help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0353
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sodré, A. K. M. B.
Right arrow Articles by Camargo, R. Y. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sodré, A. K. M. B.
Right arrow Articles by Camargo, R. Y. A.
Related Collections
Right arrow Thyroid
Right arrow Endocrine Oncology
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 10 4141-4145
Copyright © 2008 by The Endocrine Society

BRIEF REPORT

Association of Low Sodium-Iodide Symporter Messenger Ribonucleic Acid Expression in Malignant Thyroid Nodules with Increased Intracellular Protein Staining

Ana Karina M. B. Sodré, Ileana G. S. Rubio, Ana Luiza R. Galrão, Meyer Knobel, Eduardo K. Tomimori, Venâncio A. F. Alves, Cristina T. Kanamura, Carlos A. Buchpiguel, Tomoco Watanabe, Celso U. M. Friguglietti, Marco A. V. Kulcsar, Geraldo Medeiros-Neto and Rosalinda Y. A. Camargo

Thyroid Study Unit (LIM-25) (A.K.M.B.S., I.G.S.R., A.L.R.G., M.K., E.K.T., G.M.-N., R.Y.A.C.), Division of Endocrinology, Division of Pathology (V.A.F.A.), and Nuclear Medicine Center (C.A.B., T.W.), Department of Radiology, University of São Paulo Medical School, 05403-900 São Paulo, Brazil; Adolfo Lutz Institute (C.T.K.), São Paulo Public Health Service, 01246-903 São Paulo, Brazil; and Department of Head and Neck Surgery (C.U.M.F., M.A.V.K.), University of Santo Amaro, 04743-003 São Paulo, Brazil

Address all correspondence and requests for reprints to: Rosalinda Y. A. Camargo, Hospital das Clínicas, FMUSP, Av. Enéas Carvalho de Aguiar, 255, 2 A Bl 7, CEP 05403-900, São Paulo, Brazil. E-mail: ryasato{at}uol.com.br.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The expression of sodium iodide symporter (NIS) is required for iodide uptake in thyroid cells. Benign and malignant thyroid tumors have low iodide uptake. However, previous studies by RT-PCR or immunohistochemistry have shown divergent results of NIS expression in these nodules.

Objective: The objective of the study was to investigate NIS mRNA transcript levels, compare with NIS and TSH receptor proteins expression, and localize the NIS protein in thyroid nodules samples and their surrounding nonnodular tissues (controls).

Design: NIS mRNA levels, quantified by real-time RT-PCR, and NIS and TSH receptor proteins, evaluated by immunohistochemistry, were examined in surgical specimens of 12 benign and 13 malignant nodules and control samples.

Results: When compared with controls, 83.3% of the benign and 100% of the malignant nodules had significantly lower NIS gene expression. Conversely, 66.7% of the benign and 100% of malignant nodules had stronger intracellular NIS immunostaining than controls. Low gene expression associated with strong intracellular immunostaining was most frequently detected in malignant (100%) than benign nodules (50%; P = 0.005). NIS protein was located at the basolateral membrane in 24% of the control samples, 8.3% of the benign, and 15.4% of the malignant nodules. The percentage of benign nodules with strong TSH receptor positivity (41.6%) was higher than malignant (7.7%).

Conclusion: We confirmed that reduced NIS mRNA expression in thyroid malignant nodules is associated with strong intracellular protein staining and may be related to the inability of the NIS protein to migrate to the cellular basolateral membrane. These results may explain the low iodide uptake of malignant nodules.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Iodide uptake by epithelial thyroid cells requires expression of sodium iodide symporter (NIS), a transmembrane glycoprotein comprised of 643 amino acids (1). It also requires NIS migration and retention at the basolateral plasma membrane of the follicular cells. NIS expression is largely modulated by expression and functional activity of the TSH receptor (TSHR) (2).

Although most studies have shown NIS mRNA reduction or even absence of NIS transcript in malignant tumors (3, 4, 5, 6), immunostaining studies (7, 8) have demonstrated that NIS protein is overexpressed in many thyroid tumors, with localization predominantly intracellular. This suggests that defective targeting of the protein to the plasma membrane or its retention at the cytoplasm may be causing low iodide uptake in these tumors (7, 8).

