# To Live or to Die: Prosurvival Activity of PPARγ in Cancers

Authors: Y. Lynn Wang; Qi Miao
Journal: PPAR Research
 (2008)

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## Abstract

The role of PPARγ in tumorigenesis is controversial. In this article, we review and analyze literature from the past decade that highlights the potential proneoplastic activity of PPARγ. We discuss the following five aspects of the nuclear hormone receptor and its agonists: (1) relative expression of PPARγ in human tumor versus normal tissues; (2) receptor-dependent proneoplastic effects; (3) impact of PPARγ and its agonists on tumors in animal models; (4) clinical trials of thiazolidinediones (TZDs) in human malignancies; (5) TZDs as chemopreventive agents in epidemiology studies. The focus is placed on the most relevant in vivo animal models and human data. In vitro cell line studies are included only when the effects are shown to be dependent on the PPARγ receptor.


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## Body

## 1. Introduction

PPARγ is a nuclear hormone receptor that requires
ligand binding for activation. In 1995, it
was discovered that PPARγ is the molecular target of thiazolidinediones
(TZDs, [1]), a class of synthetic
compounds that are effective for the treatment of type 2 diabetes. This
discovery spurred great interest in these agents, as well as in the receptor. Besides its function as an insulin sensitizer
in diabetes, PPARγ was found to have a variety of roles in immunoregulation,
atherosclerosis, angiogenesis, and tumorigenesis.With regards to carcinogenesis,
debate continues as to whether PPARγ is pro- or antineoplastic, despite very active
research over the past few years. At the cellular level, PPARγ was found to be involved in cancer cell
survival/apoptosis, proliferation, and differentiation. While the apoptotic functions
of PPARγ and its agonists are addressed by others in
this special issue, we will conduct a critical review of the literature that
suggests that PPARγ has a prosurvival activity. The review is mainly focused on data derived
from in vivo models and/or human studies. In vitro cell line-based studies are
included only when the effects are shown to be dependent on the PPARγ receptor.One important
lesson learned from the past several years of research is that effects observed
with agonists of PPARγ are not necessarily intrinsic effects of the
nuclear hormone receptor. In tumor cell survival, the proapoptotic activities of
PPARγ agonists in various tumors act through both
receptor-dependent and receptor-independent mechanisms. When reviewing the
literature, we advise that the readers carefully consider the following to
distinguish drugs or TZDs versus receptor effects: (1) are high or low doses
used in the studies? High or low doses
should be defined with respect to EC50 of glitazones in the PPARγ transactivation assays (Table 1) or plasma
concentrations that can be reached in humans (Table 2). Effects observed with
high concentrations may not be relevant due to toxicities of certain TZDs, such
as hepatotoxicity of troglitazone and potential cardiotoxicity of rosiglitazone
(see below). (2) Are multiple pharmacological agents used? If a pharmacological
approach is the only one used, claims of a receptor-dependent effect require
demonstration with agonists of different chemical structures, such as TZDs,
tyrosine analogues, 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2),
and so forth. Beware that 15d-PGJ2 possesses many PPARγ-independent activities, including inhibition
of the NFκB pathway, that are known to have
prosurvival and anti-inflammatory properties, as well as other effects [2–4]. (3) Are any antagonists
included in the study? Do antagonists GW9662 or T0070907 block or reverse the
observed effects? (4) Are there any experiments in the study utilizing a genetic
approach to confirm the pharmacological findings? Does the study involve cell lines or primary
cells that contain or lack PPARγ, preferably in the same genetic background? For
those cell lines with endogenous PPARγ, is the siRNA, shRNA or dominant negative form
of PPARγ used to reduce the levels of the receptor? Are
specific effects of the receptor diminished by such reduction? For readers’
convenience, these questions are summarized in Table 3.Table 1
EC50 of common PPARγ agonists in transactivation assays.
AgonistsConstructs used for transactivationEC50 (μM)ReferencesCiglitazonemPPARγ1 LBD(a)-GAL4 DBD(b)3[5]PioglitazoneWild-type mPPARγ10.4[1]Wild-type mPPARγ20.4mPPARγ1(c) LBD-GAL4 DBD0.55[6]hPPARγ1(d) LBD-GAL4 DBD0.58RosiglitazoneWild-type mPPARγ10.03[1]Wild-type mPPARγ20.1mPPARγ1 LBD-GAL4 DBD0.076[6]hPPARγ1 LBD-GAL4 DBD0.043TroglitazonemPPARγ1 LBD-GAL4 DBD0.78[6]hPPARγ1 LBD-GAL4 DBD0.5515d-PGJ2Wild-type mPPARγ12[7]mPPARγ1 LBD-GAL4 DBD(a) LBD, ligand binding domain.(b) DBD, DNA binding domain.(c) mPPARγ1, mouse PPARγ1.(d) hPPARγ1, human PPARγ1.Table 2
Peak plasma concentrations of PPARγ agonists.
AgonistsCmax(a) (μM)ReferencesCiglitazone15~30(b)[8]Pioglitazone0.2~2.5[9]Rosiglitazone0.2~1.7Avandia Prescribing Information(c)Troglitazone0.7~8.8[10]15d-PGJ2Low nanomolar to picomolar range(d)[11][12](a)Cmax, the maximum or
peak plasma concentration in human unless otherwise indicated.(b)That in dog plasma.(c)Fromhttp://us.gsk.com/products/assets/us_avandia.pdf.(d)Physiological concentrations in cerebrospinal fluid,
urine, and the interior of adipocytes.Table 3
Points to be considered to discern drugs/TZDs versus receptor effects.
(1) Are high or low doses of drugs used in the studies with respect to theirKd
values for PPARγ, or plasma concentrations?(2) Are multiple pharmacological agents of
different chemical classes used?(3) Are any antagonists included in the
study?(4) Are any genetic approaches used to
confirm the pharmacological findings?


