Bexarotene (LGD1069, Targretin), a Selective Retinoid X Receptor Agonist, Prevents and Reverses Gemcitabine Resistance in NSCLC Cells by Modulating Gene Amplification

Research Article

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 almost 80% of all cases. Chemotherapy remains the only treatment Acquired drug resistance is a major obstacle in cancer option for patients with unresectable NSCLC. However, the current therapy. As for many other drugs, this is also the case for 5-year survival rate has not improved with this therapy (2). Thus, gemcitabine, a nucleoside analogue with activity against non– identification of effective treatment regimens for late-stage disease is urgently needed. small cell lung cancer (NSCLC). Here, we evaluate the ability ¶ ¶ of bexarotene to modulate the acquisition and maintenance Gemcitabine (2 ,2 -difluorodeoxycytidine) is a nucleoside ana- of gemcitabine resistance in Calu3 NSCLC models. In the logue and has shown antitumor activity in various cancers, prevention model, Calu3 cells treated repeatedly with gemci- including lung cancer (3–6). The activity of gemcitabine is based tabine alone gradually developed resistance. However, with primarily on the formation of gemcitabine triphosphate (dFdCTP), inclusion of bexarotene, the cells remained chemosensitive. which incorporates into the DNA and acts as a DNA synthesis chain terminator (7). In addition, the diphosphate metabolite of RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3- gemcitabine (dFdCDP) was also reported to inhibit ribonucleotide GemR), a gene known to be involved in gemcitabine reductase causing a decrease in dCTP, decreased feedback resistance. In addition, the expression of genes surrounding inhibition of deoxycytidine kinase, and enhanced phosphorylation the chromosomal location of rrm1 was increased, suggesting of gemcitabine (8–10). Recent studies have suggested that that resistance was due to gene amplification at the chr11 ribonucleotide reductase large subunit 1 (RRM1) and deoxycytidine p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 kinase play important roles in resistance to gemcitabine (11, 12). gene copy number was increased over 10-fold. Correspond- Developing new treatment strategies to prevent and overcome ingly, fluorescence in situ hybridization analysis of metaphase gemcitabine resistance will greatly improve the clinical utility of chromosomes showed an intrachromosomal amplification this compound. of the rrm1 locus. In the therapeutic model, bexarotene We previously showed that the selective retinoid X receptor gradually resensitized Calu3-GemR cells to gemcitabine, agonist bexarotene (LGD1069, Targretin) is an efficacious chemo- reaching parental drug sensitivity after 10 treatment cycles. preventive and chemotherapeutic agent in a number of preclinical rodent models of breast cancer (13–16). We further showed that This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors the combination of bexarotene and cytotoxic agents produces generated from the resistant cells did not respond to synergistic growth inhibition as well as prevents and overcomes gemcitabine but were growth inhibited when bexarotene was acquired drug resistance in several preclinical cancer models added to the cytotoxic agent. The data indicate that (17–21). The encouraging preclinical results with bexarotene, alone bexarotene can resensitize gemcitabine-resistant tumor cells or in combination with various cytotoxic agents, led us to further by reversing gene amplification. This suggests that bexarotene examine its role in treatment of solid tumors. The purpose of this may have clinical utility in cancers where drug resistance by study was to evaluate the influence of bexarotene on the gene amplification is a major obstacle to successful therapy. acquisition and maintenance of gemcitabine resistance in human [Cancer Res 2007;67(9):4425–33] 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 Introduction associated with prevention or reversal of rrm1 gene amplification. Lung cancer is the leading cause of cancer death for both men Moreover, xenograft tumors generated from the gemcitabine- and women (1). The American Cancer Society estimated for 2004 resistant Calu3-GemR cells were unresponsive to gemcitabine or f173,770new cases of lung cancer in the United States, and bexarotene as single agents but showed a statistically significant f160,440 people dying of this disease. Non–small-cell lung cancer decrease in growth when treated with the bexarotene/gemcitabine (NSCLC) is the most common type of lung cancer, accounting for combination.

