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Mol Cell Biol. May 2004; 24(9): 3972–3982.
PMCID: PMC387734

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


To understand the mechanism of retinoid resistance, we studied the subcellular localization and function of retinoid receptors in human breast cancer cell lines. Retinoid X receptor α (RXRα) localized throughout the nucleoplasm in retinoid-sensitive normal human mammary epithelial cells and in retinoid-responsive breast cancer 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, RXRα was not associated with active transcription site in the presence of ligand. Similarly, ligand-dependent RXR homo- or heterodimer-mediated transactivation on RXR response element or RARE showed minimal response to ligand in MDA-MB-231 cells. Infecting MDA-MB-231 cells with adenoviral RXRα induced nucleoplasmic overexpression of RXRα and resulted in apoptosis upon treatment with an RXR ligand. This suggests that nucleoplasmic RXRα restores retinoid sensitivity. Epitope-tagged RXRα and a C-terminus deletion mutant failed to localize to the SFC. Moreover, RXRα localization to the SFC was inhibited with RXRα C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, the RXRα C terminus may play a role in the intranuclear localization of RXRα. Our results provide evidence that altered localization of RXRα to the SFC may be an important factor for the loss of retinoid responsiveness in MDA-MB-231 breast cancer cells.

Retinoids are natural and synthetic vitamin A derivatives which regulate development (36), cell proliferation (24), and differentiation (7). Retinoids also act as cancer preventive agents and are presently being used successfully to treat certain types of cancer (6, 45). Although many studies have shown retinoid effectiveness on inhibition of cancer cell growth in vitro and in vivo (18), the clinical usage of vitamin A and its derivatives is currently limited by the requirement of a large dosage to reach therapeutic efficacy. The combination of synthetic retinoid and tamoxifen inhibited the growth of estrogen-positive breast cancers in premenopausal patients; however, it failed to show any significant effect on advanced breast cancer patients (2, 3, 30). It is likely that the responsiveness of cancer cells to retinoid diminishes, along with malignant progression. Indeed, growth inhibitory effects of retinoids have been observed in estrogen receptor (ER)-positive breast cancer cell lines such as MCF-7 and T-47D (19), whereas the effectiveness of retinoid diminishes in highly malignant ER-negative breast cancer cell lines such as MDA-MB-231 and BT-20 (5, 13, 14, 35, 53). The existing hormonal and chemotherapeutic therapies have provided significant improvement for the survival of patients with localized breast cancer; however, treatment for metastatic breast cancer still remains palliative (31). The 5-year survival ratio for patients diagnosed with metastatic breast cancer is only 15%. Thus, there is an urgent need to understand the mechanism of retinoid resistance in order to develop therapeutic agents for metastatic breast cancer.

The physiological actions of retinoids are mediated through two distinct nuclear receptor families (12, 26): the retinoic acid receptors (RARα, RARβ, and RARγ), each of which binds both all-trans-retinoic acid or 9-cis-retinoic acid (9-cis-RA), and the retinoid X receptors (RXRα, RXRβ, and RXRγ), which preferentially bind 9-cis-retinoic acid. RARs and RXRs bind to a specific DNA response element (RARE and RXRE, respectively) in the 5′-flanking region of target genes as homodimers or heterodimers, thereby promoting gene transcription (25). Since RAR and RXR play a central role in mediating retinoid action in the physiology of normal cells, it is likely that they are also involved in anticancer effects of retinoids.

Retinoid receptors are members of the steroid-thyroid-vitamin D receptor superfamily. This superfamily can be subdivided into two classes according to the partitioning of the unliganded receptor in the cells. The first group of receptors, the glucocorticoid receptor (GR), the mineral corticoid receptor, the ER, the progesterone receptor, and the androgen receptor (AR) localize predominantly within the cytoplasm in the absence of ligand (34). Receptors in this group bind to hsp90 upon ligand binding and translocate to the nucleus (34). In contrast to these receptors, the thyroid and retinoid receptors tightly associate with the nucleus independent of the presence of the ligand (16, 34). The liganded nuclear receptor proteins, AR, GR, ER, and aryl-hydrocarbon receptor have been shown to localize in 250 to 400 microfoci (11, 38, 49). They recruit transcription cofactors such as SRC-1, TIF-II, and p300/CBP into microfoci, and active transcription occurs (38). With respect to spatial distribution, the mechanism by which a specific transcription factor recruits its dimerization partner and accesses the active transcription site is not clearly understood yet.

In the present study, we focused on subcellular localization of the retinoid receptors to understand the mechanism whereby breast cancer cells become refractory to retinoids.



In addition to the abbreviations introduced above, the following abbreviations are used here: PPAR, peroxisome proliferator-activated receptor; VDR, vitamin D receptor; SFC, splicing factor compartment; PML, promyelocytic 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.


