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Mol Cancer Ther. 2004;3:1011-1020
© 2004 American Association for Cancer Research

Retinoid X receptor-{gamma} and peroxisome proliferator-activated receptor-{gamma} expression predicts thyroid carcinoma cell response to retinoid and thiazolidinedione treatment

Joshua P. Klopper1, William R. Hays1, Vibha Sharma1, Margaret A. Baumbusch1, Jerome M. Hershman2 and Bryan R. Haugen1

1 Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado and 2 Department of Medicine, West Los Angeles VAMC and University of California School of Medicine, Los Angeles, California

Requests for reprints: Joshua P. Klopper, Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Colorado Cancer Center, University of Colorado Health Sciences Center, Box B151, 4200 East 9th Avenue, Denver, CO 80262. Phone: 303-315-8443; Fax: 303-315-4525. E-mail: joshua.klopper{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Poorly differentiated, metastatic thyroid cancer is difficult to treat. These tumors often do not concentrate radioactive iodine and may require chemotherapy, which is suboptimal and toxic. Nuclear hormone receptors peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and retinoid X receptor (RXR) are variably expressed in thyroid carcinoma cell lines. Expression of these receptors may predict thyroid cancer cell response to treatment with rexinoids and thiazolidinediones. We studied three thyroid carcinoma cell lines: BHP 5-16 (PPAR{gamma}–/RXR{gamma}+), BHP 2-7 (PPAR{gamma}±/RXR{gamma}–), and DRO-90 (RXR{gamma}+/PPAR{gamma}+). BHP 5-16 (RXR{gamma}+) cells treated with rexinoid had decreased proliferation to 69 ± 6% growth compared with vehicle. BHP 2-7 (PPAR{gamma}+) cells treated with thiazolidinedione had no decrease in cellular proliferation. DRO-90 (RXR{gamma}+ and PPAR{gamma}+) cells had 36 ± 10%, 15 ± 3%, and 13 ± 4% growth when treated with rexinoid, thiazolidinedione, or a combination, respectively. We next investigated the role of apoptosis in the ligand-responsive BHP 5-16 and DRO-90 cells. BHP 5-16 cells underwent no significant apoptosis with rexinoid (1 µmol/L). DRO-90 cells, however, had 3.6 ± 1.3% apoptotic cells with vehicle, 13 ± 3.5% with rexinoid (1 µmol/L), 18 ± 4% with thiazolidinedione (1 µmol/L), and 28 ± 6% with combination treatment (1 µmol/L), suggesting that apoptosis plays a major role in this anaplastic cell line and that the effects of the two ligands are additive. We conclude that receptor expression is necessary for inhibition of thyroid carcinoma growth with ligand treatment but may not be sufficient for response. Additionally, expression of both RXR{gamma} and PPAR{gamma} may be necessary for maximal growth inhibition by ligands and may be required for the increased apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most thyroid carcinomas are well differentiated and respond well to surgery, radioactive iodine therapy, and levothyroxine suppression therapy. However, a significant minority of thyroid cancers are poorly differentiated and often metastatic and do not maintain their capacity to concentrate radioactive iodine (1). These tumors lead to the majority of thyroid carcinoma–related deaths. The tumors may be aggressive papillary or follicular thyroid carcinomas or the almost uniformly fatal anaplastic thyroid carcinoma. Although in vitro and in vivo studies, including small human trials, have showed modest efficacy in treating anaplastic thyroid carcinoma with agents such as paclitaxel, there is by no means a cure for this deadly disease (2, 3). A novel approach to thyroid cancer therapy, based on features at a molecular as opposed to a histologic level, may help direct novel therapies to patients who would most benefit.

Retinoids are vitamin A derivatives that activate retinoic acid receptors and retinoid X receptors (RXR; ref. 4). Retinoids have been shown to inhibit cellular growth and to induce redifferentiation in some poorly differentiated thyroid cancer cell lines (5–8). Retinoid receptors have six separate isotypes encoded on six separate genes (RAR{alpha}, RARß, RAR{gamma}, RXR{alpha}, RXRß, and RXR{gamma}; ref. 9). We have shown that the RXR{gamma} isotype is expressed in a subset of thyroid carcinoma tissue and cell lines but is not expressed in normal thyroid tissue. We have also showed that expression of this receptor predicts growth suppression by treatment with retinoids (10).

Another class of nuclear hormone receptors is the peroxisome proliferator-activated receptors (PPAR). Similar to the RXRs, three isotypes exist (PPAR{alpha}, PPARß, and PPAR{gamma}; ref. 11). PPAR{gamma} is most well known as a regulator of adipocyte differentiation and the target for the thiazolidinedione class of drugs used for the treatment of insulin resistance in type II diabetes mellitus (12). PPAR{gamma} acts via formation of a heterodimer with RXR. This heterodimer complex interacts with peroxisome proliferator response elements and regulates the expression of target genes (13–15). Some studies suggest that the combination of thiazolidinedione and RXR selective retinoids may redifferentiate tumors by a synergistic or additive mechanism (16, 17).

