Bexarotene Via CBP/P300 Induces Suppression of NF-Kb– Dependent Cell Growth and Invasion in Thyroid Cancer

Published OnlineFirst December 5, 2011; DOI: 10.1158/1078-0432.CCR-11-0510

Clinical Cancer Cancer Therapy: Preclinical Research

Bexarotene via CBP/p300 Induces Suppression of NF-kB– Dependent Cell Growth and Invasion in Thyroid Cancer

Audrey Cras1,2,3,Beatrice Politis1,2, Nicole Balitrand1,2,3, Diane Darsin-Bettinger1,2,3, Pierre Yves Boelle4,5,6, Bruno Cassinat1,2,3, Marie-Elisabeth Toubert3, and Christine Chomienne1,2,3

Abstract Purpose: Retinoic acid (RA) treatment has been used for redifferentiation of metastatic thyroid cancer with loss of radioiodine uptake. The aim of this study was to improve the understanding of RA resistance and investigate the role of bexarotene in thyroid cancer cells. Experimental Design: A model of thyroid cancer cell lines with differential response to RA was used to evaluate the biological effects of retinoid and rexinoid and to correlate this with RA receptor levels. Subsequently, thyroid cancer patients were treated with 13-cis RA and bexarotene and response evaluated on radioiodine uptake reinduction on posttherapy scan and conventional imaging. Results: In thyroid cancer patients, 13-cis RA resistance can be bypassed in some tumors by bexarotene. A decreased tumor growth without differentiation was observed conﬁrming our in vitro data. Indeed, we show that ligands of RARs or RXRs exert different effects in thyroid cancer cell lines through either differentiation or inhibition of cell growth and invasion. These effects are associated with restoration of RARb and RXRg levels and downregulation of NF-kB targets genes. We show that bexarotene inhibits the transactivation potential of NF-kB in an RXR-dependent manner through decreased promoter permissiveness without interfering with NF-kB nuclear translocation and binding to its responsive elements. Inhibition of transcription results from the release of p300 coactivator from NF-kB target gene promoters and subsequent histone deacetylation. Conclusion: This study highlights dual mechanisms by which retinoids and rexinoids may target cell tumorigenicity, not only via RARs and RXRs, as expected, but also via NF-kB pathway. Clin Cancer Res; 18(2); 1–12. 2011 AACR.

Introduction ablative doses of radioiodine, and suppressive treatment. The follow-up is based on measuring serum thyroglobulin Thyroid cancers are the most common malignancy of (TG) and imaging with radioiodine (RI) therapeutic scans. endocrine organs (1). Well-differentiated thyroid carcino- It has been estimated that about 20% of these patients will mas (DTC) derived from follicular cells account for around develop a local or distant recurrence. In some cases, TG and 80% of thyroid cancers and include papillary thyroid cancer RI scan are discordant, suggesting the presence of thyroid (PTC) and follicular thyroid cancer (FTC), respectively, cancer metastases which have lost the ability to trap RI (2). which are differentiated on the basis of histologic para- This is challenging for thyroid cancer management, as meters. Most of these cancers have an excellent prognosis anatomical localization of recurrences cannot be assessed and can be effectively managed by total thyroidectomy, and treatment with RI therapeutic doses is not effective, predicting a poorer outcome. Signaling through growth factors and their receptors and Authors' Afﬁliations: 1INSERM U490, F-75475, Paris, 2Universite Paris Diderot, Sorbonne Paris Cite, IUH, UMRS 940, F-75475, Paris, 3AP-HP, defects in regulation of cell-cycle control elements are Hôpital Saint Louis, F-75475, Paris, 4INSERM U707, F-75475, Paris, considered essential for cancer cell proliferation. Recent 5Universite Pierre et Marie Curie, Sorbonne Universites, UMRS 707, advances have improved the understanding of the thyroid F-75005 Paris, 6AP-HP, Hopital^ Saint Antoine, F-75005, Paris, France oncogenesis. Inappropriate activation of the mitogen-acti- Note: Supplementary data for this article are available at Clinical Cancer vated protein kinase pathway effectors may lead to tumor Research Online (http://clincancerres.aacrjournals.org/). initiation and transformation. Indeed, mutations or rear- Corresponding Author: Christine Chomienne, UMR-S 940, INSERM, rangements of tyrosine kinase receptor (RET and NTRK1), Universite Denis Diderot, Institut Universitaire d'Hematologie, Hopital^ Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France. Phone: BRAF, and Ras are found in most of thyroid cancers (3). 33-0-1-42-49-42-34; Fax: 33-0-1-53-72-40-16; E-mail: Cancer progression is associated with a loss of the differ- [email protected] entiated characteristics that deﬁne mature follicular thyroid doi: 10.1158/1078-0432.CCR-11-0510 cells with the full capacity for normal physiologic function 2011 American Association for Cancer Research. such as iodine uptake. Iodine is transported across the

