonists alone, which in fact appears to be the case (Table 1).
Endothelial cells also coexpress PPARα and PPARγ [20], and
synergy between PPARα and PPARγ in the endothelium has been described [19], but
hemangiosarcoma frequencies appear comparable between mice treated with
dual-acting PPARα+γ agonists and selective PPARγ agonists (Table 1). This may
relate to differences between mouse urothelium and endothelium in PPARα and PPARγ
expression, signalling, and/or cross-talk.Specifically, we are not aware of any data that a priori disqualifies a
receptor-mediated carcinogenicity mechanism for dual-acting PPARα+γ agonists in
the rat urothelium [48]. For
example, (human) urothelium does not appear to receive growth/differentiation
cues from the urine [39] and
hence low urinary excretion of PPAR agonists does not rule out
receptor-mediated carcinogenic effects. Also, the well known in vitro cytotoxic effects of PPAR
agonists [5, 6, 36, 62],
including ragaglitazar [61], are
generally mediated by nongenomic (off-target) mechanisms, that is, do not rule
out receptor-mediated carcinogenic effects in vivo. In fact, our current working hypothesis for ragaglitazar is that exaggerated pharmacology and
nongenomic cytotoxicity may occur simulaneously and together promote cancer
development in the rat urothelium (Figure 3). Finally, the overrepresentation
of bladder cancers in male rats seen with some dual-acting PPARα+γ agonists is
sometimes presented as an argument against receptor-mediated carcinogenic
effects [48].
However, current data suggests that there are gender differences in the
expression of all PPAR isoforms, in a variety of species and tissue, including the
urinary bladder [108–113].
Moreover, our hypothesis implies that the male rat may represent an accelerated
tumour model, as any cytotoxicity/damage to the urothelium will provoke
urothelial regeneration and thus promote a receptor-mediated carcinogenic
effect (Figure 3).A key issue for the future will be how to distinguish
between “receptor-mediated” and “nonreceptor-mediated”
urinary bladder carcinogenicity mechanisms in rat experiments. Most simply, we
suggest that receptor-mediated (exaggerated pharmacology) carcinogenicity
mechanisms may be suspected for dual-acting agonists that induce
carcinogenicity-relevant biomarkers in the rat urothelium with rapid kinetics
(i.e., following a minimum of repeated oral doses) and with equal distribution
in the dorsal and ventral bladder domes [28–30]. The
maximal doses in this type of study could logically be the same as those used
for 2-year rat carcinogenicity studies (heart weight increases of approx. 25%
at 13 weeks have been suggested to identify the maximum tolerated dose for
2-year rodent oncogenicity studies) [8], and
lower doses may allow evaluation of nongenomic (off-target) effects on the biomarker endpoints [30].
Further refinement may be accomplished by including PPARα and PPARγ antagonists
[15, 116, 117],
inactive analogs [98], or,
more speculatively, modulation of PPAR expression in the bladder by siRNA
approaches [118].Adding 1% NH4Cl
to rat feed induces systemic acidosis, measurable directly by reduced blood pH,
reduced blood [HCO3-], and
increased blood [H+]  as well as indirectly by, for example,
increased urinary Ca++ and phosphorus excretion due to bone
resorption [119]. As
urine acidification also occurs, adding 1% NH4Cl to rat feed is sometimes used to evaluate whether rat
bladder carcinogenesis is urolith-mediated [29, 48, 114, 115].
However, urine acidification by feeding rats NH4Cl has also been
reported to reduce the occurence of bladder tumours, where the mechanism is not
thought to be urolith-mediated [120–122], and
NH4Cl feeding of rats can also influence the occurrence of tumours
outside of the urinary bladder [123]. In
fact, systemic acidosis induced by NH4Cl would be expected to have
profound effects on cellular and organ function in the whole organism,
including the bladder [123–129].
Therefore, while some aspects of bladder function are unaffected by NH4Cl
feeding [130], the
impact of systemic acidosis on bladder cancer development may be unrelated to
urolith formation. Further, it is possible that induction of acidosis may
directly interfere with the action of some PPAR agonists [131–134]. In
short, induction of systemic acidosis
may not specifically discriminate between mechanisms of PPAR carcinogenicity in
the rat urothelium (Figure 1).


## 8. Current Mechanism Hypothesis for Urothelial Cancers Induced by Dual-Acting PPAR Agonists in the Rat

We have attempted to integrate the urothelial changes
observed in ragaglitazar-treated rats [28–30],
results from ragaglitazar-treated urothelial cell cultures [61],
knowledge about PPARs in urothelial biology (see Figure 3), and new data about
PPAR isoform expression in rat and human bladder [18] into a
mode of action hypothesis for urothelial carcinogenesis by dual-acting PPAR
agonsts in rat urothelium (Figure 3).The hypothesis is completely speculative (Figure3), but
to our knowledge, does not conflict with current knowledge of PPAR biology (see
Figure 3). The main predictions of the hypothesis are that (i) coactivation of PPARα and PPARγ
in the rat urothelium can produce effects different from those observed with
specific activation of either PPARα or PPARγ, (ii) the effects of dual-acting PPARα+γ agonists on early
biomarkers (e.g., Egr-1) depend on structural aspects such as PPAR selectivity,
affinity, and activating effect of the agonist, and (iii) early biomarker changes (e.g., Egr-1 induction,
phosphorylation of c-jun and S6) are causally involved in later urothelial
cancer development.For practical reasons, our focus is on early (precancerous) changes in the rat
bladder urothelium (Figure3), but involvement of PPARs in later stages of
urothelial cancer progression is also possible by paracrine [51] or
immunological mechanisms [35].


## 9. Future Directions

In practical experimental terms, based on the mode of
action hypothesis presented in Figure3, we currently prioritize (i) evaluating cross-talk between
PPARα and PPARγ signalling in urothelial cells, by treating rats orally with
rosiglitazone and fenofibrate either separately or in combination with short-term
studies [28], (ii) evaluating the causal role of
Egr-1 in urothelial cancer development by, for example, chromatin
immunoprecipitation experiments from rat bladder, and (iii) comparing the findings between rat urothelium in vivo and finite cultures of normal
rat and human urothelial cell in vitro.Specifically, we believe that establishing cause-effect
relationships between early biomarkers and later cancer development is key to
understand the mode of action for carcinogenic effects of dual-acting PPARα+γ
agonists in the rat urothelium (Figure 3). Validating early carcinogenicity
biomarkers in rats should also allow developing simple preclinical assays to
rank the carcinogenic potential of developmental PPAR agonists (Figure 3).
Additionally, understanding the mechanisms in rats (Figure 3) would aid in
assessing the human relevance of the rat bladder cancer findings [135, 136].Finally, a recent study showed that the specific PPARγ
agonist rosiglitazone is a strong promoter of hydroxybutyl(butyl)nitrosamine-induced
bladder cancer in rats [49]. It is
tempting, but obviously highly speculative, to integrate this obervation into
the proposed mode of action hypothesis for ragaglitazar-induced bladder cancers
in rats (Figure 3). The prediction would be that in the rat urothelium in vivo, PPARα activation may provide
cancer initiation and PPARγ activation cancer promotion signals. A plausible
cancer initiation mechanism by PPARα activation is peroxisome formation and
free radical production. Thus, exploring the effects of specific PPARα agonists
in the rat urothelium would seem a
highly worthwhile  undertaking.



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*Source: 103167-2009-01-28.xml*