# Clinical Use of PPARγ Ligands in Cancer

Authors: Jennifer L. Hatton; Lisa D. Yee
Journal: PPAR Research
 (2008)

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## Abstract

The role of PPARγ in adipocyte differentiation has fueled intense interest in the function of this steroid nuclear receptor for regulation of malignant cell growth and differentiation. Given the antiproliferative and differentiating effects of PPARγ ligands on liposarcoma cells, investigation of PPARγ expression and ligand activation in other solid tumors such as breast, colon, and prostate cancers ensued.  The anticancer effects of PPARγ ligands in cell culture and rodent models of a multitude of tumor types suggest broad applicability of these agents to cancer therapy.  This review focuses on the clinical use of PPARγ ligands, specifically the thiazolidinediones, for the treatment and prevention of cancer.


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## Body

## 1. Introduction

The peroxisome proliferator-activated receptorγ (PPARγ) is a ligand-activated transcription factor in
the superfamily of the steroid/thyroid nuclear hormone receptors, one of three PPAR isotypes (α, β/δ, and γ) [1–3]. PPARs share a highly conserved DNA-binding domain that matches with specific DNA sequences known as peroxisome
proliferator response elements (PPREs), binding as a heterodimer with retinoid
X receptor (RXR) to initiate transcription of target genes [4–6]. Highly
expressed in adipose tissue, PPARγ is best known for its important role in
adipocyte differentiation [7–9].PPARs were first shown to be
activated by compounds inducing peroxisome proliferation [10, 11], then by a variety of polyunsaturated fatty acids (PUFAs) in the micromolar range [12]. PPARγ has a more restricted list of
activators compared to the other two isotypes, being more selective to PUFAs
compared to other fatty acids [13, 14]. Fatty acid derivatives, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2)
and 13- and 9-hydroxyoctadecadienoic acid (HODE), have also been identified as activators of PPARγ [15–17]. Synthetic ligands for PPARγ include the thiazolidinediones, a class of
oral hypoglycemic drugs that reduce hyperglycemia and hyperinsulinemia in
insulin-resistant states [18]. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are also PPARγ ligands, but exhibit much lower binding
affinities in comparison to thiazolidinediones [19].A wealth of preclinical data supports a role for PPARγ ligand therapy in many different types of solid malignancies. Experimental studies
of human cancer cells and PPARγ have focused primarily on the γ-activating effects of thiazolidinediones, identifying PPARγ agonists as negative regulators of cell growth and tumor progression. As a putative
natural ligand for PPARγ, 15d-PGJ2 also appears to have anticancer
effects. Treatment with PPARγ ligands inhibits malignant cell proliferation,
with evidence for cell cycle arrest and induction of apoptosis [20–25]. With the pivotal role of PPARγ in adipocyte differentiation, PPARγ ligands have been tested for differentiating effects on malignant cells. Changes consistent
with induction of a more differentiated cancer cell phenotype were detected in
several tumor model systems, including breast, colon, and liposarcoma [9, 26–30].The anticancer effects of PPARγ agonists may also be mediated in part via
suppression of an angiogenic tumor phenotype or angiogenic, inflammatory tumor
microenvironment. PPARγ ligands appear to elicit antiangiogenic
effects, via modulation of endothelial cell function and growth [31–35]. The anti-inflammatory actions of PPARγ ligands [36–38], which are of relevance to the
treatment of atherosclerosis and cardiovascular disease, may also prove
important for the treatment and prevention of cancer given the association of
chronic inflammation with increased cancer risk [39].Approved by the Food and Drug
Administration for treatment of type 2 diabetes mellitus, thiazolidinediones
have been evaluated for use as investigational cancer therapies. The initial studies utilized troglitazone
(Rezulin), which was the first thiazolidinedione in clinical use but ultimately
withdrawn from the US market in 2000 because of instances of severe idiopathic
liver disease [40]. Rosiglitazone (Avandia) and pioglitazone
(Actos) were thereafter used without evidence of similar hepatotoxicity. The majority of clinical trials of
thiazolidinediones have been conducted in advanced stages of disease. The following review examines the clinical
trial experience to date with thiazolidinediones in cancer.


## 2. Monotherapy

Searches of the PubMed andwww.clinicaltrials.gov databases
identified nine clinical trials testing the efficacy of thiazolidinediones as
single agent therapy in cancer patients, with the majority conducted in
subjects with advanced and/or metastatic stages of disease refractory to
treatment. Troglitazone was the drug of
choice for human studies initiated prior to 2000, replaced by rosiglitazone or
pioglitazone in trials conducted after the withdrawal of troglitazone from
clinical use because of rare instances of hepatotoxicity. Thiazolidinediones have been generally
administered at the highest recommended dose for treatment of diabetes
mellitus, or slightly higher as with troglitazone at 800 mg/day. Rather than mimic the generalized cytoxicity
of chemotherapeutic regimens, such doses might selectively target PPARγ-mediated effects such as cellular
differentiation, growth inhibition, and induction of apoptosis.


### 2.1. Liposarcomas

As mesenchymal malignancies that
arise from adipose tissue [41, 42], liposarcomas express PPARγ at
levels comparable to normal adipose tissue [26]. PPARγ expression is a distinguishing feature
of liposarcomas relative to other soft tissue sarcomas [43]. Treatment of primary cell strains  derived from human liposarcomas (two well-differentiated
liposarcomas and one intermediate grade myxoid/round cell liposarcoma) with
PPARγ and/or RXR ligands induced morphologic and molecular changes consistent
with adipocyte differentiation [26]. These in
vitro findings led to consideration of thiazolidinediones for
differentiation therapy of liposarcomas.Demetri et al. conducted an open
label phase II clinical trial to determine the effects of troglitazone (800 mg/day) on tumor differentiation in patients with advanced and/or metastatic
liposarcoma [44]. In a report of three study subjects, tumor
samples obtained after the six week intervention showed marked cytoplasmic
lipid accumulation in comparison to pretreatment biopsies, as well as
morphologic changes suggestive of mature adipocytes. The expression of markers of adipocyte differentiation such as adipsin, aP2, and PPARγ by Northern analysis increased following treatment with troglitazone. Furthermore, cell proliferation as assessed by the percentage of
liposarcoma cells with Ki67 immunostaining decreased by two- to fourfold with
the study intervention. Three additional
patients enrolled in the study also had post-treatment biopsies with histologic
evidence of adipocyte differentiation and decreased cell proliferation. The histologic subtypes treated in this study
ranged from intermediate to high grade, and the authors raise the possibility
that differentiation may prove difficult to assess in more differentiated
tumors. This small pilot trial of six
subjects demonstrated a differentiating effect of short-term troglitazone
therapy in advanced stage liposarcomas providing the rationale for further
clinical evaluation of troglitazone and other PPARγ ligands in the management
of liposarcomas.A second phase II trial evaluated
the effects of the thiazolidinedione rosiglitazone (8 mg administered as 4 mg
twice daily) in nine patients with advanced, unresectable liposarcomas [45]. Analyses of tumor samples obtained at 0, 6,
and 12 weeks did not reveal changes in histopathology or gene expression to
support a differentiating effect. By
quantitative RT-PCR, levels of adipsin, fatty acid-binding protein (FABP), and
PPARγ increased in only two of nine tumors although in a somewhat inconsistent
fashion. In one of the patients with a
dedifferentiated liposarcoma, expression of PPARγ increased at week 12, FABP at
week 6 and adipsin at weeks 6 and 12. However, disease progression had been detected in this patient at 10
weeks of treatment, such that the expression of differentiation markers did not
correlate with clinical benefit. For the
other subject, upregulation of adipsin at week 12 was noted in a myxoid
liposarcoma, and treatment continued until progression of disease at six
months. Cell proliferation assessed by
Ki67 immunostaining did not decrease significantly with the study intervention,
although the low baseline levels of proliferation decreased further with
rosiglitazone. Taken together the data
fail to support a role for rosiglitazone in the treatment of liposarcoma at
advanced stages.The discrepant findings between the
two phase II trials of thiazolidinedione therapy may relate to differences in
the patient cohorts, tumors, and/or study agents. Whether methodological issues contributed to
the differences is not clear. As surgery
remains the mainstay of treatment for liposarcomas, with limited therapeutic
options for unresectable, advanced disease, and investigation of PPARγ ligands for adjuvant therapy of liposarcomas
or control of microscopic or minimal residual disease may still be
warranted.


