# PPAR Ligands as Potential Modifiers of  Breast Carcinoma Outcomes

Authors: Ancha Baranova
Journal: PPAR Research
 (2008)

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## Abstract

Chemically synthesized ligands for nuclear receptors of the PPAR family modulate a number of physiological functions, particularly insulin resistance in the context of energy homeostasis and the metabolic syndrome. Additionally, these compounds may treat or prevent the development of many secondary consequences of the metabolic syndrome. Many PPAR agonists are also known to influence the proliferation and apoptosis of breast carcinoma cells though the experiments were carried out at suprapharmacological doses of PPAR ligands. It is possible that the breast epithelium of diabetics exposed to PPAR agonists will experience perturbation of the corresponding signaling pathway. Consequently, these patients' lifetime breast carcinoma risks could be modified, as their breast lesion incidence or the rates of the conversion of these lesions to carcinomas might vary upward or downward. PPAR activating treatment may also influence the progression of existing, undiagnosed invasive lesions. In this review, we attempt to summarize the possible influence of chemical PPAR ligands on the molecular pathways involved in the initiation and progression of breast carcinoma, with a major emphasis on PPARγ agonists thiazolidinediones (TZDs).


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## Body

## 1. Introduction

Breast carcinoma is the most common nonskin
cancer among women worldwide, responsible for about 375 000 deaths per year [1].
The probability of the development of breast carcinoma increases before
menopause (ages 40–50) and then
gradually decreases, possibly due to diminishing levels of circulating
estrogens [2]. In developed countries, the prevalence of breast carcinoma is
higher due to the frequency of known risk factors for the disease, including
early age at menarche, nulliparity, late age at first birth, late menopause,
and brief duration of breastfeeding [2]. All of these risk factors are tightly
linked to hormonal background, particularly to lifelong exposure of breast
tissue to endogenous estrogens [3]. Exogenous factors influencing breast
carcinoma development include the use of oral contraceptives [4] and hormone
replacement therapy [5, 6] as well as dietary or lifestyle-related variables.
The latter category is rather vague, as it includes many factors detrimental to
general health, such as high body-mass index [7], high fat intake [8], high red
meat consumption [9], excessive alcohol consumption [10], and reduced physical
activity [11].A number of chemoprevention strategies for
breast carcinoma are developed or under development. The noteworthy example is
a tamoxifen chemoprevention in high-risk premenopausal women, which heralded
the success of selective estrogen receptor modulators (SERMs) [12]. A new
agent, raloxifene (Evista, Eli Lilly, IN, USA)
also competes with endogenous estrogen for ER binding and shows similar
promises with fewer side effects [13]. Interestingly, many potential breast
carcinoma preventive agents studied earlier are also available over-the-counter
and widely used by target populations. Examples of this kind include aspirin [14],
soy isoflavones [15], and Vitamin D [16].Recently, the universe of chemical compounds
commonly encountered by current and future breast carcinoma patients has been
enriched by a number of pharmacotherapeutic agents being prescribed as a
lifelong support for common chronic diseases. Depending on the particular
molecular pathways which these agents modulate, they may contribute to initial immortalization
of breast epithelia, stimulate proliferation and invasion of existing tumor cells,
or on the contrary, prevent the tumor’s development. For example, type II
diabetes patients are routinely treated with  chemically synthesized ligands for
PPARγ, thiazolidinedione (TZD), namely pioglitazone (Actos, Takeda/Lilly), and
rosiglitazone (Avandia, GlaxoSmithKline). The glucose-lowering effects of these
compounds are mediated primarily by decreasing insulin resistance and
increasing glucose uptake by the skeletal muscles [17]. In addition, TZDs
suppress glucose production in the liver [17]. These and other beneficial
effects rapidly made TZDs a mainstream diabetes therapy [18].In addition to their antidiabetic effects, TZDs
are known to suppress the proliferation and induce apoptosis of breast
carcinoma cells in vitro [19, 20]. It is likely that the breast epithelium of diabetics exposed to
TZDs will also experience perturbation of the PPAR signaling pathway.
Consequently, current or past TZD users’ lifetime breast carcinoma risks may be
modified, as their breast lesion incidence or rates of the conversion of these lesions
to carcinomas might change upward or downward. TZD treatment may also influence
the progression of existing undiagnosed invasive lesions.In addition to PPARγ ligands, PPARα [21] and
PPARδ [22] are currently being explored as potential
cardiovascular therapeutics and metabolic syndrome alleviation agents. If these
agents will be approved by FDA, it is very possible that in the next two or
three decades the number of women exposed to one or another type of PPAR
ligands may reach 10–15 million in the
USA alone. Possible modifications of the breast carcinoma incidence and
outcomes resulted by the chronic exposure to these compounds might translate
into statistically significant changes visible in epidemiological survey data,
similar to those seen in cohorts taking hormone replacement therapy [5, 6].In this review, we attempt to summarize the
possible influence of chemical PPAR ligands on the molecular pathways involved in
the initiation and progression of breast carcinoma. Major emphasis will be on
PPARγ, as small molecular agonists of this nuclear receptor are widely used in
the treatment of type II diabetes all over the world.


