Background
Lung cancer is a leading cause of cancer-related deaths worldwide, and non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all cases of lung cancer [
1]. The majority of patients with advanced lung cancer are treated with chemotherapy [
2]. Platinum-based drugs, particularly cisplatin (DDP), are used in the treatment of many cancers, including NSCLC [
3]. Initially, cisplatin treatment demonstrates favorable outcomes, but often chemo-resistance develops later on and results in failure of this therapy [
4]. Thus, a better understanding of the molecular mechanisms underlying cisplatin resistance is mandatory to achieve advancement in NSCLC therapy.
Annexin A2 is a calcium-dependent, phospholipid-binding cell surface protein which has diverse cellular functions, including membrane-cytoskeleton organization, vesicular trafficking, and regulation of ion channel activity [
5]. Accumulating evidence suggested a correlation between the deregulation of Annexin A2 expression and tumor progression in many cancers [
6]. High level of Annexin A2 was detected in various malignant tumors, including brain tumors [
7], colorectal cancer [
8], gastric cancer [
9], pancreatic cancer [
10], breast cancer [
11], hepatocellular cancer [
12], and lung cancer [
13], and this abnormal expression of Annexin A2 was positively correlated with the differentiation status, histological type, lymph node metastasis and distant metastasis, as well as poor prognosis [
14,
15]. Annexin A2 has been shown to play an important role in cancer cell adhesion, proliferation, invasion, and metastasis, thus playing a crucial role in tumor development. Moreover, recently studies showed that Annexin A2 appears to be involved in the drug resistance phenotype of cancer cells. Quantitative proteomics results indicated that Annexin A2 was up-regulated in Adriamycin-resistant MCF-7 cells compared to drug-sensitive MCF-7 cells [
16], and up-regulation of Annexin A2 played a critical role in enhanced invasiveness of MCF-7/ADR cells [
17]. Annexin A2 overexpression was also significantly associated with rapid recurrence after gemcitabine-adjuvant chemotherapy in patients with resected pancreatic cancer [
18,
19]. In our previous studies, we found that Annexin A2 is up-regulated in the cisplatin-resistant non-small cell lung cancer cell line A549/DDP [
20]. However, the role of Annexin A2 in cisplatin resistance has not been determined.
In this study, we investigated the role of Annexin A2 in cisplatin resistance of NSCLC cells by analyzing its function both in vitro and in vivo. Our research demonstrated for the first time that Annexin A2 contributes to cisplatin resistance by activation of JNK-p53 pathway in lung cancer cells, and suggested promise as a marker for patients likely to benefit from cisplatin-based chemotherapy.
Materials and methods
Cell lines and cell culture
A549 and cisplatin-resistant A549/DDP cells were purchased from the Cancer Institute & Hospital, Chinese Academy of Medical Sciences (Beijing, China). H460 and H1650 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cell lines were tested for authenticity by short tandem repeats (STR) genotyping. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, New York State, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin. Cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO2. A549/DDP cells were cultured in medium supplemented with cisplatin at a final concentration of 2 μM, to maintain drug resistance.
Reagents and antibodies
Cisplatin was purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, USA). Specific inhibitors of AKT (LY294002), JNK (SP600125), and p38 (SB203580) were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO. The list of antibodies used in this study is summarized in Additional file
1: Table S1.
RNA interference, lentivectors, and plasmid transfection
Negative control siRNA and siRNAs against Annexin A2 (human Annexin A2 siRNA) were produced by RiboBio Co. Ltd. (Guangzhou, China). And targeting sequences were 5′-CAAGCCCCTGTATTTTGCTGAT-3′ (Annexin A2 siRNA-1), 5′-CGGGAT GCTTTGAACATTGAA-3′ (Annexin A2 siRNA-2), 5′- GCAGGAAATTAACAGAG TCTA-3′ (Annexin A2 siRNA-3). The small-interfering RNA (siRNA) targeting p53 (SignalSilence® p53 siRNA I #6231) were from Cell Signaling Technology (Beverly, MA, USA). A549/DDP cells were plated into 6-well plate at a density of 1.5 × 105 cells per well. After 24 h of incubation, negative control siRNA, siRNAs targeting human Annexin A2 or p53 were transiently transfected to A549/DDP cells with Lipofectamine 2000 Transfection Reagent (Invitrogen, California, USA) and incubated for 48 h. Annexin A2 shRNA-lentivirus vectors and a non-targeting control shRNA were obtained from GeneCopeia (Guang zhou, China). HEK293FT cells were co-transfected with lentivirus and packaging vectors using Lipofectamine 2000. After 48 h, media containing lentiviruses were harvested and filtered through 0.45 μm syringe filters. Lentiviruses were transduced in 50% confluent A549/DDP cells. Annexin A2 stable knockdown A549/DDP cells were selected by puromycin (1 μg/mL). For Annexin A2 rescue experiments, 1 μg of empty vector (pCMV6) or pCMV6-Annexin A2 (Origene, Rockville, MD, USA) were transfected in A549 cells using Fugene (Roche, Indianapolis, IN, USA), following the manufacturer’s instructions.
