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[Cancer Research 63, 3531-3538, July 1, 2003]
© 2003 American Association for Cancer Research

Carcinogenesis

Regulation of Retinoic Acid Receptor ß Expression by Peroxisome Proliferator-activated Receptor {gamma} Ligands in Cancer Cells1

Sharon Y. James, Feng Lin, Siva Kumar Kolluri, Marcia I. Dawson and Xiao-kun Zhang2

Cancer Center, The Burnham Institute, La Jolla, California 92037


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR{gamma} ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) ß expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR{gamma}, in the presence of RXR, formed a strong complex with a retinoic acid response element (ß retinoic acid response element) in the RARß promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR{gamma} ligand 15d-PGJ2, cooperatively promoted the transcriptional activity of the ß retinoic acid response element. Ciglitazone, but not 15d-PGJ2, strongly induced RARß expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RARß expression by the ciglitazone and SR11237 combination was diminished by a PPAR{gamma}-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RARß in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RARß-mediated signaling pathway of PPAR{gamma} action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR{gamma} ligands in potentiating antitumor responses.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPAR{gamma}3 is a ligand-activated transcription factor belonging to the steroid/thyroid receptor superfamily, which plays a critical role in the control of adipogenesis (1, 2, 3, 4) . Specific ligands of PPAR{gamma}, including the thiazolidinedione class of antidiabetic agents, the prostanoid 15d-PGJ2, and certain polyunsaturated fatty acids, have been identified (5 , 6) . PPAR{gamma} expression is not limited to adipocytes because activation of PPAR{gamma} by its ligands has been shown to promote growth inhibition, differentiation, and/or apoptosis of various cancer cells (5 , 7, 8, 9) , including breast (10, 11, 12, 13, 14, 15, 16, 17, 18) and non-small cell lung carcinoma tissues (19, 20, 21, 22) .

Retinoids, comprising the native and synthetic derivatives of vitamin A, are promising agents for the prevention and treatment of human cancers, including those of breast and lung (23 , 24) . The biological effects of retinoids are mainly mediated by their nuclear receptors, RAR and RXR, which each exist as {alpha}, ß, and {gamma} isoforms (1 , 25 , 26) . PPAR{gamma} heterodimerizes with RXR, and the resulting heterodimer binds strongly to its DNA-specific sequence, the PPRE (3 , 4) . Recent studies have shown that PPAR{gamma}/RXR also binds to the estrogen response element (27) . PPAR{gamma} and RXR ligands have been found to cooperatively induce differentiation and apoptosis of breast and colon cancer cells through interaction with PPAR{gamma}/RXR (17 , 18 , 23 , 28) .

RARß plays a critical role in mediating the growth-inhibitory effects of retinoids in various cancer cells (29 , 30) . The aberrant expression or loss of RARß in a variety of cancer cell lines suggests that decreased RARß expression may contribute to retinoid resistance (31, 32, 33, 34, 35, 36, 37, 38) , implying that RARß may act as a tumor suppressor. Regulation of RARß gene expression is principally mediated by the retinoic acid response element (ßRARE) in its promoter, to which RAR/RXR heterodimers bind strongly. Activation of RAR/RXR heterodimers is principally mediated via RAR, whereas RXR serves as a silent partner (25) . The TR3/RXR heterodimer also binds to the ßRARE, which is activated by RXR ligands (39) . Thus, the ßRARE can be transcriptionally controlled by various heterodimers and their ligands. Other factors also influence RARß gene expression. The COUP-TF, which is not expressed in many cancer cells, is required for induction of RARß by ATRA (40) . Lack of RARß expression in cancer cells has also been attributed to abnormal regulation of histone acetylation/deacetylation, which modulates chromatin structure and gene transcription, or hypermethylation of the RARß promoter (41, 42, 43, 44, 45) .