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

Cell culture.

Human mammary epithelial cells (HMEC) were purchased from Cambrex (Rockland, Minn.) and maintained in mammary epithelial growth medium. MCF-7 and MDA-MB-231 breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, Md.) and maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum in a humidified atmosphere supplemented with 5% CO2 at 37°C. The subconfluent cells were treated with trypsin and seeded onto two-well glass chamber slides. On the 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 by two washes in PBS and permeabilization with 0.2% Triton X-100. After a brief wash with PBS, the slides were blocked with 10% goat serum for 1 h at room temperature and then incubated overnight at 4°C or for 1 h at room temperature with the following primary antibodies diluted in goat serum: rabbit anti-RXRα (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, Cleveland, 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 isothiocyanate (FITC)-coupled mouse anti-Flag (1:500; Invitrogen, Carlsbad, Calif.). The same amount of affinity-purified normal immunoglobulin G (IgG) from the corresponding species was used as a negative control. Slides were washed with PBS extensively and incubated with goat anti-rabbit FITC and/or goat anti-mouse Texas red, donkey anti-goat TRITC (tetramethyl rhodamine isothiocyanate; 1:300; Jackson Laboratories, West Grove, Pa.) in 0.5% IgG-free bovine serum albumin for 1 h at room temperature in the dark. The slides were then washed with PBS and rinsed with deionized water. Nuclear counterstaining was performed with Hoechst 33342 (Sigma) for 5 min at room temperature. Paraffin-embedded blocks of human breast tissue from the Cooperative Human Tissue Network of the University of Pennsylvania Medical Center, Philadelphia, were sliced to obtain 6-μm-thick sections for immunohistochemistry of RXRα according to the method of Lawrence et al. (22). Antigen was retrieved in 10 mM citrate buffer (pH 6.0) in a microwave for 15 min past boiling. Rabbit anti-RXRα and mouse anti-SC-35 antibodies were applied in a 1:50 and 1:1,000 dilution, respectively. Each section was stained with hematoxylin and eosin for pathological diagnosis. Confocal images were captured by using a Zeiss confocal microscope and processed by using Zeiss LMS 5 Image. Series of images were obtained under the same conditions when fluorescence intensity needed to be compared.

Western blotting.

The cell pellets were lysed in Laemmli buffer and centrifuged, and the lysates were loaded onto 4 to 12% bis-Tris polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% blocking milk, probed with rabbit anti-RXRα, RARα, p21, and mouse anti-hnRNP K (Santa Cruz Biotechnology), bcl-2, and Bax (Calbiochem, La Jolla, Calif.). The blots were washed with PBS-Tween, incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse antibody, and visualized by chemiluminescence (Pierce, Rockford, Ill.).

Plasmid construct and transfection.

RXRα insert was excised from pCMX-hRXRα and then subcloned into phrGFP-N1 (Stratagene) and pcDNA 3.1 (N-terminus Flag tag). C-terminus GFP-tagged RXRα was generated by PCR with sense primer (5′-GGATCCATGGACACCAAACATTTC-3′), antisense primer (5′-CTCGAGAGTCATTTGGTGCGGCGCCTC-3′) ligated into phrGFP-C (Stratagene, La Jolla, Calif.), and C-terminus HA tag pMH (Roche). The full-length DNA sequence of RXRα was confirmed by using a dye terminator reaction (ABI Prism, Foster City, Calif.). Then, 106 of trypsin-treated MCF-7 and MDA-MB-231 cells were mixed with 5 μg of supercoiled plasmid DNA, followed by electroporation at 180 V for ca. 70 ms in a 4-mm cuvette by using ECM 830 (BTX, Holliston, Mass.). The electroporated cells were seeded in a two-well slide and cultured for 24 to 48 h. Live cell image was taken 24 h after transfection. Nontagged RXRα, Flag-tagged RXRα, and HA-tagged RXRα transfected cells were fixed and used for immunofluorescence with corresponding antibody as described above.

Labeling of transcription site.

The cells were grown in two-well slides for 2 days. 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 mM phenylmethylsulfonyl fluoride [PMSF]), followed by permeabilization with 0.05% Triton X-100 for 10 min on ice. The cells were washed with glycerol buffer to remove excess detergent and then incubated for 10 min at 37°C with transcription buffer (50 mM Tris-HCl [pH 7.4], 100 mM KCl, 25% glycerol, 5 mM MgCl2, 1 mM PMSF, 25 U of RNase inhibitor/ml) supplemented with 2 mM ATP, 0.5 mM CTP and GTP, and 0.2 mM BrUTP. After incorporation, the cells were fixed with 4% paraformaldehyde in PBS for 20 min and evaluated by immunofluorescence with mouse anti-BrdU and rabbit anti-RXRα antibodies.