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toma by various natural and synthetic retinoids (10), and Translational Relevance the transcriptional control of the normal RARA gene expression by all-trans RA (ATRA) treatment in acute promyelo- After thyroidectomy, monitoring and treatment of cytic leukemia cells (11). RA efficacy in patients with thyroid thyroid cancer metastases relies on the uptake of radio- cancer is supported by in vitro data, showing that retinoids active iodine by the malignant thyroid cells. Unfortu- induce upregulation of RARb and differentiation of cells, nately, some patients have signs of relapse with no accompanied by the upregulation of NIS and TG expression detectable metastases on radioiodine whole-body scan. and DIO1 activity (12, 13). Treatment with 13-cis RA was This has been attributed to an absence of sodium iodide shown to restore RI uptake in thyroid cancer patients in symporter (NIS) and subsequent loss of iodine uptake. clinical trials (14–16). However, only one-third of the Retinoids, inducers of differentiation during embryo- patients respond, and further trials have failed to confirm genesis, are equally important for thyroidogenesis and 13-cis RA efficacy in these patients (17, 18). A clinical study NIS and the 50-deiodase genes are retinoic acid target showed that treatment with bexarotene, a RXR agonist can genes. We confirm previous reports of the ability of induce a radioiodine uptake by metastases in some patients retinoids to restore differentiation in thyroid cancer in (19, 20). These data prompted for a better understanding of vitro and in vivo and present novel data showing that RA signaling pathways and resistance in thyroid cancer cells. retinoid-resistant cells and patients may respond to We have previously reported that lack of RA responsive- rexinoids via inhibition of cell growth and invasion. ness in a FTC cell line was attributable to an altered histone This results from the decrease of NF-kB target gene acetylation pattern and could be relieved by combination of expression by modification of promoter permissiveness. ATRA and trichostatin A, a histone deacetylase inhibitor, This highlights a novel mechanism by which bexarotene underscoring the ongoing clinical trials based on differen- may control cancer progression. tiation and transcriptional therapy combination (21). To investigate the role of RXR agonist in these patients, we took advantage of the RA-resistant thyroid cancer cell line, FTC238, derived from the metastasis of the patient whose basolateral plasma cell membrane of thyrocytes via the cells, at diagnosis, allowed the establishment of the RA- þ NA /I (sodium iodine) symporter (NIS; ref. 4). NIS and sensitive FTC133 cell line (22). In this study, we show that other proteins linked to the synthesis of thyroid hormones, ligands of RARs or RXRs exert different effects in thyroid such as TG, thyroid peroxydase (TPO), and type I iodothyr- cancer cells through either differentiation or inhibition of onine 50-deiodinase (DIO1) have been reported to be growth and invasion and may thus provide different ther- poorly expressed in thyroid cancer patients (5). Reduced apeutic approaches in DTC patients. Drug effects were expression of these differentiation proteins results from Ras associated with restoration of RARB and RXRG levels. Inter- mutations and alterations of transcription factors involved estingly, bexarotene inhibits the transactivation potential of in thyroid differentiation such as loss of TTF1 expression or NF-kB through the involvement of nuclear coactivator chromosomal translocation of paired box 8 (PAX8) with p300, without interfering with either nuclear translocation peroxisome proliferator-activated receptor gamma (PPARg; or binding of p65 to NF-kB–responsive elements. This study refs. 6, 7). highlights in a given cancer dual mechanisms by which Retinoids, a group of structural and functional derivatives retinoids may target cell tumorigenicity, not only via targets of vitamin A are known to regulate a large number of of retinoids such as RARs and RXRs, as expected, but also via essential biological processes, such as cell growth, differen- targets of the NF-kB pathway. tiation, and death. The effects are mainly mediated by 2 types of nuclear receptors—retinoic acid (RA) receptors Materials and Methods (RARs) and retinoid X receptors (RXRs)—which act as ligand-dependent transcription factors. By qualitative Reagents and chemicals mRNA detection assays, expression of retinoid receptors Bexarotene (LGD1069) was kindly provided by Ligand (RARA, RARB, RARG, and RXRA and RXRB) has been Pharmaceuticals. ATRA and 13-cis RA were purchased from detected in normal thyrocytes, suggesting that absence of Sigma-Aldrich. The powdered retinoid was dissolved in a tissue-specific retinoid receptor may participate in differ- ethanol at an initial stock concentration of 10 2 mol/L, entiation arrest and thyroid oncogenesis (8). Reduced stored protected from light at 80C, and further diluted expression of RARb has indeed been observed in human before use. JSH-23 was purchased from Santa Cruz Biotech- thyroid carcinomas (9). RA therapy in malignant cells is nology and was dissolved in dimethyl sulfoxide at an initial based on functional alternative RA signaling pathways stock concentration of 10 2 mol/L. which, upon pharmacologic concentrations of a given retinoid, restore control of cell death, differentiation, and Cell culture and treatment proliferation. Various mechanisms are involved including Human FTC cell lines FTC133 and FTC238 were kindly the transcriptional control of the tissue-specific retinoid provided by C. Schmutzler (Dusseldorf, Germany). Human receptor gene via endogenous receptors. We have shown breast cancer cell line MCF7 and FTC cell lines were prop- the upregulation of RARb in neuroblastoma and glioblas- agated in Dulbecco’s modified Eagle’s medium/Ham’s

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F12 (1:1; DMEM-F12; Invitrogen) supplemented with 10% done for each gene of interest and the porphobilinogene (v/v) or 5% (v/v) heat-inactivated FBS and penicillin (100 deaminase (PBGD) reference gene to normalize for input units/mL), streptomycin (100 ng/mL), and L-glutamine (2 cDNA. To quantify the gene expression profile, we used the mmol/L). Human acute promyelocytic leukemia NB4 was comparative threshold cycle (Ct) method. propagated in RPMI-1640 medium supplemented with 10% (v/v) FBS and penicillin (100 units/mL) and strepto- Western blot analysis mycin (100 ng/mL) and L-glutamine (2 mmol/L). All cul- Nuclear extracts were obtained as previously described tures were incubated at 37 C in a 5% CO2 humidified (23) and were quantified by the BCA protein assay (Pierce). atmosphere. For retinoids treatment, cells were split the Proteins were run in SDS-PAGE gels, transferred to nitro- day before treatment and cultured with serum-containing cellulose membranes. Membranes were blocked in 5% medium. On the next day, the medium was replaced nonfat dry milk in 1 PBS at room temperature. The blots with retinoid-containing or retinoid-free medium with were incubated overnight at 4C with the primary antibody serum. anti-p65 (Upstate; Millipore). After washes with 1 PBS with 0.1% Tween 20, antigen–antibody complexes were Cell viability assay (MTS assay) detected by means of peroxidase-conjugated secondary Cells were plated in triplicate at 500 cells for FTC238 cells, antibody and an enhanced fluorochemiluminescence at 1,000 cells for FTC133 and MCF7 cells and at 10,000 cells system (ECL-plus; Amerscham Biosciences). Equivalent for NB4 cells per well in 96-well plates. Cell viability was loading of lanes was controlled by an anti-actin antibody measured by the Cell Titer 96 Aqueous One Solution Assay (Sigma-Aldrich). Band intensities were quantified using a (Promega) according to the manufacturer’s instructions. ChemiDoc XRS and Quantity One version 4.4.0 software.

