The Selective Retinoid X Receptor Agonist Bexarotene (LGD1069, Targretin) Prevents and Overcomes Multidrug Resistance in Advanced Breast Carcinoma

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 carcinoma 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

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 was vincristine, doxorubicin, cisplatin, methotrexate, and 3-(4,5- measured by MTT assay as described previously. dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Measurement of Pgp Activity (MTT) were from Sigma Chemicals (St. Louis, MO). The degree of Pgp efflux activity was quantified by Cell Line measuring the accumulation of a fluorescent Pgp substrate The human breast cancer cell line MDA-MB-231 and calcein in the cytosol according to the manufacturer’s human umbilical vein endothelial cells (HUVEC) were instruction (Molecular Probes, Eugene, OR). The intracel- obtained from the American Type Culture Collection (Manassas, VA) and Cambrex Bioscience, respectively. lular accumulation of free calcein was measured with MDA-MB-231 cells were routinely cultured in RPMI 1640 a fluorescence microplate reader (BioTek Instruments, supplemented with 10% fetal bovine serum and 2 mmol/L Winnoski, VT) at excitation/emission wavelength of 485/ 530 nm. glutamine in 95% air-5% CO2. HUVECs were maintained in EGM-2 supplemented with basic fibroblast growth factor, RNA Preparation and Quantitative Real-time PCR heparin, epidermal growth factor, hydrocortisone, vascular Total RNA was isolated using RNeasy Mini kit (Qiagen, endothelial growth factor, ascorbic acid, and 5% fetal Valencia, CA) according to the manufacturer’s instructions. bovine serum. RNA samples were eluted in RNase-free water and stored In vitro Drug Sensitivity Assay at À80jC. Total RNA (1 Ag) was reverse transcribed into To determine the drug effect after a single exposure, cDNA in a 50 AL reaction volume containing 1Â reverse MDA-MB-231 cells were seeded in 96-well tissue culture transcription buffer, 5.5 mmol/L MgCl2, 2 mmol/L plates. Stock solutions of each cytotoxic compound or deoxynucleotide triphosphates, 2.5 Amol/L random hex- bexarotene were dissolved in DMSO, and the final amer, 0.4 unit RNase inhibitor, and 1.25 unit MuLV reverse

Mol Cancer Ther 2005;4(5). May 2005

in an ABI PRISM 7700 sequence detection system (Applied Biosystems) for 40 cycles of 15 seconds at 95jC and 60 seconds at 60jC. The level of expression of the target gene was normalized to the expression level of the housekeeping gene 36B4. Primers and Probes for Real-time PCR The primers and probes used in this study are as follows: mdr1 (Genbank accession no. M14758) forward 5V- AGGAAGCCAATGCCTATGACTTTA-3V, reverse 5V- CAACTGGGCCCCTCTCTCTC-3V, and probe 6-carboxyfluorescein-ATGAAACTGCCTCATAAATTTGACACCCT- GG-TAMRA (23); human 36B4 (Genbank accession no. M17885) forward 5V-GCAGATCCGCATGTCCCTT-3V, reverse 5V-TGTTTTCCAGGTGCCCTCG-3V, and probe 6-carboxyfluorescein-AGGCTGTGGTGCTGATGG-TAMRA; human abcb11 (Genbank accession no. NM_003742) forward 5V-GACATGCTTGCGAGGACCTT-3V, reverse 5V- GAGCGTTGCCGGATGG-3V, and probe 6-carboxyfluorescein-AGCCCTTAAACTATCCTGGTAGCTCCCTCTGCT- TAMRA; abcc1 (Genbank accession no. NM_004996) forward 5V-TCATGGTGCCCGTCAATG-3V,reverse5V- CGATTGTCTTTGCTCTTCATGTG-3V, and probe 6-carboxyfluorescein-ACCTGATACGTCTTGG TCTTCATCGCCA- TAMRA; abcc2 (Genbank accession no. NM_000392) forward 5V-GTTCGATATACCAATCCAAGCCTC-3V, reverse 5V-CCAGAATAGGGACAGGAACCAG-3V,andprobe6- carboxyfluorescein-TCTGTACACACCATTGTCTGCTG- TATTGGATCAG-TAMRA; abcc3 (Genbank accession no. NM_003786) forward 5V-GCACCATTGTCGTGGCTACA-3V, reverse 5V-GCAGGACACCCAGGACCAT-3V, and probe 6- carboxyfluorescein-CATCCTCTCCCACCTGTC- CAAGCTCA-TAMRA (24); bcrp (Genbank accession no. AF098951) forward 5V-AGATGGGTTTCCAAGCGTTCAT- 3V,reverse5V-CCAGTCCCAGTACGACTGTGACA-3V, Figure 1. Treatment scheme for the bexarotene/cytotoxic combination. and probe 6-carboxyfluorescein-TGCTGGGTAATCCC- (1) Cytotoxic agent alone regimen: the cells were exposed to a single CAGGCCTCTATAGC-TAMRA (25); and mvp (Genbank cytotoxic agent for 3 d followed by 7 d in control medium. (2) Sequential combination: the cells were treated with a single cytotoxic agent for 3 d accession no. NM_017458) forward 5V-AGCTCGCAAG- followed by 1 Amol/L bexarotene for 7 d. (3) Simultaneous combination: GAACTTTTGGA-3V, reverse 5V-CCTTGGCAGTCCCGGTG- the cells were exposed to a single cytotoxic agent in the presence of 3V, and probe 6-carboxyfluorescein-ACGGCCATGCTCA- 1 Amol/L bexarotene for 3 d followed by a 7-d drug holiday. (4) Intermittent cytotoxic agent with continuous bexarotene: the cells were GAGCCTCCA-TAMRA. Brain standard was used for exposed to a single cytotoxic agent and 1 Amol/L bexarotene for 3 d 36B4, abcc1, bcrp, and mvp and liver standard for followed by 1 Amol/L bexarotene for 7 d. Not shown: control cells were 36B4, mdr1, abcb11, abcc2,andabcc3.Theprobeswere treated similarly with fresh medium containing 0.1% solvent or cell growth in the presence of 1 Amol/L bexarotene either given continuously obtained from Integrated DNA Technologies (Coralville, IA). or on a 7-d on, 3-d off cycle. Cell Invasion and Angiogenesis Assays The invasion assay was carried out in a Matrigel-coated 24-well Transwell unit (BD Biosciences, Bedford, MA). transcriptase (Applied Biosystems, Foster City, CA). Hu- Briefly, MDA-MB-231 parental and drug-resistant cells man brain and liver cDNA were used as standards were grown in culture medium until 80% confluence. Cells (Ambion, Austin, TX). The resulting cDNA was diluted in were trypsinized, resuspended in serum-free medium, RNase-free water, aliquoted, and stored at À80jC. Real- and seeded at 1 Â 105 in triplicate wells. The lower time PCR was done using a dual-fluorescent nonextendable chambers were filled with culture medium containing probe containing a 5V 6-carboxyfluorescein reporter dye 5% fetal bovine serum as a chemoattractant. The fraction and a 3V 6-carboxytetramethylrhodamine (TAMRA). of cells invading into and through the Matrigel matrix cDNAs (50 ng each) were used for real-time PCR in a final after 18 to 20 hours was quantified by MTT assay. To volume of 50 AL containing 1Â Taqman buffer (Applied determine the ability of MDA-MB-231 parental and drug- Biosystems), 300 nmol/L of each forward and reverse resistant cells to modulate angiogenesis, 3 Â 106 parental primer, and 100 nmol/L probe. Reactions were carried out cells or drug-resistant variants were cultured in 10 mL