### 2.2. Colon Cancer

Considerable in vitro evidence exists to support
the differentiating and antiproliferative effects of PPARγ ligand therapy in colon cancer. PPARγ is highly expressed in human colon tumor
specimens and cancer cell lines [27, 46]. In addition to growth inhibition, treatment
of colon cancer cells with PPARγ ligands promotes tumor cell differentiation as
assessed by increased cytoplasmic to nuclear ratio and higher levels of
differentiation markers such as carcinoembryonic antigen (CEA), villin,
intestinal alkaline phosphatase, GOB-4, and keratin 20 [27, 29, 47]. Although in vitro and in vivo
studies demonstrate a role for PPARγ in modulating growth and differentiation of
human colon cancer cell lines [27], thiazolidinedione treatment with rosiglitazone
(BRL49653) or troglitazone stimulated rather than inhibited the development of
colon tumors in APCmin/+ mice [48, 49]. The loss of adenomatous polyposis coli (APC)
tumor suppressor gene function, which leads to accumulation of β-catenin and enhanced Wnt pathway signaling,
may account for the contradictory findings of enhanced tumorigenesis in this
genetic versus xenograft model of colon cancer [50]. Interestingly, treatment with pioglitazone
over a wide range of doses suppressed intestinal tumor formation and
hyperlipidemia in APC deficient mice (APCmin/+ and Apc1309) [51, 52]. Whether the use of different thiazolidinediones or experimental conditions led to the discrepant findings is unclear.Loss of function mutations inPPARG in colon cancer may also account for variability in response to PPARγ therapy. Four unique mutations of PPARγ were identified in 55 sporadic colon cancers
in exons 3 or 5; one yielding a truncated protein with loss of the ligand
binding domain (c.472delA) and three causing defects in the binding of
synthetic or natural ligands (Q286P, K319X, and R288H) [53]. A point mutation in exon 6, K422Q, was
detected in four human colon cancer cell lines that were resistant to the
antiproliferative and differentiating effects of PPARγ ligands [54]. However, K422Q may have limited clinical
relevance, as a study of 170 primary human colorectal cancers and 12 liver
metastases failed to detect the presence of K422Q mutations [55].A phase II trial evaluated
troglitazone therapy (800 mg/day as a single dose) in 25 colon cancer patients
with lung and/or liver metastases [56]. Although well-tolerated, troglitazone did not elicit objective tumor responses. All
patients had progression of disease with a median progression-free survival of
1.6 months and median overall survival of 3.9 months. Troglitazone does not seem to have activity
in advanced colon cancer, although the lack of response may relate at least in
part to the biologically aggressive nature of Stage IV colon cancer that is refractory to cytotoxic chemotherapy (the median number of prior chemotherapy regimens was 3). Although the study was not designed
to assess troglitazone for possible tumor promoting effects, Kulke et al. urged caution in the use of
PPARγ agonists in colon cancer. In the future, advances in the genomic
profiling of colon cancer might allow for identification of susceptible colon
cancer subtypes.Colorectal cancer provides an
excellent model of multistep progression in carcinogenesis, based on the
acquisition of biochemical, molecular, and genetic alterations that transform
normal epithelium to adenomatous polyps to invasive cancer [57]. PPARγ ligands may have a role in chemoprevention of
colon cancer, depending on the specific molecular and genetic subtype. Thiazolidinediones suppress the development
of aberrant crypt foci (ACF), a putative precancerous lesion of the colon [58]. However, activation of certain oncogenic
signaling pathways at later stages of carcinogenesis may override this
regulatory role, as suggested by the APCmin/+
mouse studies [50]. Timing of PPARγ therapy prior to certain initiating events in
colon carcinogenesis may thus prove to be useful for chemoprevention of colon
cancer. As suggested by the in vivo studies of thiazolidinediones
in min mice, use of PPARγ agonists in colon cancer prevention may
require identification of high-risk individuals based on genetic susceptibility
as well as aberrant gene expression and signal transduction.Although the differentiating and
antiproliferative effects of PPARγ activation form the basis for evaluating PPARγ ligands for cancer therapy, the
anti-inflammatory effects of PPARγ may also be relevant to colon
carcinogenesis. Chronic inflammation is
linked to carcinogenesis, and diagnoses of inflammatory bowel disease (IBD),
which includes Crohn's disease and ulcerative colitis, carry increased risk for
colorectal cancer [59]. PPARγ ligands repress cytokine production by colon
cancer cells and inhibit chemically-induced colitis in murine models of inflammatory bowel disease [60–62]. These findings led to an open-label pilot
trial of rosiglitazone therapy (4 mg taken twice daily) in patients with active
ulcerative colitis for 12 weeks, showing clinical improvement in 8/15 (53%) patients
of which 4 (27%) subjects had clinical remission [63]. Two subjects required hospitalization for
worsening disease; other serious adverse events included severe pharyngitis
requiring hospitalization and nephritic syndrome leading to withdrawal.  The increased risk for colon cancer in
patients with ulcerative colitis relates to the severity and duration of
disease; by suppressing inflammation in the colon, drugs such as the
thiazolidinediones or other PPARγ agonists might play a role in colon cancer
prevention.


