Altered Localization of Retinoid X Receptor Coincides with

MOLECULAR AND CELLULAR BIOLOGY, May 2004, p. 3972�3982 Vol. 24, No. 90270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.9.3972–3982.2004

Altered Localization of Retinoid X Receptor � Coincides with Loss ofRetinoid Responsiveness in Human Breast Cancer MDA-MB-231 Cells

T. Tanaka, B. L. Dancheck, L. C. Trifiletti, R. E. Birnkrant, B. J. Taylor,S. H. Garfield, U. Thorgeirsson, and L. M. De Luca*

National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255

Received 4 September 2003/Returned for modification 26 September 2003/Accepted 16 January 2004

To understand the mechanism of retinoid resistance, we studied the subcellular localization and function ofretinoid receptors in human breast cancer cell lines. Retinoid X receptor � (RXR�) localized throughout thenucleoplasm in retinoid-sensitive normal human mammary epithelial cells and in retinoid-responsive breastcancer cell line (MCF-7), whereas it was found in the splicing factor compartment (SFC) of the retinoid-resistant MDA-MB-231 breast cancer cell line and in human breast carcinoma tissue. In MDA-MB-231 cells,RXR� was not associated with active transcription site in the presence of ligand. Similarly, ligand-dependentRXR homo- or heterodimer-mediated transactivation on RXR response element or RARE showed minimalresponse to ligand in MDA-MB-231 cells. Infecting MDA-MB-231 cells with adenoviral RXR� induced nucle-oplasmic overexpression of RXR� and resulted in apoptosis upon treatment with an RXR ligand. This suggeststhat nucleoplasmic RXR� restores retinoid sensitivity. Epitope-tagged RXR� and a C-terminus deletionmutant failed to localize to the SFC. Moreover, RXR� localization to the SFC was inhibited with RXR�C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, theRXR� C terminus may play a role in the intranuclear localization of RXR�. Our results provide evidence thataltered localization of RXR� to the SFC may be an important factor for the loss of retinoid responsiveness inMDA-MB-231 breast cancer cells.

Retinoids are natural and synthetic vitamin A derivativeswhich regulate development (36), cell proliferation (24), anddifferentiation (7). Retinoids also act as cancer preventiveagents and are presently being used successfully to treat certaintypes of cancer (6, 45). Although many studies have shownretinoid effectiveness on inhibition of cancer cell growth invitro and in vivo (18), the clinical usage of vitamin A and itsderivatives is currently limited by the requirement of a largedosage to reach therapeutic efficacy. The combination of syn-thetic retinoid and tamoxifen inhibited the growth of estrogen-positive breast cancers in premenopausal patients; however, itfailed to show any significant effect on advanced breast cancerpatients (2, 3, 30). It is likely that the responsiveness of cancercells to retinoid diminishes, along with malignant progression.Indeed, growth inhibitory effects of retinoids have been ob-served in estrogen receptor (ER)-positive breast cancer celllines such as MCF-7 and T-47D (19), whereas the effectivenessof retinoid diminishes in highly malignant ER-negative breastcancer cell lines such as MDA-MB-231 and BT-20 (5, 13, 14,35, 53). The existing hormonal and chemotherapeutic thera-pies have provided significant improvement for the survivalof patients with localized breast cancer; however, treatmentfor metastatic breast cancer still remains palliative (31). The5-year survival ratio for patients diagnosed with metastaticbreast cancer is only 15%. Thus, there is an urgent need tounderstand the mechanism of retinoid resistance in order todevelop therapeutic agents for metastatic breast cancer.

The physiological actions of retinoids are mediated through

two distinct nuclear receptor families (12, 26): the retinoic acidreceptors (RAR�, RAR�, and RAR�), each of which bindsboth all-trans-retinoic acid or 9-cis-retinoic acid (9-cis-RA),and the retinoid X receptors (RXR�, RXR�, and RXR�),which preferentially bind 9-cis-retinoic acid. RARs and RXRsbind to a specific DNA response element (RARE and RXRE,respectively) in the 5�-flanking region of target genes as ho-modimers or heterodimers, thereby promoting gene transcrip-tion (25). Since RAR and RXR play a central role in mediatingretinoid action in the physiology of normal cells, it is likely thatthey are also involved in anticancer effects of retinoids.

Retinoid receptors are members of the steroid-thyroid-vita-min D receptor superfamily. This superfamily can be subdi-vided into two classes according to the partitioning of theunliganded receptor in the cells. The first group of receptors,the glucocorticoid receptor (GR), the mineral corticoid recep-tor, the ER, the progesterone receptor, and the androgenreceptor (AR) localize predominantly within the cytoplasm inthe absence of ligand (34). Receptors in this group bind tohsp90 upon ligand binding and translocate to the nucleus (34).In contrast to these receptors, the thyroid and retinoid recep-tors tightly associate with the nucleus independent of the pres-ence of the ligand (16, 34). The liganded nuclear receptorproteins, AR, GR, ER, and aryl-hydrocarbon receptor havebeen shown to localize in 250 to 400 microfoci (11, 38, 49).They recruit transcription cofactors such as SRC-1, TIF-II, andp300/CBP into microfoci, and active transcription occurs (38).With respect to spatial distribution, the mechanism by which aspecific transcription factor recruits its dimerization partnerand accesses the active transcription site is not clearly under-stood yet.

In the present study, we focused on subcellular localization

* Corresponding author. Mailing address: National Institutes ofHealth, Bldg. 37, Rm. 4054C, 37 Convent Dr., Bethesda, MD 20892-4255. Phone: (301) 496-2698. Fax: (301) 496-8709. E-mail: [email protected].

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of the retinoid receptors to understand the mechanism where-by breast cancer cells become refractory to retinoids.

MATERIALS AND METHODS

Abbreviations. In addition to the abbreviations introduced above, the follow-ing abbreviations are used here: PPAR, peroxisome proliferator-activated recep-tor; VDR, vitamin D receptor; SFC, splicing factor compartment; PML, promy-elocytic leukemia; NPC, nuclear pore complex; TSA, trichostatin A; TTNPB,(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl-]benzoic acid.

