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NR1D1 enhances oxidative DNA damage by inhibiting PARP1 activity
Accepted Manuscript NR1D1 enhances oxidative DNA damage by inhibiting PARP1 activity Na-Lee Ka, Tae-Young Na, Mi-Ock Lee PII:
S0303-7207(17)30331-3
DOI:
10.1016/j.mce.2017.06.004
Reference:
MCE 9976
To appear in:
Molecular and Cellular Endocrinology
Received Date: 27 February 2017 Revised Date:
27 May 2017
Accepted Date: 4 June 2017
Please cite this article as: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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NR1D1 enhances oxidative DNA damage by inhibiting PARP1 activity
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Na-Lee Ka, Tae-Young Na, and Mi-Ock Lee
College of Pharmacy and Bio-MAX Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
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Seoul 08826, Korea
Corresponding Author: Mi-Ock Lee, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
Seoul
08826,
Korea.
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mail:[email protected]
Phone:
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The authors declare no potential conflicts of interest.
1
82-2-880-9331;
Fax:
82-2-887-2692;
E-
ACCEPTED MANUSCRIPT ABSTRACT
Cancer cells exhibit an elevated intracellular level of reactive oxygen species (ROS) because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit. The oxidative stress in
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cancer cells may provide clinical benefits, which can be associated with a better response to anticancer therapies. Therefore, identifying the regulatory pathway of oxidative stress in cancer cells is important in the development of therapeutic targets that enhance sensitivity to ROS-generating
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anticancer therapies. Here, we report that nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) inhibited DNA repair of ROS-induced DNA damage in breast cancer cells. NR1D1
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interacted with poly(ADP-ribose) polymerase 1 (PARP1) and subsequently inhibited catalytic activity of PARP1. NR1D1 enhanced accumulation of DNA damage, which increased sensitivity of breast cancer cells to oxidative stress. Our findings suggest that NR1D1 could be a therapeutic target for breast cancer treatment, especially in those patients treated with ROS-inducing chemotherapeutic
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Keywords: NR1D1; PARP1; DNA damage; DNA repair; breast cancer
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ACCEPTED MANUSCRIPT 1. Introduction
Breast cancer is the most commonly diagnosed cancer in women worldwide (Ferlay et al., 2015). Cancer cells produce higher levels of intracellular reactive oxygen species (ROS) than their normal
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counterparts, because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit (Trachootham et al., 2009). ROS can cause DNA damage, which may trigger DNA repair to eliminate the ROS-mediated DNA damage to protect genome integrity. However, ROS tend to induce
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loss of p53 function, resulting in a defect in DNA repair that leads to accumulation of DNA damage that would promote cancer development and progression (Gorrini et al., 2013; Trachootham et al.,
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2009). Meanwhile, recent studies suggest that this vicious cycle of ROS stress in cancer cells could provide therapeutic benefits, because high levels of ROS could lead to growth arrest, senescence, and cell death, which could eliminate cancer cells by radiation or pharmacological ROS stimuli (Nogueira and Hay, 2013). Therefore, the mechanism elucidating the regulatory pathway of oxidative stress in
therapies.
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cancer cells could provide a tool that modulates both cancer development and response to anticancer
Accumulation of ROS can lead to multiple DNA lesions, including oxidized purines and pyrimidines, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) (Kryston et al., 2011).
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In response to the oxidative stress-induced DNA damage, cells have evolved a variety of DNA repair
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mechanisms, such as base excision repair/single-strand break repair (BER/SSBR), nucleotide excision repair, and mismatch repair pathways. The BER/SSBR pathway is the main repair pathway for repair of SSBs that arises directly from ROS-induced disintegration of deoxyribose or that transiently formed during the process of DNA repair (Hegde et al., 2012). During the BER/SSBR process, poly(ADP-ribose) polymerase 1 (PARP1) plays a critical role. PARP1 detects SSBs, and subsequently triggers massive synthesis of poly(ADP-ribose) (PAR) polymers that modify this polymerase and other DNA damage response (DDR) factors (Wei and Yu, 2016). The PARylation allows the DDR factors to be recruited to the site of DNA damage, which is one of the most important steps for efficient DNA repair (Woodhouse and Dianov, 2008). Therefore, the enzymatic 3
ACCEPTED MANUSCRIPT activity of PARP1 would affect the BER capacity that modulates the sensitivity of cells to oxidative stress-induced DNA damage. Nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) is a member of the nuclear receptor family that links circadian rhythms to metabolic homeostasis (Everett and Lazar,
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2014). The NR1D1 gene is located in the ERBB2 amplicon (17q12–q21), a predictor of aggressive tumor phenotype, and the expression level of this receptor is correlated with poor clinical outcome (Chin et al., 2006; Davis et al., 2007). Recently, we reported a novel function of NR1D1 in DNA
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repair, which enhanced sensitivity to doxorubicin-induced cell death in breast cancer cells. NR1D1 inhibited both nonhomologous end-joining and homologous recombination double-strand break
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(DSB) repair, which is probably linked to the enhanced chemosensitivity in breast cancer cells (Ka et al., 2017). NR1D1 was upregulated in response to oxidative stress, which suggests a potential involvement of NR1D1 in the oxidative DNA damage (Yang et al., 2014). Therefore, in this study, we investigated the role of NR1D1 in DNA repair pathways that respond to oxidative stress-induced
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DNA damage.
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ACCEPTED MANUSCRIPT 2. Materials and Methods
2.1. Cell culture and reagents MCF7, a human breast cancer cell line, was obtained from the American Type Culture Collection.
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The MCF7 stable cell lines expressing shNR1D1, or NR1D1, were described previously (Ka et al., 2017). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The cells were grown in an incubator with 5% CO2/95% air at 37°C. Hydrogen
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peroxide (H2O2), paraquat, GSK4112, 4',6-diamidino-2-phenylindole (DAPI), and 3-(4,5-
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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Merck.
