Role of PARP1-mediated autophagy in EGFR-TKI resistance in non-small cell lung cancer

Resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has become the main clinical challenge of advanced lung cancer. This research aimed to explore the role of PARP1-mediated autophagy in the progression of TKI therapy. PARP1-mediated autophagy was evaluated in vitro by CCK-8 assay, clonogenic assay, immunofluorescence, and western blot in the HCC-827, H1975, and H1299 cells treated with icotinib (Ico), rapamycin, and AZD2281 (olaparib) alone or in combination. Our results and GEO dataset analysis confirmed that PARP1 is expressed at lower levels in TKI-sensitive cells than in TKI-resistant cells. Low PARP1 expression and high p62 expression were associated with good outcomes among patients with NSCLC after TKI therapy. AZD2281 and a lysosomal inhibitor reversed resistance to Ico by decreasing PARP1 and LC3 in cells, but an mTOR inhibitor did not decrease Ico resistance. The combination of AZD2281 and Ico exerted a markedly enhanced antitumor effect by reducing PARP1 expression and autophagy in vivo. Knockdown of PARP1 expression reversed the resistance to TKI by the mTOR/Akt/autophagy pathway in HCC-827IR, H1975, and H1299 cells. PARP1-mediated autophagy is a key pathway for TKI resistance in NSCLC cells that participates in the resistance to TKIs. Olaparib may serve as a novel method to overcome the resistance to TKIs.

Scientific Reports | (2020) 10:20924 | https://doi.org/10.1038/s41598-020-77908-z www.nature.com/scientificreports/ resistance to TKIs is therefore an important clinical issue. TKIs combined with radiotherapy or other conventional cancer therapies do not improve prognosis 3 . Different strategies have been suggested to overcome this resistance, such as combining TKIs with thermal therapy 4 , anti-angiogenic agents 5,6 , or c-Met inhibitors 7 . These strategies seem to improve prognosis, but the results are far from perfect. Thus, novel targets are still required to overcome resistance. Autophagy is a process in which damaged organelles, protein aggregates, and bacteria are digested in autophagic vesicles 8 . The aim of autophagy is to maintain a homeostatic state by degrading organelles and proteins 8 . Autophagy is a protective, cellular response to stress that is upregulated following hypoxia, starvation, and drug exposure 9 . Autophagy is upregulated in cancer cells because of their high metabolic state to maintain energy balance and prevent apoptosis 10 . Autophagy is upregulated in the presence of a number of cancer drugs, including TKIs [11][12][13][14][15] , and plays a critical role in the resistance to TKIs through Atg5 but not Beclin 1 16,17 . Treatment with autophagy inhibitors combination with TKIs may be an effective approach when basal autophagy is upregulated in cancer cells [18][19][20] . Inhibiting autophagy overcomes TKI resistance through the modulation of endoplasmic reticulum stress and apoptosis 9 .
We detected the levels of different molecules downstream of EGFR and found that the expression of mTOR/p-Akt was significantly different in cancerous and paracancerous tissues, but the expression of PI3K was not different. Poly(ADP)-ribose polymerase-1 (PARP1) repairs single-strand breaks in DNA and is important in autophagy regulation 21 . Reactive oxygen species (ROS) induce PARP1 activity and autophagy, and PARP1 knockout cells display low levels of autophagy, suggesting a prosurvival role for autophagy and PARP1 activity 22 . PARP1 is an important, positive regulator of autophagy induced by DNA damage, ROS, and starvation 22,23 . PARP1 activation and ATP depletion mediate cell death and autophagy via AMPK activation and mTOR suppression 24 . Furthermore, PARP1 inhibitors have antitumor activity in some types of cancer 25 . The poly-ubiquitin receptor p62 is also involved in the regulation of PARP1-mediated autophagy 26 . Therefore, treatment with a combination of TKIs and PARP1 inhibitors could be a potential approach to overcome TKI resistance and improve prognosis, but the exact involvement of PARP1 in the autophagy of NSCLC cells is still poorly understood. Therefore, the aim of the present study was to explore the role of the PARP1-mediated autophagy pathway in TKI resistance in NSCLC.

