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Cancer Research 68, 6578, August 15, 2008. doi: 10.1158/0008-5472.CAN-08-0855
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Peroxisome Proliferator-Activated Receptor-{delta} Induces Cell Proliferation by a Cyclin E1–Dependent Mechanism and Is Up-regulated in Thyroid Tumors

Lingchun Zeng1, Yan Geng2, Maria Tretiakova1, Xuemei Yu1, Peter Sicinski2 and Todd G. Kroll1

1 Department of Pathology, University of Chicago School of Medicine, University of Chicago Cancer Research Center, Chicago, Illinois and 2 Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Todd G. Kroll, 5841 South Maryland Avenue, AMB P323 (MC1089), Chicago, IL 60637. Phone: 773-702-3017; Fax: 773-834-5251; E-mail: tkroll{at}bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Peroxisome proliferator-activated receptors (PPARs) are lipid-sensing nuclear receptors that have been implicated in multiple physiologic processes including cancer. Here, we determine that PPAR{delta} induces cell proliferation through a novel cyclin E1–dependent mechanism and is up-regulated in many human thyroid tumors. The expression of PPAR{delta} was induced coordinately with proliferation in primary human thyroid cells by the activation of serum, thyroid-stimulating hormone/cyclic AMP, or epidermal growth factor/mitogen-activated protein kinase mitogenic signaling pathways. Engineered overexpression of PPAR{delta} increased thyroid cell number, the incorporation of bromodeoxyuridine, and the phosphorylation of retinoblastoma protein by 40% to 45% in just 2 days, one usual cell population doubling. The synthetic PPAR{delta} agonist GW501516 augmented these PPAR{delta} proliferation effects in a dose-dependent manner. Overexpression of PPAR{delta} increased cyclin E1 protein by 9-fold, whereas knockdown of PPAR{delta} by small inhibitory RNA reduced both cyclin E1 protein and cell proliferation by 2-fold. Induction of proliferation by PPAR{delta} was abrogated by knockdown of cyclin E1 by small inhibitory RNA in primary thyroid cells and by knockout of cyclin E1 in mouse embryo fibroblasts, confirming a cyclin E1 dependence for this PPAR{delta} pathway. In addition, the mean expression of native PPAR{delta} was increased by 2-fold to 5-fold (P < 0.0001) and correlated with that of the in situ proliferation marker Ki67 (R = 0.8571; P = 0.02381) in six different classes of benign and malignant human thyroid tumors. Our experiments identify a PPAR{delta} mechanism that induces cell proliferation through cyclin E1 and is regulated by growth factor and lipid signals. The data argue for systematic investigation of PPAR{delta} antagonists as antineoplastic agents and implicate altered PPAR{delta}–cyclin E1 signaling in thyroid and other carcinomas. [Cancer Res 2008;68(16):6578–86]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Peroxisome proliferator-activated receptors (PPAR) form molecular complexes with hydrophobic ligands such as fatty acids, the 9-cis-retinoic acid receptor (retinoid X receptor) and coactivators to regulate the transcription of target genes (1, 2). The three major PPARs, {alpha}, {delta}, and {gamma} (NR1C1, NR1C2, and NR1C3), are encoded by separate genes and have distinct patterns of expression and biological activities. PPAR{alpha} is expressed primarily in liver, brown adipose, kidney, and heart tissues (3). It plays a central role in fatty acid uptake and oxidation and is the target of the fibrate drugs that are used to treat patients with dyslipidemia (4). PPAR{gamma} is expressed most highly in fat and macrophages. PPAR{gamma} functions in adipogenesis, lipid storage, glucose homeostasis, inflammtion, and atherosclerosis (5, 6). Synthetic thiazolidinedione agonists for PPAR{gamma} are used to treat patients with type 2 diabetes (7). PPAR{delta} is more widely expressed than PPAR{alpha} and PPAR{gamma}, implying a more general cell function that is not yet understood. PPAR{delta} has been implicated in fatty acid β-oxidation (8), muscle fiber–type remodeling (9), lipoprotein metabolism, and glucose uptake (10, 11). A synthetic agonist for PPAR{delta} modulated HDL and triglyceride levels in healthy volunteers (12). Thus, all three PPARs function in aspects of lipid metabolism.

PPARs have also been implicated in tumorigenesis but the molecular mechanisms are not well understood. For example, PPAR{alpha} is thought to mediate the carcinogenic effects of peroxisome proliferators in hepatocellular carcinoma in rodents (13). In addition, both PPAR{delta} and PPAR{gamma} modulate colon tumorigenesis in predisposed mouse models (14–18). Furthermore, natural mutations in PPAR{gamma} have been identified in human colon (19) and thyroid carcinoma (20, 21) tissues. These findings indicate significant functions for PPARs in thyroid cancer and physiology.

