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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1627
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 5 1884-1892
Copyright © 2008 by The Endocrine Society

Retinoic Acid Stimulation of the Sodium/Iodide Symporter in MCF-7 Breast Cancer Cells Is Meditated by the Insulin Growth Factor-I/Phosphatidylinositol 3-Kinase and p38 Mitogen-Activated Protein Kinase Signaling Pathways

Takahiko Kogai, Emi Ohashi, Megan S. Jacobs, Saima Sajid-Crockett, Myrna L. Fisher, Yoko Kanamoto and Gregory A. Brent

Molecular Endocrinology Laboratory (T.K., E.O., M.S.J., S.S.-C., Y.K., G.A.B.), Department of Pathology (M.L.F.), Veterans Affairs Greater Los Angeles Healthcare System, Departments of Medicine and Physiology (T.K., E.O., G.A.B.), David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90073

Address all correspondence and requests for reprints to: Dr. Gregory A. Brent, Molecular Endocrinology Laboratory, Building 114, Room 230, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: gbrent{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The sodium/iodide symporter (NIS) is expressed at high levels in the thyroid and lactating breast and functions to concentrate iodide from the blood stream to these tissues. Thyroid hormone synthesis requires iodide and iodide uptake is regulated by TSH (1). NIS activity is reduced in most thyroid cancers, resulting in the finding of a cold lesion on a radioiodine scan. Iodide uptake after TSH stimulation, however, is sufficient in most differentiated thyroid cancer to use radioactive iodide for treatment of residual and metastatic disease.

In the thyroid, TSH increases NIS expression via the cAMP pathway, primarily by stimulating NIS transcription (2, 3, 4). In FRTL-5 rat thyroid cells, the paired domain containing transcription factor paired box protein-8 and members of the cAMP-response element binding protein family increase in response to TSH and bind to the NIS upstream enhancer (NUE), located about 9 kb upstream from the human NIS coding region (1, 5). The full activation of the NUE additionally requires activation of signal transduction pathways, including protein kinase A (PKA) (3, 4), a small GTPase Rap1 (5) and the MAPK/ERK kinase (MEK)/ERK1/2 cascade (4).

The lactating mammary gland produces milk with an iodine concentration of 20–700 µg/liter, providing substrate for thyroid hormone synthesis by the neonatal thyroid (6). Oxytocin, prolactin, and estradiol stimulate expression of NIS in the lactating breast (7). The iodide uptake in the thyroid and lactating mammary gland, however, is not correlated (1, 8), indicating differential regulation of the NIS expression in these tissues. Nonlactating mammary gland does not express NIS or concentrate iodine, but approximately 80% of breast cancers express NIS and concentrates iodine at a low level (7, 9). A variety of approaches have been used to enhance functional NIS expression in breast cancer (1), with the goal of using radioiodine therapy for breast cancer (10).

All-trans retinoic acid (tRA) significantly inhibits cell proliferation (11) and induces differentiation in breast cancer cells. tRA and its derivatives, therefore, have a potential for chemoprevention of breast cancer. tRA significantly induces expression of the differentiation marker NIS in MCF-7 breast cancer cells (12), in vivo xenografts, and genetic breast cancer models (13). Our pharmacological studies indicate that tRA stimulation of NIS is mediated by the retinoic acid receptor (RAR) and retinoid-X receptor (RXR) (14). Nuclear hormone receptors, including RAR, are thought to stimulate gene expression predominantly through genomic actions (15). RAR forms a heterodimer with RXR, and after combining with its ligand, tRA, activates a target gene as a trans-regulator on a cis-acting retinoic acid response element (RARE). The consensus sequence of an RARE contains direct repeats of the core motif, 5'-PuG[G/T][T/A]CA-3' with a space of two or five bases (DR-2 or DR-5), or everted repeats with a space of eight bases (16).