Results
TKI treatment increases the expression of PARP1 and LC3 II in TKI-resistant HCC-827IR and H1299 cells. TKI resistance is common in NSCLC, identifying the differences between TKI-sensitive and TKI-resistant cells is needed to better understand its mechanisms. HCC-827 was shown to be sensitive to icotinib (Ico) and HCC-827IR was shown to be resistant to Ico using the CCK-8 assay (Fig. 1A). Ico treatment revealed that PARP1 and LC3 II/ LC3 I ratio was decreased in HCC-827 cells but increased in HCC-827IR cells compared to that observed without treatment. The autophagy substrate, p62 expression, was negative correlated with LC3 II level (Fig. 1B). To further confirm the Fig. 1B results and show the differences of autophagy level between TKI-sensitive and TKI-resistant cells, we repeated the above process and transiently transfected HCC-827 cells and HCC-827IR cells with GFP-LC3 and treated these cells with or without Ico. The percentage of cells with punctate pattern of GFP-LC3 was counted by fluorescence microscopy. The punctate pattern (an accumulation of autophagosomes) in HCC-827IR cells were significantly higher than HCC-827 cells, and after treatment with Ico there was more punctate pattern (Fig. 1C). To identify the differences of PAPR1 expression between TKI-sensitive and TKI-resistant cells, data analysis from GEO dataset of PARP1 mRNA in TKI-sensitivity cells and TKI-resistance cells. One-way ANOVA analysis comparing the mRNA level of PARP1 in 2 TKI-sensitive cell lines and 2 TKI-resistant cell lines, with a total of 4 samples in triplicate analyzed. We found the mRNA level of PARP1 was significantly higher in TKI (Gef, Gefitinib)-resistant cell lines than that in TKI (Gef)-sensitive cell lines after TKI treatment, as confirmed by GEO dataset analysis (Fig. 1D). And, we also got similar results after Ico treatment in HCC-827 cells ( Supplementary Fig. 1B). Finally, to directly observe the differences of autophagy level, from 50 cells analyzed for each condition, electron microscope assay tested the autophagosome level after Ico treatment in HCC-827IR cells. Electron microscope confirmed that HCC-827IR cells had higher level of autophagy by Ico treatment than control group (Fig. 1E). These results suggest that the trend toward PARP1 expression and autophagy was clearly different in the HCC-827 and HCC-827IR cells after TKI treatment, which could be attributed to Ico resistance in HCC-827IR. PARP1 and p62 expression were associated with PFS and OS in TKI-treated NSCLC patients. We evaluated the expression of PARP1 and p62 proteins in 104 patients with NSCLC with an EGFR mutation, and all patients received TKI treatment. PARP1 was distributed in the cytoplasm and nucleus, while p62 was only localized in the cytoplasm. Kaplan-Meier survival curves show, compared with PFS and OS associated with the low expression of PARP1, high expression of PARP1 was associated with shorter PFS (P < 0.005) and OS (P < 0.005) ( Fig. 2A,B). Compared with PFS and OS of the low p62 expression group, the high expression of p62 was associated with longer PFS (P < 0.005) and OS (P < 0.005) (Fig. 2C,D). These data suggest that the low expression of PARP1 could be associated with better outcomes for NSCLC patients treated with TKIs, p62 expression was negatively correlated with OS and PFS in patients. These results implied that the PARP1/ autophagy may play an important role in the survival outcome of EGFR mutations in patients with NSCLC.
Role of PARP1-mediated autophagy in Ico resistance. Rapamycin (Rapa) and hydroxychloroquine (CQ) were used to investigate the role of PARP1-mediated autophagy in Ico resistance. The CCK-8 assay showed that Rap, which promotes autophagy, did not inhibit cell proliferation after Ico treatment in HCC-827 and HCC-827IR cells (Fig. 3A). In contrast, CQ, which inhibits autophagy, significantly reduced cell proliferation in HCC-827IR cells after Ico treatment (Fig. 3B). And 3-MA (a specific autophagy inhibitors) also clearly inhibited  figure A). These results suggest that autophagy plays an important role in Ico resistance in HCC-827IR cells. AZD2281 (olaparib) was used to inhibit PARP1. Ico treatment increased the ratio of LC3 II/LC3 I when used alone, while the combination of AZD2281 with Ico decreased the ratio of LC3 II/LC3 I compared to that observed in control cells (Fig. 3C). In addition, the combination of AZD2281 and Ico had a synergetic effect on cell proliferation by increased LC3 II accumulation in HCC-827IR cells (Fig. 3D), compared with that observed in the Ico or control groups (Fig. 1D). Taken together, both CQ and AZD2281 significantly decreased the resistance of HCC-827IR cells to Ico, indicating that PARP1-mediated autophagy is an important mechanism required for Ico resistance.

