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Lung cancer remains one of the most common cancer-associated mortalities worldwide, and platinum-based doublet chemotherapies are recommended as the first-line treatment for advanced non-small cell lung cancer (NSCLC). However, the frequent development of multidrug resistance, to cisplatin regimens in particular, is a major cause of chemotherapy failure in patients with aggressive NSCLC. Wnt/β-catenin signaling and sex-determining region Y box 2 (Sox2) have been implicated in the development and progression and resistance to epidermal growth factor receptor-targeting therapy in lung cancer. The present study aimed to explore the effects of Wnt/β-catenin and Sox2 signaling on the chemoresistance of cisplatin-resistant lung cancer cells by assessing the effects of Sox2 on Wnt/β-catenin signaling activity, cell migration, invasion and clonogenicity, and susceptibility to cisplatin in lung adenocarcinoma A549 cells and cisplatin-resistant A549/DDP cells. The results demonstrated that an enforced expression of Sox2 led to inhibition of Wnt/β-catenin signaling activity, potentially by upregulating glycogen synthase kinase 3 β in A549 and A549/DDP cells. An overexpression of Sox2 promoted cell migration and invasion, in addition to enhancing the clonogenic capacity in A549 cells. Notably, knockdown Sox2 using short hairpin RNA led to an enhanced susceptibility of A549 and A549/DDP cells to cisplatin, along with increased cell apoptosis. The present study thus suggests that Sox2 may be an important regulator in development of chemoresistance of lung cancer cells and may be a novel therapeutic target for treatment chemoresistant lung cancer.
Lung cancer is a respiratory system malignancy with high mortality and its incidence has increased in recent years (
The platinum-based doublet chemotherapy has been recommended as the first-line therapy for advanced non-small cell lung cancer (NSCLC) and has a 20% response rate in patients with NSCLC (
The Wnt/β-catenin signaling pathway has been recognized as an oncogenic pathway with pivotal roles in numerous types of cancer, and aberrant activation of Wnt signaling was detected in 50% of human NSCLC cell lines and resected lung cancer samples (
The sex-determining region Y box-containing (Sox) family of transcriptional factors have emerged as potent modulators in embryonic development, stem cell maintenance, tissue homeostasis and carcinogenesis in numerous processes. A previous study (
A previous study (
The present study therefore attempted to investigate the potential role of Sox2 in Wnt/β-catenin signaling and the chemoresistance of NSCLC cells to cisplatin. The results suggest that Sox2 may be involved in the chemoresistance of NSCLS to platinum-based doublet chemotherapy.
A549 lung cancer cell line (cat. no. CCL-185) was purchased from American Type Culture Collection (Mannasas, VA, USA). The cisplatin-resistant A549/DDP cell line was purchased from the Bank of Cancer Cell Lines of the Chinese Academy of Medical Science (Beijing, China) and its drug resistance phenotype was maintained in a medium containing 10 nM cisplatin (Cayman Chemical Company, Ann Arbor, MI, USA). The cells were cultured and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air in Dulbecco's modified Eagle medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.).
To generate a plasmid capable of overexpressing Sox2 in mammalian cells, human Sox2 cDNA (NM_003106) was cloned into the pcDNA3.1 backbone plasmid downstream of a cytomegalovirus (CMV) promoter (Invitrogen; Thermo Fisher Scientific, Inc.), which was referred to as pCDNA-Sox2. To construct a plasmid able to inhibit Sox2 expression, a small hairpin RNA (shRNA) construct was generated by annealing the sense oligonucleotide, 5′-CCGGCGCTCATGAAGAAGGATAACTCGAGTTATCCTTCTTCATGAGCGTTTTTG-3′, and the anti-sense oligonucleotide5′-AATTCAAAAACGCTCATGAAGAAGGATAACTCGAGTTATCCTTCTTCATGAGCG-3′. The resulting double stranded shRNA was cloned into a GV248 vector (Shanghai GenePharma Co., Ltd., Shanghai, China). The canonical Wnt reporter plasmid carrying a tandem of 7 T cell factor (TCF) binding sites upstream of a minimal c-fos promoter driving the firefly luciferase gene (BATflash) and its control plasmid (BOTflash, containing mutated TCF binding sites) were produced by EMD Millipore (Billerica, MA, USA). The transfection of control plasmid expressing Renilla luciferase (RL) from (Promega Corporation, Madison, WI, USA) was used for assessing the transfection efficiency. The plasmid DNA transfection was performed using Lipofectamine LTX reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Cells transfected with plasmid pcDNA3.1 served as the untreated control. To investigate the effect of Sox2 on the chemoresistance of lung cancer cells to cisplatin, the transfected A549 or A549/DDP cells were then exposed to culture medium containing cisplatin at a final concentration of 10 µM for 24 h prior to being harvested for analysis. Control cells were untreated with cisplatin. The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.