2.2. Plasmids, siRNA duplexes, and transient transfection
The eukaryotic expression vector for p3XFLAG10-NR1D1 was described previously (Ka et al., 2017). The siRNA duplex targeting human NR1D1 (5’-UCGGAGCAUCCAGCAGAACAU-3’), and
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the non-specific si-RNA were synthesized by ST Pharm. Transient transfection of plasmids and siRNA was performed as described previously (Kang et al., 2014).
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2.3. Western blotting, immunoprecipitation, and immunofluorescence Western blotting was performed as described previously using specific antibodies against NR1D1
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(#14506-1-AP; Proteintech), PARP1 (#1561; Santa Cruz), FLAG (#F3165; Merck), PAR (#551813; BD Biosciences), or Actin (#1616; Santa Cruz) (Ka et al., 2017). For immunoprecipitation, PARP1 antibody (#1561; Santa Cruz) or normal goat IgG (#2028; Santa Cruz) was incubated with protein G beads (Millipore) at 4°C overnight. And then cross-linking reactions were performed with 20 mM dimethylpimelimidate in 0.2 M sodium tetraborate, pH 9.0, for 30 min at room temperature. The reactions were stopped with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. Cells were harvested and lysed in a lysis buffer (20 mM HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.2 % NP40, and protease inhibitor cocktail (Roche)) and then incubated with antibody-crosslinked beads in
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ACCEPTED MANUSCRIPT the lysis buffer overnight. The immunoprecipitated beads were washed three times with the lysis buffer, and eluted with 2x SDS sample buffer by boiling for 10 min. For immunofluorescence staining, cells were fixed and stained with the γH2AX antibody (1:100) (#ab22551; Abcam) or PAR antibody (#71848; Santa Cruz). Nuclei were stained by DAPI. Images were acquired using a Zeiss
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LSM 700 confocal microscope (Carl Zeiss).
2.4. PARP enzymatic activity assay
2.5. Analysis of oxidative DNA damage
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K; Trevigen) according to the manufacturer’s instruction.
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PARP enzymatic activity was measured using the universal colorimetric PARP assay kit (#4677-096-
The alkaline comet assay was performed using a CometAssay kit (Trevigen) according to the manufacturer’s protocol. Samples were stained with SYBR green and visualized using a fluorescence
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microscope (Olympus IX71). The tail moment was quantified based on 100–150 randomly selected cells using the Comet Assay IV CometScore software (Perceptive Instruments Ltd). To quantify the oxidative DNA damage, the amount of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a marker of
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oxidative DNA damage, was quantified using an Oxiselect Oxidative DNA Damage ELISA Kit
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(#STA-320; Cell Biolabs).
2.6. Measurement of cell survival For measurement of clonogenic survival ability, cells were seeded in 35-mm plates and then treated with H2O2 for 3 days. At the end of 11 days of incubation, colonies were fixed with methanol, and stained with 0.5% crystal violet. Colonies that composed of more than 50 cells were counted. For measurement of cell growth, cells were seeded into 96-well plates (4000 cells/well) and treated with H2O2 or paraquat for 3 days. At the end of treatment, cells were continued for incubation with MTT for 4 h, and then resulting formazan crystals were dissolved in DMSO and optical density was
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ACCEPTED MANUSCRIPT measured at 570 nm with 750 nm as the reference wavelength.
2.7. Statistical analyses Statistical analyses were conducted using GraphPad Prism software. Experimental values were
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expressed as the mean ± S.D. of three independent experiments. Statistically significance between two groups was determined using the nonparametric Mann–Whitney U test. Statistical analyses of multiple groups were performed using two-way ANOVA followed by the Bonferroni posttest. P <
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0.05 was considered to be significant. Data shown as images of immunofluorescence, immunoblots, comet assay, and clonogenic survival assays were taken from a representative experiment, which was
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qualitatively similar to at least three independent experiments.
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ACCEPTED MANUSCRIPT 3. Results
3.1. NR1D1 inhibits the oxidative DNA damage-induced DNA repair in MCF7 breast cancer cells Previously, we reported that NR1D1 delayed DNA repair in response to doxorubicin-induced DSBs
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(Ka et al., 2017). To determine the effect of NR1D1 in response to oxidative DNA damage, we examined γH2AX foci clearance as an indicator of efficient DNA repair. Many reports have shown that γH2AX foci are formed under oxidative stress, such as H2O2 or paraquat treatment (Mao et al.,
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2011; Fortini et al., 2012). In agreement with the these reports, treatment with H2O2 increased the number of γH2AX foci in MCF7 cells, but there was rapid clearance of γH2AX foci after removing
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H2O2 from MCF7 cells (Fig. 1). However, γH2AX persisted longer when NR1D1 was transiently overexpressed (Fig. 1). These results show that NR1D1 inhibits the repair of DNA damage induced by oxidative stress.
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3.2. NR1D1 inhibits the enzymatic activity of PARP1 through the binding to PARP1 Because oxidative DNA damage is mainly repaired through the BER pathways, we hypothesized that NR1D1 could modulate the BER signaling component (Hegde et al., 2012). In the BER pathway,
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PARP1 plays a key role in the recognition of DNA strand breaks including intermediates of BER (Dianov and Hübscher, 2013). We found that NR1D1 physically interacted with PARP1 and that the
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interaction was further enhanced by H2O2 treatment (Fig. 2A and 2B). PARP1 becomes activated after binding to damaged DNA, synthesizing PAR chains that attached to itself and its target proteins, which results in the efficient recruitment of the DNA repair complex (Wei and Yu, 2016). Therefore, we explored whether NR1D1 affected the enzymatic activity of PARP1. MCF7 cells were exposed to H2O2, and PARylation status of intracellular proteins was assessed by western blotting. As shown in Fig. 2C, H2O2 treatment induced PARylation of proteins, but the level of PARylation was decreased in the presence of NR1D1. Next, we measured the PARP activity in the cell lysates using histone proteins as the substrate. The PARP1 activity was increased after the H2O2 treatment, but it
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ACCEPTED MANUSCRIPT significantly decreased when NR1D1 was overexpressed (Fig. 2D). Treatment of GSK4112, a ligand of NR1D1, also reduced the activity of PARP1 (Fig. 2E). Together, these results show that NR1D1 inhibits the enzymatic activity of PARP1 through binding to PARP1.
