Endogenous NIS Research Articles

Targeting GLI1 Transcription Factor for Restoring Iodine Avidity with Redifferentiation in Radioactive-Iodine Refractory Thyroid Cancers.

Simple SummaryThyroid cancers have an excellent prognosis by standard therapy of surgery followed by radioactive-iodine therapy. However, metastatic thyroid cancers do not response to radioactive-iodine therapy by losing iodine avidity. Therefore, reversing iodine avidity to metastatic thyroid cancers gives a new chance of applying radioactive-iodine therapy to the cancers. In the current study, we found that GLI1 knockdown can revert iodine non-avid thyroid cancers to iodine avid cancers by increasing expression of thyroid-specific proteins. Restoration of iodine avidity in thyroid cancers makes the cancers sensitive to radioactive-iodine therapy again. Therefore, the GLI1 can be a potential therapeutic target of radioactive-iodine resistant thyroid cancers.Radioactive-iodine (RAI) therapy is the mainstay for patients with recurrent and metastatic thyroid cancer. However, many patients exhibit dedifferentiation characteristics along with lack of sodium iodide symporter (NIS) functionality, low expression of thyroid-specific proteins, and poor RAI uptake, leading to poor prognosis. Previous studies have demonstrated the effect of GLI family zinc finger 1 (GLI1) inhibition on tumor growth and apoptosis. In this study, we investigated the role of GLI1 in the context of redifferentiation and improvement in the efficacy of RAI therapy for thyroid cancer. We evaluated GLI1 expression in several thyroid cancer cell lines and selected TPC-1 and SW1736 cell lines showing the high expression of GLI. We performed GLI1 knockdown and evaluated the changes of thyroid-specific proteins expression, RAI uptake and I-131-mediated cytotoxicity. The effect of GANT61 (GLI1 inhibitor) on endogenous NIS expression was also assessed. Endogenous NIS expression upregulated by inhibiting GLI1, in addition, increased expression level in plasma membrane. Also, GLI1 knockdown increased expression of thyroid-specific proteins. Restoration of thyroid-specific proteins increased RAI uptake and I-131-mediated cytotoxic effect. Treatment with GANT61 also increased expression of endogenous NIS. Targeting GLI1 can be a potential strategy with redifferentiation for restoring RAI avidity in dedifferentiated thyroid cancers.

MiR-181a-5p Regulates NIS Expression in Papillary Thyroid Carcinoma.

NIS is a potent iodide transporter encoded by the SLC5A5 gene. Its expression is reduced in papillary thyroid carcinoma (PTC). In this study we analyzed the impact of miR-181a-5p on NIS expression in the context of PTC. We used real-time PCR to analyze the expression of SLC5A5 and miR-181a-5p in 49 PTC/normal tissue pairs. Luciferase assays and mutagenesis were performed to confirm direct binding of miR-181a-5p to the 3′UTR of SLC5A5 and identify the binding site. The impact of modulation of miR-181a-5p using appropriate plasmids on endogenous NIS and radioactive iodine accumulation was verified. We confirmed downregulation of SLC5A5 and concomitant upregulation of miR-181a-5p in PTC. Broadly used algorithms did not predict the binding site of miR-181a-5p in 3′UTR of SLC5A5, but we identified and confirmed the binding site through mutagenesis using luciferase assays. In MCF7 and HEK293-flhNIS cell lines, transfection with mir-181a-expressing plasmid decreased endogenous SLC5A5, whereas silencing of miR-181a-5p increased it. We observed similar tendencies in protein expression and radioactive iodine accumulation. This study shows for the first time that miR-181a-5p directly regulates SLC5A5 expression in the context of PTC and may decrease efficacy of radioiodine treatment. Accordingly, miR-181a-5p may serve as an emerging target to enhance the efficacy of radioactive iodine therapy.

Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale

The sodium iodide symporter (NIS) is widely used as a reporter gene to noninvasively monitor the biodistribution and durability of vector-mediated gene expression via gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). However, the approach is limited by background signal due to radiotracer uptake by endogenous NIS-expressing tissues. In this study, using the SPECT tracer pertechnetate (99mTcO4) and the PET tracer tetrafluoroborate (B18F4), in combination with the NIS inhibitor perchlorate, we compared the transport properties of human NIS and minke whale (Balaenoptera acutorostrata scammoni) NIS in vitro and in vivo. Based on its relative resistance to perchlorate, the NIS protein from minke whale appeared to be the superior candidate reporter gene. SPECT and PET imaging studies in nude mice challenged with NIS-encoding adeno-associated virus (AAV)-9 vectors confirmed that minke whale NIS, in contrast to human and endogenous mouse NIS, continues to function as a reliable reporter even when background radiotracer uptake by endogenous NIS is blocked by perchlorate.

FOXA1 Regulation Turns Benzamide HDACi Treatment Effect-Specific in BC, Promoting NIS Gene-Mediated Targeted Radioiodine Therapy

Human sodium iodide symporter (NIS) gene mediated radio-ablation is a successful procedure in thyroid cancer clinics. In recent years, natural expression of NIS is reported in breast cancer (BC) cases but is yet to make its mark as a therapeutic procedure in BC clinics. A pre-exposure to histone deacetylase (HDAC) inhibitors to amplify endogenous NIS expression was attempted, but achieving cancer tissue-specific enhancement of NIS in patients is an important challenge to win. Here, for the first time, we show that a benzamide class of HDACi (bHDACi) can significantly induce NIS gene expression and function (p < 0.05) in BC cells with minimal off-target effects. Transcription factor (TF) profiler and promoter binding array reveals 22 TFs differentially activated by CI-994, of which FOXA1 is identified as a unique and positive regulator of NIS. Clonogenic assay shows reduced survival with bHDACi + 131I combination treatment. Further, AR-42 and MS-275 treatment shows enhanced NIS expression in an orthotopic breast tumor model. Combining bHDACi with 1 mCi 131I shows 40% drop in signal (p < 0.05), indicating enhanced radio-ablation effect. Cerenkov imaging revealed higher accumulation of 131I in MS-275-treated tumors. Thus, bHDACi-mediated selective enhancement ensuring minimal off-target effect is a step further toward using NIS as a therapeutic target for BC.

Abstract 920: p97/VCP: A novel interactor of the sodium iodide symporter, which can be pharmacologically targeted to increase radioiodine uptake in thyroid and breast cancer cells