Materials and Methods Note: Supplementary data for this article are available at Cancer Research Online Chemicals and reagents. RPMI 1640, fetal bovine serum, glutamine, and (http://cancerres.aacrjournals.org/). Current address for Wan-Ching Yen: Oncomed Pharmaceuticals, Inc., 265 North gentamicin were obtained from Life Technologies. Bexarotene was Whisman Road, Mountain View, CA 94034. synthesized at Ligand Pharmaceuticals, Inc. Gemcitabine (2¶,2¶-difluoro- Requests for reprints: Thomas W. Hermann, Department of Molecular Oncology, deoxycytidine) was purchased through Pharmaceutical Buyers, Inc. Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, CA 92121. Phone: Cytosine arabinoside (ara-C), doxorubicin, cisplatin, and 3-(4,5-dimethylth- 858-550-7826; Fax: 858-550-7730; E-mail: [email protected]. I2007 American Association for Cancer Research. iazol-2-yl)-2,5-diphenyltetrazolum bromide were obtained from Sigma doi:10.1158/0008-5472.CAN-06-4495 Chemicals. Gemcitabine and bexarotene stock solutions in sterile water www.aacrjournals.org 4425 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research or DMSO, respectively, were at concentrations that limited the final concentration of the solvent in the culture medium to <0.1%. Cell lines. The human NSCLC cell line Calu3 was obtained from the American Type Culture Collection. A gemcitabine-resistant derivative of Calu3 cells (Calu3-GemR) was generated as described in Results. All cells were routinely cultured in RPMI 1640supplemented with 10%fetal bovine serum and 2 mmol/L glutamine in 95% air, 5% CO2. In vitro treatment regimens. To determine the effect of bexarotene and/or gemcitabine on the development of gemcitabine resistance, Calu3 cells were seeded at 2 Â 106 in T-225 flasks. The treatment schemes are illustrated in Fig. 1A. Briefly, the cells were exposed to the regimens on a 10- day cycle. After a 3-day treatment with gemcitabine alone or in combination with bexarotene the cells were washed, counted, and replated followed by either a 7-day exposure to bexarotene or to control medium. At the end of each treatment cycle, the cells were trypsinized, when possible counted, and then replated onto a new flask and again exposed to the same treatment regimen. This procedure was repeated 10times. For the gemcitabine single- agent regimen (Fig. 1A, regimen 1), the cells were exposed to 50nmol/L gemcitabine for 3 days followed by 7 days in control medium. For the combination of intermittent gemcitabine with continuous bexarotene (Fig. 1A, regimen 3), the cells were exposed to 50nmol/L gemcitabine and 1 Amol/L bexarotene for 3 days followed by 1 Amol/L bexarotene for 7 days. Control cells were treated similarly with fresh medium containing 0.1% solvent or 1 Amol/L bexarotene given continuously (Fig. 1A, regimen 2). Cell numbers were monitored by trypan blue exclusion. In vitro drug sensitivity assay. To determine the sensitivity of parental Calu3 cells and their gemcitabine-resistant variants to gemcitabine, ara-C, doxorubicin, and cisplatin, cells were seeded in 96-well tissue culture plates and treated with each agent at various concentrations for 3 days. Drug effect was measured by WST-1 assay (Roche). Briefly, 10 ALofWST-1 reagent were added to each well and incubated for 2 h at 37jC; the absorbance was measured using a microplate reader (BioTek Instruments) at a wavelength of 420nm. RNA isolation and cDNA preparation. Total RNA was isolated from 2 Â 106 to 5 Â 106 cells using RNeasy Mini kits (Qiagen) according to the manufacturer’s instructions. Total RNA was eluted in RNase-free water and stored at À80jC. Total RNA (200 ng) was reverse transcribed into cDNA in a50AL reaction volume containing 1Â reverse transcription buffer, 5.5 mmol/L MgCl2, 2 mmol/L deoxynucleotide triphosphates, 2.5 Amol/L random hexamers, 0.4 unit of RNase inhibitor, and 1.25 units of MuLV reverse transcriptase (Applied Biosystems). Human reference cDNA, prepared from commercial RNA pooled from various tissues (Clontech), was used as a standard. All cDNAs were stored at À80jC. DNA isolation and purification. Total DNA was isolated from 1 Â 106 to 5 Â 106 cells using PureLink genomic DNA purification kits (Invitrogen) according to the manufacturer’s instructions. Genomic DNA was eluted with Tris-HCl (pH 8.5) and stored at À20jC until use. Quantitative real-time PCR. Real-time PCR assays were done using specific primers and a specific dual-labeled (5¶-6FAM/3¶-TAMRA) probes for Figure 1. Bexarotene prevents the development of resistance to gemcitabine in each target. For gene expression analyses, cDNAs corresponding to 10ng Calu3 cells. A, treatment regimens. Cells were treated repeatedly with 10-d cycles of (1) 50 nmol/L gemcitabine for 3 d followed by 7 d in control medium, (2) total RNA were analyzed, for relative DNA copy number determinations A A 1 mol/L bexarotene continuously for 10 d, or (3) 50 nmol/L gemcitabine in the 10ng of genomic DNA were used per assay. Reaction volumes were 50 L presence of 1 Amol/L bexarotene for 3 d followed by 1 Amol/L bexarotene as containing 1Â Universal Taqman buffer (Applied Biosystems), 300 nmol/L single agent for 7 d. Control cells were treated similarly with fresh medium of each forward and reverse primer and 100 nmol/L probe. Reactions were containing 0.1% solvent (not shown). B, comparison of total cell numbers in carried out in an ABI PRISM 7700 sequence detection system (Applied vehicle control, bexarotene alone, gemcitabine alone, and bexarotene/ j j gemcitabine combination for the first five cycles. Cell numbers were determined Biosystems) for 40cycles of 15 s at 95 C and 60s at 60 C. RNA expression as described in (C). C, effect of bexarotene on the development of gemcitabine- of the target genes was normalized to the expression level of the resistant Calu3 cells. Calu3 cells were subjected to treatments as shown housekeeping genes 36b4 or gapdh. DNA copy numbers were normalized in (A). At the end of each treatment cycle (10 d), cells were harvested by by gapdh DNA signal to account for input variation. The following primers trypsinization. The number of viable cells was determined by trypan blue ¶ exclusion and cells were reseeded and exposed to the same treatment. The and probes were used for RNA analyses: 36b4 (M17885) forward 5 - procedure was repeated 10 times (gray) after which cultures were allowed to GCAGATCCGCATGTCCCTT-3¶, reverse 5¶-TGTTTTCCAGGTGCCCTCG-3¶, recover without treatment. probe 5¶-6FAM-AGGCTGTGGTGCTGATGGCCAAGAAC-TAMRA-3¶; gapdh (NM_002046) forward 5¶-ACCACAGTCCATGCCATCACT-3¶, reverse 5¶- CATCACGCCACAGTTTCCC-3¶,probe5¶-6FAM-ACCCAGAAGACTGTG- of dck, cda, ctps, and rrm2 RNA was measured by commercial assays GATGGCCCCT-TAMRA-3¶; rrm1 (NM_001033) forward 5¶-CAGCTTTGG- obtained from Applied Biosystems (assay Ids Hs00176127_m1, Hs00156401_ TATGCCATCATTG-3¶, reverse 5¶-GCTCTTTCGATTACAGGAATCTTTG-3¶, m1, Hs00157163_m1, and Hs00357247_g1, respectively). The following probe 5¶-6FAM-CTCAGACGGAAACAGGCACCCCG-TAMRA-3¶. Expression primers and probes were used to measure relative gene copy numbers:

