Identification of COX17 As a Therapeutic Target for Non-Small Cell Lung Cancer1

[CANCER RESEARCH 63, 7038–7041, November 1, 2003] Advances in Brief

Identification of COX17 as a Therapeutic Target for Non-Small Cell Lung Cancer1

Chie Suzuki, Yataro Daigo, Takefumi Kikuchi, Toyomasa Katagiri, and Yusuke Nakamura2 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

Abstract Saccharomyces cerevisiae (7, 8). Although this small protein has been cloned or purified from mammals as well, including human, mouse, We have been investigating gene expression profiles in non-small cell and pig, its function in cancer cells or even in normal mammalian lung cancers (NSCLCs) to identify molecules involved in pulmonary somatic cells has not been established (8–10). carcinogenesis and select which genes or gene products might be useful as diagnostic markers or targets for new molecular therapies. Here we report Here we report functional characterization of COX17 in human evidence that the cytochrome c oxidase (CCO) assembly protein COX17 is lung tumors and in metabolism of normal mammalian cells. Our a potential molecular target for treatment of lung cancers. By semiquan- results suggest that overexpression of COX17 plays a significant role titative reverse transcription-PCR, we documented increased expression in development/progression of lung cancer and that this molecule of COX17 in all of 8 primary NSCLCs and in 11 of 15 NSCLC cell lines represents a potential target for development of novel therapeutic examined, by comparison with normal lung tissue. Treatment of NSCLC drugs. cells with antisense S-oligonucleotides or vector-based small interfering RNAs of COX17 suppressed expression of COX17 and also the activity of Materials and Methods CCO, and suppressed growth of the cancer cells. Because our data imply that up-regulation of COX17 function and increased CCO activity are Cell Lines. Fifteen human NSCLC cell lines were used in this study: A549, frequent features of lung carcinogenesis, we suggest that selective sup- NCI-H23, NCI-H522, LC174, LC176, LC319, PC14, PC3, PC9, NCI-H1435, pression of components of the CCO complex might hold promise for NCI-H1793, SK-LU-1, RERF-LC-AI, SK-MES-1, and NCI-H358. Human development of a new strategy for treating lung cancers. SAECs and NHDF cells were used as controls. All of the cancer cells were grown in monolayers in appropriate medium supplemented with 10% fetal Introduction bovine serum. SAECs and NHDF cells were grown in optimized medium (SAGM and FGM-2, respectively) purchased from Cambrex Bio Science Inc. Lung cancer is one of the most common cancers in the world. (Walkersville, MD). Primary NSCLCs were obtained with informed consent 3 NSCLC is by far the most common form, accounting for ϳ80% of from NSCLC patients, as described previously (5), and 8 samples were used in lung tumors (1). The overall 10-year survival rate remains as low as this study. 10% despite recent advances in multimodality therapy, because the Selection of a Candidate Gene and Analysis by Semiquantitative RT- majority of NSCLCs are not diagnosed until advanced stages (2). PCR. Using the gene expression profiles of 37 primary NSCLCs obtained Although chemotherapy regimens based on platinum are considered previously by cDNA microarray analysis (5), we selected genes that showed the reference standards for treatment of NSCLC, those drugs are able levels of expression that were 5-fold higher than in normal lung in Ͼ50% of to extend survival of patients with advanced NSCLC only ϳ6 weeks the tumors examined. The COX17 transcript was among them. To confirm (3). Numerous targeted therapies are being investigated for this dis- overexpression of COX17 by semiquantitative RT-PCR, total RNA was ex- tracted from cultured cells and clinical tissues