Transforming growth factor-β1 increases cell migration and β1 integrin up-regulation in human lung cancer cells

Lung Cancer 64 (2009) 13–21

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Transforming growth factor-␤1 increases cell migration and ␤1 integrin up-regulation in human lung cancer cells Yi-Chin Fong a,b,c,1 , Sheng-Feng Hsu d,1 , Chien-Lin Wu d , Te-Mao Li d , Shung-Te Kao a,c , Fuu-Jen Tsai e , Wen-Chi Chen f , Shan-Chi Liu g , Chi-Ming Wu d , Chih-Hsin Tang g,∗ a

Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan Department of Orthopaedic Surgery, China Medical University Hospital, Taichung, Taiwan School of Chinese Medicine, China Medical University, Taichung, Taiwan d Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan e Department of Pediatrics and Medical Genetics, China Medical University Hospital, Taichung, Taiwan f Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan g Department of Pharmacology, China Medical University, College of Medicine, No. 91, Hsueh-Shih Road, Taichung, Taiwan b c

a r t i c l e

i n f o

Article history: Received 1 April 2008 Received in revised form 14 July 2008 Accepted 22 July 2008 Keywords: TGF-␤1 Lung cancer Integrin Migration NF-␬B Akt

a b s t r a c t Transforming growth factor-␤1 (TGF-␤1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF␤1 increased the migration and cell surface expression of ␤1 integrin in human lung cancer cells (A549 cells). TGF-␤1 stimulation increased phosphorylation of p85␣ subunit of phosphatidylinositol 3-kinase (PI3K) and Ser473 of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-␤1-induced migration activities of A549 cells. Treatment of A549 cells with NF-␬B inhibitor (PDTC) or I␬B protease inhibitor (TPCK) also repressed TGF-␤1-induced cells migration and ␤1 integrins expression. In addition, treatment of A549 cells with TGF-␤1 induced I␬B kinase ␣/␤ (IKK␣/␤) phosphorylation, I␬B phosphorylation, p65 Ser536 phosphorylation, and ␬B-luciferase activity. Furthermore, the TGF-␤1-mediated increases in IKK␣/␤, I␬B␣ phosphorylation and p65 Ser536 phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85␣ and Akt mutants also reduced the TGF-␤1-induced ␬B-luciferase activity. Taken together, our results suggest that TGF-␤1 acts through PI3K/Akt, which in turn activates IKK␣/␤ and NF-␬B, resulting in the activations of ␤1 integrins and contributing the migration of human lung cancer cells. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer-related mortality in both men and women [1], with 1.2 million new cases diagnosed every year and 1 million deaths being recorded worldwide [2]. Nonsmall cell lung cancer (NSCLC) affects approximately 80% of all lung cancer patients. Most patients present with locally advanced (37%) or metastatic (38%) disease at the time of diagnosis [1], and a large percentage of those diagnosed with early-stage disease eventually experience recurrence of metastatic disease. Thus, the high invasiveness of NSCLC to regional lymph nodes, liver, adrenal glands,

Abbreviations: TGF, transforming growth factor; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; IKK␣/␤, I␬B kinase ␣/␤; NF-␬B, nuclear factor-␬B. ∗ Corresponding author. Tel.: +886 4 22053366x2228; fax: +886 4 22053764. E-mail address: [email protected] (C.-H. Tang). 1 These authors Yi-Chin Fong and Sheng-Feng Hsu contributed equally to this work. 0169-5002/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2008.07.010

contralateral lung, brain, and bone marrow, etc. may play a key role in its biological virulence [1]. Decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors [3]. The invasion of tumor cells is a complex, multistage process. To facilitate the cell motility, invading cells need to change the cell–cell adhesion properties, rearrange the extracellular matrix (ECM) environment, suppress anoikis and reorganize their cytoskeletons [4]. Integrins are a family of transmembrane adhesion receptors comprising 19 ␣ and 8 ␤ subunits that interact noncovalently to form up to 24 different heterodimeric receptors. The combination of different integrin subunits on the cell surface allow cells to recognize and respond to a variety of different ECM proteins including fibronectin, laminin, collagen and vitronectin [5]. Because integrins are the primary receptors for cellular adhesion to ECM molecules, they act as crucial transducers of bidirectional cell signaling, regulating cell survival, differentiation, proliferation, migration and tissue remodelling [6]. Integrin