Reagents. 9-cis-RA, actinomycin D, TSA, and Hoechst 33342 were obtainedfrom Sigma (St. Louis, Mo.). TTNPB was purchased from BIOMOL (PlymouthMeeting, Pa.).

Cell culture. Human mammary epithelial cells (HMEC) were purchased fromCambrex (Rockland, Minn.) and maintained in mammary epithelial growth me-dium. MCF-7 and MDA-MB-231 breast cancer cell lines were purchased fromthe American Type Culture Collection (Rockville, Md.) and maintained in Dul-becco modified Eagle medium (DMEM) containing 10% fetal bovine serum ina humidified atmosphere supplemented with 5% CO2 at 37°C. The subconfluentcells were treated with trypsin and seeded onto two-well glass chamber slides. Onthe following day, the cells were subjected to analysis by immunofluorescence.

Immunostaining and microscopy. Cells were rinsed twice with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min, followed bytwo washes in PBS and permeabilization with 0.2% Triton X-100. After a briefwash with PBS, the slides were blocked with 10% goat serum for 1 h at roomtemperature and then incubated overnight at 4°C or for 1 h at room temperaturewith the following primary antibodies diluted in goat serum: rabbit anti-RXR�(1:200; Santa Cruz Biotechnology, Santa Cruz, Calif.), mouse anti-PML (1:100;Santa Cruz Biotechnology), mouse anti-NPC (1:1,000; BabCO, Richmond,Calif.), mouse anti-SC-35 (1:2,000; Sigma), mouse anti-p150 (1:10; ICN, Cleve-land, Ohio), goat anti-adenovirus type 5 (1:50; Chemicon, Temecula, Calif.),mouse anti-bromodeoxyuridine (anti-BrdU; 1:50; Roche, Indianapolis, Ind.),mouse anti-hemagglutinin (anti-HA; 1:25; Roche), and fluorescein isothiocya-nate (FITC)-coupled mouse anti-Flag (1:500; Invitrogen, Carlsbad, Calif.). Thesame amount of affinity-purified normal immunoglobulin G (IgG) from thecorresponding species was used as a negative control. Slides were washed withPBS extensively and incubated with goat anti-rabbit FITC and/or goat anti-mouse Texas red, donkey anti-goat TRITC (tetramethyl rhodamine isothiocya-nate; 1:300; Jackson Laboratories, West Grove, Pa.) in 0.5% IgG-free bovineserum albumin for 1 h at room temperature in the dark. The slides were thenwashed with PBS and rinsed with deionized water. Nuclear counterstaining wasperformed with Hoechst 33342 (Sigma) for 5 min at room temperature. Paraffin-embedded blocks of human breast tissue from the Cooperative Human TissueNetwork of the University of Pennsylvania Medical Center, Philadelphia, weresliced to obtain 6-�m-thick sections for immunohistochemistry of RXR� accord-ing to the method of Lawrence et al. (22). Antigen was retrieved in 10 mM citratebuffer (pH 6.0) in a microwave for 15 min past boiling. Rabbit anti-RXR� andmouse anti-SC-35 antibodies were applied in a 1:50 and 1:1,000 dilution, respec-tively. Each section was stained with hematoxylin and eosin for pathologicaldiagnosis. Confocal images were captured by using a Zeiss confocal microscopeand processed by using Zeiss LMS 5 Image. Series of images were obtainedunder the same conditions when fluorescence intensity needed to be compared.

Western blotting. The cell pellets were lysed in Laemmli buffer and centri-fuged, and the lysates were loaded onto 4 to 12% bis-Tris polyacrylamide gelsand transferred to a polyvinylidene difluoride membrane. The membrane wasblocked with 5% blocking milk, probed with rabbit anti-RXR�, RAR�, p21, andmouse anti-hnRNP K (Santa Cruz Biotechnology), bcl-2, and Bax (Calbiochem,La Jolla, Calif.). The blots were washed with PBS-Tween, incubated with horse-radish peroxidase-labeled anti-rabbit or anti-mouse antibody, and visualized bychemiluminescence (Pierce, Rockford, Ill.).

Plasmid construct and transfection. RXR� insert was excised from pCMX-hRXR� and then subcloned into phrGFP-N1 (Stratagene) and pcDNA 3.1(N-terminus Flag tag). C-terminus GFP-tagged RXR� was generated by PCRwith sense primer (5�-GGATCCATGGACACCAAACATTTC-3�), antisenseprimer (5�-CTCGAGAGTCATTTGGTGCGGCGCCTC-3�) ligated into phrGFP-C (Stratagene, La Jolla, Calif.), and C-terminus HA tag pMH (Roche). Thefull-length DNA sequence of RXR� was confirmed by using a dye terminatorreaction (ABI Prism, Foster City, Calif.). Then, 106 of trypsin-treated MCF-7and MDA-MB-231 cells were mixed with 5 �g of supercoiled plasmid DNA,followed by electroporation at 180 V for ca. 70 ms in a 4-mm cuvette by usingECM 830 (BTX, Holliston, Mass.). The electroporated cells were seeded in atwo-well slide and cultured for 24 to 48 h. Live cell image was taken 24 h after

transfection. Nontagged RXR�, Flag-tagged RXR�, and HA-tagged RXR�transfected cells were fixed and used for immunofluorescence with correspond-ing antibody as described above.

Labeling of transcription site. The cells were grown in two-well slides for 2days. The cells were placed on ice and washed twice with ice-cold glycerol buffer(20 mM Tris-HCl [pH 7.4], 25% glycerol, 5 mM MgCl2, 0.5 mM EGTA, 0.5 mMphenylmethylsulfonyl fluoride [PMSF]), followed by permeabilization with0.05% Triton X-100 for 10 min on ice. The cells were washed with glycerol bufferto remove excess detergent and then incubated for 10 min at 37°C with tran-scription buffer (50 mM Tris-HCl [pH 7.4], 100 mM KCl, 25% glycerol, 5 mMMgCl2, 1 mM PMSF, 25 U of RNase inhibitor/ml) supplemented with 2 mMATP, 0.5 mM CTP and GTP, and 0.2 mM BrUTP. After incorporation, the cellswere fixed with 4% paraformaldehyde in PBS for 20 min and evaluated byimmunofluorescence with mouse anti-BrdU and rabbit anti-RXR� antibodies.