Invasion assay siRNA transfection The invasiveness of FTC cells was tested using the QCM siRNA against RXRG gene (siRXRG) and nontarget con- Collagen Cell Invasion Assay (Chemicon; Millipore). Cells trol siRNA (siCRT) were purchased from Santa Cruz Bio- were seeded in serum-free medium containing bexarotene technology. siRNAs were transfected into cells at a final to the upper collagen-coated transwell. The lower chambers concentration of 50 nmol/L by using the transfection were filled with culture medium containing 10% FBS as a reagent Lipofectamine RNAiMax (Life Technologies; Invi- chemoattractant. After a 72-hour incubation period, the trogen) according to the manufacturer’s instructions. Anal- number of cells that migrated through the filters into the yses were realized after 48 hours of transfection. lower chamber was evaluated by a colorimetric method. Briefly invaded cells on the bottom of the insert membrane Promoter activity assay are incubated with Cell Stain Solution, then subsequently For cells transfections, FTC238 cells were seeded in 24- extracted and detected on a standard Microplate reader wells plates. After reaching about 80% confluence, the cells (560 nm). were transfected with luciferase gene in the reporter plasmid controlled by a synthetic promoter that contains direct Cell-cycle and apoptosis analysis repeats of the transcription recognition sequences for For cell-cycle analysis, FTC238 cells were permeabilized NF-kB (pNF-kB-luc; Stratagene) using Lipofectamine with 0.1% triton in 1 PBS and incubated with 0.5 mL of (Lipofectamine Reagent Plus; Invitrogen) according to the a50mg/mL propidium iodide (PI) solution containing manufacturer’s instructions. Cotransfection with a b-galac- 20 U/mL RNAse-A for 30 minutes (Sigma-Aldrich). For tosidase plasmid (pSV-b-gal control vector; Promega) was apoptosis, cells were double stained with fluorescein iso- used to normalize luciferase activity. After transfection, the thiocyanate–conjugated Annexin V and PI for 15 minutes at medium was replaced by fresh normal growth medium with 4C in a calcium-enriched binding buffer (Beckman Coul- retinoid and the cells were incubated for 24 hours. Lucif- ter). Allanalysis was done on a FACS Calibur Flow Cytometer erase (Luciferase assay system; Promega) and b-galactosi- with Cell Quest software (Becton Dickinson, France). dase (b-Gal Reporter Gene Assay; Promega) assay were done by a standard procedure. RNA isolation and real-time PCR Total RNA was extracted as described by the manufacturer DNA-binding assay (RNA-PLUS; Qbiogene). Reverse transcription was done on Assay for NF-kB binding was done with the colorimetric 1 mg of total RNA using the High Capacity RT Kit (Applied Universal EZ-Transcription Factor Assay (Upstate; Milli- Biosystems). cDNAs were quantified using a fluorescence- pore) according to the manufacturer’s instructions. Briefly, based real-time detection method (ABI PRISM 7700 the capture probe, a double strand biotinylated oligonu- Sequence Detection System; Applied Biosystems) with the cleotide containing a NF-kB consensus binding site, is TaqMan Universal Master Mix. The primers and probe mixed with nuclear extract in the streptavidin-coated 96- sequences for RARs and RXRs are provided as Supplemen- well plate. Bound NF-kB was detected by incubation of tary information (Table S1). Others primers and probe were samples with primary antibody against the p65 subunit provided by Applied Biosystems (Gene Expression Assay; provided with the kit. A horseradish peroxidase (HRP)- Courtaboeuf). For each sample, parallel PCR reactions were conjugated secondary antibody is then used for detection.