Abstract By exploiting the canonical function of the sodium iodide symporter (NIS), ablative radioiodine therapy is an effective treatment for papillary thyroid cancer (PTC). Unfortunately, ~25% of PTC patients are unable to accumulate therapeutically sufficient radioiodine due to diminished expression and/or altered plasma membrane localization of NIS. Patients with radioiodine-refractory PTC have reduced mean survival times. Radioiodine therapy has been proposed as a viable treatment for breast cancer but is hampered by low levels of membranous NIS localization. Currently, however, the regulation of NIS membrane localization remains ill-defined. Mass spectrometry identified the protein p97/VCP as a novel NIS interactor, which was validated through co-immunoprecipitation and proximity ligation assays. p97 siRNA depletion increased NIS-mediated radioiodine uptake by 97% and 141% in the lentivirally-expressing NIS MDA-MB-231 breast and TPC1 thyroid cancer cell lines respectively (p&amp;lt;0.05), and significantly boosted endogenous NIS function in human primary thyrocytes (p&amp;lt;0.05). Conversely, p97 overexpression significantly repressed NIS function in all cell systems, accompanied by lowered membranous NIS localization (p&amp;lt;0.05), as quantified via cell surface biotinylation assays. We next identified five different allosteric p97 inhibitors - Eeyarestatin-1, NMS-873, Astemizole, Clotrimazole and Ebastine - which were all able to overcome p97 inhibition of NIS function in TPC1 and MDA-MB-231 cell lines lentivirally-expressing NIS, significantly increasing NIS function by at least 100%. TCGA analyses of matched PTCs revealed p97 mRNA expression is highly upregulated in PTC compared to matched normal thyroid (n=58, p&amp;lt;0.05), providing a putative explanation for repressed NIS function. In a wider cohort of 413 PTC patients, high tumoral p97 expression was associated with a worse disease-free survival when compared to low p97 expression (p&amp;lt;0.01, hazard ratio (HR): 2.924 (95% CI, 1.280-6.683)). Strikingly, in patients that did not receive radioiodine, high tumoral p97 expression had no significant effect on disease-free survival (n=136, p=NS, HR: 0.020 (95% CI, 0.000013-31.192)), whereas in patients who received radioiodine, high tumoral p97 expression resulted in a markedly worse disease-free survival (n=194, p&amp;lt;0.01, HR: 6.043 (95% CI, 1.749-20.879)). This is indicative that high p97 expression correlates with a worse response to radioiodine therapy. Our data therefore highlight a new pathway of NIS regulation. Critically, two of these p97 inhibitors are already FDA-approved, highlighting a novel potential therapeutic strategy for enhancing radioiodine uptake in patients with radioiodine-refractory PTC and increasing the feasibility of radioiodine therapy in breast cancer via the transient inhibition of p97 activity. Citation Format: Alice Fletcher, Martin L. Read, Vikki L. Poole, Vicki E. Smith, Christopher J. McCabe. p97/VCP: A novel interactor of the sodium iodide symporter, which can be pharmacologically targeted to increase radioiodine uptake in thyroid and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 920.

PO-022 Benzamide class of HDACi differentially enhance sodium iodide symporter expression in breast cancer

IntroductionSodium iodide symporter (hNIS) expression is reported across all subtypes of breast cancer (BC) and is a potential endogenous gene target for radioiodine therapy [PlosOne8(1:e54055, 2012]. Successful clinical application however relies on very high expression of functional NIS in BC cells. We have been exploiting various transcriptional regulation approaches for NIS, of which use of HDAC inhibitors (HDACi) have shown promising amplification of endogenous NIS expression in BC [Scientific Reports6:19341, 2016]. Here, we show that differential induction of NIS expression in BC is possible using Benzamide class of HDACi (bHDACi) with minimal off-target effects on other cell types.Material and methodsThe effect of bHDACi, i.e. CI-994, Chidamide, MS-275 and AR-42, on NIS gene transcription was verified on a panel of human cancer cells by RTPCR. Functional in vitro assessment was done by non-radioactive iodine uptake. I131therapeutic efficacy was measured by clonogenic assay. Promoter binding transcription factor (TF) profiler array was done to identify novel TFs responsible for differential activation in BC cells. Further, in vivo therapeutic efficacy was monitored by bioluminescence imaging (BLI) of ZR-75–1-fl2.tdt orthotopic model.Results and discussionsUsing a non-toxic, low dose of bHDACi(s) treatment on a cancer cell panel, including breast, thyroid, lung, glioma, fibrosarcoma and ovarian, a significant induction of NIS expression (p<0.05) was observed only in BC cells. Observed differences of NIS expression was confirmed by protein expression and functional assessment on BC cells. Further, novel TFs (FOXA1, FOXOF2 etc.) were identified which show differential activation in BC after CI-994 treatment. Only in case of BC cells pre-treated with bHDACi(s), cell survival assay results showed enhanced cell death on exposure to I131. Further, AR-42 drug was tested in vivo which showed similar results. AR-42 pre-treated ZR-75–1 fl2tdt orthotopic tumour showed increased NIS expression. In comparison to untreated and only I131 treated group, a combination treatment of AR-42 with 1mCi I131, BLI imaging showed 20% and 30% signal drop on day1 and day 2 respectively indicating specific radio-ablation effect.ConclusionbHDACi(s) show differentially higher regulation of endogenous NIS with minimal effect on non-breast tissues and thus may potentially benefit NIS-based radioablation therapy trials in BC patients with low/moderate expression of NIS.

