Bexarotene (LGD1069, Targretin), a Selective Retinoid X Receptor Agonist, Prevents and Reverses Gemcitabine Resistance in NSCLC Cells by Modulating Gene Amplification Patricia Tooker, Wan-Ching Yen, Shi-Chung Ng, et al. Cancer Res 2007;67:4425-4433.
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Research Article
Bexarotene (LGD1069, Targretin), a Selective Retinoid X Receptor Agonist, Prevents and Reverses Gemcitabine Resistance in NSCLC Cells by Modulating Gene Amplification Patricia Tooker, Wan-Ching Yen, Shi-Chung Ng, Andre`s Negro-Vilar, and Thomas W. Hermann Department of Molecular Oncology, Ligand Pharmaceuticals, Inc., San Diego, California
Abstract Acquired drug resistance is a major obstacle in cancer therapy. As for many other drugs, this is also the case for gemcitabine, a nucleoside analogue with activity against non– small cell lung cancer (NSCLC). Here, we evaluate the ability of bexarotene to modulate the acquisition and maintenance of gemcitabine resistance in Calu3 NSCLC models. In the prevention model, Calu3 cells treated repeatedly with gemcitabine alone gradually developed resistance. However, with inclusion of bexarotene, the cells remained chemosensitive. RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3GemR), a gene known to be involved in gemcitabine resistance. In addition, the expression of genes surrounding the chromosomal location of rrm1 was increased, suggesting that resistance was due to gene amplification at the chr11 p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 gene copy number was increased over 10-fold. Correspondingly, fluorescence in situ hybridization analysis of metaphase chromosomes showed an intrachromosomal amplification of the rrm1 locus. In the therapeutic model, bexarotene gradually resensitized Calu3-GemR cells to gemcitabine, reaching parental drug sensitivity after 10 treatment cycles. This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors generated from the resistant cells did not respond to gemcitabine but were growth inhibited when bexarotene was added to the cytotoxic agent. The data indicate that bexarotene can resensitize gemcitabine-resistant tumor cells by reversing gene amplification. This suggests that bexarotene may have clinical utility in cancers where drug resistance by gene amplification is a major obstacle to successful therapy. [Cancer Res 2007;67(9):4425–33]
Introduction Lung cancer is the leading cause of cancer death for both men and women (1). The American Cancer Society estimated for 2004 f173,770 new cases of lung cancer in the United States, and f160,440 people dying of this disease. Non–small-cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for
almost 80% of all cases. Chemotherapy remains the only treatment option for patients with unresectable NSCLC. However, the current 5-year survival rate has not improved with this therapy (2). Thus, identification of effective treatment regimens for late-stage disease is urgently needed. Gemcitabine (2¶,2¶-difluorodeoxycytidine) is a nucleoside analogue and has shown antitumor activity in various cancers, including lung cancer (3–6). The activity of gemcitabine is based primarily on the formation of gemcitabine triphosphate (dFdCTP), which incorporates into the DNA and acts as a DNA synthesis chain terminator (7). In addition, the diphosphate metabolite of gemcitabine (dFdCDP) was also reported to inhibit ribonucleotide reductase causing a decrease in dCTP, decreased feedback inhibition of deoxycytidine kinase, and enhanced phosphorylation of gemcitabine (8–10). Recent studies have suggested that ribonucleotide reductase large subunit 1 (RRM1) and deoxycytidine kinase play important roles in resistance to gemcitabine (11, 12). Developing new treatment strategies to prevent and overcome gemcitabine resistance will greatly improve the clinical utility of this compound. We previously showed that the selective retinoid X receptor agonist bexarotene (LGD1069, Targretin) is an efficacious chemopreventive and chemotherapeutic agent in a number of preclinical rodent models of breast cancer (13–16). We further showed that the combination of bexarotene and cytotoxic agents produces synergistic growth inhibition as well as prevents and overcomes acquired drug resistance in several preclinical cancer models (17–21). The encouraging preclinical results with bexarotene, alone or in combination with various cytotoxic agents, led us to further examine its role in treatment of solid tumors. The purpose of this study was to evaluate the influence of bexarotene on the acquisition and maintenance of gemcitabine resistance in human NSCLC. Our results show that inclusion of bexarotene into the gemcitabine treatment regimen prevented and overcame acquired gemcitabine resistance in NSCLC Calu3 cells. This activity was associated with prevention or reversal of rrm1 gene amplification. Moreover, xenograft tumors generated from the gemcitabineresistant Calu3-GemR cells were unresponsive to gemcitabine or bexarotene as single agents but showed a statistically significant decrease in growth when treated with the bexarotene/gemcitabine combination.
Materials and Methods Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for Wan-Ching Yen: Oncomed Pharmaceuticals, Inc., 265 North Whisman Road, Mountain View, CA 94034. Requests for reprints: Thomas W. Hermann, Department of Molecular Oncology, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, CA 92121. Phone: 858-550-7826; Fax: 858-550-7730; E-mail:
[email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4495
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Chemicals and reagents. RPMI 1640, fetal bovine serum, glutamine, and gentamicin were obtained from Life Technologies. Bexarotene was synthesized at Ligand Pharmaceuticals, Inc. Gemcitabine (2¶,2¶-difluorodeoxycytidine) was purchased through Pharmaceutical Buyers, Inc. Cytosine arabinoside (ara-C), doxorubicin, cisplatin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide were obtained from Sigma Chemicals. Gemcitabine and bexarotene stock solutions in sterile water
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