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For the past 80 years, radioiodine (131I) has been used to ablate thyroid tissue not removed by surgery or to treat differentiated thyroid cancer that has metastasized to other parts of the body. However, the Na+/I− symporter (NIS), which mediates active iodide uptake into thyroid follicular cells, is also expressed in several non-thyroidal tissues. This NIS expression permits 131I accumulation and radiation damage in these non-target tissues, which accounts for the adverse effects of radioiodine therapy. We will review the data regarding the expression, function, and regulation of NIS in non-thyroidal tissues and explain the seemingly paradoxical adverse effects induced by 131I, the self-limited gastrointestinal adverse effects in contrast to the permanent salivary dysfunction that is seen after 131I therapy. We propose that prospective studies are needed to uncover the time-course of pathological processes underlying development and progression or ultimate resolution of 131I-induced salivary ductal obstruction and nasolacrimal duct obstruction. Finally, preventive measures and early therapeutic interventions that can be applied potentially to eliminate or alleviate long-term radioiodine adverse effects will be discussed.

Background: Thyroid cancer patients with radioiodine-refractory (RAI-R) disease, resulting from insufficient RAI delivery and/or RAI resistance, have increased mortality and limited treatment options. To date, studies have largely focused on tumor mutations associated with different stages of disease, which could provide prognostic value for RAI-R disease. It was hypothesized that germline variants contributing to intrinsic differences in iodine metabolism, tumor microenvironment, and/or immune surveillance are associated with RAI-R disease. Methods: Whole-genome genotyping data analysis was performed on 1145 Caucasian (CAU) patients, 244 of whom were RAI-R, and 55 African American (AA) patients, nine of whom were RAI-R. Germline-variant association studies were conducted using candidate genes involved in iodine metabolism or DNA-damage repair, as well as genome-wide association analysis. Initial data indicated several notable variants in a small number of patients (n = 7), who were later determined to be AA patients of >80% African ancestry (n = 37). This led to the study focusing on germline single nucleotide polymorphisms uniquely associated with RAI-R AA patients. Sanger sequencing was performed to validate risk alleles and identify the incidence of the common somatic mutations BRAF(V600E), NRAS(Q61R), and HRAS(Q61R) in AA patients whose primary tumor samples were available (28/55). Results: TG, BRCA1, and NSMCE2 haplotypes were identified as being uniquely associated with RAI-R AA patients of >80% African ancestry. All patients with the TG haplotype (n = 4) had a biochemical incomplete response to RAI therapy. Patients with the NSMCE2 haplotype (n = 4) were diagnosed at a young age (13, 17, 17, and 26 years old) with distant metastatic disease at initial diagnosis. The BRCA1 haplotype co-occurred in three out of four patients with the NSMCE2 haplotype. The incidence of BRAF(V600E) appears lower in papillary thyroid carcinomas from AA patients of >80% African ancestry (3/14; 21%) than in AA patients of

Radioactive iodine (RAI) 131I is a targeted therapy for patients with RAI-avid follicular cell-derived thyroid cancer. However, the responsiveness to 131I therapy varies among thyroid cancer patients mainly owing to differential RAI uptake and RAI radiosensitivity among patients’ lesions. A personalized approach to maximize 131I therapeutic efficacy is proposed based on recent scientific advances and future opportunities.

The sodium iodide symporter (NIS) is a classical iodide pump typically localized within the cell plasma membrane in thyroid cells, where NIS expression is believed to ensure success of mainstay radioiodide therapy in thyroid cancers. Although radioiodide uptake is generally reduced in thyroid cancer tissue, intracellular nonmembranous NIS has been reported to increase, suggesting that NIS serves a pump-independent function. Thyroid cancer is one of the major component cancers of Cowden syndrome, a subset of which is caused by germline mutations in PTEN. In this study, we explored the noncanonical tumorigenic role of NIS in thyroid cancer cells in relation to PTEN signaling. PTEN knockdown in thyroid cancer cell lines stabilized intracellular NIS protein by promoting an interaction with NIS-LARG (leukemia-associated RhoA guanine exchange factor). Increased protein levels of cytoplasmic NIS enhanced RhoA activation and resulted in a promigration tumorigenic phenotype. Inhibition of NIS glycosylation through activation of the PI3K/AKT/mTOR signaling pathway contributed to mislocalization of NIS in the cytoplasm, facilitating its nonpump tumorigenic function through an interaction with LARG, which predominantly localized in the cytoplasm. Moreover, PTEN or PI3K/AKT/mTOR signaling could affect DPAGT1, a glycosylating enzyme involved in the initial step of N-linked glycosylation, to inhibit glycosylation of NIS. In summary, our results elucidate a pump-independent, protumorigenic role for NIS in thyroid cancer via its cross-talk with PTEN signaling.Significance: A novel pump-independent protumorigenic role of nonmembranous NIS challenges the presumption that radioiodine treatment of thyroid cancer is ineffective when transmembrane NIS is not expressed. Cancer Res; 78(21); 6121–33. ©2018 AACR.