### 2.3. Breast Cancer

In vitro and in vivo
research studies support a role for PPARγ agonists in breast cancer therapy. Thiazolidinedione treatment of human breast
cancer cells appears to induce changes suggestive of terminal differentiation,
resulting in the inhibition of cell proliferation, suppression of keratin 19,
mucin-1, and upregulation of maspin [28]. Troglitazone also inhibited breast cancer
growth in a xenograft animal model [20]. Another synthetic ligand of PPARγ, GW7845, had inhibitory effects in a
carcinogen-induced model of mammary tumorigenesis [64]. Furthermore, PPARγ haploinsufficiency (PPARγ+/−) conferred increased susceptibility to
dimethylbenz[a]antracene (DMBA)-induced
mammary carcinogenesis [65]. These studies suggest that PPARγ ligands could serve as negative regulators of
breast cancer development and progression.We and others have demonstrated PPARγ immunostaining in human breast cancer
specimens, as well as normal and benign proliferative breast tissue [28, 66]. PPARγ appears to be expressed in most if not all
breast cancer cell lines, as well as in normal and malignant breast tissue [20, 28, 66]. We did not find that the invasive, metastatic
phenotype of the breast cancer cells correlates with PPARγ expression levels, gel shift mobility patterns,
or relative sensitivity to PPARγ ligands [67]. All of the cell lines tested (MCF-7,
MDA-MB-436, MDA-MB-231, and MDA-MB-435) responded to thiazolidinedione
treatment in transactivation assays, which also led to inhibition of cell
growth.However, a Phase II trial of
troglitazone in metastatic breast cancer refractory to cytotoxic chemotherapy did
not demonstrate beneficial effects on disease progression [68]. In twenty two Stage IV breast cancer patients
with an 8-week median treatment duration, troglitazone at 800 mg/day did not
prevent disease progression. The median time
on study was 56 days, ranging from 11 to 134 days; 19/22 subjects were removed
from the study for objective or subjective evidence of disease
progression. There were no objective
responses, and only 3/21 (14%) subjects completing 8 weeks of troglitazone
therapy had stable disease. In patients
with elevated serum levels of CEA and/or CA27.29 at the onset of the study,
these tumor markers had continued to increase despite 8 weeks of troglitazone. Burstein et al. note that more selective criteria to target PPARγ therapy to the most susceptible tumor type might
be helpful. The lack of effect might
have also related to an advanced stage of disease that had become refractory to
cytotoxic therapies.To assess the effects of PPARγ ligand therapy on breast cancer, we conducted a
pilot study of short-term administration of rosiglitazone (4 mg taken twice a
day) to women newly diagnosed with early stage breast cancer (Stages 0 to II) during the two- to six-week period between diagnostic biopsy and surgical removal of the cancer [69]. Thirty eight women completed the study intervention without serious adverse events; thirty four subjects had
sufficient pre and posttreatment tumor tissue for correlative analyses. By Ki67 immunostaining, short-term
intervention with rosiglitazone did not lead to statistically significant
differences cell proliferation. We did not detect any somatic or germline mutations in PPARG to account for differential
tumor tissue responses or lack of effect. Comparing H&E-stained sections of tumor before and after
rosiglitazone therapy also failed to note increased tumor cell differentiation
by standard criteria of tubule formation, mitotic activity, and nuclear
morphology. PPARγ expression in tumor
samples was graded 0 to 3+ for intensity of nuclear and cytoplasmic
immunostaining, which did not reflect an overall change in expression following
rosiglitazone therapy. Interestingly, nuclear
expression of PPARγ was significantly reduced in a majority of the patients
whose pre-rosiglitazone PPARγ scores were 1+ or 2+ and therefore assessable for
increased or decreased levels of expression, which raised the possibility of a
PPARγ mediated response in the tumor tissue.Serum levels of adiponectin and
indices of insulin sensitivity increased with rosiglitazone, based on serum
samples obtained from seventeen subjects. Given the association of lower levels of adiponectin and insulin
resistance with increased breast cancer risk [70–73], therapy with rosiglitazone or
other PPARγ ligands may have a role in breast cancer
prevention. Of interest are potential effects
of rosiglitazone therapy on nonepithelial components of the mammary tumor microenvironment;
unfortunately, pretreatment core biopsy samples in our study had negligible to
minimal stromal, noncancerous tissue for any comparative analyses.Although short-term intervention
with rosiglitazone did not alter the endpoint of cell proliferation, this pilot
study indicated the potential for other tumor tissue specific effects with
evidence for downregulation of PPARγ protein expression. Breast cancer is a family of diseases with
diverse molecular, genetic features, and PPARγ ligands may affect only certain subtypes. Indeed, breast cancer therapy already
encompasses molecularly targeted strategies, such as tamoxifen for estrogen
receptor positive cancer and trastuzumab for tumors with HER-2/neu
overexpression. Variability in PPARγ-mediated effects on cell proliferation could
arise from tumor heterogeneity in nuclear receptor cross-talk, complement of
cofactors, or mutated or aberrant signaling pathways that override PPARγ signaling, leading to an apparent lack of
effect of rosiglitazone [74–77]. For example, in vitro studies indicate
that estrogen receptor α (ERα) can repress PPARγ signaling by binding to PPREs and that
PPAR/RXR heterodimers can competitively inhibit ER binding at specific estrogen
response elements (EREs) [74, 75]. In
vitro administration of the PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 inhibited estrogen-mediated
transactivation of ERE and estrogen-responsive gene expression in MCF-7 breast
cancer cells [78]. By immunohistochemical analysis, PPARγ expression has been detected in 42% (99/238)
to 58% (101/170) of human breast cancer samples, correlating positively with ER
expression and improved clinical outcome [78, 79]. Concomitant administration of other drugs,
such as estrogen in postmenopausal women or thyroid hormone in patients with
hypothyroidism, may therefore affect the interplay between nuclear receptors. Our sample size did not support subset
analyses, and further studies are needed to identify specific molecular subtypes
of breast cancer that are susceptible to PPARγ ligand therapy and crosstalk interactions. These data also raise the possibility of PPARγ-mediated modulation of systemic conditions
relevant to breast cancer risk, such as serum adiponectin and insulin
resistance.Breast cancer treatment and
prevention may benefit from future studies of PPARγ therapy that address issues of susceptible
breast cancer subtypes, duration and timing of intervention in the multistep
process of mammary carcinogenesis. Combination
of PPARγ ligands with other agents may enhance
therapeutic efficacy, as with RXR ligands for synergism in PPARγ-RXR heterodimer-mediated signaling [80]. Interestingly, inhibition of HER-2/neu
tyrosine kinase activity in a prostate cancer model prevents PPARγ degradation and thereby enhances
susceptibility to PPARγ activators such as R-etodolac [81]. Administration of a PPARγ agonist in conjunction with trastuzumab
(Herceptin), a humanized monoclonal anti-HER-2/neu antibody used in the
treatment of metastatic and high-risk HER-2/neu+ breast cancer, could also
represent a novel combination of targeted therapies.


### 2.4. Prostate Cancer

PPARγ is expressed in human prostate cancer cell
lines and human prostate cancer specimens [82]. In
vitro and in vivo
studies also demonstrate the anticancer effects of PPARγ ligands on prostate cancer, including the
reduction of prostate specific antigen (PSA) levels in androgen responsive
LNCaP cells [82–85].A phase II clinical study assessed
the effects of troglitazone at 800 mg/day in 41 men with androgen-dependent (n=12) or androgen-independent prostate cancer (n=29) that had progressed following local treatment or androgen deprivation therapy yet remained asymptomatic [82]. Four patients with androgen-dependent cancers
had a reduction in serum PSA levels after onset of therapy, which were measured
every 4 weeks, with a greater than 50% decrease in this tumor marker detected
in one patient after 16 months of troglitazone.  PSA levels decreased less than 50% in four
patients with androgen-independent prostate cancer. The median duration of troglitazone treatment
was longer in the androgen dependent (26.8 weeks) versus independent (14.3
weeks) group and could correlate with the more indolent course of disease associated
with the former, as well as greater susceptibility of this prostate cancer
subtype. As suggested by Mueller et al., variability in response might
relate to activated mitogen-activated protein (MAP) kinase signaling leading to
phosphorylation and inactivation of PPARγ as well as the possibility of somatic PPARG mutations.Following from this initial study,
a randomized placebo-controlled Phase II trial of rosiglitazone (8 mg/day) was
conducted in prostate cancer patients with progressive disease evidenced by a
rise in serum levels of PSA following local treatment with radical
prostatectomy and/or radiation therapy [86]. 106 men participated in this
multi-institutional study, with a median duration of 338 and 315 days of
treatment for rosiglitazone and placebo, respectively. Based on serum PSA levels obtained every four
weeks, rosiglitazone did not significantly increase the amount of time it took
serum levels of PSA to double or increase the time before disease progression
in men with prostate cancer. This trial
does not support a role for rosiglitazone in recurrent prostate cancer, even if
detected at the stage of biochemical, nonradiographic progression. Use of PPARγ ligands in prostate cancer may require
identification of susceptible tumor subtypes as well as consideration of
intervention at earlier stages of disease.