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3.3. NR1D1 enhances DNA damage and increases sensitivity of MCF7 cells to oxidative stress
Because defects in appropriate DNA repair can result in accumulation of DNA damage, we examined whether the activation of NR1D1, i.e., overexpression of NR1D1 or GSK4112 treatment, influenced
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oxidative stress-induced DNA damage in MCF7 cells. We performed comet assays to evaluate the degree of DNA damage. The tail moment, an indicator of DNA breakage, increased remarkably after
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treatment with H2O2. Transient transfection of NR1D1 or treatment with GSK4112 also increased the comet tail moment although to a lesser degree than H2O2. The tail moment increased dramatically when the cells were cotreated with H2O2 (Fig. 3A). Similar results were obtained when comet assays were performed with paraquat, a different oxidative stress inducer (Supplementary Fig. 1A).
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Overexpression of NR1D1 or GSK4112 treatment increased the level of 8-OHdG, the most common forms of oxidative DNA lesions, and it increased further by cotreatment with H2O2 (Fig. 3B) (Valavanidis et al., 2009). The GSK4112-induced 8-OHdG level decreased after knockdown of
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NR1D1, which indicates the involvement of NR1D1 in the GSK4112-induced DNA damage (Fig. 3B). To investigate further the effect of NR1D1 on oxidative DNA damage, we employed the MCF7
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sublines that stably expressing NR1D1 (Supplementary Fig. 1B). We found that H2O2-induced PAR formation was significantly lower in the NR1D1-overexpressing cells compared with that in the control cells (Fig. 3C). Because high intracellular ROS levels in cancer cells can lead to oxidative damage-driven cell death, we assessed whether NR1D1 makes cells sensitive to excessive ROS. NR1D1-overexpressing cells were more sensitive to H2O2 and paraquat compared with control cells (Fig. 3D and Supplementary Fig. 1C). Together, these data suggest that NR1D1 enhances oxidative DNA damage, which may increase the sensitivity of cells to oxidative stress.
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ACCEPTED MANUSCRIPT 3.4. Lack of NR1D1 decreases sensitivity to oxidative DNA damage To gain further insight into the role of NR1D1 in the sensitivity to oxidative DNA damage, we employed three stable MCF7 sublines expressing shNR1D1 (Ka at al., 2017). First, we found that PAR formation was increased significantly in the H2O2-treated shNR1D1 sublines than shGFP
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control cells (Supplementary Fig. 2A and 2B). Furthermore, the shNR1D1-MCF7 sublines were less sensitive to H2O2-induced clonal growth inhibition (Fig. 4A). Similarly, shNR1D1 was more resistant to paraquat-induced cell death when cell viability was measured using MTT assays (Fig. 4B).
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Together, these results indicated that NR1D1 enhanced the sensitivity of cancer cells to oxidative
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stress, probably by inhibiting the PARP1-mediated BER process.
4. Discussion
In the present investigation, we found that NR1D1 inhibits DNA repair caused by ROS-induced DNA
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damage in MCF7 cells. NR1D1 interacts with PARP1 under oxidative stress and thereby inhibits the catalytic activity of PARP1. Thus, NR1D1 may result in disruption of the proper DNA repair processes, including BER, which requires PAR-dependent recruitment of DDR factors to DNA
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damage sites. The disruption of DNA repair may subsequently lead to accumulation of oxidative DNA damage, which could enhance the sensitivity of cancer cells to oxidative stress-induced cell
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death.
Several chemotherapeutic agents used for treatment of breast cancer, such as taxanes,
platinum-based agents, and anthracyclines, induce ROS-induced cell death by promoting the generation of intracellular ROS (Conklin, 2004). Ionizing radiation also increases ROS production that leads to severe cellular damage and apoptosis (Azzam et al., 2012). Therefore, NR1D1 could enhance efficacy of such anticancer therapies by disrupting the ROS-associated DDR. Indeed, several public patient data sets show that NR1D1 expression correlates positively with clinical outcome in breast cancer patients who received chemotherapy. Further, NR1D1 and its ligand, GSK4112,
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ACCEPTED MANUSCRIPT increased the sensitivity of breast cancer cells to doxorubicin, probably by inhibiting DSB repair (Ka et al., 2017). Here, our data suggest that the mechanism of the chemosensitizing effect of NR1D1 may also involve, at least in part, its ability to increase ROS-induced DNA damage, because many chemotherapeutic drugs including doxorubicin generate high levels of ROS in tumor cells (Conklin,
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2004).
Recently, inhibiting PARP is considered as a promising therapeutic strategy for the treatment of breast cancer (Livraghi and Garber, 2015). As PARP is crucial in the cellular response to
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genotoxic stimuli including oxidative stress, PARP inhibition may sensitize tumor cells to chemotherapy-induced oxidative stress. Thus, combinations of a PARP inhibitor with chemotherapy
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or with radiation therapy have been under diverse clinical trials (Dréan et al., 2016). For example, veliparib in combination with carboplatin is under clinical trials for stage III–IV breast cancer susceptibility gene (BRCA)-associated breast cancer (phase 2; NCT01149083) and for human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (phase 1;
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NCT01251874). The combination of olaparib and radiotherapy is also being trialed for inoperable breast cancer (phase 1; NCT02227082) (https://clinicaltrials.gov/). Here, we speculate that specific ligands that activate NR1D1 and thereby inhibit PARP1 activity may exert the same sensitizing
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effects as PARP inhibitors when applied as combination therapy with chemotherapeutics or radiation. Together, our findings suggest that NR1D1 may provide a potential chemosensitizer to enhance the
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outcomes of adjuvant therapy that are associated with the generation of intracellular ROS. The facilitating role of NR1D1 in the oxidative stress-induced damage might be specific in
cancer cells. Sengupta et al. (2016) suggested that NR1D1 provides preconditioning for protection against oxidative stress by inducing antioxidant genes such as forkhead box protein O1 (FoxO1), superoxide dismutase 2 (SOD2; also known as MnSOD), heme oxygenase (decycling) 1 (Hmox-1), and catalase in lung fibroblasts. It was also shown that NR1D1 was upregulated in response to hyperoxia-induced oxidative stress in neonatal mouse lung fibroblasts (Yang et al., 2014). However, we observed that NR1D1 decreased the expression of SOD2 in MCF7 cells, which suggests that the
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ACCEPTED MANUSCRIPT NR1D1-induced suppression of ROS-scavenging activity of cancer cells may potentiate oxidative stress-induced cancer cell death (Supplementary Fig. 3). The causes of this discrepancy in the celltype-dependent regulation of antioxidant enzymes by NR1D1 are not currently clear; however, differences in regulation of cellular antioxidative systems in normal and cancer cells may be a reason
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(Cairns et al., 2011). Further studies are warranted to understand the context-dependent role of NR1D1 regarding regulation of cellular oxidative systems. In this regard, the role of NR1D1 in DNA repair during the development of breast cancer should be studied in future, because ROS are potential
Acknowledgment
This
work
was
supported
by
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factors that contribute to initiation, promotion, and progression of breast carcinogenesis.