Cell proliferation was determined by using an MTT cell proliferation kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). A549 or A549/DDP cells were cultured in a 6-well plate and transfected with pcDNA3.1 control plasmid or plasmids expressing Sox2 or Sox2 shRNA (shSox2) for 12 h, then the cells were divided and seeded into a 96-well plate at a density of 2×104 per well and allowed to adhere overnight. The cells were then used for MTT assay at indicated time points following the manufacturer's protocol (Beijing Solarbio Science & Technology Co., Ltd.).
Wnt/β-catenin signaling was assessed using a dual luciferase reporter assay, which was determined using a Dual-Glo Luciferase Assay System (Promega Corporation, Madison, WI, USA) on a 20/20n Luminometer (Turner Designs, Sunnyvale, CA, USA) according to the manufacturer's protocols. The A549 or A549/DDP cells were cultured in a 24-well plate and transfected with plasmid BATflash or BOTflash for 24 h. The activity of Wnt/β-catenin was assessed by determining the relative activity of firefly luciferase. The transfection efficiency was assessed by luciferase activity of the co-transfected RL plasmid, pCMV-RL (Promega Corporation).
The A549 or A549/DDP cells transfected with a plasmid expressing Sox2 or shSox2 were seeded at 80% confluence and exposed to cisplatin for 24 h (cells were cultured to confluence) in 6-well culture plates. The cells were then scratched with a 200 µl pipette tip. The resultant unattached cells were removed by washing with pre-warmed PBS three times and the wounded monolayers were cultured for an additional 24 h prior to staining with 0.1% crystal violet solution. The closure of the wounded areas was observed under a light microscope at ×40 magnification and images were captured. The distance of closure and unrecovered area were quantified with the NIH Image J image processing program version 1.46 (National Institutes of Health, Bethesda, MD, USA). The experiments were performed in triplicate. Each condition was tested in duplicate and each experiment was repeated at least three times.
A clonogenic assay was used for assessing the degree of stemness of the A549 and A549/DDP cells. For clonogenicity, 1–2×103 of cells treated under the different conditions were seeded onto separated 35 mm dishes. Cells were continuously cultured for 10 days with a refreshment of an appropriate medium (e.g., containing 10 nM cisplatin for cisplatin-resistant cells or regular medium for cisplatin-sensitive cells) at intervals of 3 days. For colony counting, the medium was removed and the cells were rinsed with PBS prior to being fixed with 4% paraformaldehyde at room temperature for 5 min. Following the removal of the fixative, the cells were then stained with 0.5% crystal violet solution and incubated at room temperature for 30 min. The staining solution was carefully removed, and the cells were rinsed with H2O to remove residual staining solution, prior to air-drying the sample at room temperature for up to a day. The number of colonies were counted and calculated under a light microscope. Each condition was tested in duplicate and each experiment was repeated three times.
In order to assess the effect of Sox2 on cisplatin-resistant lung cancer cells, the invasive capacity of A549 or A549DDP cells transfected with the plasmid either expressing Sox2 or shSox2 was ascertained by a Transwell assay using Transwell migration chambers (BD Biosciences, Franklin Lakes, NJ, USA). The 8-µm filters were coated with 100 µl Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), which was diluted to 1:2 of concentration using serum-free 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), and incubated at 37°C in a 5% CO2 atmosphere for 30 min for gelling. A total of 104 cells resuspended in 100 µl DMEM basal medium were seeded in the upper chamber, and 700 µl of DMEM medium supplemented with 10% FBS was added in the lower chamber. The culture was then incubated at 37°C in a 5% CO2 atmosphere for 12 h. The medium was then removed and the cells were washed twice with cool PBS. Cells were then fixed with 4% paraformaldehyde for 20 min, prior to being stained with 1% crystal violet for 20 min. The crystal violet was removed from the top of the membrane with a pipette tip or cotton tipped applicator, then the rinsed the Transwell membrane with distilled water to remove the excess crystal violet and allowed to dry for a day. The number of cells in 10 different fields of view was counted under a Upright light microscope (Leica DM4B, equipped with DFC450 camera, Leica, Shanghai, China) to obtain an average sum of cells that had migrated from the top of membrane toward the basolateral side of the membrane. The percentage of invasive cells was calculated as (the average sum of cells attached on basolateral membrane/the average sum of cells (attached on the top membrane + attached on basolateral membrane) ×100%.
Cell apoptosis was assessed by Annexin V analysis using flow cytometry. For flow cytometry, cells were detached and labeled using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (NeoBioscience Technology Co., Ltd., Beijing, China) according to the manufacturer's protocol. Apoptotic and necrotic cells were quantified using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) and the CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). For each sample, ≥10,000 cells were analyzed. Cells negative for Annexin V and PI were considered to be viable, and cells stained with Annexin V but not PI were considered to be apoptotic.
