Transforming growth factor-b1 promotes lung adenocarcinoma

Mol Cell Biochem (2011) 355:309–314 DOI 10.1007/s11010-011-0869-3

Transforming growth factor-b1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition Hui-Jun Zhang • He-Yong Wang • Hong-Tao Zhang • Jin-Mei Su • Jun Zhu • Hai-Bing Wang • Wen-Yong Zhou • Hui Zhang • Ming-Chuan Zhao Lei Zhang • Xiao-Feng Chen

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Received: 31 March 2011 / Accepted: 28 April 2011 / Published online: 22 June 2011 Ó Springer Science+Business Media, LLC. 2011

Abstract Lung cancer is a highly malignant carcinoma, and most deaths of lung cancer are caused by metastasis. The alterations associated with epithelial-to-mesenchymal transition (EMT) may be related to the cancer cell metastasis. Nevertheless, the mechanism of lung cancer metastasis remains unclear. We conducted a study in vitro to investigate whether transforming growth factor-b1 (TGFb1) could induce changes of, such as cell morphology, expression of relative protein markers, and cellular motile and invasive activities. In this research, the changes of cell morphology were first investigated under a phase contrast microscope, then western blotting was employed to detect the expression of E-cadherin, vimentin, and fibronectin, and finally cell motility and invasion were evaluated by cell wound-healing as well as invasion assays. The data indicated that human lung adenocarcinoma cell lines, A-549 and PC-9 cells of epithelial cell characteristics, were Hui-Jun Zhang and He-Yong Wang contributed equally to this study. H.-J. Zhang Tenth People’ Hospital of Tongji University, Shanghai, China H.-J. Zhang H.-Y. Wang J.-M. Su H.-B. Wang W.-Y. Zhou H. Zhang M.-C. Zhao L. Zhang (&) X.-F. Chen (&) Shanghai Pulmonary Hospital, Tongji University, Shanghai, China e-mail: [email protected] X.-F. Chen e-mail: [email protected] H.-T. Zhang Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou, China J. Zhu Forth People’ Hospital of Wuxi, Jiangsu, China

induced to undergo EMT by TGF-b1. Following TGF-b1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker E-cadherin and increased mesenchymal markers vimentin and fibronectin. More importantly, cell motility and invasion were also enhanced in the EMT process. These results indicated that TGF-b1 may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT. Keywords TGF-b1 EMT Lung adenocarcinoma Invasion Metastasis

Introduction Lung cancer remains the most common death cause of malignant carcinomas. Many patients with lung cancer are diagnosed as having distant metastatic disease. Furthermore, most cancer patients die of metastases rather than their primary tumors. Therefore, metastasis is a fatal step in the progression of cancer. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells [1, 2]. Greenburg and Hay [3] first described that epithelial cells cultured in vitro might acquire mesenchymal features, providing the proof of principle for the process of EMT. The transition of epithelial cells into mesenchymal cells, known as EMT, is a process during which cells undergo a morphological switch from the epithelial polarized phenotype to a highly motile fibroblastic or mesenchymal phenotype. In the EMT process, epithelial cells lose their features, gain mesenchymal properties, and become motile and invasive [1]. The feature of EMT is the reduction of cell–cell adhesion, especially the reduction of E-cadherin which is critical to maintain the