grants,
the
NRF-2017R1A2B3011870
(M.L.),
NRF-
2014R1A2A1A10052265 (M.L.), and the NRF-2013H1A2A1033512 (N.K.) from the National
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Research Foundation of Korea.
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. NR1D1 inhibits DNA repair against ROS-induced DNA damage. (A) MCF7 cells were transfected with empty vector (EV) or FLAG-NR1D1 and then treated with 500 µM H2O2 for 30 min.
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Cells were fixed at the indicated recovery time (RT) points and immunostained with anti-γH2AX (green). Nuclei were visualized by DAPI staining (blue). (B) The number of γH2AX foci shown in panel (A) was quantified at the indicated RT from 50 cells. Values represent mean ± SD (n=3). **P <
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0.01, ***P < 0.001 vs EV at each time point. (C) Expression level of NR1D1 was measured by
the western blot were presented.
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western blotting and shown as control. Images of NR1D1 obtained after short and long exposure of
Figure 2. NR1D1 is associated physically with PARP1 and inhibits its activity. (A and B) MCF7 cells (A) or MCF7 cells that transfected with FLAG-NR1D1 (B) were treated with vehicle or 500 µM
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H2O2 for 5 min. Whole cell lysates were immunoprecipitated (IP) using IgG or anti-PARP1 and probed by western blot (WB) analysis. (C) MCF7 cells transfected with empty vector (-) or FLAGNR1D1 were treated with vehicle or 500 µM H2O2 for 5 min. Expression of the PARylated proteins,
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PARP1 and FLAG-NR1D1 was analyzed by western blotting. (D) MCF7 cells transfected with empty vector (-) or FLAG-NR1D1 were treated with vehicle or 500 µM H2O2 for 5 min. PARP1 activities in
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the cell lysates were measured by ELISA. *P < 0.05 vs no treatment; #P < 0.05 vs H2O2 treatment. (E) MCF7 cells were treated with 10 µM GSK4112 for 24 h. PARP1 activities in the cell lysates were measured by ELISA. *P < 0.05 vs no treatment.
Figure 3. NR1D1 further increases ROS-induced DNA damage. (A) MCF7 cells were transfected with empty vector (EV) or FLAG-NR1D1, or the cells were treated with 10 µM GSK4112 (GSK) for 24 h. Then, the cells were treated with vehicle or 500 µM H2O2 for 5 min and were subjected to comet assay (left). Tail moment was quantified from randomly selected 100-150 cells per image
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ACCEPTED MANUSCRIPT (right). Values represent mean ± SD (n=3). ***P < 0.001 vs no treatment;
###
P < 0.001 vs H2O2
treatment. (B) MCF7 cells were transfected with EV or FLAG-NR1D1, or treated with 10 µM GSK4112 for 24 h and then treated with 500 µM H2O2 for 5 min. The amount of 8-OHdG was measured by ELISA. Values represent mean ± SD (n=3). *P < 0.05 vs no treatment; #P < 0.05 vs
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H2O2 treatment (left). Cells were transfected with siGFP or siNR1D1 and then treated with 10 µM GSK4112 for 24 h. The amount of 8-OHdG was measured by ELISA. Values represent mean ± SD (n=3). *P < 0.05 vs siGFP only; #P < 0.05 as indicated (right). (C) MCF7 stable cell lines that
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expressing EV or NR1D1 were treated with 500 µM H2O2 for 30 min. Cells were fixed and immunostained with anti-PAR (green). Nuclei were visualized by DAPI staining (blue) (left). The
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percentage of PAR-positive cells was quantified from 100 cells (right). Values represent mean ± SD (n=3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs no treatment of each cells; ##P < 0.01 as indicated. (D) The MCF7 stable cell lines were exposed to increasing concentrations of H2O2 for 72 h. Relative cell viability was measured using MTT assay. Values represent mean ± SD (n=3). **P < 0.01, ***P <
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0.001 vs control cells.
Figure 4. Lack of NR1D1 decreases sensitivity to oxidative DNA damage in MCF7 cells. (A) The
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MCF7 stable cells expressing shGFP or shNR1D1 were exposed to H2O2 for 3 days, and then further incubated for another 11 days. Representative images obtained from three independent experiments
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are shown (top). Colonies that composed of more than 50 cells were counted. Values represent mean ± SD (n=3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs shGFP as indicated (bottom). (B) The MCF7 stable cells were exposed to increasing concentrations of paraquat for 72 h. Relative cell viability was measured using MTT assay. Values represent mean ± SD (n=3). ***P < 0.001 vs shGFP as indicated.
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ACCEPTED MANUSCRIPT NR1D1 inhibits the oxidative DNA damage-induced DNA repair in MCF7 cells. NR1D1 inhibits the enzymatic activity of PARP1 through the binding to PARP1. NR1D1 enhances cellular oxidative DNA damage.