Mol Cancer Ther 2005;4(5). May 2005

serum-free medium. The conditioned medium was collect- orally once daily at 100 mg/kg. Doxorubicin and cisplatin ed 48 hours later. HUVECs (5 Â 104) were seeded into the were dissolved in sterile saline. Paclitaxel was prepared upper chamber of the Matrigel-coated Transwells. The freshly each time from the concentrated stock solution conditioned medium from MDA-MB-231 parental cells and (6 mg/mL) with sterile saline. The cytotoxic agents were drug-resistant variants was used as chemoattractant and given i.p. once weekly. Doses used for cytotoxic agents and added to the lower chambers. The fraction of cells invading bexarotene were at the maximum tolerated dose, the dose into and through the Matrigel matrix after 24 hours was that caused <10% weight loss during the study. Animals quantified by MTT assay. Results of invasiveness and receiving no drugs were given vehicle for bexarotene orally angiogenic potential were normalized with parental cells daily and saline i.p. weekly. Animals receiving bexarotene and expressed as an invasion index. only were given saline i.p. once weekly; animals receiving Fluctuation Analysis cytotoxic agent only were given vehicle for bexarotene MDA-MB-231 cells were seeded at 1,000 per flask in orally daily. The treatment continued for 6 weeks. Tumor 75 cm2 tissue culture flasks. The flasks were divided into growth was measured with an electronic caliper (Mitutoyo, Inc., Utsunomiya, Japan) twice weekly. Tumor volumes two groups, with one group of cells grown in culture 2 medium and the other group in 1 Amol/L bexarotene. Cells were calculated using the formula: 1/2ab , where a is the b were allowed to grow to 80% confluence (average of longest and is the shortest axis of the tumor. Animal 6 weights were recorded once weekly. The animals used in 4.2 Â 10 cells per flask from cells grown in the culture 6 this study were housed in a U.S. Department of Agricul- medium and 4.5 Â 10 cells per flask from cells grown in ture–registered facility in accordance with NIH guidelines the bexarotene-containing medium). The total cell popula- for the care and use of laboratory animals. tion from each flask from both treatment groups was Data Analysis seeded onto separate 96-well plates overnight. For cultures 6 6 Dose-response curves for growth inhibition were gener- that contained >3.5 Â 10 cells per flask, only 3.5 Â 10 cells ated and plotted as a percentage of untreated control. The were seeded on the plate to avoid high cell density per well. drug concentration needed to produce 50% growth Cells grown in the culture medium during the expansion inhibition, IC , was determined by nonlinear least squares period were treated with 30 nmol/L paclitaxel for 7 days, 50 regression (JMP, Cary, NC). Differences in mean values whereas cells grown in 1 Amol/L bexarotene during the between groups were analyzed by unpaired Student’s t test expansion period were treated with a combination of with two-tailed comparison. Multiple comparisons used 30 nmol/L paclitaxel and 1 Amol/L bexarotene. Drug- two-way ANOVA followed by Tukey-Kramer test. Differ- containing medium was changed every other day for 7 days ences of P < 0.05 are considered significant. Software for andthenreplacedbydrug-freemedium.Surviving statistical analysis was by SigmaStat (SPSS, Inc., colonies were counted 3 weeks later. In a control Chicago, IL). experiment, the bulk population of MDA-MB-231 cells (4 Â 107 cells at 2 Â 106 cells per plate) without expansion of the population before drug treatment was treated directly Results with paclitaxel to determine the number of drug-resistant Effect of Bexarotene/Cytotoxic Combination on the variants in the total population. Mutation rate was Development of Acquired Drug Resistance In vitro calculated according to the method of Catcheside (26). When used as a single agent, paclitaxel produced a In vivo Animal Studies sigmoidal concentration-dependent growth inhibition in For the human xenograft tumor model, MDA-MB-231 F MDA-MB-231 cells with an IC50 of 3.17 0.2 nmol/L cells in log phase were harvested and resuspended in 1:1 (mean F SD, n = 3), whereas bexarotene showed limited (v/v) mixture of culture medium and Matrigel (BD growth inhibitory activity up to 10 Amol/L. Bexarotene did Biosciences, San Diego, CA). Tumor cells were implanted not interfere or enhance paclitaxel activity in various s.c. into right and left axial regions of 6-week-old female combinations with paclitaxel after single exposure (data athymic nude mice (Harlan, Madison, WI) with 25 gauge not shown). To determine the effect of the bexarotene/ 6 needle containing 5 Â 10 cells/100 AL. Thereafter, fresh paclitaxel combination after multiple exposures, cells were tumors harvested from donor mice were minced into 1 to subjected to repeated treatment for 10 cycles (see Fig. 1). 3mm3 in size. Approximately 25 to 30 fragments in 1:1 After four treatment cycles, paclitaxel activity was not culture medium and Matrigel were injected s.c. into enhanced nor was it inhibited by the combination with recipient mice with a 16 gauge needle. This process was bexarotene when compared with cells treated with pacli- repeated twice before initiation of the study. Animals were taxel alone (Fig. 2A). Cells treated with continuous randomized 2 days after tumor injection. Treatment began bexarotene grew similarly as vehicle-treated cells (Fig. 2A). when tumors were palpable (4–5 days after tumor Repeated treatment with intermittent paclitaxel alone injection). Each group consisted of 6 to 10 animals bearing resulted in the development of paclitaxel resistance within two tumors per animal. Bexarotene was prepared in an 80 days (Fig. 2B; Fig. 1, scheme 1). Treatment with aqueous solution containing 10% (v/v) polyethylene glycol intermittent paclitaxel and continuous bexarotene (Fig. 1, (Mr 400)/Tween 80 (99.5:0.5) and 90% of 1% (w/v) scheme 4) resulted in complete growth inhibition, whereas carboxymethylcellulose (Sigma Chemicals) and dosed the sequential combination (Fig. 1, scheme 2) and the

