# The Role of the PAX8/PPARγ Fusion Oncogene in  Thyroid Cancer

Authors: Kimberly A. Placzkowski; Honey V. Reddi; Stefan K. G. Grebe; Norman L. Eberhardt; Bryan McIver
Journal: PPAR Research
 (2008)

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## Abstract

Thyroid cancer is uncommon and exhibits relatively low mortality rates. However, a subset of patients experience inexorable growth, metastatic spread, and mortality. Unfortunately, for these patients, there have been few significant advances in treatment during the last 50 years. While substantial advances have been made in recent years about the molecular genetic events underlying papillary thyroid cancer, the more aggressive follicular thyroid cancer remains poorly understood. The recent discovery of the PAX8/PPARγ translocation in follicular thyroid carcinoma has promoted progress in the role of PPARγ as a tumor suppressor and potential therapeutic target. The PAX8/PPARγ fusion gene appears to be an oncogene. It is most often expressed in follicular carcinomas and exerts a dominant-negative effect on wild-type PPARγ, and stimulates transcription of PAX8-responsive promoters. PPARγ agonists have shown promising results in vitro, although very few studies have been conducted to assess the clinical impact of these agents.


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## Body

## 1. Introduction

Thyroid
cancer is the most frequent endocrine malignancy. While relatively uncommon,
the American Cancer Society estimates that there will be over 37 000 new cases
in 2008 in the United States [1].
The majority of thyroid cancers are well-differentiated malignancies originating from
the thyroid follicular cells. Papillary thyroid carcinomas (PTCs) are the most frequent
histotype, particularly in iodine-sufficient areas, while follicular thyroid carcinoma (FTC) and
Hürthle cell carcinomas (HCCs) together represent around 20% of thyroid cancer.
However, these latter thyroid cancer types are often more aggressive, more
advanced at the time of diagnosis, less responsive to traditional therapy, and
more likely to cause both morbidity and mortality. FTC share similar cytologic
features with follicular adenomas (FAs) and are distinguished only by the
presence of invasion beyond the tumor capsule or into blood vessels on
pathology. This common histology suggests an adenoma to carcinoma sequence,
with adenomas representing a premalignant lesion, though this remains unproven.However, the peroxisome proliferator-activated receptors (PPARs), includingα,
β, δ, and γ
subtypes, are part of the ubiquitous nuclear hormone receptor superfamily that
has been the focus of considerable  research over the last two decades. PPARγ
is best known in the endocrine world for its role in adipogenesis and insulin sensitization.
However, it also plays a role in cell cycle control, inflammation, atherosclerosis,
apoptosis, and carcinogenesis through its influence on gene expression involving
multiple cell signaling pathways [2]. Research on PPARγ
as a potential thyroid proto-oncogene was accelerated by the discovery of a chromosomal
translocation involving the PPARγ-1 gene
in a subset of follicular carcinomas [3].
Since that time, considerable effort has gone into further clarifying the
possible effect of PPARγ in several
cancers, including FTC.


