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In vivo and Microarray Analysis of Rexinoid-Responsive Anaplastic Thyroid Carcinoma

Authors' Affiliations: ¹ Department of Medicine, Division of Endocrinology, Metabolism, and Diabetes, and ² Department of Pathology, ³ University of Colorado Cancer Center, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado; and ⁴ Department of Molecular Oncology, Ligand Pharmaceuticals, San Diego, California

Requests for reprints: Joshua P. Klopper, Department of Medicine, University of Colorado at Denver and Health Sciences Center, MS 8106, P.O. Box 6511, Aurora, CO 80045. Phone: 303-724-1454; Fax: 303-724-3920; E-mail: joshua.klopper{at}uchsc.edu.

	Abstract

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Purpose: Anaplastic thyroid carcinoma is rare, yet lethal despite aggressive therapy. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid X receptor–selective agonist, as a novel treatment. In this report, we describe the efficacy of LGD1069 in anaplastic thyroid carcinoma in vitro and assess the in vivo treatment effects on a responsive cancer. Additionally, we explore potential mediators of the rexinoid effect on a responsive anaplastic thyroid cancer using comparative microarray analysis.

Experimental Design: Anaplastic thyroid cancer cell lines DRO, ARO, and FRO were treated with LGD1069 in vitro. Responsive DRO xenograft tumors were treated with control chow or chow containing a low dose (30 mg/kg/d) or a high dose (100 mg/kg/d) of LGD1069. Comparative microarray analysis of DRO cells treated with LGD1069 compared with volume-equivalent control was assessed after 24 h of treatment to evaluate early gene expression changes.

Results: DRO xenograft tumor growth was inhibited by LGD1069 treatment in a dose-dependent manner. Comparative microarray analysis showed that 80 genes had a significant increase in expression and 29 genes had a decrease in expression after 24 h of treatment with LGD1069. Expression of angiopoietin-like 4 (ANGPTL4) mRNA was increased 6.5-fold. A trend towards an increase in ANGPTL4 mRNA (not statistically significant) was seen in treated tumors in vivo and this correlated with decreased tumor vascularity and increased necrosis.

Conclusions: LGD1069 therapy decreases proliferation in an anaplastic thyroid cancer cell line that expresses retinoid X receptor-, and this effect is confirmed with decreased tumor size in vivo in a nude mouse model. ANGPTL4 is increased in DRO in response to LGD1069 and may be a potential mediator of the effects of rexinoid treatment.

We have previously shown that the anaplastic thyroid carcinoma cell line DRO expresses the nuclear hormone, receptor retinoid X receptor (RXR)- (6), which predict the effects of RXR-selective ligands (rexinoids). Rexinoids, distinguished from retinoids that bind retinoic acid receptors (RAR) or both RAR and RXR receptors in a less selective manner (e.g., 9-cis retinoic acid), specifically activate RXR receptors leading to an alteration in transcription (7). We have previously shown that the rexinoid, LGD1069 (bexarotene, Targretin), significantly inhibits DRO proliferation in vitro, in part, through increased apoptosis (6, 8).

In this report, we describe the efficacy of LGD1069 therapy for anaplastic thyroid cancer in vitro and in vivo in a nude mouse model. In order to explore the mechanisms by which the rexinoid LGD1069 alters thyroid cancer proliferation in responsive cells, we used microarray gene expression analysis. Of the genes that showed significant change of expression with rexinoid treatment, angiopoietin-like 4 (ANGPTL4) had the largest fold change increase in vitro and this was correlated with observations of responsive tumors.

	Materials and Methods

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Chemicals. All thyroid cancer cells were grown in RPMI 1640 (Invitrogen Corporation) supplemented with 2% fetal bovine serum (Hyclone) and 0.05% penicillin/streptomycin. LGD1069 was provided by Ligand Pharmaceuticals.

Cell lines. DRO, ARO, and FRO (human anaplastic thyroid carcinoma) were kindly provided by Dr. G.J. Juillard (University of California at Los Angeles, Los Angeles, CA).

