Role of transcription factor FOXA1 in non‑small cell lung cancer
- Authors:
- Published online on: October 26, 2017 https://doi.org/10.3892/mmr.2017.7885
- Pages: 509-521
Abstract
Introduction
Among malignant tumours, lung cancer poses the greatest threat to human health, and non-small cell lung cancer (NSCLC) accounts for 85–90% of all lung cancer cases. Metastasis is present in the majority of patients with NSCLC upon diagnosis, and surgery is an option in only ~20% of cases. Local and distal NSCLC metastases are the major causes of treatment failure (1). There is currently no effective prophylactic treatment against NSCLC metastasis available. Therefore, it is important to investigate the mechanisms underlying the invasion and metastasis of NSCLC.
In our previous study, the invasive/metastatic potential of NSCLC cells were analysed using an in vitro tumour cell invasion assay (2). Transwell inserts were used, and a Transwell membrane with an appropriate pore size was coated with basement membrane extract (BME). Those cells with a high invasive/metastatic potential migrated to the lower surface of the membrane or to the lower chamber. By repeated screening, stable subpopulations of high/low invasive potential (H/L-INV) were obtained from the A549 lung cancer cell line, and from prostate, breast and colon cancer cell lines (Fig. 1A). Analysis of the H/L-INV A549 cells revealed that the H-INV subpopulation exhibited the typical cancer stem cell phenotype (CD24low/CD44+ and CD133), but the L-INV subpopulation did not (Fig. 1B).
In the present study, microarray analysis of the H/L-INV A549 subpopulations was performed to evaluate genes associated with high invasiveness, and Forkhead box protein A1 (FOXA1) was selected for further investigation. The expression levels of FOXA1 in primary lesions and metastatic lymph nodes were assessed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, the mRNA and protein expression levels of FOXA1 were examined in H-INV A549 cells transfected with a specific FOXA1 small interfering RNA (siRNA), and the role of FOXA1 in proliferation, invasion and metastasis in the NSCLC cells was evaluated.
Materials and methods
Cell culture
The human A549 lung cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The H/L-INV A549 cells were obtained by repeated Transwell screening and routinely cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS) and penicillin/streptomycin (all from Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The cells were incubated at 37°C in 5% CO2.
Gene microarray
Total RNA was extracted from the H/L-INV A549 cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The mRNA was purified using the RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) and reverse transcribed into cDNA, which was transcribed to biotin-labelled cRNA using T7 DNA polymerase (Invitrogen; Thermo Fisher Scientific, Inc.). The cRNA samples were fragmented into fragments of between 50 and 100 nt in fragmentation buffer (Invitrogen; Thermo Fisher Scientific, Inc.). The fragmented cRNA was dissolved in hybridization buffer (Invitrogen; Thermo Fisher Scientific, Inc.) and hybridised with the GeneChip (Illumina, Inc., San Diego, CA, USA) at 45°C for 16 h. The chip was then washed and stained according to the manufacturer's protocol and scanned using an Illumina BeadArray reader. Microarray Suite 5.0 (Affymetrix, Inc., Santa Clara, CA, USA) was used to comprehensively analyse and compare the microarray data.
To identify the genes with high invasive/metastatic potential, genes with significantly different expression levels between H-INV A549 and L-INV A549 were examined. The gene sets with ≥2-fold differences in mRNA levels are shown in Tables I and II.
NSCLC specimen collection
A total of 40 pairs of primary tumour tissues and corresponding metastatic lymph nodes were collected from patients who underwent tumour resection at Hangzhou Hospital Affiliated to Nanjing Medical University (Hangzhou, China) between 2014 and 2015. The tissues were confirmed to be NSCLC by post-operative pathological evaluation. The fresh specimens were frozen in liquid nitrogen and stored at −80°C. The present study was approved by the ethics committee of Nanjing Medical University and was performed with the provision of written informed consent from patients.