Thyroid transcription factor 1 (TTF1) regulates the tissue-specific expression of genes. However, the molecular regulation of TTF1 in thyroid normal and carcinoma cells has not been revealed. Here we identify 2 distinct ubiquitin E3 ligases that are responsible for TTF1 degradation in normal thyroid cells and carcinoma cells, respectively. Phorbol myristate acetate induced TTF1 protein degradation in the ubiquitin-proteasome system in both HTori3 thyroid follicular epithelial cells and follicular thyroid carcinoma 133 (FTC133) cells. Lysine 151 residue was identified as a ubiquitin acceptor site within TTF1 in both cell types. Overexpression of E3 ubiquitin protein ligase 1 containing HECT, C2, and WW domain (HECW1) induced TTF1 degradation and ubiquitination in Htori3 cells but not in FTC133 cells. Overexpression of ubiquitin E3 ligase subunit FBXL19 increased TTF1 ubiquitination and degradation in FTC133 cells, but it had no effect on TTF1 levels in Htori3 cells. Overexpression of TTF1 increased thyroglobulin and sodium/iodide symporter mRNA levels, cell migration, and proliferation in HTori3 cells, whereas the effects were reversed by the overexpression of HECW1. This study reveals an undiscovered molecular mechanism by which TTF1 ubiquitination and degradation is regulated by different E3 ligases in thyroid normal and tumor cells.—Liu, J., Dong, S., Wang, H., Li, L., Ye, Q., Li, Y., Miao, J., Jhiang, S., Zhao, J., Zhao, Y. Two distinct E3 ligases, SCF(FBXL19) and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively.

Purpose: Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. Experimental Design: Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. Results: BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. Conclusions: BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.

Context: Sialadenitis and xerostomia are major adverse effects of 131I therapy in thyroid cancer patients. The risk factors for these adverse effects, other than administered activity of 131I, have not been investigated. Objective: The aim of this study is to identify risk factors for 131I-induced salivary gland damage among follicular cell-derived thyroid cancer patients. Design: We enrolled 216 thyroid cancer patients who visited The Ohio State University Wexner Medical Center between April 2013 and April 2014. Symptoms of xerostomia and sialadenitis were identified via questionnaire and medical record search. To validate the findings in a large cohort, we retrospectively searched for ICD-9/10 codes for sialadenitis, xerostomia, and autoimmune disease associated with Sjogren's syndrome (AID-SS) in our existing database (n = 1507). Demographic and clinical information was extracted from medical records. Multivariate analyses were performed to identify independent predictors for salivary gland damage. Res...

The ability of thyroid follicular cells to take up iodine enables the use of radioactive iodine (RAI) for imaging and targeted killing of RAI-avid thyroid cancer following thyroidectomy. To facilitate identifying novel strategies to improveA customized mouse cradle was designed and used for microSPECT/CT image acquisition at 1 hour (t1) and 24 hours (t24) post injection ofThe customized mouse cradle facilitates consistent tissue configuration among image acquisitions such that rigid body registration can be applied to align serial images of the same mouse via the in-house CTViewer software. CTViewer is designed specifically to streamline SPECT/CT image analysis with functions tailored to quantify thyroid radioiodine uptake. Automatic segmentation of thyroid volumes of interest (VOI) from adjacent salivary glands in t1 images is enabled by superimposing the thyroid VOI from the t24 image onto the corresponding aligned t1 image. The extent of heterogeneity inMicroSPECT/CT image acquisition and analysis for thyroidal RAI uptake is greatly improved by the cradle and the CTViewer software, respectively. Furthermore, the approach of superimposing thyroid VOIs from t24 images to select thyroid VOIs on corresponding aligned t1 images can be applied to studies in which the target tissue has differential radiotracer retention from surrounding tissues.

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro, human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia.

Sialadenitis and xerostomia are major adverse effects ofThe aim of this study is to identify risk factors forWe enrolled 216 thyroid cancer patients who visited The Ohio State University Wexner Medical Center between April 2013 and April 2014. Symptoms of xerostomia and sialadenitis were identified via questionnaire and medical record search. To validate the findings in a large cohort, we retrospectively searched for ICD-9/10 codes for sialadenitis, xerostomia, and autoimmune disease associated with Sjögren's syndrome (AID-SS) in our existing database (n = 1507). Demographic and clinical information was extracted from medical records. Multivariate analyses were performed to identify independent predictors for salivary gland damage.Risk factors for

Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is l-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7-13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2-5-fold higher than that of nonaarginine (R(9)). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.