Reverting iodine avidity of radioactive-iodine refractory thyroid cancer with a new tyrosine kinase inhibitor (K905-0266) excavated by high-throughput NIS (sodium iodide symporter) enhancer screening platform using dual reporter gene system.

Radioactive-iodine (RAI) therapy is typically unprevailing as anaplastic thyroid cancer (ATC) management, owing to the decrease in the endogenous sodium iodide symporter (NIS) expression. Therefore, new strategies for NIS re-induction are required to improve the efficacy of RAI therapy in ATC. In this study, we developed a novel high-throughput NIS enhancer screening platform using a dual reporter gene system to identify a potent tyrosine kinase inhibitor (TKI) and selected a new hit compound, K905-0266 TKI. The effects of K905-0266 TKI treatment was validated as RAI accumulation, changes in signalling pathway related to thyroid pathogenesis, and cytotoxicity of RAI depending on re-induction of endogenous NIS expression in ATC. Furthermore, we evaluated enhancement of NIS promoter and therapeutic efficacy of RAI in ATC tumour xenograft mice. After K905-0266 TKI treatment, the expression of endogenous NIS was significantly increased, while phosphorylated-ERK was decreased. In addition, the thyroid-metabolising protein expressions were upregulated and increased of RAI accumulation and its therapeutic effects in ATC. Moreover, K905-0266 TKI increased therapeutic efficacy of RAI in ATC tumour in vivo. In conclusion, we successfully established a novel high-throughput NIS enhancer screening platform to excavate a NIS enhancer and identified K905-0266 TKI among TKI candidates and it's proven to increase the endogenous NIS expression and therapeutic efficacy of RAI in ATC. These findings suggest that a novel high-throughput NIS enhancer screening platform is useful for selecting of NIS promoter enhancers. In addition, K905-0266 TKI can be used to re-induce endogenous NIS expression and recover RAI therapy in ATC.

Enhancement of human sodium iodide symporter gene therapy for breast cancer by HDAC inhibitor mediated transcriptional modulation

The aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) has raised the possibility of using targeted radioiodide therapy. Here we investigate modulation of endogenous, functional NIS expression by histone deacetylase inhibitors (HDACi) in vitro and in vivo. Luciferase reporter based initial screening of six different HDACi shows 2–10 fold enhancement of NIS promoter activity in majority of the cell types tested. As a result of drug treatment, endogenous NIS transcript and protein shows profound induction in BC cells. To get an insight on the mechanism of such transcriptional activation, role of Stat4, CREB and other transcription factors are revealed by transcription factor profiling array. Further, NIS-mediated intracellular iodide uptake also enhances substantially (p < 0.05) signifying functional relevance of the transcriptional modulation strategy. Gamma camera imaging confirms 30% higher uptake in VPA or NaB treated BC tumor xenograft. Corroborating with such functional impact of NIS, significant reduction in cell survival (p < 0.005) is observed in VPA, NaB or CI994 drug and 131I combination treatment in vivo indicating effective radioablation. Thus, for the first time this study reveals the mechanistic basis and demonstrates functional relevance of HDACi pre-treatment strategy in elevating NIS gene therapy approach for BC management in clinic.

Sodium Iodide Symporter Expression in Adenoid Cystic Carcinoma of the Head and Neck.