## 2. Expression of PPARγ in Human Tumor versus Normal Tissues

It is generally believed that expression of
a gene in a particular tissue suggests that the activity of the encoded protein
is required for certain cellular functions of that tissue. In so far as cancers
are concerned, the general rule is that oncogenes are overexpressed due to
dysregulation, and tumor suppressor genes are underexpressed or absent due to
mutations or deletions. In order to clarify the roles of the PPARγ receptor,
it would be informative to review the expression levels of PPARγ in
tumors with respect to their normal tissue counterparts. In this article, expression
data from tumor cell lines are not included.A review of the current literature on human
cancers showed that expression levels of PPARγ mRNA and protein are generally higher in neoplastic
tissues than their normal counterparts (summarized in Table 4). The most convincing data came from a large
study of prostate cancer that included 156 patients with prostate cancer (PC),
15 with less aggressive prostatic intraepithelial neoplasia (PIN), 20 with
benign prostatic hyperplasia, and 12 normal prostate tissues. In this study, a high level of PPARγ expression,
by immunohistochemistry, is observed in PC and PIN cases in comparison to low
or no expression in the benign hyperplasia and normal tissues. The results were
confirmed at the mRNA level with RT-PCR on a few cases from each category of
the malignant and benign conditions [13]. A large study of 126 renal cell carcinomas
also showed significantly more extensive and intensive PPARγ staining
in tumor epithelium compared to the average staining levels seen in 20 normal
tissues [14]. Similarly, in 22 patients with nonsmall
cell lung carcinoma, higher levels of PPARγ are
expressed in tumor cells than in the surrounding normal tissue, as determined
by immunohistochemical staining. In addition, higher expression levels in tumor
cells are confirmed by Western blotting hybridization, using homogenized tissue
samples [15]. In hepatocellular carcinoma, immunostaining
also demonstrates that PPARγ is
overexpressed in all of 20 carcinoma tissues but not in normal hepatocytes [16]. For squamous cell carcinoma, 20 cases of
primary tumor and six cases of lymph node metastasis were demonstrated to have
increased PPARγ protein
expression compared to normal tongue tissue [17]. Infiltrating adenocarcinoma of the breast
also expresses higher nuclear staining of PPARγ compared
to normal ductal epithelial cells by immunohistochemical analysis. However,
only one of the three cases was shown [18]. For papillary thyroid carcinoma, six
patients were studied to determine PPARγ mRNA
expression using reverse transcription PCR. The message was found in three of
six tumor tissues while the corresponding normal tissues do not express PPARγ [19].Table 4
PPARγ expression in human tumor versus normal tissues.
Tumor versus normal tissueNo. of casesReferencesOverexpressionProstate cancer/prostatic intraepithelial
neoplasia156/15[13]Renal cell carcinoma126[14]Nonsmall-cell lung carcinoma22[15]Hepatocellular carcinoma/lymph node
metastasis20/6[16]Squamous cell carcinoma20[17]Metastatic breast adenocarcinoma6[20]Infiltrating ductal breast adenocarcinoma3[18]Papillary thyroid carcinoma6(a)[19]Increased expression during tumor
progressionBreast adenocarcinoma1(b)[20]Ovarian carcinoma28 versus 28(c)[21]Urinary bladder carcinoma100 versus 70(d)[22]Pancreatic ductal adenocarcinoma45 versus 84(e)[23]Similar expressionColorectal adenocarcinoma11[24]Gastric adenocarcinoma12[25]Liposarcoma13[26]Adrenocortical tumors32[27](a) Of the six papillary carcinoma tissues, three expressed PPARγ mRNA.(b) The primary and metastatic breast cancer cell lines were derived
from a single patient.(c) Normal, benign, or borderline versus malignant tumors (grades 1, 2, and 3).(d) Lower (≤pT1) versus higher (≥pT2) tumor stages.(e) Lower (pT1 & pT2) versus higher (pT3 & pT4) tumor stages.Follicular thyroid carcinoma, a less common
histological subtype of thyroid cancer, is characterized by a chromosomal
translocation t(2;3) that results in a fusion between paired box gene 8 on
chromosome 2 and PPARγ on
chromosome 3 (PAX8-PPARγ). The fusion protein was initially thought to
function as a dominant-negative inhibitor of the wild-type PPARγ protein
[28]. However, a recent microarray study revealed
that (1) PPARγ transcript
levels in all seven cases of PAX8-PPARγ-containing
follicular carcinomas are more than 10-fold higher than normal thyroid tissues,
as determined by both microarray and quantitative RT-PCR analyses; (2) the
expression profile of the fusion-positive follicular carcinomas shows induction
of genes that are involved in fatty acid, amino acid, and glucose metabolic
pathways. Interestingly, many of the upregulated genes are known
transcriptional targets of the wild-type receptor, suggesting that the PAX8-PPARγ
fusion protein functions similarly to wild-type PPARγ, rather
than antagonizing its activity. (3) Using cell lines transfected with PPARγ or the
fusion protein, it is shown that expression of some genes, including angiogenic
factors PGF and ANGPTL4, is specifically upregulated by the fusion protein, particularly