## 2. PPARγ and Thyroid Cancer

Chromosome analysis and microsatellite mapping techniques have revealed a number
of chromosomal changes associated with thyroid carcinogenesis, and pointed to
specific loci that might harbor oncogenes or tumor suppressor genes, including
a region spanning the fragile site on chromosome 3p14, through 3pter that is
often disrupted in follicular thyroid cancers [4].
Jenkins et al. had already described a somatic mutation involving 3p25-p21
region in three cases of follicular thyroid cancer [5, 6], and there followed a number
of reports of balanced translocations or aneusomy involving this chromosomal
region in FTC, HCC, PTC, and anaplastic thyroid cancers (ATCs) [7–11].
A specific translocation, t(2;3)(q13;p25), was described in a case of
aggressive FTC associated with bone metastases [11],
though it was also seen in some FAs [10].
The importance of this chromosomal rearrangement and the mechanism of its oncogenic
activity remained unclear until Kroll et al. mapped the involved 3p25 locus in a
number of FTC to the PPARγ gene in 2000
[3]. Thus these data were the first to convincingly
link the PPARγ gene to thyroid cancer.
Altered PPARγ activity has subsequently
been shown to have a potential role in several types of thyroid cancer.Several reports of ATC, FTC, and some PTC cell lines have demonstrated PPARγ
mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR),
without identifiable PPARγ mutations or
translocations [12–15]. In these PPARγ-positive
cell lines, PPARγ ligands (troglitazone
[15, 16],
ciglitazone [12, 14, 17],
rosiglitazone [12],
prostaglandin J2 [13, 15, 17],
and RS1303 [13])
inhibit growth of cells, while no change in growth is seen in PPARγ-negative cell
lines. Growth suppression was dose-dependent [13, 16, 17],
and one study found a correlation between PPARγ expression and
response to PPARγ ligands
[13], though another did not
[16]. Levels of Bax protein and c-myc, both
apoptosis-related proteins, were increased in a dose-dependent
fashion by treatment with PPARγ ligand
[13, 15],
although one study found increased levels of a different apoptotic protein,
Bcl-2, rather than of Bax [12].
Inhibition of cell invasion [13],
attachment [12, 17],
and anchorage-independent growth on soft agar [12],
all features of malignancy, are also seen with PPARγ
ligand treatment in thyroid cancer cell lines. Finally, evidence of cell death,
with decreased viable cell numbers and increased rates of necrosis and apoptosis,
have been reported by some groups following PPARγ
agonist treatment [12, 17].
This reduction in cell viability may be inhibited by coincubation with GW9662,
a selective PPARγ antagonist, suggesting that the
described changes in cell growth and survival were truly
PPARγ-dependent
[12].Addition of PPARγ agonist to PPARγ-positive cells
lines led to an increased portion of cells in G0/G1 with a
reduction of cells in G2/M and S phase, consistent with decreased cell
proliferation [12]. DNA synthesis appeared to
be slowed with decreased 3H-thymidine incorporation in these cells, while expression of the cell-cycle progression inhibitors p21cip1 andp27kip1 were increased [12].
Overexpression  of the PPARγ gene by
transfection into PPARγ-positive or -negative
cell lines similarly decreased colony formation and triggered nuclear condensation,
fragmentation of chromatin and apoptosis, with G0/G1 cell
cycle arrest [12, 14–16].
Together, these data provide strong support that PPARγ
has a tumor suppressive effect in thyroid follicular cells, which is consistent
with results in other nonthyroid cell lines [18–23].One of the few available animal models of follicular thyroid carcinoma was created
by generating homozygous mutations in the thyroid receptor gene TRβ,
a mutation initially described in a patient with thyroid hormone resistance syndrome
[24] and referred to as the PV/PV
mutant. Homozygous TRβPV/PV mice
develop follicular thyroid cancer with predictable progression from thyroid
