Published OnlineFirst March 19, 2013; DOI: 10.1158/1535-7163.MCT-12-0950 Molecular Cancer Chemical Therapeutics Therapeutics RAD51C-Deficient Cancer Cells Are Highly Sensitive to the PARP Inhibitor Olaparib Ahrum Min1, Seock-Ah Im1,2, Young-Kwang Yoon1, Sang-Hyun Song1, Hyun-Jin Nam1, Hyung-Seok Hur1, Hwang-Phill Kim1, Kyung-Hun Lee1,2, Sae-Won Han1,2, Do-Youn Oh1,2, Tae-You Kim1,2,4, Mark J. O'Connor5, Woo-Ho Kim1,3, and Yung-Jue Bang1,2 Abstract A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencingofRAD51Cinaresistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C- deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. Ó2013 AACR. Introduction mutations (3–5). PARP inhibitors block the repair of DNA The DNA repair system is critical for maintaining single-strand breaks (SSB); unrepaired SSBs lead to the genomic integrity. Synthetic lethality is defined as the formation of DNA double-strand breaks (DSB). If DSBs loss of cell viability when multiple genes lose their func- cannot be repaired because of homologous recombination tions altogether, especially when compensatory genes are dysfunction, genomic instability or cell death can result also defective. The concept of synthetic lethality has been (6). Therefore, PARP inhibitors may be effective against shown using the novel PARP inhibitor in patients with various human cancer cells with defective DNA repair breast and ovarian cancer harboring mutations in the genes. An example of that is CDK1 depletion increased BRCA1 or BRCA2 genes (1, 2). There is clinical evidence PARP inhibitor sensitivity in cancer cells because the showing that olaparib (AZD2281; KU-0059436), a small- DSBs induced by PARP inhibition could not be repaired molecule inhibitor of PARP, has potential as a therapeutic because of inactivation of homologous recombination– agent alone and in combination with radiotherapy or associated repair in CDK1-depleted cells (7). In addition, chemotherapy to treat cancers with BRCA1 and BRCA2 recent study suggests that nonhomologous end joining (NHEJ) plays an important role in the genomic instability and hypersensitivity to PARP inhibitors in homologous Authors' Affiliations: 1Cancer Research Institute, Seoul National Univer- recombination–deficient cells (8). 2 3 sity; Departments of Internal Medicine and Pathology, Seoul National RAD51C is a RAD51-like gene that has a key role in University College of Medicine; 4Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science maintaining genomic stability (9–12). The functional role and Technology, Seoul, Republic of Korea; and 5AstraZeneca UK Ltd., of RAD51C in DNA damage repair has also been exam- fi Maccles eld, Cheshire, United Kingdom ined (13). The results of these studies suggest that Note: Supplementary data for this article are available at Molecular Cancer RAD51C acts sequentially with RAD51 at the DNA Therapeutics Online (http://mct.aacrjournals.org/). damage site to repair DNA damage. Therefore, RAD51C Corresponding Authors: Seock-Ah Im and Yung-Jue Bang, Department depletion leads to impaired RAD51 foci formation, of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Republic of Korea. Phone: 82-2- resulting in impaired DNA repair (13, 14). In addition, 2072-0850; Fax: 82-2-762-9662; E-mail: Seock-Ah Im, [email protected]; some studies have shown that RAD51C is required for Yung-Jue Bang, [email protected] the checkpoint response to DNA damage (13, 15). Fur- doi: 10.1158/1535-7163.MCT-12-0950 thermore, recent studies have found that germline muta- Ó2013 American Association for Cancer Research. tions of RAD51C are associated with cancers. In these www.aacrjournals.org 865 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 19, 2013; DOI: 10.1158/1535-7163.MCT-12-0950 Min et al. RAD51C cancers, germline mutations of impede RAD51 (Bid-Rad); the cell survival rate and IC50 of olaparib were foci formation results in blocking the homologous determined using SigmaPlot software. recombination–mediated DNA repair. RAD51C-defec- tive cancers can therefore potentially be treated with cDNA microarray analysis olaparib because the DNA damage induced by olaparib The SNU-601 and SNU-668 cells were treated for dif- cannot be effectively repaired by homologous recombi- ferent periods of time (24, 48, and 72 hours) with 1 mmol/L nation, as RAD51C deficiency interferes with RAD51- olaparib or dimethyl sulfoxide (DMSO). The total RNA