using TRIzol reagent (Life ease, including tyrosine kinase inhibitors, but thus far promising Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s pro- results have been achieved in only a limited number of patients, and tocol. The RNA preparations were treated with DNase I (Nippon Gene, Tokyo, some recipients suffer severe adverse reactions (4). By analyzing Japan) and reverse-transcribed to single-stranded cDNAs using oligo(dT)12–18 genome-wide expression profiles of 37 primary NSCLCs on a cDNA primer with Superscript II reverse transcriptase (Life Technologies, Inc.). We microarray containing 23,040 genes, we have been attempting to prepared appropriate dilutions of each single-stranded cDNA for subsequent identify more selective molecular targets for development of strate- PCR amplification by monitoring the ␤-2-microglobulin gene (B2M)asa gies for earlier diagnosis, treatment, and/or prevention of NSCLC (5). quantitative control. The primer sets for amplification were B2M-F (5Ј- In the course of those experiments we identified COX17 as one of the TTAGCTGTGCTCGCGCTACT-3Ј), B2M-R (5Ј-TCACATGGTTCACACG- Ј Ј Ј genes that was frequently overexpressed in lung tumors. GCAG-3 ) for B2M, and COX17-F (5 -GTGGACATCTAATTGAGGCC-3 ), Ј Ј COX17 is an M 8,000 protein that is essential for assembly of COX17-R (5 -GAAGATCTTCCACTAGTAATATT-3 ) for COX17. All of r the reactions involved initial denaturation at 94°C for 2 min followed by 22 functional CCO in yeast and for delivery of copper ions to the yeast (for B2M) or 27 cycles (for COX17) of 94°C for 30 s, 58–62°C for 30 s, and mitochondrion for insertion into the enzyme (6). Mutations among the 72°C for 45 s on a GeneAmp PCR system 9700 (Applied Biosystems, Foster six conserved cysteines of COX17 lead to respiratory defects in City, CA). AS S-Oligonucleotides. Two ϫ 105 cells from NSCLC line A549 plated Received 5/26/03; revised 8/15/03; accepted 8/29/03. onto six-well dishes were transfected with synthetic S-oligonucleotides (0.2 The costs of publication of this article were defrayed in part by the payment of page ␮M) corresponding to the COX17 gene, using Lipofectamine reagent (40 nM; charges. This article must therefore be hereby marked advertisement in accordance with Invitrogen, Carlsbad, CA), and maintained for 2 days in medium containing 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a “Research for the Future” Program Grant of The Japan Society 10% fetal bovine serum. Cell numbers and cell viability were evaluated, for the Promotion of Science (#00L01402; to Y. N.). respectively, by Giemsa staining and MTT assay, each in triplicate. The 2 To whom requests for reprints should be addressed, at Laboratory of Molecular sequences of the S-oligonucleotides were as follows: AS, 5Ј-TCTCCTTTCTC- Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, GATGATA-3Ј; and R, 5Ј-ATAGTAGCTCTTTCCTCT-3Ј. MTT assays were 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: [email protected]. performed as described elsewhere (11). 3 The abbreviations used are: NSCLC, non-small cell lung cancer; CCO, cytochrome RNAi Assay. We had established a vector-based RNAi system, c oxidase; SAEC, small airway epithelial cell; NHDF, normal human dermal fibroblast; psiH1BX3.0, to direct the synthesis of siRNAs in mammalian cells, as reported RT-PCR, reverse transcription-PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet- elsewhere (12). Ten ␮g of siRNA-expression vector was transfected using 30 razolium bromide; R, reverse; AS, antisense; RNAi, RNA interference; siRNA, small ␮ interfering RNA; GFP, green fluorescent protein; EGFP, enhanced green fluorescent l of Lipofectamine 2000 (Invitrogen) into NSCLC cell lines, A549 and protein. LC319. In this assay, Ͼ90% of the transfected cells expressed this synthetic 7038