In the present study, we evaluated the growth-inhibitory effect of the PPAR{gamma} ligand ciglitazone alone and in combination with the RXR ligand (rexinoid) SR11237. Our results showed that ciglitazone and SR11237 cooperatively inhibited the growth and induced apoptosis of breast and lung cancer cell lines. In studying the possible underlying molecular mechanisms, we observed that PPAR{gamma} could bind strongly to the ßRARE as a PPAR{gamma}/RXR heterodimer. The combination of RXR ligands with ciglitazone, but not 15d-PGJ2, strongly activated the ßRARE and induced RARß expression in breast and lung cancer cells. The induction of RARß expression by rexinoids and ciglitazone was reduced by a PPAR{gamma} antagonist, BADGE (46) , indicating the involvement of PPAR{gamma}/RXR heterodimers. Together, our results demonstrate that PPAR{gamma} can bind to the ßRARE as a PPAR{gamma}/RXR heterodimer and that induction of RARß may contribute to the anticancer effect of certain PPAR{gamma} ligands.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Cell Lines.
The RXR-selective retinoids SR11237, SR11246, and SR11345 were prepared as reported previously (47, 48, 49) . ATRA and TSA were obtained from Sigma Chemicals (St. Louis, MO). The PPAR{gamma} ligands 15d-PGJ2, ciglitazone, and BADGE were obtained from Cayman Chemicals (Ann Arbor, MI). All reagents were dissolved in a 1:1 ratio of ethanol and DMSO and stored in amber containers at -20°C. Other analytical-grade reagents were obtained from Sigma Chemicals unless otherwise stated.

MDA-MB-231, T-47D, and ZR-75-1 cell lines were routinely maintained in DMEM (MDA-MB-231) or RPMI 1640 (T-47D and ZR-75-1), supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Irvine Scientific, Santa Ana, CA). Calu-6 were maintained in MEM Earle’s Salt Medium (Irvine Scientific) supplemented with 10% FCS and antibiotics. For for Western analysis studies, cells were cultured in their respective medium supplemented with 5% charcoal-treated FCS and antibiotics.

Plasmids.
The PPAR{gamma} expression vector was kindly provided by Dr. Mark Leid (Oregon State University, Corvallis, OR). Expression vectors for RXR{alpha} and reporter gene ßRARE-tk-CAT have been described previously (26 , 50) .

Transfection Assays.
MDA-MB-231 and ZR-75-1 cells were seeded at 2 x 105 cells/ml in 6-well plates for 16–24 h before transfection. Cells were transfected with ßRARE-tk-CAT plasmid (200 ng), ß-gal (200 ng) expression vector (pCH 110; Amersham Biosciences), and carrier DNA (pBluescript; Stratagene, La Jolla, CA) to a final concentration of 2000 ng total DNA/well using LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA). Cells were treated for 20 h with RXR and PPAR{gamma} ligands. CAT activity was normalized with ß-gal activity for transfection efficiency.

Western Blot Analysis.
Treated cell cultures were subjected to Western blot analysis as described previously (51) . Preblocked nitrocellulose membranes were incubated with 1 µg/ml equivalent of anti-RARß rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). RARß protein was detected by horseradish peroxidase-conjugated antirabbit secondary antibody (Amersham Biosciences), and specific bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences). Equivalent loading of samples was determined by reprobing the nitrocellulose membrane with a mouse monoclonal antibody recognizing ß-actin (Sigma Chemicals).

Gel Shift Analysis.
In vitro-synthesized RXR and PPAR{gamma} receptor proteins (Promega, Madison, WI) were incubated with RXR and PPAR{gamma} ligands alone or in combination and treated with a rabbit polyclonal anti-PPAR{gamma} antibody (Santa Cruz Biotechnology) for 20 min before the addition of 32P-labeled ßRARE oligonucleotide. Gel shift analysis was performed as described previously (35) .

Cell Proliferation and Apoptosis Studies.
Treated cells were trypsinized, pelleted by centrifugation at 2000 rpm for 5 min, resuspended in 1 ml of PBS, and fixed in 70% ice-cold ethanol. After two additional PBS washes, the cells were resuspended in PBS containing 50 µg/ml propidium iodide (Sigma Chemicals) and 100 µg/ml DNase-free RNase A (Roche Diagnostics, Indianapolis, IN). Cell suspensions were incubated for 30 min at 37°C with protection from light and analyzed using a FACScatter-Plus flow cytometer (Becton Dickinson, San Jose, CA).

To assess cell viability, cells were seeded at 1 x 103 cells/well in 96-well microtiter plates and treated with varying concentrations of SR11237 and ciglitazone, with medium and ligands replaced every 48 h. After treatment, 20 µl of MTS/phenozine methosulfate solution (Promega) were added to each well, and incubation was continued for 2–4 h at 37°C in the dark. Absorbance (490 nm) was measured on a Bio-Rad 550 microplate reader.