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NR1D1 increases sensitivity of MCF7 cells to oxidative stress.
S0303-7207(17)30331-3
DOI:
10.1016/j.mce.2017.06.004
Reference:
MCE 9976
To appear in:
Molecular and Cellular Endocrinology
Received Date: 27 February 2017 Revised Date:
27 May 2017
Accepted Date: 4 June 2017
Please cite this article as: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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NR1D1 enhances oxidative DNA damage by inhibiting PARP1 activity
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Na-Lee Ka, Tae-Young Na, and Mi-Ock Lee
College of Pharmacy and Bio-MAX Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
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Seoul 08826, Korea
Corresponding Author: Mi-Ock Lee, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
Seoul
08826,
Korea.
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mail:[email protected]
Phone:
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The authors declare no potential conflicts of interest.
1
82-2-880-9331;
Fax:
82-2-887-2692;
E-
ACCEPTED MANUSCRIPT ABSTRACT
Cancer cells exhibit an elevated intracellular level of reactive oxygen species (ROS) because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit. The oxidative stress in
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cancer cells may provide clinical benefits, which can be associated with a better response to anticancer therapies. Therefore, identifying the regulatory pathway of oxidative stress in cancer cells is important in the development of therapeutic targets that enhance sensitivity to ROS-generating
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anticancer therapies. Here, we report that nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) inhibited DNA repair of ROS-induced DNA damage in breast cancer cells. NR1D1
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interacted with poly(ADP-ribose) polymerase 1 (PARP1) and subsequently inhibited catalytic activity of PARP1. NR1D1 enhanced accumulation of DNA damage, which increased sensitivity of breast cancer cells to oxidative stress. Our findings suggest that NR1D1 could be a therapeutic target for breast cancer treatment, especially in those patients treated with ROS-inducing chemotherapeutic
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Keywords: NR1D1; PARP1; DNA damage; DNA repair; breast cancer
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ACCEPTED MANUSCRIPT 1. Introduction
Breast cancer is the most commonly diagnosed cancer in women worldwide (Ferlay et al., 2015). Cancer cells produce higher levels of intracellular reactive oxygen species (ROS) than their normal
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counterparts, because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit (Trachootham et al., 2009). ROS can cause DNA damage, which may trigger DNA repair to eliminate the ROS-mediated DNA damage to protect genome integrity. However, ROS tend to induce
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loss of p53 function, resulting in a defect in DNA repair that leads to accumulation of DNA damage that would promote cancer development and progression (Gorrini et al., 2013; Trachootham et al.,
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2009). Meanwhile, recent studies suggest that this vicious cycle of ROS stress in cancer cells could provide therapeutic benefits, because high levels of ROS could lead to growth arrest, senescence, and cell death, which could eliminate cancer cells by radiation or pharmacological ROS stimuli (Nogueira and Hay, 2013). Therefore, the mechanism elucidating the regulatory pathway of oxidative stress in
therapies.
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cancer cells could provide a tool that modulates both cancer development and response to anticancer
Accumulation of ROS can lead to multiple DNA lesions, including oxidized purines and pyrimidines, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) (Kryston et al., 2011).
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In response to the oxidative stress-induced DNA damage, cells have evolved a variety of DNA repair
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mechanisms, such as base excision repair/single-strand break repair (BER/SSBR), nucleotide excision repair, and mismatch repair pathways. The BER/SSBR pathway is the main repair pathway for repair of SSBs that arises directly from ROS-induced disintegration of deoxyribose or that transiently formed during the process of DNA repair (Hegde et al., 2012). During the BER/SSBR process, poly(ADP-ribose) polymerase 1 (PARP1) plays a critical role. PARP1 detects SSBs, and subsequently triggers massive synthesis of poly(ADP-ribose) (PAR) polymers that modify this polymerase and other DNA damage response (DDR) factors (Wei and Yu, 2016). The PARylation allows the DDR factors to be recruited to the site of DNA damage, which is one of the most important steps for efficient DNA repair (Woodhouse and Dianov, 2008). Therefore, the enzymatic 3
ACCEPTED MANUSCRIPT activity of PARP1 would affect the BER capacity that modulates the sensitivity of cells to oxidative stress-induced DNA damage. Nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) is a member of the nuclear receptor family that links circadian rhythms to metabolic homeostasis (Everett and Lazar,
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2014). The NR1D1 gene is located in the ERBB2 amplicon (17q12–q21), a predictor of aggressive tumor phenotype, and the expression level of this receptor is correlated with poor clinical outcome (Chin et al., 2006; Davis et al., 2007). Recently, we reported a novel function of NR1D1 in DNA
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repair, which enhanced sensitivity to doxorubicin-induced cell death in breast cancer cells. NR1D1 inhibited both nonhomologous end-joining and homologous recombination double-strand break
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(DSB) repair, which is probably linked to the enhanced chemosensitivity in breast cancer cells (Ka et al., 2017). NR1D1 was upregulated in response to oxidative stress, which suggests a potential involvement of NR1D1 in the oxidative DNA damage (Yang et al., 2014). Therefore, in this study, we investigated the role of NR1D1 in DNA repair pathways that respond to oxidative stress-induced
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DNA damage.
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ACCEPTED MANUSCRIPT 2. Materials and Methods
2.1. Cell culture and reagents MCF7, a human breast cancer cell line, was obtained from the American Type Culture Collection.
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The MCF7 stable cell lines expressing shNR1D1, or NR1D1, were described previously (Ka et al., 2017). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The cells were grown in an incubator with 5% CO2/95% air at 37°C. Hydrogen
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peroxide (H2O2), paraquat, GSK4112, 4',6-diamidino-2-phenylindole (DAPI), and 3-(4,5-
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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Merck.
2.2. Plasmids, siRNA duplexes, and transient transfection
The eukaryotic expression vector for p3XFLAG10-NR1D1 was described previously (Ka et al., 2017). The siRNA duplex targeting human NR1D1 (5’-UCGGAGCAUCCAGCAGAACAU-3’), and
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the non-specific si-RNA were synthesized by ST Pharm. Transient transfection of plasmids and siRNA was performed as described previously (Kang et al., 2014).
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2.3. Western blotting, immunoprecipitation, and immunofluorescence Western blotting was performed as described previously using specific antibodies against NR1D1
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(#14506-1-AP; Proteintech), PARP1 (#1561; Santa Cruz), FLAG (#F3165; Merck), PAR (#551813; BD Biosciences), or Actin (#1616; Santa Cruz) (Ka et al., 2017). For immunoprecipitation, PARP1 antibody (#1561; Santa Cruz) or normal goat IgG (#2028; Santa Cruz) was incubated with protein G beads (Millipore) at 4°C overnight. And then cross-linking reactions were performed with 20 mM dimethylpimelimidate in 0.2 M sodium tetraborate, pH 9.0, for 30 min at room temperature. The reactions were stopped with 0.2 M ethanolamine, pH 8.0, for 2 h at room temperature. Cells were harvested and lysed in a lysis buffer (20 mM HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.2 % NP40, and protease inhibitor cocktail (Roche)) and then incubated with antibody-crosslinked beads in
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ACCEPTED MANUSCRIPT the lysis buffer overnight. The immunoprecipitated beads were washed three times with the lysis buffer, and eluted with 2x SDS sample buffer by boiling for 10 min. For immunofluorescence staining, cells were fixed and stained with the γH2AX antibody (1:100) (#ab22551; Abcam) or PAR antibody (#71848; Santa Cruz). Nuclei were stained by DAPI. Images were acquired using a Zeiss
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LSM 700 confocal microscope (Carl Zeiss).
2.4. PARP enzymatic activity assay
2.5. Analysis of oxidative DNA damage
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K; Trevigen) according to the manufacturer’s instruction.
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PARP enzymatic activity was measured using the universal colorimetric PARP assay kit (#4677-096-
The alkaline comet assay was performed using a CometAssay kit (Trevigen) according to the manufacturer’s protocol. Samples were stained with SYBR green and visualized using a fluorescence
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microscope (Olympus IX71). The tail moment was quantified based on 100–150 randomly selected cells using the Comet Assay IV CometScore software (Perceptive Instruments Ltd). To quantify the oxidative DNA damage, the amount of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a marker of
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oxidative DNA damage, was quantified using an Oxiselect Oxidative DNA Damage ELISA Kit
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(#STA-320; Cell Biolabs).
2.6. Measurement of cell survival For measurement of clonogenic survival ability, cells were seeded in 35-mm plates and then treated with H2O2 for 3 days. At the end of 11 days of incubation, colonies were fixed with methanol, and stained with 0.5% crystal violet. Colonies that composed of more than 50 cells were counted. For measurement of cell growth, cells were seeded into 96-well plates (4000 cells/well) and treated with H2O2 or paraquat for 3 days. At the end of treatment, cells were continued for incubation with MTT for 4 h, and then resulting formazan crystals were dissolved in DMSO and optical density was
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2.7. Statistical analyses Statistical analyses were conducted using GraphPad Prism software. Experimental values were
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expressed as the mean ± S.D. of three independent experiments. Statistically significance between two groups was determined using the nonparametric Mann–Whitney U test. Statistical analyses of multiple groups were performed using two-way ANOVA followed by the Bonferroni posttest. P <
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0.05 was considered to be significant. Data shown as images of immunofluorescence, immunoblots, comet assay, and clonogenic survival assays were taken from a representative experiment, which was
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qualitatively similar to at least three independent experiments.
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ACCEPTED MANUSCRIPT 3. Results
3.1. NR1D1 inhibits the oxidative DNA damage-induced DNA repair in MCF7 breast cancer cells Previously, we reported that NR1D1 delayed DNA repair in response to doxorubicin-induced DSBs
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(Ka et al., 2017). To determine the effect of NR1D1 in response to oxidative DNA damage, we examined γH2AX foci clearance as an indicator of efficient DNA repair. Many reports have shown that γH2AX foci are formed under oxidative stress, such as H2O2 or paraquat treatment (Mao et al.,
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2011; Fortini et al., 2012). In agreement with the these reports, treatment with H2O2 increased the number of γH2AX foci in MCF7 cells, but there was rapid clearance of γH2AX foci after removing
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H2O2 from MCF7 cells (Fig. 1). However, γH2AX persisted longer when NR1D1 was transiently overexpressed (Fig. 1). These results show that NR1D1 inhibits the repair of DNA damage induced by oxidative stress.
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3.2. NR1D1 inhibits the enzymatic activity of PARP1 through the binding to PARP1 Because oxidative DNA damage is mainly repaired through the BER pathways, we hypothesized that NR1D1 could modulate the BER signaling component (Hegde et al., 2012). In the BER pathway,
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PARP1 plays a key role in the recognition of DNA strand breaks including intermediates of BER (Dianov and Hübscher, 2013). We found that NR1D1 physically interacted with PARP1 and that the
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interaction was further enhanced by H2O2 treatment (Fig. 2A and 2B). PARP1 becomes activated after binding to damaged DNA, synthesizing PAR chains that attached to itself and its target proteins, which results in the efficient recruitment of the DNA repair complex (Wei and Yu, 2016). Therefore, we explored whether NR1D1 affected the enzymatic activity of PARP1. MCF7 cells were exposed to H2O2, and PARylation status of intracellular proteins was assessed by western blotting. As shown in Fig. 2C, H2O2 treatment induced PARylation of proteins, but the level of PARylation was decreased in the presence of NR1D1. Next, we measured the PARP activity in the cell lysates using histone proteins as the substrate. The PARP1 activity was increased after the H2O2 treatment, but it
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ACCEPTED MANUSCRIPT significantly decreased when NR1D1 was overexpressed (Fig. 2D). Treatment of GSK4112, a ligand of NR1D1, also reduced the activity of PARP1 (Fig. 2E). Together, these results show that NR1D1 inhibits the enzymatic activity of PARP1 through binding to PARP1.
