Regulation of Sodium Iodide Symporter Gene Expression by Rac1/p38β Mitogen-activated Protein Kinase Signaling Pathway in MCF-7 Breast Cancer Cells*

Background: Induction of the iodide transporter in cancer cells confers targeted cytotoxicity with radioiodide. Results: Isoforms of p38 MAPK were identified that specifically promote iodide uptake in breast cancer cells. Conclusion: p38 isoform-specific stimulation may induce iodide uptake sufficient for radioiodide therapy in breast cancer. Significance: Study of p38 isoform-specific signaling improves understanding of cancer cell differentiation and identifies novel therapeutic targets. Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide 131I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of 131I concentration and treatment of breast cancer.

ity of p38 requires dual phosphorylation of a conserved motif, Thr-Gly-Tyr. Because p38 is activated in many types of cancer, including breast cancer and thyroid cancer, p38 has been proposed as a therapeutic target to modulate cell growth and differentiation (1). A range of stimuli, including cytokines, growth factors, hormones, and cell stress, stimulate p38 activity through selective MAPK kinases (MKKs), MKK3 and/or MKK6. Activation of p38 by small GTPases, such as Rac1 and Rho, contributes to cell differentiation and transformation.
The sodium iodide symporter (NIS, 3 or solute carrier family 5, member 5 (SLC5A5)) is expressed predominantly in the thyroid gland and lactating breast and mediates accumulation of iodide from the blood stream to these tissues (2). In the majority of differentiated thyroid cancer, after stimulation with high levels of thyroid-stimulating hormone (TSH), NIS is induced sufficiently to ablate residual tumor with ␤-emitting radioiodide-131 ( 131 I). Recent studies of NIS gene therapy have demonstrated that NIS expression sufficient for tumor shrinkage with 131 I can be achieved in several types of non-thyroidal cancer (3)(4)(5)(6).
Approximately 70% of breast cancer expresses endogenous NIS (7,8) and has been considered as a potential target of radioiodide therapy for breast cancer (9,10). The native expression level of NIS, however, is insufficient to deliver an effective dose of 131 I. Extensive experience with 131 I therapy in thyroid cancer indicates the importance of maximizing the magnitude of iodide uptake specifically in the tumor.
All-trans-retinoic acid (tRA) is the most potent NIS inducer in breast cancer models, including MCF-7 cells (11)(12)(13)(14)(15). NIS expression sufficient for iodide uptake and cytotoxicity with 131 I has been demonstrated in several in vitro breast cancer models (11,14). Our study with in vivo breast cancer models has shown that iodide uptake can be achieved but that a large dose of tRA is required. Elucidation of signaling pathways involved in the NIS induction by tRA may lead to more effective induction of NIS in some breast cancer.
The induction of NIS by tRA is primarily mediated by the heterodimer of retinoic acid receptor (RAR)-␤ and retinoid X receptor (RXR)-␣ (14,16). tRA-stimulated RAR-RXR has been shown to up-regulate NIS expression in MCF-7 cells (16,17). Although RAR-RXR can act as a transcription factor and directly stimulate gene expression, it can also activate signaling pathways, such as phosphoinositide 3-kinase and p38 (16,18).
NIS gene expression is differentially regulated in thyroid and breast tissues. In thyroid cells, stimulation with TSH, followed by cAMP accumulation, is critical for NIS expression (15,19). In contrast, cAMP does not influence NIS expression in breast cancer cells (11). tRA, the NIS inducer in breast cancer cells, reduces NIS expression in FRTL-5 rat thyroid cells (11,20). Stimulation of p38 MAPK activity is required for TSH-induced NIS expression in thyroid cells (21) as well as tRA-induced NIS expression in MCF-7 cells (17). The Rac1-p38 pathway is upregulated by TSH-cAMP stimulation in thyroid cells (21) and by tRA treatment in MCF-7 breast cancer cells (18). We hypothesized that the Rac1-p38 pathway was a common pathway for NIS induction in breast cancer cells and thyroid cells.