in the absence of ligand, indicating that the fusion protein is constitutively active.
Taken together, these experimental data suggest that the translocation enhances
the function of PPARγ in a way
that contributes to the development or progression of follicular carcinoma of the
thyroid [29].Upregulation of PPARγ has
been demonstrated during tumor progression. Mueller et al. have found
significant PPARγ staining
in six cases of metastatic breast adenocarcinoma. In cell lines established
from the primary and metastatic tumors of one of these patients, significantly higher
amounts of PPARγ transcript
are shown in the cell line derived from the metastatic tumor [20]. In ovarian cancer, intensity and location
of PPARγ immunostaining
were examined in 28 carcinoma cases along with 28 normal, benign or borderline cases.
Twenty six of 28 carcinomas showed strongly positive PPARγ staining
compared to 2 weak-staining cases in the control group. Moreover, it is noted
that PPARγ staining
was predominantly nuclear in grade 2 or 3 tumors, as compared to a predominantly
cytoplasmic staining pattern in grade 1 tumors [21]. Similar findings were made in transitional
cell carcinoma of urinary bladder. Whereas no significant PPARγ immunoreactivity
was observed in 20 normal tissues, elevated PPARγ was
found in 168 tumors. Furthermore, the intensity of staining increased as the
histological grade increased from G1 to G3 and the tumor stage increased from
early (pT1 or lower) to advanced (stage 2 or higher) [22].A recent large study of 129 cases of
pancreatic ductal adenocarcinoma convincingly showed by array-based gene
profiling that expression of PPARγ in the
tumor cells is ~7 fold higher than that in the normal ductal epithelia. This
finding was confirmed with immunohistochemical analysis of the tissue sections.
Normal ductal epithelia showed insignificant staining for PPARγ. An
early lesion, intraepithelial neoplasia showed occasional PPARγ expression
whereas more than 70% of invasive pancreatic carcinoma demonstrated weak to
strong expression. Statistical analysis indeed revealed that expression of PPARγ correlates
with high tumor stage and higher tumor histological grade. More strikingly,
expression of PPARγ in pancreatic
cancer is shown, by multivariant survival analysis, to be a significant
prognostic indicator for shortened patient survival [23].In parallel
to the above literature, levels of PPARγ mRNA
found in several well- or poorly-differentiated colorectal adenocarcinomas,
were similar to normal tissues [24]. Another group also found that the PPARγ immunostaining
in well-, moderately-, or poorly-differentiated gastric adenocarcinomas is
comparable to that in noncancerous tissue adjacent to the tumor [25]. In liposarcomas, PPARγ
transcript levels are similar to that of the adipose tissue [26]. In adrenal glands, there is, again, no significant difference in mRNA
expression among cases of carcinoma, adenoma, and normal tissues [27]. Notably, at the time of composition of
this manuscript, we have not yet found any reports stating that PPARγ expression
is downregulated or absent in human tumor versus normal tissues (Table 4).The next
question is whether or not the PPARγ
expressed in tumor tissues is functional. Are ligands of PPARγ
present in the tumor tissues? A thorough and up to date literature search yielded
few results. The English abstract of a
study published in a foreign language stated that there was no significant
difference in 15d-PGJ2 concentration between gastric cancer tissues
and controls [30].
An earlier study showed that 15d-PGJ2 promotes the proliferation of HCA-7, a cyclooxygenase 2 (COX-2)-containing
colon cancer cell line at nanomolar concentrations. Further characterization by HPLC and mass
spectrometry identified PGJ2, a chemical precursor of 15d-PGJ2 in the culture medium of HCA-7 cells [31]. COX-2 is a key enzyme in the
biochemical pathway that leads to the formation of cyclopentenone
prostaglandins including 15d-PGJ2.
Overexpression of COX-2 has been documented in many cancer types and contributes
to tumor growth [32]. Overall, these few and somewhat circumstantial evidences
suggest that 15d-PGJ2 might be present in the tumor tissues.Does PPARγ lose
or gain abnormal functions through mutations other than PAX8-PPARγ
translocation? A large survey of human tumor samples and cancer cell lines does
not support such a notion. The exon 3 and 5 mutations, once reported in
sporadic colon cancers [33], were not present in nearly 400 cell lines
and primary tumor samples including lung, breast, prostate, colon cancers, and
leukemias [34].Taken
together, several lines of evidence regarding PPARγ expression
suggest a positive contributive role of the receptor in the development,
maintenance, or progression of human malignancies: (1) PPARγ is
overexpressed in the vast majority of cancers. (2) In several types of cancer, PPARγ expression
is further increased during tumor progression. (3) The oncogenic fusion PAX8-PPARγ results
in PPARγ overexpression
and upregulation of a similar profile of transcriptional targets as the
wild-type protein. (4) Expression of PPARγ in
pancreatic cancer is associated with shorter survival.