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Quantification of binding levels was evaluated by optical received 13-cis RA for 8 weeks (1 mg/kg/d), followed by intensities at 450 nm with a Microplate reader. A specific RI treatment (3.7 GBq) after 4 weeks of LT4 withdrawal. unlabeled competitor oligonucleotide was included to Treatment was repeated when reinduction of RI uptake on ensure that the complex is binding the kB site in a posttherapy scan was obtained. In the absence of RI uptake, sequence-specific manner. a further regimen with bexarotene (300 mg/m2/d) for 8 weeks was undertaken, then followed by RI treatment (3.7 Chromatin immunoprecipitation assays GBq) after rhTSH stimulation, to avoid associated central Cells were fixed with 1% formaldehyde in media for 10 hypothyroidism (24). To become eligible to bexarotene, minutes at 37C to cross-link DNA with proteins. Then, the patients were required to have acceptable organ function cells were washed twice with PBS1X containing proteases defined as follows: adequate hematologic function, defined inhibitors. Cells were resuspended in lysis buffer and son- as WBC 3,000/mL, absolute neutrophil count 1,500/mL, icated (Bioruptor; Diagenode) to obtain DNA fragments to and platelet count 100,000/mL; normal coagulation para- 200 to 1,000 bp. After preclearing with salmon sperm DNA/ meters; bilirubin 1.5 times the upper limit of normal protein G agarose beads (Upstate; Millipore), the samples (ULN); AST/ALT 2.5 ULN; and serum creatinine 2.5 underwent immunoprecipitation with antibodies specific ULN (or creatinine clearance > 40 mL/min). In addition, against p65, p300, acetylated histone H3, acetylated histone only patients who had a normal baseline fasting triglyceride H4 (Upstate; Millipore), or rabbit/mouse IgG at 4C over- level were allowed to enter this study; triglycerides could be night. DNA–protein complexes were sequentially washed, normalized before study entry with use of an antilipemic eluted (1% SDS, 50 mmol/L NaHCO3), and cross-link agent. Response was immediately judged on RI reinduction reversed by heating at 65C overnight. DNA was recovered on posttherapy scan, and 4 to 6 months later on TG levels by proteinase K digestion, phenol-chloroforme purifica- and tumor size by conventional imaging techniques accord- tion, and ethanol precipitation. DNA was amplified by ing to RECIST criteria (25). real-time PCR using the Power master mix SYBR green on a ABI PRISM 7700 Sequence Detection System (Applied Results Biosystems). Primers sets were designed to overlap the NF-kB–binding sites of the CYR61 promoter and the RARE Distinct differentiation potential by ATRA, 13-cis RA, site of the RARB2 promoter. Specificity of the assay was and bexarotene in FTC cell lines tested by amplification of an intergenic region (between the FTC133 and FTC238 cells provide a well-established GAPDH gene and the chromosome condensation–related model for the study of differential thyroid cancer retinoid SMC-associated protein gene) not bound by transcription sensitivity based on different criteria, such as thyroid-spe- factors (negative controls). The primers sequences are pro- cific functions (DIO1 and NIS expression), cell–cell or cell– vided as Supplementary Information (Table S1). Data for matrix interaction (intercellular adhesion molecule-1 and each immunoprecipitated sample were normalized to its E-cadherin), differentiation markers (alkaline phosphatase, corresponding input (total chromatin fraction) and CD97 and TG), cell growth, and tumorigenicity (26). To expressed as fold enrichment above background (IgG con- characterize and compare the cell lines used for this study, trols) or as fold change compared with untreated sample we quantitatively assessed the expression of genes associ- using the comparative Ct method. ated with thyrocyte differentiation [PAX8, TPO, thyroid stimulating hormone receptor (TSHR), DIO1, NIS and TG; Statistical methods ref. 5]. Increased expression of these differentiation-associ- All data were obtained from at least 3 independent ated genes was observed in FTC133 cells after 24 hours of experiments conducted in triplicate, and the results are treatment with ATRA 10 6 mol/L. In contrast, no difference presented as mean and SEM. To show statistical significance, was observed in the FTC238 cell line (Fig. 1A). Thus, a 2-tailed Student t test was used ( denotes a P value of at FTC238 remains refractory to ATRA differentiation induc- least < 0.05). tion contrary to the FTC133 cell line. After treatment of FTC238 cells with 13-cis RA or bexarotene, currently used Clinical study retinoids in thyroid cancer patients, no upregulation In parallel to these in vitro studies, 17 patients (8 male and of thyroid differentiation markers was noted (Fig. 1A), 9 female, median age at diagnostic 50 years), with progres- correlating with absence of morphologic aspects of differ- sive metastatic DTC and decreased or absent RI uptake, were entiation as observed in FTC133 cells, which harbor a more included in a prospective open clinical trial (CRIC99340). homogenous and spindle-shaped aspects after ATRA or These patients (9 PTC and 8 FTC including 2 Hurthle and 1 13-cis RA treatment (Supplementary Fig. S1). insular tumor) had previously received multiple treatments: high cumulative RI dose (median 13.7 GBq), surgery of Upregulation of RARB and RXRG expression correlates metastatic sites (#8), External Beam Therapy (#3), cimen- with achievement of ATRA differentiation in FTC cell toplasty (#1), and bisphosphonate (#1). Group 1 (n ¼ 13) lines had pulmonary, bone and/or mediastinal macrometastases We have previously shown that FTC133 and FTC238 cell and group 2 (n ¼ 4) had lung and/or mediastinal infra- lines present, respectively, low and null expression of RARB centimetric metastases. After informed consent, they all mRNA levels (21). In this study, we examined the gene

