Cancer Therapy: Preclinical Therapeutic Targeting of ATP7B in Ovarian Carcinoma

Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH2-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. Conclusion: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance. Ovarian cancer has the highest mortality rate among all gynecologic malignancies (1). Following cytoreductive surgery, treatment with paclitaxel and platinum has become a recommended approach for initial chemotherapy (2). Current combination chemotherapy regimens produce complete remission in up to 80% of patients with advanced ovarian cancer. However, despite these initial high response rates, most patients suffer relapse and require treatment with multiple subsequent chemotherapy regimens (3). Successful management of advanced or recurrent gynecologic malignancies is often difficult due to both Authors' Affi l iat ions: Departments of Gynecologic Oncology, Experimental Therapeutics, and Cancer Biology, The University of Texas M. D. Anderson Cancer Center; Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston; Department of Obstetrics and Gynecology, Baylor College of Medicine; Brown Institute of Molecular Medicine, The University of Texas Health Science Center, Houston, Texas; Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon; and Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received 9/4/08; revised 2/28/09; accepted 3/4/09; published OnlineFirst 5/26/09. Grant support: Program Project Development Grant from the Ovarian Cancer Research Fund, Inc., the ZarrowFoundation, theUniversity of TexasM.D. Anderson Ovarian Cancer Specialized Program of Research Excellence grant P50 CA 083639, the Betty Ann Asche Murray Distinguished Professorship, and the Marcus Foundation (A.K. Sood); National Cancer Institute F31CA126474 Fellowship for Minority Students award (G.N. Armaiz-Pena); NIH-sponsored Women's Reproductive Health Research grant HD050128 through Baylor College of Medicine (M.M.K. Shahzad); National Cancer Institute-Department of Health and Human Services NIH 32 Training grant T32 CA101642 (Y.G. Lin and A.M. Nick); NIH grant DK071865 (S. Lutsenko, V. Zuzel, and E.S. Leshane); and NIH grant CA16672 (Z.H. Siddik). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). The sponsors had no role in the study design, data collection and analysis, interpretation of the results, the preparation of the manuscript, or the decision to submit the manuscript for publication. Requests for reprints: Anil K. Sood, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1155 Herman Pressler, Unit 1362, Houston, TX 77030. Phone: 713-7455266; Fax: 713-792-7586; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2306 3770 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from intrinsic and acquired resistance of cancer cells to chemotherapeutic agents (4–6). Therefore, novel strategies for overcoming resistance are needed. Cisplatin exerts its cytotoxicity by forming platinum-DNA adducts that arrest the cell in G1, S, or G2-M phases of the cell cycle, which ultimately lead to programmed cell death. Enhanced DNA repair, increased intracellular levels of glutathione or metallothionein, and drug accumulation may lead to resistance of cells to cisplatin (7–11). In addition, decreased influx or increased efflux of cisplatin may contribute to resistance. However, the mechanisms underlying these drug accumulation defects are poorly understood. Recent studies suggest that the transporter that mediates copper uptake and efflux may also regulate the cellular pharmacology of cisplatin (12, 13). Specifically, two copper transporters, ATP7A and ATP7B, are expressed at higher levels in platinum-resistant cell lines (14–17) and have been functionally implicated in resistance to several platinum agents, including cisplatin, carboplatin, and oxaliplatin (17). ATP7B was also shown to be overexpressed in several solid tumors, including gastric, breast, esophageal, hepatocellular, colorectal, uterine, and oral squamous cell carcinomas (18–24). ATP7A and ATP7B are members of the P-type ATPase family of transporters and are the product of genes affected in two disorders of copper homeostasis in humans, Menkes disease and Wilson's disease, respectively (25, 26). The primary function of ATP7A and ATP7B is to transport copper into the lumen of the transGolgi network (TGN) for the biosynthesis of copper-dependent enzymes and to facilitate export of excess copper from the cell by sequestering copper into exocytic vesicles (27). Cu-ATPases bind copper at their large NH2-terminal domain and then transfer copper across the membrane using the energy of ATP