BrdUrd Analysis.
Treated cells were incubated with BrdUrd (20 µM; Sigma Chemicals) for 2 h before harvesting of cells. After trypsinization and two PBS washes, cells were pelleted by centrifugation and permeabilized with 4% paraformaldehyde. After a 20-min incubation at room temperature, 0.1% saponin (Sigma Chemicals) was added to the cell suspension, and the incubation was continued for 10 min. The cells were then centrifuged, washed twice with PBS containing 0.1% saponin, and resuspended in PBS containing 30 µg of DNase I (Roche Diagnostics). After a 1-h incubation with either an anti-BrdUrd fluorescent antibody or an isotope control antibody (Becton Dickinson), cells were given a final PBS wash before being analyzed using the FACScatter-Plus flow cytometer (Becton Dickinson).

Statistical Analysis.
One-way ANOVA with the Dunnett’s post test (GraphPad Prism software) was used to assess significance of treatments at the 5% level for growth inhibition studies.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ciglitazone and SR11237 Cooperatively Inhibit the Growth of Cancer Cell Lines.
Both retinoids and PPAR{gamma} ligands are potent regulators of cancer cell growth. Because PPAR{gamma} heterodimerizes with RXR (5 , 52) , we investigated the inhibitory effect of their ligands, alone or in combination, on the growth of breast and lung cancer cells. Fig. 1ACitation illustrates the effects of the rexinoid SR11237 and the PPAR{gamma} ligand ciglitazone on the growth of the hormone-dependent breast cancer cell lines ZR-75-1 and T-47D and the lung cancer cell line Calu-6. Cell proliferation was assessed by MTS assay after 8 (ZR-75-1 and Calu-6) or 10 (T-47D) days of treatment. All three cell lines have been reported to be sensitive to growth inhibition by ATRA (35 , 53) . Treatment of these cell lines with ATRA strongly inhibited their growth in a dose-dependent manner (Fig. 1A)Citation . SR11237 did not exhibit a significant effect on T-47D cell growth, whereas in ZR-75-1 and Calu-6 cells, the rexinoid modestly inhibited cell growth when used at 1 µM (10% and 22% for ZR-75-1 and Calu-6 cells, respectively; Fig. 1ACitation ). Ciglitazone (1 µM) did not have any marked antiproliferative effects in these cancer cell lines. However, when SR11237 and ciglitazone were used in combination, cooperative effects on cell growth were observed in the three cell lines. In ZR-75-1 cells, a 2-fold growth-inhibitory effect was observed when using 1 µM SR11237 and 1 µM ciglitazone (10% with SR11237 versus 23% with the combination). In addition, cotreatment of ZR-75-1 cells with 1 µM SR11237 and 10 µM ciglitazone further enhanced growth inhibition by 47% (Fig. 1ACitation , middle graph). Cotreatment of T-47D cells with SR11237 and ciglitazone resulted in 51% growth inhibition, compared with the antiproliferative effects of each ligand used alone, whereas an additive effect was observed in Calu-6 cells.



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  		Fig. 1. PPAR{gamma} ligand ciglitazone and RXR ligand SR11237 cooperatively inhibit growth and induce apoptosis of cancer cells. A, inhibition of cell proliferation by RXR and PPAR{gamma} ligands. T-47D, ZR-75-1, and Calu-6 cells were treated for 10 (T-47D) or 8 (ZR-75-1 and Calu-6) days with the indicated concentrations of ATRA, SR11237, or ciglitazone. After treatment, inhibition of cell proliferation was assessed by MTS assay. Cell viability is expressed as a percentage of the control ± SE (n = 4). *, P < 0.05. B, rexinoid and ciglitazone cooperatively inhibit breast cancer cell proliferation. ZR-75-1 cells were treated for 72 h with ATRA alone (1 µM, as positive control) or with SR11237 (1 µM) or ciglitazone (10 µM), either alone or in combination. Cell proliferation was assessed by BrdUrd incorporation (top graphs). The data are presented as a histogram (bottom graph). C, induction of apoptosis by RXR and PPAR{gamma} ligands. ZR-75-1 cells were treated for 5 days with SR11237 (1 µM) or ciglitazone (10 µM), either alone or in combination. Apoptotic cells were determined by measuring the sub-G1 peak of cells using propidium iodide staining and flow cytometry. Results are representative of three separate experiments.