## 3. Receptor-Dependent Proneoplastic Effects of PPARγ

Is there also
cellular-level evidence suggesting that PPARγ promotes tumors? Most studies, especially
those employing high doses of TZDs, suggest that PPARγ agonists have antitumor activities through inhibition
of cell proliferation or induction of apoptosis or differentiation. However, receptor-independent pathways are
involved in most of the cases (reviewed elsewhere in this special issue). Then what does the receptor by itself do in
tumors?Schaefer et al. showed that inhibition of PPARγ induces apoptosis of hepatocellular carcinoma
cells (HCCs) by preventing their adhesion to the extracellular matrix,
suggesting that the activity of PPARγ is required for HCC cells to adhere and
survive [16]. In that study, those
particular effects were shown to be receptor-dependent. Loss of cell adhesion
requires almost complete loss of PPARγ activity achieved by either PPARγ-targeting siRNA or PPARγ inhibitor T0070907. In addition, T0070907
causes cell death at concentrations far lower than those needed for PPARγ agonists rosiglitazone and troglitazone.
Together, the data suggest that PPARγ functions to promote tumor cell adhesion and
survival in HCC cells. In line with this notion, the promoter region of
hepatocyte growth factor contains a functional PPAR response element (PPRE) that
mediates its transcriptional upregulation by PPARγ.
The growth factor plays an essential role in liver growth during embryonic
development, as well as in maintenance and renewal of cells in various organs including liver, lung, and kidney, in
adulthood [35].Our laboratory studied
human anaplastic large T-cell lymphomas, a common form of large cell lymphoma
in the pediatric population. We first demonstrated with immunohistochemical
staining that PPARγ is expressed in the malignant cells of the lymphoma
tissues [36]. We then tested the effect of
PPARγ activation in cell lines established from
patients with this lymphoma. A pair of cell lines, Karpas 299 and SUP-M2 that, respectively,
contain and lack endogenous PPARγ were selected to address the receptor-dependency
issue. Additionally, only low ligand concentrations were used, following
initial dose titration, to minimize any off-target effects. Using this system,
we have found that low doses of PPARγ agonists do not affect cell survival under
normal conditions. When cell death was induced by nutrient deprivation through
serum withdrawal, activation of the receptor with low doses of rosiglitazone
(0.5–2μM) attenuated cell death, as compared to drug
vehicle-treated cells. This result was reproducible with low doses of GW7845
(0.5–2μM) and 15d-PGJ2(0.5–1μM). The effect occurred only in PPARγ-containing Karpas 299 cells but not in PPARγ-lacking SUP-M2 cells. Moreover, reducing PPARγ in Karpas 299 cells with siRNA diminished the
prosurvival effect of the receptor. Furthermore, we showed that the prosurvival
effect is mediated through PPARγ-dependent cellular metabolic changes,
including increased cellular ATP levels, stabilized mitochondrial membrane
potential, and reduced reactive oxygen species (ROS) production that each favor
cell survival. PPARγ does so through coordinated regulation of the expression
of ROS metabolic enzymes, including the p67 subunit of NADPH oxidase, uncoupling
protein 2 (UCP2), and manganese superoxide dismutase (Mn-SOD) at both mRNA and
protein levels that lead to ROS limitation. Lastly, we showed that stable
transfection of PPARγ into SUP-M2 cells not only improved cell
survival, but also suppressed ROS accumulation during serum starvation. These
genetic manipulations have provided definitive evidence that PPARγ promotes lymphoma cell survival under
conditions of nutrient deprivation.Our group has also
made similar findings in a murine cellular model [37, 38]. FL5.12 is a murine lymphocytic
cell line that requires interleukin-3 (IL-3) for survival and proliferation. This cell
line has been extensively used to characterize tumor cell metabolism [39]. FL5.12 cells express little
PPARγ, but are killed by high concentrations of PPARγ agonists, 15d-PGJ2 (≥10μM) and ciglitazone (≥80μM). In an FL5.12 cell line stably-transfected
with PPARγ, low doses of PPARγ agonist do not affect cell viability under
normal conditions. However, when cells are induced to die by IL-3 withdrawal,
low doses of ciglitazone (10μM) and rosiglitazone (0.05–2μM) improved survival in only PPARγ-containing cells. Improved cell survival is
also accompanied by stabilized mitochondria and reduced ROS. Moreover, ATP
production is required for PPARγ to exert its prosurvival effect. In this
system, expression of a different panel of ROS metabolic enzymes including
catalase, and Cu/Zn-SOD are
involved in reduction of the cellular levels of ROS. Functional PPRE sequences were
shown to be present in the promoter regions of these two genes, suggesting that
the upregulation of their expression could be directly regulated by PPARγ [40–42]. Taken together, data from
both human and murine cell line studies suggest that PPARγ promotes tumor cell survival under conditions
of nutrient/growth factor deprivation, and that the effect is not limited to a
particular system. The mechanism by which PPARγ increases cell survival is diagrammed in
Figure 1 (Also see below).Figure 1
Schematic diagram showing how PPARγ increases cell survival in
growth factor/nutrient-deprived cells. Growth factor/nutrient withdrawal induces ROS
production. In the absence of PPARγ activation, increased levels of ROS inhibit mitochondrial
electron transport, leading to mitochondrial depolarization, caspase activation,
and cell death. When PPARγ is activated, the increase in ROS is attenuated by the
receptor through transcriptional upregulation of cell type specific antioxidant
factors, such as catalase, Cu/Zn-SOD (SOD1), Mn-SOD (SOD2), or UCP2. The
transcriptional upregulation of these genes by PPARγ may or may not be direct (shown to be direct in the diagram
for simplicity).In support of the
prosurvival activity of PPARγ in T-cell malignancies, Ferreira-Silva et al. very
recently showed that RNAi-mediated silencing of PPARγ in Jurkat T-cells caused increased DNA
fragmentation and apoptosis as well as G2/M cell cycle arrest, arguing that the
receptor, proper, promotes the viability of the tumor cells [43].In parallel to
these findings in tumors, the prosurvival activity of PPARγ has been well documented in certain nonneoplastic
pathological conditions, especially ischemia-reperfusion injury in
nutrient-sensitive tissues such as brain, heart and kidney [44–51]. Irreversible damage that results from
prolonged ischemia causes stroke, and myocardial and kidney infarction. At the cellular
level, cell death occurs as a result of nutrient deprivation and inflammatory
responses that involve the actions of proinflammatory cytokines, chemokines and
transcriptional factors. In addition,
increased production of ROS plays an important role in causing damage to macromolecules
and eventual cell death [52]. A recent study using a rat
model of cerebral focal ischemia has shown that expression of PPARγ mRNA and protein is upregulated in the areas
adjacent to infarct caused by middle cerebral artery occlusion [46]. Administration of glitazones
prior to, at the time of, or shortly after ischemia induction causes an
increase in DNA binding of the receptor. This is accompanied by a decrease in the
expression of a number of inflammatory genes, along with an increase in the
expression of antioxidant enzymes including catalase and Cu/Zn-SOD [44–47]. Consequently, these changes
lead to limited cell demise, which eventually results in significantly reduced infarct
size. This process apparently works through a PPARγ-dependent mechanism, as GW9662 can block these
effects of TZDs in animals [47]. Another PPARγ antagonist, T0070907, even increases the infarction
size, both in the presence and absence of PPARγ ligands [46].In light of both
these findings and the overexpression of PPARγ in many cancers, it is reasonable to
hypothesize that the function of PPARγ in cancer is to confer a survival advantage
upon the malignant cells, allowing them to survive in an adverse environment.
As a result of fast growth, the center of a three dimensional tumor mass is
often deprived of oxygen, growth factors, glucose, and other nutrients due to
excessive demand and insufficient vascularization. However, cancer cells
possess remarkable tolerance and are able to survive despite the adverse
conditions [53, 54]. Besides increasing
angiogenesis, increasing PPARγ might be another mechanism that allows tumor cells
to enhance their survival under these unfavorable conditions (Figure 1).