Here, we show that PPAR{delta} is the predominant PPAR that is expressed in normal human thyroid cells and tissues. Our experiments have determined that PPAR{delta} induces cell proliferation by a novel cyclin E1–dependent mechanism and is up-regulated in six different classes of human thyroid tumors. The results show that PPAR{delta} regulates epithelial cell proliferation via cyclin E1, growth factor, and lipid signals. Thus, deregulation of the PPAR{delta}-cyclin E1 signaling axis seems to be important in thyroid and other carcinomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell lines and primary cultures of human thyroid cells. Primary cultures of human thyroid follicular epithelial cells were isolated from normal thyroid tissues by mechanical dispersion and treatment with collagenase P and dispase II (Roche), as previously described (ref. 22; Supplemental Fig. S1A–D). The primary thyroid cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L of L-glutamine, and penicillin/streptomycin (Invitrogen). Fungizone (Invitrogen) and primocin (Sigma) were present in the medium for the first 2 to 3 days. The primary cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. 293T human embryonic kidney cells were obtained from American Type Culture Collection.

Immunoreagents and chemicals. PPAR{delta} (H74) antiserum, PPAR{gamma} (E8) antibody, cyclin E1 (M20) antiserum, cyclin A2 (BF683) antibody, cyclin B1 (GNS1) antibody, p21 (F5) antibody, p27 (F8) antibody, cyclin D1 (H295) antiserum, and goat anti-mouse IgG-horseradish peroxidase (HRP) secondary antibody were obtained from Santa Cruz Biotechnology. Phosphorylated retinoblastoma (phospho-Rb) antiserum was purchased from Cell Signaling. Cyclin D3 antiserum (LabVision), anti-rabbit IgG-HRP (GE Healthcare), thyroglobulin antiserum (M0781; DAKO), β-actin antibody (Sigma), and anti–bromodeoxyuridine (BrdUrd)-fluorescein (Roche) were also used. The PPAR{delta} agonist GW501516 was purchased from Cayman Chemical. Epidermal growth factor (EGF), thyroid-stimulating hormone (TSH), and insulin were purchased from Sigma.

SDS-PAGE and immunoblots. Total protein was extracted from tissues or cells with lysis buffers containing nonionic (NP40 or Triton X-100) and/or ionic (sodium dodecylsulfate and sodium deoxycholate) detergents (U.S. Biochemical or Sigma) and Complete Mini protease inhibitors (Roche). Twenty to 40 µg of protein was separated on bis/tris SDS-PAGE gels in the MOPS system (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) using a semidry apparatus (Hoeffer). Nitrocellulose membranes were blocked in 5% nonfat dry milk, incubated at 4°C with primary antibody or antiserum overnight, incubated with HRP-conjugated secondary antibodies for 1 to 2 h at room temperature and detected using enhanced chemiluminescence (Amersham). The blots were stripped and reprobed with β-actin as a loading control. Total protein in cell lysates was determined with a modified Bradford assay (Bio-Rad). Protein levels were quantified on X-ray films from immunoblots using ImageJ software.3 Values were normalized to levels of control β-actin in the same lanes. Kaleidoscope (Bio-Rad) and MagicMark (Invitrogen) markers were used as molecular weight standards.

Electroporation and transfection. Electroporations were performed using the VPI-1005 Amaxa system (Amaxa, Inc.) that achieved 60% to 80% transduction efficiency in primary thyroid cells as determined by expression of control green fluorescent protein plasmids based on fluorescence microscopy and flow cytometry.4 pCDNA3.1 or pCDNA3.2 expression vectors (Invitrogen) or small inhibitory RNAs (siRNA; Santa Cruz Biotechnology) were introduced into primary thyroid cells by electroporation, and the cells were allowed to recover for 2 days in normal growth medium. Electroporation experiments were repeated three to five times using preparations of primary thyroid cells from different patients. 293T cells were transfected with Fugene (Roche), according to the manufacturer's recommendations.

Cell proliferation assays. Cell proliferation was determined by cell counting and the incorporation of BrdUrd into cell nuclei. Cells were counted using an automated Z2 particle counter (Beckman-Coulter). For BrdUrd incorporation, cells were plated on glass coverslips and BrdUrd was added to the culture medium 3 to 5 h before fixation. The cells were fixed in absolute methanol at 4°C, denatured in 2 mol/L of HCl and stained with mouse anti–BrdUrd fluorescein conjugate (Roche), according to the manufacturer's recommendations. Propidium iodide was used as the nuclear counterstain. Microscopic fields were selected randomly and ≥200 cells were counted under each condition. BrdUrd-positive cells were calculated as the percentage of total cells in each field. For GW501516 treatment, cells were incubated in medium with 10% charcoal dextran–stripped FBS (Hyclone).