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. COX17: A NOVEL TARGET FOR NSCLC TREATMENT siRNA, and endogenous expression of COX17 was effectively suppressed. The ulations, all of which had been purified by laser-capture microdissec- transfected cells were cultured for 5 days in the presence of appropriate tion (5). We additionally examined expression of this gene using concentrations of geneticin (G418). Cell numbers and viability were measured semiquantitative RT-PCR in 8 additional NSCLC tissues, and our by Giemsa staining and MTT assay in triplicate. The target sequences of the panel of 15 NSCLC cell lines and 2 normal human cells including synthetic oligonucleotides for RNAi were as follows: control1 (EGFP: gene, a SAEC, and then confirmed its increased expression in all of the 8 mutant of Aequorea victoria GFP), 5Ј-GAAGCAGCACGACTTCTTC-3Ј; control2 (Luciferase: Photinus pyralis luciferase gene), 5Ј-CGTACGCG- primary tumors and in 11 of the 15 cell lines examined (Fig. 1). GAATACTTCGA-3Ј; and control3 (Scramble: Chloroplast Euglena gracilis Growth-Inhibitory Effect of AS S-Oligonucleotides and siRNA gene coding for the 5S and 16S rRNA), 5Ј-GCGCGCTTTGTAGGATTCG-3Ј; Designed to Reduce Expression of COX17 in NSCLC Cells. To COX17-siRNA1, 5Ј-GTGACTGCGGACGAATCGG-3Ј; COX17-siRNA2, 5Ј- assess whether COX17 plays a role in growth or survival of lung GGAGAAGAACACTGTGGAC-3Ј. To validate our RNAi system, individual cancer cells, we synthesized a pair of R (control) and AS S- control siRNAs (EGFP, Luciferase, and Scramble) were initially confirmed oligonucleotides corresponding to the COX17 sequence and trans- using semiquantitative RT-PCR to decrease the expression of the correspond- fected each of them into A549 cells, a line that had shown a high ing target genes that had been transiently transfected to COS-7 cells. Down- regulation of the COX17 expression by effective siRNA but not by controls level of COX17 expression. Introduction of the AS S-oligonucleo- was also confirmed with semiquantitative RT-PCR in the cell lines used for tide decreased the number of foci formed and also decreased cell this assay. viability, compared with the control nucleotide, suggesting that Immunocytochemical Analysis. The human gene in question had been COX17 was essential to the growth and/or survival of these cancer isolated as an orthologue of yeast COX17, which is essential for the assembly cells (Fig. 2A). To confirm that the growth suppression by AS of functional CCO in S. cerevisiae and is believed to deliver copper ions to the S-oligonucleotide was gene specific, we also synthesized two yeast mitochondrion for insertion into the enzyme (7, 8). We attempted to siRNA expression plasmids designed to suppress endogenous confirm the subcellular localization of human COX17 protein in mammalian cells by constructing pcDNA3.1(ϩ) vectors (Invitrogen) that contained c-myc- COX17 and three different controls (siRNAs for EGFP, Luciferase, His epitope sequences (LDEESILKQE-HHHHHH) at the COOH terminal of or Scramble), and transfected each of them into A549 and LC319 COX17 protein and transfecting these plasmids to COS-7 cells. Transiently cells. As shown in Fig. 2B, siRNA-2 significantly suppressed transfected COS-7 cells replated on chamber slides were fixed with PBS expression of COX17 in the transfected cells, but neither siRNA-1 containing 4% paraformaldehyde, then rendered permeable with PBS contain- nor the three control siRNAs had this effect. Growth and viability ing 0.1% Triton X-100 for 3 min at 4°C. Cells were covered with 2% BSA in of these two NSCLC cell lines were, in consequence, reduced PBS for 30 min at room temperature, to block nonspecific antibody-binding significantly in cells transfected with siRNA-2 (Fig. 2, B and C). sites. Then the cells were incubated with a mouse anti-c-myc antibody diluted 1:800 in the blocking solution. The murine antibodies were stained with a goat Localization of the Product of COX17 and Activity of CCO in antimouse secondary antibody conjugated to FITC and viewed with a micro- Mammalian Cells. To investigate the subcellular localization of pro- scope. teins encoded by the human orthologue of yeast COX17, we trans- CCO Activity in Lung Cancer Cells. LC319 cells were separated into fected COS-7 cells with plasmids that contained c-myc-His-epitope mitochondrial and other fractions using digitonin (Wako, Osaka, Japan). To sequences (LDEESILKQE-HHHHHH) at the COOH terminal of the confirm fractionation we used mouse monoclonal antibody to human mito- human COX17 protein. Anti-c-myc antibodies detected the COX17 chondria (MAB1273; Chemicon, Temecula, CA) as a positive control (13). protein mainly in mitochondria (Fig. 3A). Cytochrome c (63 ␮M) in buffer [10 mM Tris, 0.2 mM EDTA, and 0.05% Deficiencies in cytochrome oxidase, the terminal enzyme of the n-dodecyl-␤-D-maltoside, (pH 7.6)] was incubated with 12.5 ␮M L(ϩ)-ascor- bic acid for 30 min at room temperature (18°C) to convert ferric cytochrome mitochondrial respiratory chain, can be lethal to cells, and COX17 is c to ferrous cytochrome c. Twenty ␮l of the 1 mg/ml mitochondrial protein assumed to be essential for the assembly of a functional CCO complex solution was then added to 2 ml of the reaction mixture at 37°C. The reaction in yeast (6). On the basis of the information we attempted to inves- representing CCO activity was measured at 550 nm absorbance. tigate the biological importance of COX17 in regard to CCO activity in human NSCLC cells. We measured CCO activity in LC319 cells Results treated either with siRNA against COX17 or with control siRNAs. Overexpression of COX17 in NSCLC Tissues and Cell Lines. Two days after transfection, CCO activity in the cells was signifi- We had observed that the COX17 transcript was overexpressed on a cantly decreased in accordance with the suppression of COX17 ex- cDNA microarray by the majority of our primary NSCLC cell pop- pression by siRNA-2 (Fig. 3B).