The aim of this study was to quantify NIS transcript levels by real-time PCR and determine the presence and cellular localization of NIS and TSHR proteins by immunohistochemistry in benign and malignant thyroid nodules in comparison with surrounding nonnodular tissue samples. We also performed a combined analysis of NIS mRNA and protein expression for each individual tumor.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and tissue samples

Nodular (12 benign, 13 malignant) and nonnodular (control) thyroid tissue specimens were collected from 25 patients after total or partial thyroidectomy. In addition to that, specimens were obtained from a patient with hot nodule and suppressed TSH that were not included in the statistical analysis. Part of the samples was immediately frozen in liquid nitrogen, and the remaining part was maintained in formalin. Two pathologists classified the nodules according to the World Health Organization histological criteria (9). Exclusion criteria included use of antithyroid drugs, previous treatments with radioiodine or percutaneous ethanol injection, and serum TSH out of the normal ranges (0.5–4 µU/ml). Four patients with clinical hypothyroidism were on levothyroxine (LT4) therapy (Table 1Go).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Thyroid function evaluation, scintigraphy, and pathological diagnosis

 
Before surgery, all patients underwent clinical examination and evaluation with thyroid function tests, ultrasound, and ultrasound-guided fine-needle aspiration biopsy with cytology (Table 1Go). I123 thyroid scintigraphy was performed in 22 patients.

This protocol was approved by the Hospital das Clínicas Ethics Committee, and informed patient consent was obtained for all procedures.

NIS mRNA quantification by real-time PCR

Total RNA was isolated using TRIzol LS (Invitrogen, Carlsbad, CA). cDNA was synthesized with SuperScript III RNase-H reverse transcriptase (Invitrogen). Quantitative RT-PCR assays were carried out using ABsolute QPCR SYBR Green mix (Abgene, Surrey, UK) using Rotor-Gene 3000 equipment (Corbett Research, Mortlake, Australia). The intron-spanning primers and amplicon sizes were: NIS forward, 5'-ACACTGACTGCGACCCTCTCCT-3', NIS reverse, 5'-TGCTGAGGGTGCCACTGTAA-3' (141 bp) (6); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-GCCAAAAGGGTCATCATCTC-3', GAPDH reverse, 5'-CAGGGATGATGTTCTGGAG-3' (281 bp) (Abgene). Standard calibration curves were performed in all experiments. NIS mRNA levels were normalized with GAPDH expression and reported as relative arbitrary units. To confirm NIS expression results, NIS expression was also normalized with PSMC6 (proteasome 26S ATPase subunit 6) expression (10).

Immunohistochemistry analysis

Paraffin-embedded tissue samples were stained by immunoperoxidase (11) with primary NIS antibody (FP5A; Mayo Clinic, Rochester, MN) and TSHR antibody (DakoCytomation, Dako, Glostrup, Denmark). Amplification step was performed with Dako EnVision system, peroxidase kit (Dako, Glostrup, Denmark).

According to localization, NIS positivity was categorized as intracellular or basolateral membrane. An immunostaining score was given according to the percentage of follicular cells with NIS or TSHR-positive staining as follows: 0 (0%), 1+ (low, 1–20%), 2+ (moderate, 21–49%), and 3+ (high, ≥50%).

Statistical analysis

Results were expressed as median and range. P < 0.05 was considered statistically significant. NIS expression was analyzed with the nonparametric tests Wilcoxon signed-rank, Mann-Whitney, and Kruskal-Wallis tests. The Fisher exact test and {chi}2 test were used to compare sonographic characteristics of the nodules.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Clinical data, thyroid function tests, and pathological diagnoses of all patients are shown in Table 1Go. Intranodular calcifications and irregular outline of nodules were the features that helped differentiate benign from malignant nodules (P = 0.02; P = 0.03, respectively, Table 1Go).

NIS mRNA quantification

NIS mRNA was detected in 100% of the control samples, 91.7% of the benign, and 100% of the malignant nodules. NIS expression was reduced in 92% of the nodules (100% of malignant and 83.3% of benign) when compared with controls, with median reduction of 99.08% (62.05–100%). In malignant and benign nodules, NIS expression was significantly lower when compared with controls (P < 0.015, P < 0.001, respectively), with median reduction of 99.8% (73.7–100%) and 94.39% (62.1–100%), respectively.

On the other hand, when only the warm and hot nodules were considered, higher NIS mRNAs levels were observed when compared with control, with increases of up to 3- and 26-fold, respectively.

There were no significant differences in NIS transcript levels between benign and malignant nodules (P = 0.57), independently of histological subtype.