Nuclear digestion. Cells were digested, with a modification of the methoddescribed by Nickerson et al. (32). The cells were washed three times in cytoskel-eton buffer {10 mM PIPES [piperazine-N,N�-bis(2-ethanesulfonic acid); pH 6.8],100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EDTA, 0.05 mM PMSF,10 �g of aprotinin/ml} and incubated with the cytoskeleton buffer containing0.5% Triton X-100 for 10 min at 4°C. The cells were rinsed twice with RSB buffer(42.5 mM Tris [pH 8.3], 8.5 mM NaCl, 2.6 mM MgCl2, 0.05 mM PMSF, 10 �gof aprotinin/ml) and then incubated with RBS buffer containing 1% Tween 20and 0.3% of deoxycholate for 10 min at 4°C. Chromatin was removed by treat-ment with 100 U of RNase-free DNase I/ml in digestion buffer (10 mM PIPES[pH 6.8], 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EDTA, 0.05 mMPMSF, 10 �g of aprotinin/ml) for 1 h at 37°C. To complete digestion, ammoniumsulfate was added to a final concentration of 0.25 M, and the mixture wasincubated for 5 min at 4°C, followed by 2 M NaCl for 5 min. For the removal ofRNA, cells were incubated in digestion buffer containing the indicated concen-trations of RNase A for 30 min at 37°C. The cells were washed extensively withice-cold PBS, fixed with 4% paraformaldehyde for 15 min, and then used for im-munofluorescence or collected and lysed in Laemmli buffer for Western blotting.

Reporter assay. RXRE-luciferase, RARE-thymidine kinase (TK)-luciferase,or TK-luciferase reporter plasmid was transfected into MCF-7 and MDA-MB-231 cells for 5 h. Cells were treated with 10�6 M RXR-selective agonist for 20 h,lysed, spun down briefly to remove debris, and analyzed for luciferase activityaccording to the manufacturer’s instructions (Promega, Madison, Wis.). Thelight intensity was measured by a luminometer Tropix TR717 (Perkin-Elmer,Boston, Mass.). The transfection efficiency was normalized to TK. The datashown are the means of at least six separate experiments.

Peptide delivery. Peptide delivery was performed according to the manufac-turer’s instructions (GTS, San Diego, Calif.). A total of 1 �g of (i) C-terminuspeptide from RXR� (441-IDTFLMEMLEAPHQMT-456), (ii) N-terminus pep-tide from RXR�-N (1-MDTKHFLPLDFSTQVNSSLT-20), or (iii) the controlpeptide, the C-terminus peptide of RAR� (441-VPGGQGKGGLKSPA-454),which has no homology to the C-terminal sequence of RXR�, was delivered tothe cells in the presence of the delivery agent. The cells were further incubatedfor 24 to 48 h after peptide delivery. For simultaneous plasmid transfection andpeptide delivery, the cells were cultured in antibiotic-free DMEM supplementedwith 10% serum for 24 h. On the following day, the cells were preincubated with1 �g of RXR�–C-terminus, RXR�-N, or RAR�–C-terminus peptide for 5 min,followed by plasmid transfection for 3 h. The cells were recovered overnight andincubated with ligand for 24 h, and the reporter activity was measured.

TUNEL assay. Flow cytometry TUNEL (terminal deoxynucleotidyltransfer-ase-mediated dUTP-biotin nick end labeling) assay was performed according tothe manufacturer’s instructions (Pharmingen, San Diego, Calif.). Stained cells wereanalyzed by flow cytometry using the FACSCalibur (BD Biosciences, San Jose,Calif.).

Recombinant adenovirus construction and propagation. The complete codingsequence of wild-type RXR� cDNA was subcloned into shuttle vector containingcytomegalovirus promoter and simian virus 40 poly(A). To obtain recombinantplasmid, pAdEasy-1 and shuttle vector (Stratagene) were mixed and transformed.The recombinant plasmid was transfected to HEK-293 cells to generate adeno-virus. Adenovirus was purified, and the titer was measured. A day after seeding,MDA-MB-231 cells were infected with adenovirus at a multiplicity of infection(MOI) of 100 for 24 h at 37°C in 10% fetal bovine serum containing DMEM.

RESULTS

Altered localization of RXR� coincides with lack of respon-siveness to retinoid. Retinoic acid inhibited growth of HMEC

VOL. 24, 2004 RXR� IN SFC CAUSES RETINOID UNRESPONSIVENESS 3973

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and MCF-7 human breast cancer cells (not shown). Immuno-fluorescence confocal microscopy revealed that RAR� local-ized throughout the nucleoplasm diffusely and in microspeck-les in HMEC, MCF-7 cells, and MDA-MB-231 cells (Fig. 1,top panels). In sharp contrast to RXR� homogeneous nucleardistribution in retinoid-sensitive HMEC and MCF-7 cells,RXR� showed a distinct punctate pattern in the retinoid-resistant MDA-MB-231 cells (Fig. 1, bottom panels). Controlexperiments with the same amount of normal rabbit IgG orwith blocking peptides verified that the RXR� and RAR�signal was specific (data not shown).

Identification of subnuclear localization of RXR� in MDA-MB-231 cells. Confocal microscopy was used to pinpoint sub-nuclear localization of RXR� in the MDA-MB-231 cells (Fig.2A). The subnuclear structures were visualized by using Texasred-labeled secondary antibody to different nuclear proteinantibodies (Fig. 2A, panels a to d). RXR� was visualized byusing FITC-labeled secondary antibody (Fig. 2A, panels e toh). RXR� did not colocalize with NPC (Fig. 2A, panel i) orPML bodies (Fig. 2A, panel j). However, extensive colocaliza-tion was observed between RXR� and SC-35 (Fig. 2A, panelk), a splicing factor known to localize to SFC. RXR� was alsofound to colocalize with p105 (Fig. 2A, panel l), a componentof interchromatin granule cluster, which is a substructure ofthe SFC.

