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)β 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 RARβ2 promoter is effective in the presence of pharmacological concentrations of all-trans RA, and enhanced in RA-resistant FTC238 cells after ectopical expression of RARβ, suggesting a defective endogenous RARβ2 promoter in these cells. Further analyses show that whereas the RARβ2 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 RARβ 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.
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).
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 (RARα, β, γ and RXRα and β) 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 RARβ 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 RARβ 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, RARβ expression is restored both in vitro and in vivo (Sun and Lotan, 2002). We have shown the upregulation of RARβ in neuroblastoma and glioblastoma by various natural and synthetic retinoids (Carpentier et al., 1997, 1999), and the transcriptional control of the normal RARα 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 RARβ 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 RARβ are barely detectable (16±1 copies per μg of mRNA) compared to FTC133 (1643±272 copies per μg of mRNA) (Figure 2b). Transcription of the RARβ gene is controlled via specific retinoic acid response elements (RAREs) located in the 5′ regulatory sequences of the RARβ2 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, RARβ transcript levels in FTC133 increase upon ATRA treatment from two-fold (1.7±1.3) after 2 h to four-fold (3.8±1.1) after 24 h, whereas no increase in RARβ 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, RARβ transcript and protein are differentially expressed in both cell lines and their expression levels correlate with the sensitivity of the cell to RA-differentiation.
To study the functional RA pathways in these two cell lines, we used a RARβ2 promoter combined to a luciferase reporter gene (De Thé et al., 1990). After transfection of the RARβ2-luciferase reporter gene, luciferase activity was estimated under different concentrations of ATRA. Interestingly, the RARβ2 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-RARβ expression plasmid in FTC238 cells restored RARβ level (Figure 3b, inset), and increased transactivation of the RARβ2 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 RARβ levels are sufficient. These data lead us to consider that lack of RA efficacy and restoration of RARβ expression in FTC238 cells may be due to abnormalities located at the endogenous RARβ2 promoter region.
Methylation of the RARβ2 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 RARβ2 promoter (Suh et al., 2002). Thus, it is likely that, in the FTC238 cell line, a non-permissive RARβ2 promoter provides resistance to transcription initiation of RA target genes. To address this hypothesis, we first studied the methylation status of the endogenous RARβ2 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 RARβ2 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 RARβ expression superior to baseline or after ATRA treatment levels (Figure 4b). These results indicate that, in FTC238 cells, RA-refractoriness and absence of RARβ expression, whether spontaneously or after ATRA, does not result from a hypermethylated status of the RARβ2 promoter.
We then analysed the levels of acetylated histones H3 and H4 on the RARβ2 promoter by chromatin immunoprecipitation (ChIP) assay using antibodies against the critical acetylated forms of histones H3 and H4 and primers selecting the RARβ2 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 RARβ2 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 RARβ 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.
differentiated thyroid cancer
follicular thyroid carcinoma
retinoic acid receptor
retinoic acid response element
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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.
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Cras, A., Darsin-Bettinger, D., Balitrand, N. et al. Epigenetic patterns of the retinoic acid receptor β2 promoter in retinoic acid-resistant thyroid cancer cells. Oncogene 26, 4018–4024 (2007). https://doi.org/10.1038/sj.onc.1210178
- RARβ2 promoter
- thyroid cells
- histone acetylation
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