The nongenomic actions of nuclear hormone receptors are usually rapid and mediated through interactions with membrane receptors as well as modifying the signal transduction cascade (15). In addition to tRA, NIS in breast cancer cells is induced by IGFs, prolactin (17), and cAMP analogs (18). Overexpression of phosphatidylinositol 3-kinase (PI3K) increases the iodide uptake in MCF-7 cells (18). Cross talk between these signals and the retinoic acid signal, however, has not been elucidated.

We investigated the genomic and nongenomic actions of tRA on the regulation of NIS induction in MCF-7 cells by screening of putative RARE on the human NIS gene as well as a number of signal transduction inhibitors in MCF-7 cells. The pathways of NIS regulation in MCF-7 cells were compared with regulation in TSH-stimulated FRTL-5 rat thyroid cells. The relevance of these pathways to cell proliferation was also investigated in MCF-7 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and cells

MCF-7 cells and JEG-3 cells were maintained as previously described (19). FRTL-5 cells, provided by Dr. Leonard Kohn (Ohio University, Athens, OH), were maintained as described (2). Signal transduction inhibitors were purchased from EMD Biosciences (La Jolla, CA). tRA, bovine TSH, and other chemicals were purchased from Sigma (St. Louis, MO).

Sequence inspection

Sequences around the human and mouse NIS locus were inspected by MacMolly Tetra Lite (Mologen, Berlin, Germany). To determine putative RARE, consensus half-sites (16), [A/G]G[G/T][A/T]CA, as well as other reported half-sites (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org), were searched on the top and bottom strands of the human [National Center for Biotechnology Information (Bethesda, MD) accession no. NT_011295.10] and mouse NIS (NC_000074).

Vectors

Vectors for reporter assay were generated as described (supplemental data). To generate the constructs for screening of functional RARE on human NIS gene (Figs. 1Go and 2Go), NIS fragments from the clone-{lambda}II-1 (20), genomic PCR, or annealed synthetic oligonucleotides were inserted to polylinker sites of pGL3 promoter (Promega, Madison, WI), phRGB (Promega), or p{Delta}812-LUC (20). pRL-1xDR5 and pRL-1xDR2 were constructed by insertion of annealed synthetic oligonucleotides into pRL-TK (Promega). The RAR{alpha} cDNA was subcloned from pBluescript-RAR{alpha}, provided by Dr. Ronald Evans (The Salk Institute, La Jolla, CA), into pcDNA3.1 (Invitrogen).


Figure 1
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  		FIG. 1. Systemic characterization of retinoic acid responsive sequences in human NIS gene locus. A, Map of the human chromosome 19p around the NIS gene locus. The A in the translation start site of NIS is referred to as +1. The position of putative RAREs (see supplemental data for details) around the human NIS gene, between –14,254 and +29,500, are noted by asterisk. National Center for Biotechnology Information accession no. are: JAK3 (NM_000215); ribosomal protein L18a (RPL18A; NM_000980); coiled-coil domain containing 124 (CCDC124; NM_138442). B, Transient transfection of the human NIS gene segments from the 5' flanking region and between first and seventh introns, upstream of the Simian virus 40 (SV40) promoter or the human NIS basal proximal promoter controlling luciferase reporter gene in MCF-7 cells. Cells were treated with or without 10–6 M tRA for 12 h after the 48-h transfection with Effectene. The indicated constructs were cotransfected with pRL-CMV Renilla luciferase reporter vector to normalize transfection efficiency. Ratio of luciferase activity in tRA-treated MCF-7 to untreated cells is shown. Values are the mean ± SD (n = 3).