Four isoforms of p38, ␣, ␤, ␥, and ␦, have been reported in mammalian cells. There are isoform differences in tissue distribution and substrate specificity (22)(23)(24), which may confer differential activation of downstream signaling pathways. We therefore determined the role of each p38 isoform in the regulation of NIS in MCF-7 breast cancer cells and compared it with the role in FRTL-5 rat thyroid cells. We identified a p38 MAPK cascade that selectively regulates NIS expression in MCF-7 cells. The effect of tRA treatment on the NIS-selective p38 signal transduction pathway was also investigated.
Cell Culture-MCF-7 cells (lot F15100 and 205623 from the ATTC, Manassas, VA), MBA-MD-231 cells, and FRTL-5 cells were maintained as described previously (11,25,26). BT-474 cells were grown in Dulbecco's modified Eagle's medium (ATCC) with 10% fetal bovine serum (ATCC). When cells were treated with tRA and/or signal transduction inhibitors, they were maintained with 0.1% DMSO vehicle and fed with fresh media with the agents every 24 h.
Transfection of siRNA-Reverse transfection of RNAi duplex was performed with Lipofectamine RNAiMAX (Invitrogen), as recommended. Briefly, MCF-7 cells (2 ϫ 10 5 cells/well), digested with 2.5 g/liter trypsin (Invitrogen), were incubated with RNAi duplex-Lipofectamine RNAiMAX complexes, containing 20 nmol of SMARTpool siRNA (Dharmacon, Lafayette, CO) or Stealth RNAi (Invitrogen), in 6-well plates for 48 h. Cells were then fed with fresh media and treated with or without tRA for 12 h starting 60 h after the beginning of transfection.
Transfection of Plasmid-In transient expression experiments, 2 g of plasmid was transfected into ϳ10 6 MCF-7 cells with the Nucleofector system (Lonza, Gaithersburg, MD), as recommended by the manufacturer. For stable expression experiments, the transfected cells were selected in 400 mg/liter G418 (Invitrogen) for 3-4 weeks.
Iodide Uptake-The iodide uptake assay was performed as described previously (27) with minor modifications. Briefly, cells were grown in 12-well dishes, washed with Hanks' balanced salt solution, and incubated for 1 h at 37°C with 500 l of Hanks' balanced salt solution containing ϳ0.1 Ci of carrierfree Na 125 I (MP Biomedicals, Solon, OH) and 10 M NaI. The specific activity under these conditions was 20 mCi/mmol. In some experiments, 30 M KClO 4 , the specific inhibitor of NIS, was added to the Hanks' balanced salt solution to measure iodide uptake independent of NIS. After incubation, the cells were washed once with ice-cold PBS and scraped from each well, and radioactivity was measured in a ␥-counter. Cell number was determined by counting in a hemocytometer. The radioactivity was normalized to the cell number at the time of the assay. In the signal transduction inhibitor dose-response experiments, iodide uptake was normalized to the amount of cellular protein. Cells were incubated with Na 125 I and then lysed with the addition of 200 l of passive lysis buffer (Promega) to each well. The radioactivity in the cell lysate was determined by a ␥-counter, and the protein content was determined by the Bio-Rad protein assay (Bio-Rad).
Analysis of mRNA Expression-Two-step quantitative RT-PCR was performed, as described previously (14,26). Primers for human NIS, GAPDH, and rat Gapdh were designed, as described (14,26). Quantitative PCR of isoforms of human and rat p38 cDNA was performed with the QuantiTect primer assay (Qiagen). Quantitative RT-PCR of human MKK3A (NM_ 002756) and MKK3B (NM_145109) was carried out with custom primers (Invitrogen), 5Ј-GTTCTCAGTTGGCCCGT-GTG/5Ј-CCAAGTCATCAGCCTCCACCTC and 5Ј-CCA-CTTGCAGCATGGAGTCG/5Ј-CCACTTGCAGCATGGA-GTCG, respectively. Standard curves representing 6-point serial dilution of the corresponding control group were analyzed in each assay and used for calculation of relative expression values. The sample quantifications were normalized by the internal control GAPDH mRNA.