Cancer Res 2007; 67: (9). May 1, 2007 4426 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Bexarotene Prevents and Overcomes Gemcitabine Resistance rrm1 (NC_000011.8, intron 5) forward 5¶-AAGAGATTGCCTTATTGTGG- chromosomes were visible due to signal background in the green channel, ATGAT-3¶, reverse 5¶-GCGTCTAGAAAGAGAGAAACAACAAA-3¶,probe counterstaining was not necessary. Slides were evaluated using a Leitz DMRB 5¶-6FAM-TATCCACTGCGTCTTTAAAAACGGTGCTCTG-TAMRA-3¶; gapdh fluorescence microscope using filters for FITC (rrm1) and Texas red (NM_002046) forward 5¶-ACCACAGTCCATGCCATCACT-3¶, reverse 5¶- (chromosome 11). CATCACGCCACAGTTTCCC-3¶,probe5¶-6FAM-ACCCAGAAGACTGTG- In vivo animal studies. For the human xenograft tumor model, parental GATGGCCCCT-TAMRA-3¶. The probe for 36b4 was obtained from or gemcitabine-resistant Calu3 cells were harvested in log-phase growth Integrated DNA Technologies. All other probes were purchased from and resuspended in a 1:1 (v/v) mixture of culture medium and Matrigel (BD Applied Biosystems. Biosciences). Tumor cells were implanted s.c. into the right and left axial Oligonucleotide array hybridization and data analysis. Sample regions of 6-week-old female athymic nude mice (Harlan) with a 25-gauge processing and hybridization was done as described in the Affymetrix needle containing 0.5 Â 106 cells in 100 AL. Animals were randomized and GeneChip Expression Analysis Manual (Affymetrix). Briefly, double-stranded treatment began when tumors were palpable (4–5 days after tumor cDNA was synthesized from 2 Ag total RNA using the T7-(dT)24 primer injection). Each group consisted of 10animals bearing two tumors per

