Dexamethasone enhances the cytotoxic effect of radioiodine therapy in prostate cancer cells expressing the sodium iodide symporter

Recently, we have reported the induction of prostate-specific radioiodine accumulation in prostate cancer cells (LNCaP) using a prostate-specific antigen (PSA)-promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. The aim of our current study was to examine the regulation of PSA-promoter-directed NIS expression in NIS-transfected LNCaP cells (NP-1) by dexamethasone (Dex). For this purpose, NIS mRNA and protein expression levels were examined in NP-1 cells by Northern and Western blot analysis, respectively, after incubation with Dex (10(-8)-10(-6) M) in the presence of 10(-9) M mibolerone. NIS functional activity was measured by iodide uptake assay. In addition, we examined regulation of in vitro cytotoxicity of 131-I by Dex in an in vitro clonogenic assay. After incubation with Dex, iodide accumulation in NP-1 cells increased up to 1.5-fold, whereas NIS mRNA and protein expression levels were increased up to 1.7-fold. This effect of Dex was blocked by the androgen receptor antagonist casodex (10(-6) M). The killing effect of 131-I in NP-1 cells was increased from 55% when incubated with mibolerone alone to 95% when treated with Dex (10(-7) M) plus mibolerone. Treatment of NP-1 cells with Dex resulted in an additional antiproliferative effect as measured by clonogenic assay and nonradioactive proliferation assay. In conclusion, in addition to an antiproliferative effect, treatment with Dex increases androgen-dependent NIS mRNA and protein expression as well as iodide accumulation, resulting in an increased cytotoxic effect of 131-I in prostate cancer cells stably expressing NIS under the control of the PSA-promoter.     

Retinoic acid-induced stimulation of sodium iodide symporter expression and cytotoxicity of radioiodine in prostate cancer cells

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.     

Differential regulation of sodium/iodide symporter gene expression by nuclear receptor ligands in MCF-7 breast cancer cells

The sodium/iodide symporter (NIS) mediates iodide uptake in lactating breast tissue and is expressed in some breast cancers. We have previously demonstrated that all-trans retinoic acid (tRA) stimulates NIS gene expression and the selective cytotoxic effect of beta-emitting radioiodide-131 ((131)I) in both in vitro and in vivo MCF-7 breast cancer cell systems. We studied the ability of natural and synthetic retinoids, in combination with other nuclear receptor ligands, to achieve greater and more sustained induction of NIS in MCF-7 cells and enhance (131)I-mediated cytotoxicity. Selective stimulation of retinoic acid receptor (RAR) beta/gamma produced marked NIS induction; and selective stimulation of RARalpha, RARgamma, or retinoid X receptor produced more modest induction. Maximal NIS induction was seen with 9-cis retinoic acid and AGN190168, a RAR beta/gamma-agonist. Dexamethasone (Dex), but not the other nuclear receptor ligands, in combination with tRA synergistically induced iodide uptake and NIS mRNA expression, predominantly by prolonging NIS mRNA half-life. The addition of Dex reduced the EC(50) of tRA for NIS stimulation to approximately 7%, such that 10(-7) m tRA with addition of Dex enhanced iodide uptake and selective cytotoxicity of (131)I greater than 10(-6) m tRA alone. AGN190168 combined with Dex synergistically increased iodide uptake and significantly prolonged induction (5 d) of iodide uptake compared with that induced by the combination of tRA/Dex or 9-cis retinoic acid/Dex. The addition of Dex reduced the effective dose of retinoid and prolonged the induction of NIS, especially with AGN190168, suggesting higher efficacy of (131)I after combination treatment.     