 

Figure 2
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  		FIG. 2. Activity of putative RAREs and variants identified by sequence from the NIS gene 3' flanking region in MCF-7 breast cancer cells. A, Schematic representation of Renilla luciferase reporter vectors containing the putative RAREs with the heterologous herpes simplex virus thymidine kinase promoter as well as DR-5 variants with the human NIS proximal promoter (–812 to –286). B and C, Enhancer activity of the putative RAREs in MCF-7 and JEG-3 cells. The pRL-1xDR2 or pRL-1xDR5 was transfected into MCF-7 (B) and JEG-3 cells (C) with RAR{alpha} expression vector pcDNA3.1-RAR{alpha} or pcDNA3.1 empty vector. Renilla luciferase activity of pRL-1xDR2 without tRA or RAR{alpha} was set at 1 in each experiment. D, Activity of putative RAREs with DR-5-like sequence from 3' flanking region of the human NIS gene in MCF-7 cells. Renilla luciferase activity of phRGB without tRA was set at 1. In B–D, cells were treated with or without 10–6 M tRA for 12 h after the 48-h transfection with Effectene. The indicated Renilla luciferase constructs were cotransfected with β-galactosidase reporter vector, pSV-β-galactosidase control (Promega), to normalize transfection efficiency. Values are the mean ± SD (n = 3). *, P < 0.02, when compared with the group without tRA treatment in each vector combination.

 
Transfection

Plasmids were transfected into MCF-7 cells or JEG-3 cells with Effectene (QIAGEN, Valencia, CA) reagent as described (19).

Iodide uptake

The iodide uptake assay was performed as described (14) with 20 mCi/mmol Na125I. The iodide uptake was normalized to the cellular protein content measured in the same cells with Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).

Reverse transcription and quantitative real-time PCR (RT-qPCR) analysis

RT-qPCR of NIS was carried out as described (14). Standard curves representing 6- or 7-point serial dilution of mixed cDNA of the corresponding control group, MCF-7 cells treated with 10–6 M tRA for 12 h, were analyzed in each assay and used for calculation of relative expression values. The sample quantifications were normalized by the internal control glyseraldehyde-3-phosphate dehydrogenase mRNA.

Flow cytometric analysis

Cell cycle distribution was determined by staining cells with propidium iodide, as described (21).

Statistical analysis

Statistical comparison was performed using StatView 5.0 software (Abacus Concept, Berkeley, CA) with significance at P < 0.05. Comparison between groups was determined by conducting a paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Response of putative human NIS gene RAREs to tRA in MCF-7 cells

tRA up-regulates NIS expression in MCF-7 cells, in part, at the transcriptional level (12). To determine whether tRA up-regulation of the NIS gene in MCF-7 breast cancer cells is mediated through RAREs, we inspected the sequence of the human NIS gene from –14,254 to +29,500 (numbering with the A in the ATG of NIS open reading frame is +1) (Fig. 1AGo) for putative RAREs. Based on half-sites sequence and configuration, we identified 12 candidate elements, 10 DR-2, one DR-5, and one everted repeat with a space of eight bases (Fig. 1AGo and supplemental Table 2).

The putative RAREs were evaluated for function in luciferase reporter vectors containing segments from –9847 to –1578 (4, 20) as well as the first to seventh introns (Fig. 1BGo) and transfected into MCF-7 cells. The NIS gene segments tested included eight putative RAREs by sequence inspection. No tested segment responded to tRA (Fig. 1BGo), even with the addition of exogenous RAR{alpha} expression (data not shown).

Interestingly, seven identical DR-2 element sequences with typical half-sites, AGGTCAggAGTTCA, were identified between the first and 13th intron (supplemental Table 2). The first intron contained two of the DR-2 elements; however, this sequence did not confer tRA responsiveness to the heterologous Simian virus 40 promoter or the homologous NIS proximal promoter in MCF-7 cells (Fig. 1BGo). Elements with the consensus DR-2 (AGGTCAaaAGGTCA) sequence responded to tRA in JEG-3 choriocarcinoma cells with the addition of exogenous RAR{alpha} (Fig. 2BGo) but not in MCF-7 cells (Fig. 2CGo). In contrast, the consensus DR-5 from the RARβ promoter (GGTTCAccgaaAGTTCA) responded to the tRA in both MCF-7 cells (Fig. 2CGo) and JEG-3 cells (Fig. 2BGo), even without the addition of exogenous RAR{alpha}. These data indicate that in MCF-7 cells, the DR-5 RARE has greater tRA responsiveness than the DR-2.