### 2.5. Thyroid Cancer

The thyroid gland is comprised of
follicle cells, which produce thyroglobulin as well as synthesize and store thyroxine, triiodothyronine, and parafollicular C cells that produce calcitonin. Papillary and follicular thyroid cancers
arise from follicle cells, comprising 80–90% of thyroid
cancers. Differentiated thyroid cancers
usually retain the ability to take up iodine, which serves as the basis for (1)
radioactive iodine treatments to ablate residual or metastatic disease and (2)
diagnostic screening via radioiodine scans [87]. These thyroid cancers also produce
thyroglobulin, and elevated thyroglobulin levels following definitive therapy
can signify recurrent disease.Normal, benign, and malignant
thyroid tissues express PPARγ, with dysregulated expression in thyroid cancers [88, 89]. A PAX 8/PPARγ rearrangement has been noted in
half of all follicular thyroid cancers, in which the resulting protein has a
loss of PPARγ function [90]. Treatment of human thyroid cancer cells lines
with thiazolidinediones inhibits cell proliferation and induces increased
expression of markers of thyroid cell differentiation such as thyroglobulin,
sodium-iodine symporter, thyroperoxidase, and TSH receptor [91–93].These laboratory findings provided
the basis for clinical investigation of the differentiating effects of PPARγ ligands in thyroid cancer. Philips et al. conducted a pilot trial of rosiglitazone therapy in five
patients with thyroglobulin-positive and radioiodine-negative thyroid cancer, a
clinical scenario suggestive of dedifferentiation of the thyroid cancer [94]. After three months of rosiglitazone (4 mg/day
for one month, then 8 mg/day for 2 months), thyroglobulin levels increased in 4
of 5 patients but only one subject had faint radioiodine uptake to delineate two
lung metastases. Kebebew et al. also administered
rosiglitazone (4 mg/day for 1 week, then 8 mg/day for an additional 7 weeks) to
patients with recurrent or progressive papillary or follicular thyroid cancer who
had elevated serum thyroglobulin levels and negative radioiodine scans [95]. In this open label phase II trial, 4 of 10 (40%)
of the subjects had posttreatment radioactive iodine scans showing uptake in
the neck (n=3) and pelvis (n=1). At the
six month follow-up visit, serum thyroglobulin changes did not reveal a
consistent pattern, with decreased (n=2), increased (n=5), and stable (n=3)
levels. There were no complete
responses, defined as increased radioiodine uptake and decreased thyroglobulin
levels. PPARγ expression in pretreatment
tumor samples by immunohistochemisty (formalin fixed, paraffin-embedded samples
for eight patients) and real time quantitative RT-PCR (frozen samples for four
subjects) did not correlate with the biological, biochemical responses. As noted in both reports, serum thyroglobulin
level is somewhat problematic as a marker of differentiation in that increases
could reflect re-differentiation/apoptosis as well as tumor progression. Future studies will also need to address
heterogeneity of stage IV disease (e.g., some patients presented only with
elevated thyroglobulin whereas others had diffuse metastases), dose level,
duration of therapy, and intervention prior to development of metastases.


## 2.1. Liposarcomas

As mesenchymal malignancies that
arise from adipose tissue [41, 42], liposarcomas express PPARγ at
levels comparable to normal adipose tissue [26]. PPARγ expression is a distinguishing feature
of liposarcomas relative to other soft tissue sarcomas [43]. Treatment of primary cell strains  derived from human liposarcomas (two well-differentiated
liposarcomas and one intermediate grade myxoid/round cell liposarcoma) with
PPARγ and/or RXR ligands induced morphologic and molecular changes consistent
with adipocyte differentiation [26]. These in
vitro findings led to consideration of thiazolidinediones for
differentiation therapy of liposarcomas.Demetri et al. conducted an open
label phase II clinical trial to determine the effects of troglitazone (800 mg/day) on tumor differentiation in patients with advanced and/or metastatic
liposarcoma [44]. In a report of three study subjects, tumor
samples obtained after the six week intervention showed marked cytoplasmic
lipid accumulation in comparison to pretreatment biopsies, as well as
morphologic changes suggestive of mature adipocytes. The expression of markers of adipocyte differentiation such as adipsin, aP2, and PPARγ by Northern analysis increased following treatment with troglitazone. Furthermore, cell proliferation as assessed by the percentage of
liposarcoma cells with Ki67 immunostaining decreased by two- to fourfold with
the study intervention. Three additional
patients enrolled in the study also had post-treatment biopsies with histologic
evidence of adipocyte differentiation and decreased cell proliferation. The histologic subtypes treated in this study
ranged from intermediate to high grade, and the authors raise the possibility
that differentiation may prove difficult to assess in more differentiated
tumors. This small pilot trial of six
subjects demonstrated a differentiating effect of short-term troglitazone
therapy in advanced stage liposarcomas providing the rationale for further
clinical evaluation of troglitazone and other PPARγ ligands in the management
of liposarcomas.A second phase II trial evaluated
the effects of the thiazolidinedione rosiglitazone (8 mg administered as 4 mg
twice daily) in nine patients with advanced, unresectable liposarcomas [45]. Analyses of tumor samples obtained at 0, 6,
and 12 weeks did not reveal changes in histopathology or gene expression to
support a differentiating effect. By
quantitative RT-PCR, levels of adipsin, fatty acid-binding protein (FABP), and
PPARγ increased in only two of nine tumors although in a somewhat inconsistent
fashion. In one of the patients with a
dedifferentiated liposarcoma, expression of PPARγ increased at week 12, FABP at
week 6 and adipsin at weeks 6 and 12. However, disease progression had been detected in this patient at 10
weeks of treatment, such that the expression of differentiation markers did not
correlate with clinical benefit. For the
other subject, upregulation of adipsin at week 12 was noted in a myxoid
liposarcoma, and treatment continued until progression of disease at six
months. Cell proliferation assessed by
Ki67 immunostaining did not decrease significantly with the study intervention,
although the low baseline levels of proliferation decreased further with
rosiglitazone. Taken together the data
fail to support a role for rosiglitazone in the treatment of liposarcoma at
advanced stages.The discrepant findings between the
two phase II trials of thiazolidinedione therapy may relate to differences in
the patient cohorts, tumors, and/or study agents. Whether methodological issues contributed to
the differences is not clear. As surgery
remains the mainstay of treatment for liposarcomas, with limited therapeutic
options for unresectable, advanced disease, and investigation of PPARγ ligands for adjuvant therapy of liposarcomas
or control of microscopic or minimal residual disease may still be
warranted.