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(27). Upregulation of RARs and RXRs gene expression was A monitored in the 2 cells lines after incubation with ATRA 70 10 6 mol/L. ATRA induced a significant RARB and RXRG 60 NIS DIO1 TG TPO mRNA expression in only the RA differentiation–sensitive 50 TSHR PAX8 FTC133 cell line but not in RA-resistant FTC238 cells (Fig. 40 1C). Thus ATRA differentiation refractoriness of the FTC238 30 cell line may be related to absence of RARB and RXRG 20 expression and its induction by ATRA. 10 0 Bexarotene induces RARB and RXRG expression and mRNA expression (fold increase) (fold mRNA expression ATRA 13-cis RA Bexarotene ATRA 13-cis RA Bexarotene inhibits cell growth and invasion in ATRA-resistant FTC133 FTC238 FTC238 cells RARs and RXRs gene expression was monitored in both B 4 FTC133 cell lines after incubation with either 13-cis RA or bexar- 3.5 FTC238 3 otene. These retinoids were not more effective than ATRA to 2.5 induce RARs and RXRs expression in FTC133 cell line (data 2 not shown). Interestingly, in the ATRA-resistant FTC238 1.5 cells, both retinoids allowed a significant recovery of RARB 1 and RXRG expression levels (Fig. 2A). This upregulation of 0.5 retinoid receptors by bexarotene could not be linked to Relative mRNA expression 0 RARA RARB RARG RXRA RXRB RXRG induction of differentiation-associated genes (Fig. 1A) but to a decrease of cell growth in a dose-dependent manner C 10 9 FTC133 ATRA (Fig. 2B). Inhibition of cell growth by bexarotene in FTC238 FTC238 * 8 cells was not due to apoptosis, as no increase in the per- 7 centage of Annexin V–positive cells was noted after incu- 6 * 5 bation with bexarotene (Fig. 2C) but, rather, to an inhibi- 4 tion of proliferation. Cell population in the G1 phase 3 2 increased from 75.4% 1.3% to 89.8% 5.5% upon 1 bexarotene (P ¼ 0.002). This increase was accompanied by 0 mRNA expression (fold increase) (fold mRNA expression RARA RARB RARG RXRA RXRB RXRG a decrease of cell population in the S/G2/M phase (13.2% 4.3% compared with 23.4% 4.2%, P ¼ 0.01; Fig. 2D). The small increase in subG1 population was not significant Figure 1. ATRA-induced differentiation is correlated with RARB and RXRG confirming the low level of Annexin V–positive cells after expression levels in FTC cells lines. A, distinct thyroid differentiation bexarotene. As the FTC238 cell line is derived from a marker induction by retinoids in FTC cells lines after 24 hours treatment 6 6 6 pulmonary metastasis, we also investigated the invasive with ATRA 10 mol/L, 13-cis RA 10 mol/L, bexarotene 10 mol/L or vehicle. B, RARs and RXRs mRNA expression levels under basal activity of these cells by their ability to migrate through a conditions in FTC cell lines. mRNA levels were measured by quantitative collagen layer. A 30% decrease of invasion was observed RT-PCR normalized for the PBGD gene as reference. Results are upon bexarotene treatment (P ¼ 0.02; Fig. 2E). Thus, expressed as relative mRNA expression using comparative Ct method. C, bexarotene restores RARB and RXRG expression in FTC238 RARs and RXRs mRNA induction after 2 hours treatment with ATRA 10 6 mol/L in FTC133 and FTC238 cells. mRNA levels were measured by cell line and inhibits cell proliferation and invasion but quantitative RT-PCR normalized for the PBGD gene as reference. Results does not increase differentiation. are expressed as fold increase based on the expression observed in the C absence of ATRA (arbitrarily set at 1) using comparative t method. Bexarotene effectiveness in DTC patients (P < 0.05). In a clinical proof-of-concept open clinical trial, 17 DTC patients with progressive metastatic DTC and decreased or expression levels of the different RARs and RXRs by quan- absent RI uptake were first treated by 13-cis RA for 8 weeks titative reverse transcriptase PCR (RT-PCR). RARA, RARG, (1 mg/kg/d), followed by RI treatment (3.7 GBq) after 4 RXRA, and RXRB are expressed in both cells lines, whereas weeks of LT4 withdrawal. Characteristics and clinical RARB and RXRG are barely detectable. RARA mRNA is the response of patients are presented in Supplementary Table major retinoid receptor mRNA detected in FTC133 cells, S2. Clinical response was assessed on RI uptake and RECIST whereas RXRA is predominant in FTC238 cells. Globally, criteria (25). After one course of 13-cis RA, one patient had a compared with FTC133 cells, FTC238 cells have a lower partial response (PR), 7 patients a stable disease, and 9 expression of RARs and a higher expression of RXRs patients a progressive disease. Treatment with 13-cis RA (Fig. 1B). induced RI uptake in metastases of 6 of 17 (35%) patients, In cancer cells with altered RA receptors, differentiation irrespective of the clinical response, though the patient with induced by pharmacologic concentrations of RA is associ- a PR did have also induced RI uptake (patient no. 1). All ated with the upregulation of receptor expression via acti- responding patients had macroscopic metastatic tumors vation of endogenous normal receptors by the retinoids (group 1). Of these 6 patients, 3 received a second 13-cis

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A 25 ATRA Figure 2. Bexarotene restores FTC238 RARB RXRG 13 cis RA * and expression and 20 Bexarotene reduces cell proliferation and * invasion in ATRA-resistant FTC238 cells. A, RARs and RXRs mRNA 15 expression after 2 hours treatment with bexarotene 10 6 mol/L were 10 * * calculated by quantitative RT-PCR via comparative Ct method, using PBGD as reference gene. Results 5 are expressed as fold induction based on the basal expression

mRNA expression (fold increase) (fold mRNA expression 0 observed in the absence of retinoid RARARARB RARG RXRA RXRB RXRG (arbitrarily set at 1). B, cell viability at day 6 under indicated concentrations of retinoid was BC measured by an MTS assay. 0.1 µmol/L 1 µmol/L 10 µmol/L Untreated Bexarotene Positive control Results are expressed as 5 1.8 ± 1.8% 2.9 ± 1.8% 12.6 ± 2.7% 0 percentage of cell viability –5 compared with the absence of

–10 PI retinoids. C, cells were treated for 6 6 –15 days with bexarotene 10 mol/L. –20 * Cells were incubated with Annexin –25 * V and PI and then analyzed by flow –30 cytometry. For the positive control, –35 we incubate cells in 3% Viability/untreated (%) –40 ATRA Annexin V formaldehyde during 30 minutes. Bexarotene Values represent apoptotic cells defined as Annexin Vþ/PI cells. D E D, cell cycle was analyzed by flow Untreated Bexarotene 0.7 cytometry with PI after 6 days G G 1 1 treatment with bexarotene 10 6 0.6 150 150 mol/L. E, cells were plated in 0.5 120 120 * serum-free medium in a Transwell S/G /M S/G /M 2 2 0.4 and treated for 3 days with 80 80 bexarotene 106 mol/L. Invasion Counts Counts 0.3 60 60 capacity was measured by 0.2 30 30 colorimetric reaction. Optical 0.1 density (DO) at 560 nm was

0 0 Invasion (OD 560 nm) 0 200 400 600 0 200 400 600 0.0 correlated to the number of cells Untreated Bexarotene invading through PI PI the collagen layer in transwell. G1 75.4 ± 1.3% G1 89.8 ± 5.5% ( P < 0.05). S/G2/M 23.4 ± 4.2% S/G2/M 13.2 ± 4.3%