 
We also evaluated the antiproliferative effects of SR11237 and ciglitazone by BrdUrd incorporation. ZR-75-1 cells were treated for 72 h with SR11237 alone or in combination with ciglitazone. As shown in Fig. 1BCitation , SR11237 or ciglitazone alone did not inhibit BrdUrd incorporation relative to control. In contrast, the combination decreased BrdUrd incorporation by 46%. Additional studies were performed to determine whether the combination induced apoptosis (Fig. 1C)Citation . ZR-75-1 cells were treated for 5 days with SR11237 alone or in the presence of ciglitazone. After treatment, cells were harvested and stained with propidium iodide for flow cytometry. Apoptotic cells were determined by measuring sub-G1-phase cells. When the cells were treated with the combination of SR11237 and ciglitazone, 7.6% of the cells were apoptotic compared with control (Fig. 1C)Citation . Taken together, these results demonstrate that ciglitazone and a RXR ligand can cooperatively inhibit growth and induce apoptosis in cancer cells.

Induction of RARß by a PPAR{gamma} Ligand in Cancer Cell Lines.
Induction of RARß has been correlated with the growth-inhibitory and apoptosis-inducing effects of retinoids in breast and lung cancer cells (30 , 35 , 36) . We then determined whether the antiproliferative effects observed using rexinoids and PPAR{gamma} ligands could be attributed to their ability to induce RARß. Western analysis was used to investigate whether RXR and PPAR{gamma} ligands were capable of regulating RARß protein expression. ZR-75-1, T-47D, and Calu-6 cells were treated for 24 h with SR11237, in the absence or presence of ciglitazone. In these cell lines, ATRA readily induced RARß, as reported previously (35) , whereas ciglitazone did not show any effect (Fig. 2)Citation . SR11237 only weakly induced RARß in all of the cell lines. However, cotreatment with SR11237 and ciglitazone resulted in a marked expression of RARß protein. Together, these data demonstrate that ciglitazone strongly induces RARß expression in combination with a RXR ligand.



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  		Fig. 2. Regulation of RARß protein expression by RXR and PPAR{gamma} ligands. T-47D, ZR-75-1, and Calu-6 cells were treated for 24 h with SR11237 (1 µM) or ciglitazone (10 µM), either alone or in combination. Cells were also treated with ATRA (1 µM) as a positive control. Cell lysates were prepared, and RARß protein expression was assessed by Western analysis (51) .

 
Differential Effects of PPAR{gamma} Ligands on RARß Expression.
Next, we examined whether other known PPAR{gamma} ligands were able to induce RARß. We evaluated the prostanoid 15d-PGJ2 and rosiglitazone, two well-characterized PPAR{gamma} ligands (5 , 6) . T-47D, ZR-75-1, and Calu-6 cells were first treated with 15d-PGJ2 alone or with SR11237. Unlike SR11237 and ciglitazone cotreatment (Fig. 2)Citation , SR11237 and 15d-PGJ2 did not induce RARß (Fig. 3)Citation , thus highlighting the differential effects of the PPAR{gamma} ligands on modulating RARß expression in combination with a RXR-selective retinoid. We also found that rosiglitazone alone or with SR11237 did not induce RARß (Fig. 3)Citation . Thus, not all PPAR{gamma} ligands are capable of inducing RARß expression in cancer cells.



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  		Fig. 3. Differential effects of PPAR{gamma} ligands in inducing RARß expression. T47D, ZR-75-1 and Calu-6 cells were treated for 24 h with SR11237 (1 µM), 15d-PGJ2 (5 µM), or rosiglitazone (10 µM), alone or with SR11237 + 15d-PGJ2 or SR11237 + rosiglitazone in combination. Cells were also treated with ATRA (1 µM), as a positive control. RARß protein expression was assessed by Western analysis.

 
PPAR{gamma} Mediates the Effects of Ciglitazone in Inducing RARß.
To determine whether the induction of RARß by ciglitazone (in combination with SR11237) was mediated by PPAR{gamma}, we examined the effect of BADGE a PPAR{gamma} antagonist. BADGE was shown to block the ability of PPAR{gamma} ligands to activate the transcriptional and adipogenic function of PPAR{gamma} (46) . Fig. 4Citation illustrates that BADGE alone did not affect basal RARß expression in T-47D cells. However, BADGE significantly attenuated ciglitazone/SR11237-induced RARß. The effect of BADGE was specific because it did not inhibit ATRA-induced RARß expression. Similar attenuation of ciglitazone/SR11237-induced RARß by BADGE was observed in ZR-75-1 cells (data not shown). These data strongly suggest that induction of RARß expression by ciglitazone (in combination with SR11237) is mediated via activation of PPAR{gamma}.



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  		Fig. 4. A PPAR{gamma} antagonist blocks induction of RARß protein expression. T-47D cells were pretreated for 3 h with the PPAR{gamma} antagonist BADGE (20 µM), after which the cells were treated with SR11237 (1 µM) or ciglitazone (10 µM), alone or in combination. RARß protein expression was determined by Western analysis.