## 2.2. Colon Cancer

Considerable in vitro evidence exists to support
the differentiating and antiproliferative effects of PPARγ ligand therapy in colon cancer. PPARγ is highly expressed in human colon tumor
specimens and cancer cell lines [27, 46]. In addition to growth inhibition, treatment
of colon cancer cells with PPARγ ligands promotes tumor cell differentiation as
assessed by increased cytoplasmic to nuclear ratio and higher levels of
differentiation markers such as carcinoembryonic antigen (CEA), villin,
intestinal alkaline phosphatase, GOB-4, and keratin 20 [27, 29, 47]. Although in vitro and in vivo
studies demonstrate a role for PPARγ in modulating growth and differentiation of
human colon cancer cell lines [27], thiazolidinedione treatment with rosiglitazone
(BRL49653) or troglitazone stimulated rather than inhibited the development of
colon tumors in APCmin/+ mice [48, 49]. The loss of adenomatous polyposis coli (APC)
tumor suppressor gene function, which leads to accumulation of β-catenin and enhanced Wnt pathway signaling,
may account for the contradictory findings of enhanced tumorigenesis in this
genetic versus xenograft model of colon cancer [50]. Interestingly, treatment with pioglitazone
over a wide range of doses suppressed intestinal tumor formation and
hyperlipidemia in APC deficient mice (APCmin/+ and Apc1309) [51, 52]. Whether the use of different thiazolidinediones or experimental conditions led to the discrepant findings is unclear.Loss of function mutations inPPARG in colon cancer may also account for variability in response to PPARγ therapy. Four unique mutations of PPARγ were identified in 55 sporadic colon cancers
in exons 3 or 5; one yielding a truncated protein with loss of the ligand
binding domain (c.472delA) and three causing defects in the binding of
synthetic or natural ligands (Q286P, K319X, and R288H) [53]. A point mutation in exon 6, K422Q, was
detected in four human colon cancer cell lines that were resistant to the
antiproliferative and differentiating effects of PPARγ ligands [54]. However, K422Q may have limited clinical
relevance, as a study of 170 primary human colorectal cancers and 12 liver
metastases failed to detect the presence of K422Q mutations [55].A phase II trial evaluated
troglitazone therapy (800 mg/day as a single dose) in 25 colon cancer patients
with lung and/or liver metastases [56]. Although well-tolerated, troglitazone did not elicit objective tumor responses. All
patients had progression of disease with a median progression-free survival of
1.6 months and median overall survival of 3.9 months. Troglitazone does not seem to have activity
in advanced colon cancer, although the lack of response may relate at least in
part to the biologically aggressive nature of Stage IV colon cancer that is refractory to cytotoxic chemotherapy (the median number of prior chemotherapy regimens was 3). Although the study was not designed
to assess troglitazone for possible tumor promoting effects, Kulke et al. urged caution in the use of
PPARγ agonists in colon cancer. In the future, advances in the genomic
profiling of colon cancer might allow for identification of susceptible colon
cancer subtypes.Colorectal cancer provides an
excellent model of multistep progression in carcinogenesis, based on the
acquisition of biochemical, molecular, and genetic alterations that transform
normal epithelium to adenomatous polyps to invasive cancer [57]. PPARγ ligands may have a role in chemoprevention of
colon cancer, depending on the specific molecular and genetic subtype. Thiazolidinediones suppress the development
of aberrant crypt foci (ACF), a putative precancerous lesion of the colon [58]. However, activation of certain oncogenic
signaling pathways at later stages of carcinogenesis may override this
regulatory role, as suggested by the APCmin/+
mouse studies [50]. Timing of PPARγ therapy prior to certain initiating events in
colon carcinogenesis may thus prove to be useful for chemoprevention of colon
cancer. As suggested by the in vivo studies of thiazolidinediones
in min mice, use of PPARγ agonists in colon cancer prevention may
require identification of high-risk individuals based on genetic susceptibility
as well as aberrant gene expression and signal transduction.Although the differentiating and
antiproliferative effects of PPARγ activation form the basis for evaluating PPARγ ligands for cancer therapy, the
anti-inflammatory effects of PPARγ may also be relevant to colon
carcinogenesis. Chronic inflammation is
linked to carcinogenesis, and diagnoses of inflammatory bowel disease (IBD),
which includes Crohn's disease and ulcerative colitis, carry increased risk for
colorectal cancer [59]. PPARγ ligands repress cytokine production by colon
cancer cells and inhibit chemically-induced colitis in murine models of inflammatory bowel disease [60–62]. These findings led to an open-label pilot
trial of rosiglitazone therapy (4 mg taken twice daily) in patients with active
ulcerative colitis for 12 weeks, showing clinical improvement in 8/15 (53%) patients
of which 4 (27%) subjects had clinical remission [63]. Two subjects required hospitalization for
worsening disease; other serious adverse events included severe pharyngitis
requiring hospitalization and nephritic syndrome leading to withdrawal.  The increased risk for colon cancer in
patients with ulcerative colitis relates to the severity and duration of
disease; by suppressing inflammation in the colon, drugs such as the
thiazolidinediones or other PPARγ agonists might play a role in colon cancer
prevention.


## 2.3. Breast Cancer

In vitro and in vivo
research studies support a role for PPARγ agonists in breast cancer therapy. Thiazolidinedione treatment of human breast
cancer cells appears to induce changes suggestive of terminal differentiation,
resulting in the inhibition of cell proliferation, suppression of keratin 19,
mucin-1, and upregulation of maspin [28]. Troglitazone also inhibited breast cancer
growth in a xenograft animal model [20]. Another synthetic ligand of PPARγ, GW7845, had inhibitory effects in a
carcinogen-induced model of mammary tumorigenesis [64]. Furthermore, PPARγ haploinsufficiency (PPARγ+/−) conferred increased susceptibility to
dimethylbenz[a]antracene (DMBA)-induced
mammary carcinogenesis [65]. These studies suggest that PPARγ ligands could serve as negative regulators of
breast cancer development and progression.We and others have demonstrated PPARγ immunostaining in human breast cancer
specimens, as well as normal and benign proliferative breast tissue [28, 66]. PPARγ appears to be expressed in most if not all
breast cancer cell lines, as well as in normal and malignant breast tissue [20, 28, 66]. We did not find that the invasive, metastatic
phenotype of the breast cancer cells correlates with PPARγ expression levels, gel shift mobility patterns,
or relative sensitivity to PPARγ ligands [67]. All of the cell lines tested (MCF-7,
MDA-MB-436, MDA-MB-231, and MDA-MB-435) responded to thiazolidinedione
treatment in transactivation assays, which also led to inhibition of cell
growth.However, a Phase II trial of
troglitazone in metastatic breast cancer refractory to cytotoxic chemotherapy did
not demonstrate beneficial effects on disease progression [68]. In twenty two Stage IV breast cancer patients
with an 8-week median treatment duration, troglitazone at 800 mg/day did not
prevent disease progression. The median time
on study was 56 days, ranging from 11 to 134 days; 19/22 subjects were removed
from the study for objective or subjective evidence of disease
progression. There were no objective
responses, and only 3/21 (14%) subjects completing 8 weeks of troglitazone
therapy had stable disease. In patients
with elevated serum levels of CEA and/or CA27.29 at the onset of the study,
these tumor markers had continued to increase despite 8 weeks of troglitazone. Burstein et al. note that more selective criteria to target PPARγ therapy to the most susceptible tumor type might
be helpful. The lack of effect might
have also related to an advanced stage of disease that had become refractory to
cytotoxic therapies.To assess the effects of PPARγ ligand therapy on breast cancer, we conducted a
pilot study of short-term administration of rosiglitazone (4 mg taken twice a
day) to women newly diagnosed with early stage breast cancer (Stages 0 to II) during the two- to six-week period between diagnostic biopsy and surgical removal of the cancer [69]. Thirty eight women completed the study intervention without serious adverse events; thirty four subjects had
sufficient pre and posttreatment tumor tissue for correlative analyses. By Ki67 immunostaining, short-term
intervention with rosiglitazone did not lead to statistically significant
differences cell proliferation. We did not detect any somatic or germline mutations in PPARG to account for differential
tumor tissue responses or lack of effect. Comparing H&E-stained sections of tumor before and after
rosiglitazone therapy also failed to note increased tumor cell differentiation
by standard criteria of tubule formation, mitotic activity, and nuclear
morphology. PPARγ expression in tumor
samples was graded 0 to 3+ for intensity of nuclear and cytoplasmic
immunostaining, which did not reflect an overall change in expression following
rosiglitazone therapy. Interestingly, nuclear
expression of PPARγ was significantly reduced in a majority of the patients
whose pre-rosiglitazone PPARγ scores were 1+ or 2+ and therefore assessable for
increased or decreased levels of expression, which raised the possibility of a
PPARγ mediated response in the tumor tissue.Serum levels of adiponectin and
indices of insulin sensitivity increased with rosiglitazone, based on serum
samples obtained from seventeen subjects. Given the association of lower levels of adiponectin and insulin
resistance with increased breast cancer risk [70–73], therapy with rosiglitazone or
other PPARγ ligands may have a role in breast cancer
prevention. Of interest are potential effects
of rosiglitazone therapy on nonepithelial components of the mammary tumor microenvironment;
unfortunately, pretreatment core biopsy samples in our study had negligible to
minimal stromal, noncancerous tissue for any comparative analyses.Although short-term intervention
with rosiglitazone did not alter the endpoint of cell proliferation, this pilot
study indicated the potential for other tumor tissue specific effects with
evidence for downregulation of PPARγ protein expression. Breast cancer is a family of diseases with
diverse molecular, genetic features, and PPARγ ligands may affect only certain subtypes. Indeed, breast cancer therapy already
encompasses molecularly targeted strategies, such as tamoxifen for estrogen
receptor positive cancer and trastuzumab for tumors with HER-2/neu
overexpression. Variability in PPARγ-mediated effects on cell proliferation could
arise from tumor heterogeneity in nuclear receptor cross-talk, complement of
cofactors, or mutated or aberrant signaling pathways that override PPARγ signaling, leading to an apparent lack of
effect of rosiglitazone [74–77]. For example, in vitro studies indicate
that estrogen receptor α (ERα) can repress PPARγ signaling by binding to PPREs and that
PPAR/RXR heterodimers can competitively inhibit ER binding at specific estrogen
response elements (EREs) [74, 75]. In
vitro administration of the PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 inhibited estrogen-mediated
transactivation of ERE and estrogen-responsive gene expression in MCF-7 breast
cancer cells [78]. By immunohistochemical analysis, PPARγ expression has been detected in 42% (99/238)
to 58% (101/170) of human breast cancer samples, correlating positively with ER
expression and improved clinical outcome [78, 79]. Concomitant administration of other drugs,
such as estrogen in postmenopausal women or thyroid hormone in patients with
hypothyroidism, may therefore affect the interplay between nuclear receptors. Our sample size did not support subset
analyses, and further studies are needed to identify specific molecular subtypes
of breast cancer that are susceptible to PPARγ ligand therapy and crosstalk interactions. These data also raise the possibility of PPARγ-mediated modulation of systemic conditions
relevant to breast cancer risk, such as serum adiponectin and insulin
resistance.Breast cancer treatment and
prevention may benefit from future studies of PPARγ therapy that address issues of susceptible
breast cancer subtypes, duration and timing of intervention in the multistep
process of mammary carcinogenesis. Combination
of PPARγ ligands with other agents may enhance
therapeutic efficacy, as with RXR ligands for synergism in PPARγ-RXR heterodimer-mediated signaling [80]. Interestingly, inhibition of HER-2/neu
tyrosine kinase activity in a prostate cancer model prevents PPARγ degradation and thereby enhances
susceptibility to PPARγ activators such as R-etodolac [81]. Administration of a PPARγ agonist in conjunction with trastuzumab
(Herceptin), a humanized monoclonal anti-HER-2/neu antibody used in the
treatment of metastatic and high-risk HER-2/neu+ breast cancer, could also
represent a novel combination of targeted therapies.