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has been heavily implicated in tumor development [7,8], was correlated to reduced patient survival in colon carcinoma and melanoma [9,10], and has been associated with breast cancer cell metastasis to bone [11]. In addition, in vitro studies have found that integrins facilitated prostate cancer cell adhesion and migration through several ECM substrates [12,13], and transendothelial migration [14]. In the last 20 years, a large family of secreted polypeptides, known collectively as the transforming growth factor-␤ (TGF-␤) super-family has been revealed [15]. TGF-␤, originally called “sarcoma growth factor” was discovered in 1978 [16]. TGF-␤ is a potent growth inhibitor for a wide variety of cells including epithelial cells, vascular endothelial cells, hematopoietic cells, and immune lymphocytes. Perturbations of the TGF-␤ signaling pathways result in loss of cell growth regulation which is one of the most crucial steps in oncogenesis [17]. TGF-␤ family consists of three closely related isoforms (TGF-␤1, -␤2, and -␤3) that are prototypes of the larger TGF-␤ super-family. TGF-␤ family members elicit a diverse range of cellular responses including cell proliferation, migration, fibrosis, inflammation, and wound repair [15,18]. TGF-␤1 knockout mice develop diffuse mononuclear cell infiltrates that prove lethal within a few weeks of birth [19]. Recent studies have suggested a fundamental role for TGF-␤1 as a critical mediator of the metastasis activity of cancer cells [20]. Evidence for the role of TGF-␤ signaling in the complex process of cancer metastasis has recently been documented specifically in breast cancer. In mouse models of breast cancer, TGF-␤ promotes bone metastasis mediated by secreted factors such as parathyroid hormone-related peptide, interleukin-11 and CTGF [21,22]. Besides, Smad signaling is required for this TGF␤-induced bone metastasis of breast cancer cells [23,24]. Although the mechanisms underlying TGF-␤1-mediated tumor invasion have been studied in some cancers [20], the role of TGF-␤1 in the process of human lung cells migration remains large unknown. Previous studies have shown that TGF-␤1 modulates cell migration and invasion in several cancer cells [20]. TGF-␤1mediated invasion may involve activation of integrins receptors [32,33]. However, the effect of TGF-␤1 on integrins expression and migration activity in human non-small cell lung cancer cells is mostly unknown. Here we found a phenomenon that TGF␤1 increased the migration and the expression of ␤1 integrin of human lung cancer cells. In addition, phosphatidylinositol 3kinase (PI3K), Akt, IKK␣/␤ and NF-␬B signaling pathways were involved it.

2. Materials and methods 2.1. Materials Protein A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-Akt, Akt, p85␣, phosphotyrosine residues (PY20), IKK␣/␤, I␬B, p-I␬B␣, ␣-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ly294002, Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-Ooctadecylcarbonate)), TPCK and PDTC were purchased from Calbiochem (San Diego, CA, USA). Rabbit polyclonal antibody specific for phosphor-IKK␣/␤ (Ser180/181 ) and phosphor-p65 (Ser536 ) were purchased from Cell Signaling (Danvers, MA, USA). The recombinant human TGF-␤1 was purchased from PeproTech (Rocky Hill, NJ, USA). Rabbit polyclonal antibody specific for ␤1 integrin was purchased from Chemicon (Temecula, CA). The p85␣ (p85; deletion of 35 amino acids from residues 479 to 513 of p85) and Akt (Akt K179A) dominant-negative mutants were gifts from Dr. W.M.Fu (National Taiwan University, Taipei, Taiwan). The IKK␣ (KM) and IKK␤ (KM) mutants were gifts from Dr. H. Nakano