RNA isolation and reverse transcription-PCR. Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript first-strand synthesis kit (Invitrogen) from 2 µg of total RNA. PCR was performed with the following primers: PPAR{delta} forward primer 5'-ACT GAG TTC GCC AAG AGC ATC-3' and PPAR{delta} reverse primer 5'-TTA GTA CAT GTC CTT GTA GAT CT-3' (546 bp); PPAR{gamma} forward primer 5'-TGA CTT GAA CGA CCA AGT AAC TC-3' and PPAR{gamma} reverse primer 5'-CTA GTA CAA GTC CTT GTA GAT CTC-3' (508 bp); PPAR{alpha} forward primer 5'-TCC GCA TCT TTC ACT GCT GCC A-3' and PPAR{alpha} reverse primer 5'-TCA GTA CAT GTC CCT GTA GAT C-3' (600 bp); β-actin forward primer 5'-TCC TTC CTG GGC ATG GAG TC-3' and β-actin reverse primer 5'-GTA ACG CAA CTA AGT CAT AGT C-3' (361 bp). PCR products were analyzed on 1% to 1.7% agarose gels and visualized by ethidium bromide staining.

Immunohistochemistry. Tissue sections were deparaffinized in xylenes, rehydrated through a graded ethanol series and washed in TBS. Antigen retrieval was carried out by heating in DTRS buffer (pH 6; DAKO) or Tris-EDTA (pH 9) buffer for 15 min in a microwave. Endogenous peroxidase activity was blocked by 3% H2O2 in methanol for 5 min. Nonspecific binding sites were blocked by Protein Block (DAKO) for 20 min. Tissue sections were incubated for 1 h at room temperature with rabbit antiserum against PPAR{delta} (H74; Santa Cruz Biotechnology), monoclonal antibody against PPAR{gamma} (E8, Santa Cruz Biotechnology), or monoclonal antibody against Ki67 proliferation antigen (clone KiS5, DAKO). This was followed by a 30-min incubation with goat anti-mouse or goat anti-rabbit IgG conjugated to HRP-labeled polymer (EnVision, DAKO). Slides were developed for 5 min with 3-3'-diaminobenzidine chromogen and counterstained with Gil's hematoxylin. Negative controls were performed by substituting primary antibody with nonimmune mouse or rabbit immunoglobulins.

Construction of thyroid tissue microarrays. Tissue microarrays were constructed using formalin-fixed paraffin-embedded tissue blocks from 103 patients with benign and malignant thyroid tumors that were selected by two experienced endocrine pathologists (T.G. Kroll and M. Tretiakova): 18 follicular adenomas, 20 follicular carcinomas, 37 papillary carcinomas, 10 anaplastic carcinomas, 9 Hurthle cell adenomas, and 9 Hurthle cell carcinomas were chosen. Seventy-six corresponding normal thyroid tissues were selected from the same thyroid blocks as controls. A minimum of two tissue cylinders with a diameter of 1 mm were arrayed using an automated tissue microarrayer (ATA-27, Beecher Instruments). Recipient blocks were cut into 4-µm-thick sections on Surgipath silane-coated positively charged slides. PPAR{delta} expression was quantified by two methods. First, PPAR{delta} immunoreactivity was calculated as a Reiner score (23) based on the nuclear and cytoplasmic intensity and the percentage of immunoreactive cells visualized manually by bright-field microscopy. Second, PPAR{delta} expression was quantified using the Chromavision automated cellular imaging system (ACIS), with positive brown nuclear and cytoplasmic staining recorded as a numerical score between 0 and 225 for each pixel and normalized to an area of 1 µm2. Nuclear staining for Ki67 was quantified using ACIS based on three color parameters: hue, luminosity, and saturation. The ACIS software was instructed by setting color-specific thresholds to determine brown (positive stain) from blue (negative counterstain) in the nuclei within outlined areas. The percentage of positively stained nuclei (nuclear index) was calculated from 200 to 500 cells in each tissue sample.

Statistical analyses. Cell proliferation was calculated as the mean of duplicate or triplicate measurements ±SD. P values were calculated using a two-tailed Student's t test for continuous variables. Correlations were calculated using the Spearman rank correlation test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
PPAR{delta} is expressed in normal human thyroid cells and tissues. We first determined the expression of PPAR{alpha}, PPAR{delta}, and PPAR{gamma} in normal human thyroid cells and tissues. Cultures of primary thyroid cells were isolated from normal human thyroid tissues that were obtained after surgery for thyroid tumors (Supplemental Fig. S1A–C). The primary cultures were 85% to 90% pure and retained differentiated functions such as the expression of thyroglobulin and thyroperoxidase in response to TSH (Supplemental Fig. S1D). PPAR{delta} mRNA was abundant, whereas PPAR{gamma} mRNA was low and PPAR{alpha} mRNA was barely detectable in normal thyroid tissue and primary thyroid cells based on reverse transcription-PCR (Fig. 1A ). PPAR{delta} expression was estimated to be 10-fold to 15-fold higher than PPAR{gamma} expression based on Northern blots that detected the PPAR{delta} (3.7 kb) and PPAR{gamma} (1.9 kb) mRNA transcripts.4 PPAR{gamma} mRNA was highest in control adipose tissue and PPAR{alpha} mRNA was highest in control HepG2 hepatocellular carcinoma cells (Fig. 1A). The expression of β-actin was similar in all samples (Fig. 1A).