Micro-single-photon emission computed tomography (SPECT) provides a noninvasive way to evaluate the effects of genetic and/or pharmacological modulation on sodium-iodide symporter (NIS)-mediated radionuclide accumulation in mouse thyroid and salivary glands. However, parameters affecting image acquisition and analysis of mouse thyroids and salivary glands have not been thoroughly investigated. In this study, we investigated the effects of region-of-interest (ROI) selection, collimation, scan time, and imaging orbit on image acquisition and quantification of thyroidal and salivary radionuclide accumulation in mice.The effects of data window minima and maxima on thyroidal and salivary ROI selection using a visual boundary method were examined in SPECT images acquired from mice injected with (123)I NaI. The effects of collimation, scan time, and imaging orbit on counting linearity and signal intensity were investigated using phantoms filled with various activities of (123)I NaI or Tc-99m pertechnetate. Spatial resolution of target organs in whole-animal images was compared between circular orbit with parallel-hole collimation and spiral orbit with five-pinhole collimation. Lastly, the inter-experimental variability of the same mouse scanned multiple times was compared with the intra-experimental variability among different mice scanned at the same time.Thyroid ROI was separated from salivary glands by empirically increasing the data window maxima. Counting linearity within the range of 0.5-14.2 μCi was validated by phantom imaging using single- or multiple-pinhole collimators with circular or spiral imaging orbit. Scanning time could be shortened to 15 minutes per mouse without compromising counting linearity despite proportionally decreased signal intensity. Whole-animal imaging using a spiral orbit with five-pinhole collimators achieved a high spatial resolution and counting linearity. Finally, the extent of inter-experimental variability of NIS-mediated radionuclide accumulation in the thyroid and salivary glands by SPECT imaging in the same mouse was less than the magnitude of variability among the littermates.The impacts of multiple variables and experimental designs on micro-SPECT imaging and quantification of radionuclide accumulation in mouse thyroid and salivary glands can be minimized. This platform will serve as an invaluable tool to screen for pharmacologic reagents that differentially modulate thyroidal and salivary radioiodine accumulation in preclinical mouse models.

The selective increase of Na+/I−symporter (NIS)-mediated active iodide uptake in thyroid cells allows the use of radioiodine I131for diagnosis and targeted treatment of thyroid cancers. However, NIS-mediated radioiodine accumulation is often reduced in thyroid cancers due to decreased NIS expression/function. As PI3K signaling is overactivated in many thyroid tumors, we investigated the effects of inhibitors for PI3K, Akt, or mTORC1 as well as their interplay on NIS modulation in thyroid cells under chronic TSH stimulation. PI3K inhibition by LY294002 increased NIS-mediated radioiodide uptake (RAIU) mainly through upregulation of NIS expression, however, mTORC1 inhibition by Rapamycin did not increase NIS-mediated RAIU despite increased NIS protein levels. In comparison, Akt inhibition by Akti-1/2 did not increase NIS protein levels, yet markedly increased NIS-mediated RAIU by decreasing iodide efflux rate and increasing iodide transport rate and iodide affinity of NIS. The effects of Akti-1/2 on NIS-mediated RAIU are not detected in nonthyroid cells, implying that Akti-1/2 or its derivatives may represent potential pharmacological reagents to selectively increase thyroidal radioiodine accumulation and therapeutic efficacy.

Selective iodide uptake and prolonged iodine retention in the thyroid is the basis for targeted radioiodine therapy for thyroid cancer patients; however, salivary gland dysfunction is the most frequent nonthyroidal complications. In this study, we have used noninvasive single photon emission computed tomography functional imaging to quantify the temporal dynamics of thyroidal and salivary radioiodine accumulation in mice. At 60 min post radionuclide injection, radionuclide accumulation in the salivary gland was generally higher than that in thyroid due to much larger volume of the salivary gland. However, radionuclide accumulation per anatomic unit in the salivary gland was lower than that in thyroid and was comparable among mice of different age and gender. Differently, radionuclide accumulation per anatomic unit in thyroid varied greatly among mice. The extent of thyroidal radioiodine accumulation stimulated by a single dose of exogenous bovine TSH (bTSH) in triiodothyronine (T₃)-supplemented mice was much less than that in mice received neither bTSH nor T₃ (nontreated mice), suggesting that the duration of elevated serum TSH level is important to maximize thyroidal radioiodine accumulation. Furthermore, the extent and duration of radioiodine accumulation stimulated by bTSH was less in the thyroids of the thyroid-targeted RET/PTC1 (thyroglobulin (Tg)-PTC1) mice bearing thyroid tumors compared with the thyroids in wild-type (WT) mice. Finally, the effect of 17-allyamino-17-demothoxygeldanamycin on increasing thyroidal, but not salivary, radioiodine accumulation was validated in both WT mice and Tg-PTC1 preclinical thyroid cancer mouse model.

The design and fabrication of an active multiaxis probing system for high-speed atomic force microscopy is presented. The probing system employs a multiaxis compliant manipulator that is actuated by two magnetic actuators to control the tip position simultaneously along the vertical and the lateral directions. The manipulator is optimally designed to achieve high bandwidth actuation and large scanning range. A novel process to fabricate multiaxis compliant manipulators reliably by using focused ion beam milling is proposed. The fabricated active multiaxis probing system is demonstrated to have high bandwidth of actuation with the lateral and vertical resonance frequencies at 46.4 and 101.5 kHz, respectively. The lateral scanning range is estimated to be ~350 nm at a magnetic field of 20 × 10-4 T. It enables imaging rate of 10 frame/s with pixel resolution of 100 × 100 pixels.

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.