PARP1-regulated autophagy is required for TKI resistance in primary TKI-resistant cell lines.
The knockdown of PARP1 increased the ratio of LC3 II/LC3 I and mTOR expression in HCC-827IR, H1299, and H1975 (EGFR p. T790M and EGFR p. L858R) cells compared to that in control cells (Fig. 4A). In addition, the knockdown of PARP1 significantly decreased the proliferation induced by Ico in HCC-827IR, H1299, and H1975 cells compared to that in cells expressing PARP1 (Fig. 4B). These results strongly suggest that PARP1 is essential for autophagy in NSCLC and that decreasing PARP1 expression inhibits autophagy and resistance to TKI.

AZD2281 reverses Ico resistance in vivo.
The efficacy of the combination of Ico + AZD2281 against tumor growth was tested in the HCC-827IR xenograft model. The combination of Ico + AZD2281 significantly inhibited the growth of the xenograft, as measured by tumor size and weight (P < 0.01, Fig. 5A,B). The PARP1mediated autophagy signaling pathway was verified in the HCC-827IR xenograft model. Western blot analysis showed that AZD2281 treatment increased p62 expression and increased mTOR expression and the ratio of LC3 II/LC3 I compared to those in the control cells (Fig. 5C). IHC revealed p62 expression in the xenograft model, and the results were consistent with those from the western blot analysis (Fig. 5D,E). The in vivo data further showed that the combination of Ico with AZD2281 reverses the acquired resistance to TKIs.  cells, which were not sensitive to Ico, but the addition of AZD2281 increased their sensitivity to Ico (Fig. 6A,C). Immunoblotting showed that AZD2281 decreased the ratio of LC3 II/LC3 I induced by Ico in H1299 and H1975 cells compared to that observed in cells not treated with AZD2281 (Fig. 6B,D). These results suggest that the PARP1-mediated autophagy signaling pathway is probably a critical factor in the resistance to TKIs.