## 4. Impact of PPARγ and Its Agonists on Animal Tumor Models

Animal models
were employed to examine the role of PPARγ in tumors. These systems can be categorized by
how the tumor models are generated and by how the dose/activity of PPARγ is altered. With respect to the former, tumors
can be generated with xenografts, carcinogens, or genetic manipulations. Watch
for spontaneous tumor formation in certain PPARγ genetic backgrounds has also been conducted. With
respect to the dose/activity of PPARγ, it can be altered using PPARγ agonists including TZDs or GW7845, or genetic manipulations
including hemizygosity or tissue-specific overexpression or deletion of PPARγ. Results differ drastically between different
model systems, even for the same types of cancer (Tables 5 and 6). This
review focuses on models that are more relevant to human cancers. As such, animal
studies involving TZD treatment of xenografted tumors are not discussed here.Table 5
PPARγ and agonists in animal models (differentially shaded according to methods of tumor induction).Table 6
PPARγ and agonists in animal models (differentially shaded according to methods of PPARγ manipulation).


### 4.1. Colon Cancer

Apc+/Min mice possess a
nonsense mutation in one copy of the adenomatous polyposis coli (APC) gene
which truncates the protein at amino acid 850. Loss-of-function mutations in
the APC gene are common in human familial adenomatous polyposis and can
be found in sporadic colon cancers as well. Using this model, which is highly relevant
to human colon cancers, one study showed an increase in tumor number and size, as
well as worse histological grade in mice treated with troglitazone or
rosiglitazone. This is associated with a rosiglitazone-induced increase in the β-catenin protein level in the colon tissues [55]. Another study [56], which also used Apc+/Min mice, reported an
increase in the number of colon polyps in troglitazone-treated mice, but
reported no significant difference in tumor size or histology, which may be
related to the shorter TZD treatments used in this study (5 weeks as compared
to 8 weeks in the first study). Similar findings were made in Apc+/1638N:Mlh1+/− double mutant mice. In these mice, one copy of the APC gene is
truncated at amino acid position 1638 and one of the two alleles of the DNA
repair enzyme Mlh1 is absent. In the double mutant mice, troglitazone treatment
significantly increased the number of mice that developed large intestine tumors
[58]. In contrast to these reports, another
study used Apc+/1638N mice crossed with hemizygous PPARγ mice.
Because homozygous deletion of PPARγ is
embryonic-lethal, studies examining the dose effect of the gene employed either
a hemizygous Pparγ+/−
mouse strain or a conditional knock-out strategy. No differences in
survival, number of colonic tumors or β-catenin expression levels were observed
between mice of Apc+/1638N : Pparγ+/− and Apc+/1638N : Pparγ+/+ littermates [57]. Therefore, in colon cancer induced by
APC mutations, it appears that activation of PPARγ by TZDs
promotes tumor formation, while reduction of PPARγ gene
dosage has little effect on tumor formation.In stark contrast to theAPC genetic
tumor models, carcinogen-generated colon cancer models seem to yield opposite
results. In the study that evaluated PPARγ haploinsufficiency
in an Apc+/1638N background, the investigators also
determined the effect of Pparγ+/− in azoxymethane-mediated colon cancer. Compared to the Pparγ+/+ mice, a greater number of haploinsufficient mice developed tumors in
the colon. The tumor-bearing Pparγ+/− mice also had a greater number of tumors in them that led to
significantly decreased survival. In another study, mice with azoxymethane-mediated
colon cancer were treated with troglitazone, pioglitazone, or rosiglitazone.
This resulted in reduced incidence, number, and size of colorectal tumor [59]. Taken together, these data suggest that PPARγ suppress
azoxymethane-induced colon carcinogenesis.What would happen in normal mice? Spontaneous
colon tumor development was evaluated in normal mice administered with
troglitazone [58]. All nine mice fed with troglitazone
developed tumors in the large intestine, in contrast to none of the 10 mice in
the control group. An earlier study did not find any tumors in 17
troglitazone-fed normal mice, possibly due to the short duration of feeding (5
weeks in [56] versus 6 months in [58]).


### 4.2. Mammary Gland Tumors

The mammary gland tumor is another
relatively well-studied tumor in animals. Similar to colon carcinogenesis, data
on PPARγ’s
role in mammary gland carcinogensis suggest a wide range of effect depending on
the tumor models (Tables 5 and 6). Some studies indicate no effect, while
others suggest that it has a tumor promoting role, while others yet suggest a tumor
suppressing role. A murine genetic model supports a tumor-promoting role [60]. In this model, the mammary gland tumor is
induced by mammary gland-specific expression of polyoma middle T antigen
(MMTV-PyV). Mammary gland specific constitutive expression of PPARγ(MMTV-VpPPARγ) did
not yield tumor development. However, when crossed with the MMTV-PyV mice, the
double mutant progeny developed more mammary gland tumors sooner than MMTV-PyV
mice. The increased tumor burden eventually led to shorter survival.
Interestingly, hemizygosity of PPARγ in
the MMTV-PyV background did not change the time course of tumor development. Exacerbation
of tumor formation by PPARγ was
ascribed to increased Wnt-β catenin signaling as demonstrated by zebrafish
developmental models.In contrast to this genetic model,
chemically induced mammary gland tumors were inhibited by PPARγ agonists.
Both TZDs and GW7845, a tyrosine analog, have been shown to exhibit antitumor
effects. An early study using nitrosomethylurea (MNU) to induce mammary
carcinogenesis showed that GW7845 reduced the incidence, number of tumors
per animal,
and average weight of tumor at autopsy following a two-month administration of
the drug to rats [61]. In 7,12-dimethylbenzanthracene
(DMBA)-mediated mouse carcinogenesis model, the animals develop multiple types
of tumor, including mammary ductal papilloma and adenocarcinoma. Incidence of
mammary gland tumor was significantly higher in Pparγ+/− mice than in Pparγ+/+ mice. The hemizygous mice
also had increased number of tumors and a lower survival rate [62].Spontaneous
tumor formation was also examined inPparγ+/− mice. Dose reduction of PPARγ does not make animals prone to increased
carcinogenesis [62]. In concordance with this
finding, the specific deletion of PPARγ in mouse mammary epithelia failed to induce
mammary tumors in 20 mice observed for 12 months [63].