Sodium iodide symporter (NIS) expression in adenoid cystic carcinoma (ACC) has not been fully elucidated in the literature, and it is unclear whether radioactive iodine may be a potential therapeutic modality. To our knowledge, the present study includes the largest ACC tumor sample size to evaluate for NIS expression. To assess whether ACC of the head and neck expresses NIS by using immunohistochemical staining techniques, as well as assess whether the presence or intensity of staining correlates with tumor or patient variables. Immunohistochemical analysis of NIS expression was performed on 20 ACC specimens from various head and neck subsites obtained from January 1, 1988, to May 6, 2013, at a single academic tertiary care medical center. Staining intensity was graded on a scale of 0 to 3+ (higher numbers indicate greater staining intensity) and was analyzed according to multiple patient and tumor variables. Tumors were eliminated from the study if the patient had undergone prior surgical resection, chemotherapy, or radiotherapy, or was receiving thyroid hormone supplementation at the time of the operation. Presence or absence of staining was the primary outcome measured; secondary outcomes were the intensity and localization of staining. Sodium iodide symporter staining was positive in 15 of the 20 tumor specimens (75%). Staining was largely localized to the cytoplasm and was of low intensity. There was no significant association between the presence or intensity of staining and the tumor subtype, tumor location, or any of the patient variables assessed (P > .05). No association between staining intensity and tumor growth pattern was shown on χ² analysis: 1+ (P = .53), 2+ (P = .14), or a combination of 1+ and 2+ staining (P = .64). Parotid control tissue demonstrated intense membranous staining of the striated parotid gland ducts. Sodium iodide symporter was expressed in the cytoplasm with low intensity in most of the tumor specimens examined in this study. These staining characteristics are also commonly found in thyroid cancer cells. Further investigation is required to determine the significance of this finding. We are optimistic that future studies using endogenous NIS stimulation and identification of genes associated with NIS plasma membrane localization could be applied to the treatment of ACC with radioactive iodine techniques.

Effect of sodium/iodide symporter (NIS)-mediated radioiodine therapy on estrogen receptor-negative breast cancer.

Since the sodium/iodide symporter (NIS) stimulates the iodine uptake in normal lactating breast, our study aimed to study the effect of NIS-mediated radioiodide therapy on ER-negative breast cancers. A recombinant lentivirus plasmid encoding the human NIS (hNIS) gene and firefly luciferase (Fluc) was constructed. MDA-MB-231 cells were transfected with the recombinant lentivirus, and the hNIS gene expression was identified by western blot analysis and real-time PCR. Tissue-specific expression of the NIS gene was confirmed by immunohistochemical (IHC) staining. Functional NIS activity in the MDA-hNIS cells was confirmed by the uptake of 131I and cytotoxicity assays. The relative expression level of hNIS mRNA exhibited a 10-fold higher expression in the MDA-hNIS cells compared with the level in the control cells without the endogenous NIS gene. Abundant expression of hNIS protein was noted in the cell membrane compared to the cytoplasm which confirmed the efficient expression of the functional hNIS gene. Iodine uptake into the MDA-hNIS cells was rapid, reaching a maximum after 15 min, followed by a decline. Exposure of the MDA-hNIS cells with 131I resulted in a time-dependent reduction in colony formation compared with the survival of the control (MDA) cells. Our results confirmed that NIS overexpression enhances the sensitivity of ER-negative breast cancer cells to radioiodide therapy.

Sodium iodide symporter (NIS) in extrathyroidal malignancies: focus on breast and urological cancer.

BackgroundExpression and function of sodium iodide symporter (NIS) is requisite for efficient iodide transport in thyrocytes, and its presence in cancer cells allows the use of radioiodine as a diagnostic and therapeutic tool in thyroid neoplasia. Discovery of NIS expression in extrathyroidal tissues, including transformed cells, has opened a novel field of research regarding NIS-expressing extrathyroidal neoplasia. Indeed, expression of NIS may be used as a biomarker for diagnostic, prognostic, and therapeutic purposes. Moreover, stimulation of endogenous NIS expression may permit the radioiodine treatment of extrathyroidal lesions by concentrating this radioisotope.ResultsThis review describes recent findings in NIS research in extrathyroidal malignancies, focusing on breast and urological cancer, emphasizing the most relevant developments that may have clinical impact.ConclusionsGiven the recent progress in the study of NIS regulation as molecular basis for new therapeutic approaches in extrathyroidal cancers, particular attention is given to studies regarding the relationship between NIS and clinical-pathological aspects of the tumors and the regulation of NIS expression in the experimental models.

