Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated.
Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified.
Results and Discussion
NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype.
Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.
Expression of the sodium iodide symporter (NIS) is required for efficient iodide uptake in thyroid and lactating breast. Since most differentiated thyroid cancer expresses NIS, β-emitting radioactive iodide is routinely utilized to target remnant thyroid cancer and metastasis after total thyroidectomy. Stimulation of NIS expression by high levels of thyroid-stimulating hormone is necessary to achieve radioiodide uptake into thyroid cancer that is sufficient for therapy. The majority of breast cancer also expresses NIS, but at a low level insufficient for radioiodine therapy. Retinoic acid is a potent NIS inducer in some breast cancer cells. NIS is also modestly expressed in some non-thyroidal tissues, including salivary glands, lacrimal glands and stomach. Selective induction of iodide uptake is required to target tumors with radioiodide. Iodide uptake in mammalian cells is dependent on the level of NIS gene expression, but also successful translocation of NIS to the cell membrane and correct insertion. The regulatory mechanisms of NIS expression and membrane insertion are regulated by signal transduction pathways that differ by tissue. Differential regulation of NIS confers selective induction of functional NIS in thyroid cancer cells, as well as some breast cancer cells, leading to more efficient radioiodide therapy for thyroid cancer and a new strategy for breast cancer therapy. The potential for systemic radioiodide treatment of a range of other cancers, that do not express endogenous NIS, has been demonstrated in models with tumor-selective introduction of exogenous NIS.
Sodium iodide symporter; thyroid cancer; breast cancer; Transcriptional regulation; Posttranslational regulation
The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. This in turn allows radioiodine imaging and therapy for thyroid cancer. To extend the use of NIS-mediated radioiodine therapy to other types of cancer, we successfully transferred and expressed the sodium-iodide symporter (NIS) gene in prostate, colon, and breast cancer cells both in vivo and in vitro by using non-replicating adenoviral vectors.
To improve virotherapy efficiency, we developed a conditionally replicating adenovirus (CRAd) in which the transcriptional cassette RSV promoter-human NIScDNA-bGH polyA was also inserted at the E3 region. The E1a gene is driven by the tumor-specific promoter MUC-1 in the CRAd Ad5AMUCH_RSV-NIS.
In vitro infection of the MUC-1-positive breast cell line T47D resulted in virus replication, cytolysis, and release of infective viral particles. Conversely, the MUC-1-negative breast cancer cell line MDA-MB-231 was refractory to the viral cytopathic effect and did not support viral replication. The data indicate that Ad5AMUCH_RSV-NIS activity is stringently restricted to MUC-1-positive cancer cells. Radioiodine uptake was readily measurable in T47 cells infected with Ad5AMUCH_RSV-NIS 24 hours after infection, thus confirming NIS expression before viral-induced cell death.
This construct may allow multimodal therapy, combining virotherapy with radioiodine therapy to be developed as a novel treatment for breast and other MUC1-overexpressing cancers.
Iodide uptake in the thyroid and breast is mediated by the sodium/iodide symporter (NIS). NIS activation is utilized for radioiodide imaging and therapeutic ablation of thyroid carcinoma. NIS is expressed in more than 70% of breast cancers, but at a level insufficient for radioiodine treatment (1). All-trans retinoic acid (tRA) induces NIS gene expression and functional iodide uptake in human breast cancer cell lines and mouse breast cancer models. tRA usually regulates gene expression by direct interaction of Retinoic Acid Receptor (RAR) with a target gene, but it can also acts through nongenomic pathways. We report a direct influence of tRA treatment on the PI3K signal transduction pathway that mediates tRA-induced NIS expression in MCF-7 breast cancer cells. MCF-7 cells express all three RAR isoforms, α, β and γ, and RXRα. We previously identified RARβ and RXRα as important for NIS induction by tRA. Treatment with LY294002, the PI3K inhibitor, or p85α “knockdown” with siRNA, abolished tRA-induced NIS expression. Immunoprecipitation experiments and GST pull-down assay demonstrated a direct interaction between RARβ2, RXRα and p85α. RA also induced rapid activation of Akt in MCF-7 cells. Treatment with an Akt inhibitor or Akt “knockdown” with siRNA, reduced NIS expression. These findings indicate that RA-induction of NIS in MCF-7 cells is mediated by rapid activation of the PI3K pathway and involves direct interaction with RAR and RXR. Defining these mechanisms should lead to methods to further enhance NIS expression, as well as retinoid targets that influence growth and differentiation of breast cancer.
