Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition

Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667

Cancer Molecular and Cellular Pathobiology Research

Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2, and Anna Sablina1

Abstract Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identiﬁes the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream

effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-ﬁdelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efﬁcacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 1–11. 2012 AACR.

Introduction during the DNA repair process (3, 4), suggesting that phos- Genome stability is essential for the prevention of undue phatases may serve not only as negative regulators but also as cellular death and cancer development. To maintain genome active modulators of DNA damage response. integrity, cells have evolved multiple DNA repair pathways (1). Protein phosphatase 2A (PP2A) refers to a large family of One of the most powerful activators of the DNA repair response heterotrimeric Ser/Thr phosphatases. The PP2A core enzyme is double-strand breaks (DSB). consists of a catalytic C subunit and a structural A subunit. In Initiation of DSB repair is controlled by the phosphoinosi- mammals, 2 distinct genes encode closely related versions of tide 3-kinase–like kinase (PIKK) family. A wave of phosphor- both the PP2A A (Aa/PPP2R1A and Ab/PPP2R1B) and C (Ca/ ylation events radiating from PIKKs is amplified to convey the PPP2CA and Cb/PPP2CB) subunits. The AC dimer recruits a signals to a large number of substrates. Although this cascade third regulatory B subunit, which is responsible for the sub- fi has been studied in a great detail, the biologic relevance of strate speci city and function of the PP2A heterotrimeric many of these phosphorylation events and the mechanisms complex. Four unrelated families of B subunits have been identified to date: B/B55/PPP2R2A, B0/B56/PR61/PPP2R5, that control their downregulation remain unknown (2). It is 00 conceivable that Ser/Thr protein phosphatases could be B /PR72/PPP2R3, and Striatin/STRN. Approximately 100 dis- responsible for keeping proteins involved in DNA repair tinct complexes can be formed through combinatorial asso- fi response in an inactive state under normal conditions or for ciation of these subunits, and it is believed that speci c PP2A inactivating the signaling once DNA has been repaired. How- complexes mediate particular physiologic functions (5, 6). ever, phosphorylation of a number of PIKKs, including ataxia PP2A has been directly implicated in the negative regulation telangiectasia mutated (ATM), ATR, and CHK2, oscillates of DSB DNA repair proteins, including g-H2AX, ATM, CHK1, and CHK2 (2). However, consistent with the idea that protein phosphatases are not just negative regulators of DNA repair Authors' Affiliations: 1VIB Center for the Biology of Disease; Center for signaling, selective inhibition of PP2A activity impairs DNA Human Genetics, KU Leuven, Leuven, Belgium; and 2Department of repair (7–9). PP2A function is also essential for activation of Toxicology, Faculty of Preventive Medicine, Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat- cell-cycle checkpoints in response to irradiation (10, 11). One sen University, Guangzhou, China potential explanation for this apparent discrepancy is that Note: Supplementary data for this article are available at Cancer Research several distinct PP2A complexes may modulate different steps Online (http://cancerres.aacrjournals.org/). of the DNA repair process. fi Corresponding Author: Anna Sablina, CME Department, KU Leuven, O&N Here, we assessed the role of speci c PP2A complexes in I Herestraat 49, bus 602, Leuven, Belgium 3000. Phone: 32-163-30790; control of DSB repair and identify PPP2R2A as a critical Fax: 32-163-30145; E-mail: [email protected] effector of the homologous recombination (HR) repair through doi: 10.1158/0008-5472.CAN-12-1667 modulation of ATM phosphorylation. Critically, defects of HR 2012 American Association for Cancer Research. DNA repair in PPP2R2A-depleted cells dramatically increased