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3.3. NR1D1 enhances DNA damage and increases sensitivity of MCF7 cells to oxidative stress
Because defects in appropriate DNA repair can result in accumulation of DNA damage, we examined whether the activation of NR1D1, i.e., overexpression of NR1D1 or GSK4112 treatment, influenced
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oxidative stress-induced DNA damage in MCF7 cells. We performed comet assays to evaluate the degree of DNA damage. The tail moment, an indicator of DNA breakage, increased remarkably after
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treatment with H2O2. Transient transfection of NR1D1 or treatment with GSK4112 also increased the comet tail moment although to a lesser degree than H2O2. The tail moment increased dramatically when the cells were cotreated with H2O2 (Fig. 3A). Similar results were obtained when comet assays were performed with paraquat, a different oxidative stress inducer (Supplementary Fig. 1A).
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Overexpression of NR1D1 or GSK4112 treatment increased the level of 8-OHdG, the most common forms of oxidative DNA lesions, and it increased further by cotreatment with H2O2 (Fig. 3B) (Valavanidis et al., 2009). The GSK4112-induced 8-OHdG level decreased after knockdown of
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NR1D1, which indicates the involvement of NR1D1 in the GSK4112-induced DNA damage (Fig. 3B). To investigate further the effect of NR1D1 on oxidative DNA damage, we employed the MCF7
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sublines that stably expressing NR1D1 (Supplementary Fig. 1B). We found that H2O2-induced PAR formation was significantly lower in the NR1D1-overexpressing cells compared with that in the control cells (Fig. 3C). Because high intracellular ROS levels in cancer cells can lead to oxidative damage-driven cell death, we assessed whether NR1D1 makes cells sensitive to excessive ROS. NR1D1-overexpressing cells were more sensitive to H2O2 and paraquat compared with control cells (Fig. 3D and Supplementary Fig. 1C). Together, these data suggest that NR1D1 enhances oxidative DNA damage, which may increase the sensitivity of cells to oxidative stress.
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ACCEPTED MANUSCRIPT 3.4. Lack of NR1D1 decreases sensitivity to oxidative DNA damage To gain further insight into the role of NR1D1 in the sensitivity to oxidative DNA damage, we employed three stable MCF7 sublines expressing shNR1D1 (Ka at al., 2017). First, we found that PAR formation was increased significantly in the H2O2-treated shNR1D1 sublines than shGFP
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control cells (Supplementary Fig. 2A and 2B). Furthermore, the shNR1D1-MCF7 sublines were less sensitive to H2O2-induced clonal growth inhibition (Fig. 4A). Similarly, shNR1D1 was more resistant to paraquat-induced cell death when cell viability was measured using MTT assays (Fig. 4B).
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Together, these results indicated that NR1D1 enhanced the sensitivity of cancer cells to oxidative
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stress, probably by inhibiting the PARP1-mediated BER process.
4. Discussion
In the present investigation, we found that NR1D1 inhibits DNA repair caused by ROS-induced DNA
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damage in MCF7 cells. NR1D1 interacts with PARP1 under oxidative stress and thereby inhibits the catalytic activity of PARP1. Thus, NR1D1 may result in disruption of the proper DNA repair processes, including BER, which requires PAR-dependent recruitment of DDR factors to DNA
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damage sites. The disruption of DNA repair may subsequently lead to accumulation of oxidative DNA damage, which could enhance the sensitivity of cancer cells to oxidative stress-induced cell
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death.
Several chemotherapeutic agents used for treatment of breast cancer, such as taxanes,
platinum-based agents, and anthracyclines, induce ROS-induced cell death by promoting the generation of intracellular ROS (Conklin, 2004). Ionizing radiation also increases ROS production that leads to severe cellular damage and apoptosis (Azzam et al., 2012). Therefore, NR1D1 could enhance efficacy of such anticancer therapies by disrupting the ROS-associated DDR. Indeed, several public patient data sets show that NR1D1 expression correlates positively with clinical outcome in breast cancer patients who received chemotherapy. Further, NR1D1 and its ligand, GSK4112,
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ACCEPTED MANUSCRIPT increased the sensitivity of breast cancer cells to doxorubicin, probably by inhibiting DSB repair (Ka et al., 2017). Here, our data suggest that the mechanism of the chemosensitizing effect of NR1D1 may also involve, at least in part, its ability to increase ROS-induced DNA damage, because many chemotherapeutic drugs including doxorubicin generate high levels of ROS in tumor cells (Conklin,
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2004).
Recently, inhibiting PARP is considered as a promising therapeutic strategy for the treatment of breast cancer (Livraghi and Garber, 2015). As PARP is crucial in the cellular response to
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genotoxic stimuli including oxidative stress, PARP inhibition may sensitize tumor cells to chemotherapy-induced oxidative stress. Thus, combinations of a PARP inhibitor with chemotherapy
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or with radiation therapy have been under diverse clinical trials (Dréan et al., 2016). For example, veliparib in combination with carboplatin is under clinical trials for stage III–IV breast cancer susceptibility gene (BRCA)-associated breast cancer (phase 2; NCT01149083) and for human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (phase 1;
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NCT01251874). The combination of olaparib and radiotherapy is also being trialed for inoperable breast cancer (phase 1; NCT02227082) (https://clinicaltrials.gov/). Here, we speculate that specific ligands that activate NR1D1 and thereby inhibit PARP1 activity may exert the same sensitizing
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effects as PARP inhibitors when applied as combination therapy with chemotherapeutics or radiation. Together, our findings suggest that NR1D1 may provide a potential chemosensitizer to enhance the
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outcomes of adjuvant therapy that are associated with the generation of intracellular ROS. The facilitating role of NR1D1 in the oxidative stress-induced damage might be specific in
cancer cells. Sengupta et al. (2016) suggested that NR1D1 provides preconditioning for protection against oxidative stress by inducing antioxidant genes such as forkhead box protein O1 (FoxO1), superoxide dismutase 2 (SOD2; also known as MnSOD), heme oxygenase (decycling) 1 (Hmox-1), and catalase in lung fibroblasts. It was also shown that NR1D1 was upregulated in response to hyperoxia-induced oxidative stress in neonatal mouse lung fibroblasts (Yang et al., 2014). However, we observed that NR1D1 decreased the expression of SOD2 in MCF7 cells, which suggests that the
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ACCEPTED MANUSCRIPT NR1D1-induced suppression of ROS-scavenging activity of cancer cells may potentiate oxidative stress-induced cancer cell death (Supplementary Fig. 3). The causes of this discrepancy in the celltype-dependent regulation of antioxidant enzymes by NR1D1 are not currently clear; however, differences in regulation of cellular antioxidative systems in normal and cancer cells may be a reason
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(Cairns et al., 2011). Further studies are warranted to understand the context-dependent role of NR1D1 regarding regulation of cellular oxidative systems. In this regard, the role of NR1D1 in DNA repair during the development of breast cancer should be studied in future, because ROS are potential
Acknowledgment
This
work
was
supported
by
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factors that contribute to initiation, promotion, and progression of breast carcinogenesis.