The sequences flanking the NIS gene were inspected for the DR-5 motif including, the adjacent genes (RPL18A, NM_000980 and CCDC124, NM_138442, see Fig. 1AGo). No consensus DR-5 element was identified, but two DR-5-like sequences, TGTACAgtctaGGGACT (+28060 to +28076) and GAGACAaggaaAGTTCA (+36699 to +36715), consisting of half-sites with 0 or 1 base mismatch from the consensus half-sites, were found in the 3' flanking region. None of these elements, fused to Renilla luciferase reporter gene (Fig. 2AGo), however, enhanced the NIS promoter activity in MCF-7 cells in response to tRA (Fig. 2DGo).

These studies demonstrate that there is no functional RARE with consensus half-sites or variations of this sequence, in the human NIS gene locus. tRA has been reported to transcriptionally induce a homeobox protein, Nxk-2.5, and Nkx-2.5 increases the NIS gene expression in MCF-7 cells (22). The protein synthesis inhibitor puromycin, however, does not completely inhibit the induction of NIS mRNA by tRA (Kogai, T., and G. A. Brent, unpublished data), suggesting that endogenous de novo protein synthesis is not necessary for the NIS induction. We therefore hypothesized that additional signal pathway(s), likely nongenomic action(s) rather than the Nkx-2.5 or genomic action through RARE, is required for the full induction of NIS by tRA.

Effects of receptor tyrosine kinase inhibitors

We studied the influence of a number of signal transduction inhibitors on tRA induction of NIS in MCF-7 cells. We performed iodide uptake assay in MCF-7 cells treated with 1 µM tRA with or without the addition of inhibitors at conventional doses. The inhibitors that reduced iodide uptake were further tested to determine the level of NIS mRNA by RT-qPCR after treatment with inhibitors (Table 1Go). The dose response of effective inhibitors was also determined to assess the sensitivity of iodide uptake in tRA-treated MCF-7 cells. The response to an inhibitor in MCF-7 cells was compared with NIS expression with and without inhibitors in TSH-stimulated FRTL-5 rat thyroid cells (Fig. 3Go).


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  		TABLE 1. Effects of signal transduction inhibitors on the NIS expression in MCF-7 cells

 

Figure 3
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  		FIG. 3. Dose-response curves of signal transduction inhibitors on tRA-induced iodide uptake in MCF-7 cells and TSH-induced iodide uptake in FRTL-5 cells. Cells grown in 24-well plates were treated with the indicated inhibitors, as well as 10–6 M tRA in MCF-7 cells or 1 mU/ml bovine TSH in FRTL-5 cells, for 48 h. Because the induction of NIS gene by tRA is not affected by the presence of serum (14 ), MCF-7 cells were treated in the presence of 10% fetal bovine serum and 0.1% dimethyl sulfoxide vehicle. FRTL-5 cells were grown under conditions without TSH for 6–7 d as described (2 ), and TSH and/or an inhibitor was added with 0.1% dimethyl sulfoxide vehicle. Data are shown as percent of control (iodide uptake without inhibitor set at 100%). Values are the mean ± SD (n = 3).

 
IGF-I and IGF-II stimulation induces iodide uptake and NIS mRNA expression in MCF-7 cells (17). An inhibitor of IGF-I receptor, AG1024 (10 µM), as well as a pan-tyrosine kinase inhibitor, genistein (100 µM), markedly decreased the tRA-induced iodide uptake and NIS mRNA in MCF-7 cells (Table 1Go). The effect of AG1024 was dose dependent, with an IC50 of 3.87 ± 0.35 µM (Fig. 3Go). Although genistein inhibits a wide range of tyrosine kinases, no significant reduction of iodide uptake was found with inhibitors of epidermal growth factor (EGF) receptor (AG1478) or platelet-derived growth factor receptor (AG1296) (Table 1Go).