Cell viability assay
Cell viability was measured using the 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5- Diphenyltetrazolium Bromide (MTT) assay. Briefly, cells (1 × 104) were seeded in 96-well plates. Cells were allowed to adhere for 24 h and were subsequently incubated with the indicated drug concentrations for 48 h. 100 μL of 0.25 mg/mL MTT in medium culture was added to each well. The plate was incubated for 4 h at 37 °C. Then, culture medium was removed, and DMSO (100 μL) was added into each well to dissolve the dark blue crystal. The amount of MTT formazan product was analyzed by microplate reader (Spectra MAX 340, Molecular Devices Co., Sunnyvale, CA, USA) at a wavelength of 570 nm. Each individual experiment was repeated at least three times.
For colony-formation assay, about 500 cells were seeded per well in six-well-plates 48 h before the addition of indicated chemicals. After 14 days, the cells were fixed in methanol and stained with 0.2% crystal violet. Number of colonies was counted using Quantity One software (Bio-Rad, Hercules, CA, USA).
Cell apoptosis assay
Cell apoptosis was determined by AnnexinV/PI (KeyGEN, Nanjing, China), followed by flow cytometer analysis (Beckman Coulter, California, USA) according to manufacturer’s instructions.
Caspase activity was measured by Caspase-Glo 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
Western blot
Cells were harvested and proteins were extracted using RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 12000 rpm for 30 min at 4 °C, supernatants were saved, and protein concentrations were determined by by BCA protein assay (Thermo Scientific, Rockford, Illinois, USA). Protein extracts (40 μg) was resuspended and electrophoresed on 10–12% sodium dodecyl sulfate polyacrylamide gel and then blotted onto polyvinylidene fluoride membranes (Millipore A) at 200 mA for 1.5 h. Following blocking with 5% nonfat milk in TBST (TBS-1% Tween 20) for 1 h, membranes were immunoblotted with primary antibodies overnight at 4 °C and further incubated with secondary horseradish peroxidase-conjugated anti-rabbit. Finally, protein bands were detected by developing the blots with the enhanced chemiluminescence western blot detection kit (Engreen Biosystem, China).
RNA extraction and quantitative real-time PCR
Total RNA isolated from cells was used E.Z.N.A.® HP Total RNA Kit (Omega Bio-tek, Doraville, GA, USA). The reverse transcription was performed with the PrimeScript® RT reagent Kit (TakaRa, Shiga, Japan). After mixing the resulting Complementary DNA template with primers, respectively and TaKaRa SYBR® Premix Ex Taq™, Quantitative Real-Time PCR reaction was performed on a ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Gene-specific primer pairs used in this study are: p53, 5′-GCGCACAGAGGAAGAGAATCTCCG-3′ (sense) and 5′-TTTGGCTGGGGAGAGGAGCTG-3′ (antisense); GADD45A, 5′-GGATGCCCTGGAGGAAGTGCT-3′ (sense) and 5′-GGCAGGATCCTTCCATTGAGATGAATGTG-3′ (antisense); p21 5′-TGTACCCTTGTGCCTCGCTC-3′ (sense) and 5′-TGGAGAAGATCAGCCGGCGT-3′ (antisense); BAX: 5′-TTTGCTTCAGGGTTTCATCC-3′ (sense) and 5′-CAGTTGAAGTTGCCGTCAGA-3′ (antisense); Bcl2: 5′-GGATGCCTTTGTGGAACTGT-3′ (sense) and 5′-AGCCTGCAGCTTTGTTTCAT-3′ (antisense); MDM2: 5′-AGCGCAAAACGACACTTACA-3′ (sense) and 5′-ACACAATGTGCTGCTGCTTC-3′ (antisense); β-actin: 5′-ACACTGTGCCCATCTACGAGG-3′ (sense); β-actin 5′-AGGGGCCGGACTCGTCATACT-3′ (antisense). β-actin was used as the reference gene. The relative levels of gene expression were represented as ΔCt-Ct gene-Ct reference, and the fold change of gene expression was calculated by the 2−ΔΔCt Method. Experiments were repeated in triplicate.
Chromatin-immunoprecipitation
The ChIP assay was carried out using Millipore EZ-Magna ChIP kit (Millipuro) following the manufacturer’s instruction. Briefly, 5 × 106 cells were fixed with 1% formaldehyde and quenched in 0.125 M glycine. Cells were sonicated by Bioruptor Sonication System UCD-300. DNA was immunoprecipitated by either control IgG or c-Jun antibody. Precipitated DNA samples and inputs were amplified by PCR. The primers used for the amplification of c-Jun binding site in p53 promoter are: 5′-GCTGAGAGCAAACGCAAAAG-3′ (sense) and 5′-GAAATGGAGTTGGGGAGGAG-3′ (antisense).