We hypothesize that the nuclear hormone receptor complement of RXR{gamma} and PPAR{gamma} in thyroid carcinoma cell lines will predict response to receptor-specific ligand treatment. Furthermore, given the requirement for PPAR{gamma} to form a heterodimer with RXR, activation of both receptors should lead to an additive or synergistic response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Cells were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) with 2% fetal bovine serum (FBS; Hyclone, Logan, UT). LG 346 and LG 1069 were provided by Ligand Pharmaceuticals (San Diego, CA). Pioglitazone was provided by Takeda Pharmaceuticals (Lincolnshire, IL). Rosiglitazone was provided by GlaxoSmithKline Pharmaceuticals (Philadelphia, PA).

Cell Lines
BHP 5-16 and BHP 2-7 are human papillary thyroid carcinoma cell lines (10). DRO-90 human anaplastic thyroid carcinoma cell lines were kindly provided by Dr. G.J. Juillard (University of California-Los Angeles, Los Angeles, CA).

Reverse Transcription-PCR
Qualitative reverse transcription-PCR (RT-PCR) was done by generating sense strand RNA for PPAR{gamma} and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Oligonucleotides to the corresponding human ligand binding domain were synthesized by Invitrogen Life Technologies (Carlsbad, CA): for PPAR{gamma}, sense 5'-TCTGGCCCACCAACTTTGGG-3' and antisense 5'-CTTCACAAGCATGAACTCCA-3'; for GAPDH, sense 5'-CTTTGGTATCGTGGAAGGAC-3' and antisense 5'-GAAATGAGCTTGACAAAGTG-3'. Total RNA was obtained from our experimental cell lines (TriReagent, Sigma Chemical Co., St. Louis, MO), and 5 µg were reverse transcribed using random hexamers and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). The reverse transcription procedure involved incubation times and temperatures as follows: 10 minutes at room temperature, 45 minutes at 42°C, and 5 minutes at 90°C. The reverse transcription product was divided into two PCR reactions (PPAR{gamma} and GAPDH). The PCR reaction used 98°C for 5-minute denaturation temperature and 1-minute annealing temperature and 72°C for 1-minute elongation temperature. The reaction was carried out over 35 cycles and required 50°C annealing temperature for PPAR{gamma} and 60°C annealing temperature for GAPDH.

Quantitative RT-PCR was done for RXR{gamma} as described previously (10). Quantitative RT-PCR was done for PPAR{gamma} using RNA extracted from DRO-90 cell lines and followed the same methods as described previously (10). Primers and probes are available on request.

Western Blot Analysis
Protein was isolated from nuclear extracts using a commercial nuclear protein extraction kit (Active Motif, Carlsbad, CA). The protein content of lysates was measured using a commercial DC protein assay kit (Bio-Rad, Hercules, CA). Diluted samples containing equal amounts of protein were mixed with 2x Laemmli sample buffer (Bio-Rad, catalogue no. 161-0737). Proteins were separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 1x TBST [20 mmol/L Tris-HCl (pH 7.6), 8.5% NaCl, 0.1% Tween 20] containing 5% nonfat dry milk at room temperature for 2 hours and incubated in the appropriate primary antibody in 1x TBST containing 5% nonfat dry milk at 4°C overnight. RXR{gamma} (Y-20) primary antibody and PPAR{gamma} (E-8, catalogue no. 7273) monoclonal antibody were obtained (Santa Cruz Biotechnology, Santa Cruz, CA). We used 6.6 µg (1:500 dilution) of RXR{gamma} and 0.13 µg (1:1,500 dilution) of PPAR{gamma} primary antibody for overnight incubation. After washing, membranes were incubated for 1 hour at room temperature with antirabbit IgG conjugated to peroxidase at a 1:5,000 dilution for RXR{gamma} and antimouse IgG conjugated to peroxidase at 1:5,000 dilution for PPAR{gamma}. The enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ) was used for immunodectection.

Immunoprecipitation
Cells (~12–13 million) were used for each immunoprecipitation experiment. The cells were washed in cold PBS-EDTA, and solubilization buffer (1 mL) was added [buffer contents: 55 mmol/L TEA (pH 7.5), 111 mmol/L NaCl, 2.2 mmol/L EDTA, 0.44% SDS]. The solution was passed through a 20 G syringe five times, frozen at –80°C for 3 hours, and thawed on ice. Triton X-100 (10%, 80 µL) was added followed by protein A/G plus agarose beads (80 µL, sc-2003, Santa Cruz Biotechnology). The mixture was shaken at 4°C for 8 hours. The suspension was spun for 2 minutes at 10,000 rpm to pellet the beads, and the supernatant was removed. PPAR{gamma} (5 µL or 200 µg/mL, H-100) rabbit polyclonal antibody (sc-7196, Santa Cruz Biotechnology) was added followed by protein A/G plus agarose beads (80 µL). This was again shaken at 4°C for 8 hours and spun to remove the supernatant. This mixture was washed twice with a wash buffer [50 mmol/L TEA (pH 7.5), 100 mmol/L NaCl, 2 mmol/L EDTA, 0.1% SDS, 0.5% Triton X-100] and washed twice with 10 mmol/L TEA (pH 7.5). The bound antigen was eluted by adding the Western blot 1x sample buffer, heated for 5 minutes at 100°C, and loaded onto a SDS-PAGE gel for Western blot analysis. The methods are the same as above, except for the PPAR{gamma} antibody concentration at 1:500.