Figure 1
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  		Figure 1. PPAR{delta} is expressed highly and correlates with proliferation in normal thyroid cells and tissue. A, reverse transcription-PCR showed that PPAR{delta} mRNA was abundant, PPAR{gamma} mRNA was low, and PPAR{alpha} mRNA was barely detectable in normal thyroid cells and tissue. β-Actin mRNA served as the positive control. B, expression of PPAR{delta} protein was abundant in normal thyroid cells and tissue based on immunoblots using the H74 antiserum against PPAR{delta} (left). 293T cells transfected with PPAR{delta}, PPAR{delta}-V5, PPAR{gamma}, or PPAR{gamma}-V5 showed that H74 was specific for PPAR{delta} and did not cross-react with PPAR{gamma} (right). C, loss of PPAR{delta} expression paralleled that of phospho-Rb after removal of FBS from the culture medium to inhibit cell proliferation. The expression of thyroglobulin, a marker of thyroid differentiation, was increased after FBS removal. D, an induction in PPAR{delta} expression paralleled that of phospho-Rb after the addition of FBS to the culture medium to stimulate the proliferation of quiescent thyroid cells. The expression of thyroglobulin was reduced and β-actin was relatively constant after the addition of FBS.

 
Expression of PPAR{delta} protein was next determined by immunoblotting. H74 antiserum against PPAR{delta} reacted on immunoblots with the native PPAR{delta} protein (50 kDa) in normal thyroid tissues and primary thyroid cells (Fig. 1B, left). H74 also reacted with PPAR{delta} and PPAR{delta}-V5 but not with PPAR{gamma} (54 kDa) or PPAR{gamma}-V5 that were expressed by transfection in 293T kidney cells (Fig. 1B, right). Immunohistochemistry showed that H74 reacted with the nuclei and cytoplasm of normal thyroid cells in paraffin-embedded tissue sections (Supplemental Fig. S2A). This nuclear immunoreactivity was not elevated in thyroid carcinomas that expressed the PAX8-PPAR{gamma} fusion protein (Supplemental Fig. S2B; ref. 21), which reacted strongly with the E8 antibody against PPAR{gamma} in the same tissue sections (Supplemental Fig. S2C). These experiments show that (a) PPAR{delta} is the predominant PPAR expressed in normal thyroid cells and tissues and (b) the H74 antiserum is specific for PPAR{delta}.

Expression of PPAR{delta} correlates with cell proliferation and is induced by thyroid mitogens. We observed that the expression of PPAR{delta} correlated directly with proliferation in thyroid cells. For example, PPAR{delta} protein decreased on immunoblots after removal of FBS from the culture medium and this paralleled a reduction in phospho-Rb (Fig. 1C), an established marker of cell proliferation. In a complementary fashion, the expression of PPAR{delta} protein increased in parallel with phospho-Rb when quiescent primary thyroid cells were induced to re-enter the cell cycle by the addition of FBS to the culture medium (Fig. 1D). The level of PPAR{delta} in these experiments varied inversely with thyroid differentiation, as measured by the markers thyroglobulin (Fig. 1C and D) and thyroperoxidase.4 Control β-actin protein was relatively constant (Fig. 1C and D). These results show that the expression of PPAR{delta} correlates directly with proliferation and inversely with differentiation in normal thyroid cells.

We next determined the effects of thyroid mitogens on expression of PPAR{delta}. Expression of PPAR{delta} and phospho-Rb were induced in parallel in quiescent thyroid cells by treatment with TSH plus insulin or EGF plus insulin but not with insulin alone (Fig. 2A ). The TSH/cyclic AMP and EGF/mitogen-activated protein kinase (MEK/ERK) pathways induce proliferation in thyroid cells in the presence of insulin/insulin-like growth factor-I, which are permissive for proliferation but have no mitogenic activity by themselves (24). As predicted, the MEK/ERK inhibitor U1026 and the pKa inhibitor H89 each attenuated the induction of PPAR{delta} (Fig. 2B) and phospho-Rb4 by EGF and TSH treatment, respectively. Thus, PPAR{delta} is increased coordinately with proliferation that is induced in normal thyroid cells by serum, TSH, or EGF.


Figure 2
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  		Figure 2. Expression of PPAR{delta} is induced by thyroid mitogens. A, TSH + insulin and EGF + insulin but not insulin alone induced both PPAR{delta} and phospho-Rb in normal thyroid cells. B, the induction of PPAR{delta} and phospho-Rb was blocked by treatment of thyroid cells with H89 or U0126, inhibitors of the TSH and EGF pathways, respectively.