[5¶-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG(T)24-3¶]. animal. Bexarotene was suspended in an aqueous solution containing 10% From the cDNA, biotinylated cRNA was synthesized using the ENZO High (v/v) polyethylene glycol (Mr 400) / Tween 80 (99.5:0.5) and 90% of 1% (w/v) Yield BioArray IVT Kit (Affymetrix). Following chemical fragmentation, 10 carboxymethylcellulose (Sigma Chemical Co.) and dosed p.o. once daily at Ag of the fragmented cRNA probe were mixed with a hybridization cocktail 100 mg/kg. This dose of bexarotene was previously determined as the containing control oligonucleotide B2 and hybridization controls BioB, maximum tolerated dose, the dose that caused <10% weight loss over the BioC, BioD, and cre (Affymetrix), herring sperm DNA (Fisher Scientific), and course of the study (15, 22). Gemcitabine was prepared fresh each time in acetylated bovine serum albumin (Invitrogen). All samples were hybridized sterile saline and was administered at 100 mg/kg i.p. weekly. Animals to human HG-U133A GeneChip Arrays for 16 h at 45jC with rotation (60 receiving no drugs were given vehicle for bexarotene p.o. daily and saline rpm). After washing and staining (using R-phycoerythrin streptavidin with i.p. weekly. Animals receiving bexarotene only were given saline i.p. weekly. amplification by biotinylated antistreptavidin antibody) on the Affymetrix Animals receiving gemcitabine only were given vehicle for bexarotene p.o. Microfluidics Workstation according to the manufacturer’s instruction daily. The treatment continued for 6 weeks. Tumor growth was measured (Microfluidics Protocol EukGE-WS2v4), the arrays were scanned on an with an electronic caliper (Mitutoyo, Inc.) twice weekly. Tumor volumes Agilent scanner at a wavelength of 570nm. Expression intensity signals for were calculated using the formula V = 1/2AB2, where A and B represent the each probe set on each of the microarrays were generated with the longest and the shortest axis of the tumor, respectively. Animal weights Affymetrix MAS5.0software using the software’s global scaling procedure to were recorded once weekly. The animals used in this study were housed in a normalize between arrays. From each of the different Calu3 cultures U.S. Department of Agriculture–registered facility in accordance with NIH (parental, gemcitabine-resistant treated with vehicle, bexarotene, gemcita- guidelines for the care and use of laboratory animals. bine, or with the gemcitabine/bexarotene combination for 10cycles), four Data analysis. Dose-response curves for growth inhibition were replicate RNA samples were prepared and individually subjected to generated and were plotted as a percentage of untreated control. Values microarray analysis. The expression signals were averaged and transformed for IC50 (the drug concentration needed to produce 50% growth inhibition) into log 2 ratios. Genes not called consistently ‘‘present’’ by the MAS5.0 were determined by nonlinear least square regression (JMP). Differences in software in all replicates of at least one of the five conditions were excluded mean values between groups were analyzed by unpaired Student’s t test from further analysis. The complete microarray data set has been deposited with two-tailed comparison. Multiple comparisons used the one-way in National Center for Biotechnology Informations Gene Expression ANOVA test with post hoc t test comparison. Differences of P < 0.05 are Omnibus1 and is accessible through Gene Expression Omnibus Series considered significantly different. Software for statistical analysis was by accession number GSE6914. SigmaStat (SPSS, Inc.). Fluorescence in situ hybridization on metaphase chromosomes. Confluent cultures of parental, resistant, or resensitized Calu3 cells were split Results (1:5) and 24 h after passage these semisynchronized cultures were treated for 2 h with 0.1 Ag/mL colcemid for metaphase arrest. Cells were harvested, In vitro growth inhibition by bexarotene and gemcitabine. treated with a hypotonic solution of 0.075 mol/L KCl and fixed in methanol/ When used as a single agent, gemcitabine produced a sigmoidal acetic acid (3:1). The suspensions were dropped onto microscope slides and concentration-dependent growth inhibition of Calu3 cells (data not metaphase spreads were stored overnight at room temperature. Afterward, shown). The concentration needed to inhibit 50% of cell growth, slides were either processed immediately for hybridization or stored at IC50, was f3 nmol/L. On the other hand, bexarotene showed À j 20 C. Fluorescence in situ hybridization (FISH) analysis was done using a limited growth inhibitory activity up to 10 Amol/L. Bexarotene did probe for rrm1 that was generated from the bacterial artificial chromosome not interfere with or enhance gemcitabine activity in the clone CTD-2253D11 (Invitrogen) and labeled using the FISH Tag DNA Green combination with gemcitabine after single exposure or multiple Kit (Invitrogen) following the manufacturer’s protocol. A chromosome 11 centromeric probe, purchased prelabeled with Direct Red (Qbiogene), was exposures (Fig. 1B). Furthermore, after repeated treatment for five included as control. Metaphase chromosomes were washed twice with cycles, gemcitabine activity was neither enhanced nor inhibited by 2Â SSC and digested with pepsin (100 Ag/mL in 10mmol/L HCl) for 5 min at the combination with bexarotene when compared with cells 37jC. After two more washes with 2Â SSC, slides were consecutively treated treated with gemcitabine alone (Fig. 1B). Cells treated with with PBS/50mmol/L MgCl 2 (5 min), PBS/50mmol/L MgCl 2/1% formalde- continuous bexarotene grew similarly as vehicle-treated controls hyde (10min), and PBS/50mmol/L MgCl 2 (5 min), followed by ethanol (Fig. 1B) and continued to do so until the end of the 10-cycle dehydration and air drying. Before hybridization, both probes were combined regimen (data not shown). However, when the gemcitabine single- j in Hybrisol VII hybridization buffer (Qbiogene), denatured (96 C for 5 min), agent regimen (Fig. 1A, regimen 1) was continued beyond five and immediately placed on ice. The hybridization mixture was added onto cycles, the cells lost sensitivity to the agent and the cultures regrew the slides, sealed under a coverslip, and the slides were incubated at 75jCfor within 100 days (Fig. 1C). In contrast, when gemcitabine was 5 min. Hybridization was done for 18 h at 37jC followed by washes in 2Â SSC/ 0.1% NP40 (5 min at 37jC) and 0.4Â SSC/0.3% NP40 (2 min at 73jC). Because combined with bexarotene (Fig. 1A, regimen 3), the cultures stayed sensitive and only recovered after treatment was stopped (Fig. 1C). Characterization of gemcitabine-resistant cells. Surviving cells obtained at the end of treatment from cultures treated with gemcitabine alone, Calu3-GemR, or from cultures treated with the 1 http://www.ncbi.nlm.nih.gov/geo/ combination regimen were evaluated for their sensitivity to www.aacrjournals.org 4427 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research gemcitabine and other cytotoxic agents. The surviving cells isolated somal in the form of episomes or double minutes (23). To from gemcitabine-treated cultures were highly resistant to distinguish between these mechanisms, metaphase chromosomes gemcitabine with a resistance factor of >50. They also showed of parental and resistant cells were analyzed by FISH (Fig. 2D). some cross-resistance to a compound of same class, ara-C, but Single copies of rrm1 on chromosome 11 (identified by a were sensitive to other cytotoxic agents with different mechanisms chromosome-specific centromeric probe) in parental cells could of action (Table 1A). On the other hand, cells recovered after not be detected by FISH, apparently due to insufficient sensitivity treatment with the bexarotene/gemcitabine combination remained of the probe (Fig. 2D, top). However, in the resistant cells, a very chemosensitive toward all compounds tested, including gemcita- strong rrm1 signal was observed on one of the number 11 bine (Table 1A). chromosomes (Fig. 2D, bottom). These data indicate that the rrm1 To elucidate the mechanism of gemcitabine resistance in the gene is amplified intrachromosomally as extended chromosomal Calu3-GemR cells, real-time PCR was used to analyze mRNA levels region within one of the number 11 chromosomes. No signal for genes known to be involved in gemcitabine resistance: rrm1 indicative of extrachromosomal elements was observed. Taken (ribonucleotide reductase large subunit 1), rrm2 (ribonucleotide together, these results showed that intrachromosomal amplifica- reductase large subunit 2), dck (deoxycytidine kinase), cda tion of the rrm1 gene and its concomitant overexpression was most (cytidine deaminase), and ctps (CTP synthase). Our data showed likely responsible for the phenotype in gemcitabine-resistant Calu3- that rrm1 mRNA expression was low in Calu3 parental cells, in cells GemR cells. treated with bexarotene alone or with the combination regimen, Effects of bexarotene on gemcitabine-resistant cells. Because but was increased f20-fold (P < 0.0001) in the gemcitabine- addition of bexarotene to the gemcitabine regimen successfully resistant cells (Fig. 2A). In addition, there was a small but prevented the development of resistance, we investigated whether significant (P < 0.01) change in the expression of cda (60% bexarotene can also overcome preexisting gemcitabine resistance. decrease) and ctps (60% increase) in the resistant cells when Calu3-GemR cells were repeatedly treated either with vehicle, with compared with the parental cells. The expression of rrm2 and dck bexarotene or gemcitabine as single agents, or with a combination was not significantly different between parental and gemcitabine- of both agents in the same regimens used in the prevention study resistant cells. When the gene expression profiles of gemcitabine- (Fig. 1A). After four treatment cycles, the cultures treated with resistant and parental Calu3 cells were compared by microarray the combination regimen showed a loss in cell numbers, which analysis, the p15.5 locus on chromosome 11 showed a large number continued until the end of the 10th cycle (Fig. 3A). This indicated a of genes overexpressed in the resistant cells (Fig. 2B). Included in resensitization of the Calu3-GemR cells to the cytotoxic activity of the center of the overexpression locus was rrm1 (Table 2). These gemcitabine. The cultures treated with the single drug regimens data suggested that increased expression of rrm1 and surrounding expanded as the vehicle controls. However, analysis of RNA and genes may be the result of gene amplification. This was confirmed DNA samples collected over the treatment course revealed that, for rrm1 using a DNA-specific quantitative PCR assay. The copy whereas with vehicle and gemcitabine treatment rrm1 gene number of the rrm1 gene was f12-fold higher (P < 0.0001) in the amplification and expression was still increasing over time, in the resistant cells compared with the parental cells (Fig. 2C). In cultures treated with bexarotene alone they were gradually general, gene amplification is observed in two forms: intra- reduced, reaching parental levels at the end of the study (Fig. 3B chromosomal as extended chromosomal regions or extrachromo- and C). As expected from these data, the Calu3-GemR cells, after

Table 1. Sensitivity of Calu3 variants to chemotherapeutic agents

Calu3 variant and Gemcitabine Ara-C Doxorubicin Cisplatin treatment regimen

treatment regimen IC50 (nmol/L) RF IC50 (nmol/L) RF IC50 (nmol/L) RF IC50 (nmol/L) RF

