Short Communication

Oncogene (2007) 26, 4018–4024. doi:10.1038/sj.onc.1210178; published online 8 January 2007

Epigenetic patterns of the retinoic acid receptor bold italic beta2 promoter in retinoic acid-resistant thyroid cancer cells

A Cras1,4, D Darsin-Bettinger1,4, N Balitrand1,2, B Cassinat1,2, A Soulié1, M-E Toubert1,3, L Delva1,5 and C Chomienne1,2,5

  1. 1INSERM, UMR S 718, Université Denis Diderot-Paris 7, Institut Universitaire d'Hématologie, Paris, France
  2. 2Unité de Biologie Cellulaire, Hôpital Saint-Louis, AP-HP, Paris, France
  3. 3Service de Médecine Nucléaire, Hôpital Saint-Louis, AP-HP, Paris, France

Correspondence: Professor C Chomienne, INSERM, UMR S 718, IUH, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France. E-mail: christine.chomienne@sls.ap-hop-paris.fr

4These authors contributed equally to this work.

5These authors share equal responsibility for this work.

Received 23 December 2005; Revised 11 October 2006; Accepted 20 October 2006; Published online 8 January 2007.

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Abstract

Treatment with retinoic acid (RA) is effective to restore radioactive iodine uptake in metastases of a small fraction of thyroid cancer patients. In order to find predictive markers of response, we took advantage of two thyroid cancer cell lines, FTC133 and FTC238, with low RA-receptor (RAR)beta expression but differing in their response to RA. We report that in both cell lines, RA signalling pathways are functional, as transactivation of an exogenous RARbeta2 promoter is effective in the presence of pharmacological concentrations of all-trans RA, and enhanced in RA-resistant FTC238 cells after ectopical expression of RARbeta, suggesting a defective endogenous RARbeta2 promoter in these cells. Further analyses show that whereas the RARbeta2 promoter is in an unmethylated permissive status in both FTC133 and FTC238 cells, it failed to be associated with acetylated forms of histones H3 or H4 in FTC238 cells upon RA treatment. Incubation with a histone deacetylase inhibitor, alone or in combination with RA, restored histone acetylation levels and reactivated RARbeta and differentiation marker Na+/I- symporter gene expression. Thus, histone modification patterns may explain RA-refractoriness in differentiated thyroid cancer patients and suggest a potential benefit of combined transcriptional and differentiation therapies.

Keywords:

RARbeta2 promoter, RA-resistance, thyroid cells, histone acetylation

Abbreviations:

ATRA, all-trans-retinoic acid; AZA, 5-aza-2'-deoxycytidine; DTC, differentiated thyroid cancer; FTC, follicular thyroid carcinoma; NIS, Na+/I- symporter; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; TG, thyroglobulin; TPO, thyroid peroxidase; TSA, trichostatin A; 5'DI, 5'-deiodinase

Differentiated thyroid cancers (DTCs) account for around 80% of thyroid cancers. Two types of DTCs can be distinguished: differentiated follicular (FTC) and papillary (PTC) thyroid cancers. The treatment consists of complete thyroidectomy followed by radioiodine therapy (Mazzaferri and Jhiang, 1994). DTCs are of good prognosis and the overall survival rate at 5 years is of 85–90% (Hundahl et al., 1998). However, in approximately 15% of patients, the results of serum thyroglobulin (TG) levels, a tumoral marker and radioiodine whole body scan are discordant, suggesting the presence of thyroid cancer metastases, which have lost the ability to uptake iodine (Pineda et al., 1995). These patients are resistant to radioiodine therapy and have thus limited therapeutic alternatives. Treatment with 13-cis retinoic acid (RA) was shown to restore radioactive iodine uptake in thyroid cancer patients in two clinical trials (Grunwald et al., 1998; Simon et al., 2002). However, only one-third of the patients respond, prompting for a better understanding of thyroid oncogenesis and RA signalling pathways in thyroid cancer cells.

Thyroid oncogenesis is associated with altered cellular events, among which arrest of differentiation has been correlated to the reduction or loss of protein expression involved in specific thyroid function such as iodine uptake (Wynford-Thomas, 1993). Iodine uptake is a major parameter of thyrocyte differentiation, required for the biosynthesis of thyroid hormones, tri-iodothyroxine (T3) and thyroxine (T4). Iodide is transported across the basolateral plasma cell membrane of thyrocytes via the Na+/I- (sodium iodide) symporter (NIS) (Figure 1) (Filetti et al., 1999). NIS and other proteins linked to the synthesis of T3 and T4, such as TG, thyroid peroxydase (TPO) and type I iodothyronine 5'-deiodinase (5'DI), have been reported to be poorly expressed in thyroid cancer patients (Lazar et al., 1999). Lack of expression of these differentiation proteins results from absence of expression or genetic alterations of transcription factors such as thyroid transcription factor 1 and paired box domain transcription factor 8 (De Vita et al., 1998; Pasca di Magliano et al., 2000).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic representation of thyrocyte differentiation indicating key thyroid proteins (modified after Filetti et al. (1999)).