## 2. PPARγ Ligands

A gene encoding nuclear hormone receptor,
PPARγ, expresses as two different mRNA isoforms derived from the alternative promoters,
ubiquitous PPARγ1 and adipose-specific PPARγ2 [23]. Both isoforms stimulate
adipogenesis; however, PPARγ2 can be activated by lower concentrations of ligands
[23]. Activated PPARγ heterodimerizes with various coactivators [24, 25], which
modulate the expression of genes with promoters containing bi-hexametric PPRE
elements. These elements are widespread in the human genome, being present in both
fatty acid metabolism and cell cycle control genes [26]. Moreover, the list of targets
directly regulated by PPARγ includes many genes which lack PPRE [27]. Most
likely, this is due to either the binding of activated PPARγ to other proteins
that, in turn, serve as transcription factors (TFs) or the action of PPRE-containing
genes providing delayed transcriptional response to PPARγ ligation [27]. Knowledge
about endogenous ligands for PPARγ is limited. The list of these compounds
includes polyunsaturated fatty acids (PUFAs) and eicosanoids, particularly
lipoxygenase (LOX), and cyclooxygenase (COX) products [28]. An
anti-inflammatory prostaglandin, 15-deoxy-D12,14-PGJ2 (15d-PGJ2), which is
formed from PGD2 in vivo, is
probably the most potent endogenous PPARγ ligand [28]. Another powerful
physiological stimulator of PPARγ is oxidized phosphatidylcholine [29]. It
should be mentioned that synthetic ligands of PPARγ (TZDs) display stronger
binding affinity to this nuclear receptor than its endogenous ligands, thus
raising the question whether the list of natural PPARγ ligands is complete.


### 2.1. Effects of the Chronic Exposure of the Breast Epithelium to PPARγ Agonists

PPARγ is expressed in normal breast tissue
and in many primary breast carcinoma specimens [30, 31]. Comparative studies of
PPARγ expression in breast carcinoma patients so far have produced
contradictory results [32–34]. Described
associations between PPARG polymorphisms and breast carcinoma are also discrepant: some researchers see a
marginally significant increase in the risk of breast cancer among women
homozygous for the Ala allele of PPARγ (Pro12Ala), causing a reduction in the transcriptional activity
of PPARγ2 [35], while others stress that carriers of the same variant allele
are at lower risk [36]. Since complete loss of PPARγ signaling in clinical
breast tumors seems to be a rare event [37], it is likely that patients
undergoing chronic treatment with chemical ligands for PPARγ will experience
alteration in the behavior of both breast carcinoma cells and their normal
counterparts. Patients with ER-positive tumors might benefit from TZD exposure
more than those with ER-negative tumors, as the level of PPARγ expression is
significantly associated with the ER status of carcinoma cells [38].Chemically synthesized ligands for PPARγ
(thiazolidinediones, or TZDs) have actively been used as insulin sensitizers
since the late 90s [18]. In addition to their insulin resistance-alleviating
effects, TZDs may influence an incidence or a progression of breast carcinoma
lesions as they have been shown to suppress the proliferation rates of many
types of cancer cells and induce either their differentiation or apoptosis in vitro [20, 39, 40]. Responsiveness
to TZDs has been demonstrated for both normal human mammary epithelial cells
[30] and breast cancer cells [41–43], although it
was not uniformly seen in all experimental conditions [44]. TZDs suppress the
cell cycle by repressing cyclins D1 and D3 [45], by stimulating expression of
the tumor suppressor p53 and its effector p21 (WAF1/Cip1) [46], and by inhibiting
the Akt/PTEN pathway [47]. Additionally, TZDs induce marked cellular acidosis
in breast carcinoma cell lines, leading to a decrease in the number of viable
cells [48]. Some effects of TZDs are independent of the transcriptional
activities of PPARγ [48]; these effects may be mediated through interactions of
these compounds with other cellular targets.The growth-suppressive properties of TZDs
are complemented by their ability to induce apoptosis. Many breast tumors are
naturally resistant to the apoptotic action of the tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) and other similar agents. TZDs
sensitize these cells to TRAIL [45], to anti-Fas IgM (CH11), and to tumor
necrosis factor (TNF)-α [49]. It is tempting to speculate that TZDs might prevent
the spread of microscopic breast tumors by sensitizing malignant cells to these
endogenous apoptotic signals. Interestingly, TZDs also synergize with all-trans-retinoic
acid (ATRA) to induce apoptosis in MCF-7 and primary breast carcinoma cells,
but not in the normal breast epithelium [43]. Some TZDs also stimulate
expression of apoptosis related genes, such as growth arrest and DNA damage-inducible
gene 45 (GADD45) [50], BRCA1 [51] and proline oxidase encoding
gene POX [52]. In addition to
intrinsic apoptotic pathways, TZDs are also capable of the direct stimulation
of the FASL gene encoding Fas ligand
that induces an apoptosis by cross-linking with the Fas receptor located on the
membranes of the adjacent cells [53].Additionally, TZDs block the invasion of
tumor cells through upregulation of the tissue inhibitor of MMP-1/TIMP-1 and a subsequent
decrease in MMP-9 gelatinolytic activities [54]. These observations have been
supported by experiments with the murine mammary tumor cell line LMM3, which
produces less metastatic nodules in lungs of animals treated by oral
rosiglitazone [55]. It should be mentioned that the pronounced antitumor
effects described above occur only at suprapharmacological doses of TZDs. It
remains to be seen whether chronic exposure to TZDs could have therapeutic
effects in patients with established breast tumors.The effects described above are relevant
only to some TZD users, namely, patients currently with breast tumors and those
diagnosed with such tumors in the past. It is still unclear whether action of
PPARγ ligands is different within normal and tumor cells, and what would be
effects of TXD exposure in cancer free individuals. There are some indications
that PPARγ ligands may influence the initial stages of breast carcinoma
development, in particular, immortalization of the breast epithelia. One recent
study demonstrated that exposure to low nontoxic doses of rosiglitazone (10 nM)
reduces the frequency of spontaneous immortalization of Li-Fraumeni syndrome (LFS)-derived
(p53 +/−, telomerase silent) breast epithelial cells by almost four times [56].
In these experimental settings, the antimutagenic properties of this widely
prescribed TZD were superior to those of well-known chemopreventive agents such
as sulindac sulfide and celecoxib [56]. It will be interesting to see whether
exposure to TZD is capable of lowering the incidence of malignant foci in the breast
epithelia genetically predisposed to breast carcinoma development, particularly
that of carriers of mutations in BRCA,
BRCA2, or ATM.Some effects outlined above result from the
interference of PPARγ signaling with other pathways involved in breast
carcinogenesis, particularly with estrogen receptor (ER)α and NF-κB cascades. Agonists
of PPARγ may suppress NF-κB dependent transcription either through an increase
in physical interaction between PPARγ and p65 [57] or through SUMOylation-dependent
targeting of PPARγ to NCoR/histone deacetylase-3 (HDAC3) corepressor complexes which
prevent NCoR/HDAC3 clearance from NF-κB target gene promoters [58]. The interplay
between ER and PPARγ signaling seems to be more complex. Many PPARγ ligands,
particularly troglitazone and ciglitazone, inhibit ERα signaling by stimulating
proteasomal degradation of ERα [59].On the other hand, one recent study’s findings
are disturbing: in the breast cancer cell line MCF-7, commonly used as a model
for ER-positive breast carcinoma, TZD rosiglitazone has been shown to induce
both estrogen receptor response element activity and cell proliferation [44].
Even more disturbing is the fact that in dose-response assays higher concentrations
of rosiglitazone inhibited proliferation, while lower concentrations of the
same compound induced proliferation. Rosiglitazone-induced proliferation and ERE
reporter activation were mediated by ERα and the extracellular signal-regulated
kinase-mitogen activated protein kinase (ERK-MAPK) pathway [44]. The concentration-dependent
nature of rosiglitazone’s effects may have tremendous clinical importance for
the chronic users of TZDs. Moreover, these findings point at the possibility
that the effects of the rosiglitazone might vary between individuals, as the bioavailability
of rosiglitazone depends on the activity of the CYP2C9 and CYP2C8 enzymes [60],
which are substantially polymorphic in human populations.


