Do cell surface trafficking impairments account for variable cell surface sodium iodide symporter levels in breast cancer?

The Na(+)/I(-) symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into thyroid follicular cells and serves as the molecular basis of radioiodine imaging and therapy for thyroid cancer patients. The finding that NIS protein is present in 80-90% of breast tumors suggests that breast cancer patients may also benefit from NIS-mediated radionuclide imaging and targeted therapy. However, only 17-25% of NIS-positive breast tumors have detectable radionuclide uptake activity. The discrepancy between NIS expression and radionuclide uptake activity is most likely contributed by variable cell surface NIS protein levels. Apart from the prevalent view that NIS cell surface trafficking impairments account for the variability, our current study proposes that differential levels of NIS expression may also account for variable cell surface NIS levels among breast tumors. We address the need to confirm the identity of intracellular NIS staining to reveal the mechanisms underlying variable cell surface NIS levels. In addition, we warrant a quantitative correlation between cell surface NIS levels and radionuclide uptake activity in patients such that the cell surface NIS levels required for radionuclide imaging can be defined and the defects impairing NIS activity can be recognized.

Genetic alterations in thyroid cancer: the role of mouse models.

Thyroid carcinomas are the most common endocrine neoplasms in humans, with a globally increasing incidence. Thyroid follicular cells and neuroendocrine (parafollicular) C cells are each susceptible to neoplastic transformation, resulting in thyroid cancers of differing phenotypes with unique associated genetic mutations and clinical outcomes. Over the past 15 years, several sophisticated genetically engineered mouse models of thyroid cancer have been created to further our understanding of the genetic events leading to thyroid carcinogenesis in vivo. The most significant mouse models of papillary, follicular, anaplastic, and medullary thyroid carcinoma are highlighted, with particular emphasis on the relationship between the relevant oncogenes in these models and genetic events in the naturally occurring human disease. Limitations of each model are presented, and the need for additional models to better recapitulate certain aspects of the human disease is discussed.

Nasolacrimal obstruction secondary to I(131) therapy.

PURPOSE: To report the finding of nasolacrimal drainage system obstruction associated with I(131) therapy for thyroid carcinoma from an updated and expanded cohort. METHODS: Patients with a history of epithelial derived thyroid carcinoma who had tearing were offered referral for evaluation by an oculoplastic surgeon. Patients underwent nasolacrimal probing and irrigation procedures with localization of their nasolacrimal obstruction. Therapy for the site of obstruction was instituted. RESULTS: Clinically significant tearing was identified in 26 patients, all of whom had previously undergone I(131) therapy (n = 563). Nineteen patients were evaluated and confirmed to have nasolacrimal drainage system obstruction; 7 have yet to be formally evaluated. Areas of obstruction included nasolacrimal duct, common canaliculus, and, rarely, distal upper and lower canaliculi. Patients were treated with a variety of modalities including silicone intubation, balloon dacryoplasty, dacryocystorhinostomy, and conjunctival dacryocystorhinostomy. CONCLUSIONS: The use of I(131) for thyroid carcinoma is associated with a 3.4% incidence of documented nasolacrimal drainage obstruction and an overall 4.6% incidence of documented or suspected obstruction. The true incidence may be higher, since - I(131) treated individuals were neither systematically evaluated nor questioned about tearing. It has yet to be established if the obstructions result from local toxicity caused by the passive flow of radioactive iodine containing tears through these tissues or the active uptake and concentration of I(131) in lacrimal drainage system tissues through the sodium/iodide supporter.

Effects of dose, intervention time, and radionuclide on sodium iodide symporter (NIS)-targeted radionuclide therapy.