Double immunostaining demonstrated that RAR� micro-speckles colocalized with PML bodies but not with SC-35 andp105 (data not shown). Figure 2B shows that RXR� localiza-tion to the SFC was found only in retinoid-resistant MDA-MB-231 cells and not in retinoid-sensitive HMEC and MCF-7cells. MDA-MB-231 cells infected with RXR� antisense ade-novirus were immunostained for RXR� (green) and adeno-

virus (red) (Fig. 2C). The RXR� was found in the SFC inmock- and null-infected cells but absent from RXR� anti-sense-infected MDA-MB-231 cells (Fig. 2C), indicating thatRXR� localization to the SFC was specific. RXR dimerizationpartners, RAR�, RXR�, and PPAR� and PPAR� did not lo-calize to the SFC, suggesting that they do not form dimers withRXR� in the SFC (Fig. 2D). These staining patterns werecommonly observed in all cells tested. To confirm our in vitroresults, colocalization between RXR� and SC-35 was tested inhuman breast tissue. We tested tissue sections and adjacent nor-mal region from 12 patients with invasive breast cancer. RXR�was found in the SFC of mesenchymal cells of 5 of 12 invasivebreast carcinomas (Fig. 2E). However, SFC localization of RXR�was not detected in any of the normal tissue or in benign hy-perplasias of the three breast tissues tested (data not shown).Hematoxylin and eosin staining showed a representative area.

RXR� was silenced due to SFC localization. To examinewhether the localization of RXR� to the SFC depends onthe transcription status of MDA-MB-231 cells, the cells weretreated with actinomycin D or transcription activator. Some ofthe SFC became bigger when transcription was inhibited in thepresence of actinomycin D (Fig. 3A). In order to mimic activetranscription, cells were treated with TSA, 9-cis-RA, the nat-ural ligand of RXR�, or AGN194204, a pan-agonist of RXR.Against our expectation, RXR� remained in the SFC in thepresence of excess amounts of TSA, 9-cis-RA, and AGN104204(Fig. 3A). These results demonstrate that the ligand did notinduce relocalization of RXR� from the SFC to the nucleo-plasm, where the active transcriptional machinery is present. Inorder to determine whether RXR� associates with nucleic acidin the SFC, the nuclei were digested with DNase I or RNase.Interactions between chromatin, RNA, and their binding pro-

FIG. 1. Altered localization of RXR� coincides with lack of retinoid responsiveness. Cells were grown in two-well chamber slides. The slideswere fixed, and then subcellular localization of RAR� and RXR� was detected by immunofluorescence. Bar, 10 �m. The fluorescence intensitydoes not correspond to the level of protein expression.

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teins are generally disrupted during this preparation. Thespeckled pattern of RXR� remained intact after DNA andRNA removal, suggesting that RXR� does not interact withDNA or RNA in MDA-MB-231 cells (Fig. 3B, panel f). West-ern blotting confirmed that RXR� was resistant to DNase andRNase digestion. In contrast, RAR� was sensitive to DNasedigestion, suggesting that RAR� is physically associated withDNA (Fig. 3B). Concentrations of RNase up to 1,000 �g hadno effect or slightly reduced the RXR� speckled pattern (Fig.3B, panels k to n). Corresponding samples were analyzed byWestern blotting and showed that RXR� was resistant toRNase digestion, whereas hnRNP, which localizes to the SFCand tightly interacts with RNA, was removed efficiently byRNase (Fig. 3B). In contrast to MDA-MB-231 cells, RXR�was completely removed by DNase in MCF-7 cells, and nospeckled pattern was revealed (data not shown), suggestingthat RXR� interacts with DNA in MCF-7 cells. To examinewhether RXR� participates in transcription, nascent tran-scripts were labeled with BrUTP and double immunostainedwith RXR�. RXR� did not colocalize with active transcriptionsites in MDA-MB-231 cells, but it colocalized extensively inMCF-7 cells (Fig. 3C). These results demonstrate that RXR�is actively participating in transcription in MCF-7 cells but notin MDA-MB-231 cells. These staining patterns were commonlyobserved in all cells tested.

To test whether RXR� is silent in MDA-MB-231 cells,RXRE (RXR homodimer target) and RARE (the RAR/RXRheterodimer target) linked to the luciferase reporter gene weretransfected into MCF-7 and MDA-MB-231 cells. As expected,RXR selective ligand had minimal effect on transcription viathe RXRE in MDA-MB-231, whereas it activated transcrip-tion in MCF-7 cells (Fig. 3Da). MDA-MB-231 cells transfectedwith RARE also barely responded to a RAR selective ligand(TTNPB). These results support that RXR�-mediated tran-scription is significantly reduced due to sequestration of RXR�in the SFC. Cotransfection of RXRE and RXR� expressionvector restored the response to RXR ligand in the MDA-MB-231 cells and caused a 10-fold increase in transactivation.These data show that RXR� is the limiting factor for MDA-MB-231 cells in mediating transactivation via RXRE.

Subcellular localization of exogenous RXR�. To furthercharacterize RXR� localization to the SFC, we compared thelocalization of exogenous RXR� to that of endogenousRXR�. GFP was fused to the C or N terminus of RXR� andelectroporated to MCF-7 and MDA-MB-231 cells. Figure 4Ashows the live cell image of GFP-fused RXR�. GFP fusedeither to the C or N terminus of RXR� showed a diffusenucleoplasmic localization, excluding nucleoli, and a number

FIG. 2. Colocalization of RXR� with nuclear organelle proteins.(A) Fixed MDA-MB-231 cells were double immunostained withRXR� (e to h) and the indicated nuclear protein antibody anti-NPC

(a), anti-PML (b), anti-SC-35 (c), or anti-p105 (d). Confocal overlaysof double-immunostained images are shown in merged panels (i to l).(B) HMEC, MCF-7, and MDA-MB-231 cells were double immuno-stained with anti-RXR� and anti-SC-35. (C) RXR� antisense adeno-virus was infected to MDA-MB-231 and double immunostained withanti-RXR� (green) and anti-adenovirus type 5 (red). (D) The dimer-ization partner (RXR�, PPAR�, PPAR�, or RAR�) did not colocalizewith SC-35. All antibodies were diluted at 1:200. (E) Paraffin-embed-ded human breast tissue sections were deparaffinized and hydrated.Antigen was retrieved in 10 mM citrate buffer in a microwave for 15min past boiling.