Cell Growth and Proliferation
Cells were grown to ~80% confluence in 100 mm tissue culture plates. Cells were harvested using trypsin-EDTA (Invitrogen) and counted using a hemocytometer. Cells were transferred to a 96-well plate at a concentration of 500 cells per 200 µL of medium. Each row of eight wells received the same cell type and subsequently the same drug. After cells were allowed to plate down overnight, the medium was aspirated and medium with the appropriate concentration of ligand or equivalent volume of vehicle was added to each well. Fresh medium with vehicle or ligand was added every 72 hours. At the completion of each time point (3, 6, or 9 days), cell proliferation was assessed following the manufacturer's instructions using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Following a 2-hour incubation at 37°C, each plate was analyzed by a MRX Microplate Reader (Dynatech Laboratories, Chantilly, VA) using Revelation software. Cell proliferation is assessed by determining the absorbance of a 490 nm wavelength of light that is altered by a colorimetric change in solution based on the number of metabolically active and proliferating cells.

Apoptosis
BHP 5-16 and DRO-90 cells were grown to ~80% confluence in 100 mm tissue culture plates. Cells were harvested and counted as described above and transferred to a six-well tissue culture plate at a concentration of 50,000 cells per 3 mL medium for the day 3 plates and 25,000 cells per 3 mL medium for the day 6 plates. After allowing to plate down overnight, the medium was aspirated and fresh medium with the appropriate concentration of ligand or equivalent volume of vehicle was added to the wells. At days 3 and 6, all cells were collected and analyzed by flow cytomery using the Vybrant Apoptosis Assay Kit 2 (Molecular Probes, Eugene, OR) following the manufacturer's instructions.

DR1-Luciferase Transient Transfection
The DRO-90, BHP 2-7, and BHP 5-16 cells were cultured to 80% to 90% confluency (0.8 x 106 to 0.9 x 106 cells) for transfection experiments in a six-well plate. For each well, DR1-TK-luciferase (1 µg), TK-luciferase plasmid (3 µg), and LipofectAMINE 2000 reagent (8 µL; Invitrogen) were used as per the manufacturer's instructions. Each transfection also contained Renilla luciferase plasmid (10 ng; Promega) as an internal transfection control. A Rous sarcoma virus promoter luciferase plasmid and an empty TK-luciferase plasmid were transfected in parallel as positive and negative controls, respectively. DNA and the LipofectAMINE reagent were diluted separately in serum-free medium (200 µL) without antibiotics, mixed together, and incubated at room temperature for 30 minutes. The culture plates were washed with PBS and medium was added (1,600 µL). Plasmid LipofectAMINE mixture (400 µL) was added to each well, and the plates were incubated at 37°C with and without pioglitazone. Cells were harvested after 48 hours of incubation at 37°C, subjected to freeze thaw extraction, and assayed for dual firefly and Renilla luciferase activity. Luciferase activity was measured in a Monolight 3010 luminometer using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase light units were normalized to Renilla luciferase activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PPAR{gamma} in Thyroid Carcinoma Cell Lines
Amplification by RT-PCR yielded a DNA fragment of predicted size and revealed PPAR{gamma} mRNA in two cell lines: DRO-90 and BHP 2-7 (Fig. 1A). Human GAPDH was used as a positive loading control. Quantitative RT-PCR analysis on two separate samples showed relatively high expression of PPAR{gamma} mRNA in DRO-90 and BHP 2-7 (47.8 and 21.0 fg/ng rRNA, respectively). BHP 5-16, however, did not express PPAR{gamma} and supported the findings of the qualitative RT-PCR experiment (Fig. 1B). To confirm protein expression of PPAR{gamma} in these cell lines, Western blot analysis was done on nuclear protein extracts. Figure 2A shows that PPAR{gamma} protein is detectable in all three cell lines despite lack of detectable RNA in BHP 5-16 cells. The RT-PCR analysis additionally confirmed that the well-described PPAR{gamma}/PAX8 rearrangement was not present. When corrected for ß-actin protein expression, relative levels of PPAR{gamma} were higher in DRO-90 cells compared with BHP 2-7 and BHP 5-16 cells (Fig. 2B).