The sodium iodide symporter (NIS) mediates iodide uptake into thyrocytes and is the molecular basis of thyroid radioiodine therapy. We previously have shown that NIS gene transfer into the F98 rat gliomas facilitated tumor imaging and increased survival by radioiodine. In this study, we show that: (1) the therapeutic effectiveness of (131)I in prolonging the survival time of rats bearing F98/hNIS gliomas is dose- and treatment-time-dependent; (2) the number of remaining NIS-expressing tumor cells decreased greatly in RG2/hNIS gliomas post (131)I treatment and was inversely related to survival time; (3) 8 mCi each of (125)I/(131)I is as effective as 16 mCi (131)I alone, despite a smaller tumor absorbed dose; (4) (188)ReO(4), a potent beta(-) emitter, is more efficient than (131)I to enhance the survival of rats bearing F98/hNIS gliomas. These studies demonstrate the importance of radiopharmaceutical selection, dose, and timing of treatment to optimize the therapeutic effectiveness of NIS-targeted radionuclide therapy following gene transfer into gliomas.

In vivo imaging and radioiodine therapy following sodium iodide symporter gene transfer in animal model of intracerebral gliomas.

Radioactive iodide uptake (RAIU) in thyroid follicular epithelial cells, mediated by the sodium iodide symporter (NIS), is the first rate-limiting step in iodide accumulation which provides a mechanism for effective radioiodide treatment for patients with thyroid cancer. We hypothesize that NIS gene transfer to non-thyroid tumor cells will enhance intracellular radioiodide accumulation and result in better tumor control. Here, we performed non-invasive tumor imaging and (131)I therapy studies using rats bearing intracerebral F98 gliomas that have been retrovirally transduced with human NIS. Our results show that: (1) NIS is expressed in the intracerebral F98/NIS gliomas; (2) F98/NIS gliomas can be imaged by (99m)TcO(4) (whose uptake is also mediated by NIS) and (123)I scintigraphy; (3) significant amounts of radioiodide were retained in the tumors at 24 h after (123)I injection; (4) RAIU and NIS expression in the thyroid gland can be reduced by feeding a thyroxine-supplemented diet; and (5) survival time was increased in rats bearing F98/hNIS tumors by (131)I treatment. These studies warrant further investigating tumor imaging and therapeutic strategies based on NIS gene transfer followed by radioiodide administration in a variety of human cancers.

The RET proto-oncogene in human cancers.

The activation of the RET proto-oncogene contributes to the development of human cancers in two different ways. Somatic rearrangements of RET with a variety of activating genes, which contribute to unscheduled expression and constitutive dimerization of the chimeric RET/PTC oncoproteins in thyroid follicular cells, are frequently found in radiation-induced papillary thyroid carcinomas. Germ-line mutations, mainly point mutations, that lead to constitutive activation of RET tyrosine kinase activity are responsible for the development of the inherited cancer syndrome, multiple endocrine neoplasia type 2. There appears to be a correlation between specific types of RET mutation and clinical phenotypes of the cancers involved. The biological effects and the signaling pathways induced by different forms of RET activation have been investigated in a variety of cultured cells as well as in genetically engineered animal models. The identification of RET mutations in most MEN 2 families (95%) has translated into improved care for MEN 2 patients. However, further investigation of the signaling pathways contributing to tumorigenesis in relevant tissues will eventually help us to develop novel strategies to prevent or to treat human papillary thyroid carcinomas, MEN 2 disease, as well as the sporadic cancers relevant to MEN 2 disease.

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.

Hormonal regulation of radioiodide uptake activity and Na+/I- symporter expression in mammary glands.

The observation that radioiodide uptake (RAIU) activity, mediated by the Na+/I- symporter (NIS), is significantly increased in lactating breast suggests that RAIU and NIS expression in mammary gland are modulated by hormones involved in active lactation. We showed that both the NIS expression level and RAIU in rat mammary gland are maximal during active lactation compared to those in the mammary glands of virgin and pregnant rats as well as the involuting mammary gland. In the lactating mammary gland, NIS is clustered on the basolateral membrane of alveolar cells as a lesser glycosylated form than NIS in thyroid. The RAIU of lactating mammary gland was partially inhibited by treatment with a selective oxytocin antagonist or bromocriptine, an inhibitor of PRL release. These findings suggest that RAIU and NIS expression in mammary gland are at least in part modulated by oxytocin and PRL. Indeed, we showed that NIS messenger ribonucleic acid level was increased in a dose-dependent manner by oxytocin and PRL in histocultured human breast tumors.