(Juntendo University, Tokyo, Japan). pSV-␤-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, MA, USA). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture The human lung adenocarcinoma cell lines (A549) were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture Ham’s F12 (DMEM/F12) medium which was supplemented with 10% heat-inactivated FCS, 2 mM-glutamine, penicillin (100 U/ml) and streptomycin (100 ng/ml) at 37 ◦ C with 5% CO2 . 2.3. Migration assay The migration assay was performed using Transwell (Costar, NY; pore size, 8 ␮m) in 24-well dishes. Before performing the migration assay, cells were pretreated for 30 min with different concentrations of inhibitors, including the Ly294002 (10 ␮M), Akt inhibitor (10 ␮M), PDTC (30 ␮M), TPCK (3 ␮M) or vehicle control (0.1% DMSO). Those concentrations of inhibitors didn’t affect cell death or proliferation of A549 cells by cell viability assay (data not show). Approximately 1 × 104 cells in 200 ␮l of serum-free medium were placed in the upper chamber, and 300 ␮l of the same medium containing 10 ng/ml TGF-␤1 was placed in the lower chamber. The plates were incubated for 24 h at 37 ◦ C in 5% CO2 , then cells were fixed in methanol for 15 min and stained with 0.05% crystal violet in PBS for 15 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each clone was plated in triplicate in each experiment, and each experiment was repeated at least three times. The number of invading cells in each experiment was adjusted by the cell viability assay to correct for proliferation effects of the TGF␤1 treatment (corrected invading cell number = counted invading cell number/precentage of viable cells) [34]. 2.4. Flow cytometric analysis Human lung cancer cells were plated in six-well dishes. The cells were then washed with PBS and detached with trypsin at 37 ◦ C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsed in PBS, the cells were incubated with rabbit anti-human antibody against ␤1 integrin (1:100) for 1 h at 4 ◦ C. Cells were then washed again and incubated with fluorescein isothiocyanateconjugated goat anti-rabbit secondary IgG (1:150; Leinco Tec. Inc., St. Louis, MO, USA) for 45 min and analyzed by flow cytometry using FACS Calibur and CellQuest software (BD Biosciences) [35]. 2.5. Western blot analysis The cellular lysates were prepared as described previously [36]. Proteins were resolved on SDS–PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against I␬B␣, p-I␬B, IKK␣/␤ or p-Akt (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Lanes were digitized by UN-SCAN-IT gelTM (a gel & graph digitizing softwave, version 6.1 silk scientific corporation; Utah, USA).

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2.6. Transfection and reporter gene assay Human lung caner cells were co-transfected with 0.8 ␮g ␬Bluciferase plasmid, 0.4 ␮g ␤-galactosidase expression vector. A549 cells were grown to 80% confluent in 12 well plates and were transfected on the following day by Lipofectamine 2000 (LF2000; Invitrogen). DNA and LF2000 were premixed for 20 min and then applied to the cells. After 24 h transfection, the cells were then incubated with the indicated agents. After further 24 h incubation, the media were removed, and cells were washed once with cold PBS. To prepare lysates, 100 ␮l reporter lysis buffer (Promega, Madison, WI) was added to each well, and cells were scraped from dishes. The supernatant was collected after centrifugation at 13,000 rpm for 2 min. Aliquots of cell lysates (20 ␮l) containing equal amounts of protein (20–30 ␮g) were placed into wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured in a microplate luminometer. The value of luciferase activity was normalized to transfection efficiency monitored by the cotransfected ␤-galactosidase expression vector. 2.7. Selection of stably transfected clone Purified plasmid DNA (3 ␮g) was transfected into A549 cells with LF2000 transfection reagent. Twenty-four hours after transfection, stable transfectants were selected in gentamicin (G418; Invitrogen) at a concentration of 600 ␮g/ml. Thereafter, the selection medium was replaced every 3 day. After 2 weeks of selection in G418, clones of resistant cells were isolated and allowed to proliferate in medium containing G418 (100 ␮g/ml). 2.8. Statistics The values given are mean ± S.E.M. The significance of difference between the experimental groups and controls was assessed by Student’s t-test. The difference is significant if the p value is <0.05. 3. Result 3.1. TGF-ˇ1-directed lung cancer cells migration involves ˇ1 integrin up-regulation TGF-␤1 has been reported that it stimulates directional migration and invasion of human cancer cells [37]. TGF-␤1-trigered migration in lung cancer cells was examined using the Transwell assay with correction of TGF-␤1-induced proliferation effects on human lung cancer cells [34]. TGF-␤1-directed human lung cancer cells (A549 cell) migration (Fig. 1). Previous studies have shown significant expression of integrins in human cancer cells [38,39]. We therefore, hypothesized that integrins may be involved in TGF␤1-directed lung cancer cells migration. Western blot analysis and flow cytometry analysis showed that TGF-␤1-induced the protein levels and cell surface expression of ␤1 integrin dose-dependently (Fig. 2A and B). We examined the levels of TGF-␤1 (1, 3, 10, 30 ng/ml) to determine the highest activation level of TGF-␤1 in A549 cells (Figs. 1 and 2B). We found that the migration activity and expression of ␤1 integrin were most significant in 10 ng/ml of TGF-␤1, however they mildly decreased in the level of 30 ng/ml. Therefore, the TGF-␤1 (10 ng/ml) was used in this study. Pretreatment of cells for 30 min with anti-␤1 integrin monoclonal antibody (mAb) (10 ␮g/ml) markedly inhibited the TGF-␤1-induced cancer migration (Fig. 2C). These data suggest that TGF-␤1induced cancer migration may occur via activation of ␤1 integrin receptor.

