Mammary radioiodine accumulation due to functional sodium iodide symporter expression in a benign fibroadenoma

The sodium iodide symporter (NIS) has been characterized to mediate the active transport of iodide not only in the thyroid gland but also in various non-thyroidal tissues, including lactating mammary gland and the majority of breast cancers, thereby offering the possibility of diagnostic and therapeutic radioiodine application in breast cancer. In this report, we present a 57-year-old patient with multifocal papillary thyroid carcinoma, who showed focal radioiodine accumulation in a lesion in the right breast on a posttherapy (131)I scan following radioiodine therapy. CT and MR-mammography showed a focal solid lesion in the right breast suggestive of a fibroadenoma, which was confirmed by histological examination. Immunostaining of paraffin-embedded tumor tissue sections using a human NIS antibody demonstrated NIS-specific immunoreactivity confined to epithelial cells of mammary ducts. In conclusion, in a thyroid cancer patient we identified a benign fibroadenoma of the breast expressing high levels of functionally active NIS protein as underlying cause of focal mammary radioiodine accumulation on a posttherapy (131)I scan. These data show for the first time that functional NIS expression is not restricted to lactating mammary gland and malignant breast tissue, but can also be detected in benign breast lesions, such as fibroadenomata of the breast.     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.     

Estimation of sodium/iodide symporter gene expression (NIS) in thyroid cancer by RT-PCR technique (preliminary study)

NIS gene is located on chromosome 19 and encodes 643 amino acid protein. It belongs to membrane Na+ dependent glucose symporter proteins family. In normal thyroid is located in basolateral membrane of thyreocyte. It plays a main role in concentrating of iodine in thyreocyte and thus in thyroid hormones synthesis. It was proved that NIS expression influences effectiveness of radioactive iodine therapy in well-differentiated thyroid cancers. The aim of this study was to estimate the NIS expression and its dependence with gender, age and stage in thyroid papillary and follicular cancers. The frozen sections of tissue were used as a source of tumor RNA. RT-PCR technique was employed for NIS expression analysis. We did not find dependence between the presence of NIS expression in investigated thyroid cancers and stage of disease estimated according to TNM classification. We also did not find dependence between NIS expression and gender or sex of the patients. Our results suggest that there is no dependence between NIS expression and iodine uptake.     

Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells

The sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine's action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.     

Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.     

人钠/碘同向转运体基因cDNA的克隆与序列测定

为研究人钠／碘同向转运体(hNIS)的生物学性能和用于肿瘤放射性碘治疗的可能性,运用反转录聚合酶链反应(RT—PCR)从人甲状腺组织总RNA中扩增出hNIS基因cDNA序列,将其克隆至pUCm-T载体中。序列分析证实克隆片段与献报道的hNIS基因cDNA序列完全一致,说明已成功克隆到hNIS基因cDNA。     

Radioiodide uptake and sodium iodide symporter expression in breast carcinoma

Breast cancer is a common malignancy in women all over the world and novel therapeutic approaches are required for the treatment of patients who become refractory to conventional therapies. Thyroid cancer is being treated successfully with radioiodine since many years. The iodide is transported inside the thyroid epithelial cell via sodium iodide symporter (NIS) which is a trans-membrane protein. The present study was aimed to explore the uptake of radioiodide (RAI) and the expression of NIS in breast tissues of invasive ductal carcinoma patients. Breast tissues from tumor region (Tu-Br) as well as corresponding normal region (N-Br) were collected from patients of invasive ductal carcinoma. In vitro RAI uptake, its efflux and NIS expression were studied. The uptake of RAI (1.98+/-1.75 x 10(5) cpm/g) in Tu-Br was significantly higher as compared to that observed in N-Br (0.31+/-0.27 x 10(5) cpm/g) and fast efflux was observed in the tissue samples. NIS gene expression was positive in 41.66% (10/24) samples of Tu-Br. None of the N-Br samples expressed NIS gene. In 14 samples of Tu-Br, RAI uptake as well as NIS expression was studied. In 50% of these Tu-Br samples RAI uptake as well as of NIS gene expression was positive. The results indicate that RAI uptake is significantly higher in breast tumor tissues as compared to their normal counterpart and in future radioiodine may be an important agent for treatment of breast cancer.     

Induction of sodium/iodide symporter (NIS) expression and radioiodine uptake in non-thyroid cancer cells

Background

This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells.