A novel therapeutic strategy for medullary thyroid cancer based on radioiodine therapy following tissue-specific sodium iodide symporter gene expression

CONTEXT: In contrast to papillary and follicular thyroid cancer, medullary thyroid cancer (MTC) remains difficult to treat due to its unresponsiveness to radioiodine therapy and its limited responsiveness to chemo- and radiotherapy. OBJECTIVE: To investigate an alternative therapeutic approach, we examined the feasibility of radioiodine therapy of MTC after human sodium iodide symporter (hNIS) gene transfer using the calcitonin promoter to target hNIS gene expression to MTC cells (TT). DESIGN: TT cells were stably transfected with an expression vector, in which hNIS cDNA was coupled to the calcitonin promoter. Functional hNIS expression was confirmed by iodide accumulation assays, Northern and Western blot analysis, immunostaining, and in vitro clonogenic assay. RESULTS: hNIS-transfected TT cells showed perchlorate-sensitive iodide uptake, accumulating 125-I about 12-fold in vitro with organification of 4% of accumulated iodide resulting in a significant decrease in iodide efflux. NIS protein expression was confirmed by Western blot analysis using a monoclonal hNIS-specific antibody, which revealed a major band of a molecular mass of 80-90 kDa. In addition, immunostaining of hNIS-transfected TT cells revealed hNIS-specific immunoreactivity, which was primarily membrane associated. In an in vitro clonogenic assay, 84% of NIS-transfected TT cells were killed by exposure to 131-I, whereas only about 0.6% of control cells were killed. CONCLUSIONS: A therapeutic effect of 131-I has been demonstrated in MTC cells after induction of tissue-specific iodide uptake activity by calcitonin promoter-directed hNIS expression. This study demonstrates the potential of NIS as a therapeutic gene, allowing radioiodine therapy of MTC after tissue-specific NIS gene transfer.     

Application of 188rhenium as an alternative radionuclide for treatment of prostate cancer after tumor-specific sodium iodide symporter gene expression

CONTEXT: We reported recently the induction of iodide accumulation in prostate cancer cells (LNCaP) by prostate-specific antigen promoter-directed sodium iodide symporter (NIS) expression that allowed a significant therapeutic effect of (131)iodine ((131)I). These data demonstrated the potential of the NIS gene as a novel therapeutic gene, although in some extrathyroidal tumors, therapeutic efficacy may be limited by rapid iodide efflux due to a lack of iodide organification. OBJECTIVE: In the current study, we therefore studied the potential of (188)rhenium ((188)Re), as an alternative radionuclide, also transported by NIS, with a shorter half-life and higher energy beta-particles than (131)I. RESULTS: NIS-transfected LNCaP cells (NP-1) concentrated 8% of the total applied activity of (188)Re as compared with 16% of (125)I, which was sufficient for a therapeutic effect in an in vitro clonogenic assay. gamma-Camera imaging of NP-1 cell xenografts in nude mice revealed accumulation of 8-16% injected dose (ID)/g (188)Re (biological half-life 12.9 h), which resulted in a 4.7-fold increased tumor absorbed dose (450 mGy/MBq) for (188)Re as compared with (131)I. After application of 55.5 MBq (131)I or (188)Re, smaller tumors showed a similar average volume reduction of 86%, whereas in larger tumors volume reduction was significantly increased from 73% after (131)I treatment to 85% after application of (188)Re. CONCLUSION: Although in smaller prostate cancer xenografts both radionuclides seemed to be equally effective after prostate-specific antigen promoter-mediated NIS gene delivery, a superior therapeutic effect has been demonstrated for (188)Re in larger tumors.     

Retinoic acid stimulation of the sodium/iodide symporter in MCF-7 breast cancer cells is mediated by the insulin growth factor-I/phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways

CONTEXT: All-trans retinoic acid (tRA) induces differentiation in MCF-7 breast cancer cells, stimulates sodium/iodide symporter (NIS) gene expression, and inhibits cell proliferation. Radioiodine administration after systemic tRA treatment has been proposed as an approach to image and treat some differentiated breast cancer. OBJECTIVE: The objective of this work was to study the relative role of genomic and nongenomic pathways in tRA stimulation of NIS expression in MCF-7 cells. DESIGN: We inspected the human NIS gene locus for retinoic acid-responsive elements and tested them for function. The effects of signal transduction pathway inhibitors were also tested in tRA-treated MCF-7 cells and TSH-stimulated FRTL-5 rat thyroid cells, followed by iodide uptake assay, quantitative RT-PCR of NIS, and cell cycle phase analysis. RESULTS: Multiple retinoic acid response elements around the NIS locus were identified by sequence inspection, but none of them was a functional tRA-induced element in MCF-7 cells. Inhibitors of the IGF-I receptor, Janus kinase, and phosphatidylinositol 3-kinase (PI3K), significantly reduced NIS mRNA expression and iodide uptake in tRA-stimulated MCF-7 cells but not FRTL-5 cells. An inhibitor of p38 MAPK significantly reduced iodide uptake in both tRA-stimulated MCF-7 cells and TSH-stimulated FRTL-5 cells. IGF-I and PI3K inhibitors did not significantly reduce the basal NIS mRNA expression in MCF-7 cells. Despite the chronic inhibitory effects on cell proliferation, tRA did not reduce the S-phase distribution of MCF-7 cells during the period of NIS induction. CONCLUSION: The IGF-I receptor/PI3K pathway mediates tRA-stimulated NIS expression in MCF-7 but not FRTL-5 thyroid cells.     

KT5823 differentially modulates sodium iodide symporter expression, activity, and glycosylation between thyroid and breast cancer cells

Na(+)/I(-) symporter (NIS)-mediated iodide uptake into thyroid follicular cells serves as the basis of radioiodine therapy for thyroid cancer. NIS protein is also expressed in the majority of breast tumors, raising potential for radionuclide therapy of breast cancer. KT5823, a staurosporine-related protein kinase inhibitor, has been shown to increase thyroid-stimulating hormone-induced NIS expression, and thus iodide uptake, in thyroid cells. In this study, we found that KT5823 does not increase but decreases iodide uptake within 0.5 h of treatment in trans-retinoic acid and hydrocortisone-treated MCF-7 breast cancer cells. Moreover, KT5823 accumulates hypoglycosylated NIS, and this effect is much more evident in breast cancer cells than thyroid cells. The hypoglycosylated NIS is core glycosylated, has not been processed through the Golgi apparatus, but is capable of trafficking to the cell surface. KT5823 impedes complex NIS glycosylation at a regulatory point similar to brefeldin A along the N-linked glycosylation pathway, rather than targeting a specific N-glycosylated site of NIS. KT5823-mediated effects on NIS activity and glycosylation are also observed in other breast cancer cells as well as human embryonic kidney cells expressing exogenous NIS. Taken together, KT5823 will serve as a valuable pharmacological reagent to uncover mechanisms underlying differential NIS regulation between thyroid and breast cancer cells at multiple levels.     

Approaches to gene therapy with sodium/iodide symporter.

Since cloning and characterization of the sodium iodide symporter (NIS) gene, several investigators explored the possibility of a novel cytoreductive gene therapy strategy based on NIS gene transfer into non-thyroidal tumor cells followed by radioiodine therapy. NIS gene transfer has been shown to be capable of inducing radioiodine accumulation in vitro and in vivo in several non-thyroidal cancer cell lines. Following PSA promoter-mediated NIS gene delivery we were able to demonstrate prostate-specific iodide accumulation in prostate cancer cells that was high enough to elicit a therapeutic response of 131-I in vitro and in vivo. This study clearly demonstrates the potential of NIS as a novel therapeutic gene for non-thyroidal cancers, in particular prostate cancer.     

Signaling through 3',5'-cyclic adenosine monophosphate and phosphoinositide-3 kinase induces sodium/iodide symporter expression in breast cancer

The sodium/iodide symporter (NIS) is a membrane transport glycoprotein normally expressed in the thyroid gland and lactating mammary gland. NIS is a target for radioiodide imaging and therapeutic ablation of thyroid carcinomas and has the potential for similar use in breast cancer treatment. To facilitate NIS-mediated radionuclide therapy, it is necessary to identify signaling pathways that lead to increased NIS expression and function in breast cancer. We examined NIS expression in mammary tumors of 14 genetically engineered mouse models to identify genetic manipulations associated with NIS induction. The cAMP and phosphoinositide-3 kinase (PI3K) signaling pathways are associated with NIS up-regulation. We showed that activation of PI3K alone is sufficient to increase NIS expression and radioiodide uptake in MCF-7 human breast cancer cells, whereas cAMP stimulation increases NIS promoter activity and NIS mRNA levels but is not sufficient to increase radioiodide uptake. This study is the first to demonstrate that NIS expression is induced by cAMP and/or PI3K in breast cancer both in vivo and in vitro.     