hyperplasia to capsular and vascular invasion, more extensive soft tissue
invasion and ultimately lung metastasis at an early age
[25]. Analysis of gene activity by
cDNA microarray analysis in TRβPV/PV mice
demonstrates altered regulation of several genes compared to wild-type siblings.
Many of these genes are implicated in tumor-formation, metastasis, invasion, cell
cycle control and apoptosis. PPARγ-mediated
pathways, however, are downregulated in these mice, hinting at a role for PPARγ inhibition in this thyroid cancer model [26].Early in the histologic progression of thyroid disease in this TRβPV/PV mouse model expression of PPARγ mRNA, assessed by
Northern blot analysis, was diminished approximately 50% compared to wild-type
siblings. Furthermore, not only was PPARγ
activity suppressed, but the mRNA activity in mutant mice did not increase with age
as was seen in their wild-type counterparts [27]. Data from humans has also implicated a functional downregulation of PPARγ expression in human thyroid tumors that
did not carry PPARγ translocations, in studies using
semiquantitative PCR, real-time RT-PCR, and microarray analyses
[28, 29].
Furthermore, tumors with reduced PPARγ
expression showed an increased incidence of distant metastases, local invasion, and
areas of poor differentiation [28]. These findings suggest that
downregulation of wild-type PPARγ
may be a key event in thyroid carcinogenesis.Homozygous PPARγ+/− mice,
incorporating a loss of function mutation in one allele of the
PPARγ gene, have been used as an animal
model to evaluate the molecular genetic events ultimately leading to carcinogenesis
in colon, breast, and ovarian tumors [30, 31]. Deletion of both alleles of the
PPARγ gene, however, is universally
lethal to embryos. In thyroid cancer, the
TRβPV/PV mouse model was used
to further elucidate the mechanism of
PPARγ tumorigenesis.
TRβPV/PV mice were crossed
with PPARγ+/− mice to obtain TRβPV/PVPPARγ+/− offspring. In these mice,
PPARγ mRNA and lipoprotein lipase (LpL)
expression were reduced compared to wild-type mice. LpL is a downstream target
gene for PPARγ, thereby confirming
diminished PPARγ action. Furthermore,
LpL mRNA and protein expression were further reduced in the mutant
TRβPV/PVPPARγ+/− mice compared to both
wild-type and TRβ+/+PPARγ+/− mice [32].Western blot analysis demonstrated activation of the NFκB
pathway in these TRβPV/PVPPARγ+/−
mice [32], which is consistent with
data in the PPARγ+/− mouse
model [33]. Expression of cyclin D1, a
cell cycle regulator important in the progression from G1to S phase
and a downstream target of NFκB, was
significantly increased. Also, cell cycle analysis in TRβPV/PVPPARγ+/−
mice showed a shortened G0/G1 phase and decreased apoptosis
compared to TRβPV/PVPPARγ+/+ mice [32]. Cyclin D1 is
known to be overexpressed in human thyroid carcinomas
[34] and in vitro addition of a
PPARγ agonist to human ATC cell lines
suppresses cyclin D1 levels [12], confirming that
this effect is related to alterations in PPARγ rather than TRβ. Caspase-3,
a gene critical in the apoptotic signal cascade, was significantly reduced in
TRβPV/PVPPARγ+/−
mice, a finding consistent with previous data on the downstream effects of NFκB
activation in prostate cancer cells
[35]. Conversely, increased levels of caspase-3
activation were seen in vitro with PPARγ
agonist therapy of FTC [17] and ATC cell lines
[12]. Importantly, rosiglitazone therapy slowed tumor
growth and reduced capsular invasion in TRβPV/PV mice, suggesting
that activation of the PPARγ
pathway delays disease progression [32].In summary, several lines of evidence, in human tumors, cell lines, and animal
models strongly support the hypothesis that PPARγ inhibition, downregulation, or insufficiency appears to be tumorigenic in
the thyroid. This appears to be mediated through
PPARγ effects on cell cycle progression and
inhibition of apoptosis, thereby contributing to tumor development or
progression.