 
PPAR{gamma} Binds to the ßRARE as a PPAR{gamma}/RXR Heterodimer.
The ßRARE present in the RARß promoter mediates transcriptional regulation of RARß expression by RAR and RXR heterodimers (50 , 54) . To determine whether induction of RARß by ciglitazone and SR11237 was also mediated via ßRARE, we studied the possibility that PPAR{gamma} was capable of interacting with the ßRARE. In vitro-translated PPAR{gamma} protein was analyzed for binding to the ßRARE by the gel shift assay (Fig. 5A)Citation . PPAR{gamma} alone did not exhibit clear binding to the ßRARE. However, PPAR{gamma} in combination with in vitro-synthesized RXR{alpha} protein produced a prominent complex. Binding was not affected when PPAR{gamma}/RXR was preincubated with SR11237 or ciglitazone. However, when in vitro-translated RXR and PPAR{gamma} were incubated with an anti-PPAR{gamma} antibody, binding of the RXR/PPAR{gamma} heterodimer to ßRARE was attenuated, indicating that the PPAR{gamma} antibody blocked the ability of PPAR{gamma} to bind to ßRARE (Fig. 5B)Citation . These data demonstrate that PPAR{gamma} can bind to the ßRARE as a PPAR{gamma}/RXR heterodimer.



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  		Fig. 5. RXR and PPAR{gamma} bind to ßRARE as a heterodimeric complex. A, in vitro-translated RXR and PPAR{gamma} were incubated with 32P-radiolabeled ßRARE, either alone or in combination. The resulting reactions were then analyzed by gel shift analysis. B, in vitro-translated RXR{alpha} and PPAR{gamma} were incubated with SR11237 (1 µM) and/or ciglitazone (10 µM), in the absence or presence of anti-PPAR{gamma} rabbit polyclonal antibody ({alpha}-PPAR{gamma}). After a further incubation with 32P-radiolabeled ßRARE, the reactions were analyzed by gel shift analysis.

 
Differential Regulation of ßRARE Transcriptional Activity by PPAR{gamma} Ligands.
The observation that PPAR{gamma}/RXR binds to the ßRARE suggested that it represented another RXR-containing heterodimeric complex that activates the ßRARE. Therefore, we studied whether RXR and PPAR{gamma} ligands could activate the ßRARE. A reporter containing the CAT gene fused to the ßRARE (50) was transiently transfected into MDA-MB-231 cells, which were treated with SR11237 alone or in combination with ciglitazone. As revealed in Fig. 6ACitation , SR11237, but not ciglitazone, slightly induced ßRARE transcriptional activity. Moreover, induction of ßRARE was synergized by cotreatment with ciglitazone. We also examined whether the synergistic effect on activating ßRARE could be extended to other PPAR{gamma} and RXR ligands. Ciglitazone, but not 15d-PGJ2 or rosiglitazone, strongly induced ßRARE activity together with SR11237 (Fig. 6A)Citation . This correlated with the effects of these PPAR{gamma} ligands on induction of RARß protein expression (Figs. 2Citation and 3Citation ). Similar results were obtained using ZR-75-1 breast cancer cells (Fig. 6BCitation , top graph). Thus, not all PPAR{gamma} ligands that activate the PPRE similarly activate ßRARE together with SR11237. We also examined the effect of the RXR ligands SR11246 and SR11345 on ßRARE activity in ZR-75-1 cells (48 , 49) . Again, ciglitazone strikingly enhanced transcriptional activity of the ßRARE induced by SR11246 and SR11345 (Fig. 6B)Citation .



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  		Fig. 6. PPAR{gamma} ligands differentially promote ßRARE transcriptional activity. The MDA-MB-231 and ZR-75-1 breast cancer cell lines were transiently transfected with 200 ng of ßRARE-tk-CAT (50) and 200 ng of ß-gal expression vector. Transfected cells were treated for 20 h with the indicated RXR-selective ligands (1 µM) or PPAR{gamma} ligands (5 µM 15d-PGJ2, 10 µM ciglitazone, or rosiglitazone), either alone or in combination. Transcriptional activity of ßRARE was assessed by CAT assay, using ß-gal as an internal standard to evaluate transfection efficiency. Results are representative of three separate experiments, and values are expressed as relative transcriptional activity. A, effect of RXR and PPAR{gamma} ligands on inducing ßRARE in MDA-MB-231 cells. B, effect of RXR and PPAR{gamma} ligands on inducing ßRARE transcriptional activity in ZR-75-1 cells.