Western Blot Analysis-Cells grown in 10-cm Petri dishes were rinsed, scraped with PBS containing phosphatase inhibitor mixture 2 (Sigma), spun down by centrifugation, and lysed in cell lysis buffer (Sigma), containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor mixture (Sigma), and phosphatase inhibitor mixture 2 (Sigma). The cell extracts were clarified by centrifugation, and protein concentrations were determined by using a protein assay reagent (Bio-Rad). In some experiments, cells grown in 6-well plates were directly lysed in a culture dish with SDS sample buffer, containing 62.5 mM, Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromphenol blue, protease inhibitor mixture (Sigma), and phosphatase inhibitor mixture 2 (Sigma). Aliquots of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by using standard procedures. The membranes were then subjected to Western blotting, and the blots were developed with the advanced ECL Western blot detection kit (GE Healthcare).
Immunoprecipitation-Exogenous FLAG-tagged p38␤ expressed in MCF-7 cells was immunoprecipitated with the FLAG immunoprecipitation kit (Sigma), as recommended. Briefly, clarified cell lysate in the cell lysis buffer with protease inhibitor mixture (Sigma) and phosphatase inhibitor mixture 2 (Sigma) was incubated with anti-FLAG M2 agarose gel (Sigma) at 4°C for 16 h. The fraction eluted with SDS sample buffer (Sigma) and the cell lysate before the immunoprecipitation were applied to Western blot analysis.
Statistical Analysis-Unless otherwise noted, statistical significance, at a p value of Ͻ0.05, was determined by conducting a paired Student's t test. Mann-Whitney U test was performed for data that were not normally distributed.  (Fig. 1B), however, did not significantly influence tRA-stimulated iodide uptake (Fig. 1A). The p38 inhibitors, SB203580 and SB239063, inhibited iodide uptake in a dose-dependent fashion (Fig. 1C), although the IC 50 value of these inhibitors (5.32 Ϯ 0.72 and 7.73 Ϯ 0.69 M, respectively), was higher than the usual reported range in cell culture (Ͻ1 M) (28). The marked tRA induction of NIS mRNA was significantly reduced by the addition of the p38 inhibitors, SB203580 (30 M) and SB239063 (30 M) (Fig. 1D), consistent with the effects of these inhibitors on iodide uptake. The p38 inhibitors utilized for these studies target p38 ␣ and ␤ but not ␥ and ␦ (29 -31). These data indicate that p38 ␣ and/or ␤ play a major role in mediating tRA induction of NIS expression and iodide uptake in MCF-7 cells.

Effects of MAPK Cascade Inhibitors on Iodide Uptake in
Differential Effects of p38 Inhibitors on Iodide Uptake in MCF-7 Cells and FRTL-5 Cells-TSH receptor/cAMP signaling, critical for the maintenance of NIS expression in thyroid cells, up-regulates the p38 MAPK cascade (15,21). Inhibition of p38, by SB203580 and ML3403, significantly reduced NIS expression in FRTL-5 cells (17,21). Because the IC 50 for p38 inhibition of iodide uptake in MCF-7 cells was relatively high, we compared the kinetics of the inhibitory effect of SB203580 in MCF-7 cells with that in FRTL-5 rat thyroid cells. The IC 50 in FRTL-5 cells, 0.65 Ϯ 0.01 M, was significantly lower than that in MCF-7 cells, 5.17 Ϯ 0.12 M (Fig. 2).
The IC 50 of SB203580 required to inhibit p38␣ activity was more than 10 times lower than that reported for p38␤ inhibition (29). We therefore determined if the difference in IC 50 for p38␣ inhibition between MCF-7 and FRTL-5 cells was due to the differential expression of p38 isoforms. We evaluated the endogenous expression profile of p38 isoforms in these cell lines by quantitative RT-PCR. In MCF-7 cells, all p38 isoforms were abundantly expressed with a relative ranking of ␣ Ͼ ␦ Ͼ  Table 1), consistent with a previous report (24). In FRTL-5 cells, there was abundant expression of p38␣ and -␦, modest expression of p38␥, and undetectable p38␤ mRNA ( Table 1). The expression of predominantly p38␣ and -␦ isoforms in FRTL5 cells is consistent with the pattern of expression reported in rat thyroid tissue (32). These data indicate that p38␣ is most likely essential for NIS expression in FRTL-5 cells.