## 2.4. Prostate Cancer

PPARγ is expressed in human prostate cancer cell
lines and human prostate cancer specimens [82]. In
vitro and in vivo
studies also demonstrate the anticancer effects of PPARγ ligands on prostate cancer, including the
reduction of prostate specific antigen (PSA) levels in androgen responsive
LNCaP cells [82–85].A phase II clinical study assessed
the effects of troglitazone at 800 mg/day in 41 men with androgen-dependent (n=12) or androgen-independent prostate cancer (n=29) that had progressed following local treatment or androgen deprivation therapy yet remained asymptomatic [82]. Four patients with androgen-dependent cancers
had a reduction in serum PSA levels after onset of therapy, which were measured
every 4 weeks, with a greater than 50% decrease in this tumor marker detected
in one patient after 16 months of troglitazone.  PSA levels decreased less than 50% in four
patients with androgen-independent prostate cancer. The median duration of troglitazone treatment
was longer in the androgen dependent (26.8 weeks) versus independent (14.3
weeks) group and could correlate with the more indolent course of disease associated
with the former, as well as greater susceptibility of this prostate cancer
subtype. As suggested by Mueller et al., variability in response might
relate to activated mitogen-activated protein (MAP) kinase signaling leading to
phosphorylation and inactivation of PPARγ as well as the possibility of somatic PPARG mutations.Following from this initial study,
a randomized placebo-controlled Phase II trial of rosiglitazone (8 mg/day) was
conducted in prostate cancer patients with progressive disease evidenced by a
rise in serum levels of PSA following local treatment with radical
prostatectomy and/or radiation therapy [86]. 106 men participated in this
multi-institutional study, with a median duration of 338 and 315 days of
treatment for rosiglitazone and placebo, respectively. Based on serum PSA levels obtained every four
weeks, rosiglitazone did not significantly increase the amount of time it took
serum levels of PSA to double or increase the time before disease progression
in men with prostate cancer. This trial
does not support a role for rosiglitazone in recurrent prostate cancer, even if
detected at the stage of biochemical, nonradiographic progression. Use of PPARγ ligands in prostate cancer may require
identification of susceptible tumor subtypes as well as consideration of
intervention at earlier stages of disease.


## 2.5. Thyroid Cancer

The thyroid gland is comprised of
follicle cells, which produce thyroglobulin as well as synthesize and store thyroxine, triiodothyronine, and parafollicular C cells that produce calcitonin. Papillary and follicular thyroid cancers
arise from follicle cells, comprising 80–90% of thyroid
cancers. Differentiated thyroid cancers
usually retain the ability to take up iodine, which serves as the basis for (1)
radioactive iodine treatments to ablate residual or metastatic disease and (2)
diagnostic screening via radioiodine scans [87]. These thyroid cancers also produce
thyroglobulin, and elevated thyroglobulin levels following definitive therapy
can signify recurrent disease.Normal, benign, and malignant
thyroid tissues express PPARγ, with dysregulated expression in thyroid cancers [88, 89]. A PAX 8/PPARγ rearrangement has been noted in
half of all follicular thyroid cancers, in which the resulting protein has a
loss of PPARγ function [90]. Treatment of human thyroid cancer cells lines
with thiazolidinediones inhibits cell proliferation and induces increased
expression of markers of thyroid cell differentiation such as thyroglobulin,
sodium-iodine symporter, thyroperoxidase, and TSH receptor [91–93].These laboratory findings provided
the basis for clinical investigation of the differentiating effects of PPARγ ligands in thyroid cancer. Philips et al. conducted a pilot trial of rosiglitazone therapy in five
patients with thyroglobulin-positive and radioiodine-negative thyroid cancer, a
clinical scenario suggestive of dedifferentiation of the thyroid cancer [94]. After three months of rosiglitazone (4 mg/day
for one month, then 8 mg/day for 2 months), thyroglobulin levels increased in 4
of 5 patients but only one subject had faint radioiodine uptake to delineate two
lung metastases. Kebebew et al. also administered
rosiglitazone (4 mg/day for 1 week, then 8 mg/day for an additional 7 weeks) to
patients with recurrent or progressive papillary or follicular thyroid cancer who
had elevated serum thyroglobulin levels and negative radioiodine scans [95]. In this open label phase II trial, 4 of 10 (40%)
of the subjects had posttreatment radioactive iodine scans showing uptake in
the neck (n=3) and pelvis (n=1). At the
six month follow-up visit, serum thyroglobulin changes did not reveal a
consistent pattern, with decreased (n=2), increased (n=5), and stable (n=3)
levels. There were no complete
responses, defined as increased radioiodine uptake and decreased thyroglobulin
levels. PPARγ expression in pretreatment
tumor samples by immunohistochemisty (formalin fixed, paraffin-embedded samples
for eight patients) and real time quantitative RT-PCR (frozen samples for four
subjects) did not correlate with the biological, biochemical responses. As noted in both reports, serum thyroglobulin
level is somewhat problematic as a marker of differentiation in that increases
could reflect re-differentiation/apoptosis as well as tumor progression. Future studies will also need to address
heterogeneity of stage IV disease (e.g., some patients presented only with
elevated thyroglobulin whereas others had diffuse metastases), dose level,
duration of therapy, and intervention prior to development of metastases.


