Induction of sodium iodide symporter gene and molecular characterisation of HNF3β/FoxA2, TTF-1 and C/EBPβ in thyroid carcinoma cells

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 β (HNF3β)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein β (C/EBPβ). Quantitative reverse transcription-polymerase chain reaction (RT–PCR) showed decreased expression of HNF3β/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3β/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBPβ was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBPβ in the cytoplasm, suggesting that a large proportion of C/EBPβ protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells

Development of thyroid carcinoma is accompanied by a block of differentiation of these cells. Papillary and follicular thyroid carcinomas initially are relatively well-differentiated tumours that over time may de-differentiate, whereas anaplastic thyroid carcinoma is an undifferentiated tumour (Farid et al, 1994). Poorly differentiated or undifferentiated carcinomas no longer express mature thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). In normal thyroid cells, TSHR is stimulated by thyrotropin-stimulating hormone, resulting in the activation of NIS, which incorporates iodine. Thyroglobulin and iodine are catalysed into thyroid hormones by TPO (Carrasco, 1993). Investigators have predominantely attempted to induce differentiation of thyroid cancer cells by exposure to compounds associated with known differentiation of other cancers.
Retinoic acid, including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), induces differentiation of acute promyelocytic leukaemia cells and neuroblastoma cells and is used in the therapy for these cancers. Retinoids have been reported to induce the expression of TPO, TG, and NIS mRNAs in thyroid carcinoma cell lines (Schmutzler et al, 1997;Haugen, 2004). The NIS promoter contains CpG islands, and a DNA demethylating agent (such as, 5-aza-2-deoxycytidine (5-Az)) combined with a histone deacetylase inhibitor has been shown to induce NIS expression and radioactive iodine uptake in follicular and anaplastic thyroid carcinoma cell lines (Venkataraman et al, 1999;Haugen, 2004).
The transcription factors of paired box gene 8 (Pax-8) and thyroid transcription factor 1 (TTF-1) have been analysed in thyroid cells. Paired box gene 8 is necessary for the formation of thyroxine-producing follicular cells in the thyroid gland (Mansouri et al, 1998(Mansouri et al, , 1999; and fusion of the Pax-8 and peroxisome proliferator-activated receptor g (PPARg) genes occurs in approximately 30% of follicular thyroid carcinomas (Kroll et al, 2000;Dwight et al, 2003). Thyroid transcription factor 1 is required for the development of the thyroid gland, and TTF-1-deficient mice lack a thyroid gland and die at birth (Kimura et al, 1996). The expression of Pax-8 and TTF-1 is low in thyroid carcinoma (Ros et al, 1999); and stable transfection with a Pax-8 expression vector in anaplastic thyroid carcinoma cell line, ARO, caused re-expression of endogenous NIS, TG, and TPO (Presta et al, 2005). Reporter gene analysis found that the promoter region of TG, TPO, and TSHR could be activated by the forced expression of either Pax-8 or TTF-1 in the papillary thyroid carcinoma cell line NPA (Ros et al, 1999). Co-transfection of Pax-8 and TTF-1 restored TG promoter activity in WRO (follicular thyroid carcinoma) and ARO cells (Chun et al, 1998).

