Cyclic AMP-response element modulator inhibits the promoter activity of the sodium iodide symporter gene in thyroid cancer cells.

BACKGROUND: Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells. METHODS: Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines. RESULTS: Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines. CONCLUSIONS: These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

Sunitinib inhibits MEK/ERK and SAPK/JNK pathways and increases sodium/iodide symporter expression in papillary thyroid cancer.

BACKGROUND: Sunitinib malate (Sutent, Pfizer, Inc.; SU11248) is a selective, multitargeted inhibitor of receptor tyrosine kinases and has been shown to inhibit receptors for VEGF, PDGF, KIT, FLT3, and RET. The objective of this study was to determine the effects of sunitinib on signal transduction pathways and on gene expression of iodide-metabolizing proteins in papillary cancer cells with the RET/PTC1 rearrangement. METHODS: We investigated the effects of sunitinib on cell growth, signal transduction pathways, and thyroid-specific gene expression in papillary thyroid cancer (PTC) cell lines that had the RET/PTC1 rearrangement. RESULTS: Sunitinib inhibited proliferation of RET/PTC1 subclones in a time- and dose-related manner. The mean 50% lethal concentration in the RET/PTC1 subclones was 1.81 microM. Incubation of RET/PTC1 cells with 1 microM sunitinib inhibited their migration potential and transformed their morphology. Sunitinib inhibited RET autophosphorylation at Y1062 and the activation of signal transducer and activator of transcription 3 by blocking Y705 phosphorylation. Sunitinib caused cell cycle arrest in the G0/G1 phase and dephosphorylation of retinoblastoma protein, but did not induce apoptosis. Western blot analysis of the p38, MEK/ERK, and SAPK/JNK mitogen-activated protein kinase signal transduction pathways showed that sunitinib blocked ERK 1/2 and JNK phosphorylation in the cytoplasm. Sunitinib treatment of RET/PTC1 cell lines, in combination, with forskolin induced expression of the sodium (Na)/iodide (I) symporter (NIS) and the transcription factors that bind the NIS upstream enhancer. Mechanistically, the inhibition of both MEK/ERK and SAPK/JNK cytoplasmic pathways individually and in combination caused an increase in NIS gene expression. CONCLUSION: Sunitinib appears to target the cytosolic MEK/ERK and SAPK/JNK pathways in the RET/PTC1 cell lines, suggesting that blocking these pathways is at least part of the mechanism by which sunitinib inhibits cell proliferation and causes stimulation of NIS gene expression in RET/PTC1 cells.

Stability of recombinant human thyrotropin potency based on bioassay in FRTL-5 cells.

BACKGROUND: Recombinant human thyrotropin (rhTSH; Thyrogen®) is approved for use in a 0.9 mg dose/day for 2 consecutive days for diagnosis and treatment of differentiated thyroid cancer. It is recommended that it be injected immediately after reconstitution in the distilled water diluent supplied by the manufacturer. However, Thyrogen has been used off-label in doses less than the standard 0.9 mg dose for stimulation of radioiodine uptake in the treatment of multinodular goiter. To determine whether the biologic activity of Thyrogen can be preserved after dilution, we designed experiments to assess the biologic stability of Thyrogen under different durations and temperatures of storage. METHODS: rhTSH was diluted in 1% bovine serum albumin in phosphate-buffered saline to a concentration of 0.9 mg /mL and further diluted to 0.1 mg/mL. Aliquots of 0.5 mL were stored at room temperature, 4°C, -11°C, and -60°C for various lengths of time. In addition, rhTSH aliquots were also subjected to incubation for 1 hour at 50°C and to 10 cycles of freezing in dry ice alternating with thawing at 37°C. Bioassays were performed in FRTL-5 cells. rhTSH was added to the media at a final concentration of either 5 ng/mL or 20 ng/mL, and the cells were then incubated for 48 hours. Potency was assessed by measurement of ¹²5I-iodide uptake in comparison to cells treated with perchlorate to block iodide uptake. Samples were immunoassayed at day 185 of storage. RESULTS: Samples stored at 4°C, -11°C, -60°C, and room temperature retained activity after storage periods of up to 204 days. Samples subjected to 10 freeze-thaw cycles or heated to 50°C for 1 hour retained full biologic activity. Immunoassay at day 185 showed no difference in immunoactivity in relation to the storage condition. CONCLUSION: rhTSH kept at 4°C, -11°C, -60°C, and room temperature maintained good biologic potency for more than 6 months of storage when tested in vitro, indicating that the biologic activity is very stable. However, altered sialylation occurring during storage could have altered the half-life of rhTSH. Nevertheless, the data provide reassurance that storage in the cold for a few months does not result in significant loss of biologic activity.