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  		Figure 1. RT-PCR and quantitative RT-PCR analysis of PPAR{gamma} isotypes in thyroid carcinoma cell lines. A, RT-PCR. Total RNA (5 µg) was used from each of the three thyroid carcinoma cell lines (DRO-90, BHP 5-16, and BHP 2-7), reverse transcribed using random primers, and amplified by PCR using specific primers for PPAR{gamma} and GAPDH. The PCR products were resolved by electrophoresis and stained with ethidium bromide. B, quantitative RT-PCR. Total RNA (1 µg) was used for the PPAR{gamma} RT-PCR analysis (ABI PRISM 7700, Perkin-Elmer, Boston, MA), and absolute values were derived from a standard curve using a known amount of sense strand RNA (femtograms of sense strand RNA).

 


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  		Figure 2. Western blot analysis of PPAR{gamma} in thyroid cancer cell lines using a monoclonal antibody. A, Western blot. Nuclear protein extract (60 µg) from three thyroid cancer cell lines was size separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with 1:1,500 dilution of a mouse monoclonal antibody against human PPAR{gamma} (COOH terminus, Santa Cruz Biotechnology). A 1:5,000 dilution of secondary antibody was used. ß-actin was measured as a loading control. B, densitometry analysis. Densitometry was done on the Western blot in A,. density levels of PPAR{gamma} are corrected for ß-actin loading control.

 
Expression of RXR{gamma} in Thyroid Carcinoma Cell Lines
Quantitative RT-PCR was done using sense strand RNA corresponding to the ligand binding domain of the RXR{gamma} receptor as a standard (10). Total RNA (1 µg) from each cell line was analyzed for RXR{gamma} mRNA expression. BHP 5-16 and DRO-90 cell lines had high expression of RXR{gamma} mRNA (1,120 ± 112 and 890 ± 117 ag/ng rRNA, respectively). BHP 2-7 had no detectable levels of RXR{gamma} mRNA (Fig. 3).



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  		Figure 3. Quantitative RT-PCR analysis of RXR{gamma} isotype in thyroid carcinoma cell lines. Total RNA (1 µg) was used for the RXR{gamma} RT-PCR analysis (ABI PRISM 7700, Perkin-Elmer), and absolute values were derived from a standard curve using a known amount of sense strand RNA [attograms (attg) of sense strand RNA]. Isoform RNA was normalized to total input RNA (18S rRNA measured from 1 ng total RNA).

 
To confirm the expression of RXR{gamma} protein in our three thyroid cancer cell lines, Western blot analysis was done on nuclear extract protein. BHP 5-16 and DRO-90 cell lines showed clear expression of RXR{gamma} protein, whereas the BHP 2-7 cell line lacked detectable RXR{gamma} protein (Fig. 4).



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  		Figure 4. Western blot analysis of RXR{gamma} isotype in thyroid carcinoma cell lines. Nuclear extract protein (100 µg) was size separated on a 10% SDS-PAGE gel. RXR{gamma}+ control is RXR{gamma} cDNA in a PCGN2 plasmid construct transfected into CV-1 cells. The nitrocellulose membrane was incubated with RXR{gamma} primary antibody (Santa Cruz Biotechnology) or ß-actin (loading control) and developed using a horseradish peroxidase secondary antibody system.

 
Cell Line Proliferation in Response to Ligand Treatment
Cells were grown in the presence of vehicle or ligand for 9 days. BHP 5-16 (PPAR{gamma}±/RXR{gamma}+) cellular proliferation was decreased in the presence of LG 346 (1 µmol/L) and combination of LG 346-pioglitazone (1 µmol/L; Fig. 5A). There was no suppression of growth in the presence of pioglitazone (1 µmol/L) alone. Proliferation in the presence of LG 346 (1 µmol/L) was 69 ± 6% of growth compared with cells grown in vehicle. This was similar with the combination of LG 346-pioglitazone with proliferation of 78 ± 9% compared with cells grown in vehicle.



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  		Figure 5. Cell proliferation response to vehicle versus ligand. A, treatment of BHP 5-16 (RXR{gamma}+, PPAR{gamma}±) cells with vehicle versus ligand. Cells were grown in 2% FBS-RPMI in the presence of 1 µmol/L LG 346, pioglitazone (PIO), or LG 346-pioglitazone combination (500 nmol/L of each) or vehicle (DMSO) for 3, 6, and 9 days. At each time point, cells were harvested and analyzed using a nonradioactive cell proliferation assay. Cell proliferation is represented by absorbance at 490 nm. Points, average of seven experiments for LG 346 with eight data replicants in each experiment and three experiments for pioglitazone and LG 346-pioglitazone combination with eight data replicants in each experiment; bars, SE. B, treatment of BHP 2-7 (RXR{gamma}–, PPAR{gamma}±) cells with vehicle versus ligand. Cells were grown in 2% FBS-RPMI in the presence of 1 µmol/L LG 346, pioglitazone (PIO), or LG 346-pioglitazone combination (500 nmol/L of each) or vehicle (DMSO) for 3, 6, and 9 days. At each time point, cells were harvested and analyzed using a nonradioactive cell proliferation assay. Cell proliferation is represented by absorbance at 490 nm. Points, average of six experiments for LG 346 with eight data replicants in each experiment and three experiments for pioglitazone and LG 346-pioglitazone combination with eight data replicants in each experiment; bars, SE. C, treatment of DRO-90 (RXR{gamma}, PPAR{gamma}+) cells with vehicle versus ligand. Cells were grown in 2% FBS-RPMI in the presence of 1 µmol/L LG 346, pioglitazone (PIO), or LG 346-pioglitazone combination (500 nmol/L of each) or vehicle (DMSO) for 3, 6, and 9 days. At each time point, cells were harvested and analyzed using a nonradioactive cell proliferation assay. Cell proliferation is represented by absorbance at 490 nm. Points, average of eight experiments for LG 346 with eight data replicants in each experiment and four experiments for pioglitazone and LG 346-pioglitazone combination with eight data replicants in each experiment; bars, SE.