Discussion
PARP1-mediated autophagy was shown to be a key pathway for TKI resistance in NSCLC cells and to participate in the resistance to TKIs, and olaparib could be a new drug to overcome this resistance. In addition, clinical data showed that longer PFS and OS is clearly associated with lower PARP1 expression and higher p62 expression in patients with NSCLC after TKI therapy compared to their expression levels without TKI therapy. Taken together, these results suggest that the combination of olaparib and TKIs could potentially overcome TKI resistance. The present study suggests another strategy to reverse resistance to TKIs by the combination of blocking PARP1-induced autophagy and treating with TKIs. Cotreatment with CQ and Gef promoted sensitivity to TKIs by the Akt-mTOR pathway in NSCLC 27 . The inhibition of autophagy led to decreased cetuximab resistance and has been shown to be a novel clinical strategy for TKI therapy 28 . Interestingly, the present study showed that autophagy and PARP1 expression were different in HCC-827 and HCC-827IR cells following TKI treatment. These results were consistent with a study that showed that TKI increases lethal autophagy by altering the relationship between EGFR and Beclin 1 and dissociating Beclin 1/Bcl-2 in HCC-827 and H1975 cells 29 . Nevertheless, Ico treatment induced autophagy and increased PARP1 expression in HCC-827IR cells compared to that in cells without Ico treatment, suggesting that PARP1-mediated autophagy is a prosurvival signaling pathway. In addition, it was proposed that PARP1 enhances autophagy by the ATP-and NAD + -mediated www.nature.com/scientificreports/ AMPK-mTOR pathway 23 . Autophagy mediated by PARP1 activation is a cytoprotective process in response to DNA damage 23 . PARP1 depletion also results in a switch from prosurvival autophagy induced by ROS and starvation to apoptosis 22 . This finding was consistent with the results of the present study, and the clinical data showed a possible association between PARP1 expression and autophagy. A previous study showed that EGFRactivating mutations in NSCLC were associated with PARP1 inhibitor sensitivity 30 , but the detailed mechanisms by which TKIs activate PARP1 still need further investigation. The present study showed that the inhibition of PARP1 using olaparib and siRNA-PARP1 inhibited cell growth and decreased autophagy. PARP1 expression induced by TKIs was different in HCC-827 and HCC-827IR cells, suggesting that the way in which TKIs induce PARP1 activation may involve different signaling pathways. We propose that PARP1 protein and autophagy levels could be used as biomarkers to predict the emergence of resistance after TKI therapy in NSCLC, as suggested by the xenograft experiment. It was reported that PARP1 expression was negatively associated with breast, ovarian, and pancreatic cancer outcomes 31,32 . The present study suggests a role for PARP1-mediated autophagy in the resistance to TKIs seen in NSCLC. In addition, PARP1 expression was related to p62 expression, suggesting that PARP1 is an upstream molecular regulator of autophagy in human cancers. The detail mechanical between PARP1 and autophagy had been extensively studied 33 . Munoz-Gamez, J. A. etc. found that PARP1 regulated autophagy by the ATP-and NAD + -mediated AMPK-mTOR pathway in human tumor cells 23 . ROS-dependent PARP-1-LKB1-AMPK-mTOR pathway also was proposed in human endothelial cells 34 , Pancreatic ductal adenocarcinoma 35 , Lymphangioleiomyomatosis 36,37 , prostate cancer 38 . In lung cancer the PARP-1-LKB1-AMPK-mTOR pathway also take part in cell death program 39 . Furthermore, olaparib has been shown to be safe and effective in combination therapy 40,41 . The marker of autophagy may be an important indicator for cancer prognosis. Autophagy play a key role in tumorigenesis, p62 is necessary for cell survive and tumorigenicity mediated by Ras 42 . p62 deficiency through mTORC1 inhibition directly supported nutrient availability for cancer cells and promoted tumorigenicity 43 . Our results shown that autophagy substrate, p62 high expression had a longer PFS and OS in patients with NSCLC after TKI therapy. High p62 expression The data represent means ± SD of three independent experiments, *P < 0.05 versus untreated **P < 0.001 versus untreated (because of the films before imaging were cut, we couldn't further provided fuller-length original blots). (D) HCC-827IR cells were treated with 0, 2.5, 5, or 10 µM Ico; 2 or 4 µM AZD2281; or a combination of both for 48 h. Cell viability was determined by the CCK-8 assay. The columns represent means ± SD of three independent experiments.*P < 0.05 versus untreated. (P < 0.05).

Scientific Reports
| (2020) 10:20924 | https://doi.org/10.1038/s41598-020-77908-z www.nature.com/scientificreports/ in the tumor area served as a potent indicator for poor outcome of hepatocellular carcinoma patients 44 . SNP rs10898880 in ATG16L2 may be a predictor of RP, LRFS, PFS, and OS in patients with NSCLC after definitive radiotherapy 45 . Clinical trials are necessary to application of PARP1 protein and autophagy marker to the clinic. Nevertheless, the present study conflicts with previous studies. Indeed, Erguven et al. 29 showed that EGFR-TKI induced pro-apoptotic autophagy in HCC-827 cells. In addition, the role of autophagy in the induction of apoptosis is the focus of controversy 46 . Based on the present study, we believe that PARP1-mediated autophagy is protective in NSCLC. A variety of uncontrolled environmental factors could shift the delicate balance between autophagy-mediated survival and apoptosis. In addition, in the present study, the combination of TKI with olaparib overcame the resistance of the NSCLC xenografts, as supported by Sui et al. 47 ; however, in their study, this phenomenon increased autophagy 47 . Because Sui et al. 47 did not further explore the molecular mechanisms involved, these differences could be due to the different cell lines used.
Overall, PARP1-mediated autophagy was shown to be a key pathway for TKI resistance in NSCLC cells that participates in the resistance to TKIs. Olaparib may serve as a new drug to overcome NSCLC resistance to TKIs. Our study suggests that PARP1-mediated autophagy is not only a biomarker for TKI treatment outcomes but also a target for reversing TKI resistance. The combined use of a PARP1 inhibitor and an autophagy inhibitor decreased resistance to TKI to some extent, indicating a potential novel therapeutic approach for the treatment of NSCLC.   . Tumor tissues isolated from xenografts after treatment with Ico, AZD2281, or Ico + AZD2281 were subjected to western blot analysis (C) (Because of the films before imaging were cut, we couldn't further provided fuller-length original blots). The data represent means ± SD of three independent experiments, *P < 0.05 versus untreated **P < 0.001 versus untreated. Tumor tissues isolated from xenografts after treatment with Ico, AZD2281, or Ico + AZD2281 were subjected to immunohistochemical stain of PARP1 (D) and p62 expression (E). The data shown represent the mean ± standard error, n = 10. *P < 0.05 versus the vehicle group. Clonogenic assay. The assay was used to determine the survival and proliferation of the different cell lines after drug treatment 49 . Cells were plated on 60-mm plastic dishes 1 day before drug treatment in order to produce approximately 500 surviving colonies. These cells were treated for 14 days. The cells were stained and counted.

