Published OnlineFirst April 24, 2012.
Mol Cancer Ther
Hui-Chun Wang, Alan Yueh-Luen Lee, Wen-Cheng Chou, et al.
cross-link-generating agents in vitro and in vivo
its derivative sensitize cancer cells to interstrand
Inhibition of ATR-dependent signaling by protoapigenone and
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Inhibition of ATR-dependent signaling by protoapigenone and its derivative sensitize cancer cells to interstrand cross-link-generating agents in vitro and in vivo
Hui-Chun Wang1,2,7, Alan Yueh-Luen Lee3,8, Wen-Cheng Chou1,4,8, Chih-Chung Wu1,2, Chao-Neng Tseng1,2, Kevin Yen-Ting Liu1, Wen-Lien Lin1, Fang-Rong Chang1,2, Da-Wei Chuang1, Attila Hunyadi1,5, Yang-Chang Wu1,6,7
1Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung
80708, Taiwan.
2Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan.
3
National Institute of Cancer Research, National Health Research Institutes, Miaoli 35053, Taiwan. 4
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan 5
Institute of Pharmacognosy, University of Szeged, Szeged H-6720, Hungary. 6
School of Chinese Medicine, college of Chinese Medicine, China Medical University, and Natural Medicinal Products Research Center and Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan
Running title: Inhibition of ATR and FANCD2 activation by natural products
Keywords: ATR, FANCD2, checkpoint, DNA repair, natural products
Footnote:7HC. Wang and YC. Wu share senior authorship.8AY-L. Lee and WC. Chou contributed equally to this work.
Grant support
This work was supported by grants NSC-97-2314-B-037-051 and NSC-99-2320-B-037-008 (to HC. Wang), NSC 98-2311-B-037-001-MY3 (to AY-L. Lee), NSC-99-2323-B-039-002 (to YC. Wu), and NSC
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AY-L. Lee) from the National Health Research Institutes of Taiwan, and grant KMU-Q098021 (to HC. Wang) from the Kaohsiung Medical University Research Foundation. A. Hunyadi was supported by the Zoltán Magyary postdoctoral fellowship (EEA and Norway Grants, 104/2009).
Hui-Chun Wang, Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan. Phone: +(886)-7-3121101 ext. 6921; Fax:+(886)-7-311-4773; E-mail: wanghc@kmu.edu.tw.Yang-Chang Wu, Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan. Phone: +(886)-4-22053366 ext. 1012; Fax:+(886)-2-220-60248; E-mail: yachwu@mail.cmc.edu.tw.
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Abstract
DNA damage caused during cancer treatment can rapidly activate the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR)-dependent phosphorylation of Chk2 and Chk1 kinases, which are hallmarks of the DNA damage response (DDR). Pharmacological inhibition of ATR causes a synthetic lethal effect on ATM- or p53-defective cancers, suggesting that such inhibition is an effective way to improve the sensitivity of cancers to DNA-damaging agents. Here, both the natural compound protoapigenone (WYC02) and its synthetic derivative WYC0209 exhibited cytotoxic effects on various cancer cell lines.WYC02 causes chromosomal aberration in the mitotic spreads of CHO cells.
Interestingly, cancer cells did not exhibit typical DDR markers upon exposure to WYC02 and WYC0209 (WYCs). Further investigation into the molecular mechanisms of WYCs function revealed that they have a potential ability to inhibit DDR, particularly on activation of Chk1 and Fanconi anemia group D2 protein (FANCD2), but not Chk2. In this way, WYCs inhibited ATR-mediated DNA damage checkpoint and repair. Furthermore, when combined with the DNA cross-linking agent cisplatin, treatment with WYCs resulted in increased tumor sensitivity to interstrand cross-link-generating agents both in vitro and in vivo. Our results therefore especially implicate WYCs in enhancing tumor chemosensitivity when the ATR checkpoint is constitutively active in states of oncogene-driven replicative stress or tolerance to DNA-interfering agents.