// Aparna Lakshmanan 1, 2 , Daniel Scarberry 1, 2 , Jill A. Green 3 , Xiaoli Zhang 4 , Samia Selmi-Ruby 5 , Sissy M. Jhiang 1, 2, 3 1 Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH-43210, USA 2 Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH-43210, USA 3 Comprehensive Cancer Center, The Ohio State University, Columbus, OH-43210, USA 4 Center for Biostatistics, The Ohio State University, Columbus, OH-43210, USA 5 Centre de Recherche en Cancerologie de Lyon – UMR 1052- INSERM (Institut National de la Sante et de la Recherche Medicale, INSERM, Faculte de Medecine RTH Laennec, F-69372 Lyon, France Correspondence to: Sissy M. Jhiang, e-mail: jhiang.1@osu.edu Keywords: thyroid cancer, NIS, TGF-β, apigenin, GDC-0941 Received: June 02, 2015 Accepted: August 27, 2015 Published: September 09, 2015 ABSTRACT Targeted radioiodine therapy for thyroid cancer is based on selective stimulation of Na + /I - Symporter (NIS)-mediated radioactive iodide uptake (RAIU) in thyroid cells by thyrotropin. Patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absent NIS expression. To identify inhibitors that can be readily translated into clinical care, we examined oncological pipeline inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF in their ability to increase RAIU in thyroid cells expressing BRAF V600E or RET/PTC3 oncogene. Our data showed that (1) PI3K inhibitor GDC-0941 outperformed other inhibitors in RAIU increase mainly by decreasing iodide efflux rate to a great extent; (2) RAIU increase by all inhibitors was extensively reduced by TGF-β, a cytokine secreted in the invasive fronts of thyroid cancers; (3) RAIU reduction by TGF-β was mainly mediated by NIS reduction and could be reversed by Apigenin, a plant-derived flavonoid; and (4) In the presence of TGF-β, GDC-0941 with Apigenin co-treatment had the highest RAIU level in both BRAF V600E expressing cells and RET/PTC3 expressing cells. Taken together, Apigenin may serve as a dietary supplement along with small molecule inhibitors to improve radioiodine therapeutic efficacy on invasive tumor margins thereby minimizing future metastatic events.

Adenoviral gene therapy represents a novel approach for the treatment of aggressive thyroid carcinomas. Both coxsackie-adenovirus receptor (CAR) and integrins have been shown to be the major determinants for adenoviral infectivity in many types of cancer cells, yet conflicting results have been reported. In this report we examine these factors mediating adenoviral infection in thyroid cells and to evaluate CAR expression in various types of thyroid cancer. We found that neither expression levels of CAR nor integrins are solely predictive of adenoviral infectivity in thyroid cells. However, the absence of CAR was associated with poor adenoviral infectivity in immortalized rat FRTL-5 cells. Moreover, preincubation with alpha-CAR antibody decreased infectivity in FTC 238 cells, a human thyroid tumor line. These results indicate that CAR does play a role in adenoviral infection of thyroid cells. Immunohistochemical analysis revealed that CAR is expressed at the cell surface in the majority of malignant thyroid tumors. We further show that adenoviral infectivity in some thyroid cancer cells can be improved by poly-L-lysine. Our study warrants a functional method to evaluate adenoviral infectivity should be developed and instituted prior to clinical trials of adenoviral gene therapy in patients with advanced thyroid cancer.

RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.

Purpose We have previously conducted independent microarray expression analyses of the two most common types of nonmedullary thyroid carcinoma, namely papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC). In this study, we sought to combine our data sets to shed light on the similarities and differences between these tumor types. Materials and Methods Microarray data from six PTCs, nine FTCs, and 13 normal thyroid samples were normalized to remove interlaboratory variability and then analyzed by unsupervised clustering, t test, and by comparison of absolute and change calls. Expression changes in four genes not previously implicated in thyroid carcinogenesis were verified by reverse transcriptase polymerase chain reaction on these same samples, together with eight additional FTC tumors. Results PTCs showed two distinct groups of genes that were either over- or underexpressed compared with normal thyroid, whereas the predominant changes in FTCs were of decreased expression. Five genes could collectively distinguish the two tumor types. PTCs showed overexpression of CITED1, claudin-10 (CLDN10), and insulin-like growth factor binding protein 6 (IGFBP6) but showed no change in expression of caveolin-1 (CAV1) or -2 (CAV2); conversely, FTCs did not express CLDN10 and had decreased expression of IGFBP6 and/or CAV1 and CAV2. Conclusion PTC and FTC show distinctive microarray expression profiles, suggesting that either they have different molecular origins or they diverge distinctly from a common origin. Furthermore, if verified in a larger series of tumors, these genes could, in combination with known tumor-specific chromosome translocations, form the basis of a valuable diagnostic tool.

Radioiodide uptake activity mediated by the human Na(+)/I(-) symporter (hNIS) in thyroid follicular cells is the basis for effective (131)I therapy in thyroid cancer. However, radioiodide therapy is not effective in patients with thyroid cancer displaying low or absent hNIS expression. This study assessed the Cre/loxP system for enhancing thyroid-targeted hNIS expression driven by the thyroglobulin (Tg) promoter. The following three recombinant adenoviruses (rAd) were constructed: rAd-Tg-hNIS drives hNIS expression by the Tg promoter; rAd-Tg-Cre drives Cre expression by the Tg promoter; and rAd-CMV-loxP-hNIS drives hNIS expression by the cytomegalovirus (CMV) promoter after Cre-mediated excision of an intervening loxP-GFP-Zeo-loxP. Immortalized normal and malignant rat thyroid cell lines and primary cultures of normal human thyroid and human follicular adenoma cells were investigated. We found that the relative promoter activity of Tg vs. CMV is critical for the efficacy of the Cre/loxP system. In cells with weak Tg promoter activity, coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS induced higher hNIS expression than single infection of rAd-Tg-hNIS. Finally, Tg promoter activity was partially restored in malignant thyroid cells by forced expression of the paired domain-containing transcription factor (Pax-8), allowing the Cre/loxP system to mildly enhance radioiodide uptake.