Western blot analysis. Nuclear extracts were obtained from DRO, ARO, and FRO for RXR receptor proteins using a nuclear extract kit from Active Motif. PPAR receptor protein was obtained using immunoprecipitation as previously described (8). The protein content of lysates was measured using a commercial protein assay kit (DC from Bio-Rad). Diluted samples containing equal amounts of protein (60 µg) were mixed with 2x Laemmli sample buffer (Bio-Rad Laboratories). Proteins were separated on a 10% SDS-polyacrylamide 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, and 0.1% Tween 20] containing 5% nonfat dry milk at room temperature for 2 h and incubated in the appropriate primary antibody in 1x TBST containing 5% nonfat dry milk at 4°C overnight. RXR (sc Y-20), RXR (sc D-20), and RXRβ (sc C-20) receptor antibodies were used at a concentration of 1:1,000 and PPAR (H-100) rabbit polyclonal antibody (sc-7196; Santa Cruz Biotechnology) was used at 1:500. After washing, membranes were incubated for 1 h at room temperature with anti-rabbit IgG conjugated to horseradish peroxidase at a 1:5,000 dilution for RXRs and 1:1,000 for PPAR (GE Healthcare UK). β-Actin was probed for loading control. The enhanced chemiluminescence detection reagent from Amersham Biosciences was used for immunodetection.

Cell growth and proliferation. DRO, ARO, and FRO were grown to 80% confluence in 100 mm tissue culture plates. Cells were then harvested using Trypsin-EDTA (Invitrogen Corporation) and counted using a hemocytometer. Cells were then transferred to a 96-well plate at a concentration of 500 cells/200 µL of media. Each row of eight wells received the same cell type, and subsequently, the same drug. After cells were allowed to plate down overnight, media 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 h. After 6 days, cell proliferation was assessed following the manufacturer's instructions using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Following a 2-h incubation at 37°C, each plate was analyzed by a MRX Microplate Reader (Dynatech Laboratories) using Revelation software.

Nude mice. Athymic nude mice were purchased from the National Cancer Institute (NCI-NCr nu/nu 01B74). All mice were male, 6 to 7 weeks old weighing 15 to 30 g. Mice were handled in accordance with the approval of the University of Colorado at Denver and Health Sciences Center Animal Care and Use Committee. The three groups were control chow, high dose LGD1069 (100 mg/kg/d), and low dose LGD1069 (30 mg/kg/d).

Tumor injection. DRO cells were grown in RPMI supplemented with 2% fetal bovine serum and suspended at 5 x 10⁶ cells/200 µL sterile PBS. Mice were separated into groups of eight and after they were anesthetized with an i.p. injection of Avertin (0.5-0.7 mL), 5 x 10⁶ tumor cells were injected s.c. on the right flank of each mouse.

Drug administration. LGD1069 was mixed into LabDiet 5001 by TestDiet (a division of Purina Mills), at a dose estimated to deliver the desired dose based on the assumption that mice would weigh 20 g and eat 5 g of chow/d (based on previous experience). The LabDiet 5001 chow alone was used as control chow. Each diet was irradiated with Cobalt 60 gamma irradiation to sterilize the chow for nude mouse consumption. LGD1069 powder separate from the chow was included in the irradiation process and was then used for in vitro antiproliferative experiments with DRO cells. Irradiation of LGD1069 powder did not affect the ability to inhibit cancer cell growth in vitro (data not shown). Mice were weighed prior to tumor injection, and food was weighed every 2 weeks to estimate the amount of consumption per mouse. LGD1069 treatment chow was started after tumors reached a volume of 100 mm³.

Tumor assessment. Mice were observed twice per week and tumors were measured with electronic calipers. Tumor volume was estimated using the formula: tumor (length x width x height) / 0.5236. Based on previous experience, the study was designed to stop when the first group of eight mice had an average tumor size of 3,000 mm³. This was chosen to maximize the differences between groups and yet prevent significant morbidity as it had been observed that mice with tumors of this size were still mobile, able to access food and water easily, and had not lost significant weight. Tumor necrosis and vascularity were assessed by a pathologist blinded to treatment conditions. Tumor vascularity was assessed in the control chow and high dose groups with a score of 0 to 3 given to each group. The scores were averaged to allow for statistical comparison between the two groups.

Microarray analysis. DRO cells were grown in tissue culture plates in RPMI with 2% fetal bovine serum. Four million DRO cells were plated in triplicate into 100 mm plates and incubated overnight to attach. The next day, the medium was changed and vehicle (DMSO) or 1 µmol/L of LGD1069 was added in the set of cells to incubate for 24 h.