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Introduction
Ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) are 2 members of the
phosphoinositide 3-kinase (PI3K)-related protein kinase family that play a central role in DNA damage response (DDR) coordination; they also function in the signaling machinery of cell cycle arrest, DNA repair and transcription, and cell death. While ATM is predominantly activated in response to DNA strand breaks, ATR is activated in response to damage arising from ultraviolet (UV) rays or replication block; both kinases activate signaling cascades that involve 2 checkpoint kinase effectors, Chk1 and Chk2, whose roles were previously suggested to be redundant (1). In contrast to ATM, ATR has been reported to be indispensible for cell growth and for life. ATR-knockout mouse embryos died early due to mitotic catastrophe characterized by incomplete DNA replication and chromosomal fragmentation (2,3). Moreover, ATR gene mutations are rarely found in humans. The only mutation variants that can survive are heterozygous or hypomorphic variants. Furthermore, cells derived from patients with Seckel syndrome exhibit cellular features associated with ATR signaling defects. Consistent with this phenotype, Seckel-like mouse embryonic cells showed accelerated aging due to replicative stress, exhibiting an accumulation of lethal chromosomal breaks (4,5). However, with regard to its role in regulating the replication checkpoint, ATR is activated by most cancer chemotherapeutic agents that target DNA in replicating cells. Therefore, inhibition of ATR signaling is a valid and promising strategy that can improve efficiency of chemotherapy or radiotherapy (6,7). Thus far, several inhibitors of DDR-related kinases, including Chk1 and Chk2, have been successfully used alone or in combination with each other in clinical trials (8,9). Recently, several chemicals that inhibit ATR kinase activity in vitro were used to support the hypothesis that ATR kinase can be targeted to improve cancer therapy (10-14). Since most of these studies are in their initial stages, it is imperative to focus more efforts toward
investigating strategies to inhibit ATR signaling.
WYC02 (Fig. 1A) is a flavonoid that we previously isolated and identified from the whole plant extract of Thelypteris torresiana, a fern species native to Taiwan. This compound was originally screened for cytotoxicity function; using a colorimetric cytotoxicity assay, WYC02 demonstrated therapeutic effects and was a lead compound for potential anticancer drug development (15-17). In
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previous studies, WYC02 and its more potent analog WYC0209 (Fig. 1A) were shown to induce oxidative stress, consequently activating the p38 and JNK1/2 MAPK pathways following cell cycle arrest and apoptosis in several cancer cell types. These compounds were also found to reduce the size of tumor xenografts in nude mice without exerting toxic effects on the recipient (18-22). Recently, WYCs were found to induce chromosomal breakage through oxidative stress (18,20), implicating a role for WYCs in interfering with DNA metabolism. To date, the biomolecular actions and implications of this
WYCs-mediated interference are mostly undetermined. Here, we found that WYCs are capable of inhibiting DNA damage-induced activation of ATR targets Chk1 and FANCD2, which then sensitize tumor cells to chemotherapy, and finally results in tumor size reduction in mice. Our results propose that these new flavonoid compounds are of noteworthy potential to treat cancers by inducing replication stress via the inhibition of ATR signaling.
Materials and methods
Cell culture and treatment. MDA-MB-231 (breast adenocarcinoma; ATCC HTB-26, BCRC 60425) and A549 (lung adenocarcinoma; ATCC CCL-185, BCRC 60074) human cell lines were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), and were authenticated by American Type Culture Collection (ATCC, Manassas, VA). U2OS (osteosarcoma), HeLa (cervical adenocarcinoma), and HEK 293T (embryonic kidney cells) human cell lines were provided by Dr. Sheau-Yann Shieh (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco). For DDR induction, freshly diluted H2O2 (Merck) was added to the culture medium 1 h before the cells were harvested. For UV irradiation treatment, the cells were irradiated for 10 J/m2 by a cross-linker (UVP) 1 h prior to analysis. WYC02 and WYC0209 were isolated and synthesized as described previously (15-17).
In vitro chemosensitization assay. To evaluate in vitro chemosensitization, cells were seeded in 6-well
plates 1 d before the experiment at a density of 100–400 cells/well. The drugs were incubated with the cells for 6 h, after which the medium was replaced with fresh drug-free FBS-containing medium. The
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colonies became visible and were counted 7–10 d later using 0.1% crystal violet staining following image capture by a CCD camera (LAS-4000 mini; Fujifilm).
Flow cytometry. To evaluate the effect of DNA damage checkpoint activation on cell cycle distribution, the cells were harvested at indicated time points and fixed with methanol for at least 2 h. The DNA was then stained with a solution containing propidium iodide (PI) and RNase A (Sigma-Aldrich).
Fluorescently labeled cells were subsequently analyzed by the flow cytometer (LSR II; BD Biosciences) with a suitable selection of excitation and emission wavelengths. The percentages of different
fluorescent cell populations were analyzed using WinMDI Ver. 2.9 (The Scripps Research Institute).