IGF-I significantly decreased the TSH-induced iodide uptake and NIS expression in FRTL-5 cells (23, 24). The IGF-I receptor antagonist, AG1024, modestly (~40%) decreased the iodide uptake in FRTL-5 cells only at a high concentration (10 µM), whereas 5 µM of AG1024 markedly (~80%) reduced the uptake in MCF-7 cells (Fig. 3Go). A significant reduction of cell viability in response to high concentration of AG1024 (more than 3 µM) was observed in FRTL-5 cells but not MCF-7 cells, suggesting that some of the inhibitory effect on endogenous gene expression in FRTL-5 cells was due to cytotoxicity. The differential effects of the IGF-I receptor antagonist AG1024 on MCF-7 cells and FRTL-5 cells are consistent with the differential regulation of NIS by IGF-I (17, 24).

Inhibitors of receptor-associated tyrosine kinases

Prolactin is required for the full induction of NIS by oxytocin in lactating mammary glands (7). Prolactin signaling is mediated by a receptor-associated tyrosine kinases, Janus kinase (JAK)-2 and src (25). An inhibitor of JAK (AG490), but not a src inhibitor [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine], abolished the tRA-induced iodide uptake and NIS mRNA expression in MCF-7 cells (Table 1Go) with an IC50 of 19.9 ± 1.9 µM (Fig. 3Go). Another JAK selective inhibitor, pyridone 6 (30 µM), also significantly inhibited the iodide uptake in MCF-7 cells (Table 1Go). In contrast, the effects of AG490 on TSH-induced iodide uptake in FRTL-5 cells were relatively modest (Fig. 3Go). These data suggest that the JAK signaling is more relevant to NIS expression in MCF-7 cells than that in FRTL-5 cells.

Inhibitors for the PI3K/Akt pathway

IGF-I receptor stimulation leads to the activation of PI3K via insulin receptor substrate-1, in breast cancer cells (26). tRA-induced growth arrest in MCF-7 cells involves the selective regulation of the insulin receptor substrate-1/PI3K/Akt pathway (27). Overexpression of PI3K has been reported to enhance the iodide uptake significantly in MCF-7 cells (18). The tRA-induced iodide uptake and NIS mRNA were significantly inhibited by a PI3K inhibitor, LY294002, and the Akt inhibitor VIII (Calbiochem, La Jolla, CA) (Table 1Go). LY294002 inhibited the iodide uptake in MCF-7 cells in a dose-dependent fashion with an IC50 of 7.87 ± 1.1 µM (Fig. 3Go) in the range of previously reported values for in vivo inhibition (28). The inhibition of tRA-induced iodide uptake by LY294002 was also observed in the absence of serum (data not shown). Although an off-target effect of LY294002 on casein kinase 2 has been reported (29), a casein kinase 2 inhibitor, 4,5,6,7-tetrabromobenzotriazole, did not significantly inhibit the tRA-induced iodide uptake (Table 1Go), suggesting that the effect of LY294002 on the NIS expression is most likely through PI3K. tRA stimulates mammalian target of rapamycin through the PI3K/Akt activation in acute promyelocytic leukemia cells (30); however, rapamycin did not significantly affect the tRA stimulation of iodide uptake in MCF-7 cells (Table 1Go).

The effect of LY294002 on the TSH-induced iodide uptake in FRTL-5 cells was opposite of the tRA-induced uptake in MCF-7 cells; the iodide uptake was increased in a dose-dependent fashion more than 2-fold at 30 µM concentration of LY294002 (Fig. 3Go). This finding is consistent with a previous report, demonstrating that LY294002 prevents IGF-I inhibition of TSH-induced NIS mRNA and protein expression (24).