p38␤ Regulates Iodide Transporter in Breast Cancer Cells
Effects of Selective siRNA Knockdown of p38 Isoforms on the NIS mRNA Expression in MCF-7 Cells-Our analysis with pharmacologic inhibitors indicate the importance of p38␣ and/or p38␤ for NIS expression in MCF-7 cells. To determine which isoform of p38 is critical, we utilized siRNA to knock down the isoforms in MCF-7 cells. The transfection of p38␤specific siRNA, but not p38␣ siRNA, significantly decreased tRA-induced NIS mRNA expression in MCF-7 cells (ϳ60% reduction compared with non-targeting siRNA; Fig. 3A). The siRNA knockdown of p38␤ also modestly decreased (ϳ24% reduction) the basal NIS expression before tRA treatment (Fig.  3A). Although the p38␤ protein sequence has ϳ70% homology with p38␣ (22), the isoform-selective siRNAs reduced expression of only the targeted isoform (Fig. 3B). These data demonstrate that p38␤, but not p38␣, is required for full expression of NIS in MCF-7 cells.
Overexpression of p38␤ Enhances NIS Expression in MCF-7 Cells-To investigate if a gain of p38␤ function influences NIS expression, we established a stable transfectant of FLAGtagged p38␤ in MCF-7 cells with G418 as a selection marker (Fig. 4A). The constitutive expression of p38␤ consistently increased the NIS mRNA expression in MCF-7 cells, both with and without tRA treatment (ϳ1.9-and ϳ1.4-fold, respectively), although the induction without tRA was not statistically significant (Fig. 4B). The sensitivity to tRA for the induction of iodide uptake was significantly enhanced by overexpression of p38␤; the EC 50 of tRA was 9.58 ϫ 10 Ϫ8 M with pCMV6-p38␤, whereas that with empty vector was 4.34 ϫ 10 Ϫ7 M (Fig. 4C). Iodide uptake in the p38␤-overexpressing cells, after treatment with 10 Ϫ7 M tRA, was significantly higher than that in cells transfected with the empty vector (Fig. 4D).

Rac1 Regulates NIS Expression in MCF-7 Cells-
The activity of the p38 MAPK cascade is regulated by tRA through Rac1 (18). To investigate if Rac1 plays an important role in NIS expression in MCF-7 cells, we added a pharmacological Rac1 inhibitor, NSC23766. tRA-induced iodide uptake was significantly decreased by NSC23766 (ϳ66% reduction compared with tRA only), and the inhibitory effect was enhanced by the addition of the p38 inhibitor SB203580 (Fig. 5A). Induction of NIS mRNA by tRA was also significantly decreased by the Rac1 inhibitor NSC23766 (Fig. 5B) at every time point tested (76% reduction after 12 h of treatment with tRA). A constitutively active mutant of Rac1, Rac1 G12V, stably transfected in MCF-7 cells (Fig. 5C), significantly increased the tRA-induced NIS mRNA expression (3.4-fold compared with empty vector; Fig.  5D). The sensitivity of iodide uptake induction to tRA was significantly increased by Rac1 G12V. The EC 50 of tRA (3.03 ϫ 10 Ϫ7 M) was significantly decreased to 9.57 ϫ 10 Ϫ8 M by the Rac1 activation (Fig. 5E). The iodide uptake in Rac1-overexpressing cells, treated with 10 Ϫ7 M tRA, was significantly higher than that in cells transfected with the empty vector (Fig. 5F). These data indicate a critical role of Rac1 in the tRA-induced NIS expression in MCF-7 cells.     Fig. 6A). NIS mRNA expression in MCF-7 cells without tRA treatment was also reduced by MKK3 siRNA (ϳ38% reduction), but the change was not significant, consistent with the p38␤ siRNA (Fig. 3).