Full figure and legend (79K)

Retinoids play important roles in the development and differentiation of numerous cell types. By qualitative mRNA detection assays (Northern blots and reverse transcription–polymerase chain reaction (RT–PCR)), expression of RA receptors (RARalpha, beta, italic gamma and RXRalpha and beta) has been detected in normal thyrocytes (Schmutzler et al., 1998) suggesting that absence of a tissue-specific RARs may participate in differentiation arrest and thyroid oncogenesis. In human PTC (Rochaix et al., 1998) and thyroid cancer cell lines (Schmutzler et al., 1998), reduced expression of RARbeta is indeed observed. Abnormal RARs expression, through chromosomal translocation or deletion, has been described in different cancers (De Thé, 1996). However, absence of RARs in cell tumors may also result from epigenetic silencing, as previously described for the RARbeta gene in solid tumors, such as breast, head and neck or lung cancers (Widschwendter et al., 2000; Suh et al., 2002; Youssef et al., 2004b). RA-induced differentiation therapy in malignant cells is based on functional alternative RA signalling pathways that, upon pharmacological concentrations of a given retinoid, restore control of cell death, differentiation and proliferation. Various mechanisms are involved, including the transcriptional control of the tissue-specific RAR gene via endogenous receptors (Niles, 2004). Thus, in the treatment of epithelial tumors by 13-cis RA, RARbeta expression is restored both in vitro and in vivo (Sun and Lotan, 2002). We have shown the upregulation of RARbeta in neuroblastoma and glioblastoma by various natural and synthetic retinoids (Carpentier et al., 1997, 1999), and the transcriptional control of the normal RARalpha gene expression by all-trans RA (ATRA) treatment in acute promyelocytic cells (Chomienne et al., 1991). RA efficacy in DTC patients is supported by in vitro data, showing that retinoids induce upregulation of RARbeta gene and morphological modification of cells in the FTC cell line FTC133 (Schreck et al., 1994), accompanied by the upregulation of NIS mRNA levels (Schmutzler et al., 1997) and 5'DI activity (Schreck et al., 1994).

In order to explore the lack of response to RA therapy of a subgroup of DTC patients, we took advantage of a RA-resistant thyroid cancer cell line, FTC238, derived from the metastasis of the patient whose cells, at diagnosis, allowed the establishment of the RA-sensitive FTC133 cell line (Goretzki et al., 1990).

FTC133 and FTC238 cells provide a well-established model for the study of RA-differentiation based on different criteria, such as thyroid-specific functions (5'DI, NIS), cell–cell or cell–matrix interaction (intercellular adhesion molecule-1, E-cadherin), differentiation markers (alkaline phosphatase, CD97), growth and tumorigenicity (Schmutzler and Kohrle, 2000). In order to characterize and compare the cell lines used for this study, we quantitatively assessed the differentiation achieved by evaluating the level of expression of two thyrocyte genes, 5'DI and NIS, by quantitative real-time PCR. An important increase after 24 h treatment with ATRA 10-6 M of both differentiation markers is observed in FTC133 cells (25-fold increase for 5'DI and four-fold 10-fold increase for NIS) but not in FTC238 cells (four-fold increase for 5'DI and two-fold increase for NIS) (Figure 2a). In FTC238 cells, mRNA levels of RARbeta are barely detectable (16plusminus1 copies per mug of mRNA) compared to FTC133 (1643plusminus272 copies per mug of mRNA) (Figure 2b). Transcription of the RARbeta gene is controlled via specific retinoic acid response elements (RAREs) located in the 5' regulatory sequences of the RARbeta2 promoter (De Thé et al., 1990). In physiological conditions, binding of RA to the receptor activates transcription through a cascade of events, including release of corepressors, recruitment of co-activators and subsequent activation of the basal transcriptional machinery (Rochette-Egly, 2005). In cancer cells with altered RA receptors, differentiation induced by pharmacological concentrations of RA is associated with restored receptor expression via activation of endogenous normal receptors (Sun and Lotan, 2002). Accordingly, RARbeta transcript levels in FTC133 increase upon ATRA treatment from two-fold (1.7plusminus1.3) after 2 h to four-fold (3.8plusminus1.1) after 24 h, whereas no increase in RARbeta levels is detected in FTC238 cells (Figure 2c). These results are corroborated, at the protein level, by Western blot (Figure 2d) and by immunocytochemistry (Figure 2e). Thus, RARbeta transcript and protein are differentially expressed in both cell lines and their expression levels correlate with the sensitivity of the cell to RA-differentiation.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

