In Vivo and Microarray Analysis of Rexinoid-Responsive Anaplastic Thyroid Carcinoma Joshua P

Cancer Therapy: Preclinical

In vivo and Microarray Analysis of Rexinoid-Responsive Anaplastic Thyroid Carcinoma Joshua P. Klopper,1, 3 Andrew Berenz,1 William R. Hays,1 Vibha Sharma,1Umarani Pugazhenthi,1 JenniferJanssen,1Meenakshi Singh,2 Reid P. Bissonnette,4 and Bryan R. Haugen1, 2 , 3

Abstract 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. Compar- ative 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-g, 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.

Follicular cell–derived thyroid carcinoma is composed of three significant morbidity, especially relative to the benefit con- general histologic subtypes: papillary, follicular, and anaplastic ferred. Due to its aggressive invasive behavior, anaplastic thyroid cancer. The anaplastic subtype accounts for only f2% thyroid cancer may be treated with surgery, but if a complete to 5% of clinically recognized thyroid cancers but is lethal, with resection is not possible, debulking may increase morbidity most patients succumbing to the disease within 8months of with little to no palliation (2). External beam radiation may diagnosis (1). Therapy for anaplastic thyroid cancer carries offer some palliative relief, but this again is associated with significant potential side effects including xerostomia, local fibrosis, and esophageal epithelial injury (3, 4). Small clinical Authors’Affiliations: 1Department of Medicine, Division of Endocrinology, trials have shown modest efficacy for paclitaxel as a chemo- 2 3 Metabolism, and Diabetes, and Department of Pathology, University of Colorado therapeutic agent, but it has a high side effect profile relative to Cancer Center, Universityof Colorado at Denverand Health Sciences Center, Aurora, the benefit obtained (5). An alternative treatment strategy based Colorado; and 4Department of Molecular Oncology, Ligand Pharmaceuticals, San Diego, California on molecular targets may provide new therapeutic options. Received 2/1/07; revised 6/22/07; accepted 9/26/07. We have previously shown that the anaplastic thyroid Grant support: Endocrine Fellows Foundation grant, American Cancer Society carcinoma cell line DRO expresses the nuclear hormone, Institutional Research grant/University of Colorado Cancer Center Fellows grant, receptor retinoid X receptor (RXR)-g (6), which predict the 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 effects of RXR-selective ligands (rexinoids). Rexinoids, distin- Center Gene Expression Core and use of the UCDHSC Center for Comparative guished from retinoids that bind retinoic acid receptors (RAR) Medicine. or both RAR and RXR receptors in a less selective manner The costs of publication of this article were defrayed in part by the payment of page (e.g., 9-cis retinoic acid), specifically activate RXR receptors advertisement charges. This article must therefore be hereby marked in accordance leading to an alteration in transcription (7). We have previously with 18 U.S.C. Section 1734 solely to indicate this fact. Note: The studies described have been approved by the UCDHSC Institutional shown that the rexinoid, LGD1069 (bexarotene, Targretin), Animal Care and Use Committee Protocol no. 26302005(09)1E. significantly inhibits DRO proliferation in vitro, in part, through Requests for reprints: Joshua P. Klopper, Department of Medicine, University of increased apoptosis (6, 8). Colorado at Denver and Health Sciences Center, MS 8106, P.O. Box 6511, Aurora, In this report, we describe the efficacy of LGD1069 therapy CO 80045. Phone: 303-724-1454; Fax: 303-724-3920; E-mail: joshua.klopper@ in vitro in vivo uchsc.edu. for anaplastic thyroid cancer and in a nude F 2008 American Association for Cancer Research. mouse model. In order to explore the mechanisms by which doi:10.1158/1078-0432.CCR-07-0269 the rexinoid LGD1069 alters thyroid cancer proliferation in

