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The Selective Estrogen Receptor Modulator Arzoxifene and the Rexinoid LG100268 Cooperate to Promote Transforming Growth Factor ß-Dependent Apoptosis in Breast Cancer

¹ Dartmouth Medical School, Hanover, New Hampshire; ² Ligand Pharmaceuticals Inc., San Diego, California; ³ The Burnham Institute, La Jolla, California; and ⁴ Duke University Medical Center, Durham, North Carolina

	ABSTRACT

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We show that the selective estrogen receptor modulator arzoxifene (Arz) and the rexinoid LG100268 (268) synergize to promote apoptosis in a rat model of estrogen receptor-positive breast carcinoma and in estrogen receptor-positive human breast cancer cells in culture. We also show that it is not necessary to administer Arz and 268 continuously during tumor progression to prevent cancer in the rat model because dosing of these drugs in combination for relatively short periods, each followed by drug-free rests, is highly effective. This new approach to chemoprevention uses high doses of drugs that are too toxic for long-term administration. However, when given for short periods, the agents are nontoxic and still induce apoptosis in breast cancer cells. We also show that the ability of the two drugs to induce apoptosis is the combined result of induction of transforming growth factor ß by Arz, together with inhibition of the prosurvival nuclear factor B and phosphatidylinositol 3' kinase signaling pathways by 268. The new protocol we have developed for chemoprevention allows the efficacious and safe administration of 268 and Arz, and these agents now should be considered for clinical use.

	INTRODUCTION

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There is an urgent need to develop new drugs to prevent and manage breast cancer because the morbidity and mortality from this disease remain a major problem (1) . Selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene already have had a significant impact in the prevention and management of breast cancer (2) . The new SERM arzoxifene (Arz), a potent estrogen antagonist in the breast, is markedly superior to tamoxifen and raloxifene in terms of either safety or efficacy in an experimental rat model for prevention of breast cancer (3) . However, because of the proven benefit of combinations of agents for cancer prevention and therapy, it would be desirable to add a second agent to synergize with Arz (4) .

Rexinoids (selective ligands for the retinoid X receptors; Ref. 5 ) such as Targretin and LG100268 (268) are a promising class of agents for chemoprevention and therapy of cancer (6 , 7) . Targretin and 268 are superior to their parent molecule, 9-cis-retinoic acid, for the prevention of breast cancer in either estrogen receptor (ER)-positive or ER-negative animal models (6, 7, 8, 9) . However, Targretin still binds minimally to retinoic acid receptors and thus can have undesirable toxicity (9) . In contrast, the newer rexinoid 268 is specific for binding to retinoid X receptors and is more potent than Targretin (10) .

Several animal studies have found that combinations of SERMs together with 9-cis-retinoic acid, Targretin, or 268 are more effective for chemoprevention and therapy than the use of the single drugs alone (6 , 7 , 11) , as was shown with the combination of Arz plus 268 (7) . To maximize the desirable synergy of the two drugs and to decrease any long-term toxicity from chronic dosing, we have developed an intermittent dosing protocol that allows the use of high doses of both drugs for short periods, followed by rest periods between dosing. We report that such a protocol with Arz plus 268 is highly effective for the prevention of breast cancer in rats.

Importantly, we also report a novel mechanism of cooperativity between 268 and Arz that relates to their preventive and therapeutic activities, namely, the induction of apoptosis. We have studied the interactions of the two drugs with the transforming growth factor ß (TGF-ß), nuclear factor B (NFB), and phosphatidylinositol 3' kinase (PI3K) pathways, which are known regulators of apoptosis (12, 13, 14, 15) . We demonstrate here for the first time that regulation of each of these pathways either by Arz or 268 is essential for the execution of the apoptotic program induced by the combination of these agents in breast cancer cells.