### 2.2. Chronic Exposure to PPARγ Agonists Influences Nonepithelial Cells Participating in Breast
Carcinoma Development

In addition to the effects of PPARγ ligands on
premalignant and malignant breast epithelia, these compounds also produce profound
changes in noncancerous cells. Some of these changes may be relevant to breast
carcinoma outcomes. For example, PPARγ ligands demonstrate antiangiogenic effects
(reviewed in [40]), including direct suppression of the vascular endothelial
growth factor (VEGF) and the angiopoietin-1 (Ang-1) gene transcription [61, 62].
On the other hand, in some noncancerous settings, PPARγ ligands stimulate
angiogenesis [63, 64], thus pointing to their involvement in remodeling tumor
vessels rather than in suppressing angiogenesis per se.In
vitro experiments suggest that PPARγ ligands act as differentiating
agents in nonmalignant stromal cells. Malignant epithelialcells of
breast tumors secrete growth factors and cytokines to prevent the
differentiation of peri- and intratumoral stromal fibroblasts into mature
adipocytes by downregulation of adipogenic factors such as the C/EBPα and PPARγ
[65]. In turn, underdifferentiated fibroblasts provide structural and secretory
growth promoting support to tumor tissue [66]. Prolonged treatment with TZDs stimulates
the differentiation of fibroblasts into adipocytes instead of myofibroblasts and
interferes with transforming growth factor beta (TGFβ) fibrogenic pathway,
particularly, through attenuation of TGFβ-driven type I collagen protein
production [67]. Taken together, these effects of TZDs may to some degree
counteract desmoplastic proliferative response promoted by tumor proximity and
delay the formation of the scirrhous component of the breast tumors and the
subsequent spread of tumor cells.It must be taken into account that an
interference of TZDs with TGFβ signaling is a double-edged sword, since TGFβ serves
as both a tumor suppressor and a tumor promoter depending on tumor
developmental stages and cellular context [19]. During the initial phase of
breast tumorigenesis, the TGFβ signal inhibits primary tumor development and
growth by constraining cell division and possibly inducing apoptosis [68, 69]. In
the later stages of breast carcinoma development, tumors lose their sensitivity
to TGFβ, but continue overproduction of the hormone. Excess TGFβ acts upon stromal
components of the tumor promoting the metastatic process through desmoplastic
reaction, inhibiting host immune surveillance, and stimulating invasion and
angiogenesis [70]. The outcome of the crosstalk between TGFβ and PPARγ in
breast carcinoma patients should be dependent on stage of the particular breast
lesion.Last but not least, TZD therapy has been
shown to produce an average weight gain of 4-5 kg, which cannot
be explained by fluid retention [71]. The magnitude of weight gain correlates in
part with improved metabolic control, that is, better responders are more prone
to increases in body weight [72]. In turn, weight gain is associated with a
significant increase in postmenopausal ER-positive/PR-positive breast cancer [73, 74]. It remains to be seen whether TZD-associated increases in adiposity contribute
to breast carcinoma risks similarly to nonspecific weight gain.