Immunohistochemical detection of NIS protein

Intracellular NIS protein was detected in all benign and malignant nodules and control samples. NIS protein was also identified at the basolateral membrane (Fig. 1Go) in 24% of controls, 8.3% of benign, and 15.4% of malignant nodules (P = 0.51).


Figure 1
View larger version (124K):
[in this window]
[in a new window]

  		FIG. 1. Expression and localization of NIS and TSHR proteins in thyroid tissues. A, Benign hot nodule showing strong plasma membrane NIS immunostaining sample. B, A positive intracellular NIS immunostaining in classic papillary carcinoma sample. C, Low TSHR immunostaining in follicular carcinoma sample. D, Strong TSHR immunostaining in nonnodular thyroid tissue.

 
In all malignant nodules and in 66.7% (8/12) of the benign nodules, intracellular NIS protein had higher positivity when compared with control samples (P = 0.039).

The sample retrieved from the hot nodule showed strong intracellular NIS expression as well as the highest basolateral membrane NIS positivity (Fig. 1AGo).

Immunohistochemical detection of TSHR

TSHR immunostaining was positive in 88% of the control samples (22 of 25), 83.3% of the benign (10 of 12), and 92.3% of the malignant nodules (12 of 13). However, an immunostaining score of 2+ was found in 24% of the control samples (six of 25), 41.6% of the benign (five of 12), and 7.7% of the malignant nodules (one of 13) (P = 0.077), indicating that TSHR is preserved in nodules, albeit with a tendency to reduced expression in malignant ones.

Combined analysis of NIS mRNA and protein expression

A combined analysis of NIS mRNA and intracellular NIS protein data in nodular and control samples showed that six of 12 of the benign nodules (50%) had reduced mRNA concentrations associated with intracellular protein increment, four of 12 (33.4%) had reduced mRNA levels associated with similar protein levels, one of 12 (8.3%) had incremented of both NIS mRNA and protein (warm nodule), and one of 12 (8.3%) had mRNA increment and no difference in protein immunoexpression (Table 2Go).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Combined analysis of NIS mRNA concentrations and NIS and TSHR immunostaining scores of intracellular immunostaining in benign and malignant nodules, compared with nonnodular samples

 
Among the malignant nodules, reduction of NIS mRNA expression and increment of intracellular protein was found in 100% (13 of 13) of the samples.

Moreover, NIS mRNA reduction associated with higher intracellular protein expression was statistically higher in malignant than in benign nodules (P = 0.005).

Considering samples with NIS mRNA reduction associated with protein increment, exclusively in malignant nodules, there was lower TSHR positivity in the nodules than in control tissues (Table 2Go).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Driven by conflicting data in the literature, we evaluated NIS mRNA levels as well as immunohistochemistry NIS and TSHR protein expression in benign and malignant thyroid nodules and compared them with their surrounding nonnodular tissues (control). The goal of this study was the combined analysis of NIS mRNA and NIS protein expression in nodular and nonnodular samples from patients with complete clinical and radiological evaluation.

In agreement with other studies (3, 5, 6, 12), we detected reduced NIS mRNA expression in almost all benign and malignant thyroid nodules when compared with their respective control samples. Interestingly, we found that benign and malignant specimens exhibited similar NIS transcription levels.

There was a high degree of variation in mRNA transcript values in control samples with no associations with variables such as gender, age, time of disease, hormone levels, presence or absence of thyroid antibodies, sonographic or scintigraphic findings, histological diagnosis, immunohistochemical findings, or urinary iodide excretion. We could not therefore establish a normal range for NIS transcript levels in control thyroid specimens.

In some cases, we detected overlapping levels between nodular and control tissues, suggesting that NIS mRNA values per se are not able to discriminate the pathological nature of the thyroid tissue. The reported variability of NIS transcript levels in the literature (4, 5, 6) may be due to several different factors that regulate NIS expression, including circulating and local TSH levels; presence of thyroid-stimulating immunoglobulins or cytokines; iodine concentration; and infiltration of nonnodular areas by cells that do not express NIS, such as lymphocytes (13, 14, 15). However, in this study TSH may not be responsible for this variability because all patients had normal serum TSH levels.