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their sensitivity to PARP enzyme inhibition. Finally, we show polyclonal anti-phospho-CHK1 (S296), rabbit polyclonal anti- that PPP2R2A is commonly downregulated in non–small cell phospho-CHK2 (T68), rabbit polyclonal anti-phospho BRCA1 lung carcinomas (NSCLC) and therefore propose PPP2R2A (S1524), rabbit polyclonal anti-CDC25A, rabbit polyclonal anti- status as a predictive marker for sensitivity to PARP inhibition. PPP2R2A, mouse monoclonal anti-phospho-ATM (S981; clone 10H11.E12), mouse monoclonal anti-CHK2 (clone 1C12), Materials and Methods mouse monoclonal anti-PP2A A (clone 4G7), rabbit polyclonal anti-phospho-CHK1 (S317), and rabbit monoclonal anti-phos- Plasmids and cell lines pho-CHK1 (S345; clone 133D3). Lentiviral pLA CMV N-Flag vector was used to generate Flag- tagged PPP2R2A. The pLKO.1-puro shGFP, pLKO.1-puro shLu- Neutral comet assay ciferase (shLuc), and pLKO.1-puro vectors containing short Microscope slides were precoated with a thin layer of normal hairpin RNA (shRNA) targeting specific PP2A subunits were melting point agarose. Cell suspensions were placed into a provided by the RNAi Consortium (12). The RNAi experiments tube containing low melting point agarose and transferred to a were carried out using ON-TARGETplus SMARTpools against glass microscope slide. Slides were placed in 2 mol/L NaCl, PPP2R2A and PPP2CA (Thermo Scientific). 30 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, 1% Triton X-100, Cells were cultured in Dulbecco's Modified Eagle Medium and 10% dimethyl sulfoxide (DMSO), pH 8.0, for 1 hour at 4 C. or RPMI (GIBCO) supplemented with 10% FBS and penicillin/ After lysis, the slides were rinsed for 20 minutes in solution streptomycin. Lentiviral infections were carried out as 0.3 mol/L NaOH, 1 mmol/L EDTA and were subjected to described previously (12). To induce DSBs, cells were treated electrophoresis at 0.7 V/cm and 50 mA for 20 minutes. The with bleomycin (Sigma-Aldrich) or irradiated using the linear slides were then rinsed with 1 mol/L Tris-HCl, pH 7.5 and accelerator (6 MV photons, Varian Medical Systems). dried with prechilled absolute ethanol and stained with PI (2 mg/mL). Comet images were examined at 200 magnifica- Cell-cycle analysis tion with Nikon ECLIPSE Ti microscope. The tail moment fi Cells were washed with PBS, xed with 70% ethanol, treated values were quantified by CometScore software v1.5. with 100 mg/mL RNase A, and stained with 50 mg/mL propi- 0 dium iodide (PI). 5-Bromo-2 -deoxyuridine (BrdUrd) incorpo- Immunofluorescent microscopy ration was analyzed using the In Situ Cell Proliferation Kit Cells were plated on cover slips or mClear-96 well plates FLUOS (Roche) according to the manufacturer's protocol. Cell- (Greiner Bio-One) and fixed with 4% paraformaldehyde for 10 cycle distribution and BrdUrd immunostaining were assessed minutes at room temperature and permeabilized with ice-cold by FACSCanto (Becton-Dickinson). methanol for 5 minutes at 20C. The cells were incubated in blocking buffer 3% bovine serum albumin (BSA) in PBS for 30 Immunoblotting and immunoprecipitation minutes at room temperature and then with primary anti- Cells were resuspended in 0.5% Triton X-100, 100 mmol/L bodies. After 3 washes with 0.3% BSA in PBS, the secondary NaCl, 3 mmol/L MgCl2, 300 mmol/L sucrose, 1 mmol/L EGTA, antibody was added. The following antibodies were used: 10 mmol/L PIPES, pH 6.8, containing protease inhibitor and mouse monoclonal anti-g-H2AX (S139; Millipore, clone phosphatase inhibitor cocktails (Roche). The cell lysates JBW301) and mouse monoclonal anti-RAD51 (Abcam, clone were incubated on ice for 10 minutes and then centrifuged at 14B4). 500 g for 5 minutes at 4C. The supernatant and pellet were Automated Image analysis was conducted using IN Cell designated as cytoplasmic and nuclear fractions, respectively. Analyzer 2000 (GE Healthcare; Supplementary Figs. S1 and S2). The nuclear fraction was resuspended in 10 mmol/L NaCl, 5 mmol/L MgCl2, 250 mmol/L sucrose, 1 mmol/L EGTA, Quantitative real-time reverse transcription PCR 10 mmol/L Tris-HCl, pH 7.6, containing protease and phos- Total RNA was isolated using RNeasy (Qiagen), and cDNA phatase inhibitor cocktails and treated with RNase-free DNase was synthesized using the Advantage RT-PCR Kit (Clontech). I (80 mg/mL; Roche) for 30 minutes at 37C. A list of primers used for real-time quantitative PCR is pre- Immunoprecipitations were conducted 24 hours after trans- sented in Supplementary Fig. S3A. Real-time PCRs were carried fection in lysis buffer containing 50 mmol/L Tris, pH 7.4, 1% out in a Roche LightCycler-480-96 (Roche), using SYBR Green NP-40, 250 mmol/L NaCl, and proteinase inhibitor cocktail PCR Master Mix (Roche). (Roche). The protein lysates were precleaned by incubation Quantitative real-time reverse transcription PCR (qRT-PCR) with sepharose-A beads and incubated with anti-Flag M2 analysis of The Lung Cancer cDNA and Cancer Survey cDNA beads (Sigma-Aldrich) or with anti-ATM antibody (Novus arrays (OriGene) was conducted using an ABI 7500 (Applied Biologicals) and sepharose-protein A beads (GE Healthcare) Biosystems). overnight at 4C. Flag complexes were eluted using 3Flag peptide (Sigma-Aldrich). NHEJ and HR DNA repair assays The following antibodies were used: goat polyclonal anti- Nonhomologous end joining (NHEJ) repair efficiency was ATM, (Novus Biologicals), mouse monoclonal anti-PP2AC (BD analyzed using H1299dA3-1 cells, which contain a chromosom- Biosciences, clone 46), and mouse monoclonal anti-BRCA1 ally integrated copy of the pIRES-TK-EGFP DNA construct (Santa Cruz, clone D-9). The following antibodies were pur- (a generous gift of Dr. Kohno, National Cancer Center Research chased from Cell Signaling: rabbit polyclonal anti-Flag, rabbit Institute, Tokyo, Japan; ref. 13). To assess HR efficiency, we