### 2.3. Effects of TZDs on Breast Carcinogenesis in vivo

The PPARγ agonist GW7845 delays the development
of mammary tumors in immunocompetent mice treated with medroxyprogesterone
acetate followed by DMBA administration by an average of 2 months [75]. In the
classic rat model of mammary tumorigenesis employing nitrosomethylurea as a
carcinogen, GW7845 also significantly reduces both tumor incidence and tumor
weight [76]. Similarly, troglitazone, alone or in combination with RXR ligands,
prevents the induction of preneoplastic lesions in a mouse mammary gland organ
culture model treated by DMBA [77]. TZD treatment alone or in combination with
ATRA suppresses tumor growth from breast carcinoma cells MCF-7 [43]. On the
other hand, attempted rosiglitazone chemoprevention of breast carcinogenesis in
the MMTV-HER-2/neu transgenic mouse model produced no encouraging data [78]. It
is important to note that the mechanisms underlying various routes of the
tumorigenesis in rodent breast differ substantially [79]; therefore, it is
entirely possible that TZDs may modify outcomes only in some of the models
studied. It is also possible that these effects might be either compound or
dose-specific.Recently, a few epidemiological studies have
explored the association of TZD-based diabetes therapy and breast carcinoma
incidence. The largest profiled cohort was the one covered by the Integrated
Healthcare Information Services (IHCISs), Mass, USA, managed care database [80]. The
relevant part of IHCIS allowed analysis of pharmacy and doctor’s office claim
data related to 126 971 nonelderly USA diabetics with a mean followup time
of 16.6 months. Importantly, each individual case of breast carcinoma (N=513) was
matched to up to five diabetes controls (cumulative N=2557) using matched
nested case-control design. The adjusted odds ratios and 95% CI for breast
cancer from any exposure to TZD (mono- or combination therapy) compared to all
non-TZD antidiabetic agents were
0.89 (0.68–1.15) [80]. Thus,
neither a beneficial nor a deleterious effect of TZDs on the likelihood of breast
carcinoma development was found. It should be mentioned that the median
duration of followup in the studied cohort was rather short for the development
of breast tumors. Studies following patients for longer periods of time are
warranted.Another group of researchers analyzed 1003
adult diabetic patients participating in a Vermont Diabetes Information System
(VDIS) study and revealed a significant association between any cancer and the
use of any TZD (OR=1.59, 95% CI (1.03–2.44), P =
.04) [79]. When TZDs were analyzed by compound, a significant association was
found for rosiglitazone (OR = 1.89, 95% CI (1.11–3.19), P=.02), but not for pioglitazone. Stratification by gender showed a highly
significant association between cancer prevalence and TZD use for women (OR =
2.07, 95% CI (1.18–3.63), P=.01) [81], but not for men. It is important to note that the number of the
patients enrolled in this study is not allowed assessment of the risks for individual cancers.
Nevertheless, the increase of tumor incidence in TZD using women points at the
possible vulnerability of the breast epithelia.Slightly more encouraging results were
produced in the recently completed PROactive Study (PROspective pioglitAzone
Clinical Trial In macroVascular Events). This study reviewed longitudinal data of
5238 diabetic patients treated with pioglitazone or with a placebo [82]. The incidence
of breast carcinoma was nonsignificantly reduced in the pioglitazone-treated
group (3 versus 11 cases in the equally sized pioglitazone and placebo arms of
the study, resp.).Several attempts to use TZDs as a means of
therapy for breast carcinoma have been made so far. One trial of TZD as a
monotherapy ended 5 months after it started, because troglitazone was withdrawn
from the marker. This trial—performed in the cohort of patients with
advanced breast cancer refractory to at least one chemotherapy regimen—resulted in no objective responses [83]. Another
attempt at TZD monotherapy enrolled 38 women with early-stage lymph node
negative breast carcinomas. This intervention was even shorter as rosiglitazone
treatment (8 mg/d) was given between the time of diagnostic biopsy and
definitive surgery. No significant effects on breast tumor cell proliferation
were observed using Ki67 expression as an endpoint. Interestingly,
rosiglitazone treatment leads to down-regulation of nuclear PPARγ expression, as
demonstrated by immunohistochemistry. Additionally, rosiglitazone intervention
resulted in an increase of serum adiponectin concentrations (P<.001). Serum adiponectin negatively regulates breast cancer growth [84] and
inhibits angiogenesis by suppression of endothelial cell proliferation and
migration [85]. The potential therapeutic implications of rosiglitazone
modulation of adiponectin levels require further study.