Whole cell lysates were prepared in a lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40), and cell nuclear proteins were extracted with the NucBuster Protein Extraction kit following the manufacturer's protocol (Novagen; EMD Millipore, Billerica, MA, USA). Whole cell extract or nuclear extract of cells (40 µg) were resolved by a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, followed by being transferred to a PVDF membrane (EMD Millipore). The membranes were blocked with blocking buffer (5% fat-free milk in PBS-0.1% Tween-20) at room temperature for 1 h prior to being probed with the primary antibody at 4°C overnight, followed by being incubated with the appropriate horseradish peroxidase-labeled secondary antibody (Donkey anti-mouse immunoglobulin G, cat. no. 109415; donkey anti-rabbit immunoglobulin G, cat. no. 108894; or donkey anti-goat immunoglobulin G, cat. no. 109291. All secondary antibodies were applied by 1:2,000 dilution in blocking buffer. Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at room temperature for 2 h. The blots were developed using the enhanced chemiluminescence (ECL) reagent (Advansta, Menlo Park, CA, USA) after they were incubated with the appropriate peroxidase labeled secondary antibodies. Antibodies against β-actin (cat. no. sc-8432; 1:1,000 dilution), lymphoid enhancer-binding factor-1 (LEF-1; cat. no. sc-8592; 1:1,000 dilution) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); the antibody to phosphorylated (phos)-catenin (cat. no. ab75777; 1:1,000 dilution) was purchased from Abcam (Cambridge, MA, USA); antibodies against phos-glycogen synthase kinase (GSK)3β (cat. no. 05–413; 1:1,000 dilution), acetyl-Histone H3 (cat. no. 06–942; 1:1,000 dilution) and active β-catenin (cat. no. 05–665; 1:10,000 dilution) were purchased from EMD Millipore; the antibody to GSK3β (cat. no. 610202; 1:1,000 dilution) was purchased from BD Biosciences (San Jose, CA, USA); antibodies against Sox2 (cat. no. 66411; 1:500 dilution), B-cell lymphoma 2 (Bcl-2) (ct. no. 12789; 1:500 dilution), caspase-3 (cat. no. 10380; 1:1,000 dilution), Bcl-2-like protein 4 (Bax; cat. no. 15422; 1:1,000 dilution), myeloid cell leukemia sequence 1 protein (Mcl-1; cat. no. 66026; 1:1,000 dilution) and apoptosis inducing factor (AIF; cat. no. 17984; 1:1,000 dilution) were purchased from Proteintech (Wuhan Sanying Biotechnology, Wuhan, China). The expressions of proteins of interest were semi-quantified by optical densitometry using Image J software version 1.46 (National Institutes of Health, Bethesda, MD, USA). The ratio between the net intensity of each sample divided by the respective internal controls (β-actin or Histone H3) was calculated as densitometric arbitrary units, which served as an index of the relative expression of a protein of interest, the fold of induction for a specific protein under an indicated condition was calculated by comparing its relative expression over the control (
All data collected in this study were obtained from at least three independent experiments for each set of circumstances. SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) and PRISM 5 (GraphPad Software, Inc., La Jolla, CA, USA) were used for statistical analysis. Statistical evaluation of the data was performed by one-way analysis of variance when more than two groups were compared with a single control, and by t-test for comparison of differences between the two groups. P<0.05 was considered to indicate a statistically significant difference Data was presented as the mean ± standard deviation.