The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells

Thyroid iodide accumulation via the sodium/iodide symporter (NIS; SLC5A5) has been the basis for the longtime use of radio-iodide in the diagnosis and treatment of thyroid cancers. NIS is also expressed, but poorly functional, in some non-thyroid human cancers. In particular, it is much more strongly expressed in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines than in primary human hepatocytes (PHH). The transcription factors and signaling pathways that control NIS overexpression in these cancers is largely unknown. We identified two putative regulatory clusters of p53-responsive elements (p53REs) in the NIS core promoter, and investigated the regulation of NIS transcription by p53-family members in liver cancer cells. NIS promoter activity and endogenous NIS mRNA expression are stimulated by exogenously expressed p53-family members and significantly reduced by member-specific siRNAs. Chromatin immunoprecipitation analysis shows that the p53–REs clusters in the NIS promoter are differentially occupied by the p53-family members to regulate basal and DNA damage-induced NIS transcription. Doxorubicin strongly induces p53 and p73 binding to the NIS promoter, leading to an increased expression of endogenous NIS mRNA and protein in HCC and CCA cells, but not in PHH. Silencing NIS expression reduced doxorubicin-induced apoptosis in HCC cells, pointing to a possible role of a p53-family-dependent expression of NIS in apoptotic cell death. Altogether, these results indicate that the NIS gene is a direct target of the p53 family and suggests that the modulation of NIS by DNA-damaging agents is potentially exploitable to boost NIS upregulation in vivo.

Redifferentiation and induction of tumor suppressors miR-122 and miR-375 by the PAX8/PPARγ fusion protein inhibits anaplastic thyroid cancer: a novel therapeutic strategy

Anaplastic thyroid cancer (ATC) is an aggressive, fatal disease unresponsive to traditional therapies, generating a need to develop effective therapies. The PAX8/PPARγ fusion protein (PPFP) has been shown to favorably modulate tumor growth in follicular thyroid cancer, prompting our evaluation of its efficacy to inhibit ATC cell and tumor growth in vitro and in vivo. PPFP was constitutively expressed in five ATC cell lines: BHT-101, FRO, C-643, KTC-2 and KTC-3, and inhibited cell growth in four of five cell lines and xenograft tumor growth in four of four cell lines. PPFP-mediated growth inhibition involved multiple mechanisms, including upregulation of miR-122 and miR-375, associated with decreased angiogenesis and AKT pathway inactivation, respectively. Also, PPFP expression resulted in marked increase of thyroid-specific marker transcripts, including PAX8, thyroid peroxidase (TPO), sodium iodide symporter (NIS) and thyroglobulin, to varying degrees by activating their respective promoters, suggesting that PPFP induced cellular redifferentiation. Functional studies demonstrate that increased NIS messenger RNA is not associated with increased 125I uptake. However, ectopic expression of wild-type NIS-induced perchlorate-sensitive iodine uptake, suggesting that endogenous NIS in ATC cell lines is defective. As current treatment for ATC is only palliative, overexpression of PPFP may offer a novel therapeutic strategy for the treatment of ATC.

The Sodium Iodide Symporter (NIS) as an Imaging Reporter for Gene, Viral, and Cell-based Therapies

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.