## 2.1. Effects of the Chronic Exposure of the Breast Epithelium to PPARγ Agonists

PPARγ is expressed in normal breast tissue
and in many primary breast carcinoma specimens [30, 31]. Comparative studies of
PPARγ expression in breast carcinoma patients so far have produced
contradictory results [32–34]. Described
associations between PPARG polymorphisms and breast carcinoma are also discrepant: some researchers see a
marginally significant increase in the risk of breast cancer among women
homozygous for the Ala allele of PPARγ (Pro12Ala), causing a reduction in the transcriptional activity
of PPARγ2 [35], while others stress that carriers of the same variant allele
are at lower risk [36]. Since complete loss of PPARγ signaling in clinical
breast tumors seems to be a rare event [37], it is likely that patients
undergoing chronic treatment with chemical ligands for PPARγ will experience
alteration in the behavior of both breast carcinoma cells and their normal
counterparts. Patients with ER-positive tumors might benefit from TZD exposure
more than those with ER-negative tumors, as the level of PPARγ expression is
significantly associated with the ER status of carcinoma cells [38].Chemically synthesized ligands for PPARγ
(thiazolidinediones, or TZDs) have actively been used as insulin sensitizers
since the late 90s [18]. In addition to their insulin resistance-alleviating
effects, TZDs may influence an incidence or a progression of breast carcinoma
lesions as they have been shown to suppress the proliferation rates of many
types of cancer cells and induce either their differentiation or apoptosis in vitro [20, 39, 40]. Responsiveness
to TZDs has been demonstrated for both normal human mammary epithelial cells
[30] and breast cancer cells [41–43], although it
was not uniformly seen in all experimental conditions [44]. TZDs suppress the
cell cycle by repressing cyclins D1 and D3 [45], by stimulating expression of
the tumor suppressor p53 and its effector p21 (WAF1/Cip1) [46], and by inhibiting
the Akt/PTEN pathway [47]. Additionally, TZDs induce marked cellular acidosis
in breast carcinoma cell lines, leading to a decrease in the number of viable
cells [48]. Some effects of TZDs are independent of the transcriptional
activities of PPARγ [48]; these effects may be mediated through interactions of
these compounds with other cellular targets.The growth-suppressive properties of TZDs
are complemented by their ability to induce apoptosis. Many breast tumors are
naturally resistant to the apoptotic action of the tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) and other similar agents. TZDs
sensitize these cells to TRAIL [45], to anti-Fas IgM (CH11), and to tumor
necrosis factor (TNF)-α [49]. It is tempting to speculate that TZDs might prevent
the spread of microscopic breast tumors by sensitizing malignant cells to these
endogenous apoptotic signals. Interestingly, TZDs also synergize with all-trans-retinoic
acid (ATRA) to induce apoptosis in MCF-7 and primary breast carcinoma cells,
but not in the normal breast epithelium [43]. Some TZDs also stimulate
expression of apoptosis related genes, such as growth arrest and DNA damage-inducible
gene 45 (GADD45) [50], BRCA1 [51] and proline oxidase encoding
gene POX [52]. In addition to
intrinsic apoptotic pathways, TZDs are also capable of the direct stimulation
of the FASL gene encoding Fas ligand
that induces an apoptosis by cross-linking with the Fas receptor located on the
membranes of the adjacent cells [53].Additionally, TZDs block the invasion of
tumor cells through upregulation of the tissue inhibitor of MMP-1/TIMP-1 and a subsequent
decrease in MMP-9 gelatinolytic activities [54]. These observations have been
supported by experiments with the murine mammary tumor cell line LMM3, which
produces less metastatic nodules in lungs of animals treated by oral
rosiglitazone [55]. It should be mentioned that the pronounced antitumor
effects described above occur only at suprapharmacological doses of TZDs. It
remains to be seen whether chronic exposure to TZDs could have therapeutic
effects in patients with established breast tumors.The effects described above are relevant
only to some TZD users, namely, patients currently with breast tumors and those
diagnosed with such tumors in the past. It is still unclear whether action of
PPARγ ligands is different within normal and tumor cells, and what would be
effects of TXD exposure in cancer free individuals. There are some indications
that PPARγ ligands may influence the initial stages of breast carcinoma
development, in particular, immortalization of the breast epithelia. One recent
study demonstrated that exposure to low nontoxic doses of rosiglitazone (10 nM)
reduces the frequency of spontaneous immortalization of Li-Fraumeni syndrome (LFS)-derived
(p53 +/−, telomerase silent) breast epithelial cells by almost four times [56].
In these experimental settings, the antimutagenic properties of this widely
prescribed TZD were superior to those of well-known chemopreventive agents such
as sulindac sulfide and celecoxib [56]. It will be interesting to see whether
exposure to TZD is capable of lowering the incidence of malignant foci in the breast
epithelia genetically predisposed to breast carcinoma development, particularly
that of carriers of mutations in BRCA,
BRCA2, or ATM.Some effects outlined above result from the
interference of PPARγ signaling with other pathways involved in breast
carcinogenesis, particularly with estrogen receptor (ER)α and NF-κB cascades. Agonists
of PPARγ may suppress NF-κB dependent transcription either through an increase
in physical interaction between PPARγ and p65 [57] or through SUMOylation-dependent
targeting of PPARγ to NCoR/histone deacetylase-3 (HDAC3) corepressor complexes which
prevent NCoR/HDAC3 clearance from NF-κB target gene promoters [58]. The interplay
between ER and PPARγ signaling seems to be more complex. Many PPARγ ligands,
particularly troglitazone and ciglitazone, inhibit ERα signaling by stimulating
proteasomal degradation of ERα [59].On the other hand, one recent study’s findings
are disturbing: in the breast cancer cell line MCF-7, commonly used as a model
for ER-positive breast carcinoma, TZD rosiglitazone has been shown to induce
both estrogen receptor response element activity and cell proliferation [44].
Even more disturbing is the fact that in dose-response assays higher concentrations
of rosiglitazone inhibited proliferation, while lower concentrations of the
same compound induced proliferation. Rosiglitazone-induced proliferation and ERE
reporter activation were mediated by ERα and the extracellular signal-regulated
kinase-mitogen activated protein kinase (ERK-MAPK) pathway [44]. The concentration-dependent
nature of rosiglitazone’s effects may have tremendous clinical importance for
the chronic users of TZDs. Moreover, these findings point at the possibility
that the effects of the rosiglitazone might vary between individuals, as the bioavailability
of rosiglitazone depends on the activity of the CYP2C9 and CYP2C8 enzymes [60],
which are substantially polymorphic in human populations.