## 3. Combination Therapy

Despite the wealth of preclinical
evidence for the anticancer effects of PPARγ ligands in various types of cancer,
thiazolidinediones appear largely ineffective as monotherapy agents for
treating advanced, disseminated stages of cancer. However, cancer chemotherapy often involves
the concomitant and/or sequential administration of multiple drugs in order to
achieve maximal tumor cell kill and improved disease-free and overall
survival. Combinatorial drug regimens
may allow for additive if not synergistic effects which might also permit the
use of lower dosages with decreased side effects. Preclinical studies show the potential
benefits of combining PPARγ ligands with other anticancer agents. For example, rosiglitazone treatment of A549
lung cancer cells increases the expression of PTEN (phosphatase and tensin
homolog), which enhances the antiproliferative effects of the tyrosine kinase
inhibitor gefitinib [96]. The combination of rosiglitazone and
platinum-based cytotoxic drugs such as carboplatin and cisplatin synergistically
inhibits the growth of A549 lung cancer cells relative to single-agent therapy [97]. Based on gene array analysis, rosiglitazone appears
to mediate the downregulation of metallothioneins, heavy metal binding proteins
involved in platinum drug resistance.Lack of effect of PPARγ ligand therapy may also relate to repression
of ligand activation, such as by histone deacetylases (HDAC) which can form
complexes with PPARγ to repress gene transcription of specific PPARγ target genes [98]. Removal of HDAC-mediated transcriptional
silencing via HDAC inhibitors may allow activation of PPARγ, as suggested by in vitro and in vivo
studies of combined therapy of prostate cancer with HDAC inhibitors and PPARγ agonists [85]. Inactivation of PPARγ via phosphorylation could also occur in
cancers with high levels of activity of mitogen-activated protein kinase [99], and coadministration of
inhibitors of specific kinases might enhance the therapeutic potential of a
PPARγ agonist.Novel treatment regimens combining
pioglitazone with the COX-2 inhibitor rofecoxib and low-dose continuous chemotherapy drugs have been tested for efficacy in aggressive solid malignancies at advanced, progressive stages of disease. In a pilot
study of 6 patients with advanced malignant vascular tumors that had progressed
following surgery, radiotherapy, and/or chemotherapy, treatment with
pioglitazone (45 mg/day), rofecoxib (25 mg/day), and metronomic trofosfamide (3×50 mg/day) resulted in 2 subjects with complete responses, 1 with a partial
response, and 3 with stable disease [100]. The median progression-free survival was 7.7
months (range of 2 to 15 months), and the average treatment duration was 29.3,
21.7, and 23 weeks for pioglitazone, rofecoxib, and trofosfamide,
respectively. Interestingly, one subject
experienced regression of an extensive angiosarcoma of the facial skin with
initiation of therapy with pioglitazone and rofecoxib alone. The Phase II trial of this treatment regimen
in a cohort of patients with progression of previously treated metastatic
melanoma (n=19) or soft tissue sarcoma (n=21) led to decreased tumor burden in
5 subjects and stabilization of disease in 6 subjects [101]. More recently, a multi-institutional
randomized clinical trial of trofosfamide (50 mg TID) versus trofosfamide (50 mg TID) plus rofecoxib (25 mg/day) and pioglitazone (60 mg/day) of 76 patients
with metastatic melanoma demonstrated superiority of metronomic chemotherapy
combined with a COX-2 inhibitor and PPARγ ligand, with 0% versus 9% progression-free
survival [102]. Assessment of serum C reactive protein (CRP) levels
in 48 subjects participating in this melanoma trial showed that those on the
combined regimen had a greater than 30% decrease in this proinflammatory
marker, suggesting that the anti-inflammatory effects of the drug combination
may account for the improvement in progression-free survival. Interestingly, PPARγ appears to suppress the proinflammatory potential
of monocytes and macrophages [36, 103, 104], and thiazolidinediones have
been shown to decrease CRP levels [105]. Another metronomic, low-dose chemotherapy
regimen of either capecitabine (1250 mg/m2 twice daily for days 1–14 of 21 days) or temozolomide (50 mg/m2/d) combined with pioglitazone (60 mg/d) and rofecoxib
(25 mg/d) was tested in patients who had developed recurrent high-grade gliomas
(10 with gliobastoma, 4 with anaplastic astrocytoma) following chemotherapy,
surgery, and/or radiotherapy, with 21.5% overall progression-free survival at
six months (20% and 25% PFS for subjects with glioblastoma and anaplastic
gliomas, resp.) [106]. By immunostaining the expression of COX-2,
PPARγ, and CD31 in these high-grade gliomas did not
correlate with patient outcome; however, as recurrence was diagnosed by
magnetic resonance imaging, tumor tissue obtained prior to disease progression
might not accurately represent target gene expression in the recurrent tumors. The contribution of pioglitazone to the
activity of these combination regimens is difficult to assess but may relate to
anti-inflammatory effects as suggested by the CRP results in metastatic
melanoma. PPARγ ligands are also known to exert suppressive
effects on angiogenesis in cancer [34] and on malignant vascular cells [107], which may also enhance the effectiveness of metronomic chemotherapy regimens for certain malignancies.Combination with ligands to
retinoid X receptor (RXR), the heterodimer partner of PPARγ, may also augment the anticancer efficacy of
PPAR ligand therapy. Treatment with RXR
ligands alone appears efficacious in the treatment of certain malignancies, and
bexarotene (Targretin) is a synthetic RXR-selective retinoid utilized in the
treatment of refractory cutaneous T-cell lymphoma [108]. A Phase I study of bexarotene in patients
with advanced cancer did not show objective tumor responses but indicated the
potential for stable disease in 5 of 16 and 1 of 5 lung and head and neck
cancer patients, respectively [109]. In a multi-institutional Phase II trial of
bexarotene capsules (MINT or Targretin Monotherapy in non-small-cell lung
cancer trial) in advanced non-small-cell lung cancer patients who had failed
two or more prior chemotherapy regimens, bexarotene (400 mg/m2/day) did not meet the intended aim of a median survival of 6 months as third-line
therapy; however, the subset of subjects with treatment-related hypertriglyceridemia
(grade 1 to 4, 51%) and/or skin reactions such as rash, pruritis, and erythema
(grade 1 to 4, 28%) had prolonged survival at one year of 30% and 34%,
respectively, compared to 18% and 19%, respectively, of patients without these
adverse events [110]. A role for bexarotene as monotherapy is also demonstrated
in preclinical models of mammary carcinogenesis [111, 112]. A Phase II randomized trial of 200 or 500 mg/m2/day bexarotene in women with metastatic breast cancer
refractory to endocrine therapy or chemotherapy showed clinical benefit in the
form of complete/partial response and stable disease ≥6 months in 27 of 146 patients, with an
objective response rate of 3 to 6% [113].To evaluate RXR-PPARγ ligand therapy for synergism in cutaneous T-cell
lymphoma, a small uncontrolled study evaluated the addition of rosiglitazone to
bexarotene monotherapy in four patients with stable or progressive cutaneous T-cell
lymphoma, showing improvement in skin score (50%) and pruritus (75%) [114]. Preclinical studies of solid malignancies
such as liposarcomas, breast, and colon cancers also provide support for the greater
efficacy of combined regimens of PPARγ and RXR ligands compared to monotherapy [26, 80, 115]. In addition to increased therapeutic
benefits, combination therapy may also allow for lower doses and decreased
toxicity.Novel cancer agents that combine
PPARγ agonism with inhibition of other signaling pathways
are also of interest. LY293111 is a
diaryl ether carboxylic acid derivative that modulates multiple eicosanoid
pathways, acting as a leukotriene B4 antagonist, 5-lipoxygenase inhibitor, and
PPARγ agonist [116]. Phase I trials have identified the maximally
tolerated dose for continuous oral administration of LY293111, both as a single
agent and in combination with irinotecan for solid malignancies [116]. Future clinical trials with correlative
tissue analyses may provide insight into the specific contribution of PPARγ agonism to the therapeutic potential of
LY293111.