www.aacrjournals.org 589 Clin Cancer Res 2008;14(2) January 15, 2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical responsive cells, we used microarray gene expression analysis. desired dose based on the assumption that mice would weigh 20 g and Of the genes that showed significant change of expression with eat 5 g of chow/d (based on previous experience). The LabDiet 5001 rexinoid treatment, angiopoietin-like 4 (ANGPTL4) had the chow alone was used as control chow. Each diet was irradiated with largest fold change increase in vitro and this was correlated with Cobalt 60 gamma irradiation to sterilize the chow for nude mouse consumption. LGD1069 powder separate from the chow was included observations of responsive tumors. in the irradiation process and was then used for in vitro antiproliferative experiments with DRO cells. Irradiation of LGD1069 powder did not Materials and Methods affect the ability to inhibit cancer cell growth in vitro (data not shown). Mice were weighed prior to tumor injection, and food was weighed Chemicals. All thyroid cancer cells were grown in RPMI 1640 every 2 weeks to estimate the amount of consumption per mouse. (Invitrogen Corporation) supplemented with 2% fetal bovine serum LGD1069 treatment chow was started after tumors reached a volume of 3 (Hyclone) and 0.05% penicillin/streptomycin. LGD1069 was provided f100 mm . by Ligand Pharmaceuticals. Tumor assessment. Mice were observed twice per week and tumors Cell lines. DRO, ARO, and FRO (human anaplastic thyroid were measured with electronic calipers. Tumor volume was estimated Â Â carcinoma) were kindly provided by Dr. G.J. Juillard (University of using the formula: tumor (length width height) / 0.5236. Based on California at Los Angeles, Los Angeles, CA). previous experience, the study was designed to stop when the first 3 Western blot analysis. Nuclear extracts were obtained from DRO, group of eight mice had an average tumor size of 3,000 mm . This was ARO, and FRO for RXR receptor proteins using a nuclear extract kit chosen to maximize the differences between groups and yet prevent from Active Motif. PPARg receptor protein was obtained using significant morbidity as it had been observed that mice with tumors of immunoprecipitation as previously described (8). The protein content this size were still mobile, able to access food and water easily, and had of lysates was measured using a commercial protein assay kit (DC from not lost significant weight. Tumor necrosis and vascularity were Bio-Rad). Diluted samples containing equal amounts of protein (60 Ag) assessed by a pathologist blinded to treatment conditions. Tumor were mixed with 2Â Laemmli sample buffer (Bio-Rad Laboratories). vascularity was assessed in the control chow and high dose groups with Proteins were separated on a 10% SDS-polyacrylamide gel and trans- a score of 0 to 3 given to each group. The scores were averaged to allow ferred to polyvinylidene difluoride membranes. The membranes were for statistical comparison between the two groups. blocked with 1Â TBST [20 mmol/L Tris-HCl (pH 7.6), 8.5% NaCl, and Microarray analysis. DRO cells were grown in tissue culture plates 0.1% Tween 20] containing 5% nonfat dry milk at room temperature in RPMI with 2% fetal bovine serum. Four million DRO cells were for 2 h and incubated in the appropriate primary antibody in 1Â TBST plated in triplicate into 100 mm plates and incubated overnight to containing 5% nonfat dry milk at 4jC overnight. RXRg (sc Y-20), RXRa attach. The next day, the medium was changed and vehicle (DMSO) or (sc D-20), and RXRh (sc C-20) receptor antibodies were used at a 1 Amol/L of LGD1069 was added in the set of cells to incubate for 24 h. concentration of 1:1,000 and PPARg (H-100) rabbit polyclonal RNA was extracted from treated cells using the QIAGEN RNeasy Mini antibody (sc-7196; Santa Cruz Biotechnology) was used at 1:500. Kit and was quantified by standard spectrophotometry. RNA integrity After washing, membranes were incubated for 1 h at room temperature was verified by gel electrophoresis using an Agilent 2100 