Fig. 1. TGF-␤1 induced the migration activity of human lung cancer cells. A549 cells were incubated with various concentrations of TGF-␤1, and in vitro migration activity measured with the Transwell after 24 h showed all supported the A549 cells migration in a dose-dependent way. Results are expressed as the mean ± S.E. *p < 0.05 compared with control.

3.2. PI3K and Akt signaling pathways are involved in the TGF-ˇ1-mediated integrin up-regulation and migration of lung cancer cells PI3K/Akt can be activated by a variety of growth factors, such as insulin, nerve growth factors, and TGF-␤1 [40,41]. We then examined whether TGF-␤1 stimulation also enhances the activation PI3K. Stimulation of A549 cells led to a significant increase of phosphorylation of p85␣ subunit of PI3K, as assessed by the measurement of phosphotyrosine from immunoprecipitated lysates using p85␣ (Fig. 3A). TGF-␤1-induced the migration and ␤1 integrin expression of A549 cells were greatly reduced by treatment with Ly294002 (10 ␮M), a specific PI3K inhibitor (Fig. 3B and C). In addition, stable transfection of cells with p85␣ mutant (A549/DN-p85␣) also inhibited TGF-␤1-induced migration and integrin up-regulation of lung cancer cells (Fig. 3B and C). Ser473 residue phosphorylation of Akt by a PI3K-dependent signaling pathway causes enzymatic activation. To examine the crucial role of PI3K/Akt in cancer migration and integrin upregulation, we determined Akt Ser473 phosphorylation in response to TGF-␤1. As shown in Fig. 4A, treatment of A549 cells with TGF-␤1 resulted in time-dependent phosphorylation of Akt Ser473 . Pretreatment of cells with Akt inhibitor (10 ␮M) or stable transfection with Akt mutant (A549/DN-Akt) antagonized the TGF␤1-induced the migration and ␤1 integrin expression of A549 cells (Fig. 4B and C). 3.3. NF-B signaling pathways are involved in the TGF-ˇ1-mediated integrin up-regulation and migration activity As previously mentioned, NF-␬B activation is necessary for the migration and invasion of human cancer cells [42]. To examine whether NF-␬B activation is involved in TGF-␤1-induced cancer migration, an NF-␬B inhibitor, PDTC, was used. Fig. 5A shows that A549 cells pretreated with PDTC (10 ␮M) and inhibited TGF-␤1-induced lung cancer cells migration. Furthermore, A549 cells pretreated with TPCK (3 ␮M), an I␬B protease inhibitor, also reduced TGF-␤1-induced cancer cell migration (Fig. 5A). In addition, treatment of cells with PDTC or TPCK also antagonized TGF-␤1-induced the expression of ␤1 integrins (Fig. 5B). To directly determine NF-␬B activation after TGF-␤1 treatment, A549 cells were transiently transfected with ␬B-luciferase as an indicator of NF-␬B activation. As shown in Fig. 5C, TGF-␤1 (3–30 ng/ml) treat-