mediated homologous recombination. was extracted and hybridized to an Affymetrix GeneChip In the present study, we evaluated the antitumor activ- human Gene 1.0 ST array (Affymetrix, Inc.). The results ity of olaparib in cancer cell lines in vitro and observed that were normalized to the robust multi-array average (RMA) olaparib-sensitive cell lines had low levels of RAD51C and analyzed by DNALINK, Inc.. The microarray data expression using microarray analysis. Subsequently, we were deposited in the ArrayExpress database (accession evaluated whether the silencing of RAD51C-sensitize number: E-MTAB-1012). olaparib sensitivity and restoration of RAD51C cause decreased sensitivity to olaparib. We also characterized BRCA1 and BRCA2 mutation analysis the mechanisms of RAD51C silencing in human cancers. Genomic DNA (gDNA) was extracted from gastric RAD51C expression using immunohistochemistry was cancer cell lines using an Accuprep Genomic DNA Extrac- evaluated in gastric cancer tissue with paired normal tion Kit (Bioneer) according to the manufacturer’s proto- gastric mucosa. This is the first report to show that col. The BRCA1 and BRCA2 mutations were analyzed RAD51C-deficient cancer cells are selectively sensitive to using fluorescent-conformation sensitive gel electropho- PARP inhibitor olaparib and olaparib promotes cell death resis (F-CSGE), as described previously (17). by inducing G2–M cell-cycle arrest and apoptosis. Reverse transcription PCR and real-time PCR Materials and Methods Total RNA was isolated using TRI reagent (Molecular Reagents Research Center, Cincinnati, OH). cDNA was synthesized Olaparib was kindly provided by AstraZeneca Ltd.. The by conducting reverse transcription PCR (RT-PCR) with chemical structure of olaparib is shown in Fig. 1A. 5-Aza- ImProm-II reverse transcriptase (Promega) and amplified 20-deoxycytidine (5-aza-dc) was purchased from Sigma- using AmpliTaq Golf DNA polymerase (Applied Biosys- Aldrich. tems) with gene-specific primers. Quantitative real-time PCR was conducted using an iCycler iQ detection system Cell lines and cell culture (Bio-Rad Laboratories, Inc.) with SYBR Green. All data Human gastric cancer cells (SNU-1, -5, -16, -216, -484, from all samples were normalized to the actin cDNA -601, -620, -638, -668, -719, and KATO-III) were purchased levels. The sequences of the primers used for the RT-PCR from the Korean Cell Line Bank; the identities of the cell and qRT-PCR are listed in Supplementary Table S1. cDNA lines were authenticated by DNA fingerprinting analysis was synthesized at least 3 times from 3 independent sets of (16). Human breast cancer cells (BT-549 and MCF-7) samples, and all PCR analyses were conducted in authenticated using short tandem repeat analysis were triplicate. purchased from the American Type Culture Collection. All cell lines were stored in liquid nitrogen, passaged for Western blot analysis less than 6 months before use in this study, and cultured in Proteins were extracted and equal amount of proteins RPMI-1640 supplemented with 10% FBS and 10 mg/mL were separated on 8% to 15% SDS-PAGE as previously gentamicin at 37 C in a 5% CO2 atmosphere. described (18). The resolved proteins were transferred onto nitrocellulose membranes, the blots were probed Cell growth inhibition assay overnight at 4C with appropriate primary antibodies Cells were seeded in 96-well plates and exposed to [RAD51C and XRCC3 (Novus Biologicals), RAD51 and increasing concentrations of olaparib (doses ranged from RAD51D (Santa Cruz Biotechnology), caspase-3, cyclin 0–10 mmol/L) for 5 days. After drug treatment, the absor- B1, p21, phosphorylated p53, cdc2, and PTEN (Cell bance of MTT dye was measured at 540 nm with a Signaling Technology), phosphorylated histone H2AX VersaMax microplate reader (Molecular Devices). The (Millipore), RAD51B (Abcam), PARP (BD Biosciences), absorbance and IC50 of olaparib were analyzed using and a-tubulin (Sigma-Aldrich)]. Antibody binding was SigmaPlot software [Statistical Package for the Social detected using an enhanced chemiluminescence system Sciences, Inc. (SPSS)]. according to the manufacturer’s protocol (Amersham For the colony formation assay (CFA), the cells were Biosciences). seeded in 6-well plates and treated with various concen- trations (5, 1, 0.1, 0.01, and 0.001 mmol/L) of olaparib for 5 Cell-cycle analysis days and were cultured until colonies formed (14 days).