Bioanalyzer. with anti-rabbit IgG conjugated to horseradish peroxidase at a 1:5,000 Total RNA (5 Ag) was converted to ds-cDNA using the Superscript dilution for RXRs and 1:1,000 for PPARg (GE Healthcare UK). h-Actin Choice System (Life Technologies). A high-pressure liquid chromatog- was probed for loading control. The enhanced chemiluminescence raphy–purified T7-(dT)24 primer was used to initiate the cDNA reverse detection reagent from Amersham Biosciences was used for immuno- transcription. After the synthesis of both strands of DNA, the ds-cDNA detection. was extracted with phenol-chloroform-isoamyl alcohol and recovered Cell growth and proliferation. DRO, ARO, and FRO were grown to by ethanol precipitation. Next, in vitro transcription of cRNA was done f80% confluence in 100 mm tissue culture plates. Cells were then and the transcript underwent biotin labeling using a RNA Transcript harvested using Trypsin-EDTA (Invitrogen Corporation) and counted Labeling Kit (Enzo). Biotin-labeled cRNA was purified using the using a hemocytometer. Cells were then transferred to a 96-well plate at QIAGEN RNeasy Mini Kit. The RNA was then fragmented by incubating a concentration of 500 cells/200 AL of media. Each row of eight wells the cRNA at 94jC for 35 min to allow optimal hybridization to the received the same cell type, and subsequently, the same drug. After cells cDNA oligonucleotide array. We used the Affymetrix GeneChip Human were allowed to plate down overnight, media was aspirated and Genome U133A platform, and all gene chip processing and analyses medium with the appropriate concentration of ligand or equivalent occurred in the UCHSC Affymetrix microarray core facility. Each volume of vehicle was added to each well. Fresh medium with vehicle condition was run in triplicate from three independent experiments. or ligand was added every 72 h. After 6 days, cell proliferation was Data analysis, including background adjustment and normalization, assessed following the manufacturer’s instructions using the CellTiter was done using Affymetrix GeneSpring software. For microarray 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). analysis, those genes that had at least a >2-fold change in more than Following a 2-h incubation at 37jC, each plate was analyzed by a three of six gene chips between ligand and vehicle treatment of DRO MRX Microplate Reader (Dynatech Laboratories) using Revelation cells were selected as significant. Significance was determined by a one- software. way ANOVA (P < 0.05). Nude mice. Athymic nude mice were purchased from the National Reverse transcription-PCR. Total RNA was isolated from snap- Cancer Institute (NCI-NCr nu/nu 01B74). All mice were male, 6 to frozen tumors using the RNeasy Mini Kit (QIAGEN) as per the 7 weeks old weighing 15 to 30 g. Mice were handled in accordance with manufacturer’s protocol. The mRNA for ANGPTL4 was measured the approval of the University of Colorado at Denver and Health by real-time quantitative reverse transcription-PCR using ABI PRISM Sciences Center Animal Care and Use Committee. The three groups 7700. The sequences of forward and reverse primers as designed by were control chow, high dose LGD1069 (100 mg/kg/d), and low dose Primer Express (PE ABI) were 5¶-CGCCAAGAGCCTCTCTGG-3¶ and LGD1069 (30 mg/kg/d). 5¶-CGGAAGTACTGGCCGTTGA-3¶. The TaqMan fluorogenic probe used Tumor injection. DRO cells were grown in RPMI supplemented with was 5¶-6FAM-TGGCACCTGCAGCCATTCCAAC-TAMRA-3¶. 2% fetal bovine serum and suspended at 5 Â 106 cells/200 AL sterile Amplification reactions were done in MicroAmp optical tubes PBS. Mice were separated into groups of eight and after they were (PE ABI) in a 25 mL mix containing 8% glycerol, 1Â TaqMan buffer anesthetized with an i.p. injection of Avertin (0.5-0.7 mL), 5 Â 106 A (500 mmol/L KCl, 100 mmol/L Tris-HCl, 0.1 mol/L EDTA, tumor cells were injected s.c. on the right flank of each mouse. 600 nmol/L passive reference dye ROX; pH 8.3 at room temperature), Drug administration. LGD1069 was mixed into LabDiet 5001 by 300 mmol/L each of dATP, dGTP, dCTP, and 600 mmol/L of