	MATERIALS AND METHODS

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Reagents.
Arz (3 , 16) and 268 (10) were synthesized according to published procedures. Sources of other reagents were as follows: antibodies to TGF-ß (1D-11), Genzyme (Cambridge, MA); poly(ADP-ribose) polymerase (PARP), c-Jun NH₂-terminal kinase (JNK), phospho-JNK, p38, phospho-p38, AKT, and phospho-AKT, Cell Signaling Technology (Beverly, MA); IB, Santa Cruz Biotechnology (Santa Cruz, CA); and proliferating cell nuclear antigen (PCNA), Dako (Carpinteria, CA). Antibodies to Bcl-xL, c-IAP-1, and active caspase-3 have been reported (17, 18, 19) . FuGene 6 was from Roche Diagnostics (Basel, Switzerland); z-Val-Ala-Asp (zVAD) was from Enzyme Systems (Livermore, CA); SP600125 and SB203580 were from Calbiochem (San Diego, CA); and LY294002 was from Sigma (St. Louis, MO).

Prevention and Treatment of Mammary Cancer in Rats.
Mammary carcinomas were induced with nitrosomethylurea in weanling Sprague Dawley rats (20) . The protocol for prevention of tumors by intermittent administration of the combination of Arz and 268 is shown in Fig. 5A . Tumors were palpated and measured weekly with calipers. Autopsy was performed at week 24, at which time all of the tumors were counted and weighed. Statistical analysis of in vivo experiments was performed with nonparametric methods using the Wilcoxon matched-pairs test. Immunohistochemistry experiments were performed on 10 x 10-mm established tumors. When tumors reached this size, rats were given control diet, 268, Arz, or 268 + Arz diet for 7 days and then killed; their tumors were preserved in neutral-buffered formalin with added zinc ion (Z-Fix; Anatech Ltd., Battle Creek, MI) for immunohistochemistry. All of the drugs were fed in diet as described previously (7) .

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. A, intermittent chemoprevention protocol. The protocol was designed to give high doses of agents for short periods followed by drug-free rests. See text for details. B, intermittent chemoprevention is effective in a rat mammary cancer model. Drugs were fed for 2-week intervals, between which rats were fed control diet for 4 weeks. Tumors were measured weekly with calipers. Doses were as follows: arzoxifene (Arz; 20 mg/kg diet) and LG100268 (268; 200 mg/kg diet). I, control; II, one course Arz + 268; III, two courses of Arz + 268; and IV, three courses of Arz + 268. See text for additional details.

Apoptosis Assays and Western Blot Analyses.
Annexin V and propidium iodide binding (TACS annexin V-FITC Apoptosis Detection Kit; R&D Systems) was analyzed by fluorescence-activated cell sorter analysis. Terminal deoxynucleotidyl transferase-mediated nick end labeling staining (in situ Death Detection Kit, POD; Roche Molecular Biochemicals) was measured by light microscopy. To measure PARP cleavage, Western blot analyses were performed on cell lysates with PARP 9541 antibody (Cell Signaling).

Plasmids and Transfection Assays.
Dominant-negative Smad4, dominant-negative Smad2, FLAG-tagged Smad2, myc-Smad4, and super-repressor I-B have been described previously (23, 24, 25) . Cells were transiently transfected as described previously (26) .

Assays for PAI-1 Luc, NFB, and TGF-ß.
Conditions for the plasminogen activator inhibitor (PAI)-1 luc assay and the electrophoretic mobility shift analysis for NFB have been published (27 , 28) . TGF-ß1, -ß2, and -ß3 were measured by sELISA (TGF-ß isoforms sELISA; R&D Systems) in conditioned media from T47D cells treated with Arz and/or 268.