Mol Cancer Ther 2005;4(5). May 2005

Figure 2. Effect of bexarotene/cytotoxic combination on growth of MDA-MB-231 after multiple exposures. A, comparison of total cell numbers in vehicle control, bexarotene alone, paclitaxel alone, and various bexarotene/paclitaxel combinations for the first four cycles. Effect of bexarotene on the development of acquired drug resistance in MDA-MB-231 cells: (B) paclitaxel, (C) doxorubicin, and (D) cisplatin. MDA-MB-231 cells were subjected to various treatments as described in Fig. 1. At the end of each treatment cycle (10 d), cells were harvested by trypsinization. Viable cells were determined by trypan blue exclusion and reexposed to the same treatment. The procedure was repeated 10 times. Because only 5% of the cells for the vehicle control and bexarotene-treated groups were reseeded for the following cycle, and assuming the growth rate of these cells represents the growth rate of the total population, projected cell numbers were shown for these two groups in A.

simultaneous combination (Fig. 1, scheme 3) delayed the paclitaxel or doxorubicin alone were cross-resistant to other development of paclitaxel resistance for 1 and 2 months, Pgp substrates but responded to the non-Pgp substrate respectively, compared with cells treated with paclitaxel cisplatin (Table 1A and B). Cells isolated from the cisplatin- alone (Fig. 2B). Repeated exposure to bexarotene for 10 treated culture were resistant to cisplatin alone (Table 1C). cycles did not alter cell growth (data not shown). On the other hand, the surviving cells isolated from the Because the combination of intermittent paclitaxel and intermittent cytotoxic agent and continuous bexarotene continuous bexarotene prevented the development of (Fig. 1, scheme 4) remained chemosensitive toward all the paclitaxel resistance, we sought to determine whether compounds tested (Table 1). this treatment regimen could be applied to other It was apparent that the resistant cells isolated from cytotoxic agents to prevent or delay acquired drug the various bexarotene/paclitaxel combinations showed resistance. Two additional cytotoxic compounds, doxoru- a differential multidrug-resistant (MDR) phenotype bicin (a Pgp substrate) and cisplatin (a non-Pgp sub- (Table 1A). The resistant cells from the sequential combi- strate), were used to evaluate the efficacy of the nation (Fig. 1, scheme 2) showed a 20-fold resistance to bexarotene/cytotoxic combination. Similar to paclitaxel, tubulin agents and a 3-fold resistance to doxorubicin, bexarotene did not interfere with the activity of either whereas the resistant cells from the simultaneous combi- doxorubicin or cisplatin after single exposure (data not nation (Fig. 1, scheme 3) showed a 6-fold resistance to shown). Repeated exposures to the single cytotoxic agent paclitaxel but were sensitive to other tubulin agents and alone resulted in development of acquired drug resis- doxorubicin. To further examine the mechanism of the tance, whereas the combination treatment delayed the MDR phenotype in paclitaxel-resistant cells, the efflux growth of the cells (Fig. 2C and D) and remained activity of Pgp and mdr1 gene expression was determined. chemosensitive (see below). As seen in Fig. 3A, the extent of paclitaxel resistance (Table Characteristics of Resistant Variants 1A) was correlated with the efflux activity of Pgp, being The surviving cells after prolonged treatment with a highest in paclitaxel-resistant cells followed by cells single cytotoxic agent or with bexarotene were isolated and derived from the sequential combination and lowest in evaluated for their drug sensitivity. Cells isolated from cells derived from simultaneous combination. Addition of

Mol Cancer Ther 2005;4(5). May 2005

Table 1. Comparison of drug sensitivity between MDA-MB-231 parental cells and acquired drug-resistant cells

Cells Drugs

Paclitaxel RF Vincristine RF Doxorubicin RF Cisplatin RF

A. Paclitaxel Parental cells 1 1 1 1 Paclitaxel alone 34.0 F 3.8 30.8 F 2.3 5.0 F 0.8 1.1 F 0.1 Sequential combination 21.0 F 5.0 16.2 F 1.2 2.6 F 0.3 1.1 F 0.2 Simultaneous combination 6.4 F 1.3 1.1 F 0.2 1.1 F 0.2 1.1 F 0.2 B. Doxorubicin Parental cells 1 1 1 1 Doxorubicin alone 20.5 F 2.2 20.4 F 1.9 22.2 F 0.3 1.1 F 0.1 Intermittent doxorubicin + continuous bexarotene 1.1 F 0.2 1.1 F 0.2 1.1 F 0.3 1.1 F 0.1 C. Cisplatin Parental cells 1 1 1 1 Cisplatin alone 0.9 F 0.01 1.1 F 0.2 1.0 F 0.3 20.7 F 1.7 Intermittent cisplatin + continuous bexarotene 0.9 F 0.1 1.1 F 0.2 1.1 F 0.3 1.0 F 0.02