(A) Parental Calu3 None 2.7 1 22.5 1 20.5 1 816 1 Bexarotene alone 2.7 1.022.8 1.020 .9 1.1 999 1.2 Gemcitabine alone 146.054.6 77.1 3.4 34.7 1.7 1,376 1.7 Combination 3.5 1.3 38.2 1.6 28.4 1.4 1,473 1.8

(B) Calu3-GemR None 146.054.6 77.1 3.4 34.7 1.7 1,376 1.7 Gemcitabine alone 81.1 30.3 62.1 2.8 30.6 1.5 1,536 1.9 Bexarotene alone 2.2 0.8 19.4 0.9 21.8 1.1 1,157 1.4 Combination 110.9 41.5 57.9 2.6 25.4 1.2 1,384 1.7

NOTE: A, the sensitivity of cells derived from Calu3 parental cells; B, the sensitivity of cells derived from gemcitabine-resistant Calu3 cells (Calu3-GemR), each after 10cycles of treatment regimens shown in Fig. 1 A. Drug sensitivity was measured by treating the derived cells for 3 d with gemcitabine, ara-C,

doxorubicin, or cisplatin at various concentrations followed by a cell viability assay (WST-1). IC50 values were determined for each drug and cell population. Relative drug sensitivities are expressed as RF [IC50 ratio of treated or untreated parental (A) or Calu3-GemR (B) cell populations and untreated parental cells]. Data shown are averages of three separate experiments with replicate samples per experiment.

Cancer Res 2007; 67: (9). May 1, 2007 4428 www.aacrjournals.org

Figure 2. Gemcitabine-resistant cells show rrm1 amplification and overexpression. A, relative RNA levels of rrm1, rrm2, dck, cda, and ctps were determined by quantitative real-time reverse transcription-PCR. Signals were normalized to 36b4 and expression in resistant cells is shown relative to those in parental cells. *, P < 0.05, significant differences between resistant and parental cells. B, expression of genes from the chromosome 11p15locus was measured by oligonucleotide microarrays. Signals are plotted as ratios between resistant and parental cells in order of their chromosomal arrangement. Only genes that were detected above background are shown. Genes in the main overexpression cluster (gray) are listed in Table 2. C, relative rrm1 DNA copy numbers were determined by a DNA-specific real-time PCR assay and normalized to gapdh DNA. Columns, averages of three determinations; bars, SD. D, for FISH analysis, metaphase chromosome spreads were prepared from parental (top) or resistant (bottom) Calu3 cells and hybridized with a probe specific for rrm1 (green) and a centromeric probe for chromosome 11 (red). Signals were detected by fluorescence microscopy using specific filters for the two dyes. Images taken in the green and red channel were electronically merged. treatment with bexarotene alone for 10cycles, were again sensitive Effect of bexarotene/gemcitabine combination in vivo. To to gemcitabine (Table 1B). In addition, FISH analysis of metaphase evaluate the antitumor efficacy of the bexarotene/gemcitabine chromosomes of the resensitized cells resembled those of the combination in vivo, Calu3 cells were established as xenograft parental cells (data not shown), indicating a complete loss of the tumors in athymic nude mice. As seen in Fig. 4A, parental Calu3 rrm1 gene amplification. Comparison of the microarray gene tumors grew continuously throughout the course of study in both expression profiles of the resensitized cells and the resistant Calu3- vehicle-treated control and drug-treated animals. When compared GemR cells showed that not only rrm1 but the entire chr11p15.5 with vehicle control, bexarotene given alone at 100 mg/kg daily had amplicon was repressed by exposure to bexarotene (Table 2). In no significant effect on tumor growth after the 4-week treatment addition, most of the genes outside of this locus, whose expression period. In contrast, gemcitabine at 100 mg/kg once weekly was altered concomitantly with the acquisition of gemcitabine decreased tumor volume by 63% (P < 0.05 versus control). More resistance, were reverted as well (Supplementary Fig. S1 and importantly, the combination of both agents decreased tumor Table S1). In summary, bexarotene over the course of 10treatment volumes by 80% relative to gemcitabine alone (P < 0.05) and by 93% cycles reversed the phenotype of the gemcitabine-resistant cells relative to vehicle control (P < 0.05; Fig. 4A). To determine whether almost completely to that of the parental cells, with regard to drug the combination regimen can overcome gemcitabine resistance sensitivity, intrachromosomal rrm1 amplification, as well as their in vivo, animals were implanted with the gemcitabine-resistant molecular profiles. Calu3 cells (Calu3-GemR) and were treated with single agents or www.aacrjournals.org 4429 Cancer Res 2007; 67: (9). May 1, 2007

Table 2. Effect of resistance and bexarotene on genes at the center of the chr11p15.5 locus

Gene name Symbol Expression ratio

GemR/Parental GemR + Bex/GemR + Veh

RNase reductase, M1 polypeptide RRM1 8.6 0.2 Hemoglobin, b chain HBB 2.4 0.6 Hemoglobin, c A chain HBG1 15.00 .1 Hemoglobin, c G chain HBG2 5.2 0.2 Hemoglobin, epsilon chain HBE1 4.9 0.3 Tripartite motif protein 34 TRIM34 13.4 0.1 Tripartite motif protein 5 TRIM5 11.2 0.1 Tripartite motif protein 22 TRIM22 18.5 0.1 Protein kinase C, d binding protein PRKCDBP 3.9 0.4 Tripartite motif protein 3 TRIM3 1.9 0.6 ADP-ribosylation factor interacting protein 2 ARFIP2 3.3 0.4 Fracture callus protein 1 FXC1 2.1 0.5 KIAA0409 protein KIAA0409 2.3 0.5 Integrin-linked kinase-2 ILK 3.1 0.3 Transcription initiation factor TFIID subunit 10 TAF10 3.5 0.3 Mitochondrial ribosomal protein L17 MRPL17 6.00 .3

NOTE: Genes are ordered by position of their transcriptional start site corresponding to Fig. 2B (gray). Expression was measured using Affymetrix U133A Genechips (four replicates per condition) and all genes listed showed expression clearly above background. Expression ratios were calculated from the average signals of gemcitabine-resistant Calu3 cells (GemR) and parental Calu3 cells, as well as of gemcitabine-resistant Calu3 cells treated with bexarotene (GemR + Bex) or vehicle (GemR + Veh).