	RESULTS

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Combination of Arz and 268 Induces Apoptosis in Vivo.
We have reported previously that the combination of Arz and 268 causes dramatic shrinkage of rat breast cancers induced by N-nitroso-N-methylurea (NMU; Ref. 7 ). Fig. 1, A and B , shows that the combination of Arz (60 mg/kg diet) and 268 (200 mg/kg diet) induces apoptosis in rat breast cancers as measured by immunostaining for active caspase-3 (29) . Treatment for as little as 1 week at the aforementioned doses is sufficient to increase statistically significant (P < 0.001) levels of active caspase-3. Moreover, the observed tumor shrinkage appears to be caused by apoptosis because no necrotic foci were seen histologically in regressing tumors. We also found a statistically significant (P < 0.0005) decrease in PCNA expression, a marker of DNA synthesis (30) , in treated tumors (data not shown). Furthermore, Fig. 1B also shows that neither Arz alone (60 mg/kg diet) nor 268 alone (200 mg/kg diet) causes significant elevation of active caspase-3 staining. However, both of these agents, when used singly, do decrease PCNA staining (data not shown), which correlates with the arrest (as opposed to the regression) of tumor growth seen previously when these agents are used singly (7) .

View larger version (103K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Combination of LG100268 (268) and arzoxifene (Arz) induces apoptosis in vivo. Sprague Dawley rats were injected with N-nitroso-N-methylurea (50 mg/kg i.p.) and subsequently randomized into four groups when 10 x 10-mm tumors developed. Animals then were fed control diet, 268 (200 mg/kg diet), Arz (60 mg/kg diet), or the combination of the two drugs for 7 days. Rats then were killed, and tumors were fixed and stained for immunohistochemistry. A, staining for active caspase-3 (H&E, 40x). B, quantitation of staining, as measured by the product of the percentage of positive active caspase-3 cells and average absorbance. See text for details.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Effects of arzoxifene (Arz) and LG100268 (268) in T47D cells in culture. A, induction of apoptosis. Cells were treated with anisomycin (10 µg/ml; positive control), 268 (1 µM), Arz (10 nM), tamoxifen (10 nM), or Targretin (LG1069; 1 µM) for 24 h before flow cytometry analysis for FITC-annexin V and propidium iodide, shown in B. B, fluorescence-activated cell sorter analysis of apoptosis. X axis, annexin V; Y axis, propidium iodide. Conditions as in A. C, induction of transforming growth factor ß3 (TGF-ß3). Cells were treated with 268 (1 µM) and/or Arz (10 nM) for 24 h, and levels of TGF-ß3 in conditioned media were measured by sELISA (R&D Systems). First six bars are TGF-ß3 standard. D, Arz induces active TGF-ß. Conditioned media were prepared as in C and placed for 24 h on mink lung epithelial cells (C32) that had been stably transfected with a plasminogen activator inhibitor (PAI)-1 luc promoter construct (27) . Luciferase activity of the PAI-1 promoter measures active TGF-ß. The pan-TGF-ß 1D-11 antibody (0.1 mg/ml) was added to conditioned media before addition to C32 cells.