TestDiet (a division of Purina Mills), at a dose estimated to deliver the dUTP, 5.5 mmol/L of MgCl2, 300 nmol/L of forward primer, 900 nmol/L

Clin Cancer Res 2008;14(2) January15, 2008 590 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Rexinoid Treatment for AnaplasticThyroid Cancer 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 48jC for 30 min followed by activation of TaqGold at 95jC for 10 min. Subsequently, 40 cycles of amplification were done at 95jC for 15 s and 60jC 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 F 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 F SE and significance was assessed with a one-way ANOVA.

Results

Nuclear hormone receptor expression. Western blot analysis of the three isoforms of RXR protein revealed that DRO, ARO, and FRO had similar levels of RXRa and RXRh expression but only DRO expressed RXRg protein. All three cell lines also expressed PPARg (Fig. 1A). In vitro proliferation. DRO, ARO, and FRO were grown in the presence of 1 Amol/L of LGD1069 (replenished every 72 h) or volume-equivalent vehicle for 6 days. DRO showed a significant decrease in proliferation after rexinoid treatment P Fig. 1. Western blot of nuclear hormone receptors and effects of rexinoid treatment (42% decreased growth compared with control; < 0.05) on proliferation in anaplastic thyroid cancer cell lines. A, Western blot; 60 Agof whereas ARO and FRO had no response (Fig. 1B). nuclear protein extract from three thyroid cancer cell lines were size-separated on In vivo in vitro a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with tumor response to LGD1069. Based on 10% nonfat milk and incubated with a 1:1,500 dilution of a mouse monoclonal response, DRO tumor growth was assessed in a xenograft antibody against human PPARg (COOH terminus; Santa Cruz). A 1:5,000 dilution model. Athymic nude mice were injected s.c. with 5 Â 106 DRO of secondary antibody was used. h-Actin was measured as a loading control. A B, proliferation; cells were grown in 2% fetal bovine serum RPMI in the presence cells suspended in 200 L of sterile PBS and tumors were of1 Amol/L of LGD1069 vehicle (DMSO) 6 days (fresh media and drug added every 3 allowed to grow to 100 mm prior to the transition to chow 72 h). Cells were harvested and analyzed using a nonradioactive cell proliferation containing LGD1069. Mice receiving control chow consumed assay. Cell proliferation is represented by the percentage of growth compared with cells grown in vehicle. Columns, mean; bars, SE (*, P < 0.05). 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 (P = 0.0179) for the high-dose–treated tumors. The animals (average 25 g), the estimated amount of drug consumed was had no readily demonstrable adverse side effects following consistent with the desired treatment dose. By 3.5 weeks, LGD1069 treatment, except for drier skin noted in the high- the control chow tumor group reached the target end point dose LGD1069 group. (3,000 mm3) and the animals were sacrificed. LGD1069 Gene expression changes to LGD1069. The antiproliferative significantly inhibited DRO tumor growth in a dose-dependent effects of rexinoid treatment on DRO occur at or beyond 6 days manner with final average tumor sizes of 3,540 F 433 mm3 in of treatment (8). However, we chose to analyze gene expression the control group, 2,323 F 288 mm3 in the low-dose group, changes in DRO at 24 h because RXRg is a nuclear hormone and 447 F 133 mm3 in the high-dose group (Figs. 2 and 3). receptor and we would expect the direct effects of rexinoid All tumor sizes at the end point were significantly different treatment to occur early. Microarray gene expression after between control and each treatment group (P = 0.0002 control treatment of DRO cells for 24 h with 1 Amol/L of LGD1069 or versus high dose; P = 0.05 control versus low dose; P = 0.009 volume-equivalent vehicle (DMSO) revealed 109 genes with a low dose versus high dose). Tumors in either treatment 2-fold change in expression using a one-way ANOVA with group showed increased central necrosis (four of five in the P < 0.05. Eighty genes were up-regulated and 29 genes were low dose and six of eight in the high dose groups with >50% down-regulated after exposure to 1 Amol/L of LGD1069 for 24 h. necrosis) compared with the control chow group (six of eight, These genes broadly fell into the categories of cell growth, nucleic <25% necrosis) as determined by a detailed histopathologic acid binding, and cell signal transduction. The specific genes are evaluation by a pathologist blinded to treatment conditions listed in Table 1. ANGPTL4 showed the highest gene expression (Fig. 4A-C). Additionally, tumor vascularity was significantly change to rexinoid treatment with a 6.5-fold increase (Table 1A). different between control and high-dose drug treatment with To confirm the changes in ANGPTL4 expression in our in vivo the control tumors having a blinded score of 1.5, and 0.875 model, we assessed changes in ANGPTL4 mRNA expression in

