The Combination of the Rexinoid, LG100268, and a Selective Estrogen Receptor Modulator, Either Arzoxifene or Acolbifene, Synergizes in the Prevention and Treatment of Mammary Tumors in an Estrogen Receptor–Negative Model of Breast Cancer

Despite the development of selective estrogen receptor (ER) modulators (SERMs), aromatase inhibitors, and the monoclonal antibody trastuzumab (Herceptin), breast cancer still claims >40,000 lives in the U.S. each year (1). Because of the genetic and epigenetic complexities of an invasive cancer, arresting or reversing carcinogenesis at its earliest stages offers an attractive alternative to treating advanced disease (2, 3). However, the realities of the clinic mean that new drugs and new drug combinations are desperately needed for both the prevention and treatment of breast cancer.

A number of drugs have been shown to prevent or treat ER+ breast cancer. SERMs such as tamoxifen and raloxifene are effective in women for both prevention (4–8) and treatment (9). Newer SERMs such as acolbifene (Acol, EM-652) and its prodrug (EM-800) have been used to prevent the development of and to treat established mammary tumors in animal models (10–12), and caused the disappearance of 60% of human breast cancer tumors in nude mice (13). Acol also showed positive responses in women who failed tamoxifen treatment (14), suggesting the superiority of Acol over tamoxifen in a series of preclinical studies (15). The SERM arzoxifene (Arz) also prevented mammary carcinogenesis in rats (16) and decreased ER expression in humans in a phase 1 chemoprevention trial (17).

In addition to the SERMs, retinoids, such as 9-cis-retinoic acid (18, 19), or rexinoids [selective ligands for the retinoid X receptors (RXRs)], such as LGD1069 (bexarotene, Targretin; refs. 20, 21) and LG100268 (268; refs. 22, 23), have been reported to prevent and treat mammary tumors in animal models of ER+ breast cancer. When tested in patients with metastatic breast disease, bexarotene showed clinical benefit in 20% of patients, without any significant toxicity (24). However, the newer rexinoid, 268 (25), is more potent than bexarotene and has greater specificity for binding to RXRs; bexarotene can still bind to retinoic acid receptors (20), which is linked with undesirable toxicity. To date, no prevention trials with rexinoids have been initiated in humans. In addition to the ample evidence that SERMs, retinoids, and rexinoids can prevent and/or treat mammary tumors in experimental animals, preclinical studies establish that combinations of these agents are even more effective than a single drug (18, 19, 21–23).

Despite the ability of SERMs such as tamoxifen to reduce the incidence of ER+ breast cancer in the clinic, they were not effective for preventing or treating ER− disease. Currently, no drugs have been reported to prevent ER− breast cancer in women, and only the retinoid 9-cis-retinoic acid (26), the rexinoid bexarotene (27, 28), and the epidermal growth factor receptor inhibitor ZD1839 (gefitinib, Iressa; ref. 29) have been shown to inhibit the formation of ER− mammary tumors in mice. None of these compounds have been tested in combination for the prevention of ER− breast cancer.

We have previously shown that the SERM Arz and the rexinoid 268 prevent and treat mammary carcinogenesis in a rat nitrosomethylurea model of ER+ breast cancer (22, 23). Although there are no functional estrogen receptors in ER− breast cancer epithelium, early treatment with tamoxifen has been reported to decrease the incidence of ER− tumors in mice (30). Moreover, the mammary stroma contains functional estrogen receptors (31), and paracrine regulation from these receptors plays an important role in epithelial growth and differentiation (32, 33). Because the combination of Arz and 268 affects stromal cells which regulate the tumor microenvironment (22), we decided to test this drug combination in a mouse mammary tumor virus (MMTV)-neu mouse model of ER− breast cancer. In this commonly used transgenic model, mice develop tumors because of targeted expression of the neu gene (erbB2/HER2) in the mammary gland. The neu gene encodes for a receptor tyrosine kinase member of the epidermal growth factor receptor family; the neu protein is overexpressed in 20% to 30% of human breast cancers (34), and neu overexpression is inversely correlated with patient survival (35). The validity of neu as a drug target is supported by the emerging therapeutic importance of trastuzumab, a recombinant monoclonal antibody that targets erbB2, for treating women with HER2-positive breast cancer (36, 37).