## 2.2. Chronic Exposure to PPARγ Agonists Influences Nonepithelial Cells Participating in Breast
Carcinoma Development

In addition to the effects of PPARγ ligands on
premalignant and malignant breast epithelia, these compounds also produce profound
changes in noncancerous cells. Some of these changes may be relevant to breast
carcinoma outcomes. For example, PPARγ ligands demonstrate antiangiogenic effects
(reviewed in [40]), including direct suppression of the vascular endothelial
growth factor (VEGF) and the angiopoietin-1 (Ang-1) gene transcription [61, 62].
On the other hand, in some noncancerous settings, PPARγ ligands stimulate
angiogenesis [63, 64], thus pointing to their involvement in remodeling tumor
vessels rather than in suppressing angiogenesis per se.In
vitro experiments suggest that PPARγ ligands act as differentiating
agents in nonmalignant stromal cells. Malignant epithelialcells of
breast tumors secrete growth factors and cytokines to prevent the
differentiation of peri- and intratumoral stromal fibroblasts into mature
adipocytes by downregulation of adipogenic factors such as the C/EBPα and PPARγ
[65]. In turn, underdifferentiated fibroblasts provide structural and secretory
growth promoting support to tumor tissue [66]. Prolonged treatment with TZDs stimulates
the differentiation of fibroblasts into adipocytes instead of myofibroblasts and
interferes with transforming growth factor beta (TGFβ) fibrogenic pathway,
particularly, through attenuation of TGFβ-driven type I collagen protein
production [67]. Taken together, these effects of TZDs may to some degree
counteract desmoplastic proliferative response promoted by tumor proximity and
delay the formation of the scirrhous component of the breast tumors and the
subsequent spread of tumor cells.It must be taken into account that an
interference of TZDs with TGFβ signaling is a double-edged sword, since TGFβ serves
as both a tumor suppressor and a tumor promoter depending on tumor
developmental stages and cellular context [19]. During the initial phase of
breast tumorigenesis, the TGFβ signal inhibits primary tumor development and
growth by constraining cell division and possibly inducing apoptosis [68, 69]. In
the later stages of breast carcinoma development, tumors lose their sensitivity
to TGFβ, but continue overproduction of the hormone. Excess TGFβ acts upon stromal
components of the tumor promoting the metastatic process through desmoplastic
reaction, inhibiting host immune surveillance, and stimulating invasion and
angiogenesis [70]. The outcome of the crosstalk between TGFβ and PPARγ in
breast carcinoma patients should be dependent on stage of the particular breast
lesion.Last but not least, TZD therapy has been
shown to produce an average weight gain of 4-5 kg, which cannot
be explained by fluid retention [71]. The magnitude of weight gain correlates in
part with improved metabolic control, that is, better responders are more prone
to increases in body weight [72]. In turn, weight gain is associated with a
significant increase in postmenopausal ER-positive/PR-positive breast cancer [73, 74]. It remains to be seen whether TZD-associated increases in adiposity contribute
to breast carcinoma risks similarly to nonspecific weight gain.