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.

The Na+/I- symporter (NIS) is a membrane glycoprotein that facilitates the uptake of iodine into thyroid follicular cells. Recently, we and others have demonstrated the feasibility of imaging subcutaneous xenografts expressing exogenous NIS, suggesting that NIS may serve as an imaging reporter gene to monitor vector delivery and therapeutic gene expression. In this study, we established NIS-expressing pulmonary tumors in nude mice to investigate the minimal tumor size required for in vivo detection of pulmonary tumors by single photon emission computed tomography (SPECT) with pinhole collimation. In order to define the anatomic location of NIS-expressing tumor nodules detectable by SPECT, we performed simultaneous, dual-isotope imaging. We injected 1 mCi 99mTc-MAA via tail vein to image pulmonary perfusion and injected 1 mCi Na125I intraperitoneally to image NIS-expressing tumors. Fused images showed that 99mTc-MAA perfusion defects correlated with NIS-mediated 125I uptake. Post-mortem analysis revealed that tumors 3 mm in diameter could be detected by SPECT with pinhole collimation. These studies demonstrate the feasibility of SPECT to detect pulmonary tumors expressing exogenous NIS in mice.

The RET/PTC3 oncogene is a genetically rearranged and constitutively activated tyrosine kinase receptor that is common in papillary thyroid cancer. Because RET/PTC3 is chronically overexpressed in these thyroid cancer cells, and RET/PTC3-expressing tumors are associated with overactivity of tyrosine kinase signaling pathways and a more aggressive clinical course, we questioned whether chronic RET/PTC3 expression enhances cellular responses to thyroid mitogens in vitro. We stably transfected FRTL-5 cells with the RET/PTC3 gene; transfected and control cell lines were cultured without insulin, TSH, or serum. Thymidine incorporation into DNA was enhanced in the RET/PTC3 cells, but transformation was not observed. RET/PTC3 cells demonstrated higher basal and insulin-stimulated levels of activated Akt, both of which were reduced by LY294002, a PI3 kinase inhibitor, but not PD98059, a MEK inhibitor. By contrast, mitogen activated protein kinase (MAP kinase) was only minimally activated in RET/PTC3 cells before and after stimulation. Consistent with preferential activation of PI3 kinase, increased levels of total and phosphorylated IRS2 protein, relative activation of PDK-1, and enhanced IRS2-p85 interactions were identified in RET/PTC3-expressing cells. RET/PTC3 cells were also sensitized to insulin-induced thymidine incorporation; this effect was blocked by PI3 kinase (LY294002) rather than MEK 1/2 (PD98059) inhibitors. In summary, we have demonstrated that RET/PTC3 expression enhances basal and insulin-stimulated DNA synthesis through PI3 kinase, cooperatively activates Akt with insulin via PI3 kinase, and preferentially activates the Akt rather than MAP kinase pathway in FRTL-5 cells.

With the goal of optimizing adenovirus-mediated suicide gene therapy for prostate cancer, we have developed a method based on the human sodium iodide symporter (hNIS) that allows for noninvasive monitoring of adenoviral vectors and quantification of gene expression magnitude and volume within the prostate. A replication-competent adenovirus (Ad5-yCD/ mut TK SR39 rep -hNIS) coexpressing a therapeutic yeast cytosine deaminase (yCD)/mutant herpes simplex virus thymidine kinase ( mut TK SR39 ) fusion gene and the hNIS gene was developed. Ad5-yCD/ mut TK SR39 rep -hNIS and a replication-defective hNIS adenovirus (rAd-CMV-FLhNIS) were injected into contralateral lobes of the dog prostate and hNIS activity was monitored in live animals following administration of Na 99m TcO 4 using gamma camera scintigraphy. Despite the close proximity of the urinary bladder, 99m TcO 4 − uptake was readily detected in the prostate using viral dose levels (10 10 to 10 12 viral particles) that have been safely administered to humans. Due to its rapid clearance and short physical half-life (6 h), it was possible to obtain daily measurements of 99m TcO 4 − uptake in vivo, allowing for dynamic monitoring of reporter gene expression within the prostate as well as biodistribution throughout the body. High-resolution autoradiography of prostate sections coupled with 3D reconstruction of gene expression demonstrated that the magnitude and volume of gene expression could be quantified with submillimeter resolution. Implementation of the GENIS (gene expression of Na/I symporter) technology in the clinic will facilitate optimization of future human gene therapy trials.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has previously been implicated in the pathogenesis of follicular thyroid carcinoma (FTC), where a translocation with PAX8 has been reported in some 50% of tumors in three small series. The resultant fusion protein inhibits normal PPARgamma function by a dominant-negative mechanism. In a series of 19 FTCs, we identified this translocation in only two tumors (10.5%). However, microarray analysis and semiquantitative RT-PCR demonstrated greatly reduced PPARgamma expression in 13 of 17 (76%) nontranslocation tumors. Immunohistochemical analysis of 142 thyroid tumors showed a statistically significant reduction in PPARgamma immunoreactive protein, not only in FTCs but also in papillary thyroid carcinomas and Hurthle cell carcinomas. This suggests that while the overall frequency of the PAX8-PPARgamma translocation in FTCs may be lower than previously thought, functional downregulation of PPARgamma is a key event in multiple types of thyroid neoplasia and is a possible target for therapeutic intervention.