NOTE: Mean F SD, n = 3 from three separate experiments. MDA-MB-231 parent cells and cells recovered from the cytotoxic agent treatment alone or from the combination were tested for the sensitivity to paclitaxel, vincristine, doxorubicin, and cisplatin at various concentrations for 3 days. Drug effects were measured by MTT assay. IC50, the concentration needed to inhibit 50% of cell growth for each drug, was determined. Results were normalized with the parental cells and expressed as the resistant factor (RF). bexarotene had no effect on the efflux activity in the paclitaxel-resistant cells, whereas the Pgp inhibitor verapamil significantly decreased the efflux activity of Pgp in these cells (data not shown). To elucidate the mechanism of increased Pgp efflux activity in the resistant cells, real-time PCR was used to analyze mRNA levels for genes known to be involved in drug resistance: mdr1 (Pgp), abcb11 (MDR/ TAP), abcc1 (MRP1), abcc2 (MRP2), abcc3 (MRP3), bcrp (breast cancer–resistant protein), and mvp (lung-resistant protein). Our data showed that mdr1 mRNA was barely detected in MDA-MB-231 parental cells or cells treated with bexarotene but was increased f150-fold in cells treated with paclitaxel alone, 50-fold in cells treated with sequential combination, and 20-fold in cells treated with simultaneous combination (Fig. 3B). The expression of other resistant genes was not altered in the paclitaxel- resistant variants (data not shown). The level of mdr1 mRNA expression was consistent with the degree of efflux activity of Pgp and MDR phenotype (compare Fig. 3 and Table 1A). Taken together, these results showed that increased mdr1 mRNA expression was responsible for the MDR phenotype in paclitaxel-resistant cells. Our data further showed that bexarotene can influence the MDR phenotype in paclitaxel-resistant cells through modulation of mdr1 mRNA expression. Effect of Bexarotene/Cytotoxic Combinations on Overcoming Acquired Drug Resistance To determine whether acquired drug resistance can be overcome by the combination of intermittent cytotoxic Figure 3. Analysis of efflux activity of Pgp and mdr1 expression in MDA- agent and continuous bexarotene, resistant cells derived MB-231 parental cells and paclitaxel-resistant variants. A, Pgp activity from a single cytotoxic agent were repeatedly treated was quantified by the fluorescence intensity of free calcein inside the cells. intermittently with the same cytotoxic agent and contin- B, mdr1 expression was analyzed by real-time PCR. The expression of a housekeeping gene was used to normalized gene expression level. uous bexarotene (similar to Fig. 1, scheme 4). Treatment Columns, mean (n = 3); bars, SD. BEX, bexarotene; PAL, paclitaxel. with the cytotoxic agent alone or bexarotene alone had no *, P < 0.05, statistically different from parental cells.

Mol Cancer Ther 2005;4(5). May 2005

Effect of Bexarotene on Invasion and Angiogenic Ability Several lines of evidence have suggested that tumor cells selected for drug resistance are more invasive than their parental cells (27, 28). To determine whether bexarotene can influence invasiveness and angiogenesis, cells derived from single cytotoxic agents (drug-resistant) and from the combination (drug-sensitive) were subjected to Matrigel invasion and angiogenesis assays. As seen in Fig. 5, drug- resistant cells showed a 2-fold increase in invasiveness and angiogenic potential compared with the parental cells. In contrast, cells treated with the combination regimen showed a 50% reduction in invasiveness and angiogenic ability compared with the parental cells. The extent of invasiveness and angiogenesis of these cells was further correlated with the degree of drug resistance (see Table 1). Resistant cells that were treated with bexarotene alone or in combination also showed a decreased invasiveness and angiogenic ability compared with cells treated with single cytotoxic agents (data not shown). Effect of Bexarotene on Mutation Rate The ability of bexarotene to prevent and overcome multidrug resistance and reduce invasion and angiogenesis suggested that bexarotene may maintain and/or increase genomic integrity of the cells to prevent the cancer cell from modifying its genome resulting in the acquisition of drug resistance. To test this possibility, the effect of bexarotene on the frequency of developing drug resistance was evaluated using Luria-Delbru¨ ck fluctuation analysis (29). As seen in Table 2, no surviving clones were found in the bulk population of 2 Â 107 cells treated with paclitaxel in a single step (control group), indicating that preexisting paclitaxel-resistant variants in the MDA-MB-231 cell population was minimal. In the fluctuation groups, the average number of colonies per plate in paclitaxel-selected cells was 2.4, whereas the average number of colonies per plate in the bexarotene-treated cultures decreased 6-fold. Because the variance in the number of surviving colonies per plate exceeded the mean in the fluctuation groups, the above findings suggested that paclitaxel resistance in these clones arose spontaneously rather than being induced by Figure 4. Effect of bexarotene/cytotoxic combination on reversal of environmental selection. The mutation rate in the cells acquired drug resistance: (A) paclitaxel, (B) doxorubicin, and (C) cisplatin. expanded in control medium was calculated to be 7.3 Â Paclitaxel-, doxorubicin-, or cisplatin-resistant MDA-MB-231 cells were À8 repeatedly treated with vehicle, cytotoxic agent alone, bexarotene alone, 10 per cell generation, whereas those treated with or intermittent drug with continuous bexarotene. Parental cells were used bexarotene during the expansion period had a 7-fold as reference control and treated with a given cytotoxic drug for 3 d. Viable reduction in the mutation rate (P < 0.05, t test). Similar cells were determined by trypan blue exclusion at the end of each treatment cycle. Points, mean (n = 3); bars, SD. results were obtained by MSS maximum likelihood method (ref. 30; data not shown). The above findings suggest that effect on the growth of drug-resistant cells. However, the the interference with developing paclitaxel resistance by drug-resistant cells were resensitized to the cytotoxic agent the combination was due to a reduction in the spontaneous after multiple exposures to the bexarotene/cytotoxic mutation rate by bexarotene. combination (Fig. 4A–C). In general, the reversal of drug Effect of Bexarotene/Cytotoxic Combination In vivo resistance became apparent after repeated treatment for As seen in Fig. 6, MDA-MB-231 tumors grew continuous- three cycles. The remaining viable fraction of cells ly throughout the course of study in both control and drug- continued to decrease with increasing treatment cycles, treated animals. When compared with vehicle control, reaching <1% of untreated control after 8 to 10 treatment bexarotene given alone at 100 mg/kg daily had no effect on cycles. tumor growth. Treatment with doxorubicin (5 mg/kg)