 
PPAR{delta} regulates proliferation in normal thyroid cells. To determine whether PPAR{delta} itself regulates cell proliferation, we performed both gain-of-function and loss-of-function experiments. First, overexpression of PPAR{delta} by electroporation increased thyroid cell number by 40% to 45% (Fig. 3A ) and the incorporation of BrdUrd by 30% to 35% (Fig. 3B) over 5 days compared with vector controls. In contrast, overexpression of control PPAR{gamma} in the same experiment had little effect (Fig. 3A and B). Second, efficient knockdown of endogenous PPAR{delta} by siRNA decreased thyroid cell number (Fig. 3A) and the incorporation of BrdUrd by 50% to 55% (Fig. 3B) compared with oligonucleotide controls. The levels of PPAR{delta}, PPAR{gamma}, and β-actin were confirmed on immunoblots in these experiments (Fig. 3C). The E8 antibody cross-reacts with both PPAR{gamma} and PPAR{delta} (Fig. 3C, right). These data determine that PPAR{delta} regulates proliferation in normal human thyroid cells.


Figure 3
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  		Figure 3. PPAR{delta} induces proliferation in normal human thyroid cells. A, overexpression of PPAR{delta} by electroporation increased thyroid cell numbers by 40% to 45%, whereas knockdown of PPAR{delta} by siRNA decreased thyroid cell numbers by 50% to 55% over 5 d. Overexpression of control PPAR{gamma} had little effect in the same experiment. Columns, mean of duplicate or triplicate culture dishes; bars, SD (*, P < 0.0001). B, overexpression of PPAR{delta} increased the incorporation of BrdUrd by 30% to 35%, whereas knockdown of endogenous PPAR{delta} by siRNA decreased the incorporation of BrdUrd by 50% to 55% in the normal thyroid cells. Overexpression of control PPAR{gamma} had little effect in the same experiment. Columns, mean of duplicate or triplicate culture dishes; bars, SD (*, P < 0.0001). C, immunoblots verified the levels of PPAR{delta}, PPAR{gamma}, and β-actin in these experiments. The E8 PPAR{gamma} antibody cross-immunoreacts with PPAR{delta} (right).

 
PPAR{delta} induces cyclin E1 in normal thyroid cells. To establish the mechanism through which PPAR{delta} induces thyroid cell proliferation, we measured regulators of the epithelial cell cycle. Cyclin E1 protein was increased 9-fold by overexpression of PPAR{delta} and reduced 2-fold by siRNA knockdown of PPAR{delta} in primary thyroid cells (Fig. 4A ). Phospho-Rb was increased 2-fold and decreased 2.5-fold, respectively, under the same conditions (Fig. 4A). Cyclin A2 was induced 2-fold by overexpression of PPAR{delta} and reduced 4-fold by knockdown of PPAR{delta} (Fig. 4A) but no significant changes in cyclins D1, D3, or B1, the cyclin-dependent kinase (cdk) inhibitors p21 or p27, or β-actin were observed (Fig. 4A). Overexpression of control PPAR{gamma} induced cyclin E1 by less than 2-fold and did not alter phospho-Rb (Fig. 4A). These data determine that PPAR{delta} induces both cyclin E1 and proliferation in normal thyroid cells.


Figure 4
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  		Figure 4. PPAR{delta} induces cell proliferation by a cyclin E1–dependent mechanism. A, overexpression of PPAR{delta} in normal thyroid cells by electroporation increased cyclin E1 protein 9-fold and phospho-Rb 2-fold compared with vector controls. Efficient knockdown of PPAR{delta} by siRNA decreased cyclin E1 2-fold and phospho-Rb 2.5-fold compared with oligonucleotide controls. Overexpression and knockdown of PPAR{delta} increased cyclin A2 2-fold and reduced cyclin A2 4-fold, respectively, whereas the expression of cyclins D1, D3, and B1, the cdk inhibitors p27 and p21, and β-actin changed little under the same conditions. Overexpression of control PPAR{gamma} increased cyclin E1 by less than 2-fold and did not alter the levels of cyclin A2 or phospho-Rb. Measurements are normalized to β-actin and calculated as the fold change over vector or oligonucleotide controls. B, induction of proliferation by PPAR{delta} in normal thyroid cells was blocked by knockdown of cyclin E1 by siRNA. Columns, mean of duplicate culture dishes; bars, SD (*, P < 0.007). C, the induction of cell proliferation by PPAR{delta} was abrogated in cyclin E1–/– mouse embryo fibroblasts compared with wild-type mouse embryo fibroblasts or cyclin D1–/– mouse embryo fibroblasts. Black columns, cells overexpressing PPAR{delta}; white columns, vector controls. Columns, mean of duplicate dishes; bars, SD (*, P < 0.004).

 
Induction of cell proliferation by PPAR{delta} is dependent on cyclin E1. Two additional experiments showed that PPAR{delta} regulates cell proliferation through a cyclin E1–dependent mechanism. First, down-regulation of cyclin E1 protein by siRNA blocked the incorporation of BrdUrd that was induced by PPAR{delta} in primary thyroid cells (Fig. 4B). Second, induction of cell proliferation by PPAR{delta} was not observed in mouse embryo fibroblasts that had knockout of the cyclin E1 gene, in contrast to wild-type mouse embryo fibroblasts or mouse embryo fibroblasts that had knockout of the cyclin D1 gene (Fig. 4C). These experiments show that induction of cell proliferation by PPAR{delta} is dependent on cyclin E1.