Real-time reverse transcription polymerase chain reaction
Total RNA was isolated from thyroid carcinoma cell lines and normal thyroid tissues using Trizol reagent (Invitrogen), and cDNA was prepared from 1 mg of total RNA with Superscript III reverse transcriptase (Invitrogen). Expression of mRNAs was measured by real-time PCR using an iCycler iQ system (Bio-Rad, Hercules, CA, USA) as described previously (Xie et al, 2001). To determine the expression levels of NIS, C/EBPa, and C/EPBb with probes, amplification reactions were performed with the Universal Taqman PCR mastermix (Applied Biosystems, Foster City, CA, Radioactive iodine ( 125 I) uptake assay Na 125 I (100 mCi ml À1 ) stock was diluted to 0.02 mCi ml À1 using Hanks' Balanced Salt Solution (HBSS). Cells were plated at 1 Â 10 5 cells per well in 12-well plates and treated with appropriate drugs, either with or without 10 mU ml À1 thyrotropin-stimulating hormone and/or cold (unlabelled) NaI. For radioactive iodine uptake, cells were washed twice with HBSS and 500 ml of Na 125 I working solution was added. After incubation for 3 h at 371C, cells were washed with 1 ml cold HBSS and lysed with 1 ml of 95% ethanol for 1 h at 371C. Lysates were transferred to vials for counting, and total counts were normalised to number of viable cells in parallel cultures.
For colony assay, cells transfected with plasmid were plated at 1 Â 10 5 cells per well in 12-well plates in 1 ml culture media containing 500 mg ml À1 G418. After 2 weeks, cells were stained with Crystal violet dye (0.25% crystal violet dissolved in 50% methanol).
For clonogenic soft agar assays, cells were plated into 24-well flatbottomed wells using a two-layer soft agar system with 1 Â 10 3 cells per well in a volume of 400 ml per well as previously described (Luong et al, 2006). After 14 days of incubation, colonies were counted.
Methylation analysis of HNF3b/FoxA2 gene Genomic DNA was modified by sodium bisulphate using EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). The CpG island (À761 to À561, ATG codon considered as þ 1) of the HNF3b/FoxA2 gene was amplified from the bisulphate-modified genomic DNA with specific primers (sense primer: 5 0 -TTTTAGGG GATTTGTTGTGG-3 0 , anti-sense primer: 5 0 -AAATAATCAACTCAC ACC-3 0 ). For PCR amplification, a total volume of 10 ml was used containing modified genomic DNA, 0.5 mM of each primers, 5.0 ml of FailSafe PCR 2 Â PreMixe E (Epicentre Biotechnologies, Madison, WI, USA) and 1.0 U platinum Taq (Invitrogen).  Figure 1 Induction of NIS expression in anaplastic and papillary thyroid carcinoma cell lines with SAHA and ligands of nuclear hormone receptors. Thyroid carcinoma cells (ARO and BHP) were cultured with or without 1,25(OH) 2 D 3 (VD3, 1 mM), all-trans retinoic acid (ATRA, 100 nM), 9-cis retinoic acid (9-cis RA, 100 nM), troglitazone (Trog, 10 mM), or thyroid hormone T 3 (TH, 10 nM), either with or without SAHA (5 mM). Expression of NIS mRNA in these cells was measured by quantitative RT -PCR after 48 h exposure. NIS expression was compared with levels present in normal thyroid cells. Relative levels of transcripts for NIS were normalised to 18S RNA transcripts within each sample. Level of NIS expression is the mean of n ¼ 3 samples.

SAHA
Polymerase chain reaction products were subcloned into pCR 2.1 vector (Invitrogen) and sequenced.

Subcellular fractionation, Western blot analysis, and immunohistochemistry
Total cell lysates were prepared by lysing cells in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.5)) containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) as well as 1 mM NaF and 1 mM NaVO 4 . To separate nuclear and cytoplasmic fractions, cells were fractionated with NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA). These samples were subjected to sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS -PAGE) followed by an electrotransfer to polyvinylidene difluoride membrane. The signals were developed with either Supersignal West Pico Chemiluminescent or Supersignal West Dura Extended Duration Substrate (Pierce Biotechnology). Anti-PPARg, C/EBPb, b-actin, and heterogeneous nuclear ribonuclear protein (hnRNP) A1 antibodies were obtained from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Research Diagnostics Inc. (Concord, MA, USA). Normal and papillary thyroid carcinoma tissue blocks were cut at 3 mM thickness, deparaffinised and pre-treated in Tris-HCl (pH 9.0). These samples were incubated overnight with anti-C/EBPb antibody (1 : 500 dilution), washed, followed by the HRP-conjugated secondary antibody (Dako, Carpinteria, CA, USA), and DAB chromogen. The tissues were counterstained with haematoxylin and then coverslipped. Three samples of normal and three carcinomas were examined.