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epithelial structure. It has been reported that the loss of E-cadherin expression is correlated with tumor invasion and metastasis [4]. With the loss of E-cadherin expression, the expression of mesenchymal markers, vimentin, and fibronectin, can be up-regulated when EMT occurs [5]. Vimentin expression is regarded as a key marker to distinguish ‘‘true or complete EMT’’ from partial ‘‘EMT’’ [6]. Transforming growth factor-b (TGF-b) is involved in many biological processes, including embryogenesis, wound healing, cell proliferation, differentiation, control of apoptosis, and EMT [1]. However, its overactivity is relevant to various diseases, including fibrosis and cancer. In cancer, TGF-b plays a complicated role. It functions as a tumor suppressor in early stages of tumorigenesis via inhibiting cell growth and inducing cell apoptosis. However, in later stages of tumor progression, TGF-b acts as a tumor promoter, since tumor cells lose their ability to be growth arrested by TGF-b, but have their ability to undergo EMT, which correlates to increasing invasiveness and metastasis [7]. TGF-b can not only downregulate the expression of epithelial phenotype markers including E-cadherin, tight junction protein ZO-1, MUC1, desmoplakins, cytokeratin 18 but can also upregulate mesenchymal phenotype markers such as fibronectin, a-smooth muscle actin, and vimentin. TGF-b1 is a member of TGF-b superfamily, which not only contributes to EMT during embryonic development but also induces EMT during tumor progression [8]. It is identified as a main inducer of EMT in fibrosis of kidney [9] and lung [10], pancreatic cancer [11], and esophageal adenocarcinoma [12], etc. To understand the role of EMT in lung adenocarcinoma invasion and metastasis, we demonstrated in this study that TGF-b1 induced a series of EMT-associated changes in lung adenocarcinoma and promoted tumor progression and metastasis by means of EMT.

Methods Materials Recombinant human TGF-b1 was purchased from R&D system, rabbit monoclonal antibody against human E-cadherin was from Cell Signaling, mouse monoclonal antibody against human vimentin from Santa Cruz, mouse monoclonal antibody against human fibronectin from Neomarkers; DMEM (Gibco), Matrigel (BD), and 24-well Transwells (Corning) were also used. Cell culture Human lung adenocarcinoma cell lines, A549 and PC-9, were obtained from American Type Culture Collection

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(ATCC, Manassas, VA, USA). Cells were maintained in low glucose-DMEM containing 10% BSA, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. Confluent cultures of cells were maintained in serum-free DMEM for 24 h before stimulation with TGF-b1. Cells were then incubated with 5 ng/ml TGF-b1 for 48 h. Western blotting analysis Cells were incubated with 1 or 5 ng/ml TGF-b1 for 48 h. Monolayer cells were washed three times with ice-cold PBS, lysed with lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40) on ice for 30 min. Cell extracts were resolved by western blotting as described [13]. Wound-healing assay The wound-healing assay was performed to measure twodimensional movement. In six-well plates, cells were cultured until a confluent state was reached. After having been placed in serum-free DMEM for 24 h, the monolayers were caused wounding by 200 ll plastic tips and then were rinsed three times before stimulation with 5 ng/ml TGF-b1. Phase contrast images were captured after the incubation with 5 ng/ml TGF-b1 for 48 h.

Invasion assays To study cell invasion, the invasion assay was used. The upper chambers were coated with Matrigel, and then cells (1 9 105/well) were then plated in DMEM with 1% BSA in the upper chamber of 8-lm pored (24-well) Transwells. A total of 1 9 105 cells in 200 ll media were plated in the upper chambers. DMEM medium with 10% BSA was used as a chemoattractant in the lower chamber. Cells were allowed to migrate with 5 ng/ml TGF-b1. After 24 h, the nonmigrating cells were removed from the upper surface of each Transwell by a cotton swab. Transwell membranes were then stained with crystal violet. Cells that migrated through the membrane to the lower surface were counted.

Statistical analysis Values are shown as the mean ± SEM of measurements of at least three independently performed experiments to avoid possible variation of cell cultures. Student’s t test was employed, and P \ 0.05 was considered to be statistically significant.


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Fig. 1 Morphological changes induced by TGF-b1. Lung adenocarcinoma epithelial cells were incubated with 5 ng/ml TGF-b1 for 48 h. Most untreated lung adenocarcinoma epithelial cells showed that a pebble-like shape and tight cell– cell adhesion was clearly observed, but small part of cell morphology was somewhat mesenchymal without the treatment of TGF-b1 (a, c). TGF-b1-treated cells showed a decrease in cell–cell contacts and adopted a more elongated morphological shape, but some cell morphological changes were slight (b, d)

Results Morphological changes of lung adenocarcinoma epithelial cells To confirm EMT induced by TGF-b1 in lung adenocarcinoma epithelial cells, we first observed morphological transformation. Lung adenocarcinoma epithelial A549 and PC-9 cells were incubated with 5 ng/ml TGF-b1 for 48 h. In the absence of TGF-b1, small portions of cell morphology were somewhat mesenchymal, but most lung adenocarcinoma epithelial cells showed pebble-like shape and tight cell–cell adhesion. However, after TGF-b1 treatment for 48 h, A549 and PC-9 cells showed morphological changes assessed by phase contrast microscopy. Many cells assumed more elongated shape and lost contact with their neighbor, displaying fibroblast-like appearance compared to the untreated cells (Fig. 1).