RT-qPCR analysis
Total RNA was extracted from the tissues and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesised from 1 µg of total RNA and used as a template in a 50-µl reaction using TaqMan RT reagents according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). The RT-qPCR was performed to amplify genes from the cDNA template with gene-specific primer sets. The following PCR primers were used: FOXA1, forward 5′-TAATCATTGCCATCGTGTGCTT-3′ and reverse 5′-ATAATGAAACCCGTCTGGCTA-3′; GAPDH, forward 5′-ATCCCATCACCATCTTCCAGGAGCG-3′ and reverse 5′-AAATGAGCCCCAGCCTTCTCCATG-3′. To avoid amplifying genomic DNA, gene primers were selected from different exons. The reaction was performed in a total reaction volume of 50 µl, which contained 2 µl of cDNA solution, 0.2 µM sense and antisense primers, 25 µl GoTaq qPCR Master mix (Promega Corporation, Madison, WI, USA) and DEPC-treated water. The amplification conditions were as follows: Pre-denaturation at 95°C for 10 min, followed by 35–40 cycles of denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 1 min. The relative expression level of FOXA1 was calculated using the comparative Cq (ΔΔCq) method (expression fold value=2−ΔΔCq) (3), using GAPDH as the internal reference. Each sample was measured in triplicate.
H-INV A549 transfection with siRNA
FOXA1 siRNA and the negative control siRNA were purchased from Biotend (Shanghai, China). The siRNA sequences were as follows: FOXA1 siRNA-1: 5′-GUACUACCAAGGUGUGUAUdTdT-3′; FOXA1 siRNA-2: 5′-CUGUCCUUCAAUGACUGCUdTdT-3′; FOXA1 siRNA-3: 5′-CGUCCUUCAACAUGUCCUAdTdT-3′. The cells were divided into three groups: Non-transfected, Ctrl-siRNA and FOXA1-siRNA. In vitro transfections were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were seeded in 6-well plates in 1,500 µl of RPMI-O-MEM without antibiotics or FBS (1.5×106 cells/well). Upon reaching 30–50% confluence, the cells were transfected with 500 µl of transfection mixture containing 20, 30 or 50 nM siRNA. The cells were washed 6 h following transfection and harvested at 24 or 48 h post-transfection for subsequent experiments.
Western blot analysis
Total proteins were extracted from the cells of the three groups described above 48 h following transfection. The cell lysates were centrifuged at 16,000 × g for 10 min at 4°C, and the supernatant was collected and stored at −20°C. The protein concentration was determined using a BCA assay kit (Pierce; Thermo Fisher Scientific, Inc.), and 50 µg of protein was loaded into each well and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred onto a nitrocellulose membrane (Immobilon-P; EMD Millipore, Bedford, MA, USA) in an ice bath at 80 V. Subsequently, the membrane was blocked using 5% skim milk (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and incubated with 1:1,000 dilutions of either rabbit FOXA1 antibody (cat no. 58613; Cell Signaling Technology, Inc., Danvers, MA, USA) or rabbit β-actin antibody (cat no. 4970; Cell Signaling Technology, Inc.) as the primary antibody overnight at 4°C. Following washing with Tris-buffered saline solution containing 1% Tween-20 the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (cat no. 4412; dilution 1:5,000; Cell Signalling Technology, Inc.) at room temperature for 1 h. Finally, the proteins were detected using enhanced chemiluminescence (GE Healthcare Life Sciences, Upsulla, Sweden). The molecular mass (kDa) of the proteins was determined using the prestained protein marker (Bio-Rad Laboratories, Inc., Hercules, California, USA). The blot image was analysed using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). FOXA1 and β-actin IOD values were obtained, and the relative value of the target protein was indicated by the IOD ratio of the target protein to β-actin in the same sample. This experiment was repeated three times.