Mol Cancer Ther 2005;4(5). May 2005

Figure 5. Effect of bexarotene on invasiveness in MDA-MB-231 parental and drug-treated cells (A and B). Effect of bexarotene on angiogenic potential in HUVECs (C and D). A total of 1 Â 105 MDA-MB-231 parental cells and each of drug-treated cells were seeded onto the upper chamber of the Matrigel- coated Transwells. The lower chambers were filled with culture medium containing 5% fetal bovine serum as a chemoattractant. The fraction of cells invading into and through the Matrigel matrix was quantified by MTT assay. HUVECs (5 Â 104) were seeded onto the upper chamber of the Matrigel-coated Transwells. The conditioned medium of MDA-MB-231 drug-resistant variants (obtained from 3 Â 106 cells cultured in 10 mL serum-free medium) was served as chemoattractant and added to the lower chambers. The fraction of cells invading into and through the Matrigel matrix was quantified by MTT assay. Results of invasiveness and angiogenic potential were normalized with parental cells and expressed as invasion index. DOX, doxorubicin; CIS, cisplatin. *, P < 0.05, statistically different from 1; **, P < 0.05, statistically different from the resistant cells derived from the cytotoxic treatment alone.

decreased tumor volume by f40% (P < 0.05 versus control; Discussion Fig. 6B), whereas no significant difference in tumor The present study shows that the bexarotene/cytotoxic volumes were seen in animals treated with paclitaxel combination can delay or prevent the development of (20 mg/kg) or cisplatin (5 mg/kg) relative to the vehicle acquired drug resistance in MDA-MB-231 cells. The control (Fig. 6A and C). On the other hand, the combination extent of acquired paclitaxel resistance correlated with of bexarotene/cytotoxic agent reduced tumor volume by the MDR phenotype and efflux activity of Pgp and mdr1 f 40% relative to cytotoxic agents alone (P < 0.05 in all gene expression. Cells with acquired drug resistance cases) and by 60% relative to vehicle control (P < 0.05 in all (paclitaxel, doxorubicin, and cisplatin), when treated with cases). The benefit of adding bexarotene to the cytotoxic a bexarotene/cytotoxic combination, were resensitized to regimen became apparent beyond 3 weeks as the tumor the cytotoxic agent. Interestingly, cells treated with the volume in mice receiving the combination therapy began to combination regimen showed reduced invasiveness and diverge from the tumor volume in mice treated with the angiogenic ability than their resistant counterparts. The cytotoxic agent alone. In a separate study, animals were in vivo MDA-MB-231 xenograft model showed that the treated with paclitaxel for 2 weeks followed by the bexarotene/cytotoxic combination produced a statistically bexarotene/paclitaxel combination. Our data showed that significant greater antitumor efficacy than single agents. treatment with paclitaxel followed by the combination Our findings suggest that the bexarotene/cytotoxic resulted in a 40% decrease in tumor growth compared with combination may be an efficacious treatment for ad- paclitaxel alone (Fig. 6D). Taken together, the above vanced breast cancer by interfering with tumor cell findings indicated that the combination regimen produced progression to a more malignant drug-resistant, invasive significant decrease in tumor volume than single agents in phenotype. MDA-MB-231 xenograft model. Our data further showed Genetic instability of cancer cells is thought to be one of that treatment with a single cytotoxic agent followed by the the major factors giving rise to drug-resistant mutant or combination resulted in a much greater delay in tumor variant subpopulations (31, 32). Our data show that long- growth compared with the single cytotoxic agent alone. term exposure to the bexarotene/cytotoxic combination can