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used 293 DR-GFP cells, which contain a single integrated copy shRNA library that targets each of the known PP2A B regula- of the transgenic reporter DR-GFP. H1299dA3-1 and 293 DR- tory subunits (16). qRT-PCR analysis confirmed the knock- GFP cells were transfected with shRNAs against luciferase or down (KD) in each of the shRNA-expressing cell lines (Fig. 1C; PPP2R2A. Following puromycin selection, cells were trans- Supplementary Fig. S3B). Whenever specific antibodies were fected with I-SceI expression vector. The number of GFP- available, we also confirmed silencing of the PP2A subunits by positive cells was measured 48 hours later by flow cytometric immunoblotting (Supplementary Fig. S3C). analysis using a FACSCanto (Becton-Dickinson). In addition to PPP2CA, we found that suppression of 4 PP2A regulatory subunits, PPP2R2A, PPP2R2D, PPP2R5A, In vitro phosphatase assay and PPP2R3C, resulted in a statistically significant (P < 0.05 by 293T cells were transfected with Flag-tagged PPP2R2A, Student t test) increase of g-H2AX compared with cells expres- Wip1, and a kinase-dead Wip1-D314A mutant. FLAG-tagged sing shGFP (Fig. 1C). For PPP2R5B and STRN3 subunits, only proteins were immunoprecipitated in anti-Flag (M2)-coated expression of 1 of 2 shRNAs resulted in g-H2AX increase, most 96-well plates (Sigma-Aldrich). Immunoprecipitated com- probably due to off-target effects (Fig. 1C). These results indicate plexes were incubated with phosphopeptides for 30 minutes that PP2A B regulatory subunits, PPP2R2A, PPP2R2D, PPP2R5A, at 37C. Release of free phosphate was measured 20 minutes and PPP2R3C, could be involved in control of DSB repair. after addition of Malachite Green solution using a VictortmX3 Multilabel plate reader (Perkin Elmer). PPP2R2A is commonly downregulated in NSCLCs Inefficient DNA repair mediated by suppression of specific Clonogenic survival assay and cell viability PP2A B subunits suggests the contribution of these PP2A About 105 cells per 10-cm dish were plated in triplicate and subunits to tumor suppression. In consonance with this idea, then irradiated or treated with ABT-888 (Enzo Life Sciences). recent analysis of outlying expression coincident with extreme fi After 12 days, colonies were stained with crystal violet (Sigma- copy number alterations has identi ed PPP2R2A, one of our Aldrich) and counted using ImageJ software. Cell viability was hits, as a putative tumor suppressor gene in breast cancer (17). analyzed with a Vi-Cell XR counter. Moreover, high-resolution analyses of somatic copy number alterations of large-scale datasets revealed statistically significant loss-of-heterozygosity (LOH) of the PPP2R2A-containing Human xenograft assays region in human cancers (18, 19). Reduced PPP2R2A mRNA Xenografts were established in female NMRI-nu (nu/nu)/6- expression was also found in prostate adenocarcinoma sam- week mice (Janvier) with A549 cell line by s.c. injection (2.0 ples (20, 21). 106 cells per 0.2 mL/mouse). Tumors were staged to a pre- Our qRT-PCR analysis of PPP2R2A mRNA expression in 245 selected size using the following formula: volume ¼ (tumor samples covering 14 cancer types confirmed that PPP2R2A was length tumor width2)/2. ABT-888 (25 mg/kg) was adminis- downregulated in lung and thyroid carcinomas (P < 0.05; Fig. tered orally (twice daily for 9 days) in a vehicle containing 0.85% 2A; Supplementary Fig. S4A). Further analysis of PPP2R2A NaCl adjusted to pH 4.0. mRNA expression in a panel of 21 NSCLC samples with Tumors were fixed in 4% paraformaldehyde, paraffin- matched normal tissues revealed at least a 2-fold decrease in embedded, sectioned, and immunostained with rat anti-mouse PPP2R2A mRNA in 8 of 21 NSCLC samples (Fig. 2B). Ki67 antibody (Dako). Immunodetection was conducted with A significant correlation (P ¼ 0.0683) between the frequency R.T.U. Vectastain kit (Vector laboratories). Sections were of PPP2R2A LOH and the percentage of samples with down- counterstained with hematoxylin. For apoptosis detection In regulated PPP2R2A expression across different cancer types Situ Death detection kit, fluorescein (Roche) staining was (Supplementary Fig. S4B) suggests that decreased PPP2R2A conducted according to the manufacturer's protocol. expression in human cancers is due to LOH of the PPP2R2A- containing region. To test this idea, we analyzed PPP2R2A Results expression in a set of lung carcinoma cell lines with known Identification of PP2A specific complexes involved in status of PPP2R2A LOH (Supplementary Fig. S4C). In fact, DSB repair immunoblot analysis of PPP2R2A expression revealed lower Although previous studies showed that PP2A negatively levels of PPP2R2A in cell lines harboring PPP2R2A LOH (Fig. regulates PIKK-induced signaling, more recent reports have 2C). A common downregulation of PPP2R2A in NSCLCs due to shown that PP2A inhibition impairs DNA repair (7, 8). To LOH of PPP2R2A-containing region further supports the idea further investigate this apparent conundrum, we silenced the that PPP2R2A functions as a tumor suppressor. Thus, our more abundant PP2A catalytic subunit, PPP2CA. Because cells further studies were focused on PPP2R2A. expressing very low levels of PPP2CA proliferate poorly (14, 15), we used shRNAs that only partially suppress PPP2CA expres- PPP2R2A inhibits the HR repair by affecting cell-cycle sion (Fig. 1A). Consistent with a previous report (7), both progression in response to DNA damage immunoblot analysis of g-H2AX and automatic image analysis We validated the results of the loss-of-function screen by of g-H2AX immunostaining revealed that partial depletion of analyzing g-H2AX after PPP2R2A suppression at different time PPP2CA resulted in inefficient DNA repair (Fig. 1A and B). points following irradiation or bleomycin treatment (Fig. 3A To systematically identify PP2A complexes that mediate and B). Moreover, overexpression of shRNA resistant form of DSB repair, we conducted a loss-of-function screen using PPP2R2A in PPP2R2A-KD cells restored g-H2AX levels (Fig.

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A B 50 shGFP 45 shPPP2CA-1 shPPP2CA-2 40 shGFP shPPP2CA-1 shPPP2CA-2 35 N C N C N C 30 PP2A C 25 Short exposure 20 PP2A C 15 Long exposure

Total γ -H2AX intensity Total 10 γ -H2AX 5 0 Vinculin 02 8 C 3.5 Time after IR (h)

2.5

1.5 Mean+2SEM

1 Relative γ -H2AX intensity

0.5 shGFP shSTRN-1 shSTRN-2 shSTRN3-1 shSTRN3-2 shPPP2CA-1 shPPP2CA-2 shPPP2R2A-1 shPPP2R2A-2 shPPP2R2B-1 shPPP2R2B-2 shPPP2R2D-1 shPPP2R2D-2 shPPP2R3A-1 shPPP2R3A-2 shPPP2R3B-1 shPPP2R3B-2 shPPP2R3C-1 shPPP2R3C-2 shPPP2R5A-1 shPPP2R5A-2 shPPP2R5B-1 shPPP2R5B-2 shPPP2R5C-1 shPPP2R5C-2 shPPP2R5D-1 shPPP2R5D-2 Suppression 0 18% 36% 75% 85% 73% 62% 60% 53% 70% 85% 60% 56% 70% 59% 63% 72% 60% 53% 77% 70% 97% 75% 92% 91% 70% 60% level

Figure 1. shRNA screen to identify PP2A subunits involved in control of DNA damage response. A, immunoblot analysis of PP2A C and g-H2AX in nuclear (N) and cytoplasmic (C) fractions in HeLa cells expressing shGFP or shPPP2CA 8 hours after irradiation (IR) with 2 Gy. B, automated image analysis of g-H2AX immunostaining of HeLa cells expressing shGFP or shPPP2CA at different time points after 2 Gy of IR. C, automated image analysis of g-H2AX immunostaining of HeLa cells expressing shRNAs targeting the indicated PP2A B subunits 8 hours after 2 Gy of IR. Expression levels of PP2A subunits were assessed by qRT-PCR.

3C), indicating that inefficient DSB repair is triggered specif- PPP2R2A on irradiation-induced cell-cycle checkpoints. We ically by loss of PPP2R2A. Single-cell gel electrophoresis also found that 8 hours after irradiation, HeLa cells expressing revealed that unresolved DNA damage induced by camptothe- shGFP accumulated in G2 to S-phase, whereas cells with cin was significantly higher in PPP2R2A-KD cells (Fig. 3D). suppressed PPP2R2A were mostly in the G1 phase of the cell Taken together, our observations strongly support the idea cycle (Fig. 4E and F). In addition, BrdUrd incorporation after that PPP2R2A plays a key role in DSB DNA repair. irradiation was dramatically decreased in PPP2R2A-KD cells To assess a possible mechanism by which PPP2R2A mod- (Fig. 4G). We also observed that I-SceI–induced DSBs resulted ulates DSB repair, we elucidated the effect of PPP2R2A sup- in accumulation of 293DR-GFP-shPPP2R2A cells in the G1 pression on 2 major pathways of DSB repair, NHEJ and HR (22, phase of the cell cycle (Fig. 4H). In concordance with these 23). We found that although suppression of PPP2R2A facili- results, we found that in response to irradiation expression of tated the NHEJ repair, the HR pathway was dramatically the cell-cycle–promoting phosphatase, CDC25A (25) was sig- inhibited in PPP2R2A-KD cells (Fig. 4A and B). Consistently, nificantly decreased in PPP2R2A-depleted cells (Fig. 4I). Taken we found a decreased Rad51 foci formation in cells expressing together, these data indicate that PPP2R2A modulates the HR shPPP2R2A (Fig. 4C; Supplementary Fig. S2). Moreover, 8 hours repair through regulation of G1–S cell-cycle checkpoint. after irradiation, expression of both BRCA1 and RAD51 was Accelerated Cdc25A turnover after DNA damage is regulated significantly decreased in PPP2R2A-KD cells (Fig. 4D). Togeth- by the CHK1 and CHK2 kinases (26, 27). Therefore, we analyzed er, these results indicate that loss of PPP2R2A impairs the HR the effect of PPP2R2A on CHK1 or CHK2 activation. Although mechanism. inhibition of PPP2R2A did not affect CHK1 phosphorylation at Because the balance between NHEJ and HR repair depends S296, S217, and S345, we observed a significant accumulation of on the phase of cell cycle (22, 24), we examined the effect of CHK2 phosphorylated at T68 in PPP2R2A-KD cells (Fig. 4I),

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A B Mean 350 Normal lung NSCLC 1 300

250

0.1

200

0.01 150

100

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Relative expression of PPP2R2A mRNA (log) Relative expression 0.0001 Relative expression of PPP2R2A mRNA (%) Relative expression Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Cancer Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal C PPP2R2A Liver Lung Testis

Colon +++++++ ----- Ovary

Cervix LOH tissue Breast gland gland Kidney Thyroid Pacreas Adrenal Prostate Stomach Lymphoid

A549 Hop62 SKMES1 H1993 H1437 Hop92 H1755 H2172 H838 Colo699 Calu3 H1819

PPP2R2A

GAPDH

Figure 2. PPP2R2A expression is commonly downregulated in NSCLCs due to LOH of the PPP2R2A-containing region. A, qRT-PCR analysis of PPP2R2A expression in a set of 245 tumor samples across 14 cancer types. B, qRT-PCR analysis of PPP2R2A expression in a panel of 21 lung NSCLC samples with matched normal tissues. PPP2R2A expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. C, immunoblot analysis of PPP2R2A expression in NSCLC cell lines with and without LOH in the PPP2R2A-containing region. suggesting that G1–S checkpoint induced by loss of PPP2R2A is 30). In vitro studies using human recombinant ATM proteins mediated by prolonged activation of the CHK2 kinase. revealed that phosphorylation does not affect its kinase activity (31–33). In human cells, ATM phosphorylation at S1981 is also PPP2R2A directly regulates ATM phosphorylation dispensable for the ability of ATM to localize to DSBs, but it is CHK2 is a direct downstream target of ATM, whereas ATM required for ATM retention at DSBs (34). Consistently with this has been shown to be directly regulated by PP2A. In particular, report, immunofluorescent analysis of ATM revealed an a previous report (28) reveals that a specific PP2A inhibitor, increase of the size of ATM foci in PPP2R2A-KD cells (Fig. okadaic acid, induces ATM autophosphorylation at S1981. 5C), further confirming the idea that phosphorylation regulates Moreover, the authors showed interaction between ATM and ATM retention at the sites of DSBs (34). PP2A A and C subunits. However, specific PP2A regulatory These findings suggest that PPP2R2A controls DNA repair subunit(s) involved in ATM dephosphorylation has not been through regulation of the ATM signaling. To test this hypoth- identified, whereas our data suggest that PPP2R2A may affect esis, we analyzed whether PPP2R2A affects DNA repair in HeLa CHK2 activity by regulating ATM dephosphorylation. ATM SilenciX (ATM KD) cells. Indeed, analysis of g-H2AX levels Indeed, we found that suppression of PPP2R2A resulted in a in ATM-deficient cells revealed that suppression of PPP2R2A in significant increase of ATM phosphorylated at S1981 (Fig. 5A), these cells did not affect the kinetics of DNA repair (Fig. 5D). whereas restoration of PPP2R2A expression by overexpression To determine whether PPP2R2A directly regulates ATM of shRNA-resistant silent mutant of PPP2R2A rescued this dephosphorylation, we conducted a set of reciprocal immu- increase (Fig. 4B). Although ATM phosphorylation is consid- noprecipitations. We found PPP2R2A together with PP2A A ered as a sign of ATM activation, there are contradictory data and C subunits in complex with ATM (Fig. 6A). Using in vitro how it affects ATM functions. Studies of ATM-S1987A (mouse phosphatase assays, we also found that phosphopeptides homologue of human S1981) mice showed normal ATM- corresponding to human ATM pS367, pS1893, and pS1981, but dependent phosphorylation of ATM substrates, activation of not pT1885 and pS2996, were efficiently dephosphorylated by cell-cycle checkpoints, and localization of ATM to DSBs (29, PPP2R2A-specific complexes (Fig. 6B). Given that we observed

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A B C shPPP2R2A-2

Figure 3. Loss of PPP2R2A siGFP siPPP2CA siPPP2R2A impairs DSB repair. A, automated

PPP2CA shGFP/V V rPPP2R2A shGFP shPPP2R2A-1 shPPP2R2A-2 image analysis of g-H2AX PPP2R2A immunostaining of HeLa cells PPP2R2A PPP2R2A expressing shGFP or shPPP2R2A Vinculin GAPDH GAPDH at different time points after 2 Gy of IR. Suppression of PPP2R2A was shGFP ﬁ shGFP siGFP con rmed by immunoblotting. B, shPPP2R2A-2/V shPPP2R2A-1 siPPP2CA analysis of g-H2AX imunostaining shPPP2R2A-2/rPPP2R2A in HeLa cells transfected with ON- shPPP2R2A-2 siPPP2R2A 50 120 50 TARGETplus SMART pools 45 45 targeting GFP, PPP2CA, or 100 40 40 PPP2R2A at different time points 35 35 80 after treatment with bleomycin (10 30 30 mmol/L) for 1 hour. Suppression of 25 60 25 PP2A C and PPP2R2A subunits 2AX intensity

-H 20 20 was conﬁrmed by immunoblotting. γ 40 γ -H2AX intensity 15 15 C, the effect of PPP2R2A 10 10 Total Total 20 Total expression on g-H2AX as detected -H2AX positive cells positive% of γ -H2AX 5 5 by automated image analysis. 0 0 0 shPPP2R2A-resistant (rPPP2R2A) 0 2 8 0 2 8 24 0 2 8 form of PPP2R2A Time after IR (h) Time after bleomycin treatment (h) Time after IR (h) was overexpressed in HeLa- shPPP2R2A-2 cells. V, an empty μ Time after CPT (2 mol/L) (h) vector. D, comet assay of HEK TER D Untreated 2 12 cells expressing shGFP or 12 * shPPP2R2A after treatment with shGFP 2 mmol/L of camptothecin (CPT) 10 shPPP2R2A-1 for 1 hour. The comet tail moment shGFP 8 from 75 cells was quantiﬁed with P ¼ 6 ** CometScore software. , 0.05; , P ¼ 0.01, as determined by the 4 Student t test.

Comet tail moment 2

0 2 12 shPPP2R2A-1 Time after CPT (2 μmol/L ) (h)

an interaction between PPP2R2A heterotrimeric complexes the amount of ATM associated with PP2A A and C subunits and ATM, increased ATM phosphorylation in PPP2R2A-deplet- after irradiation. Interestingly, ATM mediates the irradia- ed cells, and in vitro dephosphorylation of 3 ATM autopho- tion-induced dissociation of the PPP2R2A subunit from the sphorylation sites (S367, S1893, and S1981) by PPP2R2A-spe- PP2A AC core dimer (37), implicating ATM in the disruption cific complexes, it seems likely that ATM is a direct target for of functional PPP2R2A-containing PP2A complexes. Con- PPP2R2A-specific PP2A phosphatase in vivo. sistently with this report, 15 minutes after irradiation, Wip1 phosphatase has been also shown to dephosphor- PPP2R2A-associated phosphatase activity was decreased. ylate ATM at S1981 and S367, but not at S1893 (refs. 35, Thirty minutes after irradiation PPP2R2A was found again 36; Fig. 6B), indicating that PPP2R2A and Wip1 may differ- in complex with ATM, which is also correlated with an entially regulate ATM dephosphorylation at different sites. increase of PPP2R2A-specific phosphatase activity toward Moreover, in contrast to PP2A subunit PPP2R2A, Wip1 ATM phosphopeptides (Fig. 6D and E). This kinetics is expression is extremely low in undamaged cells (Fig. 6C), consistent with the kinetics of ATM recruitment/dissocia- and depletion of Wip1 does not affect the level of phosphor- tion to the sites of DSBs (34). These data also suggest the ylated ATM under normal conditions (35). Taken together, existence of a negative feedback loop between PPP2R2A and these data suggest that PPP2R2A-containing PP2A holoen- ATM. zyme is the major ATM phosphatase in the absence of DNA damage. PPP2R2A downregulation increases sensitivity to PARP Irradiation triggered transient dissociation of the ATM– inhibitors PPP2R2A complex, allowing accumulation of phosphorylat- Inhibition of the HR repair in PPP2R2A-depleted cells ed ATM (Fig. 6D). This observation is consistent with a suggests that these cells are highly sensitive to DNA dam- previous report (28) that revealed a significant reduction in age. Indeed, colony formation assay revealed that inhibition

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The Role of PPP2R2A in DNA Repair

A shLuc BCshLuc shGFP 200 shPPP2R2A-1 120 shPPP2R2A-1 shPPP2R2A-1 9 180 shPPP2R2A-2 shPPP2R2A-2 shPPP2R2A-2 100 160 8 140 7 80 6 120 * ** 100 60 5 80 4

HR repair (%) 40 60 3 Figure 4. Loss of PPP2R2A impairs NHEJ repair (%) 40 2 the HR repair by triggering G1–S cell- 20 cycle arrest. A, the efficiency of I- 20 per nucleus Rad51 foci 1 SceI–induced NHEJ repair in 0 0 0 H1299dA3-1 cells expressing Untreated 2 h after IR 8 h after IR Untreated 8 h after IR shRNAs specific to luciferase or D E shGFP PPP2R2A. B, the efficiency of I-SceI– induced HR repair in 293 DR-GFP cells expressing the indicated Counts shGFP shGFP shGFP shPPP2R2A-1 shPPP2R2A-2 shPPP2R2A-1 shPPP2R2A-2 shRNAs. C, automated image shPPP2R2A-1 shPPP2R2A-2 analysis of Rad51 foci formation of PPP2R2A HeLa cells expressing shRNAs shPPP2R2A-1 Rad51 targeting GFP or PPP2R2A after 2 Gy of IR. , P ¼ 0.0033; , P ¼ 0.0054, as p-BRCA1 (S1524) determined by the Student t-test. D, immunoblot analysis of the indicated BRCA1 Counts proteins in HeLa cells expressing vinculin shGFP or shPPP2R2A after 2 Gy of shPPP2R2A-2 IR. E and F, cell-cycle distribution of PI-stained HeLa cells expressing the F G 40 shGFP indicated shRNAs 8 hours after 5 Gy 100 G 90 1 35 shPPP2R2A-1

S Counts of IR. G, analysis of BrdUrd shPPP2R2A-2 80 30 G2 incorporation by HeLa cells 70 25 expressing shRNAs speciﬁc to GFP 60 2n 4n or PPP2R2A 8 hours after 50 20 2n 4n 40 DNA Content 5 Gy of IR. H, the proportion of 293 15 30 DR-GFP cells expressing the 10 20 Untreated Cell-cycle phase (%) Cell-cycle I 2 h after IR 8 h after IR indicated shRNAs cells in G1 phase 10 cells (%) BrdUrd-positive 5 after transfection with yellow 0 0 ﬂuorescent protein (YFP) or I-SceI- shGFP shPPP2R2A-1 shPPP2R2A-2 YFP. I, immunoblot analysis of the shGFP shGFP shGFP shPPP2R2A-1 shPPP2R2A-2 shPPP2R2A-1 shPPP2R2A-2 indicated proteins in HeLa cells shPPP2R2A-1 shPPP2R2A-2 H CDC25A expressing shRNAs against GFP 80 shGFP 70 or PPP2R2A at different time points shPPP2R2A-2 p-CHK1 (S296) after 2 Gy of IR. 60 50 p-CHK1 (S317)

phase 40 1 p-CHK1 (S345) 30 in G 20 CHK1 10 % of YFP-positive cells % of p-CHK2 (T68) 0 V I-SceI CHK2

GAPDH

of PPP2R2A sensitized cells to IR (Fig. 7A). Moreover, HR 888 than cell lines with lower PPP2R2A expression (Figs. 2C deﬁciency predicts hypersensitivity to inhibition of PARP and 7C). (38, 39). In fact, analysis of cell survival after treatment with ABT-888 treatment also inhibited growth of PPP2R2A-KD ABT-888 (veliparib), a potent inhibitor of PARP1 and A549 xenografts (Fig. 7D). Moreover, we observed signiﬁcantly PARP2, revealed that PPP2R2A-KD cells are more sensitive reduced proliferation rate and increased apoptosis in to ABT-888 treatment (Fig. 7B). To validate this observation, PPP2R2A-KD tumors compared with shGFP-expressing we examined the sensitivity to PARP inhibition of lung tumors (Fig. 7E and F). These results strongly indicate that carcinoma cell lines with different levels of PPP2R2A both PPP2R2A expression levels and LOH in the PPP2R2A- expression. Consistently, we found that cell lines with containing region can be used to predict tumor sensitivity to higher PPP2R2A expression had lower sensitivity to ABT- treatment with PARP inhibitors.

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A B Untreated 2 h after IR 8 h after IR

Untreated 2 h after IR 8 h after IR Figure 5. PPP2R2A negatively regulates ATM phosphorylation. A, immunoblot analysis of phospho- ATM (S1981) and ATM in HeLa cells expressing shGFP or shGFP shGFP shGFP shPPP2R2A-1 shPPP2R2A-2 shPPP2R2A-1 shPPP2R2A-2 shPPP2R2A-1 shPPP2R2A-2

shGFP shPPP2R2A-2 shPPP2R2A-2/rPPP2R2A shGFP shPPP2R2A-2 shPPP2R2A-2/rPPP2R2A shGFP shPPP2R2A-2 shPPP2R2A-2/rPPP2R2A shPPP2R2A at different time points p-ATM (S1981) p-ATM (S1981) after 2 Gy of IR. B, immunoblot analysis of phospho-ATM (S1981) ATM and ATM in HeLa-shPPP2R2A-2 ATM cells and HeLa-shPPP2R2A-2/ rPPP2R2A cells at different time GAPDH Vinculin points after 2 Gy of IR. C, representative images and CDshGFP shPPP2R2A-1 shPPP2R2A-2 automated image analysis of ATM immunostaining in HeLa cells shGFP 70 shPPP2R2A-2 expressing shRNAs targeting GFP ATM KD shGFP or PPP2R2A after 2 Gy of IR. 60 ATM KD shPPP2R2A-2 , P ¼ 0.0021; , P ¼ 0.0034, as shGFP shGFP determined by the Student t test. 50 shPPP2R2A-1 shPPP2R2A-1 D, automated image analysis of 3 shPPP2R2A-2 18 shPPP2R2A-2 ** 40 g-H2AX immunostaining of HeLa 16 and HeLa ATM SilenciX (ATM KD) 2.5 * 14 γ -H2AX intensity 30 cells expressing shGFP or 2 12 shPPP2R2A at different time points

Total Total 20 10 after 2 Gy of IR. 1.5 8 10

1 6

4 0

Relative size of ATM foci 0.5 0 2 8 2

Number of ATM foci per nuclei Time after IR (h) 0 0

Discussion view that protein phosphatases serve only as negative reg- PP2A is a ubiquitously expressed family of Ser/Thr protein ulators of DNA repair. Instead, our data suggest that tight phosphatases, and the diversity of PP2A functions suggests regulation of phosphorylation events during DNA repair by that particular PP2A complexes may contribute independently specific protein phosphatases is required for timely DSB DNA to complex phenotypes, such as the DNA damage response (5, repair. 6). Indeed, we found that suppression of 4 different PP2A Analysis of PPP2R2A expression in human cancers revealed regulatory B subunits (PPP2R2A, PPP2R2D, PPP2R5A, and that PPP2R2A mRNA expression is commonly downregulated PPP2R3C) impairs the efficiency of DNA repair, suggesting in NSCLCs. PPP2R2A gene is located at 8p21 chromosome that these specific PP2A complexes are involved in control of region, which is frequently deleted in a wide range of epithelial DNA repair. In our prior study, we conducted a loss-of-function cancers (42). However, a homozygous deletion of PPP2R2A- screen using a similar shPP2A library to identify PP2A subunits containing locus is a more rare event in human cancers. As an involved in human cell transformation (16). This screen impli- example, a recent analysis of 141 clinical prostate tumors cates 3 PP2A regulatory subunits, PPP2R5A, PPP2R5C, and revealed PPP2R2A deletions in 61.7% of tumor samples with PPP2R3A, in control of cell transformation. Our present results only 3 recurrent homozygous deletions (2.1%; ref. 43). These suggest that in addition to modulating c-Myc and Wnt activity results are consistent with our observation that PPP2R2A (16), PPP2R5A could also contribute to the tumorigenic phe- expression is downregulated in NSCLC cell lines but never notype by impairing DNA repair efficiency. Consistent with this completely lost. As far as no potentially important somatic idea, it has been shown that PPP2R5A could directly dephos- mutations of PPP2R2A have been reported, these data impli- phorylate both c-Myc (40) and CHK2 kinase (41). However, the cate PPP2R2A as a haploinsufficient tumor suppressor gene in exact mechanism(s) by which PPP2R2D, PPP2R5A, and epithelial cancers. PPP2R3C contribute to DNA response needs to be further PARP inhibitors are emerging as a promising class of investigated. anticancer agents particularly effective against tumors with In summary, our study revealed that dephosphorylation of loss of functional BRCA1 and BRCA2 (38). However, BRCA1 ATM at S367, S1893, and S1981 by PPP2R2A-specific com- and BRCA2 mutation carriers comprise less than 5% of plexes regulates retention of ATM foci. Dysregulation of ATM breast cancer cases, and in lung cancer, BRCA mutations – triggered by loss of PPP2R2A induces G1 S checkpoint and are even much less common. Therefore, there is a pressing inhibits HR repair. These observations further challenge the need to identify novel biomarkers that would predict

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A B IP anti-ATM IP anti-Flag WCL

+ Phosphatase activity, pmol PO4 /min Substrate Empty PPP2R2A wt-WIP1 WIP1-D314A vector Control Ser/Thr phosphatase substrate ND 12.56 ± 0.26 80.11 ± 0.06 ND Vector Flag-PPP2R2A Vector Flag-PPP2R2A Vector Flag-PPP2R2A DLDVPIPGRFDRRV(pS)VAAE ATM-pS367 Flag Flag 5.26 ± 1.23 58.12 ± 0.54 109.6 ± 6.06 ND NS Ac-RSLEI(pS)QSYTT-amide ATM-pT1885 ND 11.56± 0.19 2.99 ± 0.02 ND ATM ATM Ac-TSRST(pT)PANLD-amide ATMpS1893 ND 78.34± 5.93 11.17 ± 0.15 ND Ac-NLDSE(pS)EHFFR-amide ATMpS1981 PP2A A ND 42.45 ± 2.51 74.33 ± 5.01 ND PP2A A Ac-AFEEG(pS)QSTTI-amide ATMpS2996 ND 3.28 ± 0.24 5.38 ± 0.04 ND Ac-CKRNL(pS)DIDQS-amide PP2A C PP2A C