Induction of TGF-ß3 by Arz Contributes to Apoptosis in Breast Cancer Cells in Culture.
Because Arz strongly induces expression of active TGF-ß3, we next determined whether the apoptosis induced by Arz plus 268 depended on TGF-ß. TGF-ß itself is known to induce apoptosis and growth inhibition in many epithelial cells (12 , 14 , 37, 38, 39, 40) . Several reports show that apoptosis induced by TGF-ß can be mediated in a Smad-dependent manner via the activation of the JNK (41) and p38 (12 , 42) cascades; this results in decreased levels of the antiapoptotic protein Bcl-xL (43 , 44) . Fig. 3A shows that induction of apoptosis, as measured by PARP cleavage in MCF-7 cells treated with Arz plus 268, is inhibited by the addition of the pan-TGF-ß antibody 1D-11 and by the pan-caspase inhibitor z-Val-Ala-Asp (z-VAD) (45) . To test whether apoptosis induced by Arz plus 268 depends on Smad signaling, we expressed dominant-negative Smad4 and Smad2 constructs in T47D cells before treatment with Arz plus 268 (23 , 24) . Introduction of dominant-negative Smad4 (Fig. 3B) reversed the ability of the combination of Arz plus 268 to induce apoptosis by 52%, and dominant-negative Smad2 yielded similar results (data not shown). To determine whether TGF-ß alone or the combination of TGF-ß plus 268 would induce apoptosis, we measured the percentage of apoptotic T47D cells after treatment with TGF-ß1 and/or 268 for 24 h. Fig. 3C shows that TGF-ß1 at 2 ng/ml alone is not sufficient to induce apoptosis, whereas this same dose of TGF-ß1 in combination with 268 (1 µM) is highly active. Thus, although a pathway other than TGF-ß could conceivably cause facilitation of apoptosis by Arz plus 268, the inhibition of apoptosis by the pan-TGF-ß antibody and by the dominant-negative Smads just described strongly suggests that TGF-ß is of primary importance.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. LG100268 (268) and arzoxifene (Arz) cooperate to promote transforming growth factor ß (TGF-ß)-dependent apoptosis. A, MCF-7 cells were treated with 268 (1 µM) and/or Arz (10 nM) for 24 h before total protein isolation and Western blot analysis for poly(ADP-ribose) polymerase (PARP) cleavage. The pan-anti-TGF-ß antibody 1D-11 (0.1 mg/ml) was added at the same time as the treatment compounds. Cells were pretreated with z-Val-Ala-Asp (z-VAD) for 1 h before addition of other drugs. B, T47D cells were transfected with myc-Smad4 (control for DN-Smad4) and DN-Smad4 and then treated with 268 (1 µM) and Arz (10 nM) for 24 h. Apoptosis was measured by FITC-annexin V staining. Transfection efficiency, as measured by fluorescence-activated cell sorting, was only 56%, which might account for incomplete reversal of apoptosis by DN-Smad4. C, 268 + TGF-ß induce apoptosis in T47D cells. Concentrations are Arz, 10 nM; 268, 1 µM; and TGF-ß1, 2 ng/ml. Treatment was for 24 h. Apoptosis was measured by FITC-annexin V. D, T47D cells were treated as in C, before total protein isolation and Western blot analysis for phospho-c-Jun NH2-terminal kinase (JNK) or phospho-p38. E, T47D cells treated as in A or C and analyzed as in D for Bcl-xL.

268 Facilitates TGF-ß-Mediated Apoptosis by Inhibiting the NFB and PI3K Pathways.
The NFB and PI3K pathways are known to regulate synthesis of several antiapoptotic proteins, including c-IAPs and Bcl-xL (48 , 49) . Because Arz (10 nM) alone is not sufficient to cause apoptosis of T47D cells, we sought to determine whether 268 could potentiate the activity of Arz by decreasing the activity of these prosurvival pathways. Fig. 4A shows that 268 (1 µM) inhibits degradation of the NFB inhibitory protein IB, thereby leading to suppression of NFB activation. Fig. 4B confirms this suppression of NFB activation, as measured by NFB binding to DNA in the nucleus. In Fig. 4, A and B , NFB was activated in T47D cells with tumor necrosis factor . Further confirmation of these results was obtained in cells transfected with a dominant-negative IB (25) . Fig. 4C shows that this construct enhances the ability of Arz alone (10 nM) to promote apoptosis, which otherwise is ineffective. These data indicate that inhibition of NFB activation, in combination with the increase in TGF-ß3 caused by Arz, is sufficient to induce apoptosis.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. LG100268 (268) inhibits the nuclear factor B (NFB) and phosphatidylinositol 3' kinase prosurvival pathways. A, T47D cells were treated with 268 at various concentrations for 6 h before addition of tumor necrosis factor (TNF-; 10 ng/ml). Thirty min after addition of TNF-, total protein was isolated, and cell lysates were analyzed by Western blot analysis for IB degradation. B, T47D cells were treated with 268 (1 µM) and arzoxifene (Arz; 10 nM) for 24 h before addition of TNF- (10 ng/ml) for 5 min. Nuclear protein then was extracted and incubated with an oligonucleotide probe specific for NFB, followed by electrophoretic mobility shift analysis. C, T47D cells were transfected with a DN-IB construct before treatment with 268 (1 µM) and Arz (10 nM) for 24 h (25) . Apoptosis then was measured by annexin V staining and fluorescence-activated cell sorter analysis. Transfection efficiency, as measured with ß-galactosidase (ß-Gal), was only 43%, which might account for incomplete induction of apoptosis by DN-IB and Arz. D, ZR-75 cells were treated with 268 (1 µM) and/or Arz (10 nM) for 6 h before 5-min stimulation with epidermal growth factor (EGF; 10 ng/ml). Total protein then was isolated, and Western blot analysis was performed for phospho-AKT expression. ZR-75 cells were used for optimal detection of phospho-AKT. E, T47D cells were treated with 268 (1 µM) and Arz (10 nM) for 24 h before total protein isolation and Western blot analysis for PTEN expression. F, T47D cells were treated as in D and E and analyzed as in E for c-IAP-1.