Endogenous NIS Expression in Triple-Negative Breast Cancers

The sodium iodide symporter (NIS) mediates iodide transport into cells and has been identified in approximately 70% of breast cancers. Functional NIS expression raises the possibility of using (131)I for therapeutic targeting of tumor cells. Treatment of triple-negative breast cancers [estrogen/progesterone receptor-negative and HER2-negative (ER-/PR-/HER2-)] is primarily limited to chemotherapy. Our aim was to characterize NIS expression in this subset of tumors. Archival tissue sections from 23 women with triple-negative breast cancer were analyzed for NIS expression using immunohistochemical methods and an anti-human NIS antibody. Tumors were evaluated for the presence of plasma membrane immunoreactivity. One patient with a NIS-expressing positive tumor underwent (123)I scintigraphic imaging with dosimetric analysis. Fifteen cases (65.2%) demonstrated NIS-positivity with 11 tumors (47.8%) exhibiting strong expression. Plasma membrane immunoreactivity was observed in four breast cancers and was equivocal in another four tumors. Tumor-specific radioiodide uptake was demonstrated by (123)I scintigraphy in a patient with a large primary breast cancer unresponsive to neoadjuvant therapy. The tumor concentrated 2.05, 1.53, and 1.96 times more isotope than normal breast tissue at 1, 5, and 21 h. The relative increased uptake is consistent with positive NIS expression in the tumor on definitive surgery; however, the cumulative concentration in the tumor was not sufficient to achieve a therapeutic effect, had the isotope been (131)I. NIS is strongly expressed in a significant proportion of triple-negative breast cancer cells, suggesting a potential role for NIS-directed (131)I-radioablative strategies in this patient population.

Phosphoinositide-3-kinase inhibition induces sodium/iodide symporter expression in rat thyroid cells and human papillary thyroid cancer cells

TSH stimulation of sodium iodide symporter (NIS) expression in thyroid cancer promotes radioiodine uptake and is required to deliver an effective treatment dose. Activation of the insulin/phosphoinositide-3-kinase (PI3K) signaling pathway in TSH-stimulated thyroid cells reduces NIS expression at the transcriptional level. We, therefore, investigated the effects of PI3K pathway inhibition on iodide uptake and NIS expression in rat thyroid cell lines and human papillary thyroid cancer cells. A PI3K inhibitor, LY294002, significantly enhanced iodide uptake in two rat thyroid cell lines, FRTL-5 and PCCL3. The induction of Nis mRNA by LY294002 occurred 6 h after treatment, and was abolished by a translation inhibitor, cycloheximide. Expression of the transcription factor, Pax8, which stimulates NIS expression, was significantly increased in PCCL3 cells after LY294002 treatment. Removal of insulin abrogated the stimulatory effects of LY294002 on NIS mRNA and protein expression, but not on iodide uptake. These findings suggest that PI3K pathway inhibition results in post-translational stimulation of NIS. Inhibition of the PI3K pathway also significantly increased iodide uptake ( approximately 3.5-fold) in BHP 2-7 papillary thyroid cancer cells (Ret/PTC1 positive), engineered to constitutively express NIS. Pharmacological inhibition of Akt, a factor stimulated by the PI3K pathway, increased exogenous NIS expression in BHP 2-7 as was seen with LY294002, but not increase the endogenous NIS expression in FRTL-5 cells. PI3K pathway inhibition increases functional NIS expression in rat thyroid cells and some papillary thyroid cancer cells by several mechanisms. PI3K inhibitors have the potential to increase radioiodide accumulation in some differentiated thyroid cancer.

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

BackgroundThe 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.MethodsNIS 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).ResultsIn 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.ConclusionThus, 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.

Der Natrium-Iodid-Symporter (NIS): Bedeutung für die Bildgebung und therapeutische Optionen

