Background
Lung cancer is the leading cause of cancer-related death and remains a major clinical challenge with increasing incidence and mortality [
1,
2]. Due to drug resistance, recurrence, and metastasis, the treatment efficacy of lung cancer remains unsatisfactory. A better understanding of the aetiology, pathogenesis, and molecular targets is required to develop novel therapeutic modalities. Somatic gene mutations, including
KRAS,
EGFR, and
TP53 mutations, is a major driver of lung cancer initiation [
3]. Accumulating evidence has shown that not all gene mutations occur equally. In particular, compelling evidence suggests that RAS mutants function in an allele-specific manner, justifying the acquirement of a RAS allele-specific approach for RAS-driven cancer therapy [
4‐
6]. Given the feature of allele specificity and the pivotal role of RAS in cellular events, including cell growth, cell survival, cell senescence, and cell death, novel strategies in a RAS allele-dependent manner are still required.
Autophagy is a cell survival-promoting mechanism following harsh stimuli and has been deeply implicated in cancer development and therapy [
7‐
9]. Recently, targeting autophagy has been in the spotlight for cancer therapy via pharmacological inhibition alone or combination with other therapeutics [
10,
11], providing insight into lung cancer therapy development. Cisplatin is one of the most frequently administered chemotherapeutic drugs for many solid tumours, including lung cancer. Mechanically, cisplatin kills cancer cells via interference with DNA synthesis and repair, subsequently inducing cell apoptosis [
12]. However, there is limited clinical efficacy for cisplatin-based therapy because of drug resistance [
13]. Several key factors contribute to cisplatin resistance, including autophagy [
14] and apurinic/apyrimidinic endonuclease 1 (APE1) [
15]. APE1 is a multifunctional protein with two major activities, DNA repair and transcriptional regulation [
16]. Importantly, APE1 is often overexpressed in many tumours, contributing to disease progression, chemo-resistance and a poor prognosis [
15,
17‐
20]. Our previous study found that APE1 is highly expressed in non-small cell lung cancer (NSCLC). Moreover, APE1 is a prognostic risk factor indicated by a poor overall survival [
15,
19]. Herein, targeting APE1 might represent a therapeutic vulnerability for lung cancer, particularly, cisplatin-resistant lung cancer.
Thus, based on the aforementioned details, we hypothesized that APE1 and autophagy may contribute to lung cancer progression and drug resistance and that combined blockade of APE1 and autophagy enhances the therapeutic effect of cisplatin and overcomes cisplatin resistance in lung cancer. In the present study, we applied quantitative proteomics to identify the proteomic responses to cisplatin treatment in KRASG12S-mutant A549 cells. Both APE1 and autophagy were involved in the cellular responses to cisplatin exposure. In A549 cells and cisplatin-resistant A549 cells, cisplatin-induced apoptosis was significantly enhanced via the combination of autophagy inhibition by chloroquine (CQ) and APE1 knockdown by siRNA with the involvement of p53 activation.
Methods
Chemicals and reagents
CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). 13C6-L-lysine, L-lysine, 13C615N4-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).
Cell line and cell culture
The human lung cancer cell line A549 (KRASG12S) was obtained from Chinese Academy of Science Cellbank (Shanghai, China) and was cultured in RPMI1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS). The cells were maintained at 37 °C in a 5% CO2/95% air humidified incubator.
Cell viability determination
The MTT assay was used to evaluate cell viability. Briefly, cells were seeded in 96-well plates at a density of 7.0 × 103 cells/well. After 24 h. of incubation, the cells were treated for 48 h. The absorbance was measured using a Synergy™H4 Hybrid microplate reader (BioTek, Winooski, VT, USA) at wavelengths of 560 nm (MTT formazan) and 670 nm (background).
Quantitative proteomics
Quantitative proteomic experiments were performed using a stable isotope labelling by amino acids in cell culture (SILAC)-based approach to identify the molecular targets of CDDP in the treatment of A549 cells as previously described [
21]. Briefly, A549 cells were cultured in DMEM/F12 medium (for SILAC) with (heavy) or without (light) stable isotope-labelled amino acids (
13C
6 L-lysine and
13C
615N
4 L-arginine) and 10% dialyzed FBS. After treatment with CDDP (5 μM) for 24 h., the cell samples were harvested, lysed, and quantified. Next, an equal amount of heavy and light protein samples were combined to reach a total volume of 50 μL containing 400 μg of protein, and the combined protein sample was digested and desalted. Next, the peptide mixtures (5 μL) were subjected to the hybrid linear ion trap. The peptide SILAC ratio was calculated using MaxQuant version 1.2.0.13. The proteins were identified using Scaffold 4.3.2, and the pathway was analysed using ingenuity pathway analysis (IPA) from QIAGEN Inc.
Quantification of cellular apoptosis
Cell apoptosis was evaluated using the Annexin V-PE apoptosis detection kit as previously described [
21]. Briefly, the cells were collected after treatment and resuspended in 1× binding buffer with 5 μL of Annexin V-PE and 5 μL of 7-amino-actinomycin D (7-AAD) at 1 × 10
5 cells/mL in a total volume of 150 μL. The cells were gently mixed and incubated in the dark for 15 min at room temperature. The binding buffer (100 μL) was then added to each tube, and the number of apoptotic cells was quantified using flow cytometry and collecting 10,000 events for analysis.
Quantification of cellular autophagy
Cell autophagy was examined using flow cytometry as previously described [
21]. Briefly, the cells were collected after treatment and resuspended in 250 μL of assay buffer containing 5% FBS, and Cyto-ID® Green stain solution (250 μL) was added to each tube and mixed gently. After 20 min of incubation at room temperature in the dark, the cells were collected by centrifugation, washed once and analysed using the green (FL1) channel of flow cytometry.
Confocal fluorescence microscopy
Confocal microscopy was performed to evaluate the cellular autophagy level in A549 cells after treatment with 5 μM CDDP, 10 μM CQ, and 5 μM CDDP + 10 μM CQ using the Cyto-ID autophagy detection kits as previously described [
21]. The fluorescence was assessed using TCS SP2 laser scanning confocal microscopy (LSCM).
Western blotting assay
The protein expression level was examined using Western blotting. Protein samples were extracted using RIPA buffer, the protein concentrations were measured using the BCA kit, and an equal amount of protein was separated by SDS-PAGE. The corresponding primary and secondary antibodies were applied to evaluate the expression levels of targeted proteins. Visualization was performed using the Bio-Rad ChemiDoc™ XRS system, and the blot bands were analysed using Image Lab 3.0.
RNA interference
Small interfering RNA-mediated gene silencing was performed to investigate the role of APE1 in cisplatin-induced apoptosis and autophagy in A549 cells according to the manufacturer’s instructions. A549 cells were transfected with the negative control siRNA and APE1-siRNA using Lipofectamine 2000. The protein samples were collected and kept at − 80 °C for further analysis.
Immunoprecipitation
The interaction between APE1 and p53 was examined using immunoprecipitation as previously described [
22]. After 24 h. of treatment, A549 cells were lysed in pre-chilled cell lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitors] for 5 min. The lysates were precleared with 20 μL of Proteins A/G (Invitrogen; Thermo Fisher Scientific, Inc.) at 4 °C for 45 min, followed by incubation with APE1 or p53 antibody overnight at 4 °C. Following immunoprecipitation, the samples were incubated with protein G for 3 h. at 4 °C. Thereafter, the samples were washed with lysis buffer five times to remove any un-precipitated proteins before boiling in SDS buffer for 5 min. The elution was analysed for precipitated APE1 or p53 protein using Western blotting analysis. Normal rabbit IgG antibody was used as a negative control. The antibodies used were as follows: APE1 (1:500), p53 (1:500), and normal rabbit IgG (1:1000).
Statistical analysis
The data were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison procedure was used for comparisons of multiple groups. The value of P<0.05 was considered statistically significant. The assays were performed at least three times independently.
Discussion
Lung cancer is the leading cause of cancer death, and a lack of efficacious therapeutics exists. Cisplatin, the most important chemotherapeutic drug in lung cancer therapy, has shown limited clinical efficacy due to drug resistance. Autophagy and other key cellular events, including the DNA damage repair response, are involved in chemo-drug resistance. Notably, there is increasing attention on the genetic context dependence in lung cancer therapy. Compelling evidence has shown that oncogenic RAS mutations vary, although they all promote cancer cell proliferation [
4‐
6]. Zhong et al. found that inhibition of RAS-AKT-mTOR signalling and blockage of late stage autophagy could synergistically enhance the cytotoxicity of a tumour suppressor gene ARHI [
33]. Specific RAS alleles exhibit differential biochemical features, displaying preferential signalling output and favouring differential downstream effectors that are subject to differential feedforward and feedback regulations. Therefore, individualized therapeutics are advocated in cancer therapy.