Inhibitors for MAPK

In thyroid cells, TSH and cAMP activate a MAPK pathway, MAPK kinase-3/6-p38 MAPK. A p38 inhibitor, SB203580, significantly decreases the TSH-induced NIS mRNA expression in FRTL-5 rat thyroid cells (31). Retinoic acid stimulates the phosphorylation of p38 in MCF-7 cells (32). We therefore tested the p38 inhibitors, ML3403 and SB203580, in both MCF-7 and FRTL-5 cells. Treatment with ML3403 (10 µM) as well as SB203580 (10 µM) significantly reduced the tRA-induced iodide uptake in MCF-7 cells (Table 1Go). ML3403 inhibited NIS in a dose-dependent fashion with an IC50 of 4.37 ± 0.12 µM (Fig. 3Go), in the range of previously reported concentrations (33). Removal of serum did not significantly affect the inhibitory effect of SB203580 on the tRA-induced iodide uptake (data not shown). The tRA-induced NIS mRNA expression in MCF-7 cells was significantly inhibited by 10 µM of ML3403 (Table 1Go). ML3403 also decreased the TSH-induced iodide uptake in FRTL-5 cells with a similar IC50 (1.50 ± 0.13 µM) as that in MCF-7 cells (Fig. 3Go). These data indicated that p38 activity is required for the NIS expression in both breast cancer and thyroid cells.

TSH activates another MAPK cascade, the MEK1/2-ERK1/2 MAPK pathway in FRTL-5 cells (34). A MEK1/2 inhibitor, PD98059, reduces the NUE activity (4). In MCF-7 cells, 10 µM of PD98059, however, did not significantly reduce the tRA-induced iodide uptake (Table 1Go), suggesting differential regulation of NIS by the MEK/ERK cascade between breast cancer and thyroid cells. Interestingly, SP600125 (30 µM), an inhibitor of another MAPK, c-Jun N-terminal kinase (JNK), significantly reduced the tRA-induced iodide uptake in MCF-7 cells, whereas no significant reduction was observed in NIS mRNA expression (Table 1Go). A target of SP600125, most likely JNK, may regulate the NIS at the posttranslational level.

Inhibitors to PKA and protein kinase C (PKC)

In thyroid cells, cAMP accumulation by TSH activates the NUE through partially PKA-dependent pathway(s), leading to the thyroid-selective maximum induction of NIS gene (3, 4). A PKA inhibitor, H89 (10 µM), however, did not significantly reduce the tRA-induced iodide uptake in MCF-7 cells (Table 1Go), consistent with the differential regulation of NIS by TSH in thyroid cells and breast tissue.

A novel PKC, PKC{delta}, can be activated by tRA in MCF-7 cells (35). We therefore evaluated whether inhibitors of PKC, bisindolylmaleimide I, an inhibitor of conventional PKCs, PKC{alpha} and PKCβ, and rottlerin, an inhibitor of PKC{delta} and the calmodulin-dependent protein kinase III (CaMK-III), affected the tRA-induced NIS expression in MCF-7 cells. Treatment with rottlerin (30 µM), but not bisindolylmaleimide I (5 µM), significantly decreased the tRA-induced iodide uptake and NIS mRNA in MCF-7 cells (Table 1Go). Rottlerin showed a dose-dependent response with an IC50 of 0.89 ± 0.01 µM. Although cross talk between TSH signaling and PKC{delta} or CaMK-III has not been reported, rottlerin significantly inhibited the TSH-induced iodide uptake in FRTL-5 cells with the IC50 of 4.87 ± 0.02 µM (Fig. 3Go). Knockdown of putative targets of rottlerin, PKC{delta} or CaMK-III, by small interfering RNA, however, did not significantly reduce the NIS mRNA expression in MCF-7 cells (Kogai, T., and G. A. Brent, unpublished observation), suggesting an off-target effect of rottlerin on the NIS expression. An in vitro study has described many other kinases inhibited by rottlerin (29).

Effects of signal transduction inhibitors on basal NIS mRNA expression

To investigate whether the effective inhibitors against the tRA-induced NIS expression affect the induction of NIS by tRA or the basal NIS expression, we performed RT-qPCR of NIS in MCF-7 cells treated with these inhibitors, but without tRA, for 24 h. Treatment with AG490, ML3403, and rottlerin, significantly reduced the basal NIS expression in MCF-7 cells, whereas AG1024 and LY294002 did not significantly affect the NIS expression (Fig. 4Go). These data indicated that the inhibition by AG1024 and LY294002, most likely the IGF-I receptor/PI3K pathway, did not change the basal NIS expression. The IGF-I receptor/PI3K pathway, therefore, is likely involved in the induction of NIS gene by tRA in MCF-7 cells.