Materials
In vivo experiment. Animal handling were approved by Ethics Committee of Army Medical University.
Female BALB/C nude mice (6-8 weeks of age) were obtained from the Daping Hospital of Army Medical University Laboratory Animal. These mice were housed, then injected HCC-827IR cells. Ico was intraperitoneally administered at 50 mg/kg/day, for 3 weeks. AZD2281 was intraperitoneally administered at 30 mg/kg/day for 3 weeks. These mice were measured tumor diameter and tumor size. Mice with tumors reaching about 100 mm 3 were randomized into four groups (10/group): control (PBS), 50 mg/kg Ico, 30 mg/kg AZD2281, and Ico + AZD2281 47 .  Immunohistochemistry (IHC). The slides were baked, blocked and incubated with PARP1 and p62 monoclonal antibody (1:100 and 1:50 dilution), and then with secondary antibody (1:50 dilution). Finally, the slides were incubated with 3,30-diaminobenzidine (DAB). Cytoplasmic PARP1 and p62 staining score was performed by three pathologists 50 . Weak staining and < 50% of stained cells were defined as low expression 51 .
Patients. The tumor blocks were from 88 patients treated at Daping Hospital of the Army Medical University (China) and 31 patients treated at Wuhan Union Hospital (China) (supplementary table. Clinical characteristics of NSCLC patients). These samples of patients were collected after TKI therapy. According to the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent, the Ethics Committee of the Daping Hospital, Army Medical University also approved it, and obtaining informed consent from all subjects.
The research was carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent. It was carried out after approval by the Ethics Committee of the Daping Hospital and Research Institute of Surgery (Medical Research Institute (2020) No. 119), Army Medical University and obtaining informed consent from all subjects. The methods in treating tissues were carried out strictly in accordance with institutional policies and approved guidelines of experiment operations. And animal handling were approved by Ethics Committee of Army Medical University.
Autophagy assay. Cells were transfected with GFP-LC3 vector and jet PEITM according to the manufacturer's protocol. Assays were performed on cells grown on glass coverslips in a six-well plate and treated with HANK buffer or/and rapamycin. The fluorescence image was observed with a Zeiss fluorescence microscope.
Statistical analysis. Categorical data were presented as frequencies and analyzed using the Fisher exact test.
OS and PFS were analyzed using the Kaplan-Meier method and the log-rank test. SPSS 20.0.0 (IBM, Armonk, NY, USA) was used for statistical analysis. Two-sided P values < 0.05 were considered statistically significant.

Ethics declaration.
The research was carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent. It was carried out after approval by the Ethics Committee of the Daping Hospital and Research Institute of Surgery (Medical Research Institute (2020) No. 119), Army Medical University and obtaining informed consent from all subjects. The methods in treating tissues were carried out strictly in accordance with institutional policies and approved guidelines of experiment operations. And animal handling were approved by Ethics Committee of Army Medical University.