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of bright microfoci in the nucleus of both MCF-7 and MDA-MB-231 cells (Fig. 4A). GFP-RXR� accumulated in thesemicrofoci when the cells were treated with ligand for 6 h (Fig.4B, panel b). Next, we examined whether GFP-fused RXR�colocalizes with SC-35 in the SFC of MDA-MB-231 cells. Thecells were permeabilized and immunostained with anti-SC-35antibody. Figure 4C shows that neither the C- nor N-terminusGFP-fused RXR� expression pattern overlaps with anti-SC-35(Fig. 4C, panels c and i). Next, the cells expressing GFP-fusedRXR� were treated with actinomycin D to take advantage ofits characteristics that facilitate SFC localization in the absenceof transcription. Actinomycin D treatment did not cause relo-calization of GFP-fused RXR� from the nucleoplasm to theSFC (Fig. 4C, panels f and l), whereas SC-35 accumulated inthe SFC (Fig. 4B, panels e and k). Further, cell nuclei fromGFP-RXR�-overexpressing cells were digested by DNase orRNase to remove nucleoplasmic GFP-RXR� signal. GFP-RXR� was not found in the SFC. Interestingly, GFP-RXR�microfoci disappeared when transcription was inhibited by acti-nomycin D and diffused throughout the nucleus, including thenucleolus (Fig. 4C, panels d and j). These staining patternswere common to all transfected cells tested.

Possible role of RXR� C terminus in SFC localization. Torule out the possibility that the GFP moiety might lead tochanges in secondary structure of RXR�, smaller epitope tag,Flag (N-terminal 8 amino acid) or HA (C-terminal 9 aminoacid) was fused to RXR�. They were found to localize homo-geneously throughout the nucleus but not in the SFC (Fig. 5A).Since exogenous epitope-tagged RXR� did not show the samelocalization pattern as endogenous RXR�, the open readingframe of RXR� of MCF-7 and MDA-MB-231 was sequencedand found to be identical to wild-type RXR� (data not shown).Thus, genetic alteration of RXR� is not responsible for thislocalization in MDA-MB-231. Next, MDA-MB-231 cells weretransfected with a nontagged RXR�. The cell in the square(Fig. 5A, right panel) shows endogenous RXR�, while the cellnext to it, showing homogeneous nuclear staining pattern inaddition to distinct speckles, represents exogenous nontaggedRXR� overexpression (Fig. 5A). Speckles found in cells over-expressing the nontagged RXR� were larger and markedly

FIG. 3. RXR� is silenced due to SFC localization. (A) The cellswere treated with vehicle alone (a), 0.5 �g of actinomycin D/ml (b),100 nM TSA (c), 1 �M 9-cis-RA (d), or 1 �M RXR agonist (e) for24 h. (B) MDA-MB-231 cells were permeabilized and then digestedwith DNase, followed by treatment with 2 M NaCl and 0.25 M

(NH4)2SO4 extraction. The cells were also digested with different con-centrations of RNase A for 30 min at 37°C. Digested cells were fixedand immunostained with RXR� (d to f and k to n), followed bycounterstaining with Hoechst 33342 (a to c and g to j). The series ofimages were taken by confocal microscope under the same conditionsetting. For Western blotting, the cells were either untreated (Un) ortreated with DNase (DN), RNase (RN), and different concentrationsof RNase (0 to 1,000 �g/ml) as indicated. The cells were lysed in theLaemmli buffer after digestion. Then, 10 �l of whole-cell lysate wasseparated on sodium dodecyl sulfate-polyacrylamide gel electrophore-sis and probed with the indicated antibodies (1:1,000). (C) MDA-MB-231 and MCF-7 cells were treated with 0.2 �M 9-cis-RA for 24 h.Active transcription sites were labeled with BrUTP, followed by dou-ble immunostaining with mouse anti-BrdU and rabbit anti-RXR�.(D) RXRE- and RARE-Luc was transfected into MCF-7 and MDA-MB-231 cells (a), and RXRE-Luc was cotransfected with RXR� ex-pression vector or empty vector (b). Cells were treated with vehiclealone or 1 �M RXR ligand for 20 h, and luciferase activity wasassessed. The data are presented as means � the standard deviationsof six replicates from two independent experiments.


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brighter than those of endogenous RXR� in the SFC. Thesespeckles colocalized with SC-35 (Fig. 5B) in the SFC. How-ever, the staining signals reached a plateau as we increased theamount of DNA used for transfection (data not shown), im-plying that SFC have limited capacity to interact with RXR�and that excess RXR� distributes throughout the nucleoplasm(Fig. 5B). These results indirectly suggest that the secondarystructure of either N- or C-terminal RXR� may be critical forSFC localization. To test this hypothesis, the C-terminus-trun-cated RXR�416 and full-length RXR�, which have the sameplasmid backbone, were generated. Interestingly, RXR�416showed speckles of smaller size and reduced fluorescence com-pared to the wild type (Fig. 5C). Approximately 100 trans-fected cells were analyzed for quantitative evaluation, and theaverage speckle fluorescence in those cells is summarized inthe graph (Fig. 5C). The average fluorescence of cells overex-pressing full-length RXR� was 119.7 � 5.6, approximatelytwofold more than that of the endogenous RXR�, whereasthat of RXR�416 was 68.4 � 7. Western blotting showedsimilar a expression level in both RXR� and RXR416 (Fig.5C). This excludes the possibility that the observed reducedfluorescence intensity of RXR412 is due to lower transfectionefficiency.