Prevention of Breast Cancer with Intermittent Drug Administration.
Because combinations of Arz and 268 were effective in inducing apoptosis in vivo and in vitro, we next determined whether this apoptotic mechanism could be applied to prevent breast cancer in an experimental animal. We had shown previously that short-term administration of high doses of the combination of Arz (60 mg/kg diet) and 268 (200 mg/kg diet) induces dramatic shrinkage of established rat tumors (7) . These are the same doses that we have shown to cause apoptosis in Fig. 1 . However, such high doses of these two agents cannot be given safely for prolonged periods in a prevention protocol. Thus, to use these doses for prevention, it was important to administer the drugs for only short intervals, followed by rest periods between doses. Such a protocol is shown diagrammatically in Fig. 5A . For these prevention studies, we deliberately chose a low dose of NMU (20 mg/kg body weight) to extend the latency period of tumor development (54) . At this dose, only approximately one-third of a cohort of rats develops grossly detectable carcinomas within 6 months.

Beginning 4 weeks after a single dose of NMU, rats were randomized to four groups, which then were fed control diet or given either one, two, or three intermittent courses of Arz plus 268. Each course was given in the diet for only 2 weeks and was followed by a 4-week rest period of control diet. Autopsies of all of the rats were done 24 weeks after the NMU injection, and tumors then were counted and weighed (Table 1) . Latency curves for development of palpable tumors are shown in Fig. 5B . The data indicate that although one or two short courses of Arz and 268 have some efficacy in preventing tumors, three courses of the combination are much more effective than one or two courses. Weights of animals in all of the four groups at autopsy were found to be equivalent (data not shown), suggesting caloric restriction did not account for the results. From the results shown in Fig. 1 , Fig. 5 , and Table 1 , we conclude that intermittent feeding of high doses of Arz plus 268 is an effective way to induce apoptosis and prevent breast cancer in this rat model.

	DISCUSSION

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Although the in vivo and in vitro results reported here with ER-positive breast cancer are encouraging, prevention and management of ER-negative breast cancer in women remain a major challenge. Preliminary studies, to be reported in the future, suggest that this combination of Arz and 268 also may be useful for prevention of ER-negative breast cancer, although more experimental work will be needed to prove this. The present studies should offer a paradigm to approach this problem, although additional agents need to be developed.

	ACKNOWLEDGMENTS

 
We thank Megan Padgett for expert assistance in the preparation of the manuscript; Anita Roberts, Albert Baldwin, and Dan Rifkin for cells and constructs; and Steven Banaras and Xianshu Huang for technical assistance.

	FOOTNOTES

 
Grant support: National Foundation for Cancer Research, NIH grant NS 36821, and Susan Komen Foundation grant 538660. M. B. Sporn is Oscar M. Cohn Professor.

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.

Requests for reprints: Michael B. Sporn, Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03755. Phone: 603-650-6557; Fax: 603-650-1129; E-mail: Michael.Sporn{at}dartmouth.edu

Received 1/23/04. Revised 2/18/04. Accepted 3/ 4/04.

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