Modulation of sodium iodide symporter expression and function by LY294002, Akti-1/2 and Rapamycin in thyroid cells

The selective increase of Na(+)/I(-) symporter (NIS)-mediated active iodide uptake in thyroid cells allows the use of radioiodine I(131) 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.     

Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.     

Enhancement of sodium/iodide symporter expression in thyroid and breast cancer

The sodium/iodide symporter (NIS) mediates iodide uptake in the thyroid gland and lactating breast. NIS mRNA and protein expression are detected in most thyroid cancer specimens, although functional iodide uptake is usually reduced resulting in the characteristic finding of a 'cold' or non-functioning lesion on a radioiodine image. Iodide uptake after thyroid stimulating hormone (TSH) stimulation, however, is sufficient in most differentiated thyroid cancer to utilize beta-emitting radioactive iodide for the treatment of residual and metastatic disease. Elevated serum TSH, achieved by thyroid hormone withdrawal in athyreotic patients or after recombinant human thyrotropin administration, directly stimulates NIS gene expression and/or NIS trafficking to the plasma membrane, increasing radioiodide uptake. Approximately 10-20% differentiated thyroid cancers, however, do not express the NIS gene despite TSH stimulation. These tumors are generally associated with a poor prognosis. Reduced NIS gene expression in thyroid cancer is likely due in part, to impaired trans-activation at the proximal promoter and/or the upstream enhancer. Basal NIS gene expression is detected in about 80% breast cancer specimens, but the fraction with functional iodide transport is relatively low. Lactogenic hormones and various nuclear hormone receptor ligands increase iodide uptake in breast cancer cells in vitro, but TSH has no effect. A wide range of 'differentiation' agents have been utilized to stimulate NIS expression in thyroid and breast cancer using in vitro and in vivo models, and a few have been used in clinical studies. Retinoic acid has been used to stimulate NIS expression in both thyroid and breast cancer. There are similarities and differences in NIS gene regulation and expression in thyroid and breast cancer. The various agents used to enhance NIS expression in thyroid and breast cancer will be reviewed with a focus on the mechanism of action. Agents that promote tumor differentiation, or directly stimulate NIS gene expression, may result in iodine concentration in 'scan-negative' thyroid cancer and some breast cancer.     

Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells

Placental iodide transport is critical for the fetal thyroid function, but the molecular mechanisms of this transport are not understood. The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied using real-time kinetic quantitative PCR and immunohistochemistry 1) in placental tissues collected at different gestational ages and 2) in primary cultures of villous cytotrophoblast cells (VCT) that differentiate and fuse over 2-3 days in vitro to form villous syncytiotrophoblast (VSCT) cells. Both NIS and PDS genes are expressed in placenta, albeit at low levels compared with those in thyroid tissue. NIS gene expression in placental samples from first trimester and term pregnancies was similar. In contrast, the expression of PDS gene was higher in term than in first trimester pregnancy samples. In vitro, NIS gene was expressed at a high level in VCT obtained from first trimester pregnancy, and its expression decreased by 3- to 4-fold during the differentiation of VCT in VSCT. Expression of NIS was lower (up to 30-fold) in VCT obtained in placental samples from third trimester than from first trimester pregnancy. In contrast, the expression of PDS gene was low in VCT and increased by 5- to 10-fold during VSCT formation; this was observed in cells isolated from placental samples of both first trimester and term pregnancies. Immunohistochemical analysis showed that NIS protein was present on the entire membrane of VCT, whereas pendrin was mainly located at the brush border membrane of VSCT, facing the mother. In conclusion, 1) NIS and PDS genes are differently expressed in the placenta during gestation; and 2) whereas pendrin is expressed at the brush border membrane of syncytiotrophoblast cells, NIS protein is mainly located in the cytotrophoblast layer.