The sodium-iodide symporter (NIS) is expressed by papillary (PTC) and follicular (FTC) thyroid carcinoma, and is essential for iodine uptake. We hypothesized that PTC and FTC with detectable NIS immunostaining would be more amenable to radioactive iodine ((131)I) treatment and follow a more benevolent course. To test this, we determined NIS expression by immunohistochemistry in 23 PTC, 9 FTC, and 12 benign thyroid lesions from children and adolescents. NIS expression was determined by two blinded examiners and graded as absent = 0, minimal = 1, moderate = 2, intense = 3, and very intense = 4. NIS was detected in 35% (eight of 23) of PTC, 44% (four of 9) of FTC, 25% (two of eight) of benign tumors, and 100% (four of four) of autoimmune lesions. The intensity of NIS expression was similar in PTC (0.61 +/- 0.24), FTC (0.56 +/- 0.24), and benign tumors (0.50 +/- 0.33) but was more intense in autoimmune lesions (3.0 +/- 0.7, p < 0.005). Distant metastases were found only among PTC with undetectable NIS (two of 15, 13%), and recurrence developed exclusively from PTC and FTC with undetectable NIS (four of 20, 20% versus zero of 12, p = 0.043). The dose of iodine 131 required to achieve remission in the five patients with PTC who had undetectable NIS (213.3 +/- 53 mCi) was greater than that required by patients with similar age and extent of disease for whom NIS expression is unknown (109 +/- 22 mCi, p = 0.06). We conclude that NIS expression is associated with a lower risk of recurrence for PTC and FTC of children and adolescents.

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.

BACKGROUND The sodium iodide symporter (NIS) mediates iodide uptake in thyroid follicular cells and provides a mechanism for effective radioiodide treatment of residual, recurrent, and metastatic thyroid cancers. This study investigated the clinical applications of NIS gene transfer for prostate cancer using the MATLyLu metastatic rat model. METHODS MATLyLu cells expressing NIS were injected subcutaneously in Copenhagen rats, which developed metastases in lymph nodes and lungs. NIS protein expression was evaluated by Western blot and immunohistochemistry, and function was measured by tissue gamma counts and whole-body imaging following radionuclide administration. RESULTS In vitro radioiodide-concentrating activity was increased up to 72-fold in a mixed population of MATLyLu-hNIS cells. NIS protein expression was confirmed in subcutaneous MATLyLu-hNIS tumors by immunohistochemistry and Western blot. Gamma counts of subcutaneous MATLyLu-hNIS tumors were 23-fold higher than parental MATLyLu tumors and radionuclide uptake in subcutaneous MATLyLu-hNIS tumors and lymph node metastases was visualized by whole-body image analysis. CONCLUSIONS NIS expression by a proportion of cells in a population was sufficient to confer radionuclide-concentrating function in subcutaneous and metastatic MATLyLu tumors. Ablation of residual normal and neoplastic prostate tissues by radioiodide after prostate-restricted NIS gene transfer might be a novel adjuvant therapy to prostatectomy for the treatment of advanced prostate cancer. Prostate 50: 170–178, 2002. © 2002 Wiley-Liss, Inc.

Back focal plane (BFP) interferometry is frequently used to detect the motion of a single laser trapped bead in a photonic force microscope (PFM) system. Whereas this method enables high-speed and high-resolution position measurement, its measurement range is limited by nonlinearity coupled with crosstalk in three-dimensional (3-D) measurement, and validation of its measurement accuracy is not trivial. This Letter presents an automated calibration system in conjunction with a 3-D quadratic model to render rapid and accurate calibration of the laser measurement system. An actively controlled three-axis laser steering system and a high-speed vision-based 3-D particle tracking system are integrated to the PFM system to enable rapid calibration. The 3-D quadratic model is utilized to correct for nonlinearity and crosstalk and, thus, extend the 3-D position detection volume of BFP interferometry. We experimentally demonstrated a 12-fold increase in detection volume when applying the method to track the motion of a 2.0 μm laser trapped polystyrene bead.