 
BHP 2-7 (PPAR{gamma}±/RXR{gamma}) proliferation did not seem affected by any ligand treatment, including PPAR{gamma}-specific treatment with thiazolidinedione (Fig. 5B).

DRO-90 (PPAR{gamma}+/RXR{gamma}+) proliferation was significantly decreased in the presence of LG 346 (1 µmol/L) and pioglitazone (1 µmol/L). Additionally, final concentration combination of LG 346-pioglitazone (1 µmol/L) provided the greatest suppression of proliferation at 13 ± 4% compared with cells grown in vehicle (Fig. 5C). Morphologically, the DRO-90 cells began to change after 72 hours of treatment (Fig. 6).



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  		Figure 6. DRO-90 cells in culture. Cells were grown in 100 mm tissue culture plates in 2% FBS-RPMI for 72 hours in the presence of vehicle (DMSO; A) or LG 346-pioglitazone combination treatment (1 µmol/L; B). A, typical polygonal, flat cell morphology. B, cells become "balled up" with drug treatment. This morphology is seen prior to the cells lifting from the plate surface.

 
Given the marked suppression of DRO-90 cell proliferation, we did a dose titration of ligands alone and in combination to assess the lowest concentration of drug that would suppress growth. Additionally, we used a different rexinoid, LG 1069, to confirm that our response to RXR ligands was a class effect of the drug. LG 1069 (50 nmol/L) caused a 77 ± 4% decreased cellular proliferation at day 9. Pioglitazone (50 nmol/L) caused an 87 ± 4% decreased cellular proliferation at day 9. Even combination doses (25 nmol/L) of each ligand (50 nmol/L total) provided 94 ± 2% suppression of cellular proliferation (Fig. 7).



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  		Figure 7. Dose titration of DRO-90 (RXR{gamma}+, PPAR{gamma}+) cells with vehicle versus ligand. Cells were grown in 2% FBS-RPMI in the presence of stepwise increases of LG 1069, pioglitazone (PIO), and LG-pioglitazone combination treatment from 1 to 100 nmol/L for 6 and 9 days. At each time point, cells were harvested and analyzed using a nonradioactive cell proliferation assay. Cell proliferation is represented by absorbance at 490 nm. Columns, average of two experiments with eight data replicants in each experiment; bars, SE.

 
The above experiments were all repeated replacing pioglitazone with rosiglitazone, and the results were similar including the dose titration experiment (data not shown).

Functional Analysis of PPAR{gamma} in Thyroid Cancer Cell Lines
Expression of RXR{gamma} mRNA and protein correlated well with functional suppression of growth in the thyroid cancer cell lines treated with rexinoids. PPAR{gamma} mRNA expression did not directly correlate with protein expression or functional growth inhibition by thiazolidinedione. To further explore this apparent discrepancy, whole cell protein extracts were subjected to immunoprecipitation (polyclonal PPAR{gamma} antibody) for the three thyroid cancer cell lines. Figure 8 shows that only the DRO-90 cells have detectable PPAR{gamma} protein using this technique. These data correlate with the suppression of growth by thiazolidinedione in Fig. 5. To determine if these cell lines contain functional PPAR{gamma} receptors, we did transient transfection using the DR1-TK-luciferase peroxisome proliferator response element reporter (gift from B. Forman, Department of Molecular Medicine, The City of Hope National Medical Center, Duarte, CA). Figure 9 shows that DR1-mediated luciferase activity was stimulated with thiazolidinedione only in DRO-90 cells (Fig. 9A), suggesting that only these cells have functional PPAR{gamma}. Figure 9D shows that this effect in DRO-90 cells is dose dependent.



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  		Figure 8. PPAR{gamma} immunoprecipitation. Immunoprecipitation used ~12–13 million cells each of DRO-90, BHP 2-7, and BHP 5-16. Whole cell extract was incubated with a polyclonal antibody against PPAR{gamma}, and the resultant protein was size separated on a 10% SDS-PAGE gel. The nitrocellulose membrane was incubated with a mouse monoclonal PPAR{gamma} primary antibody (Santa Cruz Biotechnology) and developed using a horseradish peroxidase secondary antibody system. Ab/no lysate, negative control.