Loss of p53 promotes anaplasia and local invasion in ret/PTC1-induced thyroid carcinomas.

Papillary thyroid carcinomas in humans are associated with the ret/PTC oncogene and, following loss of p53 function, may progress to anaplastic carcinomas. Mice with thyroid-targeted expression of ret/PTC1 developed papillary thyroid carcinomas that were minimally invasive and did not metastasize. These mice were crossed with p53-/- mice to investigate whether loss of p53 would promote anaplasia and metastasis of ret/PTC1-induced thyroid tumors. The majority of p53-/- mice died or were euthanized by 17 weeks of age due to the development of thymic lymphomas, soft tissue sarcomas, and testicular teratomas. All ret/PTC1 mice developed thyroid carcinomas, but tumors in p53-/- mice were more anaplastic, larger in diameter, more invasive, and had a higher mitotic index than tumors in p53+/+ and p53+/- mice. Thyroid tumors did not metastasize in any of the experimental p53+/+ and p53+/- mice

Expression and activity of human Na+/I- symporter in human glioma cells by adenovirus-mediated gene delivery.

Radioiodide concentrating activity in the thyroid, mediated by human Na+/I- symporter (hNIS), provides a mechanism for effective radioiodide treatment for patients who have invasive, recurrent, and metastatic thyroid cancers after total thyroidectomy. In an attempt to develop hNIS gene transfer for radioiodide therapy for patients with brain tumors, we have constructed recombinant adenoviruses, rAd-CMV-hNIS9 and rAd-CMV-FLhNIS, to express exogenous hNIS in U1240 and U1240Tag human glioma cells. U1240Tag differs from U1240 glioma cells in that it expresses the SV40 large T antigen oncoprotein. In both U1240 and U1240Tag cells, radioiodide uptake (RAIU) activity in the cells infected with rAd-CMV-hNIS9 or rAd-CMV-FLhNIS increases as the adenoviral MOI increases. The protein expression profile of hNIS in infected cells is generally in agreement with their RAIU activity profile. Although the expressed hNIS9 protein appeared to have a shorter half-life than FLhNIS, hNIS9 expression could be maintained by multiple infections in these cells. In addition, we show that hNIS can be expressed and function in a xenografted human glioma by intratumoral injection of rAd-CMV-hNIS9.

Monoclonal antibodies against the human sodium iodide symporter: utility for immunocytochemistry of thyroid cancer.

The recent cloning of the thyroidal protein that is responsible for iodide transport, the sodium iodide symporter (hNIS), has made possible studies designed to characterize its structure, function and expression in thyroidal tissues. Using a mannose binding protein (MBP)-hNIS fusion protein as antigen, we have developed mouse monoclonal antibodies against hNIS to utilize as tools in such studies. Twenty-four clones were initially recovered which recognized the MBP-hNIS fusion protein, but only two of them were specific for hNIS while the others recognized MBP alone. Both antibodies were found to be immunoglobulin G (IgG) 1kappa (kappa). The specificity of antibodies was tested by Western blotting using membranes prepared from COS-7 cells transiently transfected with the pcDNA3 plasmid containing the full-length hNIS cDNA, or cells transfected with the pcDNA3 vector. A major band with a molecular weight (MW) of approximately 97 kDa, and several minor bands with MW of approximately 160 kDa, approximately 68 kDa, approximately 30 kDa and approximately 15 kDa, were detected specifically in the hNIS-transfected cells. After enzymatic deglycosylation, the major band was present at 68 kDa, as expected based upon the amino acid sequence of hNIS. Immunohistochemistry was performed with several different types of thyroid tissue and non-thyroidal tissues, using the monoclonal antibodies. Strong immunostaining was observed in Graves' tissue, with intermediate staining in papillary and follicular thyroid cancers and an absence of staining in Hürthle cell cancer. The staining was specific for the follicular epithelium and was concentrated in the basolateral portion of the cell membrane. These monoclonal hNIS antibodies should prove useful in the characterization of NIS expression in benign and malignant thyroid tissue and in studies characterizing its structure and function.