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ARO and FRO have from DRO in terms of rexinoid receptors and PPARg is the lack of RXRg. 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 Fig. 2. In vivo tumor response of DRO cells to rexinoid treatment. DRO cells increased RXRa staining in the cytoplasm as compared with the (5 Â 10 6) 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 nucleus in thyroid cancer tumors. The authors postulate that LGD1069-containing chow at a 30 mg/kg/d concentration (low dose) or the overall decreased RXR expression and/or the subcellular 100 mg/kg/d (high dose).Tumors were measured twice per week. Points, mean localization of RXR receptors may play a functional role in the 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;**, pathogenesis of thyroid carcinomas. P =0.002;***,P =0.009). To our knowledge, only one clinical study assessing the efficacy of a rexinoid (bexarotene) in thyroid cancer therapy has treated tumors compared with controls. There was a trend for been published (16). The goal of the study was to assess the 131 increasing ANGPTL4 across treatment conditions but the ability of bexarotene to increase I uptake in previously differences were not statistically significant (P = 0.11, high radioiodine-resistant metastatic thyroid cancer. The results 131 dose treatment compared with control; Fig. 5). showed minimal I uptake after treatment. This was a small sample of patients (12), there was no assessment of the RXR Discussion expression status of original tumors or any metastases, and the bexarotene (300 mg/d) was given for only 6 weeks. Thus, the In this report, we have assessed the efficacy of the rexinoid LGD1069 to slow cell growth in three anaplastic thyroid cancers. We have shown that DRO is responsive as opposed to ARO and FRO. Furthermore, LGD1069 inhibits DRO tumors in a dose-dependent manner in a nude mouse model. This finding translates our previously published in vitro studies using rexinoids to inhibit DRO cell proliferation (6, 8) to an in vivo model. 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 (a, h,org) 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 RXRg isotype (6). Our current observations strengthen this hypothesis based on ARO and FRO not responding to LGD1069, although Fig. 3. Representative ATC tumors at the end of the treatment period. DRO h cells were injected s.c. and tumors were allowed to grow for 3.5 wk after tumor expression of RXRa and RXR are similar to the rexinoid- establishment (f10 0 mm3). Treatment conditions were (A) control, (B) responsive DRO cells. Indeed, the only molecular difference 30 mg/kg/d LGD1069, and (C) 100 mg/kg/d LGD1069.

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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 LGD1069 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).

results may reflect a subset of patients with little or no RXR vehicle. Microarray technology is a powerful unbiased molec- receptor expression, the dose and duration of bexarotene may ular tool to rapidly identify global gene signatures and gene have been inadequate, or the wrong treatment response (131I changes in disease states of interest (19). Numerous studies uptake) may have been chosen. have used comparative microarrays to identify genes that are This report is the first to describe the in vivo efficacy of an differentially expressed between thyroid carcinomas and RXR targeted therapy in an anaplastic thyroid carcinoma cell matched normal tissue as well as different forms of thyroid line. We observed a marked decrease in tumor size in the nude cancer (i.e., papillary versus follicular; refs. 20–22). Addition- mouse model supporting our previously published in vitro ally, many studies have used microarray technology to assess findings (8). A detailed histopathologic evaluation showed genetic changes after targeted therapy for cancer (23, 24), clear differences between the amounts of central necrosis in but this is the first study to assess changes associated with control versus treated tumors. Although larger tumors may the specific treatment effects of an anaplastic thyroid cancer outgrow their blood supply and have resultant necrosis, the cell line with a therapeutic rexinoid. We chose to analyze a smaller tumors in the rexinoid-treated groups had more 24-h time point of treatment to assess early genetic changes that necrosis. This may indicate a direct effect of LGD1069 as may identify mechanistic pathways of the rexinoid treatment indicated by a recent studies in non–small cell lung cancer and effect. breast cancer (17, 18). In the non–small cell lung cancer study, ANGPTL4 showed the greatest change in mRNA levels the investigators noted a reduction in the vascular network in (6.5-fold increase) in a carefully controlled in vitro experiment. tumors and decreased vascular endothelial growth factor ANGPTL4, also known as the peroxisome proliferator–activated induced by LGD1069. receptor-g angiopoietin-related gene, is a hypoxia-induced We sought to understand early gene expression changes that protein that has been shown to increase vascularity, presump- may result from rexinoid treatment in DRO and identify tively as a protective mechanism against hypoxia. This protein candidate genes for analysis. We employed comparative has been shown to be induced by hypoxia in endothelial cells at microarray technology to assess differences between DRO the RNA and protein level and has been postulated to be a treated with LGD1069 or exposed to volume-equivalent proangiogenic factor (25, 26). Additionally, in a study of the

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Table 1. Comparative microarray analysis of DRO Table 1. Comparative microarray analysis of DRO cells cells (Cont’d)