In vitro chromosome aberration test. In brief, 5 × 105 Chinese Hamster Ovary (CHO) cells were seeded in 60-mm dishes 1 d before the experiment. WYC02-induced structural chromosomal changes after 20 h were compared with that of the cells cultured in 2 μM MMC. At 18 h after WYC02, 0.1 μg/mL colchicine was added for 2 h, and metaphase cells were collected by shaking them off the dishes. Mitotic cells were treated with 0.5% KCl for 10 min and fixed with a 3:1 mixture of methanol: glacial acetic acid. The cells were then spread on slides for chromosome staining with 5% Giemsa solution. We then analyzed the chromosome structure of 200 well-spread metaphase cells (100 metaphase
cells/experiment) under a 100× oil immersion objective.
Plasmids and siRNAs. The plasmids ATR, ATRIP, and claspin were kindly provided by Dr. X. Wu (The Scripps Research Institute, La Jolla, CA), and TopBP1 was provided by Dr. J. Chen (University of Texas MD Anderson Cancer Center, Houston, Texas). The siRNA sequences of the target ATM
(5′-AAGCGCCTGATTCGAGATCCT-3′), ATR (5′-CCTCCGTGATGTTGCTTGATT-3′), DNA-PKcs (5′-GATCGCACCTTACTCTGTTGA-3′), and the random sequence that served as the control
(5′-AAGTCAATATGCGACTGATGG-3′) were synthesized by Sigma-Proligo (23,24). All transfections in HEK293T cells were performed by the calcium phosphate precipitation method.
Western immunoblotting. Cell lysate preparations, gel electrophoresis, and immunoblotting were performed as previously described (23). The binding of primary antibodies were detected by horseradish peroxidase-coupled secondary antibodies (Jackson ImmunoResearch) followed by enhanced chemiluminescence (ECL)(Millipore). The images of non-saturated bands were captured
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using a luminescent image analyzer (LAS-4000 mini; Fujifilm). The antibodies used in this study are listed in supplementary materials.
DNA homologous recombination repair assay. DNA constructs of the recombination substrate
pHPRT-DRGFP, in which the I-SceI site lies within 1 copy of 2 mutated tandem repeated GFP genes, and the I-SceI endonuclease expression vector pCBASceI, were originally constructed by Dr. M. Jasin (25). In brief, we generated a stable pHPRT-DRGFP construct in HeLa cells, and evaluated the chromosomal breaks generated by I-SceI endonuclease expression. Six hours after pCBASceI was delivered into the cells, complete medium with or without WYC02 or WYC0209 was replaced onto the cells. Forty-eight hours after delivery, the efficiency of chromosomal HRR was obtained as the percentage of
GFP-positive cells, which was assessed by flow cytometry.
Human xenograft tumors in nude mice. Human breast cancer MAD-MB-231 cells were harvested from the culture, resuspended in medium without serum at 1 × 108 cells/mL, and 0.1 mL of this suspension was subcutaneously injected into the right flank of female nude mice (BALB/cAnN-Foxn1nu/Crl Narl; purchased from the National Science Council Animal Center, Taiwan). Tumor-injected mice that successfully developed tumors that grew to approximately 50–100 mm3 in volume were randomly sorted into groups and used for the experiments. Control vehicle or 2 mg/kg of cisplatin with or without 0.2 mg/kg of WYC0209 was administered intraperitoneally every 4 d throughout the experiment. The drug formulations are described in supplementary materials.
Results
WYC02 induces chromosomal aberrations but does not produce marked DDR. Previously, WYC02 and WYC0209 were demonstrated to cause DNA strand breaks and apoptosis in lung and prostate cancers (18,20), suggesting that inducing DNA damage may be the potential mechanism underlying the anticancer effect of WYCs. To test this hypothesis, we investigated the cytogenetic effect of WYC02 on CHO cells (Fig. 1B). Low WYC02 concentrations produced dose-dependent increases in chromosomal structural changes, such as breakages, radials, and chromosomal polyploidy, similar to the effects seen with mitomycin C (MMC) treatment; however, the complete mitotic chromosome could not be
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obtained upon high-dose WYC02 treatment. Since MMC can induce DDR in many cancers, we
investigated what kind of DDR signaling was activated by WYC02. Surprisingly, high doses of WYC02 in HEK293T cells did not induce noticeable changes in the putative DDR signaling, which we measured by analyzing the phosphorylation of the ATM-dependent Chk2 Thr68 residue and the ATR-dependent Chk1 Ser345 residue (Fig. 1C). We did observe that WYC02 treatment caused slight accumulation of the p53 protein, which could have been the result of several posttranslational modifications. However, phosphorylation of the p53 Ser15 residue did not contribute to this WYC02-induced p53 protein accumulation, suggesting that WYC02 does not directly damage DNA because DNA damage normally stimulates ATM/ATR-dependent p53 Ser15 phosphorylation. Our result is similar to previous reports that p38 MAPK is activated by WYC02 (19,21,22), as its downstream target MAPKAPK2 was found to be phosphorylated starting as early as 2 h after WYC02 exposure (Fig. 1C). We repeated the WYCs experiment on lung and breast carcinoma cell lines A549 and MDA-MB-231 cells, respectively, and obtained similar results. Consistently, no marked changes in Chk1 and Chk2 phosphorylation signaling were detected even at high doses of either drug for as long as 8 h after drug treatment (Supplementary Fig. S1A).The cytotoxic effect by WYCs on cancer cells was determined by MTT assay at 48 h of
incubation (Supplementary Fig. S1B); our data indicated that the IC50 value range for cytotoxicity was similar to those in previous reports, confirming that WYCs are stable compounds that do not directly cause DNA damage.
WYC02 and WYC0209 inhibit Chk1 phosphorylation after DNA damage. Understanding the
mechanism by which the WYCs compounds cause chromosomal breakages and other abnormalities might aid in identifying their targets. We hypothesized that genes with functions associated with DNA damage checkpoints and/or DNA repair might be targeted by WYCs. To test this hypothesis, we assessed the effects of WYCs on DDR induced by H2O2. WYC02 was found to inhibit Chk1, but promote Chk2 phosphorylation in A594 cells treated with 0.1 mM H2O2 for 2 h; however, ATM
autophosphorylation was not affected (Fig. 2A).Pretreatment of cells with okadaic acid (OA) (a phosphatase inhibitor) or MG132 (a proteasome inhibitor) could not reverse the WYC02-induced inhibition of Chk1 phosphorylation, indicating that the inhibition does not occur due to phosphatase
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activation or proteasome degradation by other regulatory factors (Fig. 2B). Further, we investigated other sources of DNA stimuli specific for ATR activation; our results demonstrate that UV-induced Chk1 phosphorylation was dose-dependently inhibited by WYCs within different cells (Fig. 2C). In response to DNA double-strand breaks (DSBs), FANCD2 is known to be monoubiquitinated on K561 (FANCD2-Ub) in an ATR-dependent manner to stimulate repair (26,27). We showed that FANCD2-Ub was also inhibited by WYCs (Fig. 2A and C); further, ATR inhibition by WYCs was also observed in cells treated with currently prescribed chemotherapeutic agents (Supplementary Fig. S2). Collectively, these findings indicate that WYCs can modify ATR signaling after various types of DNA damage. Interestingly, WYC0209 was more potent than WYC02 in inhibiting Chk1 phosphorylation and cytotoxicity (Fig. 2C and Supplementary Fig. S1B). We speculate that the replacement of 2 hydroxyl groups on WYC02 with an additional benzene ring contributes positively to this ATR inhibition; however, the definite
pharmacophores need to be further investigated when the ATR protein structure is resolved. Target specificity of WYC02 and WYC0209 for ATR-mediated signaling inhibition. To elucidate the specificity of the WYC inhibition on ATR-mediated signaling, we compared the change between cells treated with WYCs or the ATM-specific inhibitor KU55933 (Fig. 1A) before the induction of DDR. After H2O2 damage, ATM is thought to be the principal responder, and KU55933 treatment strongly inhibited ATM-mediated Chk2 phosphorylation specifically, but its effect on ATR-mediated Chk1 phosphorylation was small (Fig. 3A Upper). In contrast, after hydroxyurea (HU; a replication blocker) damage, ATR is thought to be the principal responder, and WYCs treatment significantly inhibited Chk1
phosphorylation, but only slightly inhibited Chk2 phosphorylation (Fig. 3A Lower). Using these
pharmacological methods, we demonstrated that the specificity of DDR inhibition between WYCs and KU55933 was completely different. To strengthen the argument that WYCs specifically inhibits ATR signaling, small inhibitory RNAs against ATM, ATR, and the catalytic subunit of DNA protein kinase (DNA-PKcs) were introduced into HEK293T cells before exposure to UV or H2O2. Our results
demonstrated that WYCs completely inhibited UV- or H2O2-induced Chk1 phosphorylation in a manner similar to siRNA knockdown of ATR, but not ATM or DNA-PKcs (Fig. 3B and Supplementary Fig. S3). The siRNAs against ATM and DNA-PKcs decreased the UV- or H2O2-induced Chk2 phosphorylation, which
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were not altered by the addition of WYC02, but were increased by WYC0209 treatment. Interestingly, neither siRNA targeted to ATM or ATR nor DNA-PKcs affected theWYC0209-mediated increase in Chk2 phosphorylation. Since a high dose of WYC0209 itself slightly induces Chk2 activation (Supplementary Fig. S1), the increased Chk2 phosphorylation was likely a synergistic effect due to DNA damage. To further identify the specific mediator that contributes to the effect of WYC02 on the initiation of ATR kinase activation, we tested whether TopBp1, ATRIP, and claspin were involved, as they have been identified as mediators of ATR kinase activation (28).Our results demonstrated that overexpression of ATRIP or TopBP1 did not reverse the inhibitory effect of WYC02 on Chk1 phosphorylation, whereas overexpression of claspin or ATR did (Fig. 3C), suggesting that WYC02 might affect the function of ATR or claspin contributes to ATR signaling inhibition.
WYC02 and WYC0209 impair the functions of DNA damage checkpoints and DNA repair. Previously, it has been demonstrated that S/M and G2/M checkpoints are activated by ATR in response to different types of DNA damage (6,7). Of these, the G2/M checkpoint involves ATM and ATR in collaboration, whereas the S/M checkpoint is mediated solely by ATR. To maintain genetic integrity, ATR can prevent premature mitotic entry in the event of incomplete DNA replication or unrepaired DNA damage(6). In order to evaluate the effect of WYCs on these ATR-associated DNA damage checkpoints, we observed the effect of WYCs on mitotic entry following HU or cisplatin treatment. In MDA-MB-231 cells, HU and cisplatin significantly decreased the number of mitotic cells, indicating that the S/M and G2/M
checkpoints are intact in MDA-MB-231 cells (Fig. 4A). WYCs or KU55933 treatment increased the percentage of mitotic cells in cisplatin-treated cells, suggesting that all of these compounds inhibited the damage-induced G2/M checkpoint. However, WYCs, but not KU55933, significantly increased the HU-induced mitotic entry that is specific for the S/M checkpoint, indicating that WYCs specifically impaired this distinctive checkpoint mediated solely by ATR (Fig. 4A).
ATR function is also linked to DNA repair via its coupled targets (29). To examine the effect of WYCs treatment on DNA repair, we performed a homologous recombination repair (HRR) assay in HeLa cells. Our result demonstrated that chromosomal breaks normally repaired by HRR were dose-dependently inhibited by WYC02 at low concentrations. WYC0209 produced similar effects at doses that were
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10-fold lower than that of WYC02 (Fig. 4B). From these results, we assumed that the cells carrying unrepaired DNA would enter into mitosis following DNA damage. To verify this assumption, we analyzed the DNA-damage marker gamma-H2AX on mitotic cells using immunofluorescence staining. As expected, the numbers of large gamma-H2AX foci were increased upon addition of WYCs in both unperturbed and perturbed mitotic cells (Fig. 4C and Supplementary Fig. S8), suggesting that WYCs increase DNA damage in mitotic cells. The chromosomes became flat and aggregated after WYC treatment, differing from the three-dimensional and hair-like appearance of normal chromosomes at metaphase.
WYC02 and WYC0209 enhance chemosensitivity. Inhibition of the checkpoint and repair mechanisms leads to chemosensitization in cancers. We questioned whether WYCs could function as sensitizers for the chemotherapeutic drug cisplatin that has been shown to induce ATR activation as well as FANCD2 monoubiquitination, which is the vital step for DNA crosslink repair (26,30,31). We found that WYCs treatment decreased the cisplatin-induced Chk1 phosphorylation and FANCD2 monoubiquitination in A549, MDA-MB-231, and U2OS cells (Fig. 5A). Using the same doses, WYC0209 not only inhibits monoubiquitination of FANCD2 but also affects FANCD2 protein stability; this data emphasizes that WYC0209 has more potent inhibitory effects as compared to WYC02. We further treated individual cells with cisplatin in combination with several varying doses of WYCs, and counted survival colonies to determine their ability to survive after cisplatin-induced damage. Our results demonstrated that WYCs effectively decreased the clonogenic survival in cisplatin-treated MDA-MB-231 and A549 cells in the nanomolar dose range (Fig. 5B). To investigate the chemosensitization effect of low-dose WYCs in vivo, we established a tumor xenograft in nude mice using human MDA-MB-231 tumor cells, which are considered to be more resistant to cisplatin and are also sensitive to treatment with WYCs, at least as compared to A549 cells (Supplementary Fig. S1B and S4). When the mice were treated with 0.2 mg/kg WYC0209 in combination with 2 mg/kg cisplatin, the tumor inhibitory effect was greater than that of cisplatin treatment alone (Fig. 5C). However, WYC02 unexpectedly did not affect the cisplatin sensitivity of MDA-MB-231 tumors when a higher dose of 2 mg/kg was used in our experiments (data not shown). The pharmacokinetic data of WYC02 and WYC0209 needs to be compared in future studies to
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determine the differences in the chemical effects of these 2 compounds in vitro and in vivo.
Discussion
Many chemotherapeutic agents kill cancers by interfering with DNA metabolism and lethally damaging the DNA. ATM and ATR are defined as the key checkpoint sensors in the DDR. Prior to the recent discovery of ATR inhibitors (10-14), ATM and Chk1/2 inhibitors were proven to efficiently enhance the sensitivity of cancers to IR therapy or chemical agents (8). Since the effects of flavonoids on ATR signaling have not yet been investigated, we consider the findings of our study to be novel. The synthetic derivative of WYC02, WYC0209, was found to have similar, yet more potent, inhibitory effects on ATR signaling that further validate our findings.
Low doses of WYCs significantly slowed cancer growth, and caused S phase delay and inhibition of DNA synthesis (Supplementary Fig. S5); these events are similar to previously reported ATR defects (32). WYCs treatment exhibited selective inhibition of ATR signaling, which we determined experimentally by using different types of DNA-damage inducers. We clearly indicated that KU55933 specifically inhibits the ATM-mediated phosphorylation of Chk2, and WYCs specifically inhibit the ATR-mediated phosphorylation of Chk1 (Fig. 2A and 3A). Since the ATM activation that normally follows
replication-fork stalling or UV-induced irradiation damage depends on ATR kinase activity (33), the WYCs-mediated reduction in ATM activation upon HU, but not H2O2,treatment might result from ATR kinase dysfunction (Fig. 2A and 3A). This specificity was also supported by our observation that the WYCs-mediated inhibition of DNA damage-induced Chk1 phosphorylation was dependent on ATR but not on ATM and DNA-PKcs (Fig. 3B and Supplementary Fig. S3). Furthermore, radial changes in chromosome structure are characteristics of cells derived from FA patients but not from patients with AT or AT-like disease (34,35); therefore, the WYC02-mediated inhibition of ATR-dependent FANCD2 activation links this effect to radial changes in chromosome structure from FA patients (Fig. 1B). UV-induced Chk1 phosphorylation can be restored by overexpression of claspin and ATR but not TopBp1 or ATRIP (Fig. 3C). These data suggest that WYC02 might block the function of ATR kinase itself or the ATR-claspin-Chk1 interactions that then affect Chk1 activation. However, we could not exclude
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the possibility that WYCs target other mediators involved in ATR kinase activation. For example, FANCM has been shown to be necessary for both ATR activation and FANCD2 monoubiquitination (36).It remains unclear as to whether WYCs actually inhibit the ATR kinase enzyme activity or just abort the process of activation, and further investigations are required to clarify the precise mechanism(s) of action by WYCs. We screened a human kinome set for WYC02 involving 58 diverse in vitro kinase assays (Kinome Diversity Screen; MDS Pharma Services), and found that only PLK1 and SGK2 kinases were moderately inhibited by a high dose of WYC02 (Supplementary Fig. S6); therefore, it is clear that WYC02 does not target any of the kinases tested in this assay.
Our results supported 2 possible mechanisms by which WYCs could enhance cancer cell sensitivity to cisplatin: first, inhibition of the ATR checkpoint could lead cells that carry erroneous DNA into the next generation; second, inhibition of the FA repair pathway could result in elevated DNA DSB formation and error accumulation. We showed that WYCs compromise the cisplatin-induced checkpoint and lead to DSB accumulation within mitotic cells (Fig. 4A and C), suggesting that mitotic catastrophe might be induced in WYC-treated cells, whose DNA replication was not completed before entering into mitosis (37). While ATM inhibition by KU55933 treatment rapidly accumulates damage in the late-S and G2- phase cells rather than in the M-phase cells (38), and induces cell death via apoptosis (39), WYC02 treatment mainly causes cells to undergo necrosis-like death rather than apoptosis in our system (Supplementary Fig. S7). These results indicate that the inhibition of ATM or ATR differentially cause accumulation of DNA lesions in cells undergoing diverse fates via different death pathways.
Previous data suggests that ATR and ATM play different roles in preventing DSB formation through HRR (40). ATR and its targets, such as Chk1, FANCD2, and WRN, are required to repair cells damaged during the S-phase via HRR (26,29,40,41). We found that low doses of WYCs almost completely inhibited the HRR-mediated repair of chromosomal breaks, even in cells that possessed ATM function (Fig. 4B). Although ATM-deficient cells did not show reduced frequency of HRR to repair introduced DSBs (42), ATM-dependent FANCD2 Ser222 phosphorylation is required for the intra-S checkpoint, whereas the ATR-dependent FANCD2 Lys561 monoubiquitination is necessary for HRR (41). In the specific case of recovering from a stalled replication fork, we propose that ATM and ATR collaborate to induce
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checkpoint activation and recruit repair proteins for the initial step of HRR, whereas ATR signaling perhaps plays an indispensable role in the entire HRR process.
Our data revealed that WYCs could not only modify cancer cell responses to cisplatin but also to other therapeutic agents that induce Chk1 phosphorylation (Fig.3A, 5A, and Supplementary Fig. S2),
suggesting that WYCs may potentially function as cancer cell sensitizers for chemotherapeutic agents that activate the ATR checkpoint. Inhibition of DNA damage checkpoints and repair factors, such as ATM and BRCA1, are associated with promoting carcinogenesis as a consequence of genomic instability (43).However, there is no known cancer associated with ATR gene alterations. Specific ATR inhibition by expression of kinase-inactive ATR specifically in the skin prevents UV-induced skin carcinogenesis (44), suggesting that ATR inhibition has chemopreventive effects on non-transformed cells. Conversely, ATR has been proposed to play a critical role in directing cell death by senescence in unperturbed cells, where ATR is activated by overexpression of the oncoprotein Mos or the ATR activator TopBP1,
respectively (45,46). One plausible explanation proposed by Kawasumi et al. is that chemoprevention by ATR inhibition only works on precancerous cells whose oncogenes are not yet activated (44). In DNA-perturbed cells, the difference in the lesions induced by various DNA-damaging agents could also explain the different consequences of ATR/Chk1 inhibition, such as whether this inhibition promotes or suppresses apoptosis (47).However, while ATR activation clearly protects cells from death via
suppression of apoptosis when DNA damage is caused by oncogene-induced replication stress or DNA crosslinking, limited ATR activity selectively kills these cells (48-50), probably mediated by an
ATM-dependent mitotic catastrophe (51).Taken together, the genetic and pharmacological evidence suggests that ATR inhibition potentially plays a dual role by achieving both chemoprevention and chemotherapy. Given that the efficiency of ATR inhibition causes the opposite effect of synthetic lethality or tumorigenesis on Ras-transformed cells (52), inhibition of ATR function for anticancer therapy should be carefully evaluated by considering the genetic background and responsiveness of the tumor to this type of therapy; furthermore, the efficiency of ATR inhibition should be cautiously
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Acknowledgments
We are grateful to Dr. Chen-Yang Shen and Dr. Sheau-Yann Shieh for their support in providing some of their research material for this work and for their critical review of the manuscript.
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Treatment
Concentration
Type of structural aberrations (numbers) Average aberrant
(μM)
TB
TD TR
QR R CR DC PP PC
metaphases (%)
a
DMSO control
0.00
0
0
0
0 0 0 0
1
0
0.5
WYC02
2.17 2
0
4
3
1
0
0
3
0 6.5*
WYC02
4.35 1
1
13
9
2
0
0
1
1 14*
Mitomycin C
2.00 2
3
42
29
0
2
1
0
5 42*
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Table and Figure Legends
Table 1. WYC02 induces chromosomal aberration in CHO cells. Two hundred cells per treatment were analysized for chromosomal aberration. The total numbers of chromatid break (TB), chromatid deletion (TD), triradial (TR), quadriradial (QR), ring (R), complex rerrangement (CR), dicentric (DC), polyploid (PP) and pulverized cell (PC) were indicated. Others chromosome gap, chromosome break, chromosome deletion and chromatid gap were not be observed in this experiment. a, * indicated statistic significantly for tested vs. control group by t-test.
Figure 1. WYC02 induces chromosomal aberration with no marked DDR. A. Illustration of the chemical structure of selective ATM inhibitor KU55933, WYC02 and its derivative WYC0209 B. Mitotic spreads were prepared from WYC02-treated CHO cells at the indicated concentrations. The representative architecture of normal and aberrant chromosomes is shown. The numbers for different types of aberrations were counted from 200 mitotic cells in 2 independent slides is shown in Table 1. C. Immunoblots showing DDR by detecting phosphorylation of ATM Ser1981 (ATM-S1981p), Chk1 Ser345 (Chk1-S345p), Chk2 Thr68 (Chk2-T68p), P53 Ser15 (P53-S15p), P38MAPK Thr180/Tyr182
(P38-T180p/Y182p), or MAPKAPK2 Thr334 (MK2-T334p) following exposure of HEK293T cells to 10 μM WYC02 for the indicated times. Cells treated with 0.1 mM H2O2 for 30 min served as the positive control.
Figure 2. WYCs inhibit DNA damage-induced Chk1 phosphorylation. A. Immunoblots showing DDR by detecting phosphorylation of ATM, Chk1, Chk2, and monoubiquitination of FANCD2 following exposure of A549 cells to 0.1 mM H2O2 for 2 h with or without pre-treatment of the indicated doses of WYC02. B. A549 cells pretreated with 8 μM WYC02 combined with 50 μM okadaic acid (OA) or 20 μM MG132 for 30 min and subjected to 10 J/m2 UV for 1 h to induce DDR. C. Dose-dependent effect of WYC02 (left) and WYC0209 (right) on the inhibition of UV-induced Chk1 phosphorylation in A549 (upper) and
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MDA-MB-231 (lower) cells. Cells were pretreated with chemicals for 30 min and subjected to 10 J/m2 UV for 1 h to induce DDR.
Figure 3. WYCs specifically inhibit ATR-dependent Chk1 phosphorylation. A. DDR induced by either 0.1 mM H2O2 (upper) or 2 mM HU (lower) for 1 h in MDA-MB-231 cells pretreated with DMSO, 8 μM WYC02, 2 μM WYC0209, and 10 μM KU55933 for 30 min. B. Effects of WYC0209 on Chk1 and Chk2 phosphorylation were assayed 1 h after 10 J/m2 UV irradiation on HEK293T cells, and this exhibited decreased expression of ATM, ATR, or DNA-PKcs following the RNA interference method for 48 h. C. Effects of WYC02 on Chk1 phosphorylation were assayed 1 h after 10 J/m2 UV irradiation on HEK293T cells overexpressing ATRIP, TopBP1, claspin, or ATR following delivery of tagged full-length cDNA constructs for 48 h.
Figure 4. WYCs inhibit DNA damage checkpoint and repair. A. FACS dot-blot analysis for mitotic markers. HU (1 mM) or cisplatin (80 μM) was incubated with or without 10 μM KU55933, 4 μM WYC02, or 0.4 μM WYC0209 for 16 h in presence of 70 nM nocodazole. Cells were stained with phosphohistone H3 and PI, and the percentages of the mitotic marker for diploid cells was analyzed. B.FACS dot blot for analyzing the percentage of GFP cells denoting the HRR frequency, as described in Materials and
Methods. C. Immunohistochemical staining for the mitotic marker phosphohistone H3 (red), DNA
damage marker gamma-H2AX (green), and nucleus DAPI (blue) of cultured MDA-MB-231 cells, was conducted using the same experimental design as in A. Microscopic evaluation was carried out using fluorescence microscopy with a 100× oil immersion objective(Axiovert40; Zeiss). The yellow arrow indicates co-localization of the 3 markers in the same location, indicating a mitotic cell. The quantification result is shown in Supplementary Fig. S8.
Figure 5. WYCs enhance sensitivity of cancer cells to cisplatin. A. Effect of WYCs on cisplatin-induced DDR in A594, U2OS, and MDA-MB-231 cells. Cisplatin(5 μM) was incubated with or without 4 or 8 μM of WYC02 or WYC0209 for 4 h. Whole-cell lysates were subjected to immunoblot analyses for
23
phosphorylation of Chk1 and FANCD2. B. In vitro clonogenic survival for A549 and MDA-MB-231 cells. Cells were incubated with the indicated doses of cisplatin and WYC02 or WYC0209 for 6 h. After drug exposure, medium was replaced with drug-free medium for an additional 7–10 d. C. Effect of WYC0209 on MDA-MB-231 xenograft tumor in vivo. Mice with developed tumors were randomly sorted into 4 groups (n = 8–11per group), and received intraperitoneal injections of control vehicle or 2 mg/kg cisplatin combined with or without 0.2 mg/kg WYC0209 every 4 d throughout the experimental period. The width and length of the tumor were measured 3 times per week, and the volume was calculated as (width)2× length/2.
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