Sodium/iodide symporter; Retinoic acid; MCF-7; PI3K; RAR
To test the feasibility of using the survivin promoter to induce specific expression of sodium/iodide symporter (NIS) in cancer cell lines and tumors for targeted use of radionuclide therapy, a recombinant adenovirus, Ad-SUR-NIS, that expressed the NIS gene under control of the survivin promoter was constructed. Ad-SUR-NIS mediating iodide uptake and cytotoxicity was performed in vitro. Scintigraphic, biodistribution and radioiodine therapy studies were performed in vivo. PC-3 (prostate); HepG2 (hepatoma) and A375 (melanoma) cancer cells all exhibited perchlorate-sensitive iodide uptake after infection with Ad-SUR-NIS, ∼50 times higher than that of negative control Ad-CMV-GFP-infected cells. No significant iodide uptake was observed in normal human dental pulp fibroblast (DPF) cells after infection with Ad-SUR-NIS. Clonogenic assays demonstrated that Ad-SUR-NIS-infected cancer cells were selectively killed by exposure to 131I. Ad-SUR-NIS-infected tumors show significant radioiodine accumulation (13.3±2.85% ID per g at 2 h post-injection), and the effective half-life was 3.1 h. Moreover, infection with Ad-SUR-NIS in combination with 131I suppressed tumor growth. These results indicate that expression of NIS under control of the survivin promoter can likely be used to achieve cancer-specific expression of NIS in many types of cancers. In combination with radioiodine therapy, this strategy is a possible method of cancer gene therapy.
sodium iodide symporter; survivin promoter; radioiodine therapy
Molecular imaging, defined as the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms, can be obtained by various imaging technologies, including nuclear imaging methods. Imaging of normal thyroid tissue and differentiated thyroid cancer, and treatment of thyroid cancer with radioiodine rely on the expression of the sodium iodide symporter (NIS) in these cells. NIS is an intrinsic membrane protein with 13 transmembrane domains and it takes up iodide into the cytosol from the extracellular fluid. By transferring NIS function to various cells via gene transfer, the cells can be visualized with gamma or positron emitting radioisotopes such as Tc-99m, I-123, I-131, I-124 and F-18 tetrafluoroborate, which are accumulated by NIS. They can also be treated with beta- or alpha-emitting radionuclides, such as I-131, Re-186, Re-188 and At-211, which are also accumulated by NIS. This article demonstrates the diagnostic and therapeutic applications of NIS as a radionuclide-based reporter gene for trafficking cells and a therapeutic gene for treating cancers.
sodium iodide symporter; molecular imaging; radionuclide-based imaging; gene therapy; radionuclide.
The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. We have extended the use of NIS-mediated radioiodine therapy to other types of cancer, we transferred and expressed the sodium-iodide symporter (NIS) gene into prostate, colon, and breast cancer cells using adenoviral vectors. To improve vector efficiency we have developed a conditionally replicating adenovirus (CRAd) in which the E1a gene is driven by the prostate specific promoter, Probasin and the cassette RSV promoter-human NIScDNA-bGH polyA replaces the E3 region (CRAd Ad5PB_RSV-NIS). In vitro infection of the prostate cancer cell line LnCaP resulted in virus replication, cytolysis, and release of infective viral particles. Conversely, the prostate cancer cell line PC-3 (androgen receptor negative) and the pancreatic cancer cell line Panc-1 were refractory to the viral cytopathic effect and did not support viral replication. Radioiodine uptake was readily measurable in LnCaP cells infected with Ad5PB_RSV-NIS 24 hours post-infection, confirming NIS expression. In vivo, LnCaP tumor xenografts in nude mice injected intratumorally with Ad5PB_RSV_NIS CRAd expressed NIS actively as evidenced by 99Tc uptake and imaging. Administration of therapeutic 131I after virus injection significantly increased survival probability in mice carrying xenografted LnCaP tumors compared to virotherapy alone. The data indicate that Ad5PB_RSV_NIS replication is stringently restricted to androgen positive prostate cancer cells and results in effective NIS expression and uptake of radioiodine. This construct may allow multimodal therapy, combining cytolytic virotherapy with radioiodine treatment, to be developed as a novel treatment for prostate cancer.
prostate cancer; probasin; adenovirus; sodium iodide symporter; virotherapy; gene therapy
The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer.
NIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with pre-existing pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies).
In this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray.
Thus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer.