We found no differences in NIS mRNA levels in specimens retrieved from patients on LT4 therapy and those without medication. Similar results were also reported by Trouttet-Masson et al. (12) in four patients with papillary carcinoma on LT4 therapy; however, TSH levels in these patients were not mentioned. Conversely, Bruno et al. (16) showed significantly lower NIS mRNA levels in nonnodular samples when compared with nodular tissues in patients with suppressed TSH secondary to LT4 therapy.

Our immunostaining findings, in accordance with previous studies (7, 8), confirmed that NIS protein was more abundant in nodules than nonnodular areas and located mainly in the intracellular compartment (Fig. 1Go).

The combined analysis of NIS mRNA and NIS protein expression in paired samples showed that all of the malignant nodules (100%) had decreased NIS mRNA levels associated with intracellular NIS protein increment when compared with the surrounding tissue. Only two other studies have evaluated NIS mRNA and NIS protein cellular content simultaneously. Saito et al. (17) detected high transcript levels associated with high protein expression in papillary carcinoma. Trouttet-Masson et al. (12) were able to detect NIS mRNA and very low amount of a nonglycosylated intracellular NIS protein in only 50% of benign and malignant hypofunctioning thyroid tumors.

Our results indicated that in cells from thyroid nodules, low NIS mRNA expression coexists with abundant intracellular NIS protein and that this NIS expression pattern is more frequent in malignant when compared with benign thyroid nodular cells. NIS expression alterations may occur in transcriptional and posttranscriptional levels. Our hypothesis is that NIS protein is unable to migrate to the basolateral membrane in nodular cells, probably due to protein immaturity (18, 19) and/or aberrant low protein turnover, which has recently been observed in cancer cells. Intracellular accumulation of NIS may also promote a negative feedback on NIS mRNA synthesis, resulting in decreased expression (20). The low TSHR expression observed in malignant nodules may also contribute to reduced NIS mRNA synthesis and deficient protein migration.

In conclusion, our results confirmed low NIS mRNA expression associated with strong intracellular NIS protein staining in thyroid carcinomas, which may be due to defective targeting of NIS to the plasma membrane. These events may explain the low iodide uptake seen in malignant tumors cells.


   Acknowledgments
 
We gratefully acknowledge Dr. John Morris (Mayo Clinic, Rochester, MN) for kindly providing the NIS monoclonal antibody.


   Footnotes
 
This work was supported by Grant 03/08900-9 from Fundacão de Amparo á Pesquisa do Estado de São Paulo (FAPESP), São Paulo, Brazil, and a partial financial grant from Instituto da Tiroide, São Paulo, Brazil.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 15, 2008

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; LT4, treatment with levothyroxine; NIS, sodium iodide symporter; TSHR, TSH receptor.

Received February 21, 2007.