Figure 4
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  		FIG. 4. Effects of signal transduction inhibitors on the basal NIS mRNA expression in MCF-7 cells. Cells were treated with 100 µM genistein, 10 µM AG1024, 100 µM AG490, 10 µM LY294002, 10 µM ML3403, or 1 µM tRA for 12 h, and RT-qPCR of NIS was performed. A, Results of RT-qPCR in MCF-7 cells treated with the indicated agents. Values are means ± SD (n = 2). *, P < 0.01 when compared with the group without inhibitor. One of three representative data is shown. B, The standard graph of C(t) (threshold cycle) values, obtained from serial dilutions of the cDNA from tRA-treated MCF-7 (corresponding to data in A). The C(t) values in the exponential phase was between 27.4 and 36.6. Although the quantity of NIS mRNA in MCF-7 cells without tRA treatment was relatively low, the C(t) values of each sample with inhibitors (C(t) = 30.4–34.2) were in the exponential phase range.

 
Effects of signal transduction inhibitors on the growth of MCF-7 cells

Although chronic tRA treatment inhibits cell proliferation in MCF-7 cells (11), activation of signaling pathways that are used for the induction of NIS by tRA, such as the IGF receptor/PI3K pathway, stimulate cell growth (36). To address these findings, we analyzed cell cycle phase distribution in MCF-7 cells treated with tRA and/or the inhibitors that significantly reduced NIS expression. Although long-term (more than 48 h) treatment with tRA decreased the S-phase distribution in MCF-7 cells (11), the short-term (24 h) treatment, which is sufficient for the NIS induction, did not significantly affect the cell cycle distribution (supplemental Table 3). In contrast, all tested inhibitors, AG1024 (10 µM), AG490 (100 µM), LY294002 (10 µM), and ML3403 (10 µM), significantly decreased the S-phase distribution within 24 h, in both conditions with and without tRA (supplemental Table 3). These data indicate that longer duration is required for cell growth inhibition with tRA, compared with the signal transduction inhibitors. Durations of tRA treatment for the NIS induction and cell growth inhibition are also different. Rapid activation of the PI3K pathway by tRA, followed by a modest reduction, has been reported in another cancer cell line (37).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of signal transduction pathways in NIS induction has been suggested by several studies in thyroid and breast cancer (1). A functional RARE from the sequences around the NIS gene could not be identified; therefore, we investigated the possibility that tRA was acting through a nontraditional pathway. Our experiments with signal transduction inhibitors indicated that activities of IGF-I receptor, PI3K, JAK, and p38 MAPK are important for tRA-induced iodide uptake and NIS mRNA expression in the MCF-7 cells. Because the inhibitors of IGF-I receptor and PI3K do not significantly inhibit the basal NIS mRNA expression in MCF-7 cells, the activity of IGF-I receptor/PI3K pathway is likely required for the induction of NIS by tRA.

Based on our studies comparing breast cancer (MCF-7) and thyroid (FRTL-5) cells, we concluded that at least two pathways, IGF-I receptor/PI3K and p38 MAPK, are used for NIS regulation in both thyroid and breast cancer, whereas the MEK/ERK and PKA/cAMP-response element binding protein are selective for thyroid NIS expression, and JAK is selective for the NIS expression in breast cancer (Fig. 5Go). The IGF-I receptor/PI3K signaling pathway is associated with the NIS regulation in both breast cancer cells and thyroid cells; however, the effects are opposite: a stimulatory effect in breast cancer and an inhibitory effect in thyroid cells (Fig. 5Go).