 
Antagonistic Effects of Other PPAR{gamma} Ligands on RARß Expression.
The differential effect of PPAR{gamma} ligands on SR11237-induced RARß expression (Figs. 2Citation and 3Citation ) prompted us to investigate whether PPAR{gamma} ligands antagonized one another’s activity. The observation that ciglitazone synergized with SR11237 in inducing RARß expression suggested that binding of ciglitazone to PPAR{gamma} and binding of SR11237 to RXR resulted in transactivation of the ßRARE by PPAR{gamma}/RXR heterodimers. In contrast, binding of PPAR{gamma} with 15d-PGJ2 may either not activate or suppress the transcriptional activity of PPAR{gamma}/RXR on the ßRARE. Therefore, we determined whether 15d-PGJ2 interfered with PPAR{gamma} activation by ciglitazone. Fig. 7Citation shows that in ZR-75-1 cells, 15d-PGJ2 inhibited RARß expression induced by the combination of SR11237 and ciglitazone. Thus, 15d-PGJ2 acts as an antagonist of PPAR{gamma} with respect to the induction of RARß by ciglitazone.



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  		Fig. 7. 15d-PGJ2 antagonizes RARß expression induced by rexinoid and ciglitazone. ZR-75-1 cells were treated for 24 h with ATRA alone (1 µM) or with SR11237 (1 µM), ciglitazone (10 µM), or 15d-PGJ2 (5 µM), either alone or in combination. RARß protein expression was determined by Western analysis.

 
Mechanisms of RARß Induction in MDA-MB-231 Cells: Role of HDACs and the Orphan Receptor COUP-TF.
ATRA does not induce RARß expression in the estrogen-independent breast cancer cell line MDA-MB-231 (35 , 41, 42, 43 , 55 , 56) . Therefore, we examined whether SR11237 and ciglitazone could induce RARß in these cells. In agreement with previous observations (35) , treatment of MDA-MB-231 cells with ATRA did not induce RARß expression (Fig. 8A)Citation . SR11237 and ciglitazone, either alone or in combination, did not induce RARß expression, despite the expression of RXR{alpha} and PPAR{gamma} (data not shown). Histone acetylation and DNA methylation have been reported to contribute to silencing RARß gene expression in the MDA-MB-231 cell line (41 , 44) . HDAC inhibitors, such as TSA, have been shown to suppress the effects of corepressors (41) . Therefore, to determine whether inhibitors of histone deacetylation played a role in augmenting induction of RARß by RXR and PPAR{gamma} ligands, MDA-MB-231 cells were treated with SR11237, alone or in combination with ciglitazone and TSA. Cotreatment of cells with ATRA and TSA readily induced RARß protein expression (Fig. 8A)Citation . Cells cotreated with SR11237, ciglitazone, and TSA also induced RARß. These results suggest that histone deacetylation may represent a mechanism through which RARß expression is suppressed in MDA-MB-231 cancer cells.



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  		Fig. 8. RXR and PPAR{gamma} ligands differentially regulate RARß expression in wild-type MDA-MB-231 and MDA-MB-231 COUP-TF stable cells. A, MDA-MB-231 cells were treated for 48 h with SR11237 (1 µM) ciglitazone (10 µM), or TSA (100 ng/ml), alone or in combination. RARß protein expression was determined by Western analysis. B, MDA-MB-231 COUP-TF stable cells were treated for 24 h with SR11237 (1 µM), 15d-PGJ2 (5 µM), or ciglitazone (10 µM), alone or in combination, and analyzed for RARß expression by Western analysis.