Mol Cancer Ther 2005;4(5). May 2005

Table 2. Luria-Delbru¨ck fluctuation analysis of paclitaxel- decreased the spontaneous mutation rate compared with resistant MDA-MB-231 cells expansion without bexarotene, this suggests that bexarotene can maintain/increase genomic integrity of the tumor cells Fluctuation Control by interfering with the acquisition of spontaneous muta- group group tions that result in drug resistance. The molecular mechanism by which bexarotene mod- Plate Paclitaxel, Bexarotene + Paclitaxel, ulates drug-resistant genes and maintains genomic colonies paclitaxel, colonies per plate colonies per plate integrity is unknown at present. Several possibilities per plate exist. First, the tumor suppressor gene p53 functions to maintain genomic integrity by preventing cells with 1000unstable genomes from transiting through the cell cycle. 2200The functional role of p53 as a central mediator of the 3000DNA damage and regulation of apoptosis is well 4330established (33, 34). In addition, wild-type p53 has been 5500shown to repress mdr1 promoter activity, mdr1 expression, 6000and Pgp protein level, whereas mutant p53 stimulates such 7400 effects (35–37). Recent studies have indicated that a small 8020 9000ubiquitin-like modifier protein is involved in p53 stability, 10 2 0 0 function, and transcriptional regulation and genomic stabil- 11 3 0 0 ity (38, 39). The accumulated evidence suggests the 12 6 1 0 important role of wild-type p53 in DNA repair, regulation 13 0 0 0 of apoptosis pathways, and mdr1 gene expression level. 14 4 0 0 MDA-MB-231 human breast cancer cells contain mutant p53 15 3 1 0 (22). It is possible that bexarotene may interfere with mutant 16 1 0 0 p53-mediated cell survival after multiple exposures to 17 6 0 0 cytotoxic agents through inhibiting mutant p53 activity 18 3 0 0 and/or restoring a wild-type p53 phenotype. Second, 19 4 0 0 20 2 0 0 aneuploidy is one of the most common genomic abnormal- Total colonies 48 7 0 ities of cancer cells (40). Early studies by Duesberg et al. Mean 2.4 0.4 NA showed that genomic instability of cancer cells was Variance 4.1 0.7 NA proportional to the degree of aneuploidy (41). These Mutation rate 7.3 Â 10À8 9.8 Â 10À9 NA investigators further showed that aneuploid cells can acquire multidrug resistance by chromosome reassortments NOTE: Individual populations at 1,000 cells per flask were allowed to in the absence of multidrug resistance genes (42). Thus, expand in either the culture medium or 1 Amol/L bexarotene until 80% confluence. The total cell population from each flask was seeded onto bexarotene may maintain/increase genomic integrity separate 96-well plates and exposed to 30 nmol/L paclitaxel. Cells grown in through stabilizing DNA ploidy. Third, the nuclear factor- 1 Amol/L bexarotene during expansion period were treated with combina- nB has been shown to play an important role in controlling tion of 30 nmol/L paclitaxel and 1 Amol/L bexarotene. Treatment was continued for 7 days. The surviving clones were counted 3 weeks later. apoptotic cell death (43, 44) and the expression of the mdr1 Control group used the bulk populations of cells (4 Â 107 cells at 2 Â 106 cells gene (45). Furthermore, we have recently shown that a novel per plate) from mass cultures before drug selection. NA, not applicable. rexinoid LG100268 inhibited nuclear factor-nB activity to increase the activity of chemotherapeutic agents (46). It is delay and overcome acquired drug resistance and decrease possible that bexarotene may interfere with drug-resistant invasive and angiogenic capacity. We hypothesize that gene expression through inhibition of nuclear factor-nBto bexarotene may increase and/or maintain the genomic prevent and overcome multidrug resistance. Fourth, the integrity of cells to prevent the cancer cell from modifying steroid and xenobiotic receptor is a member of the nuclear its genome resulting in the development of acquired drug hormone receptor superfamily that heterodimerizes with resistance. This hypothesis was further supported by Luria- retinoid X receptor. It was reported that paclitaxel could Delbru¨ ck fluctuation analysis. As seen in Table 2, treatment enhance mdr1 gene expression through the steroid and with bexarotene decreased the spontaneous development of xenobiotic receptor in both primary hepatocytes and colon paclitaxel resistance. In theory, if resistance was induced by cancer cells, thereby increasing its own clearance and drug selection, the number of surviving colonies would be leading to the development of drug resistance (47). The expected to have a Poisson distribution, with the variance steroid and xenobiotic receptor was also found to be close to the mean (29). Our data showed that the variance in involved in cisplatin resistance by inducing glutathione S- the number of surviving colonies per plate was much transferase expression in endometrial cancer cells (48). greater than the mean, indicating that paclitaxel-resistant Bexarotene may directly or indirectly antagonize the steroid variants in MDA-MB-231 cells arose randomly rather than and xenobiotic receptor activity to prevent paclitaxel- and being induced by drug exposure. Because pretreatment cisplatin-induced expression of drug-resistant genes. Taken with bexarotene followed by drug selection significantly together, bexarotene may interfere with one or more of the

Mol Cancer Ther 2005;4(5). May 2005

Figure 6. Antitumor effect of bexarotene/cytotoxic combination in MDA- MB-231 xenograft: (A) bexarotene/ paclitaxel, (B) bexarotene/doxorubicin, (C) bexarotene/cisplatin, and (D) paclitaxel followed by bexarotene/paclitaxel. Nude mice bearing MDA-MB- 231 tumors were treated with vehicle, bexarotene, cytotoxic agent, or combination for 6 wks. Drug effect on tumor growth was determined twice weekly. Points, mean (n = 6 – 10); bars, SE. For some data points, bars were below the size of the symbol and were not shown in the figure. *, P < 0.05, statistically different from vehicle control at perspective time points; **, P < 0.05, statistically different from cytotoxic agent alone at perspective time points.