To establish a role of the C terminus of RXR� in SFClocalization, MDA-MB-231 cells were treated with RXR� C-terminus-specific peptide. At 24 h, RXR� was colocalized withSC-35 but was also detected in the cytoplasm (Fig. 5D, panelsd to f). At 48 h of incubation RXR� distributed throughout thecell and no longer remained in the SFC, and colocalizationbetween SC-35 and RXR� disappeared (Fig. 5D, panels g to i).The same amount of control peptide corresponding to theC-terminus RAR� did not affect the RXR� punctate pattern(Fig. 5D, panels a to c). Furthermore, cells incubated withRXR� C-terminus peptide showed a 2.5-fold increase inligand-mediated transactivation on RXRE, whereas the RXR�N-terminus peptide did not show any effect, suggesting that theRXR� C terminus may interact directly with the SFC. RAR�C-terminus peptide did not affect ligand-dependent transactiva-tion on RXRE (Fig. 5D). RXR� immunoblotting showed thatpeptide delivery did not alter the level of the RXR� protein(Fig. 5D). Therefore, it appears that the F region of RXR� isinvolved in intranuclear localization.

RXR� restored the responsiveness to retinoid in MDA-MB-231 cells. MDA-MB-231 cells were infected with RXR� ade-novirus. With an adenovirus-RXR� MOI of 100, RXR-selec-tive ligand promoted transactivation via RXRE (Fig. 6A).RXR� was overexpressed throughout the nucleoplasm in ad-dition to the SFC (Fig. 6B). MDA-MB-231 cells infected withRXR� adenovirus for 24 h, followed by incubation with RXR-selective ligand for 48 h, demonstrated a drastic decrease inthe number of viable cells by 40% of mock- and null-in-fected MDA-MB-231 cells (Fig. 6C). Apoptosis was mea-sured by TUNEL assay. Adenovirus RXR�-infected MDA-MB-231 cells increased TUNEL-positive cells by �60% in thepresence of ligand within 96 h of adenovirus infection, whereasthe cells with mock and adenovirus null infected in the pres-ence of ligand showed only 5% TUNEL positivity (Fig. 6D).These data indicate that nucleoplasmic overexpression of RXR�stimulates apoptosis in the MDA-MB-231 cells. Next, to testapoptosis-related protein expression, the cells were harvested

FIG. 4. Subcellular localization of GFP-RXR�. (A) GFP-RXR�,RXR�-GFP, or GFP vector alone was electroporated to MCF-7 (a andb) and MDA-MB-231 (c and d) cells. At 24 h after electroporation, livecell images were obtained by confocal microscopy. The images showedRXR�-GFP (a and c) and GFP-RXR� (b and d). The arrowheadpoints to nuclear microfoci. Bar, 5 �m. (B) GFP-RXR� was electropo-rated to MDA-MB-231, incubated with dimethyl sulfoxide (a) or a mix-ture of 0.5 �M TTNPB and 0.5 �M RXR-selective ligand (b) for 6 h. Thearrow points to nuclear microfoci. (C) GFP-RXR� and RXR�-GFP wereintroduced into the cell through electroporation, and then the cells weretreated with vehicle alone (a to c and g to i) or actinomycin D (d to f andj to l) for 6 h. The cells were fixed and immunostained with anti-SC-35.


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after 96 h of incubation with ligand or vehicle alone. Theinduction of ligand-induced apoptosis was correlated with theupregulation of p21 and downregulation of bcl-2, but Bax ex-pression was unchanged (Fig. 6E).

DISCUSSIONRetinoids control cell proliferation and differentiation

through the action of RARs and RXRs and have chemo-preventive effects in various tumor types, including the skin,

FIG. 5. Localization of epitope-tagged RXR� and nontagged RXRa. (A) Flag-RXR� (a), RXR�-HA (b), or nontagged RXR� (c) waselectroporated into MDA-MB-231 cells. The cells were fixed and used for immunofluorescence with corresponding antibodies. The cell in thesquare represents a nontransfected cell. (B) Nontagged-RXR�-overexpressing cells were labeled with anti-RXR� and mouse anti-SC-35 antibody.(C) RXR� deletion mutant was electroporated to MDA-MB-231 cells, followed by double immunostaining with anti-RXR� and anti-SC-35. Thedouble line represents the antibody recognition site. About 100 transfected cells were analyzed for quantitative evaluation, and average fluores-cence intensities of the speckles in the cells overexpressing the deletion mutant are summarized. To determine how much RXR�, includingendogenous and exogenous, localizes to the speckles, the fluorescence from adjacent areas was subtracted from the fluorescence at the speckles.The average intensity of the speckles in RXR�-transfected cells minus the average intensity from endogenous RXR� represents the amount ofexogenous RXR� that localizes to the SFCs. The series of images was taken by confocal microscope under the same condition setting. The valuesrepresent the means � the standard errors. The scheme shows the structure of the RXR� deletion mutant. The double line represents the antibodyrecognition site. (D) A total of 1 �g of C-terminus peptide corresponding to RAR� (a to c) or RXR� (d to i) was incubated for 12 h withMDA-MB-231 cells. The cells were subjected to immunofluorescence after 24 and 48 h with RXR� and SC-35 antibodies. The images wereobtained at the same setting for the comparison. Western blotting of lysates prepared from MDA-MB-231 cells treated with 1 �g of C-terminusRAR�, treated with RXR� C or N peptide, or left untreated was carried out. Antibody to actin was used as a control. RXRE-Luc, RARE-Luc,or TK-Luc was transfected into MDA-MB-231 cells pretreated with C- or N-terminus RXR� or RAR� peptide or into MDA-MB-231 cells leftuntreated. The cells were treated with vehicle alone or 1 �M RXR ligand for 24 h, and the luciferase activity was assessed. The data are presentedas means � the standard deviations of four replicates from two independent experiments.


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prostate, ovary, leukemia, and breast (15, 23, 37, 42, 46).The anticancer effect of retinoids is lost during tumor pro-gression; however, the mechanism underlying the loss ofretinoid responsiveness is still poorly understood. In thepresent study, our data demonstrate that RXR� localizes tothe SFC in the highly malignant ER-negative, retinoid-re-sistant MDA-MB-231 human breast cancer cells. Further-more, our histological data indicate that RXR� localizes tothe SFC in human breast carcinoma in vivo. Our data pro-vide functional evidence that most, if not all, endogenousRXR� is sequestered in the SFC and silenced and, conse-quently, retinoid signaling appears to be shut off in theMDA-MB-231 cells.

