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.     

Dexamethasone stimulation of retinoic Acid-induced sodium iodide symporter expression and cytotoxicity of 131-I in breast cancer cells

CONTEXT: The sodium iodide symporter (NIS) mediates the active iodide uptake in the thyroid gland as well as lactating breast tissue. Recently induction of functional NIS expression was reported in the estrogen receptor-positive human breast cancer cell line MCF-7 by all-trans retinoic acid (atRA) treatment in vitro and in vivo, which might offer the potential to treat breast cancer with radioiodine. OBJECTIVE: In the current study, we examined the effect of dexamethasone (Dex) on atRA-induced NIS expression and therapeutic efficacy of 131-I in MCF-7 cells. DESIGN: For this purpose, NIS mRNA and protein expression levels in MCF-7 cells were examined by Northern and Western blot analysis after incubation with Dex (10(-9) to 10(-7) m) in the presence of atRA (10(-6) m) as well as immunostaining using a mouse monoclonal human NIS-specific antibody. In addition, NIS functional activity was measured by iodide uptake and efflux assay, and in vitro cytotoxicity of 131-I was examined by in vitro clonogenic assay. RESULTS: After incubation with Dex in the presence of atRA, NIS mRNA levels in MCF-7 cells were stimulated up to 11-fold in a concentration-dependent manner, whereas NIS protein levels increased up to 16-fold and iodide accumulation was stimulated up to 3- to 4-fold. Furthermore, iodide efflux was modestly decreased after stimulation with Dex in the presence of atRA. Furthermore, in the in vitro clonogenic assay, selective cytotoxicity of 131-I was significantly increased from approximately 17% in MCF-7 cells treated with atRA alone to 80% in MCF-7 cells treated with Dex in the presence of atRA. CONCLUSION: Treatment with Dex in the presence of atRA significantly increases functional NIS expression levels in addition to inhibiting iodide efflux, resulting in an enhanced selective killing effect of 131-I in MCF-7 breast cancer cells.     

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.     

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.     

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.     

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.     

Resveratrol regulates insulin-like growth factor-II in breast cancer cells

IGF-II is a potent mitogen and inhibitor of apoptosis in breast cancer. Regulation of IGF-II is complex and includes inhibition by tumor suppressors, stimulation by oncogenes, and imprinting and hormonal regulation by estrogens. Resveratrol (RSV) is a phytoestrogen that displays estrogen-like agonistic and antagonistic activity. Recent studies have shown that RSV inhibits the growth of breast cancer cells and may represent a potent agent in chemopreventive therapy. Because 17beta-estradiol regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells. Treatment of MCF-7 and T47D cells with RSV (10(-6) M) caused stimulation of precursor IGF-II mRNA and protein; this effect was blocked by coincubation with 17beta-estradiol (10(-9) M). Cell growth stimulated by RSV (10(-6) M) was blocked by addition of a blocking IGF-I receptor antibody, or the antiestrogen tamoxifen (10(-7) M). In contrast, RSV treatment (10(-4) M) inhibited IGF-II secretion and cell growth in MCF-7 and T47D cells. No increase in IGF-II levels is seen in estrogen receptor (-) MCF-10 cells, even though cell growth was inhibited by RSV 10(-4) M and precursor IGF-II blocked the inhibitory effect of resveratrol. No change in IGF-I was observed with RSV treatment (10(-6) to 10(-4) M). Our study demonstrates that RSV regulates IGF-II and that IGF-II mediates RSV effect on cell survival and growth in breast cancer cells.     

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.     

Insulin-like growth factor I activates c-Jun N-terminal kinase in MCF-7 breast cancer cells

Insulin-like growth factor I (IGF-I) is an important mediator of breast cancer cell growth, although the signaling pathways important for IGF-I-mediated effects in breast cancer cells are still being elucidated. We had demonstrated previously that increased intracellular cAMP in MCF-7 breast cancer cells inhibited cell growth and IGF-I-induced gene expression, as determined using a reporter gene assay. This effect of cAMP on IGF-I signaling was independent of IGF-I-induced activation of the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1 and -2). To determine whether this effect of cAMP may be mediated via another mitogen-activated protein kinase, the ability of IGF-I to activate the c-Jun N-terminal kinases (JNKs) was investigated. Treatment of MCF-7 cells with 100 ng/ml IGF-I increased the level of phosphorylated JNK, as determined by Western blot analysis. JNK phosphorylation was not evident until 15 min after treatment with IGF-I, and peak levels of phosphorylation were present at 30-60 min. This was in contrast to ERK phosphorylation, which was present within 7.5 min of IGF-I treatment. Determination of JNK activity using an immune complex assay demonstrated a 3.3- and 3.5-fold increase in JNK1 and -2 activity, respectively, 30 min after treatment with 100 ng/ml IGF-I. The use of PD98059, which inhibits activation of ERK1 and -2, and LY 294002, an inhibitor of phosphatidylinositol 3-kinase, demonstrated that IGF-I-induced activation of JNK1 is independent of ERK and phosphatidylinositol 3-kinase activation. In contrast, increasing intracellular cAMP with forskolin resulted in abrogation of IGF-I-induced JNK activity. In summary, these data demonstrate that IGF-I activates the JNKs in MCF-7 breast cancer cells and, taken together with the results of our previous study, suggest that JNK may contribute to IGF-I-mediated gene expression and, possibly, cell growth in MCF-7 breast cancer cells.     

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.     

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.     

Genistein enhances insulin-like growth factor signaling pathway in human breast cancer (MCF-7) cells

Physiological concentration of genistein, a natural isoflavonoid phytoestrogen, stimulates human breast cancer (MCF-7) cells proliferation. In this study, we hypothesize that low concentration of genistein mimics the action of 17beta-estradiol in stimulation of MCF-7 cell growth by enhancement of IGF-I signaling pathway. Genistein, at 1 microM, stimulated the growth of MCF-7 cells. Cell cycle analysis showed that 1 micro M genistein significantly increased the S phase and decreased the G0G1 phase of MCF-7 cells. The protein and mRNA expression of IGF-I receptor (IGF-IR) and insulin receptor substrate (IRS)-1, but not Src homology/collagen protein, increased in response to 1 microM genistein in a time-dependent manner. These effects could be completely abolished by cotreatment of MCF-7 cells with estrogen antagonist ICI 182780 (1 microM) and tamoxifen (0.1 microM). Our results also showed that genistein induction of IGF-IR and IRS-1 expression resulted in enhanced tyrosine phosphorylation of IGF-IR and IRS-1 on IGF-I stimulation. Taken together, these data provide the first evidence that the IGF-IR pathway is involved in the proliferative effect of low-dose genistein in MCF-7 cells.     

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.