hydrolysis; elevated copper stimulates the CuATPase activity and causes intracellular trafficking of these transporters from the TGN to exocytic vesicles. To date, the ability to target chemotherapy resistance genes has been limited. We have recently developed highly efficient methods for in vivo gene silencing using small interfering RNA (siRNA) incorporated into neutral nanoliposomes, 1,2dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC; refs. 28, 29). Here, we used this technology to silence key cisplatin resistance genes and show antitumor efficacy with ATP7B gene silencing. Materials and Methods Cell lines and culture. The derivation, source, and propagation of human epithelial ovarian cancer cell lines, such as cisplatin-sensitive (A2780-PAR) and platinum-resistant (A2780-CP20 and RMG2) epithelial ovarian cancer cell lines, were maintained as previously described (30). The A2780-CP20 cell line was developed by sequential exposure of the A2780 cell line to increasing concentrations of cisplatin. All experiments were done with 70% to 80% confluent cultures. ATP7A and ATP7B gene silencing by siRNA. siRNA constructs targeted to ATP7A and ATP7B were designed and purchased from Qiagen. The target sequences were 5′-CTGGACCGGATTGTTAATTAT-3′ (for ATP7A) and 5′-CCAATTGATATTGAGCGGTTA-3′ (for ATP7B). In vitro transient transfectionwas done as described previously (28). Briefly, cells were transfected with ATP7Aand/or ATP7B-specific or scrambled (control) siRNA using RNAiFect reagent (Qiagen). At selected time intervals, cells were harvested tomeasure mRNA and protein levels of ATP7B using reverse transcription-PCR and Western blot analysis, respectively. An oligonucleotide sequence that did not have homology to any human mRNA (scrambled siRNA as determined by a National Center for Biotechnology Information BLAST search) served as a control. Reverse transcription-PCR. Total RNA was isolated by using Qiagen RNeasy kit. cDNA was synthesized by using the SuperScript First-Strand kit (Invitrogen) as per the manufacturer's instructions. cDNA was subjected to PCR using specific primers 5′-CTGGCAAGGCAGAAGTAAGG3′ (sense) and 5′-TGCAAAGTGGTGGTCCATAA-3′ (antisense) for ATP7A and 5′-GGTGTTCTCTCCGTGTTGGT-3′ (sense) and 5′GGCTGCACAGGAAAGACTTC-3′ (antisense) for ATP7B; β-actin was used as a housekeeping gene. PCR was done with 5 to 25 μg of reverse-transcribed RNA and 100 ng/μL of sense and antisense primers in a total volume of 20 μL. Each cycle consisted of 45 s of denaturation at 94°C, 1 min of annealing at 55°C, and 45 s of elongation at 72°C (22 cycles). Amplified PCR products were analyzed by electrophoresis on 1% agarose gel with Tris-borate-EDTA buffer and visualized under UV light after staining with ethidium bromide. Western blot analysis. Cells grown to 80% confluence were harvested and lysed in modified radioimmunoprecipitation assay buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 0.5% deoxycholate, 25 μg/mL leupeptin, 10 μg/mL aprotinin, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate) as previously described (29). To prepare lysate from snap-frozen tissue of in vivo tumors, ∼30 mm slices of tissue were homogenized in modified radioimmunoprecipitation assay buffer and the lysates were centrifuged at 12,500 rpm for 20 min at 4°C. Total protein concentration of the supernatant was determined using the bicinchoninic acid protein assay reagent kit (Pierce). Protein (30-50 μg) was separated by SDS-PAGE on a 6% gel and electrophoretically transferred onto a nitrocellulose membrane. The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively. ATP7B antibody recognized a band at 165 kDa, representing ATP7B protein, and also recognized an unknown band at ∼195 kDa. After being washed in TBST, blots were probed with horseradish peroxidase–conjugated goat anti-rabbit antibodies (GE Healthcare) in TBST for 1 h at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence (Perkin-Elmer). All membranes were stripped and reprobed with an anti–β-actin antibody (Sigma-Aldrich) at a dilution of 1:2,000 to ensure even loading of proteins. Cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The cytotoxicity of both sensitive and resistant cells to cisplatin was determined by measuring their ability to reduce the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] to a formazan, as described previously (31). Briefly, A2780-PAR and A2780-CP20 cells were plated at 2 × 10 per well in 96-well plate and allowed to adhere overnight. Cells were transfected with control or ATP7Aand/or Translational Relevance Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Although several potential targets have been identified, an understanding of the underlying mechanisms and practical approaches for reversing resistance has been largely lacking. Here, we used a highly efficient method of systemic small interfering RNA delivery using neutral nanoliposomes to target a key gene involved in cisplatin resistance and provide novel insights into its mechanism of action. These findings offer new opportunities for development of molecular therapies to reverse chemotherapy resistance. 