 
We reported previously that the orphan receptor COUP-TF is required for RARß expression by ATRA and is not expressed in MDA-MB-231 cells (40) . COUP-TF enhances RARß expression by increasing the recruitment of receptor coactivators to the RARß promoter. Stable expression of COUP-TF in MDA-MB-231 cells restored the ability of ATRA to induce RARß expression (40) . Therefore, we examined whether COUP-TF expression was also involved in the induction of RARß by SR11237 and ciglitazone. Treatment of MDA-MB-231 COUP-TF stable clone cells with ATRA resulted in strong expression of RARß, as reported previously (40) . SR11237 or ciglitazone alone slightly induced RARß expression in this COUP-TF stable clone (Fig. 8B)Citation . As observed previously with the T-47D, ZR-75-1, and Calu-6 cells (Fig. 3)Citation , 15d-PGJ2, either alone or in combination with SR11237, did not induce RARß expression in the MDA-MB-231 COUP-TF stable cells. In contrast, SR11237 and ciglitazone markedly induced RARß expression (Fig. 8B)Citation . Thus, the expression of COUP-TF modulates the induction of RARß by PPAR{gamma}/RXR heterodimers, as well as the RAR/RXR heterodimers.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Retinoids are effective in suppressing tumor development in many animal carcinogenesis models and are being evaluated in clinical trials for prevention and treatment of cancers (23 , 24) . As an illustration, the rexinoid LGD1069 suppresses mammary tumor development and inhibits the growth of established tumors in vivo (57 , 58) . This retinoid was recently approved for treatment of cutaneous T-cell lymphoma on the basis of clinical trial results and highlights the potential for rexinoids as effective cancer therapeutic agents. Unfortunately, a major limitation in retinoid therapy is that the concentrations needed for anticancer activity also produce adverse effects. Among recent anticancer approaches, combination therapy may lead to synergistic growth-inhibitory effects on cancer cells, thereby allowing the use of lower concentrations to reduce toxicity associated with retinoid treatment (23 , 24) . In this study, we demonstrate that the PPAR{gamma} ligand ciglitazone and the RXR ligand SR11237 cooperatively inhibited the growth of breast and lung cancer cells, whereas either one alone did not markedly inhibit growth (Fig. 1A)Citation . The combination resulted in an enhanced inhibition of cell growth, BrdUrd incorporation, and induction of apoptosis (Fig. 1)Citation . Previous studies showed that the rexinoids enhanced the antidiabetic activity of PPAR{gamma} ligands (59) . Our present results indicate that the combination of rexinoids and PPAR{gamma} ligands may represent a new approach to effectively inhibit the growth of cancer cells.

The cooperative effect of rexinoids and PPAR{gamma} ligands is likely mediated by their receptors RXR and PPAR{gamma} and their heterodimerization. In elucidation of the downstream pathways mediating the RXR and PPAR{gamma} heterodimers, we observed that induction of RARß, a potent growth inhibitor (29 , 30) , is involved in the cooperative growth inhibition of PPAR{gamma} and RXR ligands. Rexinoids and ciglitazone alone did not show an appreciable effect on RARß expression. However, their combination strongly induced RARß expression in breast cancer and lung cancer cells to a degree that was comparable with that of ATRA (Fig. 2)Citation . Thus, our results suggest that induction of RARß expression accounts for the cooperative growth-inhibitory effect of rexinoids and ciglitazone.

The effect of ciglitazone in inducing RARß is mediated by PPAR{gamma}. This was demonstrated by our observation that RARß induction was attenuated by the PPAR{gamma} antagonist BADGE (Fig. 4)Citation . Induction of RARß by ATRA is mainly mediated by RAR/RXR heterodimers that bind to the ßRARE in the RARß promoter (50 , 54) . In studying how PPAR{gamma} and RXR mediated the RARß induction by rexinoids and ciglitazone, we demonstrated that the PPAR{gamma}/RXR heterodimeric complex bound to the ßRARE (Fig. 5)Citation and induced its transcriptional activation in the presence of rexinoids and ciglitazone (Fig. 6)Citation . We demonstrated previously (39 , 60) that rexinoids could induce RARß expression through TR3/RXR heterodimers via ßRARE. The results from the present study demonstrate that the PPAR{gamma}/RXR heterodimer represents another RXR-containing heterodimer that mediates the effect of RXR ligands on RARß induction and growth inhibition.

Classical retinoids fail to induce RARß expression in certain lung cancer cell lines and in the estrogen-independent MDA-MB-231 breast cancer cells (35 , 41, 42, 43, 44 , 61, 62, 63, 64) . Lack of RARß induction has contributed to the retinoid resistance of cancer cells (29 , 30) . We reported previously (40) that the inability of RAR/RXR heterodimers to activate the RARß promoter in ATRA-resistant MDA-MB-231 cells was due to lack of the orphan receptor COUP-TF. Our present data indicate that the SR11237 and ciglitazone combination failed to induce RARß expression in wild-type MDA-MB-231 cells (Fig. 8A)Citation but strongly induced RARß in MDA-MB-231 cells stably expressing COUP-TF (Fig. 8B)Citation . These results indicate a requirement for COUP-TF in activating RARß promoter by the PPAR{gamma}/RXR heterodimer. Similar to ATRA, we observed that the combination of ciglitazone and SR11237 strongly induced RARß in wild-type MDA-MB-231 cells when the HDAC inhibitor TSA was present (Fig. 8A)Citation . These results suggest that histone deacetylation is another mechanism responsible for silencing RARß expression.