RA treatment, also followed by a new RI reinduction. metastasis heterogeneity led to differential metastases Responses remained the same. Of note 2 of these patients responses in a given patient. had a tumor size reduction of more than 50% (patients no. 1 and 3). Bexarotene treatment was undertaken in 5 patients Bexarotene inhibits transactivation and expression of who did not respond to the ﬁrst course 13-cis RA and were NF-kB target genes eligible for bexarotene. Two patients (patients no. 8 and 9) To further analyze the mechanisms by which bexarotene presented an increase in RI uptake, suggesting that resis- inhibits FTC238 cell growth and invasion, we focused our tance to 13-cis RA could be bypassed in some metastases by studies on the NF-kB pathway. NF-kB, a transcription factor bexarotene as illustrated in Fig. 3. These 2 patients had known to be a key regulator of genes involved in the control macroscopic metastatic tumors (group 1). The RI uptake of proliferation, apoptosis, and invasion, is constitutively responses noted in group 1 with macroscopic metastases activated in most of cancers (28). Moreover, members of the may well be explained by detection limitations. Tumor nuclear receptor family have been shown to interfere with regression or stabilization was noted by conventional imag- NF-kB signaling (29). We thus hypothesized that the acti- ing techniques in 3 other patients treated by bexarotene but vation of the RXR pathway by bexarotene could interfere with no increased RI uptake (patients no. 10, 14, and 15). with NF-kB and analyzed the expression of genes known to Thus, tumor response was not always correlated to RI be downstream effectors of NF-kB (Fig. 4A). Bexarotene uptake correlating with the in vitro data. Furthermore, treatment decreased the expression of MYC, CCND1,

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A B C

Figure 3. Posttherapy scan. A, before treatment. B, after 8 weeks of 13-cis RA treatment: no RI uptake at expected metastatic sites. C, after 8 weeks of bexarotene treatment: RI uptake at cervical metastatic lesion subsequently conﬁrmed by positive cytology.

Before treatment After 13-cis RA After bexarotene

CYR61, and CXCL1 genes involved in proliferation and the effect of bexarotene on different steps of the NF-kB invasion but not the expression of BCL2 and XIAP genes, pathway (Fig. 5A). First, we analyzed the translocation of correlating with the inhibition of proliferation and invasion the active p65 subunit of NF-kB to the nucleus compart- and absence of apoptosis observed upon bexarotene treatment. No significant change in p65 levels in nuclear extracts ment (Fig. 4B). This mechanism requires the presence of was noted after treatment, suggesting that bexarotene medi- RXRG expression restored by bexarotene, as the inhibition ated its effect on NF-kB repression at the transcriptional of NF-kB target genes expression by bexarotene is not level (Fig. 5B). However, p65 binding on a synthetic NF-kB– observed following downregulation of RXRG by siRNA (Fig. responsive element was not modified in nuclear extracts of 4C). To further analyze NF-kB activity in FTC238 cells, we treated cells in the presence or absence of both PMA (Fig. transiently transfected FTC238 cells with a luciferase report- 5C). Furthermore, chromatin immunoprecipitation (ChIP) er plasmid under the control of a synthetic promoter that assay using anti-p65 antibody on NF-kB–binding sites of contains direct repeats of the transcription recognition CYR61 promoter gene confirmed these results (Fig. 5D). sequences for NF-kB. Bexarotene repressed both constitu- Thus these data suggest that bexarotene represses transacti- tive basal and phorbol myristate acetate (PMA)-induced vation of NF-kB genes without interfering with either the NF-kB transactivation activity (Fig. 4D). Thus bexarotene, translocation or the binding of p65 to NF-kB–responsive through induction of RXRG, represses transcriptional activ- elements. ity and target gene expression of NF-kB and results in loss of Interestingly, by ChIP assay, the coactivator p300 was cell growth and invasion. A decrease of proliferation may detected on the p65-binding site of CYR61 promoter gene also be obtained on these cells by inhibiting NF-kB pathway (Fig. 5E). As expected, bexarotene induced p300 recruit- by a known chemical inhibitor JSH-23, confirming the ment at the RARE with increased level of histones H3 and biological effect of activated NF-kB pathway in these cells H4 acetylation (Fig. 5F). However, at p65-binding sites, (Fig. 4E). bexarotene released p300 (Fig. 5E) and decreased levels of Interestingly, bexarotene targeting of the NF-kB pathway histone acetylation (Fig. 5F) resulting in reduced promoter may also be observed in other thyroid cancer cells (FTC133) permissiveness. Thus, in thyroid cancer cells, bexarotene and in acute myeloid cells (NB4), albeit in different degrees represses NF-kB target gene expression through downregu- but not in breast cancer cells (MCF7). Inhibition of cell lation of the transactivation potential of p65 on its binding proliferation was essentially achieved in the NB4 cell line site via the bexarotene–RXR–dependent release of the (Supplementary Fig. S2A), whereas a decrease of invasion nuclear coactivator p300 and resulting in histone capacity was observed in FTC133 cell line (Supplementary deacetylation. Fig. S2B). Reduced expression of NF-kB proliferation and invasion target genes was noted in a cell-context manner Discussion (Supplementary Fig. S2C). RA therapy has been attempted in thyroid cancer patients Bexarotene represses NF-kB pathway at the essentially to restore iodine uptake for treatment of recur- transcriptional level rent metastases by radioactive iodine. We confirmed similar To explore the mechanism(s) whereby bexarotene response rate on RI uptake (35%) obtained by others groups induces NF-kB repression in FTC238 cells, we examined (30 to 40% refs. 14–16). Increased RI uptake in thyroid

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Cras et al.