3771 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Therapeutic Efficacy of ATP7B in Ovarian Cancer Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from ATP7B-targeted siRNAs. After 48 h of transfection, the cells were exposed to increasing concentrations of CDDP (cis-diamminedichloroplatinum or cisplatin; final concentration range, 0.01-32 μmol/L; LKT Laboratories). After 72 h of cisplatin exposure, cells were incubated with 0.15% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 2 h at 37°C. The supernatant was removed and cells were dissolved in 100 μL DMSO. The absorbance at 570 nm was recorded, and the IC50 was determined. Immunocytochemistry of ATP7B. Tumor cells (80-90% confluent) were grown on coverslips (1:20 dilution) for 24 to 48 h at 37°C and treated with 50 μmol/L bathocuproinedisulfonic acid disodium salt (BCS) to decrease copper levels and then treated with CuCl2 or CDDP (2 or 10 μmol/L) at 37°C for 1 h. Cells were fixed by immersing in acetone for 30 s at −20°C and then blocked overnight at 4°C in a blocking buffer containing 1% gelatin/1% bovine serum albumin in PBS. Cells were incubated with a primary antibody raised against the NH2-terminal domain of ATP7B and syntaxin-6 (TGN marker) at room temperature for 1 h (each antibody at a 1:500 dilution). After being washed thrice with PBS for 30 min, cells were incubated for 1 h with fluorescently labeled secondary antibodies (Alexa Fluor 488 donkey anti-rat for ATP7B and Alexa Fluor 555 donkey anti-mouse for syntaxin-6; Molecular Probes, Invitrogen). Cells were washed again with PBS as described above and then mounted using mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). Images were analyzed using 100× with a Zeiss confocal scanning microscope (Carl Zeiss); colocalization of ATP7B and syntaxin was evaluated using multiple images and Zeiss software package (track option). DNA adduct formation assay. Eighty percent of confluent cells were incubated with cisplatin at concentrations up to 20 μmol/L at 37°C for 4 h. Cells were centrifuged at 1,000 rpm for 5 min and washed twice with ice-cold PBS. Cell pellets were digested overnight at 55°C with 1 mol/L benzethonium hydroxide (0.075 mL). Samples were then acidified with 0.1 mL of 1 N HCl and the platinum content was determined in a flame atomic absorption spectrometer (SpectrAA300, Varian). To determine protein content, cell pellets from parallel incubations were first lysed with lysis buffer and then the protein content was determined by bicinchoninic acid method. The experiment was repeated for a total of three times, and the mean value was recorded. Binding of cisplatin to the NH2-terminal domain of ATP7B (N-ATP7B). N-ATP7B (previously referred as N-WNDP) was expressed and purified as a fusion protein with maltose binding protein as previously described (32). Before binding experiments, purified protein was fully reduced with 100 μmol/L DTT and then dialyzed overnight using the buffer containing 25 mmol/L phosphate and 150 mmol/L NaCl (pH 7.5). Copper or cisplatin was added to the N-ATP7B in increasing molar ratios up to 60-fold excess over protein (10-fold excess over metal-binding sites) for 10 min at room temperature. Next, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM; Invitrogen) was added in the dark for 5 min (in equimolar concentrations to metal-binding cysteines) and quenched with 20 μmol/L glutathione. Samples were run on a 12% Laemmli gel, and fluorescent images were taken using a FluorChem 5500 (Alpha-Innotech Corp.). Gels were then fixed, stained with Coomassie, and imaged again. The intensity of CPM labeling was normalized to protein levels by densitometry and expressed as a percentage of protein labeling in the absence of cisplatin. This experiment was replicated thrice, and the mean value was recorded. Overexpression of N-ATP7B in A2780-PAR cells. After 24 h of plating, cells were transfected with empty vector pTriEx-cDNA or pTriEx-NATP7B cDNA (ATP7B-WND) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, cDNA and Lipofectamine solution (1:2.5 ratio) were diluted with serum-free medium and two solutions were mixed and incubated for 20 min at room