with the combination. Gemcitabine-resistant tumor growth in expression, one of the two polypeptides that assemble to form animals treated with single agents was similar to that of vehicle the tetrameric RNase reductase enzyme. Other genes that play roles controls, whereas the combination regimen produced a 38% in the activity and metabolism of gemcitabine and have been decrease in tumor growth compared with gemcitabine-treated associated with development of resistance were either unaffected animals (Fig. 4B; P < 0.05 versus gemcitabine alone). The benefit of (rrm2, dck) or the effects were too small (cda, ctps) to contribute the bexarotene/gemcitabine combination became apparent beyond substantially to the strong increase in resistance [resistant factor 30days as the tumor volume in mice receiving the combination (RF), 55]. Therefore, the majority of the resistance can probably be therapy began to diverge from the tumor volume in mice treated attributed to the strong amplification and overexpression of rrm1. with gemcitabine alone. Collectively, these results showed that the RNase reductase is the key enzyme for the synthesis of bexarotene/gemcitabine combination produced a greater antitu- deoxyribonucleotides, including dCTP, and has been implicated mor effect than the single agents in the Calu3 xenograft model. in gemcitabine resistance in various models (11, 12, 24). Over- Furthermore, the data showed that, as in vitro, inclusion of expression of this enzyme leads to resistance on two levels, directly bexarotene into the treatment regimen could overcome gemcita- through competition of dCTP with the triphosphate of gemcitabine bine resistance in vivo. (dFdCTP) for incorporation into the DNA and indirectly through inhibition of the conversion of gemcitabine into the active metabolites. Because RNase reductase itself is also inhibited by Discussion one of these metabolites (dFdCDP), resistance through over- Acquired drug resistance is one of the major obstacles to expression of the enzyme is potentiated. This activity also accounts successful cancer therapy. Whatever the class of the cytotoxic for the observed cross-resistance (Table 1A) with ara-C. agent and its mechanism of action, most solid tumors eventually Increased expression of rrm1 coincided with a strong increase in become resistant to treatment. Therefore, the inclusion of agents rrm1 gene copy number. The rrm1 gene is located in the that can interfere with the development of acquired resistance to chr11p15.5 locus. Microarray analysis showed that, in addition to cytotoxic drugs may provide a substantial clinical benefit. In this rrm1, the expression of many genes in close vicinity of this gene study, we show that bexarotene can prevent and reverse resistance was increased in the resistant cells (Fig. 2B; Table 2). Although we to gemcitabine in the Calu3 NSCLC model. Parental Calu3 cells that have not directly determined whether, as for rrm1, this is also due were treated with repetitive cycles of gemcitabine acquired to increased DNA copy number, the high concentration of resistance to the cytotoxic agent within f8 weeks of treatment. overexpressed genes together with the measured increase in This was completely prevented when bexarotene was included in rrm1 DNA locus suggests a general amplification of this region. the treatment regimen. Parental Calu3 cells and cells recovered This is also in agreement with amplification of the mouse 7E1 after treatment with the combination regimen were f50-fold more locus, as measured by comparative genomic hybridization and sensitive to gemcitabine than the Calu3-GemR–resistant cells. This observed with gemcitabine resistance in a mouse colon tumor resistance was accompanied by a strong increase in rrm1 model (25). The mouse 7E1 region corresponds to the human

Cancer Res 2007; 67: (9). May 1, 2007 4430 www.aacrjournals.org

Figure 4. Antitumor effect of bexarotene and gemcitabine on parental and gemcitabine-resistant tumors. Nude mice bearing Calu3 xenograft tumors from parental cells (A) or Calu3 xenograft tumors from gemcitabine-resistant cells (B) were treated with vehicle, bexarotene, gemcitabine, or the combination of both agents for 4 to 6 wks. Drug effect on tumor growth was determined twice a week. Points, mean; bars, SE (n = 10 animals with two tumors per animal). *, P < 0.05, statistically significant from vehicle control. **, P < 0.05, statistically significant from gemcitabine alone.