 


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  		Figure 9. DR1-luciferase transient transfection. DRO-90 (A), BHP 5-16 (B), and BHP 2-7 (C) cells were cultured to 80–90% confluency (0.8 x 106 to 0.9 x 106 cells) for transfection experiments to assess the functional activity of PPAR{gamma} in response to pioglitazone (PIO). Luciferase activity was measured in a Monolight 3010 luminometer using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase light units were normalized to Renilla luciferase activity. D, dose response to pioglitazone (0.1 and 1 µmol/L) was assessed in the responsive DRO-90 cell line.

 
Measurement of Apoptosis in BHP 5-16 and DRO-90 Cells
To investigate the mechanisms of decreased cell proliferation, we measured rates of apoptosis in BHP 5-16 and DRO-90 cells treated with rexinoid. BHP 5-16 cells did not have a significant percentage of cells (only 1.7 ± 0.4%) that undergo apoptosis with LG 346 (1 µmol/L) at 6 days despite 23 ± 2% suppression of cellular proliferation at day 6 with the same treatment conditions. In contrast, DRO-90 cells had 13 ± 3.5% of cells that undergo apoptosis with LG 346 (1 µmol/L) at day 6 (Fig. 10).



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  		Figure 10. Measurement of BHP 5-16 (RXR{gamma}+, PPAR{gamma}±) versus DRO-90 (RXR{gamma}+, PPAR{gamma}+) cell apoptosis. Cells were grown in 2% FBS-RPMI in the presence of LG 346 (1 µmol/L) or DMSO for 6 days. Apoptosis was analyzed by flow cytometry using the Vybrant Apoptosis Assay. Apoptosis is indicated by % Annexin-positive cells.

 
Given the strong suppression of cellular proliferation in DRO-90 cells with all ligands used, we studied the apoptotic effects of RXR and PPAR{gamma} ligands alone and in combination (Fig. 11). As shown, the percentage of cells undergoing apoptosis was significantly higher after 6 days of treatment with either ligand alone (13 ± 3.5% for LG 346 and 18 ± 3.8% for pioglitazone) as compared with treatment with vehicle (3.6 ± 1.3%). Additionally, 0.05 µmol/L of each ligand together (0.1 µmol/L combination) generated more apoptotic cells (24 ± 5.4%) than 1 µmol/L of either ligand alone, suggesting a synergistic effect of these two ligands on apoptosis. We confirmed apoptosis in DRO-90 cells using poly(ADP-ribose) polymerase (PARP) cleavage. Although rexinoid alone did not show the appropriate PARP cleavage band on Western blot analysis, treatment with thiazolidinedione and combination thiazolidinedione-rexinoid yielded the expected PARP cleavage product (data not shown).



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  		Figure 11. Measurement of DRO-90 (RXR{gamma}+, PPAR{gamma}+) cell apoptosis. Cells were grown in 2% FBS-RPMI in the presence of 1 µmol/L LG 346, 1 µmol/L pioglitazone, 0.1 µmol/L LG 346-pioglitazone combination (50 nmol/L of each), 1 µmol/L LG 346-pioglitazone combination (500 nmol/L of each), or DMSO for 3 and 6 days. Apoptosis was analyzed by flow cytometry using the Vybrant Apoptosis Assay. Apoptosis is indicated by % Annexin-positive cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have showed that thyroid carcinoma cell line expression of RXR{gamma} and/or PPAR{gamma} seems to be necessary for response to receptor-specific ligands. While necessary, however, expression of PPAR{gamma} alone does not seem to be sufficient to provide decreased cellular proliferation in response to a thiazolidinedione. Furthermore, apoptosis seems to play a limited role in the mechanism of decreased cellular growth in response to a rexinoid ligand; however, the level of apoptosis is synergistically enhanced when a cell line expressing both RXR{gamma} and PPAR{gamma} receptors is treated with a combination of ligands.

The anticancer activity of vitamin A and its derivatives has been studied since the 1960s. The initial work was conducted on rat models of lung carcinoma (5). The prototype for retinoid-based therapies comes from the experience with using all-trans retinoic acid in combination with chemotherapy for the treatment of acute promyelocytic leukemia. This treatment combination has revolutionized the treatment of acute promyelocytic leukemia and is one of the few success stories in cancer therapy, wherein this treatment effectively eradicates acute promyelocytic leukemia (4). Retinoids have also been shown to induce growth arrest of MCF-7 breast cancer cell lines in vitro, and the RXR selective ligand LG 1069 has prevented the development of estrogen receptor–negative mammary tumors in an in vivo transgenic mouse model (6, 18). Additionally, LG 1069 is Food and Drug Administration approved (bexarotene, Targretin) for the treatment of T-cell lymphoma.