In the present experiments, we first tested the combination of Arz and 268 for the prevention of ER− mammary tumors in MMTV-neu mice. This prevention protocol was then repeated, and both Arz and Acol were tested alone and in combination with 268. Because of the striking results from these studies, we next allowed tumors to form and then treated the mice with combinations of 268 and a SERM. In both the prevention and treatment experiments, the combinations of either 268 and Arz or 268 and Acol were remarkably effective.

Reagents. The synthesis of 268 (25) and Acol (38) have been previously described. Arz (39) was provided by Lilly Research Labs (Indianapolis, IN). The structures for these drugs are shown in Fig. 1.

Transgenic mice. MMTV-neu transgenic mice (The Jackson Laboratory, Bar Harbor, ME) were developed in the laboratory of William Muller (McGill University). Wild-type neu is expressed in the mammary tissue under the control of the MMTV promoter. Focal mammary tumors begin to appear in the mice at 4 months of age, and these tumors are ER− (28, 40). All animal studies were done in accordance with an institutionally approved protocol.

Prevention of mammary tumors. Beginning at 7 weeks of age (first prevention study) or at 8 weeks of age (second prevention study), female mice were fed a powdered control diet or a diet containing compounds, as previously described (22, 41). When the mice were 4 months old, they were palpated weekly and tumors were measured with calipers. Tumor volume was calculated by multiplying tumor length (l) by width (w) by height (h = lw / 2) and dividing by 2 (lwh / 2). Animals were sacrificed if tumor volume exceeded 900 mm³ or at the end of the experiment (60 weeks on diet for the first prevention study and 52 weeks on diet for the second prevention study). Tumors were saved for analysis of transgene expression.

Treatment of mammary tumors. A separate cohort of mice was fed normal rodent chow until tumors developed. Mice were palpated weekly, beginning at 20 weeks of age. When tumors at least 32 mm³ in volume had developed, the mice were fed a powdered diet containing drugs [Arz (20 mg/kg diet), 268 (100 mg/kg diet), Arz + 268, Acol (10 mg/kg diet), or Acol + 268]. Mice were fed treatment diets for up to 4 weeks, but animals were sacrificed early if tumor volume exceeded 900 mm³. Tumors were measured weekly, and tumor regression was defined as at least a 50% decrease in tumor volume. Tumors that did not change (tumor volume increased or decreased less than 50%) were classified as growth-arrested. Active tumor growth was defined as a greater than 50% increase in tumor volume.

Evaluation of transgene expression. Transgene expression was analyzed by PCR, Western blot, and immunohistochemistry. The E18-14C-27 tumor cell line derived from mammary tumors from MMTV-erbB2 mice were maintained in RPMI + 10% charcoal-stripped fetal bovine serum. Cells were treated with drugs for up to 48 hours and then analyzed by PCR and Western blot. To evaluate transgene expression in vivo, 8-week-old MMTV-neu mice (four mice per group) were fed Arz (6 mg/kg diet), 268 (30 mg/kg diet), the combination of Arz and 268, Acol (6 mg/kg diet), or the combination of Acol and 268 for 8 weeks. When the mice were 16 weeks of age, the mammary glands were harvested and analyzed by PCR and immunohistochemistry for erbB2 expression. In separate treatment experiments, tumors at least 45 mm³ in volume were allowed to form, and the mice were then treated with the combination of Arz (20 mg/kg diet) and 268 (100 mg/kg diet) for 1 to 3 weeks, until tumor volume was reduced by ∼50%. These tumors and actively growing tumors from mice fed the control diet were harvested and analyzed by Western blot and immunohistochemistry. For Western analysis, cell extracts were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibodies against ErbB2/HER-2/neu (NeoMarkers, Fremont, CA) and actin (Sigma, St. Louis, MO).