In order to examine the potential role of Sox2 in canonical Wnt signaling, lung cancer A549 and cisplatin-resistant A549/DDP cells, enforced expression of Sox2 or shSox2, and the Wnt/β-catenin signaling activity was ascertained in terms of a dual luciferase Wnt reporter assay, in addition to the expression of key components in the Wnt/β-catenin signaling cascade. The results demonstrated decreased Wnt activity in cells transfected with Sox2 compared with control cells (P<0.01), and marginally increased activity of Wnt signaling in A549 and A549/DDP cells which overexpressed shSox2 in comparison with cells transfected with TOPflash and pcDNA3.1 plasmids (
The present study also investigated the effect of Sox2 on cisplatin-mediated cell apoptosis in lung cancer cells. The results from the MTT assay revealed that a transient expression of Sox2 or shSox2 had no effect on cell proliferation, but overexpression of Sox2 may increase the survival rate of A549 cells in the presence of cisplatin, although it had no effect on cisplatin-resistant A59/DDP cells. Notably, a suppression of Sox2 expression by transfection of shRNA led to an increase in the cisplatin-induced cell death in A549/DDP cells (P<0.05;
In order to investigate whether Sox2 has an effect on the metastatic properties of lung cancer cells, the capability of migration and invasion in A549 and A549/DDP cells introduced with a plasmid expressing Sox2 or shSox2 was evaluated by scratch assay (
Since Sox2 is a well-characterized marker of pluripotency of stem cells and CSCs (
Chemotherapy is a common treatment for lung cancer and regimens containing cisplatin remain the main treatment in clinical settings (
Wnt signaling has been recognized as serving multiple functions in cell proliferation and migration, organogenesis and tissue homeostasis (
As an important pluripotent marker of stem cells, Sox2 has been recognized as serving a crucial role in maintaining the properties of cancer stem cells that contribute to resistance to therapeutic agents (
Increasing evidence has suggested that Sox2 expression is associated with the cancer hallmarks of sustained proliferative signaling, activation of invasion and metastasis, and evasion of cell death (
Aside from its role in cancer cell migration and invasion, Sox2 also serves an important role in evading apoptotic signals. In this context, an overexpression of Sox2 may induce the increased apoptotic resistance in prostate cancer cells and xenograft models (
In summary, the present study demonstrated that an overexpression of the Sox2 gene led to the decreased activity of Wnt/β-catenin signaling in lung adenocarcinoma A549 cells and the cisplatin-resistant A549/DDP cells through an upregulation of the Wnt/β-catenin signaling negative regulator GSK3β. Notably, the increased expression of the Sox2 gene was able to promote cell migration and invasion, in addition to enhancing clonogenic capacity in A549 cells. Conversely, a knockdown of Sox2 expression by shRNA led to an enhanced susceptibility of A549 and A549/DDP cells to cisplatin, along with an increased cisplatin-induced apoptosis of cancer cells. The present study therefore suggests that the Sox2 gene may be a novel target for the treatment of chemoresistant lung cancers.
This study was supported by a grant from The Natural Science Foundation of Ningxia (grant no. NZ15277) to Jinxi He, and a starting grant (grant no. XM2015093) from the Ningxia Medical University to Juan Shi.
Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P<0.01), whereas the cells transfected with shSox2 exhibited a moderately enhanced luciferase activity, as compared with cells transfected with BATflash and pcDNA3.1 plasmids (n=9). (B) Molecular analysis by immunoblotting demonstrated a decreased expression of indicated Wnt signaling activators including ABC, LEF-1 and cyclin D1, and an increased expression of Wnt signaling inhibitor, GSK3β, and phos-BC in Sox2-transfected cells. All data are presented as the mean ± standard deviation of at least three independent triplicated experiments. **P<0.01 vs. control. Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA; ABC, active β-catenin; phos-, phosphorylated; BC, β-catenin; GSK3β, glycogen synthase kinase 3β; LEF-1, lymphoid enhancer-binding factor-1.
Sox2 inhibits cisplatin-induced apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P<0.05). All data are presented as the mean ± standard deviation of three independent triplicated experiments (n=9). *P<0.05, **P<0.01 vs. the corresponding non-cisplatin-treated group, #P<0.05 vs. the cisplatin-treated pcDNA3.1-transfected cells. Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
Apoptosis associated proteins determined by an immunoblotting analysis. A549 and A549/DDP cells were transfected with plasmid expressing Sox2 or shSox2, or control pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for additional 24 h prior to being harvested for immunoblotting analysis for indicated proteins. The values labeled on the top of each bands represented the relative expression levels of proteins over their respective pcDNA3.1 control as determined by a densitometric assay. Overexpression of Sox2 demonstrated a trend to reduce the expression of pro-apoptotic proteins (caspase-3, Bax), but increased the expression of anti-apoptotic proteins Bcl-2 in lung cancer cells. Cas 3: caspase-3; Bax, Bcl-2-like protein 4; AIF, apoptosis inducing factor; Bcl-2, B-cell lymphoma 2; Mcl-1, myeloid cell leukemia sequence 1 protein; C, control; Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA.
Effect of Sox2 on the migration of lung cancer cells
Effect of Sox2 on the invasion of lung cancer cells
Sox2 enhances the stemness of lung cancer cells determined by a clonogenic assay. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid and their capacity for clone formation was analyzed using a clonogenic assay in 35 mm dishes. (A) Representative images of clonogenic assay for A549 cells (left) and its relevant quantification of the number of colonies (right). (B) Representative images of clonogenic assay for A549/DDP cells (left) and its relevant quantification of the number of colonies (right). An overexpression of Sox2 demonstrated an ability to enhance the clone formation in A549 and A549/DDP cells, and a shRNA-mediated knockdown of Sox2 marginally reduced the clone formation. Data represented the mean ± standard deviation from three independent triplicated experiments (n=9). **P<0.01 vs. pcDNA3.1 group. Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA; C, control.