A B 1.6 1.4 NF-κB 1.2 1 0.8 * Proliferation Invasion Apoptosis Figure 4. Bexarotene represses CCND1 CYR61 BCL2 0.6 * * transactivation and expression of C-MYC CXCL1 XIAP 0.4 * NF-kB target genes. A, genes regulated by NF-kB and involved in 0.2 the control of proliferation,

mRNA expression (fold change) 0 apoptosis, and invasion. B, mRNA – CCND1 CMYC CYR61 CXCL1 Bcl-2 XIAP expression of NF-kB regulated genes after 2 hours treatment with bexarotene 10 6 mol/L measured 2.5 C by quantitative RT-PCR. Results CCND1 CMYC CYR61 CXCL1 are expressed as fold change 2.0 based on the basal expression observed in the absence of 1.5 bexarotene (arbitrarily set at 1) via C * comparative t method, using PBGD as reference gene. C, after 1.0 48 hours of transfection with a RXRG siRNA, cells were treated 0.5 with bexarotene for 2 hours and RT-qPCR for NF-kB target genes

mRNA expression (fold change) carried out. D, cells transiently 0.0 – siCRT siCRT siRXRG transfected with a NF-kB binding sites luciferase construct were Untreated Bexarotene exposed to bexarotene 106 mol/L or/and PMA 10 ng/mL for 24 hours. Luciferase activity was normalized p50 p65 with b-galactosidase activity. D Luciferase E E, cell viability at day 6 under NF-κB site indicated concentrations of JSH- 7.106 * * 23 was measured by an MTS 15 0.1 µmol/L 1 µmol/L 10 µmol/L 6 assay. Results are expressed as 6.10 10 percentage of cell viability 5.106 5 compared with the absence of 6 0 4.10 drug. ( P < 0.05). –5 3.106 * * –10 6 2.10 –15

6 Viability/untreated (%) Luciferase activity 1.10 –20 0 Bexarotene – + – + PMA – – + +

cancer may be explained by NIS expression. Induction of a novel biomarker of RA therapy. We identified, bexarotene, NIS expression by RA in breast cancer cells was reported to the first highly selective RXR ligand to be currently studied be mediated by genomic and/or nongenomic mechanism in clinical trial (33–35) as a growth inhibitor of the RA requiring RARs and RXRs expression (30, 31). Because differentiation–resistant FTC238 cell line, to an extent not bexarotene increases RARB and RXRG expression in FTC238 achieved with ATRA. In cutaneous lymphoma cell lines, cells without induction of NIS, it seems that thyroid cancer bexarotene induces arrest of cell cycle in G1 phase and cells could have other regulation mechanisms of NIS apoptosis with activation of caspase-3 and PARP cleavage, expression. as well as downregulation of survivin (36). A proof-of- Using FTC cell lines with differential response to reti- principle clinical trial with bexarotene in patients with noids, we have previously identified that one of the mech- non–small cell lung cancer showed regression of biomar- anism inherent to RA resistance may lie in the nonpermis- kers such as cyclin D1, cyclin D3 and epidermal growth sive status of the RARB gene promoter, relieved by histone factor receptor (37). In this study, we show that bexarotene deacetylase inhibitors (21). This study suggests that along downregulates MYC and CCND1 expression in the ATRA- with RARb, RXRg may equally be involved in thyroid resistant FTC238 cells, extending the notion that rexinoids oncogenesis, as previously suggested (32) and represents can bypass absence of RARb expression. Interestingly, in

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Bexarotene Represses NF-kB Signaling

A ⊕ B PMA P IkBα Proteasome Bexarotene p50 p65 0 15’ 30’ 1 h 2 h 6 h PMA

Cytoplasm p65 Nucleus Actin

p50 p65 Ratio 1 1.40.950.930.990.951.01

DNA NF-κB site

C D * * 6

0.8 5 * 4 * 0.6 3 0.4 2

0.2 1 p65 binding (fold change) p65 binding (OD 450 nm) 0 0 Bexarotene – + – + Bexarotene – + – +

PMA – – + + PMA – – + +

E F

50 * 2.5 * 10 Untreated

40 Untreated

Bexarotene * Bexarotene 35 2.0 8 30 25 1.5 6 20

1.0 4 15 *

Fold change * above background 10 *

p300 fold enrichment 0.5 2 5 0 0.0 0 CYR61 RARB2 CYR61 RARB2 CYR61 RARB2

H3 acetylation H4 acetylation

Figure 5. Bexarotene represses NF-kB pathway at the transcriptional level. A, schematic representation of NF-kB pathway. B, cells were treated with bexarotene 10 6 mol/L for the indicated times. Nuclear extracts were prepared, fractioned on SDS-PAGE and electrotransferred to nitrocellulose membranes. Immunoblots were done with an anti-p65 antibody. Anti-actin antibody was used to control equivalent protein loading. C, nuclear extracts of 2-hour bexarotene and/or PMA-treated cells were incubated with an oligonucleotide containing a NF-kB consensus binding site. Bound NF-kB was detected by incubation of samples with an anti-p65 antibody and detected by colorimetric reaction using an HRP-conjugated secondary antibody. D, cells were exposed to bexarotene 106 mol/L or/and PMA 10 ng/mL for 2 hours. Cross-linked protein–DNA complexes were immunoprecipitated with anti-p65 antibody. Primers flanking the NF-kB–binding site of the CYR61 promoter were used in quantitative PCR amplification. Samples were normalized to their respective input and values expressed relative to untreated counterparts. E, cross-linked protein–DNA complexes were immunoprecipitated with anti-p300 antibody. Primers flanking the NF-kB–binding site of the CYR61 promoter and the RARE of RARB2 promoter were used in quantitative PCR amplification. Samples were normalized to their respective input and expressed as fold enrichment above background (IgG controls). F, cross-linked protein–DNA complexes were immunoprecipitated with anti-acetylated histone H3 and anti-acetylated histone H4 antibodies. Samples were normalized to their respective input and values expressed relative to untreated counterparts. (P < 0.05).

