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Published ahead of print on December 12, 2003, doi:10.1165/rcmb.2003-0340OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 671-677, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0340OC

Stromal-Derived Factor-1{alpha}/CXCL12-CXCR 4 Axis Is Involved in the Dissemination of NSCLC Cells into Pleural Space

Ken-ichi Oonakahara*, Wataru Matsuyama*, Ikkou Higashimoto, Masaharu Kawabata, Kimiyoshi Arimura and Mitsuhiro Osame

Third Department of Internal Medicine, Kagoshima University Faculty of Medicine; and Department of Respiratory Medicine, National Minami-kyushu Hospital, Kagoshima, Japan

Address correspondence to: Dr. Wataru Matsuyama, M.D., Ph.D., Third Department of Internal Medicine, Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: vega{at}xa2.so-net.ne.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Malignant pleural effusion (PE) is one of the poor prognostic factors in non–small cell lung cancer (NSCLC), and the detailed mechanism of the malignant PE formation is not fully elucidated. Recently, CXCR4, a receptor for chemokine stromal-derived factor-1{alpha} (SDF-1{alpha}) that can induce chemotaxis of cells, was reported to be expressed on NSCLC. In this study, we hypothesized that the SDF-1{alpha}/CXCR4 axis may be involved in the dissemination of malignant cells into pleural space, and investigated its expression, function, and signaling pathway using NSCLC cell lines and clinical samples from 43 patients with NSCLC with malignant PE. We found functional expression of CXCR4 on NSCLC cell lines, and also found that SDF-1{alpha} could induce migration via phosphatidylinositol 3 (PI-3) kinase– and p44/42 mitogen-activated protein kinase–dependent manner. The SDF-1{alpha} levels in malignant PE were significantly higher than those in transudate PE and showed a significant positive correlation with PE volumes. The sensitivity and specificity for prediction of recurrence of malignant PE was 61.5% and 83.3%, respectively (cutoff SDF-1{alpha} = 2,500 ng/ml), and better than those using pH of PE. Cancer cells in malignant PE expressed CXCR4, and mesothelial cells of the pleura stained positive for SDF-1{alpha}. The SDF-1{alpha}/CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space.

Abbreviations: Dulbecco's modified Eagle medium, DMEM • fetal calf serum, FCS • non–small cell lung cancer, NSCLC • phosphate-buffered saline, PBS • pleural effusion, PE • phosphatidylinositol 3, PI 3 • stromal-derived factor-1{alpha}, SDF-1{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Malignant pleural effusion (PE) is most often caused by lung adenocarcinoma, because this type often forms a primary tumor in the periphery of the lung and invades the pleural cavity (1). Malignant PE indicates a poor prognosis in patients with advanced lung cancer, being associated with high morbidity and mortality in non–small cell lung cancer (NSCLC) (1–5). To control malignant PE, drainage followed by infusion of sclerosing agents is commonly used; however, the efficacy of this treatment is variable and does not extend the survival of patients with lung cancer (1, 6). Malignant PE can develop as a direct consequence of cancer cell dissemination into the pleural space; however, the exact mechanisms are not fully understood (1, 6, 7). Therefore, a better understanding of the molecular mechanisms that are involved in cancer cell dissemination is required to understand the process of malignant PE formation and to find ways to design new and effective therapies.

Chemokines are small cytokine-like peptides that play a pivotal role in the leukocyte chemotaxis through interaction with their G-protein–coupled receptors (8, 9). Chemokines have other functions that are either beneficial for tumor growth (10) or have an antitumor effect (11, 12). Recently, it was discovered that the ability of breast cancer cells to migrate or metastasize to particular tissues is the result of the expression of a specific chemokine receptor, CXCR4, on breast cancer cells; likewise, its ligand, stromal cell–derived factor-1{alpha} (SDF-1{alpha}/CXCL12), was expressed on target tissues (13). Although most chemokine receptors bind several chemokines, CXCR4 is a specific chemokine receptor because it only interacts with SDF-1{alpha} (14, 15). The SDF-1{alpha}/CXCR4 axis has been implicated in the migration and metastasis of prostate cancer cells to the bone (16), and more importantly, Phillips and coworkers reported the involvement of this axis in the metastasis of NSCLC (17).