Accepted July 8, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Levy O, Dai G, Riedel C, Ginter CS, Paul EM, Lebowitz AN, Carrasco N 1997 Characterization of the thyroid Na+/I– symporter with an anti-COOH terminus antibody. Proc Natl Acad Sci USA 94:5568–5573[Abstract/Free Full Text]
  2. Caillou B, Troalen F, Baudin E, Talbot M, Filetti S, Schlumberger M, Bidart JM 1998 Na+/I– symporter distribution in human thyroid tissues: an immunohistochemical study. J Clin Endocrinol Metab 83:4102–4106[Abstract/Free Full Text]
  3. Arturi F, Russo D, Schlumberger M, du Villard JA, Caillou B, Vigneri P, Wicker R, Chiefari E, Suarez HG, Filetti S 1998 Iodide symporter gene expression in human thyroid tumors. J Clin Endocrinol Metab 83:2493–2496[Abstract/Free Full Text]
  4. Ryu KY, Senokozlieff ME, Smanik PA, Wong MG, Siperstein AE, Duh QY, Clark OH, Mazzaferri EL, Jhiang SM 1999 Development of reverse transcription-competitive polymerase chain reaction method to quantitate the expression levels of human sodium iodide symporter. Thyroid 9:405–409[Medline]
  5. Lazar V, Bidart JM, Caillou B, Mahe C, Lacroix L, Filetti S, Schlumberger M 1999 Expression of the Na+/I– symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes. J Clin Endocrinol Metab 84:3228–3234[Abstract/Free Full Text]
  6. Ringel MD, Anderson J, Souza SL, Burch HB, Tambascia M, Shriver CD, Tuttle RM 2001 Expression of the sodium iodide symporter and thyroglobulin genes are reduced in papillary thyroid cancer. Mod Pathol 14:289–296[CrossRef][Medline]
  7. Dohan O, Baloch Z, Banrevi Z, Livolsi V, Carrasco N 2001 Predominant intracellular overexpression of the Na(+)/I(–) symporter (NIS) in a large sampling of thyroid cancer cases. J Clin Endocrinol Metab 86:2697–2700[Abstract/Free Full Text]
  8. Wapnir IL, van de Rijn M, Nowels K, Amenta PS, Walton K, Montgomery K, Greco RS, Dohan O, Carrasco N 2003 Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections. J Clin Endocrinol Metab 88:1880–1888[Abstract/Free Full Text]
  9. Hedinger C, Williams ED, Sobin LH 1989 The WHO histological classification of thyroid tumors: a commentary on the second edition. Cancer 63:908–911[CrossRef][Medline]
  10. de Sá SV, Corrêa-Giannella ML, Machado MC, Krogh K, de Almeida MQ, Albergaria Pereira MA, Coelho Siqueira SA, Patzina RA, Ibuki FS, Sogayar MC, Machado MC, Giannella-Neto D 2007 Serpin peptidase inhibitor clade A member 1 as a potential marker for malignancy in insulinomas. Clin Cancer Res 13:5322–5330[Abstract/Free Full Text]
  11. Castro MR, Bergert ER, Beito, TG, McIver B, Goellner JR, Morris JC 1999 Development of monoclonal antibodies against the human sodium iodide symporter: immunohistochemical characterization of this protein in thyroid cells. J Clin Endocrinol Metab 84:2957–2962[Abstract/Free Full Text]
  12. Trouttet-Masson S, Selmi-Ruby S, Bernier-Valentin F, Porra V, Berger-Dutrieux N, Decaussin M, Peix JL, Perrin A, Bournaud C, Orgiazzi J, Borson-Chazot F, Franc B, Rousset B 2004 Evidence for transcriptional and posttranscriptional alterations of the sodium/iodide symporter expression in hypofunctioning benign and malignant thyroid tumors. Am J Pathol 165:25–34[Abstract/Free Full Text]
  13. Ajjan RA, Watson PF, Findlay C, Metcalfe RA, Crisp M, Ludgate M, Weetman AP 1998 The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity. J Endocrinol 158:351–358[Abstract]
  14. Ajjan RA, Kamaruddin NA, Crisp M, Watson PF, Ludgate M, Weetman AP 1998 Regulation and tissue distribution of the human sodium iodide symporter gene. Clin Endocrinol (Oxf) 49:517–523[CrossRef][Medline]
  15. Eng PH, Cardona GR, Fang SL, Previti M, Alex S, Carrasco N, Chin WW, Braverman LE 1999 Escape from the acute Wolff-Chaikoff effect is associated with a decrease in thyroid sodium/iodide symporter messenger ribonucleic acid and protein. Endocrinology 140:3404–3410[Abstract/Free Full Text]
  16. Bruno R, Ferretti E, Tosi E, Arturi F, Giannasio P, Mattei T, Scipioni A, Presta I, Morisi R, Gulino A, Filetti S, Russo D 2005 Modulation of thyroid-specific gene expression in normal and nodular human thyroid tissues from adults: an in vivo effect of thyrotropin. J Clin Endocrinol Metab 90:5692–5697[Abstract/Free Full Text]
  17. Saito T, Endo T, Kawaguchi A, Ikeda M, Katoh R, Kawaoi A, Muramatsu A, Onaya T 1998 Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas. J Clin Invest 101:1296–1300[Medline]
  18. Levy O, De la Vieja A, Ginter CS, Riedel C, Dai G, Carrasco N 1998 N-linked glycosylation of the thyroid Na+/I– symporter (NIS). Implications for its secondary structure model. J Biol Chem 273:22657–22663[Abstract/Free Full Text]
  19. Riedel C, Levy O, Carrasco N 2001 Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem 276:21458–21463[Abstract/Free Full Text]
  20. Suzuki K, Kohn LD 2006 Differential regulation of apical and basal iodide transporters in the thyroid by thyroglobulin. J Endocrinol 189:247–255[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sodré, A. K. M. B.
Right arrow Articles by Camargo, R. Y. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sodré, A. K. M. B.
Right arrow Articles by Camargo, R. Y. A.
Related Collections
Right arrow Thyroid
Right arrow Endocrine Oncology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals