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Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1037 Cancer Molecular Cell Biology Research Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells Yuka Sasaki1,2, Hiroaki Fujimori1,2, Miyuki Hozumi2,3,Takae Onodera1,2,Tadashige Nozaki1,4, Yasufumi Murakami3, Kazuto Ashizawa5, Kengo Inoue6, Fumiaki Koizumi7, and Mitsuko Masutani1,2 Abstract Poly (ADP-ribose) glycohydrolase (PARG) is the main cancer A549, PC14, and SBC5 cells, and inhibited the enzyme responsible for catabolism of poly (ADP-ribose) PI3K/AKT/mTOR pathway in A549 cells, suggesting that (PAR), synthesized by PARP. PARG dysfunction sensitizes dual depletion of PARG and DUSP22 induced apoptosis by certain cancer cells to alkylating agents and cisplatin by upregulating PUMA and suppressing the PI3K/AKT/mTOR perturbing the DNA damage response. The gene mutations pathway. Consistently, the growth of tumors derived from that sensitize cancer cells to PARG dysfunction-induced death double knockdown A549 cells was slower compared with remain to be identified. Here, we performed a comprehensive those derived from control siRNA-transfected cells. Taken analysis of synthetic lethal genes using inducible PARG together, these results indicate that DUSP22 deficiency knockdown cells and identified dual specificity phosphatase exerts a synthetic lethal effect when combined with PARG 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction, suggesting that DUSP22 dysfunction could dysfunction. DUSP22 is considered a tumor suppressor and its be a useful biomarker for cancer therapy using PARG mutation has been frequently reported in lung, colon, and inhibitors. other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells Significance: This study identified DUSP22 as a novel reduced survival compared with single-knockdown counter- synthetic lethal gene under the condition of PARG dysfunction parts. Dual depletion of PARG and DUSP22 increased the and elucidated the mechanism of synthetic lethality in lung apoptotic sub-G1 fraction and upregulated PUMA in lung cancer cells. ADP-ribose to target proteins in a nicotinamide adenine dinu- Introduction þ cleotide (NAD )-dependent manner (1, 2). This reaction is Poly (ADP-ribosylation) is a posttranslational modification involved in various biological processes, including cell death, by which some PARP family proteins catalyze the transfer of chromatin regulation, and DNA repair of single-strand breaks (SSB) and double-strand breaks (DSB; refs. 1, 3). PARP inhibitors 1Department of Frontier Life Sciences, Nagasaki University Graduate School of were recently developed as a novel anticancer agent based on the Biomedical Sciences, Nagasaki, Japan. 2Division of Chemotherapy and Clinical concept of synthetic lethality (4, 5). PARP inhibitors selectively Research, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan. induce cell death in homologous recombination repair (HRR)– 3Department of Biological Science and Technology, Faculty of Industrial Science deficient cancers such as those associated with mutations in and Technology, Tokyo University of Science, Katsushika-ku, Tokyo, Japan. BRCA1/2 (6, 7), RAD51 (8), and PTEN (9). The development of 4 Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, novel anticancer agents based on the concept of synthetic lethality Hirakata, Osaka, Japan. 5Department of Clinical Oncology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto, Nagasaki, Japan. 6Pharma is a valuable cancer chemotherapy strategy because these drugs Valley Center, Nagaizumi-cho, Shunto-gun, Shizuoka, Japan. 7Department of show increased tumor selectivity with reduced adverse effects on Laboratory Medicine, Tokyo Metropolitan Cancer and Infectious Diseases Center normal cells (4). Komagome Hospital, Bunkyo-ku, Tokyo, Japan. Poly (ADP-ribose) (PAR) synthesized by PARP is rapidly Note: Supplementary data for this article are available at Cancer Research degraded to ADP-ribose by poly (ADP-ribose) glycohydrolase Online (http://cancerres.aacrjournals.org/). (PARG; ref. 10) and ADP-ribosyl hydrolase (ARH3; ref. 11). PARG Y. Sasaki and H. Fujimori contributed equally to this article. is the main enzyme catabolizing PAR to ADP-ribose through its endo- and exo-glycohydrolase activities (12). As reported previ- Corresponding Author: Mitsuko Masutani, Nagasaki University, Sakamoto 1-7-1, ously, PARG is required for the efficient repair of DSBs and Nagasaki 852-8588, Japan. Phone: 81-95-819-8502; Fax: 81-95-819-8502; E-mail: fi [email protected] SSBs (13). PARG de ciency induces PAR accumulation and a delay of DNA repair (14, 15). PAR accumulation induces cell Cancer Res 2019;79:3851–61 death (parthanatos) accompanied by the translocation of apo- doi: 10.1158/0008-5472.CAN-18-1037 ptosis inducing factor from mitochondria to nuclei, leading to Ó2019 American Association for Cancer Research. fragmentation of large-sized DNA in neuronal cells and cancer www.aacrjournals.org 3851 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1037 Sasaki et al. cells such as HeLa cells (16, 17). As previously reported, certain 3 days and divided into 2 populations. Cells were cultured for human cancer cell lines with PARG knockdown synergistically 6 days in the presence or absence of 40 ng/mL Tc, and genomic show higher sensitivity to alkylating agents (14, 18) and cisplatin DNA was purified from 2 populations using a DNA Purification treatment (18). PARG hypomorphic mouse embryonic stem cells Kit (Dojindo). Amplification of barcode sequences in genomic with residual 10% PARG activity did not exhibit growth defect but DNA and purification of DNA were performed using the showed higher sensitivity to alkylating agents, cisplatin, photon, Decode shRNA Negative Selection Kit (Thermo Fisher Scien- and particle beam irradiation compared with wild-type embry- tific) and Gene JET PCR Purification Kit (Thermo Fisher onic stem cells (14, 19, 20). In addition, BRCA2 (21) and Bruton Scientific), respectively, as recommended by the manufacturer. tyrosine kinase (BTK; ref. 22) defects increase PARG inhibition- Then, the genomic DNA was labeled using a Genomic DNA induced cytotoxicity (21–23). These findings led us to hypothe- Enzymatic Labeling Kit (Agilent Technologies) and purified size that PARG could serve as a novel therapeutic target for using Amicon Ultra-0.5 mL Centrifugal Filters (Millipore). The anticancer agents in both monotherapy and combination therapy labeledbarcodesequenceswerehybridizedtomicroarrayslides with radiotherapy or other DNA targeting chemotherapeutic for 17 hours, and the slides were washed according to the agents for particular types of cancers. Recently, PARG inhibitors Agilent CpG microarray protocol. such as phenolic hydrazide hydrazones (24), rhodanine-based PARG inhibitors (RBPI; ref. 25), xanthene compounds (26), siRNA transfection ADP-HPD (27), and PDD00017273 (28), which has an IC50 in Cells were seeded onto 6-well plates or 24-well plates. Trans- the sub-microM range, have been developed. However, specific fection with siRNA was performed using Lipofectamine RNAi and potent PARG inhibitors for clinical applications remain to MAX (Life Technologies) according to the manufacturer's proto- be developed. col. Individual siRNAs were used at final concentration of Here, we screened genes whose deficiency enhances sensi- 10 nmol/L in Opti-MEM. siRNAs (PARG#2, DUSP22#2, PUMA, tivity in a synthetic lethal manner to develop a novel anticancer TP63) targeting DUSP22, PARG, PUMA, and TP63 were purchased agent targeting PARG. We identified dual specificity phospha- from Integrated DNA Technologies. The siRNA sequence of tase 22 (DUSP22) as such a novel gene. Synthetic lethality DUSP22#1 is based on shRNA sequence of oligo ID: induced by PARG and DUSP22 dysfunctioninlungcancercells V2LHS_225030 in the Decode RNAi Pooled Lentiviral shRNA led to TP63-dependent apoptosis by upregulating p53 upregu- Screening Libraries and it was constructed from Integrated DNA lated modulator of apoptosis (PUMA). Double knockdown of Technologies. PARG#1 siRNA was obtained as described previ- PARG and DUSP22 inhibited tumor growth in a mouse xeno- ously (14). DS NC1 siRNA (Integrated DNA Technologies) and graft model. These results indicated that alterations in DUSP22 scrambled siRNA (Ambion/Applied Biosystems) were used as expression levels may serve as a predictive biomarker for PARG negative controls (N.C.). inhibitors. qRT-PCR RNA was prepared from each individual cell line and reverse Materials and Methods transcribed using a High Capacity Reverse Transcription Kit Cell culture and reagents (Thermo Fisher Scientific). The qRT-PCR analysis was per- The TRHmPARG#8 cell line is a tetracycline (Tc)-inducible formed using SYBR Green with the CFX96 Real-Time System PARG knockdown strain derived from the human T-REx HeLa (Bio-Rad). The mRNA levels were normalized to GUSB mRNA. cell line described previously (29). TRHmPARG#8 and PC14 were The sequences of primer pairs are listed in Supplementary cultured in Minimum Essential Medium and DMEM (Thermo Table S1. Fisher Scientific), respectively. A549 and SBC5 cells were grown in RPMI1640 (Thermo Fisher Scientific). Media were supplemented Cell proliferation assay with 10% FBS (Gibco) and 1% penicillin–streptomycin