Figure 5
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  		FIG. 5. A model of the mechanism of retinoic acid stimulation of NIS expression by modifying signal transduction pathways in breast cancer and thyroid cells, based on the results of the inhibitor studies. The IGF-I receptor/PI3K pathway is likely responsible for the tRA-induction of NIS through formation of RAR/RXR heterodimers. The p38 MAPK cascade is required for NIS expression in both cells types. Pax8, Paired box protein-8; CREB, cAMP-responsive element binding protein; Rap1, Ras-related protein-1; MKK, MAPK kinase; IRS1, insulin receptor substrate-1; RA, retinoic acid; Rac1, ras-related C3 botulinum toxin substrate 1.

 
Treatment of MCF-7 cells with insulin, IGF-I, and IGF-II significantly induces iodide uptake and NIS mRNA expression (17). tRA can directly bind to the IGF-II receptor with a high affinity to modulate cell proliferation (38). In this model, natural retinoids, but not synthetic RAR agonists, such as 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid, stimulate the IGF-II receptor signaling (39). Our previous data indicated that RAR-selective agonists, including TTNPB, induce the NIS expression in MCF-7 cells (12, 14). In addition, our preliminary experiments with small interfering RNA to the IGF-II receptor did not significantly reduce the NIS expression (Kogai, T., and G. A. Brent, unpublished data). These observations suggest that RAR, but not IGF-II receptor, mediates retinoic acid signaling to the NIS gene.

tRA stimulates the phosphorylation of p38 in MCF-7 cells through the activation of ras-related C3 botulinum toxin substrate 1 (32). Inhibition of p38 with ML3403 significantly reduced both the tRA-induced and basal NIS mRNA expressions in MCF-7 cells. The magnitude of inhibitory effects on the tRA-induced NIS mRNA expression and the basal NIS mRNA expression was equivalent (79.9 and 76.8% reduction, respectively). The p38 inhibition, therefore, does not likely affect the tRA inducibility of NIS in MCF-7 cells. Although tRA significantly induces the NIS expression in 4–6 h (12, 14), the activation of p38 by tRA is relatively slow, 12–24 h (32).

Although EGF receptor is an effective target for chemoprevention of HER/neu-positive breast cancer, some HER/neu-negative cancer cells, such as MCF-7, are resistant to EGF receptor antagonists (40). NIS induction by retinoid, followed by radioiodide treatment, therefore, has a potential as a therapeutic choice for such cancer.

Recently, agents targeting signal transduction pathways have been used to inhibit cell proliferation and metastasis in treatment of cancer. The selective induction of NIS expression in breast cancer provides the potential for targeted radioiodide therapy (10). tRA induces NIS expression selectively in breast cancer tissue in rodent models (13). The expression status of RAR isoforms during RA treatment is similar in both in vitro and in vivo models of breast cancer (13). The retinoid-stimulated NIS induction pathways identified by our in vitro inhibitor studies, therefore, are likely to reflect in vivo regulation. The effects of these inhibitors on NIS induction, however, should be confirmed using in vivo breast cancer models. The elucidation of retinoic acid effects on signal transduction pathways mediating NIS induction may lead to more selective and effective induction of NIS in some breast cancer. It may also be possible to combine NIS-induction therapy with treatments targeted to induce apoptosis and inhibit cell proliferation with signal transduction inhibitors.


   Acknowledgments
 
We thank Ken Marion and Drs. Michael Fenton and Neil Tran for providing thyroid cells, Lisa Che and Andrew Li for technical assistance, and Dr. Jerome Hershman for helpful discussions.


   Footnotes
 
This work was supported by National Institutes of Health Grant RO1 CA089364 and Veterans Affairs merit review funds (to G.A.B.).

Disclosure Information: All authors have nothing to declare.

First Published Online March 4, 2008

Abbreviations: CaMK-III, Calmodulin-dependent protein kinase III; DR, direct repeat; EGF, epidermal growth factor; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; NIS, sodium/iodide symporter; NUE, NIS upstream enhancer; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; RAR, retinoic acid receptor; RARE, retinoic acid response element; RT-qPCR, reverse transcription and quantitative real-time PCR; RXR, retinoid-X receptor; tRA, trans retinoic acid.

Received July 23, 2007.

Accepted February 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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