In vitro Transwell invasion and migration assays
Each 8-µm insert membrane (Falcon; BD Biosciences, Franklin Lakes, NJ, USA) was coated with 50 µl of BME gel (Tervigen, Gaithersburg, MD, USA) and incubated overnight at 37°C. The non-transfected, FOXA1-siRNA (24 h post-transfection) and Ctrl-siRNA cells were subjected to the assay in triplicate. The cell suspension was adjusted to 2×105 cells/ml in RPMI-1640 with 0.1% FBS, and 200 µl of cell suspension was added to each Transwell. The lower compartment contained 600 µl of RPMI-1640 with 10% FBS. After 48 h, the cells on the upper surface of the membrane were wiped off, and the membrane was fixed in methanol for 15 min, followed by staining with 1% rystal violet for 15 min. Using a CX31 microscope (Olympus Corporation, Tokyo, Japan), five fields were randomly selected (magnification, ×100) on each membrane and the number of the cells which had crossed the membrane were counted, with the average being calculated. The invasive potential of the tumour cells was measured using the relative invasion index (%), which was calculated as follows: Relative invasion index (%) = (invading cell count of transfected cells/invading cell count of non-transfected cells) ×100%. To compare the migration ability of the three groups of cells, the experiment was performed in the same manner with the same method for counting following incubation for 24 h, but without the BME gel coating on the Transwell membrane.
Scratch wound assay
The non-transfected, FOXA1-siRNA and Ctrl-siRNA cells were seeded in 6-well plates (3×106 cells/well). At 24 h post-transfection, a scratch was created across the bottom surface of each well with a sterile 200-µl pipette tip. The detached cells were gently washed off with PBS, and the remaining cells were cultured with serum-free RPMI-1640. The cells along the scratch edges were observed under a CX31 microscope (Olympus Corporation) at 0, 24 and 48 h post-scratch. The width of the scratch was measured at these time points, and the average scratch healing rate was calculated. The scratch healing rate was calculated as follows: Scratch healing rate (%) = (scratch width at 0 h-scratch width at 48 h)/scratch width at 0 h ×100%. This experiment was repeated three times.
MTS colorimetric assay
The non-transfected, FOXA1-siRNA (24 h post-transfection) and Ctrl-siRNA cells were seeded in 96-well plates at a density of 8,000 cells/100 µl/well. At 24, 48, 72 and 96 h post-seeding, 20 µl of MTS (Promega Corporation, Madison, WI, USA) solution was added to each well and incubated for 1 h. The absorbance at 490 nm was measured on a plate reader. The growth inhibition rate was calculated as follows: Growth inhibition rate = (control group absorption-experiment group absorption)/control group absorption. This experiment was repeated three times.
Cell cycle analysis using flow cytometry
The non-transfected, FOXA1-siRNA (24 h post-transfection) and Ctrl-siRNA cells (1×106 each) were collected and washed in PBS. The cells were fixed and stained using a cell cycle staining kit (Multisciences Biotech Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Flow cytometric analysis was performed using a BD FACSCalibur flow cytometer (BD Biosciences) equipped with a 488-nm argon-ion laser. This experiment was repeated three times.
Statistical analysis
Data were analysed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation. Significant differences among multiple groups were analysed using one-way analysis of variance and the significance of pair-wise differences was analysed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of FOXA1 is high in the H-INV A549 subpopulation
The microarray analysis revealed 450 differentially expressed genes with ≥2-fold changes between the H-INV and the L-INV subpopulations. Among these genes, 297 and 153 genes were expressed at low and high levels, respectively, in the H-INV subpopulation. The results of the preliminary microarray data analysis are shown in Fig. 2 and Tables I and II. FOXA1 was expressed at a high level in the H-INV subpopulation of A549 cells, and the level of expression was 3-fold higher, compared with that in the L-INV cells (P=5E-10).
Expression of FOXA1 is higher in metastatic lymph nodes, compared with NSCLC primary tumours
The mRNA expression of FOXA1 in 40 primary NSCLC tumours and 40 corresponding metastatic lymph nodes were examined using RT-qPCR analysis. FOXA1 mRNA was expressed in the primary NSCLC tumours and metastatic lymph nodes, and expression was higher in the metastatic lymph nodes, compared with that in the corresponding primary tumour tissues (P<0.05; Fig. 3).
mRNA expression of FOXA1 is reduced in FOXA1-siRNA transfected cells
The H-INV A549 cells were transfected with 20, 30 or 50 nM FOXA1 siRNA-1/2/3, and the mRNA expression of FOXA1 in each group was measured using RT-qPCR analysis 24 and 48 h following transfection. As shown in Fig. 4A, the mRNA expression level of FOXA1 was lowest in the cells transfected with FOXA1-siRNA-2 (30 nM; 24 h post-transfection; 0.485±0.007), which was significantly lower, compared with level in the non-transfected group (1.015±0.062; P<0.05) and the Ctrl-siRNA group (1.027±0.082; P<0.05). There was no significant difference between the non-transfected and Ctrl-siRNA groups. On the basis of the above results, FOXA1-siRNA-2 was selected for use in subsequent experiments at the optimal transfection concentration of 30 nM and examination duration of 24 h post-transfection.
Transfection with FOXA1-siRNA leads to a decrease in the protein expression of FOXA1
The results of the western blot analysis showed that the protein expression of FOXA1 was significantly reduced in the FOXA1-siRNA transfected H-INV A549 cells 48 h following transfection, compared with the expression levels in the non-transfected and Ctrl-siRNA-transfected cells (P<0.05; Fig. 4B). There was no significant difference between the non-transfected and Ctrl-siRNA groups. This result confirmed that FOXA1-siRNA reduced the protein expression of FOXA1 in the H-INV A549 cells.
Transfection with FOXA1-siRNA reduces the invasion and migration abilities of H-INV A549 cells
The results of the Transwell invasion assay showed that the number of invading cells in the FOXA1-siRNA group was 40.60±0.89, with an invasion index of 59±0.37%, whereas the number of invading cells in the Ctrl-siRNA group was 70.40±1.22, with an invasion index of 96±0.46%. The invasive potentials of the FOXA1-siRNA and Ctrl-siRNA-transfected cells were significantly different (P<0.05; Fig. 5A). This result indicated that downregulation of the gene expression of FOXA1 reduced the invasiveness of the metastatic A549 cells.
The Transwell migration assay showed that the numbers of cells crossing the membrane were 25.20±0.35, 82.77±0.56 and 79.72±0.28 in the FOXA1-siRNA, non-transfected and Ctrl-siRNA groups, respectively. The number of cells crossing the membrane was significantly lower in the FOXA1-siRNA group, compared with that in the Ctrl-siRNA and non-transfected groups (P<0.05; Fig. 5B). This result demonstrated that FOXA1 siRNA effectively reduced the migration ability of the H-INV A549 cells in vitro.
In the scratch wound assay, no significant differences were found in the scratch healing rates within 48 h post-scratching between the non-transfected group and the Ctrl-siRNA group (35.34±6.68 and 34.45±4.08%, respectively). By contrast, the healing rate in the FOXA1-siRNA cells was 19.66±5.05%, revealing significantly reduced migration ability (Fig. 6).
FOXA1-siRNA decreases H-INV A549 proliferation activity
The MTS assay showed that transfection with FOXA1-siRNA (24 h post-transfection) led to significant growth inhibition at 24, 48 and 72 h (P<0.05; Table III).
FOXA1-siRNA induces G0/G1 arrest in H-INV A549 cells
Cell cycle was assessed using flow cytometry 24 h following transfection. As shown in Table IV, 49.31±3.20% of the non-transfected cells and 49.69±3.51% of the Ctrl-siRNA transfected cells were in the G0/G1 phase, with no significant difference between these two groups. By contrast, the FOXA1-siRNA group exhibited a significantly higher percentage of cells in the G0/G1 phase (58.99±3.20%; P<0.05), suggesting that FOXA1-silencing induced G0/G1 arrest in the H-INV A549 cells (Fig. 7).