Early cellular abnormalities induced by RET/PTC1 oncogene in thyroid-targeted transgenic mice.

The RET/PTC1 oncogene, a rearranged form of the RET proto-oncogene, has been reported to be associated with human papillary thyroid carcinomas. We have shown that targeted expression of RET/PTC1 in the thyroid gland leads to the development of thyroid carcinomas in transgenic mice with histologic and cytologic similarities to human papillary thyroid carcinoma. To further investigate how RET/PTC1 expression contributes to the pathogenesis of papillary thyroid tumor, the time of tumor onset and the early phenotypic consequences of RET/PTC1 expression in thyrocytes were determined. All high copy transgenic mice developed bilateral thyroid tumors as early as 4 days of age. At embryological days 16-18, increased proliferation rate, distorted thyroid follicle formation and reduced radioiodide concentrating activity were identified in transgenic embryos. The reduced radioiodide concentrating activity was attributed to decreased expression of the sodium-iodide symporter. Our study showed that RET/PTC1 not only increased proliferation of thyrocytes, it also altered morphogenesis and differentiation. These findings provide a model for the role of RET/PTC1 in the formation of abnormal follicles with reduced iodide uptake ability observed in human papillary thyroid carcinoma.

Development of reverse transcription-competitive polymerase chain reaction method to quantitate the expression levels of human sodium iodide symporter.

The sodium iodide symporter (NIS) is the plasma membrane protein that mediates active iodide uptake into thyroid follicular cells. To investigate whether human NIS (hNIS) mRNA levels in papillary thyroid carcinomas (PCs) correlate with the ability of tumors to concentrate radioiodide, we developed a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method to quantify the hNIS mRNA levels in thyroid tissues. We studied 7 normal thyroid tissues, 8 PCs, and 1 follicular adenoma. hNIS mRNA levels in PCs were generally lower than those found in normal thyroid tissues. The reduced radioiodide concentrating activity of PCs is due, at least in part, to the reduced expression and/or the decreased stability of hNIS mRNA.

High prevalence of T354P sodium/iodide symporter gene mutation in Japanese patients with iodide transport defect who have heterogeneous clinical pictures.

A missense and loss of function mutation of the Na+/I- symporter (NIS) gene, T354P [Thr354-->Pro (ACA-->CCA)], was found in the homozygous state in two unrelated Japanese patients with iodide transport defect. In this study we have identified the homozygous T354P NIS germline mutation in seven Japanese patients, including one previously reported, from five unrelated families. No other nucleotide changes were found in the coding regions and the exon-intron boundaries of the NIS gene in these seven patients. These results suggest a common prevalence of the T354P mutation in Japanese patients. Although these seven patients have the identical NIS mutation, T354P, marked heterogeneity in clinical pictures, especially concerning goiter and hypothyroidism, were noted among them. Therefore, another factor(s), but not the nature of the NIS mutation, may account for the clinical heterogeneity among patients with the iodide transport defect. We have previously reported that the NIS messenger ribonucleic acid was markedly increased in the thyroid of a patient with the homozygous T354P mutation. In this study we demonstrated that the NIS proteins in the patients' thyroids were significantly increased (approximately 10-fold) by Western blot analysis of integral membrane proteins using an antibody against the C-terminal peptide of the human NIS. Furthermore, we showed by immunohistochemical staining that the T354P mutant NIS proteins were overexpressed in the basal and lateral plasma membranes of patients' thyrocytes.

An immunohistochemical study of Na+/I- symporter in human thyroid tissues and salivary gland tissues.