these cells, bexarotene upregulates RARB and RXRG which growth inhibition by RXR ligands (38). Thus in parallel with ATRA treatment fails to do. It has previously been reported, ATRA differentiation efﬁcacy, which requires upregulation albeit in another histologic type, anaplastic thyroid cancers or restoration of RARb and RXRg levels, bexarotene growth that the presence of RARb and RXRg was a prerequisite for inhibition may also require the upregulation of both

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Cras et al.

receptors. This growth inhibition cannot be attributed to receptor family, including glucocorticoid, estrogen, proges- apoptosis but rather to cell cycle arrest in G1 phase in terone, and androgen receptors and, more recently, PPARg accordance with the downregulation of CCND1 expression. have been shown to inhibit NF-kB activity and to physically In line with these in vitro studies, our clinical results show interact with p65 in vitro (29). Furthermore, competition for that bexarotene treatment of thyroid cancer patients who DNA NF-kB response elements between NF-kB and other failed to respond to 13-cis RA therapy leads, in some transcription factors may occur. This was shown for the patients, not only to radioiodine uptake but also to a same sequence in the promoter of the human GnRH II gene reduction in size and number of metastases without appar- for p65 and RARa/RXRa (47). However in FTC cells, neither ent differentiation and radioiodine uptake. could we detect RXR in the p65 transcriptional complex by Past and current concept of metastases initiating tumor ChIP nor RXR/p65 interaction by coimmunoprecipitation. cells highlights the importance of cell adhesion proteins, These results may well be explained by the low expression of proteases, and angiogenesis. Retinoids and bexarotene have endogenous RXR in these cells. However, other mechan- been shown to inhibit angiogenesis and metastasis in lung isms may be present in FTC cells. Indeed, our data suggest and thyroid cancer murine models (39, 40). RA was shown that bexarotene induces CYR61 promoter repression at the to decrease secretion of VEGF in thyroid tumor cells in vitro p65-binding site by releasing the p300 coactivator and (39). In our study, CYR61 and CXCL1, proteins known to be inducing histone deacetylation. In lipopolysaccharide-acti- involved in the invasion process, were downregulated by vated macrophages, Na and colleagues have also showed bexarotene in FTC238 cells and may explain the observed that retinoid-mediated suppression of the interleukin-12 decrease of cell invasion through a collagen layer. CXCL1 is production may involve both inhibition of NF-kB–DNA a small cytokine belonging to the CXC chemokine family interactions and competitive recruitment of p300 and SRC- involved in angiogenesis, inflammation, and tumorigene- 1 between NF-kB and RXR (48). Thus we can surmise that in sis. Overexpression of CXCL1 in melanocytes is associated FTC cells, the activation of RXR by its ligand, bexarotene, with constitutive activation of NF-kB leading to tumor leads to the release of p300 whether by competitive recruit- progression (41). CYR61, member of a family of secreted ment or by conformational change in the p65 transcrip- matrix-associated proteins, is described as a key regulator of tional complex, resulting in a histone deacetylation, pro- breast cancer invasion (42). The observed rapid downregu- moter repression, and a downregulation of NF-kB target lation of CYR61 gene expression induced by bexarotene in genes. FTC238 cells favors a direct transcriptional control. Response elements for both retinoid receptors dimers and fl NF-kB are present in the promoter region of the CYR61 Disclosure of Potential Con icts of Interest gene. As CYR61 is involved also in pathways leading to No potential conflicts of interest were disclosed. enhance NF-kB activation via integrins/PI3K/Akt (43), we may also surmise that in these cells, an autocrine loop Acknowledgments between CYR61 and NF-kB maintains tumorigenicity. NF-kB is known to be constitutively activated in thyroid The authors thank Ligand Pharmaceuticals for providing bexarotene and cancer cell lines and patient samples and linked to resistance Dr C. Schmutzler for FTC cells, as well as Dr P. Fourreau-Moreau, Dr B. Hamon, Dr MB. Hugues, Dr M. Lepage, Pr J. Orgiazzi, Dr AF. Rueff, Pr F. to apoptosis (44, 45). We found NF-kB constitutively acti- Borson-Chazot, Dr JP. Caravel, Dr M. Cavarec, Dr S. Denet, Dr B. Helal, and vated in FTC238 cells with high levels of p65 detected in the Pr M. Schlumberger for clinical study. nucleus and in protein complexes bound to NF-kB sites. Interestingly, incubation with bexarotene results in Grant Support decreased transactivation of an NF-kB reporter plasmid This work was supported by funds from the Institut National du Cancer strongly, suggesting that the inhibition of cell growth (INCa), the Ligue nationale contre le cancer (Comite de Paris), CRIC 99340 observed with bexarotene in FTC238 cells is achieved via (principal investigator M-E. Toubert), and the Association pour la Recherche repression of NF-kB and downregulation of genes such as sur le Cancer (ARC n 5397, principal investigator M-E. Toubert). The costs of publication of this article were defrayed in part by the MYC and CCND1. Although repression of NF-kB may payment of page charges. This article must therefore be hereby marked results from various mechanisms at the cytoplasmic and advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate nuclear level, in FTC238 cells, bexarotene did not inhibit this fact. p65 translocation to the nucleus nor its binding to NF-kB– responsive elements, contrary to results reported with fen- Received February 23, 2011; revised October 21, 2011; accepted retinide, a synthetic retinoid (46). Members of the nuclear November 16, 2011; published OnlineFirst December 5, 2011.

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OF12 Clin Cancer Res; 18(2) January 15, 2012 Clinical Cancer Research

Bexarotene via CBP/p300 Induces Suppression of NF-κB− Dependent Cell Growth and Invasion in Thyroid Cancer

Audrey Cras, Béatrice Politis, Nicole Balitrand, et al.

Clin Cancer Res Published OnlineFirst December 5, 2011.

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