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Fig. 2. TGF-␤1-directed migration activity of human lung cancer cells involves upregulation of ␤1 integrins. (A) Cells were incubated with TGF-␤1 (1 or 10 ng/ml) for 24 h, and ␤1 integrin expression was determined by Western blot analysis. (B) A549 cells were incubated with various concentrations of TGF-␤1, and the cell surface expression of ␤1 integrin was determined using flow cytometer. (C) A549 cells were pretreated with ␤1 monoclonal antibody (10 ␮g/ml) for 30 min, followed by stimulation with TGF-␤1 (10 ng/ml). The in vitro migration activity measured after 24 h showed that ␤1 monoclonal antibody could inhibit the activities. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

ment of A549 cells for 24 h caused a concentration-dependent increase in ␬B-luciferase activity. These results indicated that NF-␬B activation is important for TGF-␤1-induced cancer cell migration and the expression of ␤1 integrins. We further examined the upstream molecules involved in TGF␤1-induced NF-␬B activation. Stimulation of cells with TGF-␤1 induced IKK␣/␤ phosphorylation in a time-dependent manner (Fig. 6A). Furthermore, stable transfection with IKK␣ or IKK␤ mutant (A549/DN-IKK␣, A549/DN-IKK␤) markedly inhibited the TGF-␤1-induced cancer cells migration and integrin up-regulation (Fig. 6B and C). These data suggest that IKK␣/␤ activation is involved in TGF-␤1-induced the migration activity of human lung cancer cells. Treatment with lung cancer cells with TGF-␤1 also caused I␬B␣ phosphorylation in a time-dependent manner and I␬B␣ degradation (Fig. 6D). Previous studies showed that p65 Ser536 phosphorylation increases NF-␬B transactivation, and the antibody

Fig. 3. PI3K is involved in TGF-␤1-mediated migration and integrin up-regulation in human lung cancer cells. (A) Cells were incubated with TGF-␤1 (10 ng/ml) for indicated time intervals, and cell lysates were immunoprecipitated (IP) with an antibody specific for p85. Immunoprecipitated proteins were separated by SDS–PAGE and immunoblotted (WB) with anti-phosphotyrosine (PY). (B) Cells were pretreated for 30 min with Ly294002 (10 ␮M) or stably transfected with DN mutant of p85␣ by selection with gentamicin (G418). Then they were followed by stimulation with TGF-␤1 (10 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (C) Cells were pretreated for 30 min with Ly294002 (10 ␮M) or stably transfected with DN mutant of p85␣ by selection with gentamicin (G418). Then they were followed by stimulation with TGF-␤1 (10 ng/ml), and the cell surface ␤1 integrin was measured by using flow cytometry. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

specific against phosphorylated p65 Ser536 was used to examine p65 phosphorylation [43,44]. Treatment of A549 cells with TGF-␤1 for various time intervals resulted in p65 Ser536 phosphorylation which began at 15 min and was sustained to 120 min (Fig. 6E, upper panel). The protein levels of p65 were not affected by TGF-␤1 treatment (Fig. 6E, bottom panel).