In this study, we first applied SILAC proteomics to obtain a panoramic view of cisplatin treatment in KRAS
G12S-mutant A549 cells. At least 3262 protein molecules responded to cisplatin treatment and included APE1, p53, and LC3-I/II, which are involved in DNA damage repair, cell proliferation, apoptosis, and autophagy. Subsequent IPA analysis revealed 72 canonical signalling pathways including the BER pathway, DNA double-strand break repair, and autophagy pathways. Autophagy is a well-known cell-protective mechanism related to tumor progression, drug-resistance, and survive [
8], and blockade of RAS/RAF/MEK/ERK signalling flux promotes autophagy [
10], suggesting that inhibition of autophagy is beneficial. Kinsey et al. [
10] and Bryant et al. [
11] have shown synergistic antitumor effects of autophagy inhibition and MAPK inhibition in RAS-driven cancers, including pancreatic ductal adenocarcinoma, melanoma, and colorectal cancer, in preclinical settings [
10,
11]. This combined blockade of autophagy with other therapeutics revealed a novel therapeutic vulnerability to treat RAS-driven cancers, including lung cancer. Based on our previous research concerning BER pathway in platinum-resistance of lung cancer [
30] and present proteomic results, we herein aimed to investigate whether BER and autophagy have interaction upon cisplatin treatment in lung cancer cells. By methods of flow cytometry, fluorescence microscopy, Western blotting and RNA interference, we found that cisplatin markedly induced autophagy and apoptosis in A549 cells, accompanied by remarkable increase of DNA repair protein APE1. Suppression of autophagy enhanced the inhibition effect of cisplatin on cell growth, proliferation, and colony formation. The combination treatment of CQ, an autophagy inhibitor, with cisplatin dramatically enhanced cisplatin-induced apoptosis.
Moreover, APE1 is a major contributor to cisplatin resistance in lung cancer [
15]. In the present study, knockdown of APE1 enhanced cisplatin-induced apoptosis in both A549 cells and cisplatin-resistant A549 cells. Noteworthy, APE1 knockdown significantly synergized the apoptosis-inducing effect of cisplatin plus CQ. This dual inhibition of APE1 and autophagy could minimize the curative concentration of cisplatin in cisplatin-resistant A549 cells. The lower concentration of cisplatin was beneficial in reducing the side effects of chemotherapy that commonly occur in clinical settings. Besides, the specific targeting of autophagy without affecting other cellular processes has drawn a great attention of researchers. Mutations in the RAS pathway are often associated with the high levels of autophagy that are required to maintain cancer cell metabolism [
34,
35]. The optimal dosage of autophagy inhibitors and timing of inhibition are vital parameters for maximal therapeutic efficiency. Hopefully, Levy et al. reported that the treatment of CQ as an autophagy inhibitor in some cancer patients showed no adverse toxicity for extended time periods [
36]. This demonstrates that long-term treatment with lysosomal autophagy inhibitors is feasible. Provided that cancer cells are more dependent than normal tissues on autophagy, even a drug that causes some normal tissue toxicity can have a valuable therapeutic window for an effective cancer treatment [
8]. In inducible Atg7-knockout mice, the growth of KRAS-driven lung tumors was significantly inhibited before any signs of neurotoxicity [
37], indicating that therapeutic window for autophagy inhibition exists in some cancers.
Additionally, our previous data found that promoting p53 intracellular stability by interfering with APE1 is a possible mechanism in genistein-induced apoptosis [
38]. In the present study, we applied immunoprecipitation to explore the possible interactions between LC3-I/II and key proteins involved in DNA damage, including APE1 and p53. An interesting triple complex comprising APE1-p53-LC3 was formed in response to cisplatin plus CQ in A549 cells. Taken together, our results suggest that dual inhibition of APE1 and autophagy could enhance chemo-sensitivity and overcome cisplatin resistance by boosting apoptosis via the modulation of APE1-p53-LC3 complex assembly in a KRAS
G12S context. Whether the current findings regarding the cellular events recapitulate other
RAS mutations in lung cancer or
KRASG12S mutation in other cancer types in response to cisplatin treatment is unknown and warrants further investigation to better tailor specific therapeutic vulnerabilities for lung cancer treatment [
3].
Conclusions
In summary, this study revealed a proteomic response to cisplatin in KRASG12S-mutant A549 cells. APE1, p53, and LC3-I/II were identified to be involved in DNA damage repair, cell proliferation, apoptosis, and autophagy. Dual inhibition of APE1 and autophagy synergistically enhanced cisplatin-induced apoptosis via the regulation of APE1-p53-LC3 complex assembly. This novel combination strategy is of great potential to overcome cisplatin resistance in the context of KRASG12S-mutant lung cancer.
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