The effects of retinoids have also been studied with thyroid carcinoma cell lines. Van Herle et al. (19) first noted a significant and dose-dependent reduction in follicular carcinoma cell number and [3H]thymidine uptake in the presence of 13-cis-retinoic acid (10 µmol/L). Additionally, Schmutzler et al. (7) investigated the ability of all-trans retinoic acid to induce redifferentiation in human thyroid carcinoma cell lines (including follicular and anaplastic carcinoma cell lines) by increasing type I 5' deiodinase activity. Follicular thyroid carcinoma cell lines showed an increased activity, whereas anaplastic cell lines did not. The first report of retinoid therapy in a patient with advanced thyroid cancer was the use of isotretinoin (13-cis-retinoic acid, Accutane) in a patient with follicular thyroid cancer and lung metastases (20). The therapy caused symptomatic improvement in the patient's shortness of breath, decreased serum thyroglobulin level, and decreased tumor metabolism (18FDG positron emission tomography). In a small clinical trial conducted by Simon et al. (8), 20 patients with advanced thyroid carcinoma (excluding anaplastic thyroid carcinoma) were treated with 13-cis-retinoic acid for at least 5 weeks. The investigators studied markers of redifferentiation including thyroglobulin level, radioactive iodine uptake, and tumor size by standard imaging. A response in one marker was seen in ~65% of patients, whereas two or more markers indicated redifferentiation in ~40% of patients. Boerner et al. (21) studied 23 patients with advanced, progressive, metastatic follicular cell–derived thyroid cancer in what has been the longest clinical trial to date. Patients were treated with 13-cis-retinoic acid (0.3–1 mg/kg/d) for up to 9 months. Tumor glucose uptake decreased while on therapy and went back up at the cessation of therapy. Twenty-eight percent of patients had new or increased I-131 uptake in tumors. Unfortunately, there was no control group in this study and only two patients had a clinically significant response (of 21 evaluated and with I-131 therapy used in conjunction to isotretinoin). Finally, an initial clinical trial of LGD1550 (a synthetic retinoic acid receptor agonist) included seven patients with advanced thyroid cancer, in whom four had stable disease for at least 20 weeks (and up to 56 weeks) and one who had a transient decrease in serum thyroglobulin level (22).

In the current study, we specifically investigated the RXR{gamma} receptor subtype of the retinoid/rexinoid superfamily. We observed an ~30% decrease in cellular proliferation with RXR{gamma} selective ligands used alone in RXR{gamma}+ cell lines. Based on the proliferation curves, cellular growth was slowed but not stopped entirely.

The PPARs are members of the nuclear hormone receptor superfamily. The three isotypes of PPAR ({alpha}, ß and {gamma}) show distinct tissue distribution, with PPAR{gamma} being highly expressed in adipose tissue and macrophages (11). Like other nuclear hormone receptors, PPAR{gamma} contains a DNA binding domain and COOH-terminal ligand binding domain that mediates dimerization and transactivation functions (23). There are many examples of PPAR{gamma} activation promoting differentiation, most notably as the "master regulator" of adipocyte differentiation (13). The cell differentiation extends to numerous cell lines, however, including hepatocytes, fibroblasts, breast cells, and myocytes (14). The ligands for PPAR{gamma} include naturally occurring prostaglandins and most notably the thiazolidinedione class of antidiabetic drugs (12). Although PPAR{gamma} activation clearly leads to adipocyte differentiation, numerous in vitro studies have showed thiazolidinedione-induced differentiation in nonadipocyte cell lines (15).

Ligand activation of PPAR{gamma} has been studied in human prostate cancer cell lines via in vitro and in vivo studies. Segawa et al. showed that the PPAR{gamma} receptor mRNA was specifically seen in the prostate cancer cells, prostatic intraepithelial neoplasia, and was faintly seen in cells showing evidence of benign prostatic hypertrophy. Interestingly, PPAR{alpha} and PPARß mRNA was detected in all cell lines including normal prostate tissue (24). The authors concluded that PPAR{gamma} expression is induced in prostate cancer, and its presence may be a potential target for redifferentiation therapy. A trial of 41 men with histologically confirmed prostate cancer and no symptomatic metastases were treated p.o. with troglitazone (a thiazolidinedione and PPAR{gamma} activator), and a significant proportion of subjects had stabilization of their prostate-specific antigen (25). Additionally, thiazolidinedione ligands have been shown to induce apoptosis in hepatocellular cell lines and colon cancer cell lines in vitro (26, 27).