Two variants of MKK3, MKK3A and -B, have been reported (33). MKK3B is an alternatively spliced form of MKK3A with an additional 29 amino acids fused to the N-terminal end of MKK3A. MKK3A selectively activates p38␣, whereas MKK3B activates both p38␣ and -␤ (33). The siRNA used in our knockdown studies targeted both MKK3A and -B. Our quantitative RT-PCR indicated abundant expression of MKK3B as well as modest expression of MKK3A in MCF-7 cells, both with and without tRA treatment ( Table 2). These data indicate that among variants of MKK3 and MKK6, the MKK3B is most important for NIS expression in MCF-7 cells.
tRA Induces Phosphorylation of p38␤ in MCF-7 Cells-tRA induces the phosphorylation of p38 in MCF-7 cells (18). The commercially available anti-phospho-p38 Abs recognize the common activation site motif contained in all four p38 isoforms. Previous studies of phosphorylation of p38, therefore, have not identified specific isoforms. We determined that Western blot of cell extracts prepared from cell pellet identified phosphorylated p38␣ as well as other isoforms. Only phospho-p38␣ was detected when the lysis buffer was directly added to monolayer cells (Fig. 7). Our study demonstrated that tRA treatment (1 M) stimulated phospho-p38␣ at 0.5 h, whereas phosphorylation of other p38 isoforms was not induced by tRA until 12 h or later (Fig. 8A).
p38␤ and -␦ migrate at ϳ43 kDa, so we could not detect the phosphorylation of each isoform individually in the Western blot analysis using the pan-phospho-p38 Ab. In our preliminary experiments, the expression level of endogenous p38␤ in MCF-7 cells was not sufficient to immunoprecipitate with anti-p38␤ Ab (data not shown). We therefore transfected the expression vector Myc/FLAG-tagged p38␤ into MCF-7 cells and isolated the exogenous p38␤ by immunoprecipitation with anti-FLAG tag Ab after tRA treatment. The phosphorylation of exogenous p38␤ in MCF-7 cells was induced 6 h or longer after treatment with tRA (1 M) (Fig. 8B), consistent with the West-

p38␤ Regulates Iodide Transporter in Breast Cancer Cells
ern blot analysis with the pan-phospho-p38 Ab (Fig. 8A). These data indicate induction of phosphorylated p38␤ by tRA, although longer stimulation is required for the induction compared with phosphorylated p38␣. Rac1 Regulates p38␤ in MCF-7 Cells-Our gain and loss of function studies demonstrated critical roles for p38␤, MKK3B, and Rac1 in the induction of NIS expression in MCF-7 cells. Although tRA stimulates phosphorylation of p38 through Rac1 in MCF-7 cells (18), selective regulation of p38␤ by Rac1 has not been reported. We therefore studied if the Rac1 signaling regulates p38␤ activity in MCF-7 cells.
Our loss of function studies identified a requirement of MKK3B for NIS gene regulation. Although activation of p38␤ by MKK3B has been reported, physical interaction between these kinases has not been studied in MCF-7 cells. We therefore investigated if MKK3B was co-immunoprecipitated with p38␤. Endogenous MKK3B was co-immunoprecipitated by anti-FLAG Ab with FLAG-tagged p38␤ expressed in MCF-7 cells (Fig. 9B). The constitutively active Rac1 G12V or tRA treatment, however, did not clearly increase the amount of co-immunoprecipitated MKK3B, whereas the inhibitory effects by Rac1T17N were still reproduced (Fig. 9B, lanes 5 and 6). These data indicate that MKK3B physically interacts with p38␤ in MCF-7 cells. The amount of co-immunoprecipitated MKK3B, however, did not change with tRA treatment, Rac1 activator, or Rac1 inhibitor.

DISCUSSION
In this study, we demonstrated the importance of Rac1, MKK3B, and p38␤ for NIS expression in MCF-7 breast cancer cells. tRA up-regulated p38␤ phosphorylation through Rac1 and MKK3B. The induction of NIS by tRA was, at least partially, through the Rac1-MKK3B-p38␤ pathway. NIS in thyroid cells was also regulated by p38 signaling; however, p38␣, and not p38␤, was critical for the expression of thyroid NIS.