Gene symbol Fold change GenBank ID Gene symbol Fold change GenBank ID (A) Genes up-regulated by LGD1069 (A) Genes up-regulated by LGD1069 ANGPTL4 6.5 NM_016109.1 LOC115648 2.1 BC006408.1 S100A2 3.4 NM_005978.2 D4S234E 2.1 NM_014392.1 C14orf135 3.3 AK022120.1 PRKCA 2.1 AF035594.1 MYCN 3.3 BC002712.1 KLF4 2.1 BF514079 PLS1 3.1 NM_002670.1 TMCC1 2.1 AI934469 GEM 3.1 NM_005261.1 ST6GALNAC2 2.1 NM_006456.1 KCNJ10 2.9 NM_002241.1 TRA2A 2.1 AW978896 CORO2B 2.9 BF939649 NEK1 2.0 Z25431.1 FTO 2.8AK022473.1 DCT 2.0 AL139318 SQSTM1 2.8N30649 FAM63B 2.0 AU121431 PDK4 2.8NM_002612.1 FAT5 2.0 NM_006599.1 AKAP12 2.7 AB003476.1 KLF4 2.0 NM_004235.1 ALDH1A3 2.7 AF198444.1 FRMD4A 2.0 NM_018027.1 MAP1B 2.7 BG164365 PDLIM4 2.0 BE043700 MPZ 2.7 D10537.1 GNE 2.7 NM_005476.2 (B) Genes down-regulated by LGD1069 CDC73 2.7 AW970584 QPCT RAB2 2.7 AI189609 -3.3 NM_012413.2 RIS1 PHACTR1 2.6 AW054711 -3.1 BF062629 FGFR4 PTPRC 2.6 AI809341 -2.9 AF202063.1 MGAT4C 2.6 NM_013244.1 * -2.8W26966 TNFRSF11B PDE2A 2.6 NM_002599.1 -2.6 BF433902 IFITM1 WSB1 2.6 AA521269 -2.5 AA749101 IL1RAP GRAMD1C 2.6 NM_017577.1 -2.5 NM_002182.1 NEURL DCT 2.5 NM_001922.2 -2.4 AA772093 NOS2A LA2G5 2.5 AL158172 -2.3 L24553.1 D4S234E 2.5 BC001745.1 * -2.3 AA570453 HAPLN1 ITPR1 2.5 L38019.1 -2.3 U43328.1 C20orf149 ARHGAP26 2.4 BE671084 -2.3 NM_024299.1 CUGBP2 LIF 2.4 NM_002309.2 -2.3 NM_006561.1 CIC ELOVL4 2.4 NM_022726.1 -2.3 AB002304.1 ITGB5 NRG1 2.4 NM_013960.1 -2.2 AI335208 MGC4859 LOC159110 2.4 AK026667.1 -2.2 NM_024304.1 ACTN2 LAMA4 2.4 BC004241.1 -2.2 NM_001103.1 DHFR LOC643509 PDLIM4 2.4 AF153882.1 -2.2 AI144299 AASS LOC151162 2.3 AL134724 -2.2 AK023446.1 HGF SORBS1 2.3 NM_015385.1 -2.1 X16323.1 MEOX2 WSB1 2.3 NM_015626.1 -2.1 NM_005924.1 PARG COL5A2 2.3 NM_000393.1 -2.1 NM_003631.1 MAF RABGAP1L 2.3 AB019490.1 -2.1 BF508646 ASB9 PLXNA2 2.3 AI688418 -2.1 NM_024087.1 YEATS4 JUN 2.3 BE327172 -2.1 NM_006530.1 ORC4L LCN2 2.3 NM_005564.1 -2.0 NM_002552.1 TCEB1 TMEM16C 2.2 AJ300461.1 -2.0 N89607 FLJ10159 KDR 2.2 NM_002253.1 -2.0 NM_018013.1 SCC-112 DTNA 2.2 U26744.1 -2.0 AW991219 EGR4 2.2 NM_001965.1 ABHD2 2.2 AI557319 NOTE: DRO cells were treated in vitro with 1 Amol/L of LGD1069 C14orf106 2.2 NM_018353.1 for 24 h compared with volume-equivalent vehicle in triplicate with PLA2G5 2.2 NM_000929.1 a cutoff of a 2-fold change (P < 0.5, one-way ANOVA). The results ZNF675 2.2 BF308250 showed (A) 80 genes up-regulated and (B) 29 genes down- CTSL 2.2 NM_001912.1 regulated. Gene symbols were derived from the Affymetrix web EDNRA 2.2 NM_001957.1 site. TRPV4 2.2 NM_021625.2 *Genes that have no designated gene symbol. AOX1 2.2 NM_001159.2 OAS1 2.1 NM_002534.1 UTRN 2.1 NM_007124.1 PRKCA 2.1 NM_002737.1 PPARGC1A 2.1 NM_013261.1 efficacy of ZD6474, a vascular endothelial growth factor EIF5B 2.1 NM_015904.1 receptor-2 and endothelial growth factor receptor tyrosine CDS2 2.1 AL568982 kinase inhibitor, ANGPTL4 was up-regulated 2.3-fold in un- RGS7 2.1 NM_002924.1 differentiated gastric tumors implanted in mice. These tumors SATB2 2.1 AK025127.1 had decreased tumor growth, decreased intraperitoneal metas- LY6H 2.1 NM_002347.1 PLLP 2.1 NM_015993.1 tases and increased mouse survival. The authors postulated that GPX3 2.1 NM_002084.2 ANGPTL4 was up-regulated in response to the hypoxia induced as a result of vascular endothelial growth factor receptor-2 and