Methods

We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells.

Results

Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors.

Conclusions

This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers.     

Intracellular anion fluorescence assay for sodium/iodide symporter substrates

The sodium/iodide symporter (NIS) is primarily responsible for iodide accumulation in the thyroid gland for the synthesis of thyroid hormones; however, it can also transport other lyotropic anions in the thyroid gland and nonthyroid tissues. Some NIS substrates have important physiological or clinical roles, and others are environmental contaminants with health-related consequences. The aim of this study was to assess the utility of a yellow fluorescent protein variant, YFP–H148Q/I152L, as a biosensor to monitor the cellular uptake of NIS substrates, including thiocyanate (SCN⁻), nitrate (), chlorate (), perchlorate (), and perrhenate (). The fluorescence of purified YFP–H148Q/I152L was suppressed by anions with an order of potency of > = I⁻ = SCN⁻ = > ? Cl⁻. Anions also suppressed the fluorescence of YFP–H148Q/I152L expressed in FRTL-5, a thyroid cell line with high NIS expression. Quantitation of intracellular concentrations revealed differences among anions in the affinity and maximal velocity of NIS-mediated uptake as well as in the rate constant for passive efflux. These results suggest that YFP–H148Q/I152L can serve as an intracellular biosensor of NIS-transported anions and may be useful to study the physiology of endogenous anions as well as the health-related consequences of environmental anions.     

Mutations in the sodium/iodide symporter (NIS) gene as a cause for iodide transport defects and congenital hypothyroidism.

The ability to concentrate iodide actively is a characteristic feature of the thyroid gland and several other tissues. This function is mediated through the sodium iodide symporter (NIS), a protein that is located in the basolateral membrane of the thyrocyte. A defect in the NIS (iodide trapping defect) can result in hypothyroidism, the severity of which is variable and influenced, in part, by the amount of iodine supply. The molecular cloning of NIS and characterization of its genomic organization allowed the identification of NIS gene mutations in patients expressing the phenotype of iodide trapping defect. Six mutations (G93R, Q267E, C272X, T354P, Y531X and G543E) have been so far identified and their properties have been partially characterized. G93R, Q267E and Y531X were found in a compound heterozygous individual with NIS defect, C272X and G543E were detected in a homozygous state and T354P has been identified in both homozygotes and heterozygotes in combination with G93R. Heterozygous family members, expressing one normal allele, are clinically not affected. This was confirmed by in vitro analysis where all six mutants produced NISs with virtually no biological activity that did not interfere with the wild-type NIS function when cotransfected in mammalian cells. While the precise mechanisms by which mutant NISs cause iodide trapping defect are still unknown, preliminary data suggest that 354P interferes with the iodide transport function rather than targeting to the cell membrane.     

Expression of the thyroid sodium/iodide symporter in Xenopus laevis oocytes

Poly(A+) RNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells (Ambesi-Impiombato, F. S., Parks, L. A. M., and Coons, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3455-3459] was injected into Xenopus laevis oocytes, and the expression of the Na+/I- symporter in the plasma membrane was assayed by measuring the Na+-dependent ClO4--sensitive uptake of 125I. Expression of the Na+/I- symporter was detected as a 7-fold average increase in transport over background, 5-6 days after injection. Poly(A+) RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- transport activity. The poly(A+) RNA encoding the Na+/I- symporter was found in a fraction containing messages of 2.8-4.0 kilobases in length.     

Real-time imaging of myoblast transplantation using the human sodium iodide symporter

The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes an approach to evaluate the success of myoblast transplantation (i.e., survival of the transplanted cells and the muscle fibers formed) by real-time imaging. C2C12 myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. Specific uptake of the radioactive sodium pertechnetate (Na99mTcO4) by the hNIS-positive myoblasts was demonstrated in vitro, while only background level of Na99mTcO4 was observed within the control cells. The cells were then transplanted into the tibialis anterior (TA) muscle of mdx (X-linked dystrophic) mice. Following intraperitoneal administration of Na99mTcO4, scintigraphies were performed to detect hNIS-dependent Na99mTcO4 uptake within the TA. This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.     