GW501516 augments proliferation in normal thyroid cells. To determine whether proliferation by PPAR{delta} is dependent on PPAR{delta} lipid ligand, we tested the selective PPAR{delta} agonist GW501516. Proliferation increased in a dose-dependent manner by treatment of primary thyroid cells with GW501516 (10–500 nmol/L), as determined by cell number (Fig. 5A ) and the incorporation of BrdUrd.4 No significant effects on the expression of endogenous PPAR{delta} or β-actin protein were observed under these conditions (Fig. 5A). GW501516 (500 nmol/L) increased thyroid cell numbers by 35% to 40% compared with untreated thyroid cells over 6 days (Fig. 5B). We also determined the effects of GW501516 on thyroid cells after overexpression or siRNA knockdown of PPAR{delta}. Thyroid cell number (Fig. 5C) and the incorporation of BrdUrd4 were dependent on the levels of both PPAR{delta} and GW501516 in this experiment. Thus, synthetic PPAR{delta} agonist, a surrogate for natural PPAR{delta} lipid ligand, augmented proliferation by PPAR{delta} in normal thyroid cells.


Figure 5
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  		Figure 5. PPAR{delta} ligand GW501516 induces proliferation in normal thyroid cells. A, cultures of primary thyroid cells were treated with the synthetic PPAR{delta} agonist GW501516, a surrogate for natural PPAR{delta} lipid ligand. Thyroid cell numbers increased in a dose-dependent manner in response to increasing concentrations of GW501516 (10–500 nmol/L) over 3 d. Immunoblots determined that the expression of PPAR{delta} and β-actin protein was constant under these conditions. B, GW501516 (500 nmol/L) increased thyroid cell number by 35% to 40% compared with untreated cells over 6 d. C, 500 nmol/L GW501516 was also added to thyroid cells in which the expression of PPAR{delta} was modulated by overexpression or siRNA. Proliferation of the thyroid cells over 5 d depended on levels of both GW501516 ligand and PPAR{delta}. Columns, mean of duplicate or triplicate dishes; bars, SD (*, P < 0.0001).

 
Expression of native PPAR{delta} is elevated in benign and malignant human thyroid tumors. To further investigate PPAR{delta} in a natural state of increased cell proliferation, we determined PPAR{delta} levels in human thyroid tumors. PPAR{delta} protein was measured by immunohistochemistry on tissue microarrays that contained six classes of thyroid tumors: 18 follicular adenomas, 20 follicular carcinomas, 37 papillary carcinomas, 10 anaplastic carcinomas, 9 Hurthle cell adenomas, and 9 Hurthle cell carcinomas. Seventy-six normal thyroid tissues from the same paraffin blocks were used as controls. The expression of PPAR{delta} was quantified as described in Materials and Methods by (a) manual Reiner scoring (23) and (b) automated computer scanning (ACIS) from the bright-field microscope. Calculations from the two methods were consistent (Table 1 ). PPAR{delta} expression was moderate in the nuclei and low in the cytoplasm of normal thyroid tissues (mean ACIS score, 75.19; Table 1; Fig. 6A ), whereas PPAR{delta} expression was elevated above normal levels in follicular adenomas (mean ACIS score, 208.44; P < 0.0001; Table 1; Fig. 6B), follicular carcinomas (mean ACIS score, 221.63; P < 0.0001; Table 1), papillary carcinomas (mean ACIS score, 394.11; P < 0.0001; Table 1; Fig. 6C), anaplastic carcinomas (mean ACIS score, 438.60; P < 0.0001; Table 1; Fig. 6D), Hurthle cell adenomas (mean ACIS score, 352.67; P < 0.0001; Table 1), and Hurthle cell carcinomas (mean ACIS score, 293.89; P < 0.0001; Table 1). PPAR{delta} was increased predominantly in the nuclei of follicular (Fig. 6B) and Hurthle cell4 tumors and in both the nuclei and cytoplasm of papillary (Fig. 6C) and anaplastic (Fig. 6D) thyroid carcinomas. Mean expression of Ki67, an in situ marker of cell proliferation, was also elevated (P < 0.0219) in these thyroid tumors (Fig. 6A–D; Table 1) and correlated (R = 0.8571; P = 0.02381) with mean expression of PPAR{delta}. Thus, the expression of native PPAR{delta} protein correlated directly with cell proliferation in thyroid tumors in vivo as well as with thyroid cell proliferation in vitro.