ATRA-induced differentiation is correlated with RARbeta expression levels in FTC133 and FTC238 cells lines. (A) Quantitative 5'DI and NIS mRNA expression after 24 h treatment with ATRA 10-6 M. Total RNA was extracted following the manufacturer's instructions (RNA-PLUS Qbiogene, Illkirch, France). cDNA was generated from 1 mug of total RNA as described previously (Cassinat et al., 2000). Quantitative real-time PCR was performed by TaqMan Gene Expression Assays (Assays-on-demand) and TaqMan universal PCR master mix following the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). Assays were realized on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Courtaboeuf, France). The relative RNA expression levels were calculated via comparative Ct method, using the porphobilinogene deaminase gene (PBGD) as reference gene. Results are expressed as fold increase based on the basal expression in the absence of ATRA (arbitrarily set at 1). The data are expressed as meanplusminuss.d. of triplicate measurements from three independent experiments. (B) Qualitative and quantitative RARbeta mRNA expression under basal conditions. cDNAs were quantified as described previously (Bastie et al., 2004). The primers and probe sequences used were for RARbeta: primers, 5'-CTAAATACACCACGAATTCCAGTGCTGA-3' and 5'-CAGACGTTTAGCAAACTCCACGATCTTA-3', probe, 6FAM-5'-TCCGACTGGACCTGGGCCTCTGGG-3'-TAMRA; and for PBGD: primers, 5'-GGAGCCATGTCTGGTAACGGCA-3' and 5'-GGTACCCACGCGAATCACTCTCA-3', probe, 6FAM-5'-TGCGGCTGCAACGGCGGAAGAAA-3'-TAMRA. The data are expressed as meanplusminuss.d. of triplicate measurements from two independent experiments. (C) Quantitative RARbeta mRNA expression after treatment with ATRA 10-6 M. The data are expressed as meanplusminuss.d. of triplicate measurements from two independent experiments. (D) Western blot analysis of RARbeta protein expression. Cells were analysed after 2 and 4 days in the absence (-) or presence of ATRA 10-6 M (+). Nuclear extracts from cells were obtained as described previously (Delva et al., 1999) and quantified by the biocinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Proteins were run in 10% polyacrylamide gels and transferred to a nitrocellulose membrane. Blots were incubated overnight at 4°C with the polyclonal anti-RARbeta antibody (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antigen-antibody complexes were detected by means of peroxidase-conjugated secondary antibody and an enhanced fluoro-chemiluminescence system (ECL-plus, Amerscham Biosciences, Arlington Heights, IL, USA). Equivalent loading of lanes was controlled by a polyclonal anti-actin antibody (Sigma-Aldrich, Saint Quentin Fallavier, France). Western blotting analysis was repeated three times with similar results. (E) Immunocytochemical staining of RARbeta protein expression. Cells were cytospinned, fixed in acetone and incubated with BSA 2% and Tween 0.01%. The slides were incubated for 30 min with an immunoglobulin G control (a and d) or with the anti-RARbeta antibody (1:40) in untreated cells (b and e) or after 2 days with ATRA 10-6 M (c and f). Then, sequential 15 min incubations with antibody and alkaline phosphatase-labelled streptavidin (DAKO LSAB+System, Alkaline Phosphatase, Dako, Carpinteria, CA, USA) were carried out. Staining was completed after 10 min incubation with the substrate-chromogen solution. Finally, the slides were counterstained with Mayer's hematoxylin.

Full figure and legend (279K)

To study the functional RA pathways in these two cell lines, we used a RARbeta2 promoter combined to a luciferase reporter gene (De Thé et al., 1990). After transfection of the RARbeta2-luciferase reporter gene, luciferase activity was estimated under different concentrations of ATRA. Interestingly, the RARbeta2 promoter is transactivated in both cell lines, in a dose-dependent manner (Figure 3a). Thus, irrespective of their differentiation responsiveness to retinoids, both cell lines appear to possess functional RA signalling pathways. Transfection of expression plasmids for different RARs results in transactivation from exogenous RARE in various cell models, in the presence or absence of added ATRA (Idres et al., 2002). We thus performed a similar assay in FTC238 cells. The transfection of a pSG5-RARbeta expression plasmid in FTC238 cells restored RARbeta level (Figure 3b, inset), and increased transactivation of the RARbeta2 promoter (Figure 3b), even in the absence of ATRA (Figure 3c). These results suggest that, in the presence of low RA concentration (such as that present in culture medium), RA signalling pathways may be triggered in FTC238 cells when RARbeta levels are sufficient. These data lead us to consider that lack of RA efficacy and restoration of RARbeta expression in FTC238 cells may be due to abnormalities located at the endogenous RARbeta2 promoter region.