above-mentioned pathways to suppress multidrug resistance chemotherapy extends survival in late-stage non–small cell gene expression. Ongoing research focuses on elucidating the lung cancer patients (52). Ongoing research is directed mechanism of action of bexarotene in maintaining genomic toward understanding the mechanisms by which bexar- integrity to prevent and overcome multidrug resistance. otene prevents and overcomes acquired and intrinsic drug In the present study, we observed that cells that resistance. Information obtained from these studies will developed acquired drug resistance showed an increased have significant impact on the therapeutic use of bexar- invasiveness and angiogenic potential, whereas cells otene in cancer treatment. treated with the combination regimen were chemosensitive and had reduced invasive and angiogenic ability. Coex- pression of drug resistance and invasion/metastasis has Acknowledgments been observed in patient samples (49, 50). Cells that We thank Manny R. Corpuz for conducting xenograft studies and Drs. Reid coexpress these properties are due either to selection of P. Bissonnette and Tom W. Hermann for thoughtful discussions. more aggressive cells or to increase in the metastatic potential following chemotherapeutic insults (27). It is References likely that the bexarotene/cytotoxic combination interfered 1. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA with increased survival and tumor progression after drug Cancer J Clin 2004;54:8 – 29. exposure through maintaining and/or increasing genomic 2. Aszalos A, Thompson K, Yin JJ, Ross DD. Combinations of integrity. Using cDNA microarray analysis, we have P-glycoprotein blockers, verapamil, PSC833, and cremophor act differently on the multidrug resistance associated protein (MRP) and recently identified several bexarotene-regulated genes, on P-glycoprotein (Pgp). Anticancer Res 1999;19:1053 – 64. many of which were important for drug resistance, 3. Roninson IB. From amplification to function: the case of the MDR1 1 metastasis, and angiogenesis. We are currently evaluating gene. Mutat Res 1992;276:151 – 61. the functional roles of these genes. 4. Mechetner E, Kyshtoobayeva A, Zonis S, et al. Levels of multidrug Our present findings have important implications for resistance (MDR1) P-glycoprotein expression by human breast cancer correlate with in vitro resistance to taxol and doxorubicin. Clin Cancer Res patients with breast cancer as well as other diseases. For 1998;4:389 – 98. example, we have recently reported that the bexarotene/ 5. Galmarini CM, Kamath K, Vanier-Viornery A, et al. Drug resistance paclitaxel combination delayed and overcame paclitaxel associated with loss of p53 involves extensive alterations in microtubule resistance in non–small cell lung cancer (51). In addition, composition and dynamics. Br J Cancer 2003;88:1793 – 9. the results from a recent phase I/II clinical trials showed 6. Longley DB, Ferguson PR, Boyer J, et al. Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying that addition of Targretin capsules to cisplatin/vinorelbine sensitivity to TS- and non-TS-targeted chemotherapies. Clin Cancer Res 2001;7:3533 – 9. 7. Rudin CM, Yang Z, Schumaker LM, et al. Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res 2003; 1 Hermann and Lamph, unpublished results. 63:312 – 8.