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  		Table 1. Mean expression of PPAR{delta} and Ki67 in normal, benign, and malignant thyroid tissues

 

Figure 6
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  		Figure 6. Expression of native PPAR{delta} is elevated in benign and malignant human thyroid tumors and correlates with cell proliferation. The expression of PPAR{delta} was determined by immunohistochemistry on tissue microarrays that contained six different classes of human thyroid tumors. Normal thyroid tissues from the same paraffin blocks were used as controls. Levels of PPAR{delta} and the proliferation marker Ki67 were quantified as described in Materials and Methods. A, PPAR{delta} was expressed at moderate levels in the nuclei and low levels in the cytoplasm (mean ACIS score, 75.19) of normal thyroid cells. Ki67 was detected in 1.26% of nuclei in normal thyroid cells. B, PPAR{delta} was expressed at higher than normal levels (mean ACIS score, 208.44; P < 0.0001) in the nuclei of benign thyroid follicular adenomas. Ki67 was detected in 2.02% of nuclei in follicular adenomas (P = 0.0219). C, PPAR{delta} was expressed at higher than normal levels (mean ACIS score, 394.11; P < 0.0001) in the nuclei and cytoplasm of well-differentiated papillary thyroid carcinomas. Ki67 was detected in 4.53% of nuclei in papillary carcinomas (P < 0.0001). D, PPAR{delta} was expressed at the highest levels (mean ACIS score, 438.60; P < 0.0001) in poorly differentiated anaplastic thyroid carcinomas. Ki67 was detected in 31.18% of nuclei in anaplastic carcinomas (P < 0.0001). PPAR{delta} and Ki67 immunoreactivity (brown) and the nuclear counterstain (blue; see also Table 1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
PPARs are ligand-activated transcription factors that have been studied most thoroughly in lipid metabolism, adipogenesis, obesity, insulin sensitivity, and diabetes (1, 2). The PPARs have also been investigated in cancer but their mechanisms in tumorigenesis are not understood. Here, we determine a novel mechanism of PPAR{delta} that induces cell proliferation through cyclin E1 and show that PPAR{delta} is up-regulated in many human thyroid tumors.

We showed that the expression of PPAR{delta} was high compared with PPAR{gamma} and PPAR{alpha} in normal human thyroid cells and tissues, as reported recently in the mouse (3). Our engineered overexpression of PPAR{delta} in primary human thyroid cells generated a 40% to 45% increase in S phase cells in only 2 days. This is a remarkable induction because the usual transit time of primary thyroid cells through the cell cycle is 30 to 40 hours. The induction of proliferation by PPAR{delta} was augmented by synthetic PPAR{delta} agonist, which was a surrogate for natural PPAR{delta} lipid ligand, and was associated with a 9-fold increase in cyclin E1 protein, a regulator of the epithelial cell cycle (25). Three additional experiments showed that the induction of proliferation by PPAR{delta} was dependent on cyclin E1. First, knockdown of endogenous PPAR{delta} by siRNA led to reductions in both cell proliferation and cyclin E1. Second, knockdown of endogenous cyclin E1 by siRNA abrogated thyroid cell proliferation that was induced by PPAR{delta}. Third, the induction of proliferation by PPAR{delta} was not present in cyclin E1–/– mouse embryo fibroblasts, in contrast to wild-type mouse embryo fibroblasts or cyclin D1–/– mouse embryo fibroblasts. These experiments determine a novel PPAR{delta} mechanism that induces cell proliferation through cyclin E1, a hitherto unrecognized pathway of PPAR{delta}-mediated cell growth control.

Our findings are compatible with the known functions of cyclin E1, which in association with Cdk2, drives cells from G1 into S phase in part by hyperphosphorylation of retinoblastoma protein (25). In fact, PPAR{delta} protein is expressed early in G1 prior to expression and phosphorylation of retinoblastoma protein in quiescent primary thyroid cells that have been treated with serum and/or growth factors (Fig. 1).4 This kinetic pattern further supports the regulation of cyclin E1 by PPAR{delta}, although the exact molecular underpinnings of this regulation remain to be elucidated. Interestingly, PPAR{delta} knockout mice are smaller than wild-type littermate controls and this may reflect a deficiency in cell proliferation in tissues such as fat that are reduced in PPAR{delta} knockout animals (10, 26). We observed that engineered overexpression in normal thyroid cells of PPAR{delta} also induced cyclin A2, albeit to a lesser extent than cyclin E1. Cyclin A2, in complex with Cdk2 and Cdk1, promotes the passage of cells through the G1-S and G2-M transitions and thus cyclin A2 may modulate the effects of PPAR{delta} on cell proliferation as well (27).

Our findings are consistent with previous reports showing that PPAR{delta} stimulates proliferation in mouse preadipocytes, hepatocellular carcinoma cells, human endothelial cells, and breast, prostate, and colon carcinoma cells (28–31), although the mechanisms of PPAR{delta} in these systems were not shown to involve cyclin E1. On the other hand, PPAR{delta} has also been reported to inhibit proliferation in keratinocytes and mouse lung cancer cells (32, 33), raising the possibility that cell-dependent or culture-dependent responses to PPAR{delta} may exist. In addition, factors that confound the interpretation of PPAR activities are common and include: (a) the coexpression of multiple endogenous PPAR isoforms, such as PPAR{delta} and PPAR{gamma} that have differing effects on cell proliferation and overlapping functions at PPREs (34, 35); (b) the use of immortalized rodent cell lines that have altered growth responses compared with normal human cells; (c) the use of PPAR ligands that exhibit PPAR-independent effects (36, 37); and (d) the use of commercial immunoreagents that cross-react with PPAR{delta} (50 kDa), PPAR{gamma} (54 kDa), and/or PPAR{alpha} (52 kDa).4 Herein, our experiments provide a clear determination that PPAR{delta} induces cell proliferation through a novel cyclin E1–dependent mechanism in normal human cells that predominantly express PPAR{delta}.

