The Selective Retinoid X Receptor Agonist Bexarotene (LGD1069, Targretin) Prevents and Overcomes Multidrug Resistance in Advanced Breast Carcinoma
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824 The selective retinoid X receptor agonist bexarotene (LGD1069, Targretin) prevents and overcomes multidrug resistance in advanced breast carcinoma Wan-Ching Yen and William W. Lamph women (1). The American Cancer Society estimates that f215,990 women in the United States will be found to have Department of Molecular Oncology, Ligand Pharmaceuticals, Inc., San Diego, California breast cancer and 40,110 women will die from the disease in 2004. Although chemotherapy provides the major therapeutic modality for treatment of advanced breast cancer, the 5-year survival for disseminated breast carci- Abstract noma is <20% due to relapse with drug-resistant disease. Acquired drug resistance represents a major challenge in Several mechanisms of drug resistance have been charac- the therapeutic management of breast cancer patients. We terized in breast cancer cell lines. The best understood reported previously that the retinoid X receptor–selective mechanism is P-glycoprotein (Pgp)–mediated drug resis- agonist bexarotene (LGD1069, Targretin) was efficacious tance (2). Pgp has been shown to contribute to resistance to in treating animal models of tamoxifen-resistant breast natural product–based chemotherapeutic agents, including cancer. The goal of this study was to evaluate the effect of taxanes, anthracyclines, Vinca alkaloids, podophyllotoxins, bexarotene on development of acquired drug resistance and and camptothecins (3). The relationship among Pgp its role in overcoming acquired drug resistance in advanced expression, Pgp function, and drug resistance in clinical breast cancer. Paclitaxel, doxorubicin, and cisplatin were samples of breast carcinoma was best illustrated by chosen as model compounds to determine the effect of Mechetner et al. (4). These investigators showed that the bexarotene on the development of acquired drug resis- level of Pgp expression in freshly resected breast tumors tance. Human breast cancer cells MDA-MB-231 were was strongly correlated with the degree of clinical repeatedly treated in culture with a given therapeutic agent resistance to paclitaxel and doxorubicin. Their findings with or without bexarotene for 3 months. Thereafter, cells also revealed that both intrinsic and acquired expression were isolated and characterized for their drug sensitivity. of Pgp in breast cancer was likely to contribute in part to Compared with parental cells, cells treated with a single therapeutic failure and relapse. Other resistance mecha- therapeutic agent became resistant to the therapeutic nisms in breast cancer include alterations of glutathione agent, whereas cells treated with the bexarotene combi- metabolism, altered topoisomerase, increased thymidylate nation remained chemosensitive. Cells with acquired drug synthase levels, decreased reduced folate carrier, and resistance, when treated with the combination, showed mutations in the tumor suppressor p53 (5–9). Thus, increased sensitivity to the cytotoxic agent. Furthermore, developing effective treatment strategies to enhance cells treated with the combination regimen had reduced cytotoxic effect of chemotherapeutic agents while invasiveness and angiogenic potential than their resistant reduce the risk of developing drug resistance is highly counterparts. These in vitro findings were further con- desired. firmed in an in vivo MDA-MB-231 xenograft model. Our Bexarotene (LGD1069, Targretin), a selective retinoid X results suggest a role for bexarotene in combination with receptor ligand (10), has been shown to be an efficacious chemotherapeutic agents in prevention and overcoming chemopreventive and chemotherapeutic agent in preclini- acquired drug resistance in advanced breast carcinoma. cal rodent models of breast cancer (11–14). Furthermore, in [Mol Cancer Ther 2005;4(5):824–34] the rat N-nitroso-N-methylurea–induced mammary car- cinoma model, tumors that were resistant to tamoxifen Introduction responded to both bexarotene and bexarotene/tamoxifen Breast cancer is the most common cancer among women combination (15). Mechanistically, tumor regression by and is the second leading cause of cancer deaths in bexarotene in the rat N-nitroso-N-methylurea–induced mammary tumors involved differentiation induction along the adipocyte lineage leading to terminal cell division followed by cell death (16). More recently, we Received 1/13/05; revised 2/23/05; accepted 3/9/05. have shown that bexarotene when combined with The costs of publication of this article were defrayed in part by the paclitaxel produced a synergistic growth inhibition payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to (combination index < 1) in a rat N-nitroso-N-methylurea– indicate this fact. induced mammary tumor-derived cell line in vitro. In the Requests for reprints: William W. Lamph, Department of Molecular rat N-nitroso-N-methylurea–induced tumor model in vivo, Oncology, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, CA 92121. Phone: 858-550-7500; Fax: 858-550-7730. the bexarotene/paclitaxel combination regimen produced E-mail: [email protected] a statistically significant decrease, when compared with Copyright C 2005 American Association for Cancer Research. single agents alone, in total tumor burden and an Mol Cancer Ther 2005;4(5). May 2005 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Molecular Cancer Therapeutics 825 increase in overall objective response without further concentration of the solvent was <0.1%. MDA-MB-231 cells intensifying the major side effects of paclitaxel (17). Phase were exposed to the cytotoxic agent for 3 days and I/II clinical trials indicated that bexarotene is safe and bexarotene for 6 days. Drug-induced growth inhibition well tolerated (18). was measured by MTT assay as described previously (17). The encouraging results of bexarotene alone or in To determine the effect of bexarotene/cytotoxic combina- combination in preclinical models of early-stage mammary tion after multiple exposures, MDA-MB-231 cells were carcinoma led us to examine the efficacy of bexarotene in seeded at 2 Â 106 in T-225 flasks overnight. The various more advanced preclinical models of breast cancer. To combinations and treatment schemes are illustrated in evaluate the role of bexarotene in treatment of breast cancer Fig. 1. Briefly, the cells were exposed to the combina- further, we studied the efficacy of bexarotene/cytotoxic tion regimens on a 10-day cycle. Typically, exposures used combinations and determined the influence of bexarotene a 3-day treatment cycle with the cytotoxic agent (or on the development and treatment of multidrug resistance combination) and then were washed, counted, and replated in advanced human breast cancer. Bexarotene represents a followed by a 7-day exposure either to bexarotene or to good candidate for chemotherapy-based combinations due control medium. At the end of each treatment cycle, the to its nonoverlapping side effect profile with most cells were trypsinized, when possible counted, replated, chemotherapeutic agents and its unique mechanism of and exposed to the same treatment regimen again. This action. MDA-MB-231 cells was chosen as a model cell line procedure was repeated 10 times. For the cytotoxic agent because of its sensitivity to several cytotoxic agents used in alone regimen (Fig. 1, scheme 1), the cells were exposed to the treatment of breast cancer (19–21). In addition, MDA- 30 nmol/L paclitaxel, 50 nmol/L doxorubicin, or 5 Amol/L MB-231 cells do not express the estrogen or progesterone cisplatin alone for 3 days followed by 7 days in control receptors, are HER-2/neu positive, and have mutant p53 medium. For the sequential combination (Fig. 1, scheme 2), (22). Thus, MDA-MB-231 cells represent a model of the cells were treated with a cytotoxic agent for 3 days advanced breast cancer in which to examine the role of followed by 1 Amol/L bexarotene for 7 days. For the bexarotene in combination with cytotoxic agents for breast simultaneous drug combination (Fig. 1, scheme 3), the cells cancer therapy. Paclitaxel (a microtubule-stabilizing agent, were exposed to a cytotoxic agent and 1 Amol/L bexarotene Pgp substrate), doxorubicin (a topoisomerase II inhibitor, for 3 days followed by a 7-day drug holiday. For the Pgp substrate). and cisplatin (an alkylating agent, non-Pgp combination of intermittent cytotoxic agent with continu- substrate) were used to determine the effect of bexarotene ous bexarotene (Fig. 1, scheme 4), the cells were exposed to on the development of acquired drug resistance. a cytotoxic agent and 1 Amol/L bexarotene for 3 days followed by 1 Amol/L bexarotene for 7 days. Control cells Materials and Methods were treated similarly with fresh medium containing 0.1% solvent (not shown in Fig. 1). Cell growth in the Chemicals and Reagents presence of 1 Amol/L bexarotene either given continuously RPMI 1640, fetal bovine serum, glutamine, and gentami- or on a 7-day on, 3-day off cycle was also evaluated as an cin were obtained from Cambrex Bioscience (Walkersville, additional control. Drug-induced growth inhibition was MD). Paclitaxel in sterile solution was obtained from Bristol measured by trypan blue exclusion. The sensitivity of Myers Squibb (Princeton, NJ). Bexarotene was synthesized resistant variants to various cytotoxic agents was evaluated at Ligand Pharmaceuticals, Inc. (San Diego, CA). Paclitaxel, on 96-well tissue culture plates for 3 days. Drug effect