The human Na+/I- symporter (hNIS) is the plasma membrane protein that mediates active iodide uptake into several tissues, such as the thyroid and salivary glands. To study the distribution and cellular localization of the hNIS protein, we have generated a polyclonal antibody that could detect the hNIS protein by immunohistochemical staining on tissue sections. In normal thyroids, hNIS expression is heterogeneous, and it is only detected in sporadic thyrocytes of a given follicle. The hNIS protein was not detected in thyroid carcinomas, yet it was detected in the majority of thyrocytes in Graves' thyroids. In salivary glands, hNIS protein was not detected in acinar cells, but it was detected in ductal cells. The hNIS proteins are clustered in the basal and lateral membranes in cells stained positive for hNIS.

Promoter characterization of the human Na+/I- symporter.

The Na+/I- symporter (NIS) is the plasma membrane protein that mediates active iodide uptake into thyroid follicular cells. To understand the molecular mechanisms of human NIS (hNIS) gene regulation, the 2-kb immediate 5'-flanking region of hNIS was characterized. The tentative hNIS transcriptional start site was mapped to -375 nucleotide relative to the ATG site. Various 5'-genomic DNA fragments were tested for promoter activity in thyroid and nonthyroid cells. Our results indicate that the DNA regulatory elements in the 2-kb immediate 5'-flanking region were not sufficient to confer thyroid-selective transcription of the hNIS gene. In addition, hNIS minimal promoter was localized to a region of 144 bp that contains a 90-bp stretch of a highly conserved DNA fragment between hNIS and rat NIS.

Novel, missense and loss-of-function mutations in the sodium/iodide symporter gene causing iodide transport defect in three Japanese patients.

Iodide transport defect is a disorder affecting the active transport of iodide, an essential step in the synthesis of thyroid hormones. We have identified novel germ-line mutations in the Na+/I- symporter (NIS) gene from three Japanese patients with iodide transport defect. One patient had a compound heterozygous mutation of T354P/G93R (Gly93-->Arg [GGC-->CGC]), and two sibling patients had a homozygous mutation of G543E (Gly543-->Glu [GGA-->GGA]). G93R and G543E, two novel mutations, are located in the 3rd and 12th transmembrane domains of NIS which are encoded by exons 1 and 13, respectively. The NIS mutants carrying these mutations had minimal iodide uptake activity when expressed in COS-7 cells, confirming that the identified mutations are the direct cause of the iodide transport defect in these patients. Genotyping of unaffected family members and functional assays of co-transfected COS-7 cells indicate that expression of one normal NIS allele in the heterozygote (T354P, G93R, or G543E) is sufficient to maintain active iodide uptake activity. Thus, none of these NIS mutants acts as a dominant-negative mutant.

Congenital hypothyroidism due to mutations in the sodium/iodide symporter. Identification of a nonsense mutation producing a downstream cryptic 3' splice site.

A 12-yr-old hypothyroid girl was diagnosed at birth as athyreotic because her thyroid gland could not be visualized by isotope scanning. Goiter development due to incomplete thyrotropin suppression, a thyroidal radioiodide uptake of < 1%, and a low saliva to plasma ratio of 2.5 suggested iodide (I-) transport defect. mRNA isolated from her thyroid gland and injected into Xenopus oocytes failed to increase I- transport. Sequencing of the entire Na+/I- symporter (NIS) cDNA revealed a C to G transversion of nucleotide (nt) 1146 in exon 6, resulting in a Gln 267 (CAG) to Glu (GAG) substitution. This missense mutation produces an NIS with undetectable I- transport activity when expressed in COS-7 cells. Although only this missense mutation was identified in thyroid and lymphocyte cDNA, genotyping revealed that the proposita and her unaffected brother and father were heterozygous for this mutation. However, amplification of cDNA with a primer specific for the wild-type nt 1146 yielded a sequence lacking 67 nt. Genomic DNA showed a C to G transversion of nt 1940, producing a stop codon as well as a new downstream cryptic 3' splice acceptor site in exon 13, responsible for the 67 nt deletion, frameshift, and premature stop predicting an NIS lacking 129 carboxy-terminal amino acids. This mutation was inherited from the mother and present in the unaffected sister. Thus, although the proposita is a compound heterozygote, because of the very low expression (< 2.5%) of one mutant allele, she is functionally hemizygous for an NIS without detectable bioactivity.