Na+/I−symporter (NIS)-mediated radioiodide uptake (RAIU) serves as the basis for targeted ablation of thyroid cancer remnants. However, many patients with thyroid cancer have reduced NIS expression/function and hence do not benefit from radioiodine therapy. microRNA (miR) has emerged as a promising therapeutic target in many diseases; yet, the role of miRs in NIS-mediated RAIU has not been investigated.In silicoanalysis was used to identify miRs that may bind to the 3′UTR of humanNIS(hNIS). The top candidate miR-339-5p directly bound to the 3′UTR of hNIS. miR-339-5p overexpression decreased NIS-mediated RAIU in HEK293 cells expressing exogenous hNIS, decreased the levels ofNISmRNA, and RAIU in transretinoic acid/hydrocortisone (tRA/H)-treated MCF-7 human breast cancer cells as well as thyrotropin-stimulated PCCl3 rat thyroid cells. Nanostring nCounter rat miR expression assay was conducted to identify miRs deregulated by TGFβ, Akti-1/2, or 17-AAG known to modulate RAIU in PCCl3 cells. Among 38 miRs identified, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to the 3′UTR of hNISand its overexpression decreased RAIU in tRA/H-treated MCF-7 cells. miR-339-5p was modestly increased in most papillary thyroid carcinomas (PTCs), yet miR-195 was significantly decreased in PTCs. Interestingly, the expression profiles of 18 miRs could be used to distinguish most PTCs from nonmalignant thyroid tissues. This is the first report, to our knowledge, demonstrating that hNIS-mediated RAIU can be modulated by miRs, and that the same miRs may also play roles in the development or maintenance of thyroid malignancy. Accordingly, miRs may serve as emerging targets to halt the progression of thyroid cancer and to enhance the efficacy of radioiodine therapy.

Radioactive iodine (RAI) is a key therapeutic modality for thyroid cancer. Loss of RAI uptake in thyroid cancer inversely correlates with patient’s survival. In this review, we focus on the challenges encountered in delivering sufficient doses of I-131 to eradicate metastatic lesions without increasing the risk of unwanted side effects. Sodium iodide symporter (NIS) mediates iodide influx, and NIS expression and function can be selectively enhanced in thyroid cells by thyroid-stimulating hormone. We summarize our current knowledge of NIS modulation in normal and cancer thyroid cells, and we propose that several reagents evaluated in clinical trials for other diseases can be used to restore or further increase RAI accumulation in thyroid cancer. Once validated in preclinical mouse models and clinical trials, these reagents, mostly small-molecule inhibitors, can be readily translated into clinical practice. We review available genetically engineered mouse models of thyroid cancer in terms of their tumor development and progression as well as their thyroid function. These mice will not only provide important insights into the mechanisms underlying the loss of RAI uptake in thyroid tumors but will also serve as preclinical animal models to evaluate the efficacy of candidate reagents to selectively increase RAI uptake in thyroid cancers. Taken together, we anticipate that the optimal use of RAI in the clinical management of thyroid cancer is yet to come in the near future.

Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells.

Radioiodine-concentrating activity in thyroid tissues has allowed the use of radioiodine as a diagnostic and therapeutic agent for patients with thyroid disorders such as well-differentiated thyroid cancer. However, some extrathyroidal tissues also take up radioiodine, contributing to unwanted side effects of radioiodine therapy. Now that the molecule that mediates radioiodine uptake, the sodium iodide symporter (NIS), has been cloned and characterized, it may be possible to develop novel strategies to differentially modulate NIS expression and/or activity, enhancing it in target tissues and impeding it in others. In addition to restoring NIS expression/activity to ensure sufficient radioiodine uptake for the diagnosis and treatment of advanced thyroid cancers, we envision that it may be possible to selectively increase or confer NIS expression/activity in tumors of nonthyroidal tissues to facilitate the use of radioiodine in their diagnosis and treatment. We also consider the molecular basis of thyroid and nonthyroid disorders that may be complicated by NIS deregulation. Finally, we explore the use of NIS as an imaging reporter gene to monitor the expression profile of the transgene in transgenic mouse animal models and in patients undergoing gene therapy clinical trials.

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.

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.

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.

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.

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 markedl...

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.

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.

Background: Selectively increased radioiodine accumulation in thyroid cells by thyrotropin (TSH) allows targeted treatment of thyroid cancer. However, the extent of TSH-stimulated radioiodine accumulation in some thyroid tumors is not sufficient to confer therapeutic efficacy. Hence, it is of clinical importance to identify novel strategies to selectively further enhance TSH-stimulated thyroidal radioiodine accumulation. Methods: PCCl3 rat thyroid cells, PCCl3 cells overexpressing BRAFV600E, or primary cultured tumor cells from a thyroid cancer mouse model, under TSH stimulation were treated with various reagents for 24 hours. Cells were then subjected to radioactive iodide uptake, kinetics, efflux assays, and protein extraction followed by Western blotting against selected antibodies. Results: We previously reported that Akt inhibition increased radioiodine accumulation in thyroid cells under chronic TSH stimulation. Here, we identified Apigenin, a plant-derived flavonoid, as a reagent to further enhance the iodide influx rate increased by Akt inhibition in thyroid cells under acute TSH stimulation. Akt inhibition is permissive for Apigenin's action, as Apigenin alone had little effect. This action of Apigenin requires p38 MAPK activity but not PKC-δ. The increase in radioiodide accumulation by Apigenin with Akt inhibition was also observed in thyroid cells expressing BRAFV600E and in primary cultured thyroid tumor cells from TRβPV/PV mice. Conclusion: Taken together, Apigenin may serve as a dietary supplement in combination with Akt inhibitors to enhance therapeutic efficacy of radioiodine for thyroid cancer.