Thyroid iodide uptake through the sodium-iodide symporter (NIS) is not only an essential step for thyroid hormones biosynthesis, but also fundamental for the diagnosis and treatment of different thyroid diseases. However, part of patients with thyroid cancer is refractory to radioiodine therapy, due to reduced ability to uptake iodide, which greatly reduces the chances of survival. Therefore, compounds able to increase thyroid iodide uptake are of great interest. It has been shown that some flavonoids are able to increase iodide uptake and NIS expression in vitro, however, data in vivo are lacking. Flavonoids are polyhydroxyphenolic compounds, found in vegetables present in human diet, and have been shown not only to modulate NIS, but also thyroperoxidase (TPO), the key enzyme in thyroid hormones biosynthesis, besides having antiproliferative effect in thyroid cancer cell lines. Therefore, we aimed to evaluate the effect of some flavonoids on thyroid iodide uptake in Wistar rats in vivo. Among the flavonoids tested, rutin was the only one able to increase thyroid iodide uptake, so we decided to evaluate the effect of this flavonoid on some aspects of thyroid hormones synthesis and metabolism. Rutin led to a slight reduction of serum T4 and T3 without changes in serum thyrotropin (TSH), and significantly increased hypothalamic, pituitary and brown adipose tissue type 2 deiodinase and decreased liver type 1 deiodinase activities. Moreover, rutin treatment increased thyroid iodide uptake probably due to the increment of NIS expression, which might be secondary to increased response to TSH, since TSH receptor expression was increased. Thus, rutin might be useful as an adjuvant in radioiodine therapy, since this flavonoid increased thyroid iodide uptake without greatly affecting thyroid function.
The selective increase of Na+/I− symporter (NIS)-mediated active iodide uptake in thyroid cells allows the use of radioiodine I131 for 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.
AIM: To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter (NIS) gene.
METHODS: A recombinant baculovirus plasmid carrying the NIS gene was constructed, and the viruses (Bac-NIS) were prepared using the Bac-to-Bac system. The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein (GFP) expressing baculovirus (Bac-GFP) at different multiplicities of infection (MOI) with various concentrations of sodium butyrate was determined by flow cytometry. An in vitro cytotoxicity assay was also conducted after infection of SW1116 cells with Bac-NIS. Iodine uptake of Bac-NIS infected SW1116 cells and inhibition of this uptake by sodium perchlorate was examined, and the effect of Bac-NIS-mediated 131I in killing tumor cells was evaluated by cell colony formation tests.
RESULTS: Infection and transgene expression in SW1116 with Bac-GFP were significantly enhanced by sodium butyrate, as up to 72% of SW1116 cells were infected with the virus at MOI of 400 and sodium butyrate at 0.5 mmol/L. No obvious cytotoxicity was observed under these conditions. Infection of SW1116 with Bac-NIS allowed uptake of 131I in these tumor cells, which could be inhibited by sodium perchlorate. The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP, suggesting that NIS gene-mediated 131I uptake could specifically kill tumor cells.
CONCLUSION: Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer.
Colon cancer; Baculovirus; Sodium iodide symporter; Radionuclide therapy; Iodine radioisotopes
Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers may be treated with radio-iodine following transfection with the human sodium/iodide symporter (hNIS) gene. The glial fibrillary acidic protein (GFAP) promoter is an effective tumor-specific promoter for gene expression and thus may be useful in targeted gene therapy of malignant glioma. The present study used GFAP promoter-modulated expression of the hNIS gene in an experimental model of radioiodine-based treatment for malignant glioma. Cells were transfected using a recombination adeno-virus and evaluated in cells by studying the transfected transgene expression through western blot analysis, 125I uptake and efflux, clonogenicity following 131I treatment and radioiodine therapy using a U87 xenograft nude mouse model. Following transfection with the hNIS gene, the cells showed 95–70-fold higher 125I uptake compared with the control cells transfected with Ad-cytomegalovirus (CMV)-enhanced green fluorescent protein (EGFP). The western blotting revealed bands of ∼70, 49 and 43 kDa, consistent with the hNIS, GFAP and β-actin proteins. The clonogenic assay indicated that, following exposure to 500 μCi of 131I-iodide for 12 h, >90% of cells transfected with the hNIS gene were killed. Ad-GFAP-hNIS-transfected and 2 mCi 131I-injected U87 xenograft nude mice survived the longest of the three groups. The hNIS-expressing tumor tissue accumulated 99mTcO4 rapidly within 30 min of it being intraperitoneally injected. The experiments demonstrated that effective 131I therapy was achieved in the malignant glioma cell lines following the induction of tumor-specific iodide uptake activity by GFAP promoter-directed hNIS gene expression in vitro and in vivo. 131I therapy retarded Ad-GFAP-hNIS transfected-tumor growth following injection with 131I in U87 xenograft-bearing nude mice.