Fig. 2 Effects of TGF-b1 on the expression of E-cadherin, vimentin, and fibronectin in human lung adenocarcinoma epithelial cells. A549 and PC9 cells were treated with various concentrations of TGF-b1 (0, 1, and 5 ng/ml) for 48 h. a The expression of E-cadherin, the epithelial phenotype marker, was significantly decreased and of vimentin and fibronectin, mesenchymal phenotype markers was upregulated. b PC9 cells did neither decrease the expression of E-cadherin nor increase the expression of vimentin, but only upregulated the expression of fibronectin

Expression changes of EMT-related protein markers To better confirm morphological changes in lung adenocarcinoma epithelial cells, A549 and PC9 cells, represent EMT, western blotting was used to examine the changes of EMT-related protein markers. The results indicated that the expression of E-cadherin, the epithelial phenotype marker, was significantly decreased, and those of mesenchymal phenotype markers, fibronectin and vimentin, were greatly increased in a concentrationdependent meaner in A549 cells. However, the upregulated extent of vimentin was not as profound as that of fibronectin in A549 cells (Fig. 2a).

Unlike A549 cells, neither the decrease of E-cadherin nor the increase of vimentin was found in PC9 cells treated with TGF-b1, while the expression of fibronectin was elevated (Fig. 2b). Promotion of cell motility and invasion induced by TGFb1 Another important characteristic of EMT involves the acquisition of motile behavior and the ability to invasion. Therefore, we first investigated the effect of TGF-b1 on the motility of cells. The wound-healing assay was employed

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Fig. 3 TGF-b1 promotes cell motility. Cell motility was assessed by the wound-healing assay. Changes were observed under a phase contrast microscope after 5 ng/ml TGFb1 treatment for 48 h. a 0 ng/ml A549, b 5 ng/ml A549, c 0 ng/ ml PC9, and d 5 ng/ml PC9

to measure the motility of A549 and PC9 cells. As shown in Fig. 3, the phase contrast images captured after a 48-h incubation indicated that 5 ng/ml TGF-b1 enhanced cell motility compared with the untreated control. Transwell invasion assay was employed measure the ability of cell invasion. A549 and PC9 cells (1 9 105/well) were plated in the upper chamber of 8 lm pore Transwells. The cells were incubated with 5 ng/ml TGF-b1 for 24 h and the cells that migrated to the bottom of the Transwell filter were counted. The results showed that TGF-b1 significantly enhanced the invasion of both type of cells (Fig. 4).

Discussion Lung cancer is a disease of uncontrolled cell growth in lung tissues. This uncontrolled growth may lead to metastasis, which includes invasion of neighboring tissues and infiltration beyond the primary tumor. One mechanism of enhancing the dissemination of cancer cells is EMT [14], which is defined as the transition of polarized epithelial cells to migratory mesenchymal cells. EMT is evidenced by changes in cell morphology, cell behavior, and expression of EMT-related protein markers [5]. Recent studies provide evidence that EMT in various cell lines including A549 cells can be induced in vitro [10, 15, 16]. In agreement with previous reports, we showed that lung adenocarcinoma cells, A549, and PC-9 underwent EMT in response to TGF-b1 stimulation, indicating that TGF-b1 stimulus plays key roles in the EMT process. EMT is first identified as a change in cell morphology. Recent