### 4.3. Other Cancers

In a murine prostate
cancer model, generated using tissue-specific SV40 T antigen, reducedPparγ+/− had no effects on tumor
incidence, latency, size, histopathology, or disease progression [64]. However, in a murine follicular
thyroid cancer model containing a dominant-negative mutant form of thyroid
hormone receptor β⁢(TRβPV/PV), loss of one PPARγ allele led to increased weight of
tumor-bearing thyroid gland, increased lung metastasis, and shortened survival.
In addition, rosiglitazone treatment of TRβPV/PV mice reduced
thyroid weight, and tumor progression [65], suggesting a
tumor-suppressing role for PPARγ. Lastly, in gastric carcinoma, induced with
MNU, PPARγ haploinsufficient mice had increased tumor
incidence and shorter survival. Troglitazone treatment significantly reduced
tumor incidence in mice with wild-type PPARγ background [66].In summary, results
from animal studies regarding the role of PPARγ are conflicting and difficult to assess. For
the purpose of clarification, we attempted to analyze the published data
according to the cancer types, tumor induction models, PPARγ activation/reduction methods, and tumor
characteristics (Tables 5 and 6). Our extensive analysis revealed no
clear pattern. However, some trends have been noted: (1) in multiple types of carcinogen-induced tumor (Table 5, light grey shaded rows), PPARγ seems
to have a tumor-suppressing function. This appears to be independent of how PPARγ is
activated or reduced, whereas in genetic tumor models (Table 5, un-shaded rows), the receptor exhibited all possible different effects. As to spontaneous
tumors (Table 5, dark grey shaded rows), long-term use of troglitazone
increased tumor formation, whereas PPARγ reduction
had no effect; (2) a reduction of PPARγ dose
by itself (Table 6, light grey shaded rows) is insufficient to induce spontaneous
tumor formation, but in existing tumors, it either exacerbates tumor formation
or have no effect at all; (3)
TZDs (Table 6, un-shaded rows), in most cases, inhibits tumor
formation with a rare exception of Apc+/Min mice.The activity of the Wnt/β-catenin signaling pathway might account for
these seemingly discrepant results, as tumor models generated by APC mutation
or polyoma middle T antigen all involve overly active Wnt/β-catenin signaling. TZDs are shown to induce β-catenin in colon [55]. Paradoxically, reduction of PPARγ (Pparγ+/−) also increases β-catenin expression in colon [57]. The appropriate activation of PPARγ signaling
might also be important. Ligand-independent constitutive activation of PPARγ is
involved in the development of mammary gland tumors [60] as well as in the action of PAX8-PPARγ in
follicular thyroid carcinoma [29].


## 4.1. Colon Cancer

Apc+/Min mice possess a
nonsense mutation in one copy of the adenomatous polyposis coli (APC) gene
which truncates the protein at amino acid 850. Loss-of-function mutations in
the APC gene are common in human familial adenomatous polyposis and can
be found in sporadic colon cancers as well. Using this model, which is highly relevant
to human colon cancers, one study showed an increase in tumor number and size, as
well as worse histological grade in mice treated with troglitazone or
rosiglitazone. This is associated with a rosiglitazone-induced increase in the β-catenin protein level in the colon tissues [55]. Another study [56], which also used Apc+/Min mice, reported an
increase in the number of colon polyps in troglitazone-treated mice, but
reported no significant difference in tumor size or histology, which may be
related to the shorter TZD treatments used in this study (5 weeks as compared
to 8 weeks in the first study). Similar findings were made in Apc+/1638N:Mlh1+/− double mutant mice. In these mice, one copy of the APC gene is
truncated at amino acid position 1638 and one of the two alleles of the DNA
repair enzyme Mlh1 is absent. In the double mutant mice, troglitazone treatment
significantly increased the number of mice that developed large intestine tumors
[58]. In contrast to these reports, another
study used Apc+/1638N mice crossed with hemizygous PPARγ mice.
Because homozygous deletion of PPARγ is
embryonic-lethal, studies examining the dose effect of the gene employed either
a hemizygous Pparγ+/−
mouse strain or a conditional knock-out strategy. No differences in
survival, number of colonic tumors or β-catenin expression levels were observed
between mice of Apc+/1638N : Pparγ+/− and Apc+/1638N : Pparγ+/+ littermates [57]. Therefore, in colon cancer induced by
APC mutations, it appears that activation of PPARγ by TZDs
promotes tumor formation, while reduction of PPARγ gene
dosage has little effect on tumor formation.In stark contrast to theAPC genetic
tumor models, carcinogen-generated colon cancer models seem to yield opposite
results. In the study that evaluated PPARγ haploinsufficiency
in an Apc+/1638N background, the investigators also
determined the effect of Pparγ+/− in azoxymethane-mediated colon cancer. Compared to the Pparγ+/+ mice, a greater number of haploinsufficient mice developed tumors in
the colon. The tumor-bearing Pparγ+/− mice also had a greater number of tumors in them that led to
significantly decreased survival. In another study, mice with azoxymethane-mediated
colon cancer were treated with troglitazone, pioglitazone, or rosiglitazone.
This resulted in reduced incidence, number, and size of colorectal tumor [59]. Taken together, these data suggest that PPARγ suppress
azoxymethane-induced colon carcinogenesis.What would happen in normal mice? Spontaneous
colon tumor development was evaluated in normal mice administered with
troglitazone [58]. All nine mice fed with troglitazone
developed tumors in the large intestine, in contrast to none of the 10 mice in
the control group. An earlier study did not find any tumors in 17
troglitazone-fed normal mice, possibly due to the short duration of feeding (5
weeks in [56] versus 6 months in [58]).