## 3. PAX8/PPARγ Gene Translocation and Thyroid Cancer

Following the identification of a frequent translocation in follicular thyroid
cancer, involving 3p25 and 2q13, Kroll confirmed not only that the 3p25
breakpoint lies within the PPARγ gene, but also
that the 2q13 breakpoint lies within the PAX8 gene. Indeed, the translocation
brings together these two genes to form a neogene, which expresses a fusion protein
(PAX8/PPARγ
fusion protein, designated
PPFP) [3].Native PAX8 is a transcription factor important in thyroid follicular cell
differentiation and in the regulation of a number of thyroid-specific genes
[36]. The chromosomal translocation described by Kroll
fuses the promoter and proximal 5’ coding sequence of PAX8 inframe with a nearly
full-length PPARγ, resulting in the production of the
fusion protein PPFP, whose expression is under the transcriptional regulation
of the PAX8 promoter [3]. Several splice variants have been
identified for PPFP, which appear to be frequently coexpressed
[3, 36, 37]. Variants described to date are shown in Figure
1. In each case these PAX8,
fragments are fused to PPARγ exons 1 to 6
[3, 36, 37]. All of the known variants include the paired and
partial homeobox DNA binding domains of PAX8, as well as the DNA binding, ligand
binding, RXR dimerization and transactivation domains of PPARγ-1 [3].
These attributes make it likely that PPFP will retain at least some of the DNA
and ligand binding properties of each of the native transcription factors, with
the potential for significant impact on either the PAX8- or
PPARγ-mediated pathways, or both.Figure 1
PAX8/PPARγ rearrangement illustrating the genomic
structure with exon arrangement and sites of fusion. The PAX8 activation domain
(AD) is eliminated in all fusion events. The protein structure of the predicted
fusion protein is shown and contains the PAX8-paired domain, containing the DNA
binding domain (DBD), the octapeptide motif (OP), and the truncated homeodomain
(HD). All of the functional domains of PPARγ gene, including activation domains 1
and 2 (AD1 and AD2), DBD, and ligand binding domain are retained in the fusion
protein.A number of studies, using RT-PCR, nested PCR, FISH, or Western analysis,
have confirmed the relatively high prevalence of the PAX8/PPARγ rearrangement or expression of PPFP in
follicular thyroid lesions, though the precise incidence varies by cell type
and method of detection (Table 1). FTCs have the highest incidence at 36%
(range 0–63%) with FAs exhibiting lower rates of around 11% (range 0–55%).
Only one case of HCC has shown the PAX8/PPARγ rearrangement. PPFP does not appear to be expressed in classical PTC,
but karyotyping has shown at (2;3)(q13;p25) in one case of follicular variant of
papillary thyroid cancer (FVP) [49] and intense
PPARγ immunostaining was seen in three
PPFP-negative FVP [50], perhaps arguing for an
alternative mechanism of PPARγ
overexpression in these tumors. Although Castro et al.
[44, 47]
found that 37–50% of FVP were positive for PPFP by RT-PCR and FISH, three
other studies have found no such rearrangements
[36, 38, 39]. It is, nevertheless, tempting to speculate
whether those few cases of FVP that develop lung metastasis
[51] or display an encapsulated growth pattern
[52] might behave with a phenotype
more reminiscent of a follicular carcinoma than papillary because of PPFP
expression. Larger studies are needed to determine the frequency of expression
of PPFP in this tumor variant, and to assess the possible coexpression of genes
known to be associated with PTC, such as RAS and BRAF mutations and RET/PTC
rearrangements.Table 1
Occurrence of PAX8/PPARγ rearrangements in differentiated
thyroid carcinomas.
AuthorMethodBenign nodular hyperplasiaFollicular adenomaFollicular carcinomaHürthle cell carcinomaPapillary carcinomaFollicular variant of papillaryAnaplastic carcinomaKroll [3]RT-PCR0/100/205/8 (63%)—0/10——Martelli [14]RT-PCR——0/5—0/41—0/5Zhu [38]RT-PCR————0/460/30—Nikiforova [39]RT-PCR and
nested PCR0/162/25 (8%)8/15 (53%)0/120/230/120/2Marques [36]RT-PCR and
nested PCR0/22/16 (13%)5/9 (56%)—0/90/4Aldred [29]RT-PCR——2/19 (11%)————Lacroix [40]RT-PCR—1/16 (6%)4/26 (15%)0/4——0/5Hibi [41]RT-PCR0/120/120/6—0/12——Marques [28]RT-PCR and
FISH0/286/36 (17%)6/24 (25%)—0/38——Sahin [42]RT-PCR—4/31 (13%)31/54 (57%)1/23 (4%)———Nikiforova [43]RT-PCR and
FISH—1/23 (4%)13/33 (36%)0/19———Castro [44]RT-PCR and
FISH—————4/8 (50%)—Cheung [37]RT-PCR—6/11 (55%)6/17 (35%)————Foukakis [45]RT-PCR—1/8 (13%)5/25 (20%)————Dwight [46]RT-PCR, FISH
and Western—1/40 (3%)10/34 (29%)———0/13Castro [47]RT-PCR and
FISH—3/9 (33.3%)10/22 (45.5%)—0/29/24 (37.5%)—Giordano [48]RT-PCR——7/13 (54%)————TOTAL0/6827/247 (11%)112/310 (36%)1/58 (2%)0/17213/83 (16%)0/29