chr11p15.5 locus and also contains rrm1 as well as several other homologues contained in the human chr11p15.5 locus. Other than rrm1 itself, chr11p15.5 genes with the highest induction of expression in Calu3-GemR–resistant cells are several hemoglobins as well as members of the TRIM (tripartite motif) cluster. Based on current knowledge, there is no functional association of these genes with drug resistance, chromosomal instability, growth, or other activities that favor them for selection. Thus, their overexpression is likely due to bystander coamplification with the rrm1 locus. In addition to the genes at the chr11p15.5 locus, microarray Figure 3. Bexarotene reverses gemcitabine resistance and rrm1 gene analysis showed that several hundred other genes throughout the amplification and overexpression. A, gemcitabine-resistant Calu3 cells derived genome are also significantly higher or lower expressed in the from the prevention study were repeatedly treated with vehicle, gemcitabine alone, bexarotene alone, or with the combination regimen as outlined in Fig. 1A. resistant cells when compared with the parental cells (Supplemen- Numbers of viable cells were determined by trypan blue exclusion at the end of tary Table S1). Some of these genes are involved in DNA replication each treatment cycle, and cells were reseeded and exposed to the same treatment. The procedure was repeated 10 times. Points, mean, represented and repair and one might be tempted to speculate whether these relative to control (vehicle); bars, SD. DNA and RNA from cells harvested during genes might causatively be involved in the development of the resensitization experiment (A) were analyzed for relative rrm1 DNA (B)or resistance (or its reversal; see below). However, at this point, it is RNA (C). All data were normalized to the corresponding gapdh signals. Two independent cultures for each condition were analyzed. Points, average; bars, difficult to distinguish between cause and effect. The observed SD. Signals of resistant cells at the outset of the experiment are shown as black expression changes for most of these genes are most likely a columns and all data are normalized to parental Calu3 cells (broken columns). *, P < 0.05, time points of bexarotene treatment with rrm1 DNA or RNA consequence of the resistance and adaptation to the molecular significantly reduced compared with cultures at the outset of the experiment. changes caused by amplification of a large gene set. Further studies www.aacrjournals.org 4431 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research are necessary to identify the genes that are the active players and not know whether this intrachromosomal amplification had been possible mediators of the activity of bexarotene in preventing generated through a multistep mechanism that involves initial development of drug resistance. formation of episomes (29), in which case extrachromosomal Treatment of the Calu3-GemR cells with bexarotene resensitizes amplification may still play a role in the prevention of drug them to the cytotoxic activity of gemcitabine. This was not only resistance by bexarotene. However, the reversal of gene amplifica- observed in vitro but also in vivo. Xenograft tumors generated from tion in the form of intrachromosomally extended regions would gemcitabine-resistant cells started to respond to the combination involve more complex processes and no studies have been treatment after f30days of therapy, whereas no effect on tumor published that specifically address such mechanisms. Conceptually, growth was observed with single agent. We have not tested whether the amplified locus or even the whole chromosome carrying the the tumors treated with bexarotene as single agent for an extended amplification could be replaced by the normal alleles. This would period regained sensitivity to gemcitabine. However, Calu3-GemR resemble mechanisms described for processes leading to loss of cells treated in vitro with bexarotene alone were as sensitive as heterozygosity. Studies of RNA-guided sequence-specific DNA parental cells at the end of the treatment period. This was not rearrangements in Tetrahymena thermophila have suggested a role simply due to lack of selective pressure because it did not occur in for targeted DNA deletion in genome surveillance (30) and the cultures treated with vehicle over the same time course. In observed activity of bexarotene may involve similar processes. addition, even in the presence of selective pressure (i.e., in the Whatever the mechanism, bexarotene could either induce cellular combination treatment), bexarotene caused resensitization, as changes that selectively favor those cells that reverse the gene indicated by the dramatic cell loss in these cultures. Not amplification or, alternatively, specifically induce such processes surprisingly, due to selective pressure by the cytotoxic agent, the involved in genome surveillance. The latter would also be in small number of cells remaining alive in the cultures treated with concordance with previous data from fluctuation analysis showing the combination regimen represented the not yet resensitized that bexarotene can maintain/increase genomic integrity of tumor fraction, which still contained amplified rrm1 (Fig. 3) and showed cells by interfering with the acquisition of spontaneous mutations the resistant phenotype (Table 1B). The resensitization by bexa- resulting in drug resistance (18, 19). rotene was accompanied by a gradual loss of rrm1 gene copy As mentioned above, bexarotene also reversed the resistance- number and expression over the treatment time course, resulting in associated changes for the vast majority of the genes outside the a parental rrm1 genotype and phenotype after 3 months of chr11p15.5 locus (Supplementary Fig. S1). Some of these may be treatment. Furthermore, genes that are colocalized with rrm1 on directly or indirectly involved in the reversal or prevention of the chr11p15.5 were also normalized. DNA amplification. A potential candidate is XRCC4, which is Gene amplification has been observed either intrachromoso- reduced in the resistant cells and normalized by treatment with mally as extended chromosomal regions or extrachromosomally as bexarotene (Supplementary Table S1). This gene is involved in episomes or double minutes. Conceptually, reversal of not only nonhomologous end-joining of DNA, which is an important rrm1 amplification but of the whole locus could be most easily process in DNA repair and maintenance of genome integrity. We explained by loss or removal of double minutes containing the have observed similar effects of bexarotene on XRCC4 in other amplified genes. Such a mechanism is independent of the amplified resistance models.2 Again, currently, we cannot clearly distinguish genes, does not require any sequence-specific recombination between cause and effect. Future time course analyses of the events, and therefore could also account for the broad activities molecular events in the resensitization process should help to of bexarotene in drug resistance, including resistance to paclitaxel delineate the primary effects of bexarotene. in models of breast and lung cancer, which involve the over- In summary, we have shown that bexarotene can prevent and expression and its reversal of the mdr1 multidrug resistance gene overcome amplification of rrm1 and associated resistance to (18, 19). Studies have shown that double minutes can be eliminated gemcitabine, in vitro and in vivo. It is likely that the mechanism of from cells through a process that involves increased micronuclei action is related for both prevention and reversal of drug formation and that this process can be induced by the resistance, and future research is directed toward understanding differentiating agent hydroxyurea (26–28). However, not all of the molecular events involved. Hopefully, these findings can differentiating agents show such activity and therefore differenti- serve as a basis for innovative therapeutic strategies in the ation per se is insufficient for the reversal of amplification. Our treatment of cancer. FISH studies, however, indicate that the rrm1 overexpression is caused by intrachromosomal amplification of the rrm1 locus in Acknowledgments one of the number 11 chromosomes (Fig. 2D). At this point, we do Received 12/6/2006; revised 2/2/2007; accepted 2/28/2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 Resistance to paclitaxel involving mdr1 amplification and its reversal by We thank Rene Prudente, Tracy Cooke, Manny Corpus, Jen Sanders, Jorge Valencia, bexarotene, unpublished data. and Wen Luo for technical assistance.

References 3. Burris HA III, Moore MJ, Andersen J, et al. Improve- in patients with locally advanced or metastatic non- ments in survival and clinical benefit with gemcitabine small-cell lung cancer. J Clin Oncol 2000;18:122–30. 1. Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, as first-line therapy for patients with advanced 5. ten Bokkel Huinink WW, Bergman B, Chemaissani A, 2004. CA Cancer J Clin 2004;54:8–29. pancreas cancer: a randomized trial. J Clin Oncol et al. Single-agent gemcitabine: an active and better 2. Cortes-Funes H. New treatment approaches for lung 1997;15:2403–13. tolerated alternative to standard cisplatin-based che- cancer and impact on survival. Semin Oncol 2002;29: 4. Sandler AB, Nemunaitis J, Denham C, et al. Phase III motherapy in locally advanced or metastatic non-small 26–9. trial of gemcitabine plus cisplatin versus cisplatin alone cell lung cancer. Lung Cancer 1999;26:85–94.