temperature. This mixture was added to cells and serum-free medium was replaced with regular serum-containing medium after 6 to 8 h of incubation. After 48 h, cells were trypsinized and plated in 96-well plates. After attachment of cells, cisplatin was added to the cells and incubated for 72 h at 37°C. Cytotoxicity was determined by MTT assay Fig. 1. A, Western blot analysis of ATP7A and ATP7B in A2780-CP20 cells following treatment with targeted and control siRNA for 24, 48, and 72 h. β-Actin was used as loading control. Protein levels were quantified by densitometry and expression is shown as arbitrary units. B, effect of ATP7A and ATP7B silencing on cisplatin sensitivity in ovarian cancer cell lines. A2780-PAR and A2780-CP20 cells were transfected with ATP7A or ATP7B or control siRNA. After 24 h, cells were treated with cisplatin (0.01-32 μmol/L). Following 72 h of cisplatin exposure, MTT assay was done to determine the effects on cell viability. Columns, mean values for the IC50 of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, compared with cisplatin alone. 3772 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Cancer Therapy: Preclinical Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from as described above. The extent of protein overexpression was also confirmed by Western blot analysis. Liposomal siRNA preparation. For in vivo delivery, siRNA was incorporated into DOPC as previously described (28). Briefly, siRNA and DOPC were mixed at a ratio of 1:10 (w/w) siRNA/DOPC in excess tertiary butanol. Tween 20 was added to the mixture at the ratio of 1:19 (Tween 20:siRNA/DOPC). After vortexing, the mixture was frozen in an acetone/dry ice bath and lyophilized. Before in vivo administration, this Fig. 2. Effect of cisplatin on intracellular localization of ATP7B. A, A2780-CP20 cells were immunostained with the anti-ATP7B and anti–syntaxin-6 antibodies. Overlay images showing colocalization (white) of ATP7B (green) and syntaxin-6 (pink). B, HepG2 and A2780-CP20 cells were treated with copper chelator BCS (50 μmol/L) to deplete cells of copper and then either kept in BCS or treated with CuCl2 or cisplatin. To verify the lack of ATP7B trafficking in response to cisplatin in A2780-CP20 cells, we evaluated colocalization between ATP7B and syntaxin using multiple images and Zeiss software package. Trafficking in response to copper in HepG2 was used as a positive control. Arrows indicate direction and length of the densitometry scan. 3773 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org Therapeutic Efficacy of ATP7B in Ovarian Cancer Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from mixture was hydrated with 0.9% saline to a concentration of 25 μg/mL and 200 μL of mixture were used per injection. Orthotopic model of ovarian cancer and tissue processing. Female athymic nude mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). All mice were housed and maintained under specific pathogenfree conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and NIH. All studies were approved and supervised by the University of Texas M. D. Anderson Cancer Center Institutional Animal Care and Use Committee. All mice were used in these experiments when they were 8 to 12 wk old. Before injection, tumor cells were washed twice with PBS, detached by 0.1% cold EDTA, centrifuged for 7 min, and reconstituted in HBSS (Invitrogen). Cell viability was confirmed by trypan blue exclusion. Tumors were established by i.p. injection of either 1.0 × 10 A2780-CP20 or 3.0 × 10 RMG2 cells. Once established, this tumor model reflects the growth pattern of advanced ovarian cancer (33, 34). Long-term therapy experiments were done using two platinum-resistant ovarian cancer cell lines: A2780-CP20 and RMG2. To assess the effects of siRNA therapy on tumor growth, treatment was initiated 1 wk after i.p. injection of tumor cells. Mice were divided into five groups (n = 10 mice per group): (a) empty liposome (DOPC; vehicle), (b) control siRNA-DOPC (150 μg/kg i.p. twice weekly), (c) control siRNADOPC + cisplatin (160 μg/mouse i.p. weekly), (d) ATP7B siRNA-DOPC (150 μg/kg i.p. twice weekly), and (e) ATP7B siRNA-DOPC + cisplatin (doses same as individual treatments). Treatment was continued until control mice became moribund (typically 4-6 wk following tumor cell injection). At the time of sacrifice, mouse weight, tumor weight, number of nodules, and distribution of tumors were recorded. Tissue samples were snap frozen for lysate preparation or fixed in formalin for paraffin embedding. The individuals who did the necropsies, tumor collections, and tissue processing were blinded to the treatment group