Nuclear digestion.

Cells were digested, with a modification of the method described by Nickerson et al. (32). The cells were washed three times in cytoskeleton 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 μg of aprotinin/ml} and incubated with the cytoskeleton buffer containing 0.5% 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 μg of aprotinin/ml) and then incubated with RBS buffer containing 1% Tween 20 and 0.3% of deoxycholate for 10 min at 4°C. Chromatin was removed by treatment 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 mM PMSF, 10 μg of aprotinin/ml) for 1 h at 37°C. To complete digestion, ammonium sulfate was added to a final concentration of 0.25 M, and the mixture was incubated for 5 min at 4°C, followed by 2 M NaCl for 5 min. For the removal of RNA, cells were incubated in digestion buffer containing the indicated concentrations of RNase A for 30 min at 37°C. The cells were washed extensively with ice-cold PBS, fixed with 4% paraformaldehyde for 15 min, and then used for immunofluorescence 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 activity according to the manufacturer's instructions (Promega, Madison, Wis.). The light intensity was measured by a luminometer Tropix TR717 (Perkin-Elmer, Boston, Mass.). The transfection efficiency was normalized to TK. The data shown are the means of at least six separate experiments.

Peptide delivery.

Peptide delivery was performed according to the manufacturer's instructions (GTS, San Diego, Calif.). A total of 1 μg of (i) C-terminus peptide from RXRα (441-IDTFLMEMLEAPHQMT-456), (ii) N-terminus peptide from RXRα-N (1-MDTKHFLPLDFSTQVNSSLT-20), or (iii) the control peptide, the C-terminus peptide of RARγ (441-VPGGQGKGGLKSPA-454), which has no homology to the C-terminal sequence of RXRα, was delivered to the cells in the presence of the delivery agent. The cells were further incubated for 24 to 48 h after peptide delivery. For simultaneous plasmid transfection and peptide delivery, the cells were cultured in antibiotic-free DMEM supplemented with 10% serum for 24 h. On the following day, the cells were preincubated with 1 μg of RXRα-C-terminus, RXRα-N, or RARγ-C-terminus peptide for 5 min, followed by plasmid transfection for 3 h. The cells were recovered overnight and incubated with ligand for 24 h, and the reporter activity was measured.

TUNEL assay.

Flow cytometry TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay was performed according to the manufacturer's instructions (Pharmingen, San Diego, Calif.). Stained cells were analyzed by flow cytometry using the FACSCalibur (BD Biosciences, San Jose, Calif.).

Recombinant adenovirus construction and propagation.

The complete coding sequence of wild-type RXRα cDNA was subcloned into shuttle vector containing cytomegalovirus promoter and simian virus 40 poly(A). To obtain recombinant plasmid, pAdEasy-1 and shuttle vector (Stratagene) were mixed and transformed. The recombinant plasmid was transfected to HEK-293 cells to generate adenovirus. 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.


Altered localization of RXRα coincides with lack of responsiveness to retinoid.

Retinoic acid inhibited growth of HMEC and MCF-7 human breast cancer cells (not shown). Immunofluorescence confocal microscopy revealed that RARα localized throughout the nucleoplasm diffusely and in microspeckles in HMEC, MCF-7 cells, and MDA-MB-231 cells (Fig. (Fig.1,1, top panels). In sharp contrast to RXRα homogeneous nuclear distribution in retinoid-sensitive HMEC and MCF-7 cells, RXRα showed a distinct punctate pattern in the retinoid-resistant MDA-MB-231 cells (Fig. (Fig.1,1, bottom panels). Control experiments with the same amount of normal rabbit IgG or with blocking peptides verified that the RXRα and RARα signal was specific (data not shown).

FIG. 1.
Altered localization of RXRα coincides with lack of retinoid responsiveness. Cells were grown in two-well chamber slides. The slides were fixed, and then subcellular localization of RARα and RXRα was detected by immunofluorescence. ...

Identification of subnuclear localization of RXRα in MDA-MB-231 cells.

Confocal microscopy was used to pinpoint subnuclear localization of RXRα in the MDA-MB-231 cells (Fig. (Fig.2A).2A). The subnuclear structures were visualized by using Texas red-labeled secondary antibody to different nuclear protein antibodies (Fig. 2A, panels a to d). RXRα was visualized by using FITC-labeled secondary antibody (Fig. 2A, panels e to h). RXRα did not colocalize with NPC (Fig. (Fig.2A,2A, panel i) or PML bodies (Fig. (Fig.2A,2A, panel j). However, extensive colocalization was observed between RXRα and SC-35 (Fig. (Fig.2A,2A, panel k), a splicing factor known to localize to SFC. RXRα was also found to colocalize with p105 (Fig. (Fig.2A,2A, panel l), a component of interchromatin granule cluster, which is a substructure of the SFC.

