A retinoid X receptor (RXR)-selective retinoid reveals that RXR-alpha is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands

Abstract

Introduction: Certain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment.

Methods: We determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro.

Results: PPAR-alpha and PPAR-gamma ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-alpha-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound.

Conclusion: RXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands.

Figure 1

Expression of functionally interacting peroxisome proliferator-activated receptors (PPARs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) in human breast cancer cell lines. (a) Whole cell lysates from the indicated breast cancer cell lines were subjected to western blot analysis using the anti-PPAR, anti-RAR, anti-RXR, and anti-β-actin antibodies shown on the left. Relative expression of each protein was determined using the same membrane. (b) Human RXR-α protein was immunoprecipitated (IP RXRα) from the indicated breast cancer cell lines. Immunoprecipitated complexes were subjected to western blotting using anti-PPAR-α and anti-PPAR-γ antibodies. Blots were stripped and incubated with anti-RXR-α antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were performed three times, with similar results. Representative blots are shown. (c) An RXR-selective compound potentiates transcriptional activation by a PPAR ligand. The indicated human breast cancer cell lines were transiently transfected with a heterologous PPAR-responsive promoter/reporter construct and treated with 100 μmol/l of the PPAR ligand γ-linolenic acid (LA), 100 nmol/l of the pan RAR agonist all-trans retinoic acid (RA), 100 nmol/l of the RXR-selective ligand AGN194204 (AGN), γ-linolenic acid and retinoic acid (LA+R), or γ-linolenic acid plus AGN194204 (LA+A) for 24 hours before determination of promoter activity. Promoter activity was represented as relative light units from the luciferase reporter. These experiments were performed three times with similar results. Error bars indicate the standard error of the mean.

Figure 2

Greater peroxisome proliferator-activated receptor (PPAR)-selective antiproliferative and apoptotic responses potentiated by AGN194204 in retinoid X receptor (RXR)-α-overexpressing human breast cancer cell lines. (a) Triplicate cultures of estrogen receptor positive/retinoic acid (RA)-sensitive T47D cells were treated for 6 days with vehicle (con), 100 μmol/l of the PPAR-γ ligand γ-linolenic acid (LA), 100 nmol/l AGN194204 (AGN), or both compounds simultaneously (LA+AGN). Proliferation was monitored by cell counting at 2-day intervals using a hemacytometer. (b) Triplicate cultures of estrogen receptor negative/RA-resistant MDA-MB-468 cells were treated for 6 days with vehicle (con), 100 μmol/l LA, 100 nmol/l AGN194204, or both compounds simultaneously (LA+AGN). Proliferation was monitored by cell counting at 2-day intervals using a hemacytometer. (c) LA and AGN194204 inhibit S-phase progression in human breast cancer cell lines. The indicated cell lines were treated with 100 μmol/l LA or 100 nmol/l AGN194204 (AGN). The percentage of S-phase cells was determined by bromodeoxyuridine (BrdU) incorporation assay. (d) PPAR- and RXR-selective ligands alter expression of nonoverlapping sets of cell cycle regulatory proteins. Whole cell lysates from the indicated human breast cancer cell lines treated with 100 μmol/l LA for up to 24 hours were subjected to western blot with the antibodies shown on the left. Relative expression of each protein was determined using the same membrane. (e) Whole cell lysates from the indicated human breast cancer cell lines treated with 100 nmol/l AGN194204 for up to 24 hours were subjected to western blot with the antibodies shown on the left. Relative expression of each protein was determined using the same membrane.

Figure 3

The peroxisome proliferator-activated receptor (PPAR)-α-selective ligand hydroxyeicosatetraenoic acid (HETE) induces apoptosis in human breast cancer cell lines, which is potentiated by AGN194204. (a) The indicated breast cancer cell lines were treated with vehicle (con), 100 μmol/l HETE, 100 nmol/l AGN194204 (AGN), or both compounds simultaneously (HETE+AGN) for 24 hours. The percentage of apoptotic cells was determined by dUTP nick-end labeling (TUNEL) assay and fluorescence microscopy. These experiments were performed three times, with similar results. Error bars indicate the standard error of the mean. (b) The PPAR-α-selective ligand HETE activates the intrinsic apoptotic pathway in human breast cancer cell lines. Whole cell lysates from the indicated breast cancer cell lines treated with 100 μmol/l HETE for up to 24 hours were subjected to western blot, with anti-caspase (casp) and anti-β-actin antibodies shown on the left. Relative expression of each protein was determined using the same membrane. These experiments were performed three times, with similar results. Representative blots are shown.

Figure 4

Retinoid X receptor (RXR)-α overexpression induces ligand-selective antiproliferative and apoptotic responses in human breast cancer cell lines. (a) RXR-α expression in T47D and MCF7 G418 resistant control clones (neo) and RXR-α stable clones (RXR-1, RXR-2). Whole cell lysates were subjected to western blot with anti-RXR-α and anti-β-actin antibodies. These experiments were repeated three times with independent clones. Representative blots are shown. (b) RXR-α overexpression induces antiproliferative response to the RXR-selective ligand AGN194204 in T47D stable clones. Triplicate cultures of G418 resistant control cells (neo) and RXR-α overexpressing clones (RXR1) were treated with vehicle or 100 nmol/l AGN194204 (AGN) for 6 days. Cells were counted at 2-day intervals using a hemacytometer. (c) RXR-α overexpression induces antiproliferative response to the RXR-selective ligand AGN194204 in MCF7 stable clones. Triplicate cultures of G418 resistant control cells (neo) and RXR-α overexpressing clones (RXR1) were treated with vehicle or 100 nmol/l AGN194204 (AGN) for 6 days. Cells were counted at 2-day intervals using a hemacytometer. (d) RXR-α overexpression potentiates the hydroxyeicosatetraenoic acid (HETE) apoptotic response to the RXR-selective ligand AGN194204 in T47D and MCF7 clones. G418 resistant control cells (neo) and RXR-α overexpressing clones (RXR-1, RXR-2) were treated with vehicle (con), 100 μmol/l HETE, 100 nmol/l AGN194204, or both compounds simultaneously (H+AGN) for 24 hours. The percentage of apoptotic cells was determined by dUTP nick-end labeling (TUNEL) assay using fluorescence microscopy. These experiments were performed three times, with similar results. Error bars indicate the standard error of the mean.