Figure 3.
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ATRA induces transactivation of the RARbeta2 promoter in both FTC133 and FTC238 cell lines. (a) Cells were transiently transfected with the RARbeta2-luciferase reporter gene (De Thé et al., 1990), using the calcium chloride procedure, as described previously (Rousselot et al., 1994). Co-transfection with the TK-beta-galactosidase plasmid was used to normalize luciferase activity. Cells were treated for 24 h with different concentrations of ATRA or vehicle. Luciferase and beta-galactosidase activity were determined in a luminometer by a standard procedure. (b) FTC238 cells grown in 24-well culture plates were transfected with 0.25 mug of the RARbeta2-luciferase reporter gene, 0.20 mug of TK-beta-galactosidase plasmid and different amounts of the pSG5-RARbeta expression plasmid using lipofectamine (Lipofectamine Reagent Plus, Invitrogen, France) according to the manufacturer's instructions. At 24 h after transfection, cell extracts were assayed for RARbeta expression by Western blot analysis (Inset. lane 1: FTC238, lane 2: RARbeta transfected FTC238, lane 3: RARbeta transfected COS as control) and for luciferase activity. (c) Transactivation from the RARbeta2-luciferase reporter gene in the presence of 1 mug of pSG5-RARbeta with or without ATRA in FTC238 cells. All results are expressed as fold increase based on the basal activity of the reporter gene observed in the absence of ATRA (arbitrarily set at 1). Meanplusminuss.d. of triplicate measurements from three independent experiments are shown.

Full figure and legend (83K)

Methylation of the RARbeta2 promoter is described in head and neck, breast, colon and lung cancers (Virmani et al., 2000; Widschwendter et al., 2000; Youssef et al., 2004a, 2004b), and suggests that epigenetic modifications may result in lack of RARs expression in some cancers (Momparler, 2003). Histone acetylation is also required for the transcription of RA target genes (Rochette-Egly, 2005). For example, in lung cancer cell lines, RA-refractoriness has been attributed to aberrant histones acetylation levels on the RARbeta2 promoter (Suh et al., 2002). Thus, it is likely that, in the FTC238 cell line, a non-permissive RARbeta2 promoter provides resistance to transcription initiation of RA target genes. To address this hypothesis, we first studied the methylation status of the endogenous RARbeta2 promoter by methylation-specific PCR (MSP) focusing on a sequence (+105 to +251) (Figure 4a) rich in cytosine guanine dinucleotides (CpG) and known to be hypermethylated in solid tumors (Cote et al., 1998). Interestingly, the endogenous RARbeta2 promoter is in a permissive unmethylated status in both FTC133 and FTC238 cells (Figure 4b). Concordingly, treatment with 5-aza-2'-deoxycytidine (AZA), a DNA methyltransferase inhibitor, does not induce an increase of RARbeta expression superior to baseline or after ATRA treatment levels (Figure 4b). These results indicate that, in FTC238 cells, RA-refractoriness and absence of RARbeta expression, whether spontaneously or after ATRA, does not result from a hypermethylated status of the RARbeta2 promoter.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Epigenetic status of the RARbeta2 promoter in FTC133 and FTC238 cells lines. (a) Schematic representation of the RARbeta2 promoter and the first exon region. Black circles represent the CpG dinucleotides. Arrows and numbers denote MSP and ChIP primer locations relative to the RARE and the transcription start site (+1) (De The et al., 1990). (b) MSP analysis. Genomic purified DNA was denatured and treated with a combination of sodium metabisulfite and hydroxyquinone. DNA was then desalted, desulfonated in the presence of NaOH and precipitated by ethanol. Modified bisulfite DNA was amplified by nested PCR using first the primer set sense (5'-GAAGTGAGTTGTTTAGAGGTTAGGA-3') and anti-sense (5'-CTATAATTAATCCAAATAATCATTTACC-3'). PCR products were then reamplified with the U-sense (5'-GATGTTGAGAATGTGAGTGATTT-3'), U-antisense (5'-AACCAATCCAACCAAAACA-3') and M-sense (5'-GTCGAGAACGCGAGCGATTC-3'), M-antisense (5'-CGACCAATCCAACCGAAACG-3') primers. Positive and negative controls were generated by bisulfite modifying CpG Genome universally methylated and unmethylated DNA respectively (Chemicon, Temecula, CA, USA). U: unmethylated. M: methylated. Quantitative RT–PCR analysis of RARbeta mRNA expression. Cells were treated for 48 h with AZA 10-6 M alone or in combination with ATRA 10-6 M for 16 h. RT–PCR was realised as described in Figure 2. Results are expressed as fold increase based on the basal expression in the absence of drugs (arbitrarily set at 1). Meanplusminuss.d. of triplicate measurements from two independent experiments are shown. (c) ChIP analysis of histone H3 and H4 acetylation status on the RARbeta2 promoter. ChIP assays were performed using reagents from Upstate (Lake Placid, NY, USA). Cells were treated for 6 and 16 h with ATRA 10-6 M and/or TSA 75 ng/ml, fixed with 1% formaldehyde to cross-link DNA with proteins, lysed and sonicated to obtain DNA fragments to 200–1000 bp. After pre-clearing with salmon sperm DNA/protein A agarose beads, the samples underwent immunoprecipitation with 5 mug of antibodies specific for acetylated histone H3 (Ac-H3; lysine 9 and 14) or for acetylated histone H4 (Ac-H4; lysine 5, 8, 12 and 16) at 4°C overnight. DNA–protein complexes were sequentially washed, eluted and crosslink reversed by heating at 65°C overnight. DNA was recovered by proteinase K digestion, phenol–chloroform purification and ethanol precipitation. DNA was amplified by quantitative real-time PCR using the Power master mix SYBR green (Applied Biosystems, Foster City, CA, USA) and primers sense (5'-TCATTTGAAGGTTAGCAGCCCGGGTA-3') and anti-sense (5'-TATTCTTTGCCAAAGGGGGG-3'). Control ChIP was performed without antibody. Results are represented as the fold change in H3 or H4 acetylation following treatment compared to untreated cells using the Ct method. Meanplusminuss.d. of three PCR analyses of an IP are shown. The result is representative of two independent experiments. (d) Quantitative RT–PCR analysis of RARbeta and NIS mRNA expression. RT–PCR was realised as described in Figure 2. Results are expressed as fold increase based on the basal expression in the absence of drugs (arbitrarily set at 1). Meanplusminuss.d. of triplicate measurements from two independent experiments are shown.