## 2.3. Effects of TZDs on Breast Carcinogenesis in vivo

The PPARγ agonist GW7845 delays the development
of mammary tumors in immunocompetent mice treated with medroxyprogesterone
acetate followed by DMBA administration by an average of 2 months [75]. In the
classic rat model of mammary tumorigenesis employing nitrosomethylurea as a
carcinogen, GW7845 also significantly reduces both tumor incidence and tumor
weight [76]. Similarly, troglitazone, alone or in combination with RXR ligands,
prevents the induction of preneoplastic lesions in a mouse mammary gland organ
culture model treated by DMBA [77]. TZD treatment alone or in combination with
ATRA suppresses tumor growth from breast carcinoma cells MCF-7 [43]. On the
other hand, attempted rosiglitazone chemoprevention of breast carcinogenesis in
the MMTV-HER-2/neu transgenic mouse model produced no encouraging data [78]. It
is important to note that the mechanisms underlying various routes of the
tumorigenesis in rodent breast differ substantially [79]; therefore, it is
entirely possible that TZDs may modify outcomes only in some of the models
studied. It is also possible that these effects might be either compound or
dose-specific.Recently, a few epidemiological studies have
explored the association of TZD-based diabetes therapy and breast carcinoma
incidence. The largest profiled cohort was the one covered by the Integrated
Healthcare Information Services (IHCISs), Mass, USA, managed care database [80]. The
relevant part of IHCIS allowed analysis of pharmacy and doctor’s office claim
data related to 126 971 nonelderly USA diabetics with a mean followup time
of 16.6 months. Importantly, each individual case of breast carcinoma (N=513) was
matched to up to five diabetes controls (cumulative N=2557) using matched
nested case-control design. The adjusted odds ratios and 95% CI for breast
cancer from any exposure to TZD (mono- or combination therapy) compared to all
non-TZD antidiabetic agents were
0.89 (0.68–1.15) [80]. Thus,
neither a beneficial nor a deleterious effect of TZDs on the likelihood of breast
carcinoma development was found. It should be mentioned that the median
duration of followup in the studied cohort was rather short for the development
of breast tumors. Studies following patients for longer periods of time are
warranted.Another group of researchers analyzed 1003
adult diabetic patients participating in a Vermont Diabetes Information System
(VDIS) study and revealed a significant association between any cancer and the
use of any TZD (OR=1.59, 95% CI (1.03–2.44), P =
.04) [79]. When TZDs were analyzed by compound, a significant association was
found for rosiglitazone (OR = 1.89, 95% CI (1.11–3.19), P=.02), but not for pioglitazone. Stratification by gender showed a highly
significant association between cancer prevalence and TZD use for women (OR =
2.07, 95% CI (1.18–3.63), P=.01) [81], but not for men. It is important to note that the number of the
patients enrolled in this study is not allowed assessment of the risks for individual cancers.
Nevertheless, the increase of tumor incidence in TZD using women points at the
possible vulnerability of the breast epithelia.Slightly more encouraging results were
produced in the recently completed PROactive Study (PROspective pioglitAzone
Clinical Trial In macroVascular Events). This study reviewed longitudinal data of
5238 diabetic patients treated with pioglitazone or with a placebo [82]. The incidence
of breast carcinoma was nonsignificantly reduced in the pioglitazone-treated
group (3 versus 11 cases in the equally sized pioglitazone and placebo arms of
the study, resp.).Several attempts to use TZDs as a means of
therapy for breast carcinoma have been made so far. One trial of TZD as a
monotherapy ended 5 months after it started, because troglitazone was withdrawn
from the marker. This trial—performed in the cohort of patients with
advanced breast cancer refractory to at least one chemotherapy regimen—resulted in no objective responses [83]. Another
attempt at TZD monotherapy enrolled 38 women with early-stage lymph node
negative breast carcinomas. This intervention was even shorter as rosiglitazone
treatment (8 mg/d) was given between the time of diagnostic biopsy and
definitive surgery. No significant effects on breast tumor cell proliferation
were observed using Ki67 expression as an endpoint. Interestingly,
rosiglitazone treatment leads to down-regulation of nuclear PPARγ expression, as
demonstrated by immunohistochemistry. Additionally, rosiglitazone intervention
resulted in an increase of serum adiponectin concentrations (P<.001). Serum adiponectin negatively regulates breast cancer growth [84] and
inhibits angiogenesis by suppression of endothelial cell proliferation and
migration [85]. The potential therapeutic implications of rosiglitazone
modulation of adiponectin levels require further study.


## 3. PPARα Ligands

The nuclear receptor PPARα regulates lipid
metabolism in general and β-oxidation of fatty acids  in particular. Its gene, PPARA, expresses mainly in tissues with
high energy requirements, particularly in the skeletal muscle, the heart, and
the liver [86]. PPARα is activated by a number of natural ligands, including
various derivatives of fatty acids and leukotriene B4, and by common lipid-lowering
drugs, particularly fenofibrate and gemfibrozil. Activated PPARα exerts
beneficial effects on lipid metabolism, raising cardioprotective high-density
lipoprotein (HDL) cholesterol and lowering cardiovascular mortality [87]. In
addition, activation of PPARα may limit inflammation, both in the vessel
endothelium and in other tissues as well as inhibit the fibrotic response. The
apparent uniformly beneficial action of PPARα agonists prompted the development
of a number of these compounds. Among them, some exert dual affinity to PPARα
and PPARγ. Dual agonists hold considerable promise in the management of insulin
resistance, serving as major confounders for cardiovascular diseases and other
comorbidities associated with metabolic syndrome.Experimental data describing the effects of
PPARα agonists on tumor initiation and progression are limited. Long-term
administration of PPARα ligands clofibrate and WY-14643 in the rodent model
induces hepatocellular neoplasms including adenomas and carcinomas [88]. PPARα
suppresses apoptosis in liver tissue in response to various peroxisome
proliferator carcinogens, especially in the presence of TNFα [89]. As levels of
TNFα are substantially elevated in obesity and in metabolic syndrome, it could
be hypothesized that hepatocarcinogenesis may be an issue for long-term fibrate
medicated patients. So far, epidemiological observations in fibrate treated
populations have not produced any evidence that fibrates are associated with
elevated risk of liver cancer or any other neoplasms in humans. As PPARα-humanized
mice are resistant to hepatocarcinogenic effects of fibrates, it seems that the
response described in mouse models is species specific [90].Studies of the nonhepatic tumorigenesis
models indicate that in other tissues PPARα agonists exert antiproliferative
effects [91]. In the mouse model of skin carcinogenesis, an animal topically
treated with PPARα ligands exhibited an approximately 30% lower skin tumor
yield compared with mice treated with vehicle, thus indicating that the activation
of PPARα may suppress the earliest stages of tumor development [92]. Additionally,
PPARα ligands possess strong antiangiogenic properties, as they suppress
endothelial cell proliferation and VEGF production, upregulate TSP-1 and
endostatin, and inhibit neovascularization [93, 94].Studies concerning PPARα activation in
breast carcinomas are scarce. It is known that PPARα is expressed and
dynamically regulated in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) human
breast cancer cells. PPARα activation significantly increases proliferation of
both cell lines, and this increase is proportional to the endogenous level of
PPARα [95]. On the other hand, one recent study pointed at PPARα as a possible
contributor to the growth inhibitory effect of n-6 PUFA arachidonic acid
exerted in the same pair of breast carcinomas cell lines [96].PPARα also reduces the sensitivity of
MCF-7cells to histone deacetylase inhibitors [97]. Interestingly, there is an
inverse relationship between mean PPARα and ERα mRNA levels in ER-positive
breast cancer cells [97]. These observations point to the possible involvement
of PPARα activation in mammary gland tumorigenesis and vouch for a longitudinal
study of breast carcinoma incidence and progression in patients using fibrate
therapy.