grants,
the
NRF-2017R1A2B3011870
(M.L.),
NRF-
2014R1A2A1A10052265 (M.L.), and the NRF-2013H1A2A1033512 (N.K.) from the National
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Research Foundation of Korea.
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. NR1D1 inhibits DNA repair against ROS-induced DNA damage. (A) MCF7 cells were transfected with empty vector (EV) or FLAG-NR1D1 and then treated with 500 µM H2O2 for 30 min.
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Cells were fixed at the indicated recovery time (RT) points and immunostained with anti-γH2AX (green). Nuclei were visualized by DAPI staining (blue). (B) The number of γH2AX foci shown in panel (A) was quantified at the indicated RT from 50 cells. Values represent mean ± SD (n=3). **P <
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0.01, ***P < 0.001 vs EV at each time point. (C) Expression level of NR1D1 was measured by
the western blot were presented.
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western blotting and shown as control. Images of NR1D1 obtained after short and long exposure of
Figure 2. NR1D1 is associated physically with PARP1 and inhibits its activity. (A and B) MCF7 cells (A) or MCF7 cells that transfected with FLAG-NR1D1 (B) were treated with vehicle or 500 µM
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H2O2 for 5 min. Whole cell lysates were immunoprecipitated (IP) using IgG or anti-PARP1 and probed by western blot (WB) analysis. (C) MCF7 cells transfected with empty vector (-) or FLAGNR1D1 were treated with vehicle or 500 µM H2O2 for 5 min. Expression of the PARylated proteins,
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PARP1 and FLAG-NR1D1 was analyzed by western blotting. (D) MCF7 cells transfected with empty vector (-) or FLAG-NR1D1 were treated with vehicle or 500 µM H2O2 for 5 min. PARP1 activities in
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the cell lysates were measured by ELISA. *P < 0.05 vs no treatment; #P < 0.05 vs H2O2 treatment. (E) MCF7 cells were treated with 10 µM GSK4112 for 24 h. PARP1 activities in the cell lysates were measured by ELISA. *P < 0.05 vs no treatment.
Figure 3. NR1D1 further increases ROS-induced DNA damage. (A) MCF7 cells were transfected with empty vector (EV) or FLAG-NR1D1, or the cells were treated with 10 µM GSK4112 (GSK) for 24 h. Then, the cells were treated with vehicle or 500 µM H2O2 for 5 min and were subjected to comet assay (left). Tail moment was quantified from randomly selected 100-150 cells per image
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ACCEPTED MANUSCRIPT (right). Values represent mean ± SD (n=3). ***P < 0.001 vs no treatment;
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P < 0.001 vs H2O2
treatment. (B) MCF7 cells were transfected with EV or FLAG-NR1D1, or treated with 10 µM GSK4112 for 24 h and then treated with 500 µM H2O2 for 5 min. The amount of 8-OHdG was measured by ELISA. Values represent mean ± SD (n=3). *P < 0.05 vs no treatment; #P < 0.05 vs
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H2O2 treatment (left). Cells were transfected with siGFP or siNR1D1 and then treated with 10 µM GSK4112 for 24 h. The amount of 8-OHdG was measured by ELISA. Values represent mean ± SD (n=3). *P < 0.05 vs siGFP only; #P < 0.05 as indicated (right). (C) MCF7 stable cell lines that
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expressing EV or NR1D1 were treated with 500 µM H2O2 for 30 min. Cells were fixed and immunostained with anti-PAR (green). Nuclei were visualized by DAPI staining (blue) (left). The
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percentage of PAR-positive cells was quantified from 100 cells (right). Values represent mean ± SD (n=3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs no treatment of each cells; ##P < 0.01 as indicated. (D) The MCF7 stable cell lines were exposed to increasing concentrations of H2O2 for 72 h. Relative cell viability was measured using MTT assay. Values represent mean ± SD (n=3). **P < 0.01, ***P <
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Figure 4. Lack of NR1D1 decreases sensitivity to oxidative DNA damage in MCF7 cells. (A) The
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MCF7 stable cells expressing shGFP or shNR1D1 were exposed to H2O2 for 3 days, and then further incubated for another 11 days. Representative images obtained from three independent experiments
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are shown (top). Colonies that composed of more than 50 cells were counted. Values represent mean ± SD (n=3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs shGFP as indicated (bottom). (B) The MCF7 stable cells were exposed to increasing concentrations of paraquat for 72 h. Relative cell viability was measured using MTT assay. Values represent mean ± SD (n=3). ***P < 0.001 vs shGFP as indicated.
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ACCEPTED MANUSCRIPT NR1D1 inhibits the oxidative DNA damage-induced DNA repair in MCF7 cells. NR1D1 inhibits the enzymatic activity of PARP1 through the binding to PARP1. NR1D1 enhances cellular oxidative DNA damage.
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NR1D1 increases sensitivity of MCF7 cells to oxidative stress.