## 4. PAX8/PPARγ and PPARγ Function

To further investigate the function of PAX8/PPARγ−1, Kroll evaluated
PPARγ response element (PPRE) activity
in the presence of the fusion gene and wild-type PPARγ in
a PPARγ-null osteosarcoma U2OS cell line
[3]. In contrast to the wild-type PPARγ
gene, the fusion gene was ineffective in stimulating ligand-induced gene expression.
Furthermore, the coexpression of the fusion gene with wild-type
PPARγ abrogated the
PPARγ-mediated gene expression, in an
apparently dominant negative fashion [3].In vitro studies using an immortalized human thyroid cell line have shown
accelerated growth in cells transiently transfected with PPFP compared to
wild-type PPARγ or vector only [53].
Increased proliferation was confirmed with cell cycle transit studies, which
showed a lower proportion of PPFP-transfected cells in the resting-phase
(G0/G1) compared to vector and diminished rates of apoptosis in
the PPFP-positive cells. Similar results were seen with stable transfection
experiments, in which cells stably expressing PPFP demonstrated a growth advantage
over vector-transformed cells. These data are consistent with the hypothesis that
PPFP, acting as a dominant negative inhibitor of wild-type
PPARγ, inhibits the normal tumor suppressor
mechanism of PPARγ, and consequently
acts as an oncogene. Furthermore, PPFP stable cell lines showed
improved colony-formation on soft agar, a characteristic associated with malignant
transformation [53].Consistent with Kroll’s results, cotransfection of PPFP and wild-type
PPARγ in immortalized human thyroid cells
led to a significant decline in PPARγ
transactivation [53]. In these studies, GW9662, a
potent PPARγ inhibitor, demonstrated a
dose-dependent increase in cell growth of vector-transfected cells, but did not
increase growth further in the PPFP-transfected cells. Similar effects were
observed upon expression of a dominant negative
PPARγ mutant in these cells.
Loss of contact inhibition and anchorage dependence, which also correlate with
malignant transformation, were also observed upon overexpression of PPFP. The studies
of Powell et al. [53]
provided the first direct evidence for the oncogenic potential of the
PAX8/PPARγ fusion gene, confirming increased
proliferation, decreased apoptosis, and a dominant negative effect of PPFP on
wild-type PPARγ.The influence of PPFP on PPRE-dependent transcription appears to be cell
line-dependent. Au et al. [54] demonstrated that the
fusion gene not only had a dominant negative  effect on PPRE expression in HeLa cells, but also stimulated the expression of the
PPRE-dependent promoter in a PPARγ
ligand-dependent manner in FRTL-5 and Nthy-ori cells, immortalized rat and human
thyroid cell lines, respectively. These differences might be related to differences
in the ways these cells have been immortalized. Thus HeLa cells utilized the HPV E6 gene and Nthy-ori cells
were immortalized with the SV40 large T antigen, whereas FRTL5 cells are a
continuous line of functional, nontransformed rat thyroid cells that depend on
thyroid-stimulating hormone (TSH) for sustained growth. Whatever the mechanism,
the results of Au et al. [54] are in direct
contradiction to Powell’s findings, in which
PPARγ agonists did not augment the PPFP
response [53]. The reasons for this discrepancy are
not known. Nevertheless, PPFP expression in FRTL-5 cells showed increased
proliferation by 3H-thymidine incorporation and soft agar assays
[54], findings that are fully consistent with
Powell’s data [53]. Although further study is
clearly warranted to clarify the mechanism of PPFP action in the cell lines, the
data indicate that PPFP can act as a dominant negative inhibitor as well as an independent ligand-responsive transcription
factor in a promoter-dependent manner.It is still not clear whether PPFP alone is sufficient to promote tumorigenesis,
or whether additional genetic events are a prerequisite for this fusion gene to
exhibit an oncogenic impact. One strong candidate, RAS gene mutations, which
are seen in up to 50% of follicular tumors, rarely occur within the same tumor
as PAX8/PPARγ rearrangements,
suggesting that these putative oncogenes form two distinct pathways of
carcinogenesis [43]. For each of these pathways,
an additional step or series of steps may be required before the development of the
full malignant phenotype.