RNA was extracted from treated cells using the QIAGEN RNeasy Mini Kit and was quantified by standard spectrophotometry. RNA integrity was verified by gel electrophoresis using an Agilent 2100 Bioanalyzer.

Total RNA (5 µg) was converted to ds-cDNA using the Superscript Choice System (Life Technologies). A high-pressure liquid chromatography–purified T7-(dT)₂₄ primer was used to initiate the cDNA reverse transcription. After the synthesis of both strands of DNA, the ds-cDNA was extracted with phenol-chloroform-isoamyl alcohol and recovered by ethanol precipitation. Next, in vitro transcription of cRNA was done and the transcript underwent biotin labeling using a RNA Transcript Labeling Kit (Enzo). Biotin-labeled cRNA was purified using the QIAGEN RNeasy Mini Kit. The RNA was then fragmented by incubating the cRNA at 94°C for 35 min to allow optimal hybridization to the cDNA oligonucleotide array. We used the Affymetrix GeneChip Human Genome U133A platform, and all gene chip processing and analyses occurred in the UCHSC Affymetrix microarray core facility. Each condition was run in triplicate from three independent experiments.

Data analysis, including background adjustment and normalization, was done using Affymetrix GeneSpring software. For microarray analysis, those genes that had at least a >2-fold change in more than three of six gene chips between ligand and vehicle treatment of DRO cells were selected as significant. Significance was determined by a one-way ANOVA (P < 0.05).

Reverse transcription-PCR. Total RNA was isolated from snap-frozen tumors using the RNeasy Mini Kit (QIAGEN) as per the manufacturer's protocol. The mRNA for ANGPTL4 was measured by real-time quantitative reverse transcription-PCR using ABI PRISM 7700. The sequences of forward and reverse primers as designed by Primer Express (PE ABI) were 5'-CGCCAAGAGCCTCTCTGG-3' and 5'-CGGAAGTACTGGCCGTTGA-3'. The TaqMan fluorogenic probe used was 5'-6FAM-TGGCACCTGCAGCCATTCCAAC-TAMRA-3'.

Amplification reactions were done in MicroAmp optical tubes (PE ABI) in a 25 mL mix containing 8% glycerol, 1x TaqMan buffer A (500 mmol/L KCl, 100 mmol/L Tris-HCl, 0.1 mol/L EDTA, 600 nmol/L passive reference dye ROX; pH 8.3 at room temperature), 300 mmol/L each of dATP, dGTP, dCTP, and 600 mmol/L of dUTP, 5.5 mmol/L of MgCl₂, 300 nmol/L of forward primer, 900 nmol/L of reverse primer, 200 nmol/L of probe, 1.25 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer), 12.5 units of Moloney Murine leukemia virus reverse transcriptase (Life Technologies), 20 units of RNasin RNase inhibitor (Promega Corp.), and the template RNA. Thermal cycling conditions were as follows: reverse transcription was done at 48°C for 30 min followed by activation of TaqGold at 95°C for 10 min. Subsequently, 40 cycles of amplification were done at 95°C for 15 s and 60°C for 1 min.

A standard curve was generated using the fluorescent data from the 10-fold serial dilutions of control RNA. This was then used to calculate the relative amounts of ANGPTL4 in test samples. Quantities of ANGPTL4 in test samples were normalized to the corresponding 18S rRNA (PE ABI, P/N 4308310).

Statistics. Cell growth between control and LGD1069 treatment and DRO tumor growth was quantified using the group mean ± SE and significance was compared between control and treatment conditions with a Student's t test between conditions (SISA online statistical tool). The quantitative reverse transcription-PCR of ANGPTL4 mRNA was quantified as the group mean ± SE and significance was assessed with a one-way ANOVA.