Luciferase assays
The p53 promoter (−540 to +160 regions) was inserted into the pGL3-basic vector (Promega, Madison, WI, USA) at KpnI/XbaI sites to construct pGL3-p53 luciferase reporter plasmid (wild-pGL3-p53). The PF-1 site of the p53 promoter was mutated (from 5′-TGACTCT-3′ to 5′-TGAATTC-3′) using the QuikChange Site-directed mutagenesis kit (Stratagene, La Jolla, CA), and then the PF-1 site-mutated p53 promoter was cloned into the pGL3-basic vector (mut-pGL3-p53). All constructs were verified by sequencing. For the luciferase reporter assay, HEK293T cells and A549 cells were co-transfected with pGL3-p53-Luc, or mut-pGL3-p53 and pCMV-Annexin A2 using Lipofectamine 2000. The Renilla luciferase was as internal control. 48 h after transfection, cells were harvested and assayed by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The relative firefly luciferase activity was calculated by normalizing transfection efficiency according to the Renilla luciferase activity.
Animal study
24 female BABL/c nude mice (4–5 weeks old) from the Guangdong Province Laboratory Animal Center (Guangzhou, China) were used. A549/DDP/Control shRNA cells and A549/DDP/Annexin A2 shRNA cells (1 × 106) suspended in 100 μl cold PBS were subcutaneously injected to the right shoulder of the mice, all of which developed tumors with a size of ~30 mm3 within 7 days. Both A549/DDP/Control shRNA cells-bearing mice and A549/DDP/Annexin A2 shRNA cells-bearing mice were randomly allocated to two groups and treated with either PBS or Cisplatin (3 mg/kg body weight per day). The tumor size was measured in two dimensions with a caliper. Tumor volume was calculated based on the following formula: volume = (greatest diameter) × (smallest diameter)2/2. After treatment for 4 weeks, the tumors were extracted from sacrificed mice. Our animal study was approved by the Institutional Animal Care and Use Committee of Guangzhou medical University.
Immunohistochemistry
152 tumor tissues and 36 adjacent normal tissues were obtained from NSCLC patients who underwent complete resection in the Affiliated Tumor Hospital of Guangzhou Medical University between 2008 and 2013. In particular, adjacent normal tissues were taken 5–10 cm away from the tumor tissues. Follow-up information was obtained from review of the patients’ medical record. This study was approved by the Ethics Committee of Guangzhou Medical University.
Immunostaining was performed using the avidin-biotin-peroxidase complex method (UltrasensitiveTM, MaiXin, Fuzhou, China). The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min with an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with Annexin A2 rabbit polyclonal antibody (1:100 dilutions). Staining for antibody was performed at room temperature for 2 h. Biotinylated goat antimouse serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride.
The intensity of Annexin A2 staining was scored as 0 (no signal), 1 (weak), 2 (moderate), and 3 (marked). Percentage scores were assigned as 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The scores of each tumor sample were multiplied to give a final score of 0–12, and the tumors were finally determined as negative (−), score 0; lower expression (+), score ≤ 4; moderate expression (++), score 5–8; and high expression (+++), score ≥ 9. An optimal cutoff value was identified: a staining index of five or greater was used to define tumors of high expression, and four or lower for low expression.
The prognostic value of the Annexin A2 was analyzed by a Web-based Kaplan–Meier plotter (
http://www.kmplot.com/lung), which is a meta-analysis tool of gene expression and survival data of 2437 lung cancer patients (2015 version) using multiple microarray data [
21,
22].
Statistical analysis
All experiments were performed in triplicate at least. The results of this study are presented as mean ± SD and analyzed by Student’s t test with statistical analysis of data using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA).
Discussion
Development of drug resistance remains the major therapeutic barrier in lung cancer [
31]. Therefore, identification of the molecular mechanisms underlying drug resistance is mandatory to achieve advancement in lung cancer therapy. Using a proteomic approach, we previously demonstrated that Annexin A2 might be the important factor of cisplatin resistance [
20]. In this study, we showed that overexpression of Annexin A2 enhanced cisplatin resistance of A549, H460 and H1650 cells, whereas inhibition of Annexin A2 could selectively increase cisplatin sensitivity of A549/DDP cells both in vitro and in vivo, which suggested an important role of Annexin A2 in cisplatin resistance in NSCLC cells.