FIG. 2.
Colocalization of RXRα with nuclear organelle proteins. (A) Fixed MDA-MB-231 cells were double immunostained with RXRα (e to h) and the indicated nuclear protein antibody anti-NPC (a), anti-PML (b), anti-SC-35 (c), or anti-p105 (d). Confocal ...

Double immunostaining demonstrated that RARα microspeckles colocalized with PML bodies but not with SC-35 and p105 (data not shown). Figure Figure2B2B shows that RXRα localization to the SFC was found only in retinoid-resistant MDA-MB-231 cells and not in retinoid-sensitive HMEC and MCF-7 cells. MDA-MB-231 cells infected with RXRα antisense adenovirus were immunostained for RXRα (green) and adenovirus (red) (Fig. (Fig.2C).2C). The RXRα was found in the SFC in mock- and null-infected cells but absent from RXRα antisense-infected MDA-MB-231 cells (Fig. (Fig.2C),2C), indicating that RXRα localization to the SFC was specific. RXR dimerization partners, RARα, RXRγ, and PPARβ and PPARγ did not localize to the SFC, suggesting that they do not form dimers with RXRα in the SFC (Fig. (Fig.2D).2D). These staining patterns were commonly observed in all cells tested. To confirm our in vitro results, colocalization between RXRα and SC-35 was tested in human breast tissue. We tested tissue sections and adjacent normal region from 12 patients with invasive breast cancer. RXRα was found in the SFC of mesenchymal cells of 5 of 12 invasive breast carcinomas (Fig. (Fig.2E).2E). However, SFC localization of RXRα was not detected in any of the normal tissue or in benign hyperplasias of the three breast tissues tested (data not shown). Hematoxylin and eosin staining showed a representative area.

RXRα was silenced due to SFC localization.

To examine whether the localization of RXRα to the SFC depends on the transcription status of MDA-MB-231 cells, the cells were treated with actinomycin D or transcription activator. Some of the SFC became bigger when transcription was inhibited in the presence of actinomycin D (Fig. (Fig.3A).3A). In order to mimic active transcription, cells were treated with TSA, 9-cis-RA, the natural ligand of RXRα, or AGN194204, a pan-agonist of RXR. Against our expectation, RXRα remained in the SFC in the presence of excess amounts of TSA, 9-cis-RA, and AGN104204 (Fig. (Fig.3A).3A). These results demonstrate that the ligand did not induce relocalization of RXRα from the SFC to the nucleoplasm, where the active transcriptional machinery is present. In order to determine whether RXRα associates with nucleic acid in the SFC, the nuclei were digested with DNase I or RNase. Interactions between chromatin, RNA, and their binding proteins are generally disrupted during this preparation. The speckled pattern of RXRα remained intact after DNA and RNA removal, suggesting that RXRα does not interact with DNA or RNA in MDA-MB-231 cells (Fig. (Fig.3B,3B, panel f). Western blotting confirmed that RXRα was resistant to DNase and RNase digestion. In contrast, RARα was sensitive to DNase digestion, suggesting that RARα is physically associated with DNA (Fig. (Fig.3B).3B). Concentrations of RNase up to 1,000 μg had no effect or slightly reduced the RXRα speckled pattern (Fig. 3B, panels k to n). Corresponding samples were analyzed by Western blotting and showed that RXRα was resistant to RNase digestion, whereas hnRNP, which localizes to the SFC and tightly interacts with RNA, was removed efficiently by RNase (Fig. (Fig.3B).3B). In contrast to MDA-MB-231 cells, RXRα was completely removed by DNase in MCF-7 cells, and no speckled pattern was revealed (data not shown), suggesting that RXRα interacts with DNA in MCF-7 cells. To examine whether RXRα participates in transcription, nascent transcripts were labeled with BrUTP and double immunostained with RXRα. RXRα did not colocalize with active transcription sites in MDA-MB-231 cells, but it colocalized extensively in MCF-7 cells (Fig. (Fig.3C).3C). These results demonstrate that RXRα is actively participating in transcription in MCF-7 cells but not in MDA-MB-231 cells. These staining patterns were commonly observed in all cells tested.

FIG. 3.
RXRα is silenced due to SFC localization. (A) The cells were treated with vehicle alone (a), 0.5 μg of actinomycin D/ml (b), 100 nM TSA (c), 1 μM 9-cis-RA (d), or 1 μM RXR agonist (e) for 24 h. (B) MDA-MB-231 cells were ...

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

Subcellular localization of exogenous RXRα.