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Fig. 4. Akt is involved in TGF-␤1-mediated migration and integrin up-regulation in human lung cancer cells. (A) Cells were incubated with TGF-␤1 (10 ng/ml) for indicated time intervals, and p-Akt expression was determined by Western blot analysis. TGF-␤1 activated the Akt pathway in A549 cells. (B) Cells were pretreated for 30 min with Akt inhibitor (10 ␮M) or stably transfected with DN mutant of Akt by selection with gentamicin (G418). Then they were followed by stimulation with TGF-␤1 (10 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (C) Cells were pretreated for 30 min with Akt inhibitor (10 ␮M) or stably transfected with DN mutant of Akt by selection with gentamicin (G418). Then they were followed by stimulation with TGF-␤1 (10 ng/ml), and the cell surface ␤1 integrin was measured by using flow cytometry. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

3.4. PI3K/Akt signal transduction-mediated TGF-ˇ1-induced IKK˛ˇ, IBa phosphorylation, p65 phosphorylation and B luciferase activity To further investigate whether TGF-␤1-induced IKK␣/␤, I␬B␣ phosphorylation, p65 Ser536 phosphorylation, and NF-␬B activation occur through the PI3K/Akt pathway, A549 cells were pretreated for

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Fig. 5. TGF-␤1 induces cells migration and integrin up-regulation through NF-␬B. (A) Cells were pretreated for 30 min with PDTC (10 ␮M) or TPCK (3 ␮M) followed by stimulation with TGF-␤1 (10 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (B) Cells were pretreated for 30 min with PDTC (10 ␮M) or TPCK (3 ␮M) followed by stimulation with TGF-␤1 (10 ng/ml) for 24 h, and the cell surface ␤1 integrin was measured by flow cytometry. (C) Cells were transfected with ␬B promoter plasmid for 24 h, and were then incubated with TGF-␤1 for 24 h. Luciferase activity was measured, and the results were normalized to the ␤-galactosidase activity. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

30 min with Ly294002 (10 ␮M) and Akt inhibitor (10 ␮M), which inhibited the TGF-␤1-induced increase in IKK␣/␤ phosphorylation as shown in Fig. 7A. Moreover, the TGF-␤1-induced increase in I␬B␣ and p65 Ser536 phosphorylation was also attenuated by Ly294002 and Akt inhibitor (Fig. 7A). In addition, the TGF-␤1-

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Fig. 6. TGF-␤1 induces IKK␣/␤ phoshorylation, I␬B␣ phosphorylation and p65 Ser536 phosphorylation in human lung cancer cells. (A) Cells were incubated with TGF-␤1 (10 ng/ml) for indicated time intervals, and p-IKK␣/␤ expression was determined by Western blot analysis. (B) Cells were stably transfected with DN mutant of IKK␣ or IKK␤ by selection with gentamicin (G418) and then followed by stimulation with TGF-␤1 (10 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (C) Cells were stably transfected with DN mutant of IKK␣ or IKK␤ by selection with gentamicin (G418) and then followed by stimulation with TGF-␤1 (10 ng/ml), and the cell surface ␤1 integrin was measured by flow cytometry. (D and E) Cells were incubated with TGF-␤1 (10 ng/ml) for indicated time intervals, and p-I␬B␣, I␬B␣ and p-p65 Ser536 expression was determined by Western blot analysis. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

induced increase in ␬B-luciferase activity was also inhibited by treatment with Ly294002, Akt inhibitor, PDTC and TPCK (Fig. 7B). These inhibitors did not affect the basal B-luciferase activity in A549 cells (Fig. 7B). Co-transfection with p85␣, Akt, IKK␣ and IKK␤ mutants also reduced the TGF-␤1-induced ␬B-luciferase activity (Fig. 7C). Taken together, these data suggest that activation of PI3K/Akt is required for TGF-␤1-induced IKK␣/␤, I␬B␣ phosphorylation, p65 Ser536 phosphorylation, and NF-␬B activation in lung cancer cells. 4. Discussion By far, lung cancer is the most common cause of cancer-related death in the world [45]. Since surgery remains the gold standard treatment for locoregional NSCLC, unfortunately, only 15–20% of these tumors can be radically resected, and overall surgicallytreated patient is only around 40% survival at 5 years [46]. Even in early stages, the 5-year survival rate remains at only 60–65% after complete resection. This high mortality is probably attributable to early metastasis, principally spreading of malignant cells to many tissues including bone, especially for NSCLC [47]. Therefore, early to detect cancer and avoid cancer metastasis demand immediate attention clinically. On the other hand, to determine the mechanism of metastasis activity of cancer cells is an important issue fundamentally. To achieve metastasis, cancer cells must there-