One interesting observation in the present study is that PPAR{gamma} ligands differentially regulate ßRARE activity and induction of RARß expression. Ciglitazone, troglitazone, rosiglitazone, and 15d-PGJ2 act as potent agonists of the PPAR{gamma}/RXR heterodimer on the PPRE (3, 4, 5, 6, 7, 8) . However, only ciglitazone activated ßRARE when used with rexinoids (Fig. 6)Citation . Combination of rosiglitazone or 15d-PGJ2 with a rexinoid failed to induce RARß in these cancer cell lines (Fig. 3)Citation . Thus, different PPAR{gamma} ligands exhibit opposing effects on transactivation of the PPAR{gamma}/RXR heterodimer. Why there is such disparity among the PPAR{gamma} compounds and their ability to cooperate with RXR ligands to induce RARß is presently unclear. One obvious explanation would be the differences in ligand structure, which may bind in an alternate conformation when bound to PPAR{gamma}/RXR heterodimers complexed with the ßRARE. This difference in binding may then impair or fail to initiate efficient transcription, perhaps through inappropriate recruitment of corepressors or coactivators. Regulation of retinoid signaling by receptor polarity and allosteric control of ligand binding has been well demonstrated for RAR/RXR heterodimers. Binding of RAR/RXR heterodimers with RAR ligand strongly activates the DR5 element, whereas the binding suppresses RXR agonist activity on the DR1 element (65 , 66) . The differential effects of ligands to activate RAR on response elements was shown to result from opposite polarities of the RXR/RAR heterodimer to asymmetrically oriented half-sites (65) . Previous studies demonstrated that PPAR{gamma} binds to the 5'-half-site position of the PPRE, whereas RXR occupies the 3'-half site (67 , 68) . It is likely that similar receptor polarity and allosteric control of transcription of RXR ligand activity by PPAR{gamma} ligand binding exists with respect to the PPRE, which is a DR1 response element, and ßRARE, a DR5 element. Whether such an allosteric mechanism exists for the PPAR{gamma}/RXR heterodimer requires further investigation. Regardless of the underlying molecular mechanisms, our observation provides an opportunity to identify specific PPAR{gamma} ligands for inhibiting cancer cell growth through inducing RARß expression in combination with rexinoids.

In summary, we have demonstrated that rexinoids and ciglitazone can synergistically inhibit the growth of breast and lung cancer cells through their induction of RARß. Our results demonstrate that PPAR{gamma}/RXR heterodimers can bind to the ßRARE and promote its transcriptional activity in response to rexinoids and certain PPAR{gamma} ligands. Further characterization of the effect of PPAR{gamma} ligands on the transactivation of ßRARE by PPAR{gamma}/RXR heterodimers and the underlying molecular mechanisms may lead to the identification of potent and specific PPAR{gamma} ligands that inhibit cancer cell growth through this novel signaling pathway.


   ACKNOWLEDGMENTS
 
We thank Laura Frazer for preparation of the manuscript and John Kim for proofreading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 S. J. and S. K. K. are supported by Postdoctoral Research Fellowships from the California Breast Cancer Research Program (BCRP 7FB-0062) and the United States Army Medical Research Program (DAMD17-00-1-0173), respectively. M. I. D. and X-k. Z. are supported by National Cancer Institute Grant PO1 CA51993. X-k. Z. is also supported by grants from the NIH (5RO1 CA 60988-09 and 7PO1 CA 87000-01A1) and the United States Army Medical Research Program (DAMD17-00-1-0172). Back

2 To whom requests for reprints should be addressed, at Cancer Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3141; Fax: (858) 646-3195; E-mail: xzhang{at}burnham-inst.org Back

3 The abbreviations used are: PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; 15d-PGJ2, 15-deoxy-{Delta}12,14 prostaglandin J2; ATRA, all-trans-retinoic acid; ßRARE, ß retinoic acid response element; BADGE, bisphenol A diglycidyl ether; COUP-TF, chick ovalbumin upstream promoter transcription factor; DR, direct repeat; HDAC, histone deacetylase; PPRE, peroxisome proliferator response element; RAR, retinoic acid receptor; RXR, retinoid X receptor; TSA, trichostatin A; CAT, chloramphenicol transferase; ß-gal, ß-galactosidase; MTS, 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; BrdUrd, 5-bromo-2'-deoxyuridine; tk, thymidine kinase. Back

Received 10/28/02. Accepted 4/24/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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