To further characterize RXRα localization to the SFC, we compared the localization of exogenous RXRα to that of endogenous RXRα. GFP was fused to the C or N terminus of RXRα and electroporated to MCF-7 and MDA-MB-231 cells. Figure Figure4A4A shows the live cell image of GFP-fused RXRα. GFP fused either to the C or N terminus of RXRα showed a diffuse nucleoplasmic localization, excluding nucleoli, and a number of bright microfoci in the nucleus of both MCF-7 and MDA-MB-231 cells (Fig. (Fig.4A).4A). GFP-RXRα accumulated in these microfoci when the cells were treated with ligand for 6 h (Fig. (Fig.4B,4B, panel b). Next, we examined whether GFP-fused RXRα colocalizes with SC-35 in the SFC of MDA-MB-231 cells. The cells were permeabilized and immunostained with anti-SC-35 antibody. Figure Figure4C4C shows that neither the C- nor N-terminus GFP-fused RXRα expression pattern overlaps with anti-SC-35 (Fig. 4C, panels c and i). Next, the cells expressing GFP-fused RXRα were treated with actinomycin D to take advantage of its characteristics that facilitate SFC localization in the absence of transcription. Actinomycin D treatment did not cause relocalization of GFP-fused RXRα from the nucleoplasm to the SFC (Fig. 4C, panels f and l), whereas SC-35 accumulated in the SFC (Fig. 4B, panels e and k). Further, cell nuclei from GFP-RXRα-overexpressing cells were digested by DNase or RNase to remove nucleoplasmic GFP-RXRα signal. GFP-RXRα was not found in the SFC. Interestingly, GFP-RXRα microfoci disappeared when transcription was inhibited by actinomycin D and diffused throughout the nucleus, including the nucleolus (Fig. 4C, panels d and j). These staining patterns were common to all transfected cells tested.

FIG. 4.
Subcellular localization of GFP-RXRα. (A) GFP-RXRα, RXRα-GFP, or GFP vector alone was electroporated to MCF-7 (a and b) and MDA-MB-231 (c and d) cells. At 24 h after electroporation, live cell images were obtained by confocal microscopy. ...

Possible role of RXRα C terminus in SFC localization.

To rule out the possibility that the GFP moiety might lead to changes in secondary structure of RXRα, smaller epitope tag, Flag (N-terminal 8 amino acid) or HA (C-terminal 9 amino acid) was fused to RXRα. They were found to localize homogeneously throughout the nucleus but not in the SFC (Fig. (Fig.5A).5A). Since exogenous epitope-tagged RXRα did not show the same localization pattern as endogenous RXRα, the open reading frame of RXRα of MCF-7 and MDA-MB-231 was sequenced and found to be identical to wild-type RXRα (data not shown). Thus, genetic alteration of RXRα is not responsible for this localization in MDA-MB-231. Next, MDA-MB-231 cells were transfected with a nontagged RXRα. The cell in the square (Fig. (Fig.5A,5A, right panel) shows endogenous RXRα, while the cell next to it, showing homogeneous nuclear staining pattern in addition to distinct speckles, represents exogenous nontagged RXRα overexpression (Fig. (Fig.5A).5A). Speckles found in cells overexpressing the nontagged RXRα were larger and markedly brighter than those of endogenous RXRα in the SFC. These speckles colocalized with SC-35 (Fig. (Fig.5B)5B) in the SFC. However, the staining signals reached a plateau as we increased the amount of DNA used for transfection (data not shown), implying that SFC have limited capacity to interact with RXRα and that excess RXRα distributes throughout the nucleoplasm (Fig. (Fig.5B).5B). These results indirectly suggest that the secondary structure of either N- or C-terminal RXRα may be critical for SFC localization. To test this hypothesis, the C-terminus-truncated RXRαΔ416 and full-length RXRα, which have the same plasmid backbone, were generated. Interestingly, RXRαΔ416 showed speckles of smaller size and reduced fluorescence compared to the wild type (Fig. (Fig.5C).5C). Approximately 100 transfected cells were analyzed for quantitative evaluation, and the average speckle fluorescence in those cells is summarized in the graph (Fig. (Fig.5C).5C). The average fluorescence of cells overexpressing full-length RXRα was 119.7 ± 5.6, approximately twofold more than that of the endogenous RXRα, whereas that of RXRαΔ416 was 68.4 ± 7. Western blotting showed similar a expression level in both RXRα and RXRΔ416 (Fig. (Fig.5C).5C). This excludes the possibility that the observed reduced fluorescence intensity of RXRΔ412 is due to lower transfection efficiency.

FIG. 5.
Localization of epitope-tagged RXRα and nontagged RXRa. (A) Flag-RXRα (a), RXRα-HA (b), or nontagged RXRα (c) was electroporated into MDA-MB-231 cells. The cells were fixed and used for immunofluorescence with corresponding ...