Immunohistochemistry and terminal nucleotidyl transferase–mediated nick end labeling staining. Mammary glands or tumors were fixed in 4% paraformaldehyde or neutral-buffered formalin containing additional zinc (Z-fix, Anatech, Ltd., Battle Creek, MI), embedded in paraffin, sectioned, and stained for erbB2, proliferating cell nuclear antigens (PCNA) or terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) as previously described (23, 29). To calculate the percentage of PCNA- or TUNEL-positive cells, two sections on the periphery of each tumor (four tumors per group and >1,000 cells per treatment group) were counted in a blinded fashion.

Statistical analysis. The in vivo experiments summarized in Tables 1,Table 2-3 were analyzed with the Kruskal-Wallis one-way ANOVA on ranks followed by Dunn's method for multiple comparisons (SigmaStat 3.1). Results from the PCNA and TUNEL staining experiments were analyzed by ANOVA and the post hoc Student-Newman-Keuls test or the Mann-Whitney rank sum test, respectively.

The combination of 268 and a SERM, Arz or Acol, prevents development of ER− mammary tumors. Beginning at 7 weeks of age, female MMTV-neu mice were fed a powdered control diet or a diet containing Arz (20 mg/kg diet, ∼5 mg/kg body weight), 268 (30 mg/kg diet, ∼7.5 mg/kg body weight), or the combination. As shown in Fig. 2, 100% of the control mice developed mammary tumors by the 48th week. Notably, no tumors were ever observed in the combination group. At week 38, 25% of the mice in both the 268 and Arz groups had developed tumors compared with 81% in the control group. Although all of the mice on the 268 diet eventually developed tumors, the time required for 100% tumor incidence was delayed by 3 months. After 60 weeks on the diet, only 50% of the mice on the Arz diet had developed tumors. Tumor multiplicity was also reduced in the treated groups (Table 1), and the average number of tumors per mouse at the end of 60 weeks dropped from 2.1 in the control group to 1.4 in the 268 group, 0.9 in the Arz group, and 0 in the combination group (P < 0.05 for Arz and Arz + 268 groups versus control).

In order to confirm these striking results, a similar experiment was done with an independent cohort of mice. In this second experiment, the MMTV-neu mice were fed a diet containing Arz (6 mg/kg diet), Acol (3 mg/kg diet), 268 (30 mg/kg diet), the combination of Arz and 268, or the combination of Acol and 268. At the end of 1 year on the diet, 100% of the control mice and 58% of the mice on the 268 or Arz diet had developed tumors (Fig. 3A). When Acol was used in the diet, 67% of the mice developed tumors after 1 year (Fig. 3B); Acol was used at a lower concentration than Arz (3 versus 6 mg/kg diet) because of a lack of weight gain when mice were fed Acol at the higher dose. Remarkably, no tumors were observed in mice fed either Arz + 268 (Figs. 2A and 3A) or Acol + 268 (Fig. 3B) in either of these year-long prevention studies. The average number of tumors per mouse was again significantly lower (P < 0.05) in the groups treated with Arz alone or with the combination of a SERM and 268 compared with the control group (Table 2). The concentrations of drugs used in both of these experiments were well-tolerated by the mice, as determined by continued weight gain throughout the studies.