fore evade or co-opt multiple rules and barriers. Several discrete steps are discernible in the biological cascade of metastasis: loss of cellular adhesion, increased motility and invasiveness, entry and survival in the circulation, exit into new tissue, and eventual colonization of a distant site [3]. The mechanism of metastasis is a complicated and multistage process, however our study presented that TGF-␤1 promoted the cell migration and the expression of ␤1 integrins of human lung cancer cells. We provide an evidence that ␤1 integrin acts as crucial transducers of cell signaling, regulating cell migration and TGF-␤1 performs a critical mediator of the metastasis activity of cancer cells in tumor microenvironment. Integrins play critical roles in cell migration and adhesion. Integrins link the ECM to intracellular cytoskeletal structures and signaling molecules and are implicated in the regulation of a number of cellular processes, including adhesion, signaling, motility, survival, gene expression, growth and differentiation [48]. Previous studies have shown that TGF-␤1 modulates cell migration and invasion in several cancer cells [20,37]. However, the expression of integrins by TGF-␤1 in human lung cells is mostly unknown. Here we found that TGF-␤1 increased ␤1 integrins expression by using flow cytometry analysis, which plays an important role during tumor migration. In the present study, we used ␤1 integrin antibody to determine the role of ␤1 integrin and found that it inhibited TGF-␤1-induced cell migration, indicating the possible

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Fig. 7. PI3K/Akt involved the TGF-␤1-induced IKK␣/␤, I␬B␣ phoshorylation, p65 Ser536 phosphorylation and ␬B luciferase activity in human lung cancer cells. (A) A549 cells were pretreated with Ly294002 (10 ␮M), Akt inhibitor (10 ␮M) for 30 min before treatment with TGF-␤1 (10 ng/ml) for another 30 min, after which IKK␣/␤ phosphorylation, p65 Ser536 phosphorylation were determined by immunoblotting with antibodies specific for phospho-IKK␣/␤, I␬B␣ and phospho-p65, respectively. Equal loading in each lane is shown by the similar intensities of IKK␣/␤ and p65, respectively. (B and C) A549 cells transiently transfected with ␬B-luciferase plasmid for 24 h were either cotransfected with p85␣, Akt, IKK␣ and IKK␤ mutants or pretreated with Ly294002 (10 ␮M), Akt inhibitor (10 ␮M), TPCK (10 ␮M) or PDTC (3 ␮M) for 30 min, before incubation with TGF-␤1 (10 ng/ml) for 24 h. Luciferase activity was measured, and the results were normalized to the ␤-galactosidase activity. Results are expressed as the mean ± S.E. *p < 0.05 compared with control; # , p < 0.05 compared with TGF-␤1-treated group.

involvement of ␤1 integrin in TGF-␤1-induced migration in lung cancer cells. The TGF-␤1 signaling pathway acts through a complex of singlepass transmembrane receptors that contain an intracellular kinase domain that phosphorylates serine and threonine residues. This serine–threonine kinase receptor complex consists of two distinct transmembrane proteins, known as the type I and type II receptors [25]. Ligand binding to TGF-␤II recruits and activates the TGF-␤I receptor, which then signals to the family of intracellular mediators, called Smad-dependent signaling pathway [26,27]. Plentiful evidence clarifies that Smads are critical for TGF-␤ family signaling, however accumulating data suggests that Smad-independent pathways also exist, including, PI3K, and Akt [28–31]. In study, we did not used Smad siRNA to examine the role in TGF-␤1-induced migration and integrin expression. Whether the Smad signaling is involved needs further investigation. A variety of growth factors stimulate the expression of integrin via signal-transduction pathways that converge to activate NF-␬B complex of transcription factors [42]. The PI3K/Akt pathway is a major cascade mediating activation of the NF-␬B signaling pathway in human cancer cells [49,50]. PI3K is composed of one of five regulatory p85␣, p55␣, p50␣, p85␤ or p55␥ subunit attached to a p110␣, ␤ or ␦ catalytic subunit [51]. It has reported that TGF␤1 induced cell migration and integrin up-regulation via p85␣ subunit.[52] Phosphorylation of the p85␣ subunit is required for activation of the p110 catalytic subunit of PI3K [52,53]. We did not have mutants of the other subunits to examine the role after TGF-␤1