Fig. 1. Expression of COX17 in NSCLC tissues and cell lines measured by semiquantitative RT-PCR. In- creased expression was observed in all of 8 clinical samples and in 11 of the 15 cancer cell lines examined.

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Fig. 2. Growth-inhibitory effects of AS S-oligonucleotides and siRNAs designed to suppress COX17 in NSCLC cells. A, MTT assay using A549 cells transfected with S-oligonucleotides corresponding to a part of the COX17 sequence. R, control. B, focus-formation assays using A549 and LC319 cells transfected with siRNA to COX17. The level of COX17 expression detected by semiquantitative RT-PCR in cells treated with either control or COX17-siRNAs is shown in the bottom panel for each cell line. Luc, Luciferase. C, summarized results of the MTT assays.

Discussion Although advances have been made in development of molecular- targeting drugs for cancer therapy, the ranges of tumor types that respond as well as the effectiveness of the treatments are still very limited. Hence, it is urgent to develop new anticancer agents that target molecules highly specific to malignant cells and are likely to cause minimal or no adverse reactions. To achieve the goal we need to identify molecules of which the physiological mechanisms are well defined (14). A powerful strategy toward these ends would combine screening of up-regulated genes in cancer cells on the basis of genetic information obtained on cDNA microarrays with high-throughput screening of their effect on cell growth by inducing loss-of-function phenotypes with AS S-oligonucleotides and/or RNAi systems (5, 15–17). By pursuing such a strategy, we have shown here that COX17 is not only frequently overexpressed in clinical NSCLC samples and cell lines derived from that kind of tumor but also that the gene product is indispensable for growth of NSCLC cells. CCO, the terminal enzyme of the mitochondrial respiratory chain, contributes to the proton motive force that drives synthesis of ATP (18). This multisubunit enzyme is conserved from lower to higher eukaryotes and requires three copper atoms for proper function (19). A number of genes encode assembly factors to constitute CCO function, and some of them play roles in utilization of copper. Mutations of COX17, which contains six conserved cysteines, cause a respiratory defect in yeast (7, 8). Because this defect can be rescued by addition of exogenous copper, COX17 has been assumed to function Fig. 3. Characterization of COX17 function in mammalian and NSCLC cells. A, subcellular localization of transiently expressed COX17 protein in COS-7 cells; staining in the copper-trafficking pathway of cytochrome oxidase. For exam- is visible mainly in mitochondria. B, CCO activity in LC319 cells treated with COX17- ple, COX17 may shuttle copper ions to the mitochondria and transfer siRNA or various controls (see text). 7040

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. COX17: A NOVEL TARGET FOR NSCLC TREATMENT copper to yeast Sco1, an inner mitochondrial membrane protein (an 5. Kikuchi, T., Daigo, Y., Katagiri, T., Tsunoda, T., Okada, K., Kakiuchi, S., Zembutsu, intermediate step in copper donation to cytochrome oxidase; Ref. 7), H., Furukawa, Y., Kawamura, M., Kobayashi, K., Imai, K., and Nakamura, Y. Expression profiles of non-small cell lung cancers on cDNA microarrays: identifica- but this hypothetical interaction has not been proven in either yeast or tion of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer mammals. drugs. Oncogene, 22: 2192–205, 2003. 6. Huffman, D. L., and O’Halloran, T. V. Function, structure, and mechanism of To elucidate the function of COX17 in mammals, we examined the intracellular copper trafficking proteins, Annu. Rev. Biochem., 70: 677–701, 2001. subcellular localization of COX17 protein in COS-7 cells and found it 7. Glerum, D. M., Shtanko, A., and Tzagoloff, A. Characterization of COX17, a yeast mainly in mitochondria. To clarify whether native COX17 protein is gene involved in copper metabolism and assembly of cytochrome oxidase, J. Biol. Chem., 271: 14504–14509, 1996. required for CCO activity in human NSCLC cells, we transfected 8. Amaravadi, R., Glerum, D. M., and Tzagoloff, A. Isolation of a cDNA encoding the COX17-siRNA vectors to LC319 cells, a line in which the COX17 human homolog of COX17, a yeast gene essential for mitochondrial copper recruit- gene is highly expressed. In those lung cancer cells, endogenous ment. Hum. Genet., 99: 329–333, 1997. 9. Takenouchi, T., Fujimoto, M., Shimamoto, A., and Munekata, E. Isolation and COX17 expression was suppressed significantly by siRNA, and CCO characterization of Cox17p from porcine heart by determining its survival-promoting activity was reduced. Our results suggest that COX17 is likely to have activity in NIH3T3 cells, Biochim. Biophys. Acta., 1472: 498–508, 1999. an important role in the mitochondrial respiratory chain in humans, 10. Kako, K., Tsumori, K., Ohmasa, Y., Takahashi, Y., and Munekata, E. The expression of Cox17p in rodent tissues and cells. Eur. J. Biochem., 267: 6699–707, 2000. being essential moreover for CCO activity and, when overexpressed, 11. Akashi, H., Han, H. J., Iizaka, M., and Nakamura, Y. Growth-suppressive effect of for growth/survival of NSCLC cells. These assumptions imply a non-steroidal anti-inflammatory drugs on 11 colon-cancer cell lines and fluorescence differential display of genes whose expression is influenced by sulindac, Int. 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Chie Suzuki, Yataro Daigo, Takefumi Kikuchi, et al.

Cancer Res 2003;63:7038-7041.

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