## 5. PAX8/PPARγ and PAX8 Function

Relatively few studies have assessed the impact of
PAX8/PPARγ rearrangements on
wild-type PAX8 function. Au et al. [54] and
Espadinha et al. [55] evaluated the impact of
PAX8/PPARγ on genes containing PAX8
response elements: sodium-iodine symporter (NIS), thyroid peroxidase (TPO), thyroid
stimulating hormone receptor (TSHR), and thyroglobulin (Tg). Each of these
promoters is regulated by PAX8, while the Tg promoter is regulated by both PAX8
and thyroid transcription factor-1 (TTF1), which exhibit a synergistic effect
when both promoters are combined in
vitro. In human thyroid cancer cell lines, PPFP expression resulted in a
complex mixture of stimulatory and inhibitory effects on PAX8-responsive genes,
including in PPARγ ligand-dependent
and -independent effects. NIS gene expression was stimulated in response to PPFP
expression alone in one study [54], although this apparently
stimulatory effect required cotransfection of PPFP with wild-type PAX8 in
another study [55]. TPO transcription was also
increased by PPFP [54], while TSHr expression was
inhibited [55]. Repression of the Tg
promoter was also seen in response to PPFP [54],
but again one study found that cotransfection with PPFP and PAX8 was necessary for
this inhibitory effect to be seen [55]. However, in both studies,
the fusion gene inhibited PAX8-mediated transcription of Tg in a
dominant-negative fashion [54, 55], while the addition of ciglitazone did
not reverse this dominant negative effect [54].Consequently, the effects of the PAX8-PPARγ gene translocation on PAX8 function seem to be complex. TSHr and Tg
expressions, both of which are associated with highly differentiated thyroid tissue,
are downregulated by PPFP, though it is not clear whether the reduced expression of these genes truly alters cell
differentiation status in vivo.
In contrast, NIS and TPO expression are enhanced by the expression of PPFP,
although once again the impact on cell function is not known. Whether any of these findings relates
directly to the oncogenic actions of PPFP, and consequently the impact on the
behavior and biology of human FTC, remains to be determined.


## 6. PAX8/PPARγ Rearrangements: Bench to Bedside

On the basis of the data discussed above, PPFP appears to be important in the
development of at least a subset of thyroid follicular neoplasms, and has,
therefore, been proposed as a possible oncogene in follicular thyroid carcinoma.
The most obvious direct clinical utility of this discovery is the possibility
that PPFP status could provide a presurgical test of malignancy within the
troublesome group of biopsy specimens currently described as “suspicious for follicular
neoplasm.” These lesions represent approximately 20% of all fine needles
aspiration biopsies of thyroid nodules, and create a diagnostic challenge
because the minority (10–15%) that prove
ultimately to be malignant cannot currently be distinguished by cytological
criteria from those that prove to be benign. Consequently, the recommendation
for all such patients would be to undergo surgery
[56], something that might be
avoided with a preoperative test of sufficient accuracy. Unfortunately, the
finding of a subset of PPFP-positive adenomas reduces the negative predictive
value of a preoperative PPFP biopsy finding to 47.4%, while the presence of
PPFP in a minority of FTC reduces the positive predictive value to 80.6%
(Table 1).RT-PCR, real-time RT-PCR, and FISH have all been used experimentally to detect
translocations, though none is yet proven in a prospective, clinical setting
[3, 36, 39, 42].
PCR, probably the technique most easily adapted to a rapid turnover, clinical
setting, is concordant with PPARγ immunohistochemistry in up to 80% of cases
[36]. When the definition of “positive” PPFP is
restricted to only strong, diffuse staining, immunohistochemistry concordance
improves to 100% in some studies [7, 39], and it may be that RT-PCR techniques will
actually prove superior to the immunohistochemistry “gold standard” for the
detection of the fusion event (Algeciras-Schimnich, A. and Grebe, S.K.G., unpublished
data). The possibility of false-positive immunostaining is real since normal
thyroid tissue, chronic lymphocytic thyoiditis, or benign tissue adjacent to
malignancies may show moderate to strong nuclear staining for
PPARγ expression.
[39, 57],
so the RT-PCR-based approach may prove to have a better negative predictive
value. There are a number of possible explanations for this apparently “false-positive”
staining, including alternate PAX8/PPARγ
breakpoints, 3p25 aneusomy, overexpression of wild-type
PPARγ, or rearrangements involving PPARγ and a non-PAX8 partner [57].Despite these challenges, Sahin et al. demonstrated that a reliable preoperative
assay for PPFP might improve the accuracy of intraoperative frozen section by
significantly reducing the false-negative rate of this technique, and therefore
reducing the need for second (completion) surgeries for patients with follicular
carcinoma who undergo primary thyroid lobectomy [42].
Immunohistochemistry formed the basis for this retrospective study, so clinical
implementation would require confirmation with a more practical preoperative or
intraoperative technique (most likely RT-PCR). In this archival tissue
analysis, however, the sensitivity was improved from 85% with frozen section
alone to 97% with the combination of frozen section and
PPARγ status. Several false-positive PPARγ staining results led to a positive
predictive value of only 72%, but the overall negative predictive value of
frozen section plus immunostaining at this institution was 99%, meaning that
five additional cases of carcinoma in this series of 39 cancers could have been
identified intraoperatively, reducing the need for completion thyroidectomy to
a single patient (3%).Currently, there is no evidence that PPFP status predicts outcome in follicular thyroid
cancer, with no correlation with proven predictive factors of gender, age,
regional nodal spread, or tumor size [28, 39, 42].
The same is true for PPARγ
aneusomy or other PPARγ rearrangements
found in follicular cancers, which is not correlated with TNM stage
[7]. However, patients with
PPARγ rearrangements may have a higher
prevalence of previous nonthyroid cancers [7] and
PPFP rearrangements may be associated with an increased incidence of multifocal
capsular invasion or vascular invasion [39],
although all of these findings remain in dispute [28, 42]. Larger and more comprehensive outcome analysis will be necessary to resolve
these differences in the findings of multiple small studies.