## 4. PPARδ Ligands

The nuclear
receptor PPARδ, also known as PPARβ, is expressed ubiquitously. It controls a
number of physiological functions, particularly cell proliferation and
differentiation as well as inflammation and energy homeostasis [22].
Interestingly, PPARδ is the only PPAR isoform that maintains repressor activity
when bound to DNA. When unligated, PPARδ can act as an intrinsic transcription
repressor and inhibit the trans-activation activity of other PPARs [98]. It was
suggested that PPARδ serves as a gateway receptor capable of modulating PPARα
and PPARγ activity [98]. The ligand binding pocket domain of PPARδ is larger
than that of other PPARs and is believed to accommodate the binding of various
fatty acids and their derivatives [99]. A number of synthetic agonists are being
developed for the same purpose with nanomolar affinities [100, 101], although
none is currently marketed for clinical use in humans yet.The physiological effects of activated PPARδ
have been studied extensively [22, 102]. The results of these studies
suggest that sooner or later high-affinity PPARδ synthetic drugs which uniquely
target multiple components of the metabolic syndrome, including obesity,
insulin resistance, hyperglycemia, dyslipidemia, and atherosclerosis will enter
the market. Some of these compounds are already being subjected to phase I/II
clinical trials. In light of this fact, it is important to establish
experimental systems allowing rapid evaluation of the potential carcinogenic or
chemopreventive effects of the synthetic PPARδ ligands. Given that the
prevalence of the metabolic syndrome and comorbidities associated with the disease
is on the rise in both developed and developing countries, it is extremely
important to watch for possible effects of anticipated chronic exposure to
PPARδ ligands upon common types of cancer, particularly upon breast carcinoma.Alarmingly, PPARδ selective agonists
stimulate the growth of the hormone-dependent breast carcinoma cell lines T47D
and MCF-7. In T47D cells, activation of PPARδ stimulates expression of the
proliferation marker Cdk2. In addition, an increase in the production of both
VEGF and its receptor, FLT-1 has been noted, suggesting that PPARδ may initiate
an autocrine loop for cellular proliferation and possibly angiogenesis. Similar
pro-proliferative effects of activated PPARδ have been observed in endothelial
cell cultures [103]. Further studies of angiogenic and growth-inducing
properties of PPARδ agonists in breast epithelia are warranted.


## 5. General Remarks

It should be mentioned that breast carcinoma
is not a single disease entity, but rather an extremely polymorphic spectrum of
neoplastic pathologies which are fairly diverse in their molecular portraits.
It is likely that both chemoprevention and treatment by PPAR ligands as well as
their possible tumorigenic side effects will be selective to particular
molecular subtypes of tumor, or will be relevant to certain stages of carcinoma
progression (Figure1). Therefore, much larger cohorts of patients followed for longer
periods of time will have to be studied in order to reveal statistically
significant modifications of the disease’s outcome. Chemoprevention studies of
this type are prohibitively expensive, for example, the recently completed National
Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene
(STAR) trial with an endpoint of cancer incidence required the enrollment of 19 747 subjects from near 200 clinical centers throughout North America took 8
years before initial data analysis, and cost approximately $200 million [104, 105]. Before initiating large-scale efforts, a comparative study of the
molecular portraits of breast carcinomas developed in chronic TZD users and in
the general population needs to be completed. This kind of study could be
performed using microarrays as a primary profiling means which should be
complemented by validation efforts through the methods of immunohistochemistry,
in situ hybridization of mRNA, and phosphoproteomics. The design of this study
could be a challenge due to the difficulties with proper matching of groups
compared and with eliminating common confounders. One of the possible ways to
overcome this problem is to profile both malignant and normal breast epithelia
samples of current TZD users to that of recently diagnosed diabetics never
exposed to TZDs. Confirmed differences between the molecular portraits of
tumors which initiated or progressed despite an exposure to PPAR ligand and
subtype-matched tumors that arose on TZD free background may give some
important clues to the design of a clinical trial aimed at
chemoprevention-related endpoints.Figure 1
A summary of influence of PPAR ligands on the process of breast
carcinogenesis.



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*Source: 230893-2008-07-15.xml*