Down-regulation of the Sodium/Iodide Symporter Explains

I radiation therapy of differentiated thyroid cancer may be compromised by thyroid stunning (i.e., a paradoxical inhibition of radioiodine uptake caused by radiation from a pretherapeutic diagnostic examination). The stunning mechanism is yet uncharacterized at the molecular level. We therefore investigated whether the expression of the sodium/ iodide symporter (NIS) gene is changed by irradiation using I. Confluent porcine thyroid cells on filter were stimulated with thyroid-stimulating hormone (TSH; 1 milliunit/mL) or insulin-like growth factor-I (IGF-I; 10 ng/mL) and simultaneously exposed to I in the culture medium for 48 h, porcine NIS mRNA was quantified by real-time reverse transcription-PCR using 18S as reference, and transepithelial iodide transport was monitored using I as tracer. TSH increased the NIS expression >100-fold after 48 h and 5to 20-fold after prolonged stimulation. IGF-I enhanced the NIS transcription at most 15-fold but not until 5 to 7 days. I irradiation (7.5 Gy) decreased both TSH-stimulated and IGF-I–stimulated NIS transcription by 60% to 90% at all investigated time points. TSH and IGF-I stimulated NIS synergistically 15to 60-fold after 5 days. NIS expression was reduced by I also in costimulated cells, but the transcription level remained higher than in nonirradiated cells stimulated with TSH alone. Changes in NIS mRNA always correlated with altered I transport in cultures with corresponding treatments. It is concluded that down-regulation of NIS is the likely explanation of I-induced thyroid stunning. Enhanced NIS expression by synergistically acting agents (TSH and IGF-I) partly prevents the loss of iodide transport expected from a given absorbed dose, suggesting that thyroid stunning might be pharmacologically treatable. [Cancer Res 2007;67(15):7512–7] Introduction Radioiodine therapy with I has two main indications: toxic diffuse goiter (i.e., Graves’ disease) and differentiated thyroid cancer. Successful outcome of treatment relies on the ability of the thyroid tissue to concentrate and retain I . In both normal and pathologic thyroid tissues, this is primarily achieved by the action of the sodium/iodide symporter (NIS) present at the thyroid cell surface (1). Loss of NIS expression leads to insufficient radioiodine uptake, which is the main reason why I therapy is not applicable to patients with poorly differentiated thyroid cancer. Diagnostic scanning with low amounts of I is often used to estimate the mass of thyroid remnants after tumor surgery. Numerous studies have reported that diagnostic exposure to I may diminish subsequent uptake of the therapeutic dose, a phenomenon known as thyroid stunning, which potentially may compromise the therapeutic efficiency and outcome of radioiodine treatment (reviewed in refs. 2–5). The stunning mechanism has for long been obscure mainly because of lack of experimental data. However, recent studies in our laboratory, using an in vitro model of internal radiation of normal thyroid cells, show that I indeed reduces thyroidal iodide transport in a dose-dependent manner (6, 7) at absorbed doses equivalent to those estimated to induce stunning in vivo (3). Importantly, as quiescent (G0) cells were irradiated and the cell number did not change, it could be excluded that the inhibited transport was related to compromised cell cycle progression or apoptosis otherwise readily induced when similar amounts of I were administered to proliferating thyrocytes (6, 7). Considering NIS as the key molecule responsible for the iodideconcentrating mechanism in the thyroid gland and in thyroid carcinoma cells with preserved iodide uptake, it can be hypothesized that the expression or function of NIS might be affected by ionizing radiation and that this could explain thyroid stunning. However, this possibility has not been experimentally investigated. The NIS expression in thyroid cells is transcriptionally regulated by both positive and negative signals. Most importantly, thyroidstimulating hormone (TSH) up-regulates the NIS mRNA level multifold via activation of a cyclic AMP (cAMP)-responsible element in the NIS promoter (8, 9). This favorable effect is likely responsible for the fundamental increase in tumor radioiodine uptake in thyroid cancer patients given recombinant human TSH before radioiodine therapy (10). Conversely, down-regulation of NIS mRNA expression can be experimentally induced by excess iodine (11, 12), which requires organification (i.e., iodide-protein binding) and thus explains the so-called escape of the Wolff-Chaikoff effect, a thyroid protective mechanism against toxicity of iodine overload. In this study, we investigated with real-time reverse transcriptionPCR (RT-PCR) analysis whether I irradiation influences the NIS transcript levels in primary cultured normal thyrocytes stimulated with TSH and insulin-like growth factor-I (IGF-I). Materials and Methods Cell culture and hormone treatment. Pig thyroid glands obtained from the local abattoir were prepared for isolation and culture of thyroid epithelial cells as described (13). Briefly, purified thyroid follicle segments were plated on collagen type 1–coated filters in Transwell bicameral chamber inserts (3413 or 93413, Corning Costar) at 37jC in a CO2 (5%) incubator. The cells were grown in Earle’s MEMwith 5% FCS, penicillin (200 units/mL), streptomycin (200 Ag/mL), and fungizone (2.5 Ag/mL; all reagents were purchased from PAA Laboratories GmbH) until forming a continuous monolayer resembling the native thyroid follicular epithelium Requests for reprints: Madeleine M. Nordén, Institute of Biomedicine, The Sahlgrenska Academy at Göteborg University, Box 440, SE-405 30 Göteborg, Sweden. Phone: 46-31-786-3140; Fax: 46-31-786-3322; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0823 Cancer Res 2007; 67: (15). August 1, 2007 7512 www.aacrjournals.org Research Article American Association for Cancer Research Copyright © 2007 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-07-0823