malignant glioma; sodium iodide symporter; glial fibrillary acidic protein promoter; radioiodine therapy
The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. We have extended the use of NIS-mediated radioiodine therapy to prostate cancer. We have developed a prostate tumor specific conditionally replicating adenovirus (CRAd) that expresses hNIS (Ad5PB_RSV-NIS). For radiovirotherapy to be effective in humans, the radioiodine dose administered in the pre-clinical animal model should scale to the range of acceptable doses in humans. We performed 131I dose-response experiments aiming to determine the dose required in mice to achieve efficient radiovirotherapy. Efficacy was determined by measuring tumor growth and survival times. We observed that individual tumors display disparate growth rates which preclude averaging within a treatment modality indicating heterogeneity of growth rate. We further show that a statistic and stochastic approach must be used when comparing the effect of an anti-cancer therapy on a cohort of tumors. Radiovirotherapy improves therapeutic value over virotherapy alone by slowing the rate of tumor growth in a more substantial manner leading to an increase in survival time. We also show that the radioiodine doses needed to achieve this increase scaled well within the current doses used for treatment of thyroid cancer in humans.
prostate cancer; probasin; adenovirus; sodium iodide symporter; virotherapy; gene therapy; allometry
This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells.
We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells.
Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors.
This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers.
Common causes of unilateral breast uptake of Tc-99m pertechnetate are predominant breast-feeding on one side, mastitis, and breast cancer. Uptake of Tc-99m pertechnetate in the epithelial cells of the breast, like that of iodide, depends on the transmembrane sodium/iodide symporter (NIS), the expression of which is known to be greatly enhanced during lactation and in breast cancer. The authors present an interesting case of predominant left breast uptake of Tc-99m pertechnetate detected incidentally during thyroid scintigraphy, for the evaluation of a nodule in the left lobe of thyroid.
Cold nodule; Tc-99m pertechnetate; thyroid; unilateral breast uptake
Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model.
GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 μCi of 124I-iodide.
Viral infectivity, measured by green fluorescent protein (GFP) expression, was time- and concentration-dependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( <10,000-fold increase from the initial viral dose ) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm3 versus 168 mm3 in untreated controls (P < 0.05).
This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.
Recovery of iodide uptake in thyroid cancer cells by means of obtaining the functional expression of the sodium/iodide symporter (NIS) represents an innovative strategy for the treatment of poorly differentiated thyroid cancer. However, the NIS gene expression alone is not always sufficient to restore radioiodine concentration ability in these tumour cells.
In this study, the anaplastic thyroid carcinoma ARO cells were stably transfected with a Pax8 gene expression vector. A quantitative RT-PCR was performed to assess the thyroid specific gene expression in selected clones. The presence of NIS protein was detected by Western blot and localized by immunofluorescence. A iodide uptake assay was also performed to verify the functional effect of NIS induction and differentiation switch.
The clones overexpressing Pax8 showed the re-activation of several thyroid specific genes including NIS, Pendrin, Thyroglobulin, TPO and TTF1. In ARO-Pax8 clones NIS protein was also localized both in cell cytoplasm and membrane. Thus, the ability to uptake the radioiodine was partially restored, associated to a high rate of efflux. In addition, ARO cells expressing Pax8 presented a lower rate of cell growth.
These finding demonstrate that induction of Pax8 expression may determine a re-differentiation of thyroid cancer cells, including a partial recovery of iodide uptake, fundamental requisite for a radioiodine-based therapeutic approach for thyroid tumours.