## 7. PPARγ Agonist Therapy in Thyroid Cancer

Follicular cell-derived thyroid cancers carry a generally good prognosis.
Standard therapy involves near-total resection of the thyroid with
adjuvant131I radioablation of remnant thyroid tissue in most cases.
A subset of tumors exhibits a more aggressive clinical course, and may show features
of dedifferentiation, which has been associated with decreased expression of
thyroid-specific genes such as Tg, TPO, NIS, and TSHR
[15, 58].
These dedifferentiated thyroid cancers may consequently lose their ability to
accumulate and concentrate radioiodine, making these tumors unresponsive to
further 131I therapy. Traditionally, patients with
iodine-insensitive tumors have had few therapeutic options and further basic
and applied research is needed to identify suitable therapeutic targets for
treatment of these patients; PPARγ
provides one such target.Thiazolidinediones, including troglitazone, rosiglitazone, and pioglitazone, are
PPARγ agonists used in the treatment of type
2 diabetes. These and other PPARγ agonists have been investigated in vitro in various cancer cell lines
with evidence of growth inhibition and tumor cell apoptosis
[18–23, 59]. Small clinical trials in liposarcoma and
prostate cancer, which exhibit PPARγ
expression, have also been promising in these malignancies,
which exhibit PPARγ expression
[60, 61].
Recent in vitro evaluations in thyroid cancer cell lines have hinted at a possible
role for PPARγ agonist therapy in
redifferentiating neoplastic tissue, potentially enhancing the response to
currently available therapies [62–67]. Expression of CD97, a marker of cell
dedifferentiation, decreased in one follicular carcinoma cell line after
therapy with troglitazone [16]. In the same experiment,
expression of NIS, a gene associated with well-differentiated thyroid tissue, whose
protein product is responsible for iodine concentration within thyroid cells,
increased with agonist treatment compared to control in both a papillary and
follicular carcinoma cell lines. Such an approach of “redifferentiation” might
open up the possibility to restore radioactive iodine sensitivity in some tumors.
Anaplastic cancer cell lines have been studied in similar experiments, and
expressions of Tg, TSHR, NIS, and TPO were all increased after treatment with
rosiglitazone [12]. These findings imply that
PPARγ agonists may prove to be an effective
therapy for improving response to 131I radiotherapy, even in
patients without known PAX8/PPARγ
rearrangements.To date, three reports have been published assessing rosiglitazone therapy in
patients with recurrent thyroid cancer as indicated by elevated Tg levels, but
negative pretreatment whole body iodine scans (Tg-positive, scan-negative
thyroid cancer). Philips et al. [68] treated 2
follicular cancer and 3 papillary cancer patients with rosiglitazone 4 mg daily
for one month and then 8 mg daily for three months. Whole body 131I
scanning (WBS) using recombinant human TSH (rh-TSH) was negative for all 5 patients
at the onset of the study. Basal Tg levels rose in 3 patients while the
rhTSH-stimulated Tg increased in two of the 3 patients after rosiglitazone
therapy. Posttreatment rhTSH-stimulated WBS was faintly positive in one patient.
Elias and Lizotte [69] reported a single case of
papillary thyroid cancer in which rosiglitazone caused a marked increase in
131I uptake on WBS. Following treatment with 250 mCi 131I, the
WBS became negative, suggesting that the cancer had been effectively treated.
Kebebew et al. [70] have reported the largest
number of patients to date treated with a thiazolidenedione: 8 PTC, 1 FVP, and 1 FTC.
These patients received rosiglitazone 4 mg daily for 7 days, then 8 mg
daily for 49 days. Four patients (40%) had conversion from negative to positive WBS
after rosiglitazone, suggesting a possible redifferentiation effect. At 6 months of followup, 2
patients had improved Tg levels (one follicular and one FVP patient), 3 had
stable levels (all papillary cancer patients), and 5 patients had increased Tg
levels compared to baseline. After 11 months of followup, 4 patients had a
partial response to rosiglitazone, exhibiting decreased Tg levels or
increased 131I uptake, 4 had stable disease, and 2 had progression of disease as indicated by
increased Tg levels. In all cases, rosiglitazone was apparently well tolerated
with no significant adverse events. Overall, 6 of 16 patients (5 papillary, 1
FVP) showed uptake on WBS after rosiglitazone therapy, indicating that a subset
of patients may have experienced redifferentiation of their cancers. Whole body
iodine scanning did not always correlate with Tg levels and caution should be
exercised in interpreting Tg levels in these studies because an increase in Tg
could indicate improved differentiation, rather than being a sign of increasing
tumor mass. Only one patient in this study received
additional 131I radiotherapy, although therapy was apparently successful,
with a low Tg post-radiotherapy. Larger studies with longer followup are needed to see if
thyroid cancers treated with PPARγ
agonists show improved response to 131I radioablation or decreased
mortality, the truly important clinical outcome.Very little data is currently available on combination therapy with PPARγ and
other chemotherapeutic agents. Aiello et al. [12]
evaluated the in vitro response of rosiglitazone plus doxyrubicin, a standard
agent used in anaplastic thyroid cancer, in ATC cell lines. They found a markedly
increased effect of doxyrubicin in PPARγ-positive
cell lines when combined with rosiglitazone, but no effect in a
PPARγ-negative cell line.
Copland et al. [71] combined paclitaxel with a novel
PPARγ agonist, RS5444, in a variety of ATC
cell lines. This combination demonstrated synergistic effects on inhibition of
proliferation and stimulation of apoptosis in vitro. When athymic
nude mice were implanted with the responsive ATC cell lines, tumor growth was
inhibited by monotherapy with RS5444. No clinical studies or case reports have
yet addressed combination therapy in humans with thyroid cancer, though a
clinical trial is currently under development by our group.