The combination of 268 and a SERM is effective for the treatment of ER− mammary tumors. Because of the observed synergistic effects when 268 was combined with a SERM in these ER− mammary tumor prevention studies and because this combination was reported to induce apoptosis in breast cancer, partially by inducing transforming growth factor β (23), we next attempted to treat established ER− mammary tumors. For these studies, mice were maintained on a control diet until they had developed tumors with volumes of at least 32 mm³. The mice were then fed a control diet or a diet containing Arz (20 mg/kg diet), 268 (100 mg/kg diet), Arz + 268, Acol (10 mg/kg diet), or Acol + 268; higher concentrations of drugs were used for the treatment studies in order to inhibit tumor cell proliferation or induce apoptosis. All tumors from mice fed the control diet continued to grow (data not shown). As shown in Table 3, 83% of the tumors (n = 84) in the mice treated with the combination of Arz + 268 regressed, and tumor volume was reduced by 76% to 100% in 75% of these tumors. Another 12% of the tumors were growth-arrested, leaving only 5% of the tumors that were resistant to treatment with Arz + 268. In the Acol + 268 combination group, 74% of the tumors regressed, 9% were growth-arrested, but 18% of these tumors continued to grow. As expected, neither Arz nor Acol alone was effective for treatment of ER− tumors, as 90% of the tumors in the Arz group and 88% of tumors in the Acol group continued to grow. Surprisingly, 268 alone caused regression in 68% of the tumors, although 16% of the tumors were resistant to treatment with 268. Moreover, the 75% of tumors that were reduced in volume by 76% to 100% were significantly (P = 0.03) higher in the Arz + 268 combination group than the 52% of tumors that regressed in the 268 alone group.

268, alone or in combination with a SERM, does not reduce c-neu transgene expression in vitro or in vivo. To verify that these significant results did not occur because of a reduction in transgene expression, c-neu expression was analyzed by PCR, Western blot, and immunohistochemistry. First, E18-14C-27 tumor cells from MMTV-erbB2 mice were treated with various concentrations (0.01-1 μmol/L) of 268, Arz, Acol, or their combinations for 24 to 48 hours. Transgene expression did not change either at the mRNA or protein level, as shown by PCR (data not shown) or Western blot (Fig. 4A). Moreover, MMTV-neu mice from the prevention study were fed Arz, 268, or the combination in diet for 8 weeks, and PCR (data not shown) and immunohistochemistry (Fig. 4B) revealed no apparent changes in neu expression in the mammary glands of these mice. Finally, tumors from mice that were treated with the combination of Arz and 268 for 1 to 3 weeks, and that had regressed by at least 50% were harvested and analyzed by Western blot and immunohistochemistry for neu expression. The erbB2 protein was expressed in 11 tumors from the mice fed Arz + 268 and was indistinguishable from the levels in 7 control tumors. An additional 17 tumors were analyzed by immunohistochemistry, and the average erbB2 immunostaining score from eight control tumors was 3 versus 2.5 for nine tumors from the combination group; this difference is not statistically different (P = 0.13). Representative Western blot and immunohistochemistry staining results are shown in Fig. 4C and D, respectively. Taken together, these experiments suggest that these compounds do not inhibit the expression of the neu transgene.

The combination of 268 and Arz inhibits proliferation and induces apoptosis in mammary tumors. Because of the dramatic reduction in tumor volume in mice treated with the combination of Arz and 268, tumors were analyzed by PCNA and TUNEL staining. As shown in Fig. 5A and C, the percentage of PCNA-positive cells was similar in tumors from the control and Arz groups (20.5% and 18.9%, respectively). However, the percentage of cells that were positive for PCNA staining was significantly lower (P < 0.05) in both the 268 group (12.0%) and in the Arz + 268 tumors (6.2%). Moreover, 9.6% of the cells in the Arz + 268 tumors stained positive for TUNEL versus only 2.5% (P < 0.05) of cells in the control group (Fig. 5B and D).

The results described here clearly show that the combination of a SERM (either Arz or Acol) and the rexinoid 268 is extremely effective for the prevention and treatment of tumorigenesis in a mouse model of ER− breast cancer. Although all three individual compounds significantly delayed the development of mammary tumors in the prevention studies, the combination was remarkably potent, as no tumors were detected in any mouse fed a combination diet in two independent, year-long studies. Moreover, the combination of a SERM and 268 also induced marked regression of established ER− mammary gland tumors.