Mol Cancer Ther 2005;4(5). May 2005

8. Moscow JA, Connolly T, Myers TG, Cheng CC, Paull K, Cowan KH. 29. Luria SE, Delbuck M. Mutations of bacteria from virus sensitivity to Reduced folate carrier gene (RFC1) expression and anti-folate resistance in virus resistance. Genetics 1943;28:491 – 511. transfected and non-selected cell lines. Int J Cancer 1997;72:184 – 90. 30. Rosche WA, Foster PL. Determining mutation rates in bacterial 9. Zhou Z, Zwelling LA, Kawakami Y, et al. Adenovirus-mediated human populations. Methods 2000;20:4 – 17. topoisomerase IIa gene transfer increases the sensitivity of etoposide- 31. Karran P, Hampson R. Genomic instability and tolerance to alkylating resistant human breast cancer cells. Cancer Res 1999;59:4618 – 24. agents. Cancer Surv 1996;28:69 – 85. 10. Boehm MF, Zhang L, Badea BA, et al. Synthesis and structure-activity 32. Duesberg P, Fabarius A, Hehlmann R. Aneuploidy, the primary cause relationships of novel retinoid X receptor-selective retinoids. J Med Chem of the multilateral genomic instability of neoplastic and preneoplastic cells. 1994;37:2930 – 41. IUBMB Life 2004;56:65 – 81. 11. Gottardis MM, Bischoff ED, Shirley MA, Wagoner MA, Lamph WW, 33. Adimoolam S, Ford JM. p53 and regulation of DNA damage Heyman RA. Chemoprevention of mammary carcinoma by LGD1069 recognition during nucleotide excision repair. DNA Repair 2003;2: (Targretin): an RXR-selective ligand. Cancer Res 1996;56:5566 – 70. 947 – 54. 12. Wu K, Zhang Y, Xu XC, et al. The retinoid X receptor-selective 34. El-Deiry WS. The role of p53 in chemosensitivity and radiosensitivity. retinoid, LGD1069, prevents the development of estrogen receptor- Oncogene 2003;22:7486 – 95. negative mammary tumors in transgenic mice. Cancer Res 2002;62: 6376 – 80. 35. Chin KV, Ueda K, Pastan I, Gottesman MM. Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science 1992; 13. Wu K, Kim HT, Rodriquez JL, et al. Suppression of mammary 255:459 – 62. tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069. Cancer Epidemiol Biomarkers Prev 2002;11:467 – 74. 36. Thottassery JV, Zambetti GP, Arimori K, Schuetz EG, Schuetz JD. p53-dependent regulation of MDR1 gene expression causes selective 14. Bischoff ED, Gottardis MM, Moon TE, Heyman RA, Lamph WW. resistance to chemotherapeutic agents. Proc Natl Acad Sci U S A 1997; Beyond tamoxifen: the retinoid X receptor-selective ligand LGD1069 94:11037 – 42. (TARGRETIN) causes complete regression of mammary carcinoma. Cancer Res 1998;58:479 – 84. 37. Zastawny RL, Salvino R, Chen J, Benchimol S, Ling V. The core promoter region of the P-glycoprotein gene is sufficient to confer dif- 15. Bischoff ED, Heyman RA, Lamph WW. Effect of the retinoid X ferential responsiveness to wild-type and mutant p53. Oncogene 1993;8: receptor-selective ligand LGD1069 on mammary carcinoma after tamox- 1529 – 35. ifen failure. J Natl Cancer Inst 1999;91:2118. 38. Melchior F, Hengst L. SUMO-1 and p53. Cell Cycle 2002;1:245 – 9. 16. Agarwal VR, Bischoff ED, Hermann T, Lamph WW. Induction of adipocyte-specific gene expression is correlated with mammary tumor 39. Muller S, Ledl A, Schmidt D. SUMO: a regulator of gene expression regression by the retinoid X receptor-ligand LGD1069 (Targretin). Cancer and genome integrity. Oncogene 2004;23:1998 – 2008. Res 2000;60:6033 – 8. 40. Harris H. Cells of the body: a history of somatic cell genetics. New 17. Yen WC, Prudente RY, Lamph WW. Synergistic effect of a retinoid X York: Cold Spring Laboratory Press; 1995. receptor-selective ligand bexarotene (LGD1069, Targretin) and paclitaxel 41. Duesberg P, Rausch C, Rasnick D, Hehlmann R. Genetic instability of (Taxol) in mammary carcinoma. Breast Cancer Res Treat 2004;88:141 – 8. cancer cells is proportional to their degree of aneuploidy. Proc Natl Acad 18. Duvic M, Hymes K, Heald P, et al. Bexarotene is effective and safe for Sci U S A 1998;95:13692 – 7. treatment of refractory advanced-stage cutaneous T-cell lymphoma: 42. Duesberg P, Stindl R, Hehlmann R. Origin of multidrug resistance in multinational phase II-III trial results. J Clin Oncol 2001;19:2456 – 71. cells with and without multidrug resistance genes: chromosome reassort- 19. Hardman WE, Moyer MP, Cameron IL. Efficacy of treatment of colon, ments catalyzed by aneuploidy. Proc Natl Acad Sci U S A 2001;98: lung and breast human carcinoma xenografts with: doxorubicin, cisplatin, 11283 – 8. irinotecan or topotecan. Anticancer Res 1999;19:2269 – 74. 43. Grimm S, Bauer MK, Baeuerle PA, Schulze-Osthoff K. Bcl-2 down- n 20. Kurbacher CM, Wagner U, Kolster B, Andreotti PE, Krebs D, Bruckner regulates the activity of transcription factor NF- B induced upon HW. Ascorbic acid (vitamin C) improves the antineoplastic activity of apoptosis. J Cell Biol 1996;134:13 – 23. doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells 44. Jones DR, Broad RM, Comeau LD, Parsons SJ, Mayo MW. Inhibition in vitro. Cancer Lett 1996;103:183 – 9. of nuclear factor nB chemosensitizes non-small cell lung cancer through 21. Wang Q, Yang W, Uytingco MS, Christakos S, Wieder R. 1,25- cytochrome c release and caspase activation. J Thorac Cardiovasc Surg Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer 2002;123:310 – 7. cells to chemotherapy-induced cell death. Cancer Res 2000;60:2040 – 8. 45. Bentires-Alj M, Barbu V, Fillet M, et al. NF-nB transcription factor 22. Nieves-Neira W, Pommier Y. Apoptotic response to camptothecin induces drug resistance through MDR1 expression in cancer cells. and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell Oncogene 2003;22:90 – 7. lines of the NCI Anticancer Drug Screen: multifactorial relationships with 46. Rendi MH, Suh N, Lamph WW, et al. The selective estrogen receptor topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspase modulator arzoxifene and the rexinoid LG100268 cooperate to promote pathways. Int J Cancer 1999;82:396 – 404. transforming growth factor h-dependent apoptosis in breast cancer. 23. Nakamura T, Sakaeda T, Horinouchi M, et al. Effect of the mutation Cancer Res 2004;64:3566 – 71. (C3435T) at exon 26 of the MDR1 gene on expression level of MDR1 47. Synold TW, Dussault I, Forman BM. The orphan nuclear receptor SXR messenger ribonucleic acid in duodenal enterocytes of healthy Japanese coordinately regulates drug metabolism and efflux. Nat Med 2001;7: subjects. Clin Pharmacol Ther 2002;71:297 – 303. 584 – 90. 24. Steinbach D, Lengemann J, Voigt A, Hermann J, Zintl F, Sauerbrey A. 48. Takami N, Sakamoto H, Yamamoto T. Steroid and xenobiotic receptor Response to chemotherapy and expression of the genes encoding the (SXR) is a key system for the acquisition of cisplatin resistance in multidrug resistance-associated proteins MRP2, MRP3, MRP4, MRP5, and endometrial cancer cells. J Int Med Res 2003;31:59 – 68. SMRP in childhood acute myeloid leukemia. Clin Cancer Res 2003;9: 49. Goldstein LJ, Galski H, Fojo A, et al. Expression of a multidrug 1083 – 6. resistance gene in human cancers. J Natl Cancer Inst 1989;81:116 – 24. 25. Steinbach D, Sell W, Voigt A, Hermann J, Zintl F, Sauerbrey A. BCRP 50. Weinstein RS, Jakate SM, Dominguez JM, et al. Relationship of the gene expression is associated with a poor response to remission induction expression of the multidrug resistance gene product (P-glycoprotein) in therapy in childhood acute myeloid leukemia. Leukemia 2002;16:1443 – 7. human colon carcinoma to local tumor aggressiveness and lymph node 26. Catcheside D. The genetics of microorganisms. London: Isaac Pitman metastasis. Cancer Res 1991;51:2720 – 6. and Sons; 1951. 51. Yen WC, Corpuz MR, Prudente RY, et al. A selective retinoid X 27. De Larco JE, Wuertz BR, Manivel JC, Furcht LT. Progression and receptor agonist bexarotene (Targretin) prevents and overcomes acquired enhancement of metastatic potential after exposure of tumor cells to paclitaxel (Taxol) resistance in human non-small cell lung cancer. Clin chemotherapeutic agents. Cancer Res 2001;61:2857 – 61. Cancer Res 2004;10:8656 – 64. 28. Liang Y, Meleady P, Cleary I, McDonnell S, Connolly L, Clynes M. 52. Khuri FR, Rigas JR, Figlin RA, et al. Multi-institutional phase I/II trial of Selection with melphalan or paclitaxel (Taxol) yields variants with different oral bexarotene in combination with cisplatin and vinorelbine in previously patterns of multidrug resistance, integrin expression and in vitro untreated patients with advanced non-small-cell lung cancer. J Clin Oncol invasiveness. Eur J Cancer 2001;37:1041 – 52. 2001;19:2626 – 37.

Mol Cancer Ther 2005;4(5). May 2005

Downloaded from mct.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. 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

Mol Cancer Ther 2005;4:824-834.

Updated version Access the most recent version of this article at: http://mct.aacrjournals.org/content/4/5/824

Cited articles This article cites 49 articles, 23 of which you can access for free at: http://mct.aacrjournals.org/content/4/5/824.full#ref-list-1

Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/4/5/824.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/4/5/824. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.