## 4. Cancer Prevention

PPARγ may function as a tumor suppressor during
early events of carcinogenesis. Thiazolidinediones
or other PPARγ ligands may have efficacy during early stages
of cancer development, by targeting carcinogenesis prior to onset of initiating
events that are no longer subject to PPARγ repression. Indeed, mutant APC function and dysregulated β-catenin signaling in the APC+/min mice model of colon carcinogenesis appear to abrogate responsiveness to PPARγ activation [50]. Susceptibility to PPARγ ligands is possibly limited to only certain
premalignant conditions or individuals, and development of genomic strategies
to properly apply this chemopreventive option in an otherwise heterogeneous
high-risk population is needed.In a phase II clinical trial of pioglitazone therapy for
dysplastic leukoplakia, a precursor lesion to invasive oral cancer,
pioglitazone administered for 3 months at 45 mg/day elicited a response in 70%
of subjects (15/21) based on bidimensional measurements of the oral lesions [117]. Although the degree of dysplasia increased in three subjects, pioglitazone did not alter the degree of dysplasia in the
majority of patients. These findings
raise the possibility that PPARγ ligands may prove efficacious for
chemoprevention of oral cancer by decreasing the extent of leukoplakia. The lack of effect on dysplasia suggests that
pioglitazone may not function as a differentiating agent for oral leukoplakia
or at least within the time frame of a three month intervention.


## 5. Safety and Toxicity Issues

Even though the risk-benefit ratio
of thiazolidinediones for cancer therapy seems favorable for advanced stages of
disease, thiazolidinediones appear problematic as candidate agents for
chemoprevention rather than for treatment of advanced, metastatic cancer. Use of thiazolidinediones in adjuvant therapy
for cancer patients who are otherwise healthy but at increased risk for cancer
recurrence would also raise concerns if administered on a long-term basis as
for a chronic disease such as diabetes mellitus.  Although generally well-tolerated, thiazolidinediones
such as rosiglitazone and pioglitazone are associated with body weight gain as
a result of increased adiposity and fluid retention, with the latter resulting
in peripheral edema, anemia on the basis of hemodilution, and increased risk of
developing congestive heart failure [118–120]. Controversy currently exists regarding the
cardiovascular safety of rosiglitazone, with a recent meta-analysis of different
treatment trials of rosiglitazone suggesting that rosiglitazone was associated
with a significant increase in the risk of myocardial infarction and
cardiovascular death [121]. However, other studies have not shown an
increased risk of cardiovascular mortality, including the interim analysis of
the Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycaemia in
Diabetes (RECORD) trial, a prospective trial of rosiglitazone in type 2 diabetes
mellitus [122, 123]. For postmenopausal women, rosiglitazone
therapy has also been linked to osteoporosis and risk of fracture [124]. Such side effects are concerning for healthy
individuals contemplating cancer risk reduction rather than treatment of
disease. Defining the risks of
thiazolidinediones will allow for avoidance of these drugs in subjects at
higher risk for these associated complications.The carcinogenic potential of thiazolidinediones and other
PPARγ agonists is another point of controversy [125, 126]. Upon review of the carcinogenicity of PPARγ and dual PPARα/γ agonists in development, the US Food and Drug
Administration has recommended two-year carcinogenicity studies of new PPAR
agonist drugs in rodents prior to initiation of clinical trials longer than six
months [127, 128]. As discussed above, contradictory in vivo and in vitro findings may relate
to specific animal models or molecular, genetic subtypes of certain
cancers. Additionally, as diabetes and insulin resistance have been linked to
higher risk for certain malignancies such as breast and colon cancer [72, 129, 130], thiazolidinediones might even exert preventive effects on
carcinogenesis by ameliorating the chronic inflammation associated with
dysregulated metabolism.Postmarketing experience with
pioglitazone and rosiglitazone has not shown significantly increased cancer
risk with these drugs. The PROactive
Study (PROspective pioglitazone clinical
trial In macrovascular events), which randomized 5238 patients with type 2
diabetes mellitus and cardiovascular or peripheral vascular disease to
pioglitazone or placebo for an average of 34.5 months, did not detect
differences in the overall number of malignancies [131]. Interestingly, the
pioglitazone treated group had more bladder tumors (14 versus 6) and fewer breast
cancers (3 versus 11); however, the timing of the cancer diagnoses relative to
pioglitazone use and the presence of confounding risk factors did not support a
causal relationship to the study drug. Use
of thiazolidinediones was associated with 33% decreased risk for lung cancer in
a large retrospective database analysis of over 87,000 diabetic patients of 10 veteran affairs medical centers, without significant risk reduction for colorectal and
prostate cancer [132]. Whether lung cancer
is uniquely susceptible to thiazolidinediones or other PPARγ agonists remains to be determined; preclinical studies suggest a potential role
for PPARγ ligand therapy in the management of this
disease [133, 134]. In the preliminary report
of a more recent analysis of this Veteran Affairs database, thiazolidinedione
use also correlated with 41 to 55% reduced risk for head and neck squamous cell
carcinoma [135]. Conversely, a
smaller retrospective study of 1003 diabetic subjects in the Vermont diabetes
information system database showed a possible association of cancer with use of
thiazolidinediones and particularly rosiglitazone, based on self reported
history of malignancy of any type [136]. Prospective studies
with attention to confounding influences such as risk factors or timing and
duration of drug use relative to cancer diagnosis are needed to investigate
these possible associations and basis for the discrepant findings.


## 6. Conclusions

Based on the clinical experience to
date, use of PPARγ agonists in cancer therapy will likely require
a targeted approach, with restriction to specific types of malignancies and/or
stage(s) of disease susceptible to enhanced PPARγ signaling. Studies of PPARγ ligand therapy in cancer patients have focused
largely on thiazolidinediones approved for treatment of type 2 diabetes
mellitus, administered at the high end of doses tested for tolerability and
insulin sensitizing effects. In phase II
clinical trials, thiazolidinediones do not have significant activity in most cancers
at advanced stages, which may relate to the extent and burden of disease and
aggressive tumor phenotypes resistant to cytotoxic therapies. The lack of efficacy of thiazolidinediones in
advanced, metastatic disease may relate to the use of noncytotoxic dose levels;
alternatively, these drugs may not exert effects at late stages of cancer
progression at any dose level. However, the
demonstration of differentiating effects in some patients suggests that a
subset of patients might benefit from PPARγ ligand therapy. Thiazolidinediones and other PPARγ agonists may ultimately prove effective in
combination with standard cytotoxic agents or other molecularly targeted
therapies. Interventions at earlier
stages of disease, including premalignant or predisposing high-risk conditions,
should also be considered. Therapy
administered in the adjuvant setting, such as following surgical resection, or
in situations of microscopic rather than measurable disease may also have
greater efficacy. The use of PPARγ ligands in earlier stages of cancer or cancer
prevention will require additional research and/or drug development to address
the questions regarding the carcinogenic potential and other adverse effects of
these drugs. Genetic and molecular profiling
of human cancers may in the future enable selection of a tumor subtype
susceptible to PPARγ agonists, as specific genotypes and patterns
of nuclear receptor and cofactor expression might predict resistance or
responsiveness to PPARγ signal transduction.



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*Source: 159415-2008-12-18.xml*