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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 carcino- Fig. 5. In vivo ANGPTL4 mRNA expression. One microgram of total RNAwas used for the ANGPTL4 quantitative reverse transcription-PCR analysis (ABIPRISM 7700; ma based on the growth-inhibitory effects that we have shown Perkin-Elmer) and absolute values were derived from a standard curve using a here both in vitro and now in vivo. It is important to appreciate known amount of sense strand RNA (ag, attograms of sense strand RNA). Isoform RNA was normalized to total input RNA (18s rRNA measured from 1ng of total that bexarotene is a targeted therapy to the RXR nuclear RNA). hormone receptor and that other factors such as heterodimer partners and coregulators may alter its efficacy. Further research endothelial growth factor receptor inhibition which resulted in is warranted to utilize this therapy in clinical trials of poorly decreased vascularity and increased hypoxia (27). However, a differentiated thyroid cancer as this disease currently has no study by Ito and colleagues showed that a mouse that overex- clearly efficacious treatment modalities. pressed ANGPTL4 showed inhibition of colorectal xenograft tumor growth and decreased vascularity within the tumors (26). Note Added in Proof The authors showed additional in vitro experiments with ANGPTL4 overexpression that resulted in reduced proliferation Recent genetic profiling by short tandem repeat analysis and migration of endothelial cells. Finally, Galaup and (29) has revealed that DRO shares the same profile as the colleagues showed that low (<1 Ag) but persistent levels of melanoma cell line A375. This discovery was made 5 months ANGPTL4 in mice, induced by DNA electrotransfer of ANGPTL4 after the revised submission of this manuscript. We are in skeletal muscle, reduced the number of tumor emboli in the currently seeking original tissue to assess the origins of these lungs of mice injected with 3LL lung carcinoma cells compared cell lines. Given that our study is targeted at the molecular with control (28). profile of the different cell lines and the resultant molecular We found increased ANGPTL4 mRNA levels in response to responses with targeted RXR therapy, we feel the results LGD1069 therapy in the rexinoid-responsive anaplastic thyroid presented in this article remain relevant and suitable to direct cancer DRO. To our knowledge, this is the first report of the future studies of rexinoid therapy for poorly differentiated up-regulation of this potential antitumor modulator in thyroid carcinoma.

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Correction: Article on In vivo and Microarray Analysis of Rexinoid-Responsive Anaplastic Thyroid Carcinoma

The article on in vivo and microarray analysis of rexinoid-responsive anaplastic thyroid carcinoma in the January 15, 2008, issue of Clinical Cancer Research contained a post-proof note describing the author’s concern regarding the origins of DRO. The authors have since confirmed that DRO does represent a subline of the melanoma cell line A375 and that ARO matches the short tandem repeat (STR) profile of HT-29 and is believed to be a subline of this colon cancer–derived cell line. FRO has a unique STR profile and is likely of thyroid origin.

Klopper JP, Berenz A, Hays WR, et al. In vivo and microarray analysis of rexinoid- responsive anaplastic thyroid carcinoma. Clin Cancer Res 2008;14:589–96.

F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-15-1-COR1

Clin Cancer Res 2009;15(1) January 1, 2009 416 www.aacrjournals.org In vivo and Microarray Analysis of Rexinoid-Responsive Anaplastic Thyroid Carcinoma

Joshua P. Klopper, Andrew Berenz, William R. Hays, et al.

Clin Cancer Res 2008;14:589-596.

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