To establish a role of the C terminus of RXRα in SFC localization, MDA-MB-231 cells were treated with RXRα C-terminus-specific peptide. At 24 h, RXRα was colocalized with SC-35 but was also detected in the cytoplasm (Fig. 5D, panels d to f). At 48 h of incubation RXRα distributed throughout the cell and no longer remained in the SFC, and colocalization between SC-35 and RXRα disappeared (Fig. 5D, panels g to i). The same amount of control peptide corresponding to the C-terminus RARγ did not affect the RXRα punctate pattern (Fig. 5D, panels a to c). Furthermore, cells incubated with RXRα C-terminus peptide showed a ~2.5-fold increase in ligand-mediated transactivation on RXRE, whereas the RXRα N-terminus peptide did not show any effect, suggesting that the RXRα C terminus may interact directly with the SFC. RARγ C-terminus peptide did not affect ligand-dependent transactivation on RXRE (Fig. (Fig.5D).5D). RXRα immunoblotting showed that peptide delivery did not alter the level of the RXRα protein (Fig. (Fig.5D).5D). Therefore, it appears that the F region of RXRα is involved in intranuclear localization.

RXRα restored the responsiveness to retinoid in MDA-MB-231 cells.

MDA-MB-231 cells were infected with RXRα adenovirus. With an adenovirus-RXRα MOI of 100, RXR-selective ligand promoted transactivation via RXRE (Fig. (Fig.6A).6A). RXRα was overexpressed throughout the nucleoplasm in addition to the SFC (Fig. (Fig.6B).6B). MDA-MB-231 cells infected with RXRα adenovirus for 24 h, followed by incubation with RXR-selective ligand for 48 h, demonstrated a drastic decrease in the number of viable cells by 40% of mock- and null-infected MDA-MB-231 cells (Fig. (Fig.6C).6C). Apoptosis was measured by TUNEL assay. Adenovirus RXRα-infected MDA-MB-231 cells increased TUNEL-positive cells by >60% in the presence of ligand within 96 h of adenovirus infection, whereas the cells with mock and adenovirus null infected in the presence of ligand showed only 5% TUNEL positivity (Fig. (Fig.6D).6D). These data indicate that nucleoplasmic overexpression of RXRα stimulates apoptosis in the MDA-MB-231 cells. Next, to test apoptosis-related protein expression, the cells were harvested after 96 h of incubation with ligand or vehicle alone. The induction of ligand-induced apoptosis was correlated with the upregulation of p21 and downregulation of bcl-2, but Bax expression was unchanged (Fig. (Fig.6E6E).

FIG. 6.
RXRα restores responsiveness to retinoid in MDA-MB-231 cells. MDA-MB-231 cells were infected with RXRα adenovirus. (A) RXRE was transfected into MDA-MB-231 cells, followed by adenovirus infection, and then the cells were treated with either ...


Retinoids control cell proliferation and differentiation through the action of RARs and RXRs and have chemopreventive effects in various tumor types, including the skin, prostate, ovary, leukemia, and breast (15, 23, 37, 42, 46). The anticancer effect of retinoids is lost during tumor progression; however, the mechanism underlying the loss of retinoid responsiveness is still poorly understood. In the present study, our data demonstrate that RXRα localizes to the SFC in the highly malignant ER-negative, retinoid-resistant MDA-MB-231 human breast cancer cells. Furthermore, our histological data indicate that RXRα localizes to the SFC in human breast carcinoma in vivo. Our data provide functional evidence that most, if not all, endogenous RXRα is sequestered in the SFC and silenced and, consequently, retinoid signaling appears to be shut off in the MDA-MB-231 cells.

Splicing factors localize in 20 to 40 nuclear domains referred to as the SFC in the mammalian cell nucleus (28). SFC is composed of two distinct structures, the interchromatin granule clusters and perichromatin fibrils (9, 27, 43). Splicing factor localization in the SFC is generally highly dynamic and is influenced by transcriptional activity, cell cycle, and phosphorylation (44). Vitamin D receptor B1 (VDRB1), a heterodimerization partner of RXR, is also found in the SFC (47), and it is redistributed throughout the nucleoplasm upon exposure to its ligand, 1,25-dihydroxyvitamin D3. We found that, in contrast to the ligand-induced dynamic intranuclear mobility of VDRB1, the ligand failed to redistribute RXRα from the SFC to the nucleoplasm in MDA-MB-231 cells. This finding allowed us to hypothesize that RXRα might be sequestered in the SFC, leading to retinoid unresponsiveness.