RESULTS
Induction of expression of thyroid-specific genes: effect of 5-Az, SAHA, valproic acid, and nuclear hormone receptor ligands in thyroid carcinoma cell lines Silencing of genes can be associated with epigenetic change including abnormal methylation of CpG islands and/or deacetylation of histones. Therefore, papillary (BHP sublines 2 -7, 7 -13, 10-3, and 18 -21) and two anaplastic thyroid carcinoma (ARO and FRO) cell lines were cultured either with or without 5-Az (1 mM) and/or histone deacetylase inhibitors (SAHA (5 mM) or valproic acid (1 mM)). Quantitative reverse transcription-polymerase chain reaction (RT -PCR) showed that expressions of thyroid-specific genes (TPO, TG, and TSHR) were either extremely low or at undetectable levels compared with normal thyroid tissue (data not shown).
We also examined the effects of several ligands of nuclear hormone receptors either with or without SAHA. As shown in  Figure 1, 1,25(OH) 2 D 3 , ATRA, 9-cis RA, troglitazone, or thyroid hormone T 3 alone was able to induce the expression of NIS mRNA in ARO and BHP (sublines 2 -7 or 10 -3) cells. The combination of SAHA with ATRA, 9-cis RA, troglitazone, or thyroid hormone T 3 did not enhance the expression of NIS compared with SAHA alone in BHP papillary thyroid carcinoma cells (sublines 2 -7 and 10 -3).
Thyroid-related transcription factors are poorly expressed in papillary and anaplastic thyroid carcinoma cell lines The above data showed that the four thyroid-specific genes (NIS, TPO, TG, and TSHR) were negligibly expressed in the thyroid carcinoma cell lines; therefore, common transcription factor(s) that regulate these genes might not be expressed in these cell lines. The promoter regions of these genes contain putative transcription factor binding sites for HNF3b/FoxA2, C/EBPs, PPARg, Pax-8, and TTF-1. Real-time PCR showed that HNF3b/FoxA2, TTF-1, and Pax-8 were expressed in normal thyroid tissue; in contrast, levels were either very low or undetectable in thyroid carcinoma cell lines ( Figure 2). Western blot analysis showed that the protein expression of PPARg was barely detectable in BHP sublines, but easily found in ARO and FRO cell lines (data not shown).

Forced expression of either HNF3b/FoxA2 or TTF-1 in thyroid carcinoma cells
Next, we transfected either the HNF3b/FoxA2 or the TTF-1 expression vector into papillary thyroid carcinoma cell lines. Neither HNF3b/FoxA2-nor TTF-1-expressing BHP cells (subline 2 -7) had an increase in 125 I uptake, when compared with normal FRTL-5 thyroid cells (Figure 3). Nevertheless, the forced expression of either HNF3b/FoxA2 or TTF-1 resulted in growth inhibition compared with cells transfected with an empty vector as measured by MTT assay (data not shown), suggesting that these transcription factors have antiproliferative activity in papillary thyroid carcinoma cells.

