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Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system (RAS). ACE2 plays a critical counterbalancing role by degrading angiotensin II (Ang II) to Ang 1–7. Recent studies suggest that RAS influences tumor growth and development by its paracrine effects on the tumor microenvironment. Epithelial-mesenchymal transition (EMT) is now thought to be a process that plays a fundamental role in tumor progression and metastasis. In the present study, we investigated the role of ACE2 in lung cancer metastasis and the mechanism of EMT. This is the first study to elucidate the mechanism through which the overexpression of ACE2 in the A549 lung cancer cell line decreases metastasis formation
Lung cancer continues to be the leading cause of death due to cancer in Western countries. In 2012, lung cancer was estimated to account for approximately 26% of female and 29% of male deaths due to cancer (
The process of epithelial-mesenchymal transition (EMT) plays a fundamental role in tumor progression and formation of metastasis. In EMT, epithelial tumor cells with a cobblestone phenotype acquire mesenchymal cell characteristics with a spindle/fibroblast-like morphology (
In recent years, the role of the renin-angiotensin system (RAS) in tumor progression or metastasis has been extensively characterized (
As we previously reported, decreased local expression of ACE2 was shown to correlate with the progression of lung cancer while transcriptional overexpression of ACE2, using an adenoviral-mediated plasmid, reduced invasion and angiogenesis in A549 cells
F-12K nutrient mixture (F-12K) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-β-actin antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mouse monoclonal anti-E-cadherin and mouse monoclonal anti-vimentin were from BD Biosciences (San Diego, CA, USA). Goat polyclonal antibody to ACE2 was from R&D Systems. Rabbit polyclonal antibody to αSMA was from Abcam (Cambridge, MA, USA). DX600 was from AnaSpec (San Jose, CA, USA).
A549 cells were purchased from the Cytology Center of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in F-12K nutrient mixture (F-12K; Gibco, Carlsbad, CA, USA) containing 10% FBS and 100 U/ml each of penicillin/streptomycin at 37°C in 5% CO2. The media were replaced every 48 h. In order to examine cell survival, cells were cultured up to 70% confluence and serum-starved overnight. They were then treated with TGF-β1 in culture medium containing 1% FBS for 72 h. DX600, which is a specific inhibitor of ACE2, was added to the culture 30 min before the addition of TGF-β1.
To analyze the function of ACE2, we established stable clones of A549 cells overexpressing ACE2 (A549-ACE2). The transfection procedure was performed as previously reported (
The mRNA levels of Twist, Snail2, ZEB1 and E-cadherin were examined by RT-PCR. The procedure of RT-PCR was performed as previously reported (
Cultured A549 cells (106–107) were washed with cold phosphate-buffered saline (PBS; Mediatech) three times and disrupted in 0.2 ml ice-cold cell lysis buffer containing 10% phenylmethanesulfonyl fluoride (PMSF). After incubation for 5 min at room temperature, cells were scraped from the 6-well plates. Total cell lysates were sonicated, and insoluble materials were removed by centrifugation at 13,000 rpm for 15 min at 4°C. Protein concentrations were determined by the Bradford method (Bio-Rad, Herts, UK). Equal amounts of protein (10 μg) were separated on an 8% SDS-polyacrylamide gel (Bio-Rad). After electrophoresis, separated proteins were transferred onto immunoblot polyvinylidene difluoride (PVDF) membranes (Merck Millipore, USA). Membranes were then blocked with 5% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. Primary antibodies were added to TBST, and membranes were incubated overnight at 4°C on a rocking platform. Primary antibodies used were; mouse anti-β-actin (1:3,000) from Santa Cruz Biotechnology Inc., mouse monoclonal anti-E-cadherin (1:1,500) and mouse anti-vimentin (1:1,500) from BD Biosciences. Rabbit polyclonal to αSMA (1:500) was from Abcam. After three washing steps with TBST (15 min each), membranes were probed with the corresponding anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. Membranes went to a second stage of washing in TBST, three times each for 15 min. Immunoblots were visualized by enhanced chemiluminescence (GE Healthcare, Chalfont St. Giles, UK). β-actin band density was used as a loading control, and results were digitalized and quantified using ImageJ software.