Splicing factors localize in 20 to 40 nuclear domains referredto as the SFC in the mammalian cell nucleus (28). SFC iscomposed of two distinct structures, the interchromatin gran-ule clusters and perichromatin fibrils (9, 27, 43). Splicing factorlocalization in the SFC is generally highly dynamic and isinfluenced by transcriptional activity, cell cycle, and phosphor-ylation (44). Vitamin D receptor B1 (VDRB1), a heterodimer-ization partner of RXR, is also found in the SFC (47), and it is

redistributed throughout the nucleoplasm upon exposure to itsligand, 1,25-dihydroxyvitamin D3. We found that, in contrast tothe ligand-induced dynamic intranuclear mobility of VDRB1,the ligand failed to redistribute RXR� from the SFC to thenucleoplasm in MDA-MB-231 cells. This finding allowed us tohypothesize that RXR� might be sequestered in the SFC,leading to retinoid unresponsiveness.

We demonstrated that RXR� was not localized to activetranscription sites in MDA-MB-231 cells but showed extensivecolocalization with nascent transcripts in MCF-7 cells. Thisresult was further confirmed by reporter assays when ligandpromoted RXRE (RXR homodimer target) or RARE (RAR/RXR heterodimer target) transactivation in MCF-7 cells butfailed to do so in MDA-MB-231 cells. The absence of ligand-dependent transcriptional activation in MDA-MB-231 cellswas not due to the reduction of RXR� protein expression levelbecause RXR� protein level in retinoid-sensitive HMEC wasthe same as in MDA-MB-231 cells (data not shown). Thus, ourinterest was then to investigate whether altered localization ofRXR� could explain the loss of RXR� activity and retinoidunresponsiveness of MDA-MB-231. When MDA-MB-231

FIG. 6. RXR� restores responsiveness to retinoid in MDA-MB-231 cells. MDA-MB-231 cells were infected with RXR� adenovirus. (A)RXRE was transfected into MDA-MB-231 cells, followed by adenovirus infection, and then the cells were treated with either vehicle or ligand for24 h. (B) Immunofluorescence of MDA-MB-231 cells infected with adenovirus-null or adenovirus-RXR� at an MOI of 100. (C) Adenovirus-infected MDA-MB-231 cells were harvested and fixed in 70% ice-cold ethanol and labeled for 2 h at 37°C for TUNEL assay. (E) Adenovirus-infected MDA-MB-231 cells were harvested, and 30 �g of total cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. The results for mock infection (Mock, lanes 1 and 2), adenovirus-null (Null, lanes 3 and 4), and adenovirus-RXR� (RXR�, lanes 5 and6) infection in the presence of vehicle alone or ligand are presented.


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cells were infected with adenoviral RXR�, exogenous RXR�localized throughout the nucleus in addition to the SFC. Nu-cleoplasmic overexpression of RXR� induced apoptosis in ac-cordance with p21 upregulation and bcl-2 downregulation inthe presence of ligand. Taken together, RXR� nucleoplasmiclocalization appears to be one of the major factors determiningthe retinoid sensitivity in the MDA-MB-231.

Several studies have reported that RXR-selective ligands aremore effective than RAR-selective ligands in particular celltypes (50, 53). In addition, these ligands may be also effec-tive against cancer cells; for instance, the RXR pan-agonistLGD1069 has been reported to cause complete regression ofmammary carcinoma (1). These data implicate that there maybe more than one malignant phenotype whose retinoid respon-siveness is RXR dependent. RXR regulates multiple hormonalpathways through heterodimerization with several nuclear re-ceptors, including RAR, PPAR, VDR, LXR, and nur77 (4, 8,17, 33, 52). In MDA-MB-231, RXR� was found in the SFC,and this altered localization was associated with reducedRXR�-mediated transcription. In this respect, other receptorfamily members, which require heterodimer formation withRXR� for transcriptional activation, might be silenced in asimilar fashion if RXR� is the favorable isotype as an het-erodimerization partner. RXR isotypes �, �, and � are ex-pressed in most breast cancer cells, and their expression isindependent of retinoid sensitivity (51). MDA-MB-231 cellsexpress all three RXR isotypes, and RXR� and RXR� localizeto the nucleoplasm (Fig. 2). Reporter assays showed that li-gand-mediated transactivation on RXRE and RARE was notcompletely silenced (Fig. 3D), although RXR� did not colo-calize with active transcription sites in the presence of ligand(Fig. 3C). This incomplete silencing suggests that functionalRXR� and/or RXR� participate in transcription.

RAR� and RAR� protein expression is suppressed inMDA-MB-231 cells (51); thus, RXR� sequestration does notappear to be the only factor in the development of loss ofretinoid sensitivity, but rather multiple factors may be in-volved. In support of this idea, RAR� and RAR� overexpres-sion restores retinoid sensitivity in MDA-MB-231 cells (40,41). Wu et al. have shown that an RXR pan-agonist activatesRXR/nur77 heterodimer binding to �RARE and that RAR�is activated by an RAR�-selective ligand and induces apoptosisin MDA-MB-231 (53). The difference between Wu et al.’sresults and ours can be explained on the basis that our dataspecifically refer to RXR� as being sequestered in the SFC andleave open the possibility that RXR� and/or RXR� may stillbe available for transcription. To date, differential roles orpreferential heterodimerization partners for each RXR isotypehave not been comprehensively understood. To further evalu-ate the role of RXR isotypes, development of each RXR-isotype-selective ligand and conditional gene knockout ap-proaches are essential.

Altered localization of functional proteins occurs by variousmechanisms, including mutation (21), posttranslational modi-fication, and selection of scaffold proteins (54), and results inthe modification of cellular response. The reduction of retinoidresponsiveness is observed due to altered localization ofRXR� when NGFI-B (Nur77) shuttles RXR to the cytoplasmfrom the nucleus (54). Kumar et al. (21) reported that mutatedMTA1s, which is expressed at a high level in ER-negative

breast cancer cells, sequesters ER� in the cytoplasm andcauses loss of ER signaling in ER-negative breast cancer cells.Similarly, mutant E-cadherin is sequestered in the cell mem-brane instead of the nucleus, resulting in silencing Wnt signal-ing pathways (48). Although RXR� mutation is not responsi-ble for SFC localization (data not shown), RXR� may have adifferent posttranslational modification in highly malignantcancer cells because it might have acquired a different set ofinteracting proteins that may shuttle RXR� to the SFC. On theother hand, scaffold or chaperone proteins that do not interactwith RXR� in normal cells could be altered in highly malig-nant cancer cells and misdirect RXR� to the SFC.