In vivo imaging and tumor therapy with the sodium iodide symporter

There has been great progress in the design of vectors for cancer gene therapy. However, it has been difficult to translate success in the laboratory into clinical practice. A major hurdle in understanding these failures has been the relative difficulty in monitoring repeatedly and non-invasively the biodistribution, gene expression and replication of these viral vector systems. With the advent of molecular imaging technology, this deficiency is being rapidly rectified. A number of reporter genes have been used to monitor gene expression. In this review, we discuss the role of the sodium iodide symporter (NIS) as a reporter and therapeutic gene for cancer gene therapy when combined with various radioactive isotopes.     

The sodium iodide symporter (NIS) and potential regulators in normal, benign and malignant human breast tissue

Introduction

The presence, relevance and regulation of the Sodium Iodide Symporter (NIS) in human mammary tissue remains poorly understood. This study aimed to quantify relative expression of NIS and putative regulators in human breast tissue, with relationships observed further investigated in vitro.

Methods

Human breast tissue specimens (malignant n = 75, normal n = 15, fibroadenoma n = 10) were analysed by RQ-PCR targeting NIS, receptors for retinoic acid (RARα, RARβ), oestrogen (ERα), thyroid hormones (THRα, THRβ), and also phosphoinositide-3-kinase (PI3K). Breast cancer cells were treated with Retinoic acid (ATRA), Estradiol and Thyroxine individually and in combination followed by analysis of changes in NIS expression.

Results

The lowest levels of NIS were detected in normal tissue (Mean(SEM) 0.70(0.12) Log₁₀ Relative Quantity (RQ)) with significantly higher levels observed in fibroadenoma (1.69(0.21) Log₁₀RQ, p<0.005) and malignant breast tissue (1.18(0.07) Log₁₀RQ, p<0.05). Significant positive correlations were observed between human NIS and ERα (r = 0.22, p<0.05) and RARα (r = 0.29, p<0.005), with the strongest relationship observed between NIS and RARβ (r = 0.38, p<0.0001). An inverse relationship between NIS and PI3K expression was also observed (r = −0.21, p<0.05). In vitro, ATRA, Estradiol and Thyroxine individually stimulated significant increases in NIS expression (range 6–16 fold), while ATRA and Thyroxine combined caused the greatest increase (range 16–26 fold).

Conclusion

Although NIS expression is significantly higher in malignant compared to normal breast tissue, the highest level was detected in fibroadenoma. The data presented supports a role for retinoic acid and estradiol in mammary NIS regulation in vivo, and also highlights potential thyroidal regulation of mammary NIS mediated by thyroid hormones.     

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.     

Identification of in vivo phosphorylation sites and their functional significance in the sodium iodide symporter

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.     

Effect of oxygen concentrations on sodium iodide symporter expression and iodide uptake and hCG expression in human choriocarcinoma BeWo cells

Normal human fetal development requires an adequate supply of thyroid hormone from conception. Until about 16 wk gestation this is supplied entirely by placental transfer of maternal hormone. Subsequently, the fetal thyroid synthesizes thyroid hormones, requiring a supply of maternal iodide. Trophoblast iodide transfer is mediated by the apical sodium iodide symporter (NIS). Placental oxygen levels are low in early pregnancy (~1%), rising with placental vascularisation to a plateau of ~8% at about 16 wk. Although the impact of these changing oxygen levels on placental implantation is well recognized, effects on trophoblast materno-fetal exchange are less understood. We investigated expression of the NIS regulator hCG, NIS mRNA expression, and I(125) uptake in choriocarcinoma BeWo cells (a model of the trophoblast) cultured in 1 and 8% oxygen and in room air (21% oxygen). Expression of NIS and hCG mRNA and protein was low at 1% oxygen but rose significantly at 8 and at 21%. This was reflected in significant increases in I(125) uptake. Desferrioxamine, an iron chelator and hypoxia mimic, decreased NIS and hCG expression and I(125) uptake in BeWo cells. NIS expression and I(125) uptake in cells grown at 1% oxygen were not increased by addition of hCG (2,500 IU/l). We infer that placental NIS mRNA and protein expression are regulated by oxygen, rising with vascularization of the placenta in the late first trimester, a time when fetal iodide requirements are increasing.     

Conserved charged amino acid residues in the extracellular region of sodium/iodide symporter are critical for iodide transport activity

Background  

Sodium/iodide symporter (NIS) mediates the active transport and accumulation of iodide from the blood into the thyroid gland. His-226 located in the extracellular region of NIS has been demonstrated to be critical for iodide transport in our previous study. The conserved charged amino acid residues in the extracellular region of NIS were therefore characterized in this study.