Effect of sunitinib on growth and function of FRTL-5 thyroid cells.

BACKGROUND: Sunitinib, a multitargeted vascular endothelial growth factor and receptor tyrosine kinase inhibitor, causes hypothyroidism in patients who take it for treatment of cancer. Although the pathophysiologic mechanism of the hypothyroidism is unclear, it has been claimed that it is due to inhibition of iodide uptake. METHODS: To evaluate the pathologic mechanism of induction of the hypothyroidism, we studied the effect of sunitinib on FRTL-5 rat thyroid cells. We measured the effect of sunitinib on cell growth, (125)I-iodide uptake and efflux, TSH receptor (TSH-R), and sodium-iodide symporter (NIS) message. RESULTS: At 48 hours, sunitinib caused a dose-related inhibition of growth with LC(50) of 14.6 muM, but there was no apparent inhibition of growth at 24 hours at concentrations of 0.1-25 microM. Preincubation with sunitinib did not impair the response to TSH, indicating that it did not affect the TSH-R. Incubation with sunitinib for 24 hours caused a dose-related increase of (125)I-iodide uptake and did not reduce iodide efflux or NIS mRNA expression. CONCLUSION: The data indicate that sunitinib is unlikely to cause hypothyroidism by inhibition of iodide uptake.

Thyroid-stimulating hormone increases active transport of perchlorate into thyroid cells.

Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct measurement techniques. To demonstrate intracellular perchlorate accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1-10 microM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 microM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 microM) did not inhibit cell proliferation after 72 h of incubation. In conclusion, perchlorate is actively transported into thyroid cells and does not inhibit cell proliferation.

Identification of cyclic adenosine 3',5'-monophosphate response element modulator as an activator of the human sodium/iodide symporter upstream enhancer.

The lack of Na(+)/I(-) symporter (NIS) gene expression in some thyroid cancer patients has been a major hurdle that limits the efficacy of standard radioactive iodide therapy. The molecular mechanism that contributes to low NIS expression is not well understood. Activated NIS gene expression is stimulated by thyroid-stimulating hormone-mediated cAMP/protein kinase A signaling through a NIS upstream enhancer (NUE). The cAMP pathway is also stimulated by forskolin. In the current work, we studied the mechanism of transcriptional activation of NIS in normal thyroid cells and thyroid cancer cells. We identified the cAMP response element modulator (CREM) activator as a new component of the transcription complex that is important for NIS gene expression. The CREM complex is seen in the normal thyroid cells and BRAF (V600E) thyroid cancer cells (BHP 17-10) but is missing in rearranged in transformation/papillary thyroid carcinoma-1 rearrangement thyroid cancer cells (BHP 2-7). This complex is believed to be responsible for the loss of NUE activity and reduced NIS expression in the BHP 2-7 cell line. In BHP 2-7 cells, forskolin stimulated the thyroid-specific transcription factor Pax 8, but CREM activator mRNA did not increase, and this produced a small increase in NUE activity. Ectopic expression of CREM activator enhanced activity of the NUE, indicating that CREM is an essential regulator of NIS gene expression.

Roles for inhibitory interactions in the use of the -10 promoter element by sigma 70 holoenzyme.

A panel of seven -10 region DNA mutants was tested for holoenzyme binding against a panel of 13 region 2 mutants of sigma 70. No patterns were noticed that would indicate unique interactions between individual amino acids and individual -10 region bases. Instead, certain amino acid substitutions led to increased holoenzyme binding to DNA, implying that the wild type interactions are associated with an inhibitory component. These inhibitory interactions were stronger on DNA containing non-consensus sequences, like those of typical promoters. In addition, the DNA segment downstream from the -10 element was also inhibitory to binding when in duplex form but stimulated binding when in single strand form. Overall, the data suggest that -10 region duplex recognition and melting have a large component of overcoming unfavorable protein:DNA base interactions, particularly when the bases are non-consensus, and that this contributes to setting physiologically appropriate variations in transcription rate.

Effect of DNA bases and backbone on sigma70 holoenzyme binding and isomerization using fork junction probes.

Abasic substitutions in the non-template strand and promoter sequence changes were made to assess the roles of various promoter features in sigma70 holoenzyme interactions with fork junction probes. Removal of -10 element non-template single strand bases, leaving the phosphodiester backbone intact, did not interfere with binding. In contrast these abasic probes were deficient in promoting holoenzyme isomerization to the heparin resistant conformation. Thus, it appears that the melted -10 region interaction has two components, an initial enzyme binding primarily to the phosphodiester backbone and a base dependent isomerization of the bound enzyme. In contrast various upstream elements cooperate primarily to stimulate binding. Features and positions most important for these effects are identified.