PTTG Binding Factor (PBF or PTTG1IP) is a little characterised proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goitres (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Further, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.
radioiodine; NIS; hyperplasia; Akt; thyroid cancer
The sodium/iodide symporter (NIS) is a plasma membrane protein that mediates active iodide transport in thyroid and mammary cells. It is a prerequisite for radioiodide treatment of thyroid cancer and a promising diagnostic and therapeutic tool for breast cancer. We investigated the molecular mechanisms governing NIS expression in mammary cells. Here we report that Nkx-2.5, a cardiac homeobox transcription factor that is also expressed in the thyroid primordium, is a potent inducer of the NIS promoter. By binding to two specific promoter sites (N2 and W), Nkx-2.5 induced the rNIS promoter (about 50-fold over the basal level). Interestingly, coincident with NIS expression, Nkx-2.5 mRNA and protein were present in lactating, but not virgin, mammary glands in two human breast cancer samples and in all-trans retinoic acid (tRA)-stimulated MCF-7 breast cancer cells. A cotransfected dominant-negative Nkx-2.5 mutant abolished tRA-induced endogenous NIS induction, which shows that Nkx-2.5 activity is critical for this process. Remarkably, in MCF-7 cells, Nkx-2.5 overexpression alone was sufficient to induce NIS and iodide uptake. In conclusion, Nkx-2.5 is a novel relevant transcriptional regulator of mammary NIS and could thus be exploited to manipulate NIS expression in breast cancer treatment strategies.
Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter.
Gene therapy; imaging; NIS; oncolytic virus; PET; reporter gene; SPECT; sodium iodide symporter.
The BRAFV600E mutation is present in 62% of radioactive iodine resistant (RAIR) thyroid tumors and is associated with down-regulation of the sodium-iodide-symporter (NIS) and TSH-receptor (TSHr). We sought to evaluate the combined effect of BRAF inhibition and TSH supplementation on 131I uptake of BRAFV600E-mutant human thyroid cancer cells.
MATERIALS AND METHODS
WRO cells (a BRAFV600E-mutant follicular-derived papillary thyroid carcinoma cell line) were transfected with siRNA targeting BRAF for 72 hours in a physiologic TSH environment. NIS and TSHr expression were then evaluated at three levels: gene expression, protein levels, and 131I uptake. These three main outcomes were then reassessed in TSH-depleted media and media supplemented with supratherapeutic concentrations of TSH.
NIS gene expression increased 5.5-fold 36 hours after transfection (p=0.01) and TSHr expression increased 2.8-fold at 24 hours (p=0.02). NIS and TSHr protein levels were similarly increased 48 and 24 hours post-transfection, respectively. Seventy two hours after BRAF inhibition, 131I uptake showed was unchanged in TSH-depleted media, increased by 7.5-fold (p<0.01) in physiologic TSH media, and increased by 9.1-fold (p<0.01) in supratherapeutic TSH media.
The combined strategy of BRAF inhibition and TSH supplementation results in greater 131I uptake than when either technique is utilized alone. This represents a simple and feasible approach that may improve outcomes in patients with RAIR thyroid carcinomas for which current treatment algorithms are ineffective.
Papillary thyroid cancer; BRAF(V600E) mutation; radioactive iodine; radioactive iodine resistance; thyroid stimulating hormone; sodium-iodine symptorer
The sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates iodide (I-) transport in the thyroid, lactating breast, salivary glands, and stomach. Whereas NIS expression and regulation have been extensively investigated in healthy and neoplastic thyroid and breast tissues, little is known about NIS expression and function along the healthy and diseased gastrointestinal tract.
Thus, we investigated NIS expression by immunohistochemical analysis in 155 gastrointestinal tissue samples and by immunoblot analysis in 17 gastric tumors from 83 patients.
Regarding the healthy Gl tract, we observed NIS expression exclusively in the basolateral region of the gastric mucin-producing epithelial cells. In gastritis, positive NIS staining was observed in these cells both in the presence and absence of Helicobacter pylori. Significantly, NIS expression was absent in gastric cancer, independently of its histological type. Only focal faint NIS expression was detected in the direct vicinity of gastric tumors, i.e., in the histologically intact mucosa, the expression becoming gradually stronger and linear farther away from the tumor. Barrett mucosa with junctional and fundic-type columnar metaplasia displayed positive NIS staining, whereas Barrett mucosa with intestinal metaplasia was negative. NIS staining was also absent in intestinalized gastric polyps.
That NIS expression is markedly decreased or absent in case of intestinalization or malignant transformation of the gastric mucosa suggests that NIS may prove to be a significant tumor marker in the diagnosis and prognosis of gastric malignancies and also precancerous lesions such as Barrett mucosa, thus extending the medical significance of NIS beyond thyroid disease.