Methylation status of HNF3b/FoxA2 gene in papillary thyroid carcinoma cells
The HNF3b/FoxA2 gene has a CpG island in its promoter; and the region is often methylated in breast and lung cancers (Halmos et al, 2004;Miyamoto et al, 2005) prompting us to examine thyroid carcinoma cells. The great majority of the 21 CpG sites in the promoter were methylated in BHP (subline 2-7) and NPA papillary thyroid carcinoma cells ( Figure 4A), as well as in the anaplastic  thyroid carcinoma cell line FRO (data not shown). In contrast, the region was unmethylated in normal thyroid tissues ( Figure 4B). Real-time PCR showed that the expression of HNF3b/FoxA2 mRNA was induced after the treatment of BHP cells (subline 2-7) with the demethylating agent 5-Az (1 mM, 96 h) ( Figure 4C). Taken together, these results suggest that the expression of the HNF3b/FoxA2 gene is epigenetically repressed in thyroid carcinoma cell lines.
Forced expression of C/EBPb in thyroid carcinoma cells Next, we placed a Zn-inducible C/EBPb expression vector into BHP cells (sublines 2-7 and 7-13) ( Figure 5A). CCAAT/enhancer binding protein b has two isoforms, LAP (liver-enriched transcriptional activating protein) and LIP (liver-enriched transcriptional inhibitory protein). The smaller form of C/EBPb (LIP) clearly increased in the cells treated with zinc. Induction of C/EBPb expression resulted in a 60% growth reduction compared to the non-induced cells ( Figure 5B, left panel). The more sensitive clonogenic soft agar assay showed that clonogenic growth decreased even in the absence of zinc, suggesting that this vector was 'leaky' resulting in C/EBPb expression even in the absence of zinc. Nevertheless, clonogenic growth decreased 50% in the presence of zinc compared with the absence of zinc ( Figure 5B, right panel). Similarly, crystal violet staining demonstrated that C/EBPb had anti-growth activity in another subline, BHP 17-3 ( Figure 5C). Taken together, these results suggested that forced expression of C/EBPb can cause growth inhibition in BHP papillary thyroid carcinoma cells.

Cellular localisation of C/EBPb in human normal and papillary thyroid carcinoma tissues and cell lines
To examine expression of C/EBPb in human thyroid tissues, normal and papillary thyroid carcinoma tissues were stained with anti-C/EBPb antibody. Immunohistochemistry revealed that C/EBPb signal was strongly detected in the nucleus in normal thyroid cells ( Figure 6A). Interestingly, C/EBPb was detected in the cytoplasm and to a lesser extent the nucleus of papillary thyroid carcinoma cells ( Figure 6B). The subcellular localisation of C/EBPb in four thyroid carcinoma cell lines (BHP2-7, NPA, FRO and ARO) was also determined by fractionation ( Figure 6C). CCAAT/ enhancer binding protein b-LAP was detected in both the nucleus and the cytoplasm. CCAAT/enhancer binding protein b-LIP, which has a dominant-negative activity against LAP, was expressed in NPA and FRO cell lines, and localised in the nucleus.