Six-week-old male athymic nu/nu mice were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China) and maintained under specific pathogen-free conditions. Animals were maintained in a temperature-controlled room (at 22°C) and supplied with food and water. A single-cell suspension containing 2×106 cells in 0.1 ml phosphate-buffered saline was injected into the lateral tail veins of nu/nu mice. There were six mice per group. Three weeks after treatment, mice were sacrificed and then lung, liver and brain tissues were extracted, fixed with 4% formaldehyde, and the number of metastatic colonies was counted under a dissecting microscope. For the lung cancer xenograft model, wild-type or ACE2 overexpressing A549 cells (2×107 in 0.1 ml PBS) were transplanted subcutaneously. Four weeks later, mice were sacrificed, and tumor tissues were harvested and fixed with 4% formaldehyde. Tumor and lung tissue extracts were analyzed by hematoxylin and eosin (H&E) staining and immunohistochemical analysis.
Immunohistochemical analyses were performed as previously described. Briefly, all tissue samples were fixed in phosphate-buffered neutral formalin, embedded in paraffin and cut into 5-μm serial sections. After being deparaffinized in xylene, tissue sections were rehydrated in graded ethanol solutions, permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate and incubated overnight with primary antibodies. Immunohistochemical staining with antibodies to E-cadherin (1:50), vimentin (1:25, both from BD Biosciences) and ACE2 (1:50, R&D Systems) was performed according to standard procedures. Results were observed and photographed under a fluorescence microscope (Leica, Germany) with Image-Pro Plus 6.0 software (Media Cybernetics). Specimens were classified as positive when >10% of cancer cells were stained. The intensity of each type of staining was graded as negative or positive microscopically as previously reported (
Data are presented as averages and their respective standard deviation (means ± SEM). All statistical analyses were performed with the SPSS Statistical Program (version 17.0; SPSS, Chicago, IL, USA). Comparisons of data between two groups were conducted with the Student’s t-test. P-values of <0.05 were considered to indicate statistically significant results.
Our previous study showed that the overexpression of ACE2 inhibited the proliferation of lung cancer cells
EMT has been recently found to play a critical role in tumor development, particularly during the induction of metastasis (
We then evaluated the doubling times of A549-C and A549-ACE2 cells
We aimed to ascertain whether A549 cells overexpressing ACE2 had a differential expression pattern of genes involved in the induction of EMT. ACE2 overexpression was correlated with decreased mRNA levels of transcripts such as Snail2, ZEB1 and Twist, which are causally involved in the EMT process (
The upregulatory effect of ACE2 on E-cadherin expression in lung cancer cells led us to explore the direct impact of ACE2 on EMT. To further investigate whether ACE2 suppresses the EMT process, a classical EMT model was developed using A549 cells stimulated with TGF-β1. The presence of ACE2 in A549 cells attenuated the decrease in the levels of E-cadherin due to TGF-β1 treatment. In addition, ACE2 significantly abrogated the upregulation of mesenchymal markers, including vimentin and αSMA (
An ACE2 antagonist was used to further confirm the effect of ACE2 on EMT. Pretreatment of A549 cells with DX600 (at 106 M) abolished the increase in E-cadherin expression caused by ACE2. Furthermore, the upregulation of αSMA caused by TGF-β1 treatment was recovered in A549-ACE2 cells following treatment with DX600 (
In order to mechanistically investigate the role of ACE2 in the regulation of transcriptional repressors, including Snail2 and ZEB1 during the process of EMT, we determined the transcriptional repertoire of A549 cells following treatment with or without TGF-β1. We found that the expression levels of ZEB1, Snail2 and Twist were markedly higher in A549-ACE2 cells treated with TGF-β1 and DX600 when compared to TGF-β1 treatment alone. This was consistent with the increased expression of mesenchymal markers (
In the present study we investigated the role of ACE2 in lung cancer metastasis and EMT. To the best of our knowledge, this is the first demonstration of how ACE2, in a A549 cell lung cancer model, decreases metastasis
Components of the RAS system have been found to influence tumor growth and development (
In lung cancer, we found that decreased expression of ACE2 correlates with poor clinical outcomes, which suggested that ACE2 acts as a suppressor of lung cancer progression. It has also been reported that the overexpression of ACE2 in human pancreatic carcinoma cells decreased tumor growth, both
Several key factors have been noted to be involved in the malignant behavior of cancer cells. For example, loss of E-cadherin has been considered an early event in cancer development, which is well known to promote cancer cell invasion and lymph node metastasis. Recently, EMT has been reported to play a role in tumor progression (
In the present study, we found that ACE2, which is a newly identified component of the RAS system, attenuated lung cancer metastasis through its inhibition of the EMT process. This finding suggests that ACE2 may be a potential therapeutic target of lung cancer where EMT contributes to the development of tumor metastasis.