Aberrant Annexin A2 expression has oncogenic effects in several tumor types [
7‐
12]. Previous studies provided evidence that in patients with lung cancer, a poor prognosis for survival is correlated with Annexin A2 expression, and this observation is consistent with the results of Annexin A2 tissue staining in lung cancer [
13]. Our present data confirmed through Annexin A2 immunohistochemical staining of NSCLC tissues that Annexin A2 is overexpressed in NSCLCs and is correlation with advanced TNM stage. More important, we found that high levels of Annexin A2 is positively correlated with poor prognosis, as well as correlated with short disease-free survival for patients who received chemotherapy after surgery, which was further confirmed the specific role of Annexin A2 in chemotherapy resistance to NSCLCs.
Several mechanisms that mediate cisplatin resistance have been identified, including decreased import, pronounced activity of efflux pumps, increased detoxification, and increased efficiency of DNA repair systems [
32‐
35]. Since DNA damage and the induction of mitochondrial apoptosis are the most critical mechanisms of cisplatin action, evasion of apoptosis might be a key feature of acquired cisplatin resistance in tumor cells [
36]. Annexin A2 is involved in multiple cellular processes, including cell survival, growth, division, and differentiation. Interestingly, recent findings suggested that Annexin A2 serves as a ligand for C1q on apoptotic cells [
37]. It has been showed that apoptotic stimuli induced Annexin A2 cleavage, which contributes to cell cycle inhibition and apoptosis [
38], and knockdown expression of Annexin A2 made cells susceptible to chemotherapy- or radiation-induced apoptosis [
38,
39]. Consistent with these results, we found that knockdown of Annexin A2 significantly increased Caspase 3/7 activity, cleaved PARP levels, as well as cisplatin-induced cell apoptosis in A549/DDP cells, which suggested that Annexin A2 enhanced cisplatin resistance of NSCLC cells by a mechanism of inhibiting cell apoptosis.
The tumor suppressor p53 is a transcription factor that regulates several genes with a broad range of functions, including DNA repair, metabolism, cell cycle arrest, apoptosis and senescence [
40]. Most chemotherapeutic agents, including cisplatin, induce p53-dependent cell growth arrest and apoptosis [
41]. However, when mutation or deletion of p53 renders it non-functional, drug resistance can follow [
24]. Alternatively, abnormal expression of p53 regulators, such as bcl-2 and PIG3, can also lead to drug resistance [
42,
43]. Based on our present results, Annexin A2 facilitates cisplatin resistance in part by inhibiting p53 expression in NSCLC cells. Consistent with this notion, Annexin A2 degradation is correlated with cellular apoptosis induced by p53-mediated pathways [
44]. In response to genotoxic agents, cells depleted of Annexin A2 protected DNA from damage by enhancing phospho-histone H2Ax and p53 levels, increasing numbers of p53-binding protein 1 nuclear foci and increasing levels of nuclear 8-oxo-2′-deoxyguanine [
45].
MAPK pathway activation is a common event in tumorigenesis, and plays a key role in cancer progression and invasion by regulating cell migration, proteinase induction, and apoptosis [
46,
47]. In this study, we found that Annexin A2 had an effect on regulating JNK phosphorylation activation and subsequent cisplatin resistance in A549/DDP cells. We found that JNK, but not ERK1/2, was phosphorylated in A549 cells that were activated by overexpression of Annexin A2, whereas p38MAPK phosphorylation was suppressed by Annexin A2. Unfortunately, inhibition of p38MAPK is not required for Annexin A2-mediated cisplatin resistance, because p38MAPK inhibitor had no effect on the expression of p53 in Annexin A2-knockdown A549/DDP cells. The results were different from previous findings that upon loss of Annexin A2 tumor cells undergo apoptosis through activation of both JNK and p38MAPK signaling under hydrogen peroxide stimulation [
48]. Moreover, similar to our results, Wang et al. found that aberrant JNK inactivation was observed in Annexin A2-knockdown cells [
13]. Although JNK activation can be a positive step in the events leading a cell towards apoptosis, there are also many reports stating the opposite that activation of JNK can inhibit apoptosis and promote proliferation [
49]. Specially, activation of JNK confers drug resistance of DNA-damaging agents [
50‐
52]. Moreover, JNK is often activated in lung cancer and promotes oncogenic transformation via negatively regulating p53 through c-Jun [
26,
27,
53]. Indeed, we found that activation of JNK/c-Jun signaling is essential for Annexin A2-mediated p53 suppression, as well as drug resistance. In addition, it has been shown that epidermal growth factor receptor (EGFR) activation ultimately activates JNK signaling [
54,
55]. Notably, Annexin A2 interacts with EGFR at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking Annexin A2 function suppressed the EGF-induced EGFR tyrosine phosphorylation, as well as inhibited the EGFR-dependent downstream signaling pathways [
56]. Therefore, EGFR may be a candidate to mediate Annexin A2-assocated JNK activation. This hypothesis needs further investigation.