Der Natrium-Iodid-Symporter (NIS) vermittelt als intrinsisches Membranprotein den aktiven Transport von Iodid in die Schilddrüse sowie eine Reihe extrathyreoidaler Organe, insbesondere die laktierende Brustdrüse. Neben seiner Schlüsselrolle in der Schilddrüsenphysiologie bildet NIS die Grundlage der diagnostischen Schilddrüsenszintigrafie sowie der therapeutischen Anwendung von Radioiod bei der Behandlung benigner und maligner Schilddrüsenerkrankungen, und stellt damit eines der ältesten Targets molekularer Bildgebung und Therapie dar. In Anlehnung an die seit mehr als 60 Jahren mit großem Erfolg beim Schilddrüsenkarzinom eingesetzten Radioiodtherapie, ermöglicht die Klonierung und Charakterisierung des NIS-Gens die Entwicklung eines neuen, zytoreduktiven Gentherapieansatzes auf dem Boden der gezielten NIS-Expression in thyreoidalen und extrathyreoidalen Tumoren gefolgt von der therapeutischen Applikation von 131I oder alternativer Radionuklide, wie 188Re oder 211At. Darüber hinaus erlaubt die Möglichkeit des direkten, nicht-invasiven Imagings funktioneller NIS-Expression mittels 123I- und 99mTc-Szintigrafie oder 124I-PET-Imaging den Einsatz von NIS als neuem Reportergen. Zusammenfassend eröffnet die Doppelfunktion von NIS als diagnostisches und therapeutisches Gen sowie der Nachweis extrathyreoidaler endogener NIS-Expression in Mammakarzinomen viel versprechende Perspektiven in der Nuklearmedizin und molekularen Onkologie für die diagnostische und therapeutische Applikation von NIS auch außerhalb der Schilddrüse.

A perspective view of sodium iodide symporter research and its clinical implications

The sodium iodide symporter (NIS) is an intrinsic plasma membrane protein that mediates active iodide transport into the thyroid gland and into several extrathyroidal tissues, in particular the lactating mammary gland. Cloning and molecular characterization of the NIS have allowed the investigation of its key role in thyroid physiology as well as its potential pathophysiological and therapeutic implications in benign and malignant thyroid diseases. Similarly, elucidating the mechanisms underlying the regulation of NIS in lactating mammary gland and breast cancer, in which more than 80% of cases express endogenous NIS, may lead to findings that have novel implications for pathophysiology and therapy. Two approaches may, in the future, pave the way to extend the use of radioiodide treatment to nonthyroidal cancer. One is based on the reinduction of endogenous NIS expression in thyroid and breast cancer by targeting the main mechanisms involving tumoral transformation and dedifferentiation. The other is based on the application of NIS as a novel cytoreductive gene therapy strategy. NIS offers the unique advantage that it can be used both as a reporter and as a therapeutic gene, so that it is possible to image, monitor, and treat the tumor with radioiodide, just as in differentiated thyroid cancer. This review summarizes the main recent findings in NIS research that have a direct impact on diagnosis and therapeutic management.

Effective cancer therapy with the alpha-particle emitter [211At]astatine in a mouse model of genetically modified sodium/iodide symporter-expressing tumors.

The sodium/iodide symporter (NIS) gene is currently explored in several trials to eradicate experimental cancer with radiodine ((131)I) by its beta-emission. We recently characterized NIS-specific cellular uptake of an alternative halide, radioastatine ((211)At), which emits high-energy alpha-particles. The aim of this study was to investigate in vivo effects of the high linear energy transfer (LET) emitter (211)At on tumor growth and outcome in nude mice. We administered radioastatide in a fractionated therapy scheme to NMRI nude mice harboring rapidly growing solid tumors established from a papillary thyroid carcinoma cell line genetically modified to express NIS (K1-NIS). Animals were observed over 1 year. Tumor growth, body weight, blood counts, survival, and side effects were measured compared with control groups without therapy and/or lack of NIS expression. Within 3 months, radioastatide caused complete primary tumor eradication in all cases of K1-NIS tumor-bearing nude mice (n = 25) with no tumor recurrence during 1 year follow-up. Survival rates of the K1-NIS/(211)At group were 96% after 6 months and 60% after 1 year, in contrast to those of control groups (maximum survival 40 days). Our study indicates that (211)At represents a promising substrate for NIS-mediated therapy of various cancers either with endogenous or gene transfer-mediated NIS expression.