This study was supported by a grant from the National Natural Science Foundation of China (81071925).
Effect of ACE2 on the metastasis formation of A549 cells. ACE2 suppressed the metastasis of A549 lung cancer cells
Effects of ACE2 overexpression on epithelial (E-cadherin and β-catenin) and mesenchymal (vimentin) markers in A549 cells
Effect of ACE2 on the transcription levels of EMT markers in a lung cancer xenograft model. (A) E-cadherin mRNA levels in tumor tissues of a mice xenograft model; *P<0.01 when compared to the A549-C group. (B) RT-PCR results of EMT-associated transcription. mRNA levels of Snail2, ZEB1 and Twist were significantly lower in the A549-ACE2 mouse group when compared to the A549-C group (*P<0.01). (C) Expression analysis of ACE2, E-cadherin and vimentin by immunohistochemistry in nu/nu mice was performed, and representative images of primary tumor sections are shown. Top panel, immunohistochemistry detecting ACE2 in subcutaneous tumors of nu/nu mice transplanted with A549 cells overexpressing ACE2 (A549-ACE2) or control A549 (A549-C) cells; middle panel, immunohistochemistry detecting E-cadherin; bottom panel, immunohistochemistry detecting vimentin. Original magnification, ×400.
ACE2 overexpression inhibits EMT in A549 cells. (A) Western blot analysis of EMT markers in control (A549-C) and ACE2-overexpressing cell lysates (A459-ACE2). Cells were treated for 3 days with or without 10 ng/ml TGF-β1. The experiments were repeated three times and a representative blot is shown. The blot was stripped and re-probed with the β-actin antibody to detect the total amount of the respective proteins. Protein band intensities were quantified by densitometric analysis using the ImageJ software (National Institutes of Health). Columns, mean of triplicate experiments; bars, SE; *P<0.05. (B) Cell morphologic phenotypes of A549-C and A549-ACE2 cells were examined using a phase contrast microscope. TGF-β1-treated A549-C cells showed a spindle-like shape and a loss of cell-to-cell attachments. Untreated A549-C and A549-ACE2 cells with or without TGF-β1 treatment retained the morphological appearance of epithelial cells.
Effect of DX600 on the expression of EMT markers. (A) Western blot analysis of the epithelial marker E-cadherin and the mesenchymal marker αSMA was carried out in A549-ACE2 cells with or without TGF-β1 or DX600 treatment. Mean percentage values were obtained from the results of three independent experiments. (B) Transcription levels of Snail2, ZEB1 and Twist in A549-ACE2 cells treated either with TGF-β1 alone or in combination with DX600 using RT-PCR. The relative expression of mRNAs was normalized to that of the endogenous control (β-actin). All the experiments were performed in triplicate. Data represent the means ± SEM.