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Fig. 4 TGF-b1 promotes cell invasion. a, b A549, and PC cells were added to the Transwell chambers where the Matrigel was presented with incubation in 5 ng/ml TGF-b1 for 24 h. Cells invading the Matrigel-coated filter were stained with crystal violet and counted. c Each bar represents the relative levels observed with controls (mean ± SEM). There were significant increases in invading cells induced by 5 ng/ml TGF-b1 (P \ 0.05). These data are the mean of three independent experiments

studies have demonstrated that cell morphological elongation was obvious in cells undergoing EMT with TGF-b1 treatment, and cell scattering is evident in cells undergoing


EMT induced with EGF [1]. After treatment with TGF-b1, lung adenocarcinoma cells became more elongated and presented a spindle-shape, fibroblast-like phenotype. In our study, most lung adenocarcinoma epithelial cells showed epithelial shape except that small part of cell morphology was somewhat mesenchymal in the absence of TGF-b1. After TGF-b1 treatment for 48 h, most A549 and PC-9 cells showed obviously morphological changes compared with the untreated lung adenocarcinoma epithelial cells, but small part of cell morphology changes were slight. Our study demonstrated that all cells exposed to TGF-b1 did not undergo the same extent of morphological changes. A reduction in E-cadherin level is considered as a hallmark of EMT [1]. However, loss of E-cadherin is not the sole pivotal event of EMT, because suppressing E-cadherin expression with transfection of antisense RNA did not lead to a full EMT [7]. E-cadherin plays a key role in maintaining the epithelial structural integrity and polarization, loss of which consequently destabilizes the structural integrity of epithelium and makes cells dissociate from their neighbors. Recent results have shown that the suppression of E-cadherin expression, by the transcription factor twist, induces EMT in carcinoma cells [17–19]. Concomitant with the loss of epithelial marker such as E-cadherin, cells undergoing EMT acquire mesenchymal markers such as vimentin and fibronectin [20]. We therefore investigated the effect of TGF-b1 on the expression of EMT-related protein markers by western blotting. The results indicated that the expression changes of three EMTrelated proteins occurred in A549 cells under stimulation of TGF-b1 in vitro. However, only one of EMT-related proteins, fibronectin, changed in PC9 cells. Brown et al. [15] reported that only two of 20 human and mouse cell lines underwent a complete EMT, defined by the loss of E-cadherin, dissolution of tight junctions, and elongated spindle-shaped morphology. In our study, only A549 cell induced by TGF-b1 decreased the expression of E-cadherin. We concluded that A549, but not PC9, was prone to undergo a complete EMT. EMT may contribute to greater motility and higher invasiveness of tumor cells. To evaluate these biological functions of the cells undergoing EMT, we used cell motility and invasion assays in our study, showing cells undergoing EMT by the stimulation with 5 ng/ml TGF-b1 were more motile and invasive. Bhowmick et al. [21] reported that cell motility and invasiveness enhanced by TGF-b-induced EMT were associated with RhoA activation and the PI3kinase/AKT signaling pathway. Up-regulation of N-cadherin induced by TGF-b1 was indispensable for increasing cell motility [13]. In hepatocellular carcinoma, down-regulation of E-cadherin can often predict invasiveness and metastasis in many forms of carcinoma, including invasive ductal breast carcinoma [22], esophageal adenocarcinoma [23], and

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gastric adenocarcinoma [17]. Twist expression is able to suppress E-cadherin expression and promote migration and invasion by inducing EMT [24, 25]. Recent studies indicated that tumor cell metastasis may be regulated by miR200 expression, which inhibits the capacity of tumor cells to undergo EMT, invade, and metastasize [26, 27]. However, further studies are still needed to reveal the mechanism of cell invasion in the process of EMT. In summary, we show that the exposure of human lung adenocarcinoma cell lines to TGF-b1 results in EMT, marked by changes in cell morphology, cell behavior, and expression of EMT-related protein markers. TGF-b1 may promote invasion and metastasis by means of EMT. Further studies are required to reveal the mechanism of cell invasion in the process of EMT, which may provide novel therapeutic targets and strategies for cancer invasion and metastasis. Acknowledgments This work was supported by Grants from National Science Foundation of China (No. 30872553 and 30800631) and Shanghai Science and Technology Committee (No. 10JC1419200). Conflict of interest statement conflicts of interest.

The authors declare that there are no

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