Vinculin

C Time after IR (min) D IP anti-Flag 0 15’ 30’ 60’ 120’ 240’ Time after IR (min) 0 15’ 30’ 60’ p-ATM (S1981) ATM ATM Flag

PPP2R2A WCL Time after 0 15’ 30’ 60’ Wip1 IR (min) ATM E 120 Time after IR (min) Flag 0

/min 15’ + 100 Vinculin 4 60’ 120’ 80

Phosphatase activity, pmol PO Phosphatase activity, 0 ATM-pS367 ATM-pS1893 ATM-pS1981

Figure 6. PPP2R2A-containng complexes directly dephosphorylates ATM at S367, S1893, and S1981. A, reciprocal immunoprecipitations of ATM or Flag- tagged PPP2R2A expressed in HEK TE cells followed by immunoblotting using antibodies specific for Flag, ATM, PP2A C, and PP2A A. NS, nonspecific band. B, in vitro phosphatase activity of PPP2R2A-specific complexes and Wip1 toward ATM phosphopeptides. ND, not detected. C, immunoblot analysis of the indicated proteins at different time points after 2 Gy of IR. D, immunoprecipitations of PPP2R2A-containing complexes at different time points after 2 Gy of IR followed by immunoblotting with antibodies specific for Flag, ATM, and PP2A C. E, PPP2R2A-associated phosphatase activity of at different time points after 2 Gy of IR toward the indicated ATM phosphopeptides. responsiveness to PARP inhibitors. Here, we show that PARP sion is observed in about 40% of lung carcinomas, these data inhibition suppresses growth of tumors with decreased implicate PPP2R2A as a novel predictive marker in BRCA- PPP2R2A expression. Together with the finding that LOH proficient NSCLCs for the efficiency of treatment with PARP of PPP2R2A-containing region or decreased PPP2R2A expres- inhibitors.

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Cells with high PPP2R2A expression A B C A549 shGFP shGFP Colo699 120 120 120 shPPP2R2A-1 shPPP2R2A-1 H838 100 shPPP2R2A-2 100 shPPP2R2A-2 100 Cells with low PPP2R2A expression H1755 80 80 80 HOP92 HOP62 60 60 60 H2172

40 40 40 Cell Survival (%) Cell Survival 20 20 20

0 0 0 012 5 04080 120 04080 120 Dose of IR (Gy) Concentration of ABT-888 (μmol/L) Concentration of ABT-888 (μmol/L)

Ki67 DEA549-shGFP vehicle (n = 6) 1,400 shGFP shPPP2R2A-1 A549-shPPP2R2A-1 vehicle (n = 6) A549-shGFP ABT-888 (n = 12) 1,200 A549-shPPP2R2A-1 ABT-888 (n = 9)

1,000

8,00 *

600 F TUNNEL

Tumor volume (mm) volume Tumor shGFP shPPP2R2A-1 400 * 200

0 0245678910 Days of treatment with ABT-888

Figure 7. Decreased expression of PPP2R2A increases tumor sensitivity to PARP inhibition. A, colony formation assay of HeLa cells expressing shRNAs speciﬁc to GFP and PPP2R2A after exposure to increasing doses of IR. B, cell survival of HeLa cells expressing shGFP or shPPP2R2A-1 after ABT-888 treatment. C, cell survival of lung carcinoma cell lines treated with increasing concentrations of ABT-888. D, growth of A549 xenograft tumors expressing the indicated shRNAs in response to ABT-888 treatment. , P ¼ 0.0011, as determined by ANOVA test. E, Ki67 immunohistochemistry of A549 xenografts expressing shGFP or shPPP2R2A-1 after treatment with ABT-888. F, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analysis of A549 xenografts expressing the indicated shRNAs after 7 days of ABT-888 treatment.

Disclosure of Potential Conﬂicts of Interest Administrative, technical, or material support (i.e., reporting or orga- No potential conﬂicts of interest were disclosed. nizing data, constructing databases): P. Kalev, T. Soin, N. Danda Study supervision: P. Kalev, A. Sablina Authors' Contributions Conception and design: P. Kalev, A. Sablina Development of methodology: P. Kalev, I. Vazquez, S. Munck, T. Soin, W. Chen, Grant Support A. Sablina Acquisition of data (provided animals, acquired and managed patients, This work was supported by FWO (ZKC2592-00_WO1) and a Distinguished provided facilities, etc.): P. Kalev, M. Simicek, I. Vazquez, S. Munck, L. Chen, Young Scholar of National Nature Science Foundation of China (NSFC 30925029). T. Soin, N. Danda The costs of publication of this article were defrayed in part by the payment of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, page charges. This article must therefore be hereby marked advertisement in computational analysis): P. Kalev, M. Simicek, I. Vazquez, S. Munck, L. Chen, N. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Danda Writing, review, and/or revision of the manuscript: P. Kalev, M. Simicek, T. Received May 9, 2012; revised August 8, 2012; accepted September 8, 2012; Soin, A. Sablina published OnlineFirst October 18, 2012.

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Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition

Peter Kalev, Michal Simicek, Iria Vazquez, et al.

Cancer Res Published OnlineFirst October 18, 2012.

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