Our experiments indicate unappreciated functions for PPAR{delta} and cyclin E1 in thyroid physiology and show that PPAR{delta} is up-regulated in many thyroid tumors including papillary carcinoma, the most common thyroid cancer. In fact, all classes of thyroid tumors in our study exhibited elevated mean levels of PPAR{delta} and increased mean expression of Ki67, an in situ marker of cell proliferation. Thus, PPAR{delta} promoted thyroid cell proliferation in vitro and was associated with increased proliferation in thyroid tumors in vivo. Most previous studies have shown increased cyclin E levels in transformed thyroid cells (38) and thyroid carcinoma tissues (39–43). The deregulation of the PPAR{delta}-cyclin E1 signaling axis is likely important in thyroid tumors because (a) both PPAR{delta} and cyclin E1 control thyroid cell proliferation, (b) cyclin E is rate-limiting in the G1-S transition in many cell types (44), (c) deregulated expression of cyclin E induces chromosome instability (45), and (d) the loss of cyclin E in knockout mice confers a resistance to oncogenic transformation (25). Furthermore, the expression of cyclin E is abnormal in some human breast carcinomas (46) and PPAR{delta} ligand has been shown to change the histology and growth rate of breast carcinomas in a mouse tumor model (47). Thus, PPAR{delta}-cyclin E1 signaling may be important in thyroid, breast, and other carcinomas.

We previously identified, in a subset of thyroid follicular carcinomas, a family of mutant PPAR{gamma} gene fusions (20, 21). The early expression (20) and biological activities (21, 48) of the encoded PPAR{gamma} fusion proteins are consistent with an important role in the pathogenesis of these thyroid cancers. Interestingly, the PPAR{gamma} fusion proteins induce proliferation in primary thyroid cells without an apparent increase in cyclin E1.4 In addition, wild-type PPAR{gamma} has little ability to stimulate proliferation in normal thyroid cells, in sharp contrast to PPAR{delta} (Figs. 3 and 4). These observations suggest that PPAR{delta} and PPAR{gamma} exhibit distinct mechanisms in cell proliferation and neoplasia in the thyroid gland, a systemic endocrine regulator of growth and metabolism.

The thyroid has proven to be a valuable model to determine the mechanisms of nuclear receptors and altered signal transduction in epithelial cells and tumors. Here, we have shown that the expression of PPAR{delta} is up-regulated in thyroid cells by serum, TSH, or EGF mitogenic signals. TSH stimulates both proliferation and differentiation in thyroid cells by activation of cyclic AMP and pKa (24). EGF stimulates proliferation and inhibits differentiation in thyroid cells by activation of MEK/ERK (24). MEK/ERK are activated constitutively by mutations in subsets of follicular thyroid adenomas, follicular carcinomas, papillary carcinomas, and anaplastic/poorly differentiated thyroid carcinomas (49–52). In fact, 80% of papillary carcinomas possess RET, NTRK1, BRAF, or RAS mutations that induce MEK/ERK signaling. Our strong correlation between mean PPAR{delta} expression and mean Ki67 expression in these thyroid tumors and normal thyroid cells (R = 1; P = 0.01667) supports a fundamental mechanistic connection between PPAR{delta} and MEK/ERK signaling (53) that requires additional investigation.

Our experiments support a model in which PPAR{delta} induces cyclin E1 and cell proliferation depending on growth factor and lipid ligands in the local environment. Growth factors up-regulate the expression of PPAR{delta} during cell proliferation and PPAR{delta} ligands modulate the proliferation response. This model provides an explanation for the varied effects of PPAR{delta} activation in cell and tumor models—the requisite growth factor and/or ligand signals that produce a robust PPAR{delta} proliferation response may not be present in each case. Our data also argue that new selective antagonists for PPAR{delta} (54) should be investigated for antitumorigenic properties in preclinical and perhaps clinical models. The overall findings indicate that a coordinated interplay between PPAR{delta}, PPAR{gamma}, lipid ligands, and growth factors regulate thyroid cell proliferation and is altered by different PPAR mechanisms in different thyroid carcinomas.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA75425 and the Louis Block Fund of the University of Chicago.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Don Steiner, Graham Bell, and Sam Refetoff for sharing laboratory equipment, and Pablo Michalewicz for technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

3 http://rsb.info.nih.gov/ij/ Back

4 Unpublished data. Back

Received 3/ 6/08. Revised 6/ 2/08. Accepted 6/ 5/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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