In this study, we hypothesized that the SDF-1{alpha}/CXCR4 axis may be involved in the dissemination of NSCLC cells into pleural space during the formation of malignant PE, and investigated SDF-1{alpha} levels in PE and the expression of CXCR4 on cancer cells in PE. We found that SDF-1{alpha} stimulation of CXCR4 on NSCLC cells led chemotaxis via a phosphatidylinositol 3 (PI-3) kinase– and p44/42 mitogen-activated protein (MAP) kinase–dependent manner. We also found that human pleural mesothelial cells can induce chemotaxis of NSCLC cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was reviewed and approved by the Kagoshima University Faculty of Medicine Committee on Human Research.

Cell Lines and Cell Culture
NSCLC cell lines (CRL5848, A549, CRL5985, CCL256) were purchased from American Type Culture Collection (Rockville, MD). CRL5848 and A549 cells were from a patient with NSCLC without malignant PE; CRL5985 and CCL256 cells were from patients with NSCLC with malignant PE. Cells were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT), 1 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (complete medium).

FACS Analysis
The expression of cell surface CXCR4 of NSCLC cell lines was evaluated by flow-cytometry analysis. One hundred thousand cells were suspended in 50 µL of cold phosphate-buffered saline (PBS) containing 0.1% sodium azide, 10 ng/ml bovine serum albumin and 20 µg/ml of human IgG, incubated for 10 min on ice, and with antibodies against CXCR4 (PharMingen, San Diego, CA) for an additional 15 min on ice. Cells were washed with PBS and incubated with fluorescein isothiocyanate–conjugated goat anti-mouse IgG for 15 min on ice. At the end of the incubation, PI was added to each tube (100 µM). The cells were washed with PBS, and subsequently analyzed by flow cytometry using a FACScan (Becton Dickinson). Dead cells, determined by the incorporation of PI, were gated out. Results were processed using the CellQuest software (Becton Dickson).

Calcium Flux
Each NLCLC cell was suspended at 107 cells/ml in DMEM supplemented with 10% FCS and incubated for 30 min at 37°C with 2.5 µM fura-2 acetoxymethyl ester, as described previously (18). The fluorescence of a 2-ml cellular suspension was monitored with a Photon Technology International Delta Scan Fluorometer (dual excitation {lambda}340 and 380 nm, single emission {lambda}510 nm) at 37°C. Cytosolic free calcium concentration ([Ca2+]i) was determined by fluorescence using Photon Technology International software program (South Brunswick, NJ).

Western Blot Analysis
To determine whether MAP kinases (p44/42 MAP kinase, P38, SPAK/JNK) were activated in NSCLC cells by stimulation with SDF-1{alpha}, 1 x 107 NSCLC cells were plated on dishes, serum-starved in DMEM containing 1% FCS for 10 h, and subsequently activated with SDF-1{alpha} for various times. Cells were washed three times with PBS, and 1 x 107 cells were lysed on ice for 20 min in 1 ml of lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and a cocktail of protease inhibitors (Roche, Indianapolis, IN). The lysates were spun and 20 µl of supernatants were collected and added to 20 µl double-strength sample buffer (20% glycerol, 6% SDS, 10% 2-mercaptoethanol) was added. The samples were boiled for 10 min. Eluted proteins were analyzed on 10% polyacrylamide gels by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes at 150 mA for 1 h using a semi-dry system. The membranes were incubated with rabbit polyclonal antibodies against phosphorylated or nonphosphorylated p38, SPARK/JNK, and p44/42 MAP kinase protein (Cell Signaling Technology, Beverly, MA), followed by a sheep anti-rabbit IgG coupled with horseradish peroxidase (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Peroxidase activity was visualized by the Enhanced Chemiluminescence Detection System (Amersham).