## 4.2. Mammary Gland Tumors

The mammary gland tumor is another
relatively well-studied tumor in animals. Similar to colon carcinogenesis, data
on PPARγ’s
role in mammary gland carcinogensis suggest a wide range of effect depending on
the tumor models (Tables 5 and 6). Some studies indicate no effect, while
others suggest that it has a tumor promoting role, while others yet suggest a tumor
suppressing role. A murine genetic model supports a tumor-promoting role [60]. In this model, the mammary gland tumor is
induced by mammary gland-specific expression of polyoma middle T antigen
(MMTV-PyV). Mammary gland specific constitutive expression of PPARγ(MMTV-VpPPARγ) did
not yield tumor development. However, when crossed with the MMTV-PyV mice, the
double mutant progeny developed more mammary gland tumors sooner than MMTV-PyV
mice. The increased tumor burden eventually led to shorter survival.
Interestingly, hemizygosity of PPARγ in
the MMTV-PyV background did not change the time course of tumor development. Exacerbation
of tumor formation by PPARγ was
ascribed to increased Wnt-β catenin signaling as demonstrated by zebrafish
developmental models.In contrast to this genetic model,
chemically induced mammary gland tumors were inhibited by PPARγ agonists.
Both TZDs and GW7845, a tyrosine analog, have been shown to exhibit antitumor
effects. An early study using nitrosomethylurea (MNU) to induce mammary
carcinogenesis showed that GW7845 reduced the incidence, number of tumors
per animal,
and average weight of tumor at autopsy following a two-month administration of
the drug to rats [61]. In 7,12-dimethylbenzanthracene
(DMBA)-mediated mouse carcinogenesis model, the animals develop multiple types
of tumor, including mammary ductal papilloma and adenocarcinoma. Incidence of
mammary gland tumor was significantly higher in Pparγ+/− mice than in Pparγ+/+ mice. The hemizygous mice
also had increased number of tumors and a lower survival rate [62].Spontaneous
tumor formation was also examined inPparγ+/− mice. Dose reduction of PPARγ does not make animals prone to increased
carcinogenesis [62]. In concordance with this
finding, the specific deletion of PPARγ in mouse mammary epithelia failed to induce
mammary tumors in 20 mice observed for 12 months [63].


## 4.3. Other Cancers

In a murine prostate
cancer model, generated using tissue-specific SV40 T antigen, reducedPparγ+/− had no effects on tumor
incidence, latency, size, histopathology, or disease progression [64]. However, in a murine follicular
thyroid cancer model containing a dominant-negative mutant form of thyroid
hormone receptor β⁢(TRβPV/PV), loss of one PPARγ allele led to increased weight of
tumor-bearing thyroid gland, increased lung metastasis, and shortened survival.
In addition, rosiglitazone treatment of TRβPV/PV mice reduced
thyroid weight, and tumor progression [65], suggesting a
tumor-suppressing role for PPARγ. Lastly, in gastric carcinoma, induced with
MNU, PPARγ haploinsufficient mice had increased tumor
incidence and shorter survival. Troglitazone treatment significantly reduced
tumor incidence in mice with wild-type PPARγ background [66].In summary, results
from animal studies regarding the role of PPARγ are conflicting and difficult to assess. For
the purpose of clarification, we attempted to analyze the published data
according to the cancer types, tumor induction models, PPARγ activation/reduction methods, and tumor
characteristics (Tables 5 and 6). Our extensive analysis revealed no
clear pattern. However, some trends have been noted: (1) in multiple types of carcinogen-induced tumor (Table 5, light grey shaded rows), PPARγ seems
to have a tumor-suppressing function. This appears to be independent of how PPARγ is
activated or reduced, whereas in genetic tumor models (Table 5, un-shaded rows), the receptor exhibited all possible different effects. As to spontaneous
tumors (Table 5, dark grey shaded rows), long-term use of troglitazone
increased tumor formation, whereas PPARγ reduction
had no effect; (2) a reduction of PPARγ dose
by itself (Table 6, light grey shaded rows) is insufficient to induce spontaneous
tumor formation, but in existing tumors, it either exacerbates tumor formation
or have no effect at all; (3)
TZDs (Table 6, un-shaded rows), in most cases, inhibits tumor
formation with a rare exception of Apc+/Min mice.The activity of the Wnt/β-catenin signaling pathway might account for
these seemingly discrepant results, as tumor models generated by APC mutation
or polyoma middle T antigen all involve overly active Wnt/β-catenin signaling. TZDs are shown to induce β-catenin in colon [55]. Paradoxically, reduction of PPARγ (Pparγ+/−) also increases β-catenin expression in colon [57]. The appropriate activation of PPARγ signaling
might also be important. Ligand-independent constitutive activation of PPARγ is
involved in the development of mammary gland tumors [60] as well as in the action of PAX8-PPARγ in
follicular thyroid carcinoma [29].