We demonstrated that NIS expression in MCF-7 breast cancer cells was dependent on p38␤ but not p38␣. MKK6 activates the NIS-inducible CHOP in thyroid cells (34), whereas MKK3B, but not MKK6, was critical for NIS expression in MCF-7. Moreover, our preliminary study showed that overexpression of CHOP did not significantly increase NIS expression in MCF-7 cells. 4 Although the Rac1-p38 pathway stimulates NIS expression in both thyroid cells (21,34) and MCF-7 breast cancer cells, the Rac1 signals diverge into at least two pathways through distinct isoforms of p38, probably resulting in differential regulation of NIS in these cell types (Fig. 10).
Our previous pharmacological study has demonstrated various regulatory mechanisms for NIS expression involving several signaling pathways in MCF-7 cells (17). A phosphoinositide 3-kinase inhibitor, LY294002, significantly reduces tRAinduced NIS expression, but not basal expression, in MCF-7       (24), confirmed by our RT-PCR. The size of p38␣, ϳ40 kDa, is smaller than those of other isoforms, 43-46 kDa. Because activation of p38 is dependent on dual phosphorylation of the Thr-Gly-Tyr motif in the T-loop (35), anti-phospho-p38 Abs have been developed to recognize the Thr-Gly-Tyr dual phosphorylation site, common among all p38 isoforms. A previous study using such a pan-phospho-p38 Ab, however, showed a single band for the phosphorylated p38 in MCF-7 cells (18), probably corresponding to p38␣. Our Western blot analysis, performed with MCF-7 cell extracts prepared from the cell pellet, showed multiple bands, corresponding to each isoform, with a polyclonal pan-phospho-p38 Ab, as well as a monoclonal pan-phospho-p38 Ab. In contrast, experiments with cell extracts prepared directly from monolayer cells produced a single band, corresponding to p38␣, even with the same polyclonal phospho-p38 Ab. To analyze the phosphorylated p38 ␤-␦ in MCF-7 cells by Western blotting, cells need to be pelleted before lysing. Caution is required in analysis of p38 phosphorylation with anti-phospho-p38 Ab, especially if the cells express multiple p38 isoforms.
The treatment of MCF-7 cells with tRA followed by ␤-emitting 131 I results in significant cytotoxicity in vitro (11,14,36). Our study with mouse breast cancer models, however, showed that the dose of tRA for the maximum induction of NIS in the tumors is probably higher than would be tolerated for human treatment (13). Retinoic acid signaling regulates a wide variety of physiological events by stimulating signal transduction pathways as well as transcription of a large number of target genes. Indeed, tRA activated not only the NIS-inducing p38␤ but also other isoforms of p38, which were not required for the full induction of NIS. Targeted stimulation of signaling pathways for NIS expression should promote more efficient induction of radioiodide uptake, with less toxicity, in some breast cancer tissues. The Rac1-MKK3B-p38␤ signaling is one such pathway, as suggested by our overexpression experiments of Rac1 and p38␤ in MCF-7 cells.
Most studies on p38 signaling have focused on the p38␣ isoform, especially its responses to stress and inflammation. p38␤ is thought to be a minor pathway in immune system or stress signaling (37). Deficiency of p38␤ in mice actually causes no apparent phenotype, probably due to redundant functions among the isoforms (37). Increasing evidence points to an association of p38␣ with cell proliferation, malignant transformation, and tumor invasion in cancer cells, whereas the role of p38␤ in breast cancer is not established (1). A number of in vitro studies, however, have demonstrated that each isoform of p38 activates different downstream effectors (22)(23)(24). We demonstrate here that a retinoic acid-regulated p38␤ signal transduction pathway stimulates NIS expression, a promising target for breast cancer therapy. Confirmation of this p38␤ isoform specificity in additional in vitro and in vivo breast cancer models will be important. Dissection of isoform-specific signaling pathways is a potential novel strategy for breast cancer treatment.