Migration Assay
SDF-1{alpha} induced NSCLC migration was assessed using a 48-well microchemotixis chamber technique as previously described (19). Before conducting the chemotaxis assay, a 10-um polycarbonate filter was coated with collagen (Sigma, St Louis, MO) and then different concentrations of SDF-1{alpha} (R&D Systems Inc., Minneapolis MN) were placed in the wells of lower compartment wells of the chamber (Neuroprobe, Cabin John, MD), and NSCLC cells (106/well) were then added to the wells of the upper compartment wells. The lower and upper compartments were separated by a 10-um polycarbonate filter (Osmonics, Livermore, CA). After incubation at 37°C in humidified air with 5% CO2 for 1.5 h, the filters were removed and stained, and the cells migrating across the filter were counted using the Baioquant semiautomatic counting system. The results were presented as number of cells per high-power field. To evaluate the effect of a p44/42 inhibitor, G-coupled–protein inhibitor or PI-3 kinase inhibitor, cells were pre-treated with PD98059, PTX (100 ng/ml), LY294002 (Biochem-Novabiochem, San Diego, CA) for 30 min and subsequently stimulated with SDF-1{alpha}.

NSCLC Cell Migration Assay Against Human Pleural Mesothelial Cell
We also performed a migration assay of NSCLC cell lines against human pleural mesothelial cells (HTB-54, graciously donated by Dr. Teizo Yoshimura, National Cancer Institute, Frederick, MD). Varying numbers of human pleural mesothelial cells were plated on 24-well plates with complete medium and were cultured for 24 h. Cell culture inserts containing 3-µm porous polycarbonate membranes (BD Falcon Cell culture inserts; BD Biosciences, San Jose, CA) were coated with human type IV collagen (Sigma). Fifty thousand NSCLC cells in were put in the cell culture inserts with complete medium, and the cell culture inserts were subsequently put on the 24-well dish, where human pleural mesothelial cells had already been cultured and incubated for 8 h. After incubation, all cells in the bottom well were collected and cell numbers were counted. The migrated cell number was calculated according to the following formula: Migrated cell number = {cell number of the well where both human pleural cells and NSCLC cells were cultured} – {cell number of the well where only human pleural cells were cultured} – {cell number of the well where only NSCLC cells were cultured}.

Measurement of SDF-1{alpha} in PE
We measured SDF-1{alpha} levels in PE of 43 patients with NSCLC with malignant PE (adenocarcinoma: squamous cell carcinoma = 32:11, male:female = 35:8, mean age = 69.5 ± 10.2 yr), 19 patients with nonmalignant exudate PE (male:female = 13: 6, mean age = 63.2 ± 12.3 y old), and 22 patients with transudate PE (all patients were suffering from heart failure, male:female = 14:8, mean age = 70.1 ± 11.3 yr). SDF-1{alpha} concentrations in PE were measured in duplicate for each sample using a commercial enzyme-linked immunosorbent assay kit (R&D Systems) that recognizes recombinant and natural SDF-1{alpha}. This assay is sensitive from 10 pg/ ml and does not crossreact with other homologous cytokines. Optical density at 450 nm was measured on a Titertek Multiskan MC plate reader (Flow Laboratories, Helsinki, Finland), and SDF-1{alpha} concentration was determined by linear regression from a standard curve using GraphPad software (Flow Laboratories) for analysis. We also measured the pH of malignant PE of each patient.