The effects of liganded PPAR{gamma} receptors on thyroid carcinoma cell lines have been studied as well. Martelli et al. (28) studied numerous human thyroid carcinoma cell lines with regard to PPAR{gamma} expression. In this study, RNA extracted from a normal thyroid cell line did have low-level expression of PPAR{gamma} by RT-PCR analysis. Five of six carcinoma cell lines expressed PPAR{gamma} and had decreased cellular growth at 48 hours, increased G1 cell cycle arrest, and increased apoptosis when treated with ciglitazone (a thiazolidinedione). Interestingly, when PPAR{gamma} was overexpressed in the PPAR{gamma}-negative cell line, its growth kinetics slowed and apoptosis increased even without liganded treatment. Ciglitazone caused marked decrease in tumor cell colony growth. Ohta et al. (29) used troglitazone to study its effects on papillary carcinoma cells lines that differentially expressed PPAR{gamma} receptors. Troglitazone (10 µmol/L) caused a significant decrease in cell number in culture in PPAR{gamma}-positive papillary carcinomas compared with those that were PPAR{gamma} negative at 72 hours. This work was carried to a nude mouse model, wherein 500 mg/kg/d of troglitazone given over 5 days/wk for 7 weeks visibly inhibited the growth of implanted tumor cells to a significant degree. These investigators also showed that the BHP 2-7 cells, which express PPAR{gamma} mRNA, showed significant growth inhibition with 10 µmol/L pioglitazone. We did not observe this effect in these cells using 1 or 10 µmol/L pioglitazone. While we confirmed that the BHP 2-7 cells express PPAR{gamma} mRNA, the level of protein expression was relatively low (Fig. 2B) and these cells did not have functional PPAR{gamma} (Fig. 9C). Additionally, BHP 2-7 is RXR{gamma} negative; therefore, heterodimerization of RXR{gamma} and PPAR{gamma} cannot occur in this cell line.

RXRs and PPAR{gamma} preferentially form heterodimers. The exploitation of this PPAR/RXR heterodimer has occurred in breast cancer research. Rubin et al. showed that a combination of LG10135 (a RXR ligand) and troglitazone inhibited breast aromatase activity (which activates estrogen biosynthesis in the breast and contributes to breast carcinogenesis). The combination showed a greater effect than either ligand alone (both of which decreased aromatase activity; ref. 30). Elstner et al. (31) showed that a thiazolidinedione-rexinoid combination therapy for inhibition of breast cancer cell growth was seen only with those cells that were bcl-2 protein positive. This study shows that a nuclear hormone receptor complement alone does not completely predict a cellular response to liganded treatment and that a variety of other factors, coactivators and/or corepressors, may influence cellular response.

We have identified a thyroid carcinoma cell line, DRO-90, that expresses RXR{gamma} and PPAR{gamma} mRNA and protein. Interestingly, this cell line showed a more substantial decrease in cellular proliferation with either RXR{gamma} or PPAR{gamma} ligands than the other cell lines that only expressed one nuclear hormone receptor type. We believe this shows an accentuated response due to the heterodimerization of receptors even with only one receptor-specific ligand. More impressively, however, was the effect of treatment with both thiazolidinediones and rexinoids. The decrease in cellular proliferation was at least additive as a combination of each ligand at 50% of the dose of either ligand alone had a greater reduction in DRO-90 cellular proliferation. Furthermore, expression of both receptors was associated with increased apoptosis in response to each ligand. We used PARP cleavage to verify apoptosis and did not find the expected band on Western blot analysis for cells treated with rexinoid alone. This may represent that the amount of apoptosis initiated by the rexinoid ligand did reach the threshold for detection required to be seen using the PARP antibody. Another possibility is that PARP cleavage detects apoptosis associated with the caspase enzyme system (primarily caspase-3) and that rexinoid treatment induces apoptosis through different caspase enzymes or different mechanisms. DRO-90 cells had a much greater percentage of cells in apoptosis after treatment with either ligand alone when compared with the BHP 5-16 (RXR{gamma}+) cells that had a minimal response when treated with a rexinoid. Combination therapy seemed to have a synergistic effect as a 10-fold smaller concentration of ligands together pushed more cells into apoptosis at 6 days than either ligand alone.

To our knowledge, this is the first published set of experiments on human thyroid carcinoma cell lines using a molecularly based rationale with combination treatment of two different nuclear hormone receptor ligands. The lack of cellular response to ligands without its appropriate receptor lends support to the fact that this is a receptor driven, ligand-activated effect and not a drug effect through a secondary pathway. However, it is important to note that other factors such as coactivator/corepressor activity, DNA methylation, and histone acetylation all probably play a role in liganded effect (32, 33). These effects and pathways will still need to be investigated further.

In summary, expression of RXR{gamma} and/or PPAR{gamma} seems to predict response to ligand treatment. However, it seems that, for PPAR{gamma} activation and effect to occur, it must happen in the setting of a heterodimer coupling with RXR{gamma}. Finally, coactivation of a RXR{gamma}-PPAR{gamma} heterodimer complement lends an additive effect to cause decreased cellular proliferation in vitro, and a large part of this effect is the result of cellular apoptosis.


   Acknowledgments
 
We gratefully acknowledge the use of the Gene Expression Core Facility and Flow Cytometry Core Facility of the University of Colorado Cancer Center.


   Footnotes
 
Grant support: NIH grant DK54383, research grant from Takeda Pharmaceuticals, and (in part) by National Cancer Institute grant #R25 CA49981.

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.

Received 3/ 3/04; revised 5/24/04; accepted 6/17/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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