In addition to our important results with the combination treatments, we also show that 268 alone caused tumor regression. The efficacy of the 268 treatment was unexpected, based on our previous studies with this drug in the nitrosomethylurea rat model of ER+ breast cancer. In these studies (22, 23), 268 alone reduced tumor volume, but the combination of Arz and 268 was markedly synergistic. Bexarotene, the only rexinoid approved for use in the clinic, inhibited the development of both ER− and ER+ mammary tumors (20, 27) and caused the regression of ER+ mammary tumors (21, 42), but it was unable to reduce the size of ER− MDA-MB-231 xenografts unless used in combination with standard chemotherapeutic agents (43). Our studies also show that 268 decreased the percentage of PCNA-positive cells in the tumors, and a recent study reports that 268 inhibits the growth of breast epithelial cells by decreasing cyclin D1 protein and thus Rb phosphorylation (44). It will be important to examine cyclin D1 levels and proteins in the apoptotic pathway in tumors from mice treated with 268; these studies will be described in a future report. Furthermore, the receptors (RXR-α, RXR-β, and RXR-γ) for rexinoids such as 268 heterodimerize with many other members of the nuclear receptor superfamily (45, 46). Because the rexinoids interact with numerous networks that regulate growth and apoptosis, our results are likely the integration of diverse signals from these multiple pathways and thus will be difficult to define.

The ability of Arz or Acol alone to inhibit the formation of ER− tumors in the prevention studies, whereas having no effect in the treatment studies is also notable. Tamoxifen has been reported to reduce tumor incidence (47) or to delay the development of tumors (30) in two different mouse models of ER− breast cancer. In both of these models, the beneficial effects of tamoxifen were only observed if the treatment was started early, before tumors could be detected. Tamoxifen, however, had no effect on established tumors. These animal studies support our findings and suggest either that SERMs inhibit cell proliferation and thus decrease the number of cells available to form a tumor or that ER− tumors require estrogen during their early development. Therefore, it may be possible to prevent both ER+ and ER− breast cancer with a SERM, alone or in combination, if given at an appropriately early time before cells transition to an ER− phenotype.

Although we did not observe any decrease in PCNA staining in tumors from the Arz group, SERMs can induce the expression of transforming growth factor β in breast cancer cells or fibroblasts (22, 23, 48–50). Transforming growth factor β has growth-inhibitory and proapoptotic effects on cancer cells (51–54) and might contribute to the chemopreventive properties of the SERMs in vivo. Moreover, we have previously reported that the combination of Arz and 268 synergistically enhances transforming growth factor β production in Swiss-3T3 cells and inhibits iNOS expression in primary rat embryo fibroblasts (22). The marked synergy between the SERMs and 268 in a number of previous studies (22, 23) and in the current experimental model of ER− breast cancer probably involves several different signaling pathways, including the transforming growth factor β network (55) and stromal-epithelial interactions (33, 56). Defining these pathways and mechanisms will require additional experiments; these studies are important but will require a systems approach (55, 57, 58) to the tissue networks and thus will be described in future publications. Moreover, a recent article reported that bexarotene inhibits angiogenesis (59), and future studies will examine the effects of 268 and the combination of 268 and a SERM on angiogenesis in tumors in the MMTV-neu model. Finally, the ability of multifunctional drugs such as SERMs and rexinoids to act on multiple pathways has important clinical implications, and our data suggests that the combination of a SERM and the rexinoid 268 should be tested in patients for both the prevention and/or treatment of breast cancer.

We thank Megan Padgett for her assistance with the manuscript; Steven Banares, Xianshu Huang, and Eunice Kwon for skillful team work in histology and PCNA immunocytochemistry; and Dr. Ronald Lubet for scientific and logistical support. Preliminary results from this investigation were presented at the 4th Annual AACR International Conference “Frontiers in Cancer Prevention Research” in Baltimore, MD in 2005. This article is dedicated to Emerson Day, M.D., pioneer in chemoprevention of cancer, on the occasion of his 93rd birthday.