RAR� was found in both nucleoplasm and PML bodies, andthis localization pattern was common to all of the cells tested.PML bodies do not share the same intranuclear spatial parti-tioning with SFC. PML bodies are a cluster of proteins, includ-ing PML itself, p53, CBP, and pRb, but do not contain DNA inthe structure and are thought to be involved in transcriptionalregulation, as well as posttranslational modification, or com-partmentalization (57). In the nucleoplasm, PML acts as co-activator in the RAR/RXR heterodimer complex (56). Wefound a part of RAR� localized in the PML bodies, implyingthat RAR� may be temporarily stored in the PML bodies torecruit essential coactivators such as PML into a complex priorto active transcription.

Liganded nuclear receptors fused to GFP (AR, GR, ER,VDR, mineral corticoid receptor, and aryl hydrocarbon recep-tor) and their coactivators have been shown to localize tointranuclear microfoci where active transcription occurs (11,38, 49). Here we showed a ligand-stimulated microfocal local-ization of GFP-RXR� and that this localization was preventedby the transcription inhibitor actinomycin D. This suggests thatthe microfoci are a common site for intranuclear compartmen-talization and active transcription for RXR�, as well as othernuclear receptors and their coactivators. Even though GFP-RXR� localizes to the microfoci, it was not found in the SFCin MDA-MB-231 cells. It is possible that the large GFP moietycauses hindrance of the structural proteins of the scaffold,which interact with RXR�, or the lack of specific posttransla-tional modification(s) that may be required for SFC localiza-tion. Since the microfoci and SFC are separate nuclear com-partments (49, 55), sequestered RXR� in the SFC in MDA-MB-231 cells is unable to take part in active transcription.

In order to test the commonality of the RXR� SFC local-ization pattern, we tested additional cell lines, including theMDA-MB-453, MDA-MB-435, BT-20, and BT-549 breast can-cer cell lines; the SK-OV-3 ovarian cancer cell line; and the I-7,HT-144, S1 cells, Sp1, A2058 keratinocyte, HeLa, and ras NIH3T3 cell lines. We could not find the same RXR� localizationpattern in the SFC, suggesting that RXR� localization in theSFC in MDA-MB-231 cells may represent a phenomenon oc-curring in a small subset of tissue-specific cell types. Interest-ingly, however, immunohistochemistry studies revealed thatRXR� SFC localization was found in mesenchymal cells ofhuman invasive breast tumor tissue. Generally, the generationof a reactive stroma environment occurs in many human can-cers and is likely to promote tumorigenesis (39), and molecularalterations in breast stroma during malignant progression havebeen reported (39). The link between alteration in RXR�


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localization in the SFC and breast microenvironment duringmalignant progression needs further investigation.

Signal peptide for targeting the SFC was recently describedand characterized by a region rich in arginine/serine dipeptides(RS domain) or multiple threonine-proline (TP) repeats (10,20). However, many non-SR proteins that are reported tolocalize to the SFC do not carry a TR repeat or a RS domain,suggesting that more complex mechanisms might be involvedin this localization (29). Even though RXR� does not containthe RS domain or the TP repeats, it remained in the SFC afterDNase or RNase digestion. In addition to nuclease resistance,the RXR� C-terminus peptide caused the release of RXR�from the SFC to the nucleoplasm, and thus this nucleoplas-mic localization increased RXR homodimer-mediated ligand-dependent transactivation (Fig. 5D). This suggests that theRXR� association with SFC may be mediated by a protein-protein interaction between the RXR� C terminus and a com-ponent of the SFC. The secondary structure or posttransla-tional modification in the RXR� C terminus may be importantfor the interaction between RXR� and an SFC component andthe consequent SFC localization. We also could not rule outthat a specific set of molecular chaperones may be involved toguide RXR� to SFC in malignant cancer cells. We initiallyhypothesized that both the C terminus and the N terminus areequally important because epitope tags fused to each terminusinterfered with exogenous RXR� to localize to the SFC (Fig.5A). However, RXR� N-terminus peptide did not restore li-gand-dependent transactivation (Fig. 5D), suggesting thatRXR� N terminus presumably is not interacting directly withthe SFC. Nevertheless, RXR� N terminus may be required tocoordinate the correct folding of the whole protein for SFClocalization. Retinoid receptors are categorized into class IIreceptors which do not require ligand for their nuclear import(16, 34). In fact RXR� was found in the nucleus without ligandstimuli. Controversially, RXR� was found in the nucleoplasmand cytoplasm upon incubation with C-terminus peptide. C-terminus peptide not only rescued RXR� from the SFC butalso may block nuclear import of newly synthesized RXR�from the cytoplasm to the nucleus by masking chaperones thatmight be involved in nuclear import of RXR�. The mechanis-tic details underlying the RXR� localization to various cellularorganelles need further investigation.

In conclusion, RXR� was found in the splicing factor com-partment of the highly malignant breast cancer MDA-MB-231cell line and in mesenchymal cells of tissue sections of severalinvasive carcinomas. These findings suggest that localization ofRXR� changes during malignant progression since RXR� isnormally homogeneously distributed in the nucleoplasm. Ourstudies define a possible mechanism, i.e., sequestration ofRXR�, for retinoid unresponsiveness as frequently encoun-tered in breast cancer cells. These observations offer new in-sights into the silencing of molecular mechanisms possibly in-volved in retinoid signaling pathways.

ACKNOWLEDGMENTS

We thank Ronald M. Evans for generously providing RXR� expres-sion vector. We also thank David J. Mangelsdorf for RXRE andRARE-luc reporter plasmid and Keiko Ozato for help with variousRXR expression vectors. We are grateful to Rosh Chandraratna ofAllergan for the RXR pan-agonist.

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Altered Localization of Retinoid X Receptor Coincides with

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