Radioiodide is an effective therapy for thyroid cancer. This treatment modality exploits the thyroid-specific expression of the sodium iodide symporter (NIS) gene, which allows rapid internalization of iodide into thyroid cells. To test whether a similar treatment strategy could be exploited in nonthyroid malignancies, we transfected non–small cell lung cancer (NSCLC) cell lines with the NIS gene. Although the expression of NIS allowed significant radioiodide uptake in the transfected NSCLC cell lines, rapid radioiodide efflux limited tumor cell killing. Because thyroperoxidase (TPO) catalyzes iodination of proteins and subsequently causes iodide retention within thyroid cells, we hypothesized that coexpression of both NIS and TPO genes would overcome this deficiency. Our results show that transfection of NSCLC cells with both human NIS and TPO genes resulted in an increase in radioiodide uptake and retention and enhanced tumor cell apoptosis. These findings suggest that single gene therapy with only the NIS gene may have limited efficacy because of rapid efflux of radioiodide. In contrast, the combination of NIS and TPO gene transfer, with resulting TPO-mediated organification and intracellular retention of radioiodide, may lead to more effective tumor cell death. Thus, TPO could be used as a therapeutic strategy to enhance the NIS-based radioiodide concentrator gene therapy for locally advanced lung cancer.
Gene therapy; NIS/TPO; lung cancer
It has been reported that the sodium/iodide symporter (NIS) gene is expressed in several breast cancer tissues, suggesting the possibility of radionuclide imaging and therapy. However, the regulatory mechanism of NIS gene expression in breast cancer is not yet understood. To assess the relationship between the hormonal status and the NIS expression in breast cancer tissue, we investigated the NIS expression and correlated it to the expression of the thyrotropin receptor (thyroid stimulating hormone receptor, TSH-R), the estrogen receptor (ER) and the progesterone receptor (PR) in human breast cancer tissues.
Materials and Methods
Breast cancer tissues were obtained from 44 patients. Pathological examination showed 2 cases of Grade I, 17 of Grade II, 22 of Grade III, and 3 of unknown grade. We measured the expression of NIS and TSH-R genes by using RT-PCR and we measured the status of ER and PR by using immunohistochemistry.
The NIS gene was expressed in 15 (34%) of the 44 breast cancer tissues. The NIS gene was expressed in 32% of the cases with TSH-R gene expression. The NIS gene was expressed in 40% of the breast cancer tissues with a positive PR and in 31% with a negative PR (p>0.05). It was positive for PR in 18% of the cases and negative for PR in 39% of the cases (p>0.05).
The NIS gene is expressed in approximately one-third of the human breast cancer tissues. Its expression was not related to the presence of the TSH-R gene or hormonal receptors, ER and PR.
Breast neoplasms; Estrogen receptors; Progesterone receptors; Sodium/iodide symporter; Thyrotropin receptors
We present a paediatric case of Papillary Ca thyroid under evaluation for elevated Thyroglobulin (Tg) level with negative 131I wholebody scintigraphy. Differentiated thyroid cancer (DTC) arises from follicular epithelium and retains basic biological features like expression of sodium iodide symporter (NIS), which is the cellular basis of radio iodine (131I) concentration during thyroid ablation. Once dedifferentiation of thyroid cells occurs, cells fail to concentrate 131I, posing both diagnostic and therapeutic problems in DTC and one may have to resort to other imaging techniques for disease localization. As DTC progression is slow, patients have a relatively good prognosis. However children with thyroid malignancies need aggressive management, as initial presentation itself maybe with nodal metastases. It is well known that FDG PET CT apart from its oncological applications, is also used in the evaluation of vascular inflammation especially Takayasu’s arteritis. It is also reported in literature, that 18F-FDG uptake can be seen relatively frequently in the arterial tree of cancer patients. Dunphy et al reported the association of vascular FDG uptake in inflammation as well as in normal arteries. This study typically describes FDG uptake in a patchwork of normal vessel, focal inflammation and or calcification of vessels. The other plausible reasons for significant vascular 18F-FDG uptake are drugs such as potent non steroidal anti-inflammatory agents, dexamethasone, prednisone and tacrolimus. Our patient showed false positive 18F Fluorodeoxyglucose (FDG) uptake in spinal cord at D11/12 and D12/L1 vertebral levels in FDG PET CT imaging performed as part of raised Thyroglobulin workup. This intra spinal FDG uptake is attributed to physiological uptake and inadequate FDG clearance from artery of Adamkiewicz, which can be added as a new physiological cause of FDG uptake unreported in literature as yet.
Artery of Adamkiewicz; Dedifferentiated thyroid cancer; FDG PET scan; papillary Ca thyroid; radioiodine I 131 scan; thyroglobulin