stimulation. Whether the other subunits are involved needs further investigation. We found TGF-␤1-enhanced p85␣ subunit phosphorylation in human lung cancer cells. Pretreatment of cells with PI3K inhibitors LY294002 antagonized the increase of migration and integrin expression by TGF-␤1 stimulation. This was further confirmed by the result that the dominant-negative mutant of p85␣ inhibited the enhancement of migration and integrin expression by TGF-␤1. Moreover, we also found that TGF-␤1 activated Akt Ser473 phosphorylation, while Akt inhibitor and Akt mutant inhibited TGF␤1-mediated cell migration. Our data indicates that PI3K/Akt might play an important role in the expression of integrin and migration of human lung cancer cells. A lot of NF-␬B activation pathways have been revealed, and all of them rely upon sequentially activated kinase cascades [44]. The classical pathway is triggered by various pro-inflammatory cytokines such as IL-1␤ and TNF-␣ [44]. These extracellular signals activate the IKK complex which phosphorylates I␬B␣ at Ser32 and Ser36 and signals for ubiquitin-related degradation. The released NF-␬B is then translocated into the nucleus where it promotes NF␬B-dependent transcription [44]. Besides the phosphorylation and degradation of the I␬B signal pathway, an I␬B-independent pathway such as p65 phosphorylation for optimal NF-␬B activation has been defined [44]. p65 Ser276 is phosphorylated by the protein kinase A catalytic subunit and mitogen- and stress-activated protein kinase-1, and this phosphorylation increases p65 transcriptional activity [54,55]. In addition, p65 is phosphorylated at Ser536 by a variety of kinases through various signaling pathways, and

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Fig. 8. Schematic presentation of the signaling pathways involved in TGF-␤1-induced migration and integrins expression of human lung cancer cells. TGF-␤1 activates PI3K and Akt pathway, which in turn induces IKK␣/␤ and I␬B␣ phosphorylation, p65 Ser536 phosphorylation, and NF-␬B activation, which leads to ␤1 integrins expression and increases the migration of human lung cancer cells.

this enhances the p65 transactivation potential. TNF-␣ induces rapid p65 phosphorylation at Ser536 through IKKs, resulting in increased transcriptional activity of p65 [56]. The results of this study showed that the PI3K/Akt pathway contributed to TGF-␤1induced p65 Ser536 phosphorylation in A549 cells. TGF-␤1-induced IKK␣/␤, I␬B␣ phosphorylation as well as an increase in p65 phosphorylation at Ser536 which began at 15 and 120 min, respectively, and Ly294002 and Akt inhibitor inhibited TGF-␤1-induced IKK␣/␤, I␬B␣ and p65 phosphorylation at Ser536 . These results indicate that PI3K/Akt may act through IKK␣/␤ to increase p65 phosphorylation at Ser536 and enhance NF-␬B transactivation. To conclude, we present a novel mechanism of TGF-␤1-directed migration of lung cancer cells by up-regulation of ␤1 integrin. TGF␤1 increases cells migration and integrin expression by activation of PI3K, Akt, IKK␣/␤, and NF-␬B-dependent pathway (Fig. 8). Conflict of interest All authors have no financial or personal relationships with other people or organizations that could inappropriately influence our work. Acknowledgments This study was supported by grants from grants from the National Science Council of Taiwan (96-2320-B-039-028-MY3) and China Medical University (No. CMU-95-148 and CMU-95-152). We thank Dr. W.M. Fu for providing for providing p85 and Akt mutants; Dr. H. Hakano for providing IKK␣ and IKK␤ mutants.

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