Cancer Res 2007; 67: (9). May 1, 2007 4432 www.aacrjournals.org

6. Harris H. Cells of the body: A history of somatic cell of estrogen receptor-negative mammary tumors in selective ligand LGD1069 (Targretin) causes complete genetics. New York: Cold Spring Laboratory Press; 1995. transgenic mice. Cancer Res 2002;62:6376–80. regression of mammary carcinoma. Cancer Res 1998;58: 7. Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. 15. Gottardis MM, Bischoff ED, Shirley MA, Wagoner 479–84. Action of 2¶,2¶-difluorodeoxycytidine on DNA synthesis. MA, Lamph WW, Heyman RA. Chemoprevention of 23. Stark GR, Debatisse M, Giulotto E, Wahl GM. Recent Cancer Res 1991;51:6110–7. mammary carcinoma by LGD1069 (Targretin): an RXR- progress in understanding mechanisms of mammalian 8. Heinemann V, Schulz L, Issels RD, Plunkett W. selective ligand. Cancer Res 1996;56:5566–70. DNA amplification. Cell 1989;57:901–8. Gemcitabine: a modulator of intracellular nucleotide 16. Boehm MF, Zhang L, Badea BA, et al. Synthesis and 24. Bergman AM, Eijk PP, Ruiz van Haperen VW, et al. and deoxynucleotide metabolism. Semin Oncol 1995;22: structure-activity relationships of novel retinoid X In vivo induction of resistance to gemcitabine results in 11–8. receptor-selective retinoids. J Med Chem 1994;37: increased expression of ribonucleotide reductase subunit 9. Heinemann V, Xu YZ, Chubb S, et al. Inhibition 2930–41. M1 as the major determinant. Cancer Res 2005;65:9510–6. of ribonucleotide reduction in CCRF-CEM cells by 17. Hermann TW, Yen WC, Tooker P, et al. The retinoid 25. van de Wiel MA, Costa JL, Smid K, et al. Expression 2¶,2¶-difluorodeoxycytidine. Mol Pharmacol 1990;38: X receptor agonist bexarotene (Targretin) synergisti- microarray analysis and oligo array comparative ge- 567–72. cally enhances the growth inhibitory activity of nomic hybridization of acquired gemcitabine resistance 10. Bergman AM, Pinedo HM, Jongsma AP, et al. cytotoxic drugs in non-small cell lung cancer cells. in mouse colon reveals selection for chromosomal Decreased resistance to gemcitabine (2¶,2¶-diflourodeox- Lung Cancer 2005;50:9–18. aberrations. Cancer Res 2005;65:10208–13. ycytidine) of cytosine arabinoside-resistant myeloblastic 18. Yen WC, Corpuz MR, Prudente RY, et al. A selective 26. Eckhardt SG, Dai A, Davidson KK, Forseth BJ, Wahl murine and rat leukemia cell lines: role of altered retinoid X receptor agonist bexarotene (Targretin) GM, Von Hoff DD. Induction of differentiation in HL60 activity and substrate specificity of deoxycytidine prevents and overcomes acquired paclitaxel (Taxol) cells by the reduction of extrachromosomally amplified kinase. Biochem Pharmacol 1999;57:397–406. resistance in human non-small cell lung cancer. Clin c-myc. Proc Natl Acad Sci U S A 1994;91:6674–8. 11. Davidson JD, Ma L, Flagella M, Geeganage S, Gelbert Cancer Res 2004;10:8656–64. 27. Valent A, Benard J, Clausse B, et al. In vivo LM, Slapak CA. An increase in the expression of 19. Yen WC, Lamph WW. The selective retinoid X elimination of acentric double minutes containing ribonucleotide reductase large subunit 1 is associated receptor agonist bexarotene (LGD1069, Targretin) pre- amplified MYCN from neuroblastoma tumor cells with gemcitabine resistance in non-small cell lung vents and overcomes multidrug resistance in advanced through the formation of micronuclei. Am J Pathol cancer cell lines. Cancer Res 2004;64:3761–6. breast carcinoma. Mol Cancer Ther 2005;4:824–34. 2001;158:1579–84. 12. Jordheim LP, Cros E, Gouy MH, et al. Characteriza- 20. Yen WC, Lamph WW. A selective retinoid X receptor 28. Tanaka T, Shimizu N. Induced detachment of tion of a gemcitabine-resistant murine leukemic cell agonist bexarotene (LGD1069, Targretin) prevents and acentric chromatin from mitotic chromosomes leads line: reversion of in vitro resistance by a mononucleo- overcomes multidrug resistance in advanced prostate to their cytoplasmic localization at G(1) and the tide prodrug. Clin Cancer Res 2004;10:5614–21. cancer. Prostate 2006;66:305–16. micronucleation by lamin reorganization at S phase. 13. Wu K, Kim HT, Rodriquez JL, et al. Suppression of 21. Yen WC, Prudente RY, Lamph WW. Synergistic effect J Cell Sci 2000;113:697–707. mammary tumorigenesis in transgenic mice by the of a retinoid X receptor-selective ligand bexarotene 29. Wahl GM. The importance of circular DNA in mam- RXR-selective retinoid, LGD1069. Cancer Epidemiol (LGD1069, Targretin) and paclitaxel (Taxol) in mamma- malian gene amplification. Cancer Res 1989;49:1333–40. Biomarkers Prev 2002;11:467–74. ry carcinoma. Breast Cancer Res Treat 2004;88:141–8. 30. Yao MC, Fuller P, Xi X. Programmed DNA deletion as 14. Wu K, Zhang Y, Xu XC, et al. The retinoid X receptor- 22. Bischoff ED, Gottardis MM, Moon TE, Heyman RA, an RNA-guided system of genome defense. Science 2003; selective retinoid, LGD1069, prevents the development Lamph WW. Beyond tamoxifen: the retinoid X receptor- 300:1581–4.

www.aacrjournals.org 4433 Cancer Res 2007; 67: (9). May 1, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. 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.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/67/9/4425

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2007/05/01/67.9.4425.DC1

Cited articles This article cites 29 articles, 18 of which you can access for free at: http://cancerres.aacrjournals.org/content/67/9/4425.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/67/9/4425.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://cancerres.aacrjournals.org/content/67/9/4425. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.