	Results

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Nuclear hormone receptor expression. Western blot analysis of the three isoforms of RXR protein revealed that DRO, ARO, and FRO had similar levels of RXR and RXRβ expression but only DRO expressed RXR protein. All three cell lines also expressed PPAR (Fig. 1A ).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Western blot of nuclear hormone receptors and effects of rexinoid treatment on proliferation in anaplastic thyroid cancer cell lines. A, Western blot; 60 µg of nuclear protein extract from three thyroid cancer cell lines were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with a 1:1,500 dilution of a mouse monoclonal antibody against human PPAR (COOH terminus; Santa Cruz). A 1:5,000 dilution of secondary antibody was used. β-Actin was measured as a loading control. B, proliferation; cells were grown in 2% fetal bovine serum RPMI in the presence of 1 µmol/L of LGD1069 vehicle (DMSO) 6 days (fresh media and drug added every 72 h). Cells were harvested and analyzed using a nonradioactive cell proliferation assay. Cell proliferation is represented by the percentage of growth compared with cells grown in vehicle. Columns, mean; bars, SE (*, P < 0.05).

In vivo tumor response to LGD1069. Based on in vitro response, DRO tumor growth was assessed in a xenograft model. Athymic nude mice were injected s.c. with 5 x 10⁶ DRO cells suspended in 200 µL of sterile PBS and tumors were allowed to grow to 100 mm³ prior to the transition to chow containing LGD1069. Mice receiving control chow consumed an average of 7.3 g/mouse/d and mice receiving LGD1069 consumed an average of 5.5 g/mouse/d of low dose chow and 6.7 g/mouse/d of high dose chow. Based on mouse weights (average 25 g), the estimated amount of drug consumed was consistent with the desired treatment dose. By 3.5 weeks, the control chow tumor group reached the target end point (3,000 mm³) and the animals were sacrificed. LGD1069 significantly inhibited DRO tumor growth in a dose-dependent manner with final average tumor sizes of 3,540 ± 433 mm³ in the control group, 2,323 ± 288 mm³ in the low-dose group, and 447 ± 133 mm³ in the high-dose group (Figs. 2 and 3 ). All tumor sizes at the end point were significantly different between control and each treatment group (P = 0.0002 control versus high dose; P = 0.05 control versus low dose; P = 0.009 low dose versus high dose). Tumors in either treatment group showed increased central necrosis (four of five in the low dose and six of eight in the high dose groups with >50% necrosis) compared with the control chow group (six of eight, <25% necrosis) as determined by a detailed histopathologic evaluation by a pathologist blinded to treatment conditions (Fig. 4A-C ). Additionally, tumor vascularity was significantly different between control and high-dose drug treatment with the control tumors having a blinded score of 1.5, and 0.875 (P = 0.0179) for the high-dose–treated tumors. The animals had no readily demonstrable adverse side effects following LGD1069 treatment, except for drier skin noted in the high-dose LGD1069 group.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. In vivo tumor response of DRO cells to rexinoid treatment. DRO cells (5 x 106) were injected s.c. on the flanks of athymic nude mice. Tumors were allowed to establish and grow to a 100 mm3 volume prior to the change to LGD1069-containing chow at a 30 mg/kg/d concentration (low dose) or 100 mg/kg/d (high dose). Tumors were measured twice per week. Points, mean tumor volume/group; bars, SE. Student's t test was used to assess the significance of the difference of tumor sizes at the end of the experiment (*, P = 0.05; **, P = 0.002; ***, P = 0.009).

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Representative ATC tumors at the end of the treatment period. DRO cells were injected s.c. and tumors were allowed to grow for 3.5 wk after tumor establishment (100 mm3). Treatment conditions were (A) control, (B) 30 mg/kg/d LGD1069, and (C) 100 mg/kg/d LGD1069.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. DRO xenograft tumor histopathology. Representative H&E stained sections show that in the control/untreated tumors, despite their greater size, there is less necrosis (arrow) and vacuolization of tumor cells (A) as compared with the low LGD 1069 treatment condition (B). The high LGD1069 treatment condition shows almost complete necrosis with only a narrow rim of viable tumor cells at the periphery (C).

View larger version (7K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. In vivo ANGPTL4 mRNA expression. One microgram of total RNA was used for the ANGPTL4 quantitative reverse transcription-PCR analysis (ABI PRISM 7700; Perkin-Elmer) and absolute values were derived from a standard curve using a known amount of sense strand RNA (ag, attograms of sense strand RNA). Isoform RNA was normalized to total input RNA (18s rRNA measured from 1 ng of total RNA).

