Altered localization of retinoid X receptor alpha coincides with loss of retinoid responsiveness in human breast cancer MDA-MB-231 cells

Abstract

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 alpha (RXR alpha) 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, RXR alpha 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 RXR alpha induced nucleoplasmic overexpression of RXR alpha and resulted in apoptosis upon treatment with an RXR ligand. This suggests that nucleoplasmic RXR alpha restores retinoid sensitivity. Epitope-tagged RXR alpha and a C-terminus deletion mutant failed to localize to the SFC. Moreover, RXR alpha localization to the SFC was inhibited with RXR alpha C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, the RXR alpha C terminus may play a role in the intranuclear localization of RXR alpha. Our results provide evidence that altered localization of RXR alpha to the SFC may be an important factor for the loss of retinoid responsiveness in MDA-MB-231 breast cancer cells.

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. Bar, 10 μm. The fluorescence intensity does not correspond to the level of protein expression.

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 overlays of double-immunostained images are shown in merged panels (i to l). (B) HMEC, MCF-7, and MDA-MB-231 cells were double immunostained with anti-RXRα and anti-SC-35. (C) RXRα antisense adenovirus was infected to MDA-MB-231 and double immunostained with anti-RXRα (green) and anti-adenovirus type 5 (red). (D) The dimerization partner (RXRγ, PPARα, PPARγ, or RARα) did not colocalize with SC-35. All antibodies were diluted at 1:200. (E) Paraffin-embedded human breast tissue sections were deparaffinized and hydrated. Antigen was retrieved in 10 mM citrate buffer in a microwave for 15 min past boiling.

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 permeabilized and then digested with DNase, followed by treatment with 2 M NaCl and 0.25 M (NH₄)₂SO₄ extraction. The cells were also digested with different concentrations of RNase A for 30 min at 37°C. Digested cells were fixed and immunostained with RXRα (d to f and k to n), followed by counterstaining with Hoechst 33342 (a to c and g to j). The series of images were taken by confocal microscope under the same condition setting. For Western blotting, the cells were either untreated (Un) or treated with DNase (DN), RNase (RN), and different concentrations of RNase (0 to 1,000 μg/ml) as indicated. The cells were lysed in the Laemmli buffer after digestion. Then, 10 μl of whole-cell lysate was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with the indicated antibodies (1:1,000). (C) MDA-MB-231 and MCF-7 cells were treated with 0.2 μM 9-cis-RA for 24 h. Active transcription sites were labeled with BrUTP, followed by double immunostaining with mouse anti-BrdU and rabbit anti-RXRα. (D) RXRE- and RARE-Luc was transfected into MCF-7 and MDA-MB-231 cells (a), and RXRE-Luc was cotransfected with RXRα expression vector or empty vector (b). Cells were treated with vehicle alone or 1 μM RXR ligand for 20 h, and luciferase activity was assessed. The data are presented as means ± the standard deviations of six replicates from two independent experiments.

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. The images showed RXRα-GFP (a and c) and GFP-RXRα (b and d). The arrowhead points to nuclear microfoci. Bar, 5 μm. (B) GFP-RXRα was electroporated to MDA-MB-231, incubated with dimethyl sulfoxide (a) or a mixture of 0.5 μM TTNPB and 0.5 μM RXR-selective ligand (b) for 6 h. The arrow points to nuclear microfoci. (C) GFP-RXRα and RXRα-GFP were introduced into the cell through electroporation, and then the cells were treated with vehicle alone (a to c and g to i) or actinomycin D (d to f and j to l) for 6 h. The cells were fixed and immunostained with anti-SC-35.

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 antibodies. The cell in the square represents a nontransfected cell. (B) Nontagged-RXRα-overexpressing cells were labeled with anti-RXRα and mouse anti-SC-35 antibody. (C) RXRα deletion mutant was electroporated to MDA-MB-231 cells, followed by double immunostaining with anti-RXRα and anti-SC-35. The double line represents the antibody recognition site. About 100 transfected cells were analyzed for quantitative evaluation, and average fluorescence intensities of the speckles in the cells overexpressing the deletion mutant are summarized. To determine how much RXRα, including endogenous 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 RXRα-transfected cells minus the average intensity from endogenous RXRα represents the amount of exogenous RXRα that localizes to the SFCs. The series of images was taken by confocal microscope under the same condition setting. The values represent the means ± the standard errors. The scheme shows the structure of the RXRα deletion mutant. The double line represents the antibody recognition site. (D) A total of 1 μg of C-terminus peptide corresponding to RARγ (a to c) or RXRα (d to i) was incubated for 12 h with MDA-MB-231 cells. The cells were subjected to immunofluorescence after 24 and 48 h with RXRα and SC-35 antibodies. The images were obtained at the same setting for the comparison. Western blotting of lysates prepared from MDA-MB-231 cells treated with 1 μg of C-terminus RARγ, treated with RXRα 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 RXRα or RARγ peptide or into MDA-MB-231 cells left untreated. The cells were treated with vehicle alone or 1 μM RXR ligand for 24 h, and the luciferase activity was assessed. The data are presented as means ± the standard deviations of four replicates from two independent experiments.

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 vehicle or ligand for 24 h. (B) Immunofluorescence of MDA-MB-231 cells infected with adenovirus-null or adenovirus-RXRα 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 μg of total cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results for mock infection (Mock, lanes 1 and 2), adenovirus-null (Null, lanes 3 and 4), and adenovirus-RXRα (RXRα, lanes 5 and 6) infection in the presence of vehicle alone or ligand are presented.