Background: Familial medullary thyroid cancer (MTC) is a form of type 2 multiple endocrine neoplasia in which individuals develop MTC as the sole phenotypic manifestation of their disease. A previous study has suggested that patients with familial MTC may have a later age of onset (and more indolent course) of MTC than is observed in individuals with multiple endocrine neoplasia type 2A. Methods and Results: The age-related penetrance of MTC, C-cell hyperplasia, and a positive pentagastrin test for carriers of a codon 609 mutation of the RET gene in a large MTC family was determined. Pentagastrin testing and surgical pathology findings for patients who had thyroidectomies were correlated with RET sequence analysis findings. The penetrance of this mutation for the development of MTC was 0% at age 20, 10% at age 30, 50% at age 45, and approximately 100% at age 60. The ages of onset of C-cell hyperplasia and a positive pentagastrin stimulation test were similar, and both preceded the age of onset of MTC. Carriers of the mutated gene in this family had a later age of onset of disease than has been reported for families with multiple endocrine neoplasia type 2A and 2B syndromes. Conclusions: These results may have implications for the clinical management of MTC families with a 609 mutation.

The Na+/I- symporter is the molecule that mediates active iodide uptake in the thyroid gland. A 16.4 kb genomic DNA fragment of the rat Na+/I- symporter gene (rNIS) was isolated, restriction mapped and a 2 kb region immediate 5' to the ATG site was sequenced. The transcription start site for rNIS was mapped to -98 nucleotide (nt) relative to the ATG translation initiation site. A series of 5' genomic DNA fragments ranging from 233 bp to 8 kb were tested for promoter activity in both thyroid and non-thyroid cells. Our results indicate that the DNA regulatory elements within 8 kb of the 5' flanking region of rNIS are not sufficient to confer thyroid-selective transcription. However, consistent with the clinical observation that NIS expression is reduced in thyroid tumors, the rNIS promoter activity is suppressed in ret/PTC1 transformed NIH3T3 cells, compared to non-transformed NIH3T3 cells.

This paper presents a real-time visual sensing system, which is created to achieve high-speed three-dimensional (3D) motion tracking of microscopic spherical particles in aqueous solutions with nanometer resolution. The system comprises a complementary metal–oxide-semiconductor (CMOS) camera, a field programmable gate array (FPGA), and real-time image processing programs. The CMOS camera has high photosensitivity and superior SNR. It acquires images of 128×120 pixels at a frame rate of up to 10,000 frames per second (fps) under the white light illumination from a standard 100 W halogen lamp. The real-time image stream is downloaded from the camera directly to the FPGA, wherein a 3D particle-tracking algorithm is implemented to calculate the 3D positions of the target particle in real time. Two important objectives, i.e., real-time estimation of the 3D position matches the maximum frame rate of the camera and the timing of the output data stream of the system is precisely controlled, are achieved. Two sets of experiments were conducted to demonstrate the performance of the system. First, the visual sensing system was used to track the motion of a 2 μm polystyrene bead, whose motion was controlled by a three-axis piezo motion stage. The ability to track long-range motion with nanometer resolution in all three axes is demonstrated. Second, it was used to measure the Brownian motion of the 2 μm polystyrene bead, which was stabilized in aqueous solution by a laser trapping system.

Professor and physician Dr. Ernest L. Mazzaferri Sr. passed away on May 14, 2013, at 76 years of age ( 1 , 2 ). Ernie is remembered as a caring and talented physician, an accomplished scholar and educator, as well as a loving husband, father, and grandfather. He was a luminary figure, and few people have had a greater impact in thyroidology in recent decades. Here we include reflections from a few of us that knew him, as well as commentaries from people who did not. Ernie's passion was caring for patients and how to improve their care. Our goal is to pay tribute and memorialize the person we knew and the impact that he had on patients around the world through his dedication to research, lecturing, and writing that achieved remarkable global influence.

The Na+/I−symporter (NIS (SLC5A5)) is a transmembrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. NIS-mediated iodide uptake in thyroid cells is the basis for targeted radionuclide imaging and treatment of differentiated thyroid carcinomas and their metastases. Furthermore, NIS is expressed in many human breast tumors but not in normal non-lactating breast tissue, suggesting that NIS-mediated radionuclide uptake may also allow the imaging and targeted therapy of breast cancer. However, functional cell surface NIS expression is often low in breast cancer, making it important to uncover signaling pathways that modulate NIS expression at multiple levels, from gene transcription to posttranslational processing and cell surface trafficking. In this study, we investigated NIS regulation in breast cancer by MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling, an important cell signaling pathway involved in oncogenic transformation. We found that MEK inhibition decreased NIS protein levels in all-trans retinoic acid/hydrocortisone-treated MCF-7 cells as well as human breast cancer cells expressing exogenous NIS. The decrease in NIS protein levels by MEK inhibition was not accompanied by a decrease inNISmRNA or a decrease inNISmRNA export from the nucleus to the cytoplasm. NIS protein degradation upon MEK inhibition was prevented by lysosome inhibitors but not by proteasome inhibitors. Interestingly, NIS protein level was correlated with MEK/ERK activation in human breast tumors from a tissue microarray. Taken together, MEK activation appears to play an important role in maintaining NIS protein stability in human breast cancers.