## 5. Clinical Trials of TZDs in Human Malignancies

As discussed above, TZDs have been shown in many
preclinical studies to possess antitumor effects that have prompted several
early-phase clinical studies to evaluate their efficacies in various types of
cancers. In this review, we analyze these studies both in terms of clinical
responses and biological responses, focusing on recently published studies that
include more than 10 patients (Table7).Table 7
Clinical trials of TZDs in cancer patients.
Cancer typePhaseTZDsNo. of ptsTumor responseReferencesLiposarcomaIIRosiglitazone12All patients progressed, no sign of
differentiation by histology[68]Thyroid cancerI, IIRosiglitazone104 pts with partial response, 2 with
stable disease, and 4 with progressed disease[69]Metastatic colorectal cancerI, IITroglitazone25All patients progressed[70]Refractory breast cancerIITroglitazone22Most patients progressed with increased
serum tumor markers[71]Early-stage breast cancerIIRosiglitazone38No
reduction in Ki-67 staining on tissue biopsies[72]Metastatic prostate cancerIITroglitazone41Decrease or stabilization of PSA[73]Recurrent prostate cancerIIIRosiglitazone106Similar
to placebo in both PSADT and time-to-disease-progression[74]A phase II clinical trial of rosiglitazone in 12
patients with liposarcoma was recently conducted. Eight of 12 patients were
fully evaluated for up to 16 months. As to clinical response, all patients
progressed while on treatment with a mean time-to-progression of 5.5 months.
Histological appearance of repeated biopsy materials did not show any signs of
tumor differentiation. In one of the 8 patients, PPARγ and fatty acid binding protein (FABP) were
induced after 12-week rosiglitazone therapy, but disease in this patient
progressed similarly to the others [68]. Ten patients with thyroid
cancers were treated with rosiglitazone. Among them, 4 had partial response, 2
had stable disease, and the remaining 4 progressed. No correlation was found
between the clinical response and levels of PPARγ mRNA and protein in these patients. PAX8-PPARγ status was not assessed [69]. An early study evaluated
efficacy of troglitazone in 25 patients with metastatic colorectal carcinoma.
All 25 patients progressed with a median time-to-progression of 1.6 months and
a median survival time of 3.9 months [70].In breast cancer, data from two human trials have
been published. An early trial on 22 women with refractory breast cancer showed
no objective response to troglitazone in 18 of the 21 evaluable patients at 8
weeks after treatment. The therapy was terminated in 16 patients due to
progression of their tumors. At 8 weeks, only three patients had stable
disease. All patients were evaluated for serum tumor markers, CEA and CA27.29,
which showed increased levels within 8 weeks of treatment. Expression of PPARγ was not determined in the study [71]. A short-term pilot trial of
rosiglitazone in 38 women with early stage breast cancer was conducted.
Clinical response was not assessed in this short-term (<6 week) study.
Biological response, as assessed by Ki-67 staining on biopsy tissues before and
after treatment, was not detected in treated patients, either. Decreased
insulin levels and increased insulin sensitivity were noted in these patients,
suggesting that the rosiglitazone did affect metabolism as expected [72].An early phase II trial of troglitazone in 41
patients with metastatic prostate cancer showed a decrease in levels of
prostate-specific antigen (PSA) in 20% of patients enrolled in the study.
Prolonged stabilization of PSA was seen in 39% of patients [73]. However, these encouraging
results were not reproduced in a large double-blind, randomized, placebo-controlled
trial of rosiglitazone in 106 patients with recurrent prostate cancer [74]. The time-to-disease-progression
was not significantly different between the rosiglitazone and placebo groups.
Moreover, the PSA doubling time, a predictor of clinical recurrence, was also
not prolonged by the treatment.Taken together, TZDs
appear to show little benefit, both in terms of clinical response and
biological response, in treating various types of human cancers despite
promising results from preclinical animal studies. It is worth noting that most
of the studies use low doses of TZDs which are sufficient to activate PPARγ and control diabetes. It remains possible that
higher doses, even via receptor-independent pathways, would be beneficial for
cancer patients. However, one should keep in mind that TZDs are not a class of
drugs without dose-limiting toxicities. Troglitazone was withdrawn from the
market by the FDA in 2002 due to liver toxicity. Most recently, increased
cardiovascular risk has been associated with rosiglitazone in the diabetic
patient population [75, 76] which has prompted the FDA to
issue label warnings.


## 6. TZDs as Chemopreventive Agents in Epidemiology Studies

The clinical
trials discussed above suggest that TZDs have questionable efficacy as
chemotherapeutic agents in patients who already have cancers. Do they have the
potential to act as chemopreventive agents? Recently, a large epidemiologic
study, involving a population of 87,678 veteran men with diabetes, attempted to
answer that question [77]. In this retrospective study,
incidence of lung, prostate, and colon cancer in TZD users was compared to
incidence in non-TZD users and risk of cancer development was analyzed. Only
patients who obtained a cancer diagnosis after the date of TZD initiation were
included. TZD usage significantly reduced risk of lung cancer by 33%. It also
reduced risk of colon and prostate cancer, though without statistical
significance. Interestingly, although the risk of prostate cancer is not significantly
influenced by TZDs in the entire population, when examining distinct populations,
TZDs are associated with an increased incidence of prostate cancer in both Caucasians
and African Americans. These data suggest that the overall reduced risk is accounted
for by the non-Caucasian, non-African Americans populations in the study. These
data suggest that TZDs may be beneficial for reducing certain cancers in
certain populations. Specific molecular abnormalities in specific cancers and
the genetic background of different populations may account for these apparently
different results.Although this
study was quite strong, we suggest the following for future investigations: (1) separate
TZD-users into those using rosiglitazone and those using pioglitazone. In the
cardiovascular risk studies, it was shown that rosiglitazone increases the risk
while pioglitazone decreases the risk [78]. (2) Evaluate the impact of
the duration of TZD exposure on risk of cancer development. (3) Determine the
influence of TZDs on the behavior of existing cancers.


## 7. Conclusions

In this article,
we reviewed literature on the roles of PPARγ in cancer with an emphasis on those that
suggest a proneoplastic function for the receptor. PPARγ, unlike MYC, RAS, or p53, is neither a strong
tumor promoter nor a tumor suppressor. However, it may function as a
“conditional tumor promoter” or a “conditional tumor suppressor” that modulates
the tumorigenic process depending upon cellular conditions, tumor types, or
genetic background of an animal strain or human individuals. TZDs, as a class
of pharmacological agent, may have receptor-independent antineoplastic effects,
especially at doses higher than diabetic doses or after long-term use and
accumulation. It remains possible that their antitumor activities would be
enhanced when in combination with other drugs. Further investigation is needed
to address that possibility. To help clarify the roles of PPARγ in cancer, future large epidemiological studies
of diabetic populations with concurrent cancers would be helpful. In addition,
investigations relating PPARγ activities to the clinical outcomes of cancer
patients would also be informative.



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*Source: 209629-2008-09-08.xml*