	Discussion

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Retinoids are vitamin A derivatives that have been broadly studied as therapeutic agents in multiple cancer types and in various in vitro, in vivo, and clinical models (7). Rexinoids are RXR receptor-selective retinoids, and one rexinoid (LGD1069, bexarotene, Targretin) is currently approved for the treatment of cutaneous T-cell lymphoma (9). Research is under way investigating the efficacy of bexarotene in the treatment of non–small cell lung cancer (10, 11).

Numerous studies have looked into the expression of RARs in thyroid carcinoma in vitro and in vivo, and the response to retinoic acid therapies in terms of tumor cell growth and/or properties of redifferentiation (primarily factors associated with improved ¹³¹I uptake; refs. 12–14).

Our previous work has shown that certain thyroid cancers have differential RAR and RXR receptor expressions and even different RXR isotype (, β, or ) expressions that may predict response to retinoid or rexinoid therapies (6). In an in vitro model of DRO proliferation, our data suggests that the antiproliferative effect of a rexinoid occurs through RXR (not RAR) and may require the expression of the RXR isotype (6). Our current observations strengthen this hypothesis based on ARO and FRO not responding to LGD1069, although expression of RXR and RXRβ are similar to the rexinoid-responsive DRO cells. Indeed, the only molecular difference ARO and FRO have from DRO in terms of rexinoid receptors and PPAR is the lack of RXR. An alternative hypothesis is that there is a threshold or "dose response" of total RXR receptor (of all isotypes) that is required for rexinoid responsiveness. In this case, however, one may expect a partial response in ARO and FRO to LGD1069 and this was not seen. The observed variability of RXR isotypes in human thyroid carcinomas has been investigated by Takiyama and colleagues (15). In this study, immunohistochemistry was used to assess RXR isotype expression on a variety of human thyroid lesions including papillary, follicular, anaplastic, and medullary carcinomas as well as follicular adenomas. Staining was categorized into nuclear or cytoplasmic and showed that there was less RXR staining of all isotypes in thyroid carcinomas compared with normal tissue. Additionally, they observed increased RXR staining in the cytoplasm as compared with the nucleus in thyroid cancer tumors. The authors postulate that the overall decreased RXR expression and/or the subcellular localization of RXR receptors may play a functional role in the pathogenesis of thyroid carcinomas.

To our knowledge, only one clinical study assessing the efficacy of a rexinoid (bexarotene) in thyroid cancer therapy has been published (16). The goal of the study was to assess the ability of bexarotene to increase ¹³¹I uptake in previously radioiodine-resistant metastatic thyroid cancer. The results showed minimal ¹³¹I uptake after treatment. This was a small sample of patients (12), there was no assessment of the RXR expression status of original tumors or any metastases, and the bexarotene (300 mg/d) was given for only 6 weeks. Thus, the results may reflect a subset of patients with little or no RXR receptor expression, the dose and duration of bexarotene may have been inadequate, or the wrong treatment response (¹³¹I uptake) may have been chosen.

This report is the first to describe the in vivo efficacy of an RXR targeted therapy in an anaplastic thyroid carcinoma cell line. We observed a marked decrease in tumor size in the nude mouse model supporting our previously published in vitro findings (8). A detailed histopathologic evaluation showed clear differences between the amounts of central necrosis in control versus treated tumors. Although larger tumors may outgrow their blood supply and have resultant necrosis, the smaller tumors in the rexinoid-treated groups had more necrosis. This may indicate a direct effect of LGD1069 as indicated by a recent studies in non–small cell lung cancer and breast cancer (17, 18). In the non–small cell lung cancer study, the investigators noted a reduction in the vascular network in tumors and decreased vascular endothelial growth factor induced by LGD1069.

We sought to understand early gene expression changes that may result from rexinoid treatment in DRO and identify candidate genes for analysis. We employed comparative microarray technology to assess differences between DRO treated with LGD1069 or exposed to volume-equivalent vehicle. Microarray technology is a powerful unbiased molecular tool to rapidly identify global gene signatures and gene changes in disease states of interest (19). Numerous studies have used comparative microarrays to identify genes that are differentially expressed between thyroid carcinomas and matched normal tissue as well as different forms of thyroid cancer (i.e., papillary versus follicular; refs. 20–22). Additionally, many studies have used microarray technology to assess genetic changes after targeted therapy for cancer (23, 24), but this is the first study to assess changes associated with the specific treatment effects of an anaplastic thyroid cancer cell line with a therapeutic rexinoid. We chose to analyze a 24-h time point of treatment to assess early genetic changes that may identify mechanistic pathways of the rexinoid treatment effect.