Full figure and legend (86K)

We then analysed the levels of acetylated histones H3 and H4 on the RARbeta2 promoter by chromatin immunoprecipitation (ChIP) assay using antibodies against the critical acetylated forms of histones H3 and H4 and primers selecting the RARbeta2 promoter (Figure 4a). In RA-sensitive FTC133 cells, ATRA significantly increased the levels of acetylated histone H3 from 6–16 h incubation with ATRA reaching seven-fold increase. In FTC238 cells, levels barely reached two-fold and only transiently (Figure 4c). In both cell lines, irrespective of their sensitivity to ATRA, levels of acetylated histone H4 on the RARbeta2 promoter remained low. An inhibitor of histone deacetylase such as trichostatin A (TSA) alone resulted in a significant increase of the acetylated histone H3 levels after 16 h incubation in FTC133 cells but had no effect on the levels of histone H4. In FTC238 cells, TSA increased by two-fold the acetylated levels of histone H3 and H4 (Figure 4c) reaching a five-fold increase in the presence ATRA and TSA (Figure 4c). A concomittant time-dependent increase of RARbeta and NIS expression was noted (Figure 4d), confirming transcriptional activity of the RA on these target genes. Thus, altered histone modification may explain RA-refractoriness in FTC238 cells and may be relieved by combining ATRA with histone deacetylase inhibitors.

The promising results of retinoid therapy in DTC relapse patients, which can no longer benefit from radioiodine therapy, prompts to further study combined therapeutical strategies for enhanced activation of RA signalling pathways. In this respect, this study based on an ATRA-resistant thyroid cancer cell line, provides the evidence that lack of RA-sensitivity in these patients may relate from altered transcription pathways involving histone acetylation, which may be revealed by combining differentiation and transcriptional therapies.

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Acknowledgements

We gratefully acknowledge C Schmutzler for providing FTC cells and H de Thé for plasmids. We express our thanks to J Maes and M Goodhardt for helpful discussions on chromatin immunoprecipitation assay. We thank E Savariau, member of the 'Service d'Infographie' of the IUH, for excellent artworks. This work was supported by funds from the Association pour la Recherche sur le Cancer (ARC) and the Ligue nationale contre le cancer (Comité de Paris). AC holds an INSERM Researcher Grant position.