We demonstrated that RXRα was not localized to active transcription sites in MDA-MB-231 cells but showed extensive colocalization with nascent transcripts in MCF-7 cells. This result was further confirmed by reporter assays when ligand promoted RXRE (RXR homodimer target) or RARE (RAR/RXR heterodimer target) transactivation in MCF-7 cells but failed to do so in MDA-MB-231 cells. The absence of ligand-dependent transcriptional activation in MDA-MB-231 cells was not due to the reduction of RXRα protein expression level because RXRα protein level in retinoid-sensitive HMEC was the same as in MDA-MB-231 cells (data not shown). Thus, our interest was then to investigate whether altered localization of RXRα could explain the loss of RXRα activity and retinoid unresponsiveness of MDA-MB-231. When MDA-MB-231 cells were infected with adenoviral RXRα, exogenous RXRα localized throughout the nucleus in addition to the SFC. Nucleoplasmic overexpression of RXRα induced apoptosis in accordance with p21 upregulation and bcl-2 downregulation in the presence of ligand. Taken together, RXRα nucleoplasmic localization appears to be one of the major factors determining the retinoid sensitivity in the MDA-MB-231.

Several studies have reported that RXR-selective ligands are more effective than RAR-selective ligands in particular cell types (50, 53). In addition, these ligands may be also effective against cancer cells; for instance, the RXR pan-agonist LGD1069 has been reported to cause complete regression of mammary carcinoma (1). These data implicate that there may be more than one malignant phenotype whose retinoid responsiveness is RXR dependent. RXR regulates multiple hormonal pathways through heterodimerization with several nuclear receptors, including RAR, PPAR, VDR, LXR, and nur77 (4, 8, 17, 33, 52). In MDA-MB-231, RXRα was found in the SFC, and this altered localization was associated with reduced RXRα-mediated transcription. In this respect, other receptor family members, which require heterodimer formation with RXRα for transcriptional activation, might be silenced in a similar fashion if RXRα is the favorable isotype as an heterodimerization partner. RXR isotypes α, β, and γ are expressed in most breast cancer cells, and their expression is independent of retinoid sensitivity (51). MDA-MB-231 cells express all three RXR isotypes, and RXRβ and RXRγ localize to the nucleoplasm (Fig. (Fig.2).2). Reporter assays showed that ligand-mediated transactivation on RXRE and RARE was not completely silenced (Fig. (Fig.3D),3D), although RXRα did not colocalize with active transcription sites in the presence of ligand (Fig. (Fig.3C).3C). This incomplete silencing suggests that functional RXRβ and/or RXRγ participate in transcription.

RARα and RARβ protein expression is suppressed in MDA-MB-231 cells (51); thus, RXRα sequestration does not appear to be the only factor in the development of loss of retinoid sensitivity, but rather multiple factors may be involved. In support of this idea, RARα and RARβ overexpression restores retinoid sensitivity in MDA-MB-231 cells (40, 41). Wu et al. have shown that an RXR pan-agonist activates RXR/nur77 heterodimer binding to βRARE and that RARβ is activated by an RARβ-selective ligand and induces apoptosis in MDA-MB-231 (53). The difference between Wu et al.'s results and ours can be explained on the basis that our data specifically refer to RXRα as being sequestered in the SFC and leave open the possibility that RXRβ and/or RXRγ may still be available for transcription. To date, differential roles or preferential heterodimerization partners for each RXR isotype have not been comprehensively understood. To further evaluate the role of RXR isotypes, development of each RXR-isotype-selective ligand and conditional gene knockout approaches are essential.

Altered localization of functional proteins occurs by various mechanisms, including mutation (21), posttranslational modification, and selection of scaffold proteins (54), and results in the modification of cellular response. The reduction of retinoid responsiveness is observed due to altered localization of RXRβ when NGFI-B (Nur77) shuttles RXR to the cytoplasm from the nucleus (54). Kumar et al. (21) reported that mutated MTA1s, which is expressed at a high level in ER-negative breast cancer cells, sequesters ERα in the cytoplasm and causes loss of ER signaling in ER-negative breast cancer cells. Similarly, mutant E-cadherin is sequestered in the cell membrane instead of the nucleus, resulting in silencing Wnt signaling pathways (48). Although RXRα mutation is not responsible for SFC localization (data not shown), RXRα may have a different posttranslational modification in highly malignant cancer cells because it might have acquired a different set of interacting proteins that may shuttle RXRα to the SFC. On the other hand, scaffold or chaperone proteins that do not interact with RXRα in normal cells could be altered in highly malignant cancer cells and misdirect RXRα to the SFC.

RARα was found in both nucleoplasm and PML bodies, and this localization pattern was common to all of the cells tested. PML bodies do not share the same intranuclear spatial partitioning with SFC. PML bodies are a cluster of proteins, including PML itself, p53, CBP, and pRb, but do not contain DNA in the structure and are thought to be involved in transcriptional regulation, as well as posttranslational modification, or compartmentalization (57). In the nucleoplasm, PML acts as coactivator in the RAR/RXR heterodimer complex (56). We found a part of RARα localized in the PML bodies, implying that RARα may be temporarily stored in the PML bodies to recruit essential coactivators such as PML into a complex prior to active transcription.