ANGPTL4 showed the greatest change in mRNA levels (6.5-fold increase) in a carefully controlled in vitro experiment. ANGPTL4, also known as the peroxisome proliferator–activated receptor- angiopoietin-related gene, is a hypoxia-induced protein that has been shown to increase vascularity, presumptively as a protective mechanism against hypoxia. This protein has been shown to be induced by hypoxia in endothelial cells at the RNA and protein level and has been postulated to be a proangiogenic factor (25, 26). Additionally, in a study of the efficacy of ZD6474, a vascular endothelial growth factor receptor-2 and endothelial growth factor receptor tyrosine kinase inhibitor, ANGPTL4 was up-regulated 2.3-fold in undifferentiated gastric tumors implanted in mice. These tumors had decreased tumor growth, decreased intraperitoneal metastases and increased mouse survival. The authors postulated that ANGPTL4 was up-regulated in response to the hypoxia induced as a result of vascular endothelial growth factor receptor-2 and endothelial growth factor receptor inhibition which resulted in decreased vascularity and increased hypoxia (27). However, a study by Ito and colleagues showed that a mouse that overexpressed ANGPTL4 showed inhibition of colorectal xenograft tumor growth and decreased vascularity within the tumors (26). The authors showed additional in vitro experiments with ANGPTL4 overexpression that resulted in reduced proliferation and migration of endothelial cells. Finally, Galaup and colleagues showed that low (<1 µg) but persistent levels of ANGPTL4 in mice, induced by DNA electrotransfer of ANGPTL4 in skeletal muscle, reduced the number of tumor emboli in the lungs of mice injected with 3LL lung carcinoma cells compared with control (28).

We found increased ANGPTL4 mRNA levels in response to LGD1069 therapy in the rexinoid-responsive anaplastic thyroid cancer DRO. To our knowledge, this is the first report of the up-regulation of this potential antitumor modulator in thyroid cancer. Our in vitro model indicates that increased ANGPTL4 is likely a direct rexinoid effect and not secondary to hypoxia in this controlled in vitro system. The in vivo tumors showed a trend toward increasing ANGPTL4 levels after LGD1069 treatment. It is possible that there were no significant differences based on the levels in the control tumors. These tumors were much larger than both treatment tumors and likely had growth-induced hypoxia that may be a potential confounder. Interestingly, even though the high-dose–treated tumors were smaller, they had decreased vascularity and increased necrosis compared with controls. Changes in ANGPTL4 expression are a possible mediator of this observation as a component of direct LGD1069 tumor inhibition in DRO.

In summary, LGD1069 (bexarotene) offers a potential therapeutic alternative in poorly differentiated thyroid carcinoma based on the growth-inhibitory effects that we have shown here both in vitro and now in vivo. It is important to appreciate that bexarotene is a targeted therapy to the RXR nuclear hormone receptor and that other factors such as heterodimer partners and coregulators may alter its efficacy. Further research is warranted to utilize this therapy in clinical trials of poorly differentiated thyroid cancer as this disease currently has no clearly efficacious treatment modalities.

	Note Added in Proof

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Recent genetic profiling by short tandem repeat analysis (29) has revealed that DRO shares the same profile as the melanoma cell line A375. This discovery was made 5 months after the revised submission of this manuscript. We are currently seeking original tissue to assess the origins of these cell lines. Given that our study is targeted at the molecular profile of the different cell lines and the resultant molecular responses with targeted RXR therapy, we feel the results presented in this article remain relevant and suitable to direct future studies of rexinoid therapy for poorly differentiated carcinoma.

	Footnotes

 
Grant support: Endocrine Fellows Foundation grant, American Cancer Society Institutional Research grant/University of Colorado Cancer Center Fellows grant, American Cancer Society MRSG-06-193-01-TBE, and NIH CA100560. This research was made possible by the support of the University of Colorado Cancer Center Gene Expression Core and use of the UCDHSC Center for Comparative Medicine.

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.

Note: The studies described have been approved by the UCDHSC Institutional Animal Care and Use Committee Protocol no. 26302005(09)1E.

Received 2/ 1/07; revised 6/22/07; accepted 9/26/07.

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