## 8. Conclusion

Emerging genetic and molecular information acquired over the last 2 decades has
begun to unravel the pathogenesis of thyroid cancer and in the future may open the
door to potential novel therapies for patients with previously untreatable disease.
Research focusing on PPARγ in a
variety of cancer cell lines has implied a tumor suppressor function for wild-type
PPARγ, while PPARγ downregulation or inhibition may be one
factor in the development of at least some thyroid cancer types.Chromosomal alterations of PPARγ,
resulting in the expression of the fusion protein PPFP, may be an early event in the
development or progression of follicular thyroid cancer and perhaps the follicular
variant of papillary cancer. The detection of these alterations in FAs may support a stepwise
adenoma to carcinoma sequence, or indicate the presence of “carcinoma in situ.” However, the PAX8/PPARγ rearrangement in itself may not be
sufficient for the development of a malignant phenotype: additional genetic or
epigenetic events may be required to enable the full phenotypic expression of
follicular thyroid carcinoma.Several lines of data suggest that PPFP, either through PPARγ
inhibition, PAX8-dependent gene expression modulation, or both, leads to
downstream effects, which are at least in part mediated by the NFκB
pathway. These altered pathways stimulate cell proliferation and inhibit apoptosis,
but there may be several other paths through which PPFP modulates the tumor phenotype, including
alteration of cell differentiation status and expression of the sodium iodide
transporter NIS, which may have an impact on the efficacy of our current therapeutic
options.PPARγ represents an attractive
therapeutic target in a variety of thyroid cancers, including anaplastic, follicular,
and papillary thyroid cancers. Although in vitro
data is promising, early studies using PPARγ agonists to treat iodine-insensitive
recurrent thyroid cancer are promising, but inconclusive so far. Larger studies
with longer followup will be needed to clarify the potential for
PPARγ agonists to act as
“redifferentiation” agents, Nevertheless, the availability of a number of approved, orally
administered, well-tolerated agents makes this group of drugs an attractive
option for study.



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