Liganded nuclear receptors fused to GFP (AR, GR, ER, VDR, mineral corticoid receptor, and aryl hydrocarbon receptor) and their coactivators have been shown to localize to intranuclear microfoci where active transcription occurs (11, 38, 49). Here we showed a ligand-stimulated microfocal localization of GFP-RXRα and that this localization was prevented by the transcription inhibitor actinomycin D. This suggests that the microfoci are a common site for intranuclear compartmentalization and active transcription for RXRα, as well as other nuclear receptors and their coactivators. Even though GFP-RXRα localizes to the microfoci, it was not found in the SFC in MDA-MB-231 cells. It is possible that the large GFP moiety causes hindrance of the structural proteins of the scaffold, which interact with RXRα, or the lack of specific posttranslational modification(s) that may be required for SFC localization. Since the microfoci and SFC are separate nuclear compartments (49, 55), sequestered RXRα in the SFC in MDA-MB-231 cells is unable to take part in active transcription.

In order to test the commonality of the RXRα SFC localization pattern, we tested additional cell lines, including the MDA-MB-453, MDA-MB-435, BT-20, and BT-549 breast cancer cell lines; the SK-OV-3 ovarian cancer cell line; and the I-7, HT-144, S1 cells, Sp1, A2058 keratinocyte, HeLa, and ras NIH 3T3 cell lines. We could not find the same RXRα localization pattern in the SFC, suggesting that RXRα localization in the SFC in MDA-MB-231 cells may represent a phenomenon occurring in a small subset of tissue-specific cell types. Interestingly, however, immunohistochemistry studies revealed that RXRα SFC localization was found in mesenchymal cells of human invasive breast tumor tissue. Generally, the generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis (39), and molecular alterations in breast stroma during malignant progression have been reported (39). The link between alteration in RXRα localization in the SFC and breast microenvironment during malignant progression needs further investigation.

Signal peptide for targeting the SFC was recently described and 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 to localize to the SFC do not carry a TR repeat or a RS domain, suggesting that more complex mechanisms might be involved in this localization (29). Even though RXRα does not contain the RS domain or the TP repeats, it remained in the SFC after DNase or RNase digestion. In addition to nuclease resistance, the RXRα C-terminus peptide caused the release of RXRα from the SFC to the nucleoplasm, and thus this nucleoplasmic localization increased RXR homodimer-mediated ligand-dependent transactivation (Fig. (Fig.5D).5D). This suggests that the RXRα association with SFC may be mediated by a protein-protein interaction between the RXRα C terminus and a component of the SFC. The secondary structure or posttranslational modification in the RXRα C terminus may be important for the interaction between RXRα and an SFC component and the consequent SFC localization. We also could not rule out that a specific set of molecular chaperones may be involved to guide RXRα to SFC in malignant cancer cells. We initially hypothesized that both the C terminus and the N terminus are equally important because epitope tags fused to each terminus interfered with exogenous RXRα to localize to the SFC (Fig. (Fig.5A).5A). However, RXRα N-terminus peptide did not restore ligand-dependent transactivation (Fig. (Fig.5D),5D), suggesting that RXRα N terminus presumably is not interacting directly with the SFC. Nevertheless, RXRα N terminus may be required to coordinate the correct folding of the whole protein for SFC localization. Retinoid receptors are categorized into class II receptors which do not require ligand for their nuclear import (16, 34). In fact RXRα was found in the nucleus without ligand stimuli. Controversially, RXRα was found in the nucleoplasm and cytoplasm upon incubation with C-terminus peptide. C-terminus peptide not only rescued RXRα from the SFC but also may block nuclear import of newly synthesized RXRα from the cytoplasm to the nucleus by masking chaperones that might be involved in nuclear import of RXRα. The mechanistic details underlying the RXRα localization to various cellular organelles need further investigation.

In conclusion, RXRα was found in the splicing factor compartment of the highly malignant breast cancer MDA-MB-231 cell line and in mesenchymal cells of tissue sections of several invasive carcinomas. These findings suggest that localization of RXRα changes during malignant progression since RXRα is normally homogeneously distributed in the nucleoplasm. Our studies define a possible mechanism, i.e., sequestration of RXRα, for retinoid unresponsiveness as frequently encountered in breast cancer cells. These observations offer new insights into the silencing of molecular mechanisms possibly involved in retinoid signaling pathways.


We thank Ronald M. Evans for generously providing RXRα expression vector. We also thank David J. Mangelsdorf for RXRE and RARE-luc reporter plasmid and Keiko Ozato for help with various RXR expression vectors. We are grateful to Rosh Chandraratna of Allergan for the RXR pan-agonist.


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