DISCUSSION
We attempted to induce differentiation and inhibit proliferation of thyroid carcinoma cells with various compounds and transcription factors; and in addition, we explored the abnormalities in endogenous expression of these transcription factors in thyroid carcinoma cells. Suberoylanilide hydroxamic acid modestly induced the expression of TPO, TG and TSHR; and the combination of SAHA and 1,25(OH) 2 D 3 further enhanced the expression of NIS in BHP cell line. However, these agents were not potent stimulators of NIS expression level, when compared with expression levels found in normal thyroid tissues. Induced expression of these transcripts was about 10-to 100-fold lower than those found in normal thyroid cells. Therefore taken together, our data suggest that these compounds had little differentiation inducing activity and would be unlikely candidates to enhance the therapeutic value of radioactive iodine ( 131 I) for the treatment of thyroid tumours. Notably, two histone deacetylase inhibitors, depsipeptide and Trichostatin A, have been shown to induce the expression of NIS and 125 I uptake in several follicular and anaplastic thyroid carcinoma cell lines (Haugen, 2004).  Survey of the thyroid-specific genes showed that each promoter had transcription factor binding sites for HNF3b/FoxA2, TTF-1, C/EBPb, and Pax-8. Earlier studies using either the TG or TPO promoter found that they were activated by TTF-1 and HNF3b/FoxA2 in thyroid carcinoma cell lines (Sato and Di Lauro, 1996;Ros et al, 1999;Shimura et al, 2001). In addition, Pax-8 leads to the re-expression of NIS, TPO, TG, and TTF-1 mRNAs in ARO cells (Presta et al, 2005). Our present study demonstrated that forced expression of either HNF3b/FoxA2 or TTF-1 was unable to induce differentiation of the thyroid cancer cells as measured by NIS mRNA expression and radioiodine uptake. Similarly, co-transfection of HNF3b/FoxA2 and TTF-1 did not induce the expression of TPO, TG or TSHR mRNAs in BHP cells (data not shown), indicating that other molecule(s) might be required to induce endogenous mRNA expression of these thyroid-related differentiation genes. Interestingly, HNF3b/FoxA2 is a methylated gene in breast and lung cancer cells; and overexpression of HNF3b/FoxA2 in a lung cancer cell line leads to growth arrest and apoptosis (Halmos et al, 2004;Miyamoto et al, 2005). Here, we report for the first time that the presence of aberrant methylation of HNF3b/FoxA2 in thyroid carcinoma cell lines, and forced expression of the gene, resulted in growth inhibition.
Recently, Pomérance et al (2005) detected cytoplasmic localisation of C/EBPb in papillary thyroid carcinoma tissues. Our immunohistochemical analysis also showed cytoplasmic localisation of C/EBPb in papillary thyroid carcinoma tissues. In addition, we demonstrated by cell fractionation that C/EBPb-LAP was present in both the nucleus and the cytoplasm; in contrast, C/EBPb-LIP, a dominant-negative form of C/EBPb, was localised in the nucleus in NPA and FRO cells. Nucleocytoplasmic distribution of C/EBPb has been found in several other types of cancer. Human acute myeloid leukaemic cell line HL-60 showed cytoplasmic localisation of C/EBPb-LAP when Thr235 was phosphorylated, and the induction of differentiation and the inhibition of proliferation of these cells by 1,25(OH) 2 D 3 resulted in nuclear translocation of the transcription factor (Marcinkowska et al, 2006). In other experiments, C/EBPb phosphorylation at Ser288 was associated with cytoplasmic localisation of the protein in human liver cancer cells; in contrast, normal liver cells had neither phosphorylation of Ser288 nor cytoplasmic C/EBPb (Buck et al, 2001). CCAAT/enhancer binding protein b-LAP and -LIP contain both nuclear localisation signal and nuclear export signal in their common C-terminal region (Williams et al, 1997). The N-terminal region, which is specific for C/EBPb-LAP, might contain a motif that causes cytoplasmic retention in thyroid cancer cells. In general, transcription factors including C/EBPb function in the nucleus, suggesting that deregulation of nuclear localisation of C/EBPb leads to functional deficiency and result in cell abnormalities.
In summary, we found that the thyroid cancer cells had decreased the expression of TTF-1 and HNF3b/FoxA2; and their forced re-expression was associated with decreased cell growth. In addition, methylation of HNF3b/FoxA2 and inappropriate cellular localisation of C/EBPb were identified as novel abnormalities. Future studies will screen for small molecules that can induce expression of these transcription factors resulting in a unique therapy for thyroid cancer.  Figure 6 Cellular localisation of C/EBPb by immunohistochemistry in normal and papillary thyroid carcinoma tissues. Normal thyroid (A) and papillary thyroid carcinoma (B) tissues were immunohistochemically stained with anti-C/EBPb antibody. Photomicrographs are representative of three different samples of both normal thyroid and papillary thyroid carcinoma (data not shown). (C) Cellular fractionation of C/EBPb in papillary and anaplastic thyroid carcinoma cells. Papillary (BHP subline 2 -7 and NPA) and anaplastic thyroid carcinoma (FRO and ARO) cells were fractionated into nuclear and cytoplasmic lysates, and the localisation of C/EBPb was determined by electrophoresis followed by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase and hnRNP A1 are cytoplasm and nucleus markers, respectively. N, nuclear fraction; C, cytoplasmic fraction; LAP, liver-enriched transcriptional activating protein; LIP, liver-enriched transcriptional inhibitory protein.