Discussion
In terms of lung cancer-associated mortality, ~90% of cases are due to tumour cell invasion and metastasis (3). Distal metastasis is already present in ~40–50% of patients with lung cancer patients at the time of diagnosis and develops in the remaining 50–60% of patients during the course of treatment (4). Clinical data indicate that ~30% of patients with late-stage NSCLC who receive the targeted drug epidermal growth factor receptor tyrosine kinase inhibitor develop intracranial metastasis during the course of treatment (5,6), representing one of the major causes of treatment failure of late-stage NSCLC-targeted molecules. Although there has been progress in elucidating the molecular mechanisms underlying lung cancer metastasis, successful translation into clinical application has been limited. Therefore, it is important to investigate the molecular mechanisms underlying lung cancer metastasis in a stable and effective model to identify biomarkers potentially associated with lung cancer metastasis, and to ensure effective prevention and treatment of lung cancer metastasis.
Based on its significantly high expression in the H-INV subpopulation of A549 cells, FOXA1 was selected in the present study for investigation in subsequent experiments. FOXA1 contains a forkhead (or winged helix) DNA-binding domain of ~100 amino acids and is a member of the pioneer FOXA transcription factor family. The transcription factor FOXA1 binds to the chromosome and induces nucleosome remodelling to facilitate the binding of other transcription factors on the chromosome to initiate tissue-specific transcriptional programmes (7–11). Previous studies have identified FOXA1 as either a pro- or anti-tumourigenic factor in specific human malignancies. For example, 40% of breast carcinoma cases and up to 80% of estrogen receptor-positive breast carcinoma are positive for FOXA1, and the expression of FOXA1 is associated with improved prognosis (12). In endometrial cancer, FOXA1 also functions as a tumour suppressor in cancer progression (13). By contrast, the expression levels of FOXA1 in prostate cancer are positively correlated with tumour size, extraprostatic extension and lymph node metastasis, and negatively correlated with patient survival rates (14). In pancreatic cancer, the loss of FOXA1 is necessary and sufficient for epithelial to mesenchymal transition during cancer progression (15). The overexpression and amplification of FOXA1 have also been observed in oesophageal, colorectal and thyroid cancer, and FOXA1 is considered a potential oncogene (16–18). In addition, Deutsch et al reported that the expression of FOXA1 in squamous cell carcinoma of the lung was associated with distant metastasis and an unfavourable survival rate; it was also found that the expression of FOXA1 in brain metastasis samples from patients with squamous cell cancer was marginally higher, compared with that in non-matched primary tumours (56 vs. 43%) (19). In the present study, the combined analysis of all tumour samples confirmed that FOXA1 mRNA was expressed in the primary lesions and metastatic lymph nodes, with higher expression levels in the metastatic lymph nodes, compared with the primary lesions. This suggested that FOXA1 is important in the tumourigenesis and progression of NSCLC.
The present study further demonstrated the role of FOXA1 in the invasion, migration and proliferation of NSCLC cells in vitro. Using the A549 NSCLC cell line, the importance of FOXA1 in NSCLC metastasis was confirmed. In addition, the proliferation assay and flow cytometric analysis revealed the reduced proliferation of FOXA1-siRNA cells due to cell cycle arrest at the G0/G1 phase, suggesting that FOXA1 affected the transformation of tumour cells. FOXA1 has also been shown to promote epithelial to mesenchymal transition in A549 NSCLC cells (20), and the overexpression of FOXA1 inhibits the pro-apoptotic, anti-invasive and anti-migratory capacities of miR-194 in H1299 and A549 NSCLC cells (21). FOXA1 also promotes the migration and invasion of H1299, PC9 and A549 lung adenocarcinoma cancer cells (22).
In conclusion, the results of the present study suggested that FOXA1 is a potential oncogene in NSCLC; therefore, specific interference of the expression of FOXA1 may represent a novel approach for the treatment of NSCLC.
Acknowledgements
The present study was supported by the Major Science and Technology Innovation Project of Hangzhou (grant no. 20112312A01 to Professor Shenglin Ma), the Zhejiang Medical Science Foundation of China (grant no. 2014KYA178 to Mrs. Shirong Zhang), the Hangzhou Key Disease and Discipline Foundation of China (grant no. 20140733Q15 to Mrs. Shirong Zhang) and the Zhejiang Provincial Natural Science Foundation of China (grant no. LY15H160010 to Mrs. Shirong Zhang).
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