January 2006
Volume 5
|
January 2006
|
|
|
|
|
|||||||
|
 |
|||||||
|
Contents
*To download a copy |
Altered Localization of RXRα Coincides with Loss of Retinoid Responsiveness in Human Breast Cancer
The physiological actions of retinoids are mediated through two distinct nuclear receptor families, the retinoic acid receptors (RAR α, β, and γ), each of which binds all-trans-retinoic acid or 9-cis-retinoic acid, and the retinoid X receptors (RXR α, β, and γ), which preferentially bind 9-cis-retinoic acid. RARs and RXRs bind to a specific DNA response element (RARE) in the 5′-flanking region of target genes as homodimers or heterodimers, thereby promoting gene transcription. We studied the loss of retinoid responsiveness from the perspective of subcellular localization of the retinoid receptors. In sharp contrast to RXRα homogeneous nuclear distribution in estrogen receptor–positive HMEC and MCF-7 cancer cells, RXRα localized to the splicing factor compartment (SFC) in estrogen receptor–negative MDA-MB-231 cancer cells. We also found that RXRα localized to the SFC in the connective tissue of invasive breast carcinoma tissue, but not in the epithelial cells. SFC localization was not detected in connective tissues of normal or benign hyperplasia. Vitamin D receptor B1 (VDRB1), a heterodimerization partner of RXR, is also found in SFC and is redistributed throughout the nucleoplasm upon exposure to its ligand 1,25-dihydroxyvitamin D3. Unlike the ligand-induced dynamic intranuclear mobility of VDRB1, we found that 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, thereby contributing to loss of retinoid responsiveness. 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 the RXR-selective ligand promoted RXRE (RXR-homodimer 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 was not attributable to the reduced RXRα protein expression level because the RXRα level in retinoid-sensitive HMEC cells was the same as that in MDA-MB-231 cells. Thus, we decided to investigate whether altered localization of RXRα could explain the loss of RXRα activity and retinoid responsiveness in the MDA-MB-231 cell line. When MDA-MB-231 cells were infected with adenoviral RXRα, exogenous RXRα was 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 (Figure 1). Figure 1. Retinoid X receptor-α (RXRα) intranuclear localization is a critical factor in retinoid responsiveness. RXRα localizes throughout the nucleoplasm in retinoid-sensitive normal cells and MCF-7 cells of low malignancy. In contrast, RXRα is sequestered in the splicing factor compartment (SFC) and silenced in MDA-MB-231 cells; consequently, retinoid signaling is shut off in these cells. To reverse the lack of responsiveness to retinoid, which itself is attributable to the sequestration of RXRα, two separate approaches were taken. RXRα C-terminus–specific peptide to MDA-MB-231 cells facilitated redistribution of RXRa throughout the nucleus, increasing RXR-homodimer–mediated transactivation upon RXR-ligand treatment. Also, nucleoplasmic overexpression of RXRα in MDA-MB-231 cells infected with RXRα adenovirus resulted in apoptosis in accordance with increased p21 and decreased Bcl-2 expression, restoring the retinoid sensitivity. RA, retinoids; RXRE and RARE, response elements. Epitope-tagged and a C-terminus deletion mutant of RXRα failed to localize to the SFC, whereas exogenous full-length RXRα did so heavily, indicating that the RXRα C-terminus might play a critical role in shuttling RXRα to the SFC. Delivering RXRα C-terminus–specific peptide to MDA-MB-231 cells facilitated redistribution of RXRα throughout the nucleus, increasing RXR-homodimer–mediated transactivation upon RXR-ligand treatment and ultimately enabling MDA-MB-231 cells to respond to retinoids. In conclusion, RXRα was found in the SFC in highly malignant breast cancer MDA-MB-231 cells and invasive carcinoma of human breast tissue. These findings suggest that RXRα appears to change its subcellular localization as cells become increasingly malignant. Our study clarifies one possible pathway that participates in loss of retinoid signaling during breast cancer progression and provides the new concept that loss of RXRα activity due to altered localization leads to the loss of retinoid responsiveness in highly malignant breast tumor cells.
|
|||||||
|
 |
etinoids
are natural and synthetic vitamin A derivatives, which regulate development,
cell proliferation, and differentiation. Retinoids also act as cancer
preventive agents and are presently being used successfully to treat certain
types of cancer. Although many studies have shown retinoids to be effective
in inhibiting cancer cell growth in vitro and in vivo, the
clinical usage of vitamin A derivatives is currently limited by the requirement
of relatively large dosages to reach therapeutic efficacy. It is also
likely that the responsiveness of cancer cells to retinoids diminishes
in relation to malignant progression. Indeed, the growth inhibitory effects
of retinoids have been observed in estrogen receptor–positive breast
cancer cells of low malignancy, whereas the effectiveness of retinoids
has been observed to diminish in highly malignant breast cancer cells
that were estrogen receptor–negative. The existing hormonal, chemotherapeutic
therapies have provided a substantial improvement for the survival of
patients with localized breast cancer; however, treatment for metastatic
breast cancer remains palliative. Thus, there is an urgent need to understand
the mechanism of retinoid resistance to develop therapeutic agents for
metastatic breast cancer. 