Immunocytotochemical Staining
To determine whether CXCR4 is positive on the cancer cells in malignant PE, immunocytochemical staining for CXCR4 on cancer cells in malignant PE was performed using a mouse monoclonal anti-CXCR4 antibody (R&D Systems) employing the Diaminobenzidine (DAB) method. After obtaining PE, a quick cytospin was performed and the cells were mounted on poly-L-lysine–coated slides. Endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in methanol for 10 min. After washing twice in PBS 1% saponin, slides were blocked using 5 mg of bovine serum albumin per ml in PBS. Slides were again washed in PBS 1% saponin. Sections were incubated with primary antibody solution for 2 h at room temperature using a 1:150 concentration of a working dilution of the antibody. Negative control slides were incubated with muse monoclonal antibody (R&D Systems). Slides were rinsed twice in PBS 1% saponin. Secondary biotynated anti-immunoglobulin antibody (PharMingen) was added, and the mixture was incubated for 30 min at room temperature. The sections were again rinsed twice with PBS 1% saponin. Streptavidin conjugated to horseradish peroxidase (Amersham) was incubated for 30 min and then rinsed off with deionized water. DAB substrate solution was then added, and the mixture was incubated for 10 min. A brown colored reaction represented a positive result. The cytologic diagnosis was made according to the morphology of the cells by a professional pathologist and cytologist.

Immunohistochemistry
Three patients underwent autopsy, and their pleural tissues were examined by immunohistochemical staining for SDF-1{alpha} using a mouse monoclonal antibody (R&D Systems) employing the DAB method as described previously (20). Four-micrometer-thick sections were mounted on poly-L-lysine-coated slides and were then dewaxed and washed in Tris-buffered saline (pH 7.4) for 10 min. For optimal antigen retrieval, sections were pressure cooked in 0.01 M citrate buffer (pH 6.0) for 90 s. Endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in methanol for 10 min. After washing twice in PBS 1% saponin, blocking reaction was performed as reported previously (20). Sections were incubated with primary antibody solution for 2 h at room temperature using a 1:50 concentration of a working dilution of the antibody. Negative control slides were incubated with mouse monoclonal IgG (R&D Systems). Secondary biotynated anti-immunoglobulin antibody (R&D Systems) was added, and the mixture was incubated for 30 min at room temperature. After washing, the sections were incubated with streptavidin conjugated to horseradish peroxidase (Amersham) and then rinsed off with deionized water. DAB substrate solution was then added, and the mixture was incubated for 10 min. A brown color reaction represented a positive result.

Statistical Analysis
We used one-way factorial ANOVA with the Bonferroni-Dunn test. We also used the Student's t test and Pearson's correlation coefficient test. A P value < 0.05 was considered significant. Most values were expressed as the mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CXCR4 on NSCLC
As shown in Figure 1, all NSCLC cell lines expressed CXCR4 on cell surface. The intensity of CXCR4 expression was significantly higher on CRL5985 and CCL256 cell lines than on CRL5848 and A549 cell lines.



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  		Figure 1. All NSCLC cell lines expressed CXCR4 (A). The intensity of CXCR4 expression was significantly higher in cell lines with PE (CRL5985 and CCL256) than in cell lines without PE (CRL4858 and A549) (B). *P < 0.05, **P < 0.01 by Bonferroni-Dunn with one-way factorial ANOVA test.

 
SDF-1{alpha} Stimulation on NSCLC Cell Lines Induced Calcium Flux and Chemotaxis
As shown in Figure 2, SDF-1{alpha} stimulation induced calcium flux (10 ng/ml SDF-1{alpha}) and chemotaxis for all NSCLC cell lines. Migrated cell numbers were significantly higher in CRL5985 and CCL256 cell lines than in CRL5848 and A549 cell lines. We repeated this experiment three times and obtained the same results.




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  		Figure 2. All NSCLC cell lines expressed functional CXCR4. All cell lines showed calcium flux and chemotaxis through stimulation with SDF-1{alpha} (A). Migrated cell numbers were significantly higher in cell lines with PE than in the cell lines without PE (B). Human pleural mesothelial cells induced the chemotaxis of NSCLC cells (C) and produced SDF-1{alpha} (D). *P < 0.05, **P < 0.01 by Bonferroni-Dunn with one-way factorial ANOVA test. Open bars, CRL5848; vertically striped bars, A549; filled bars, CRL5985; diagonally striped bars, CCL256.

 
Human Pleural Mesothelial Cells Induced Chemotaxis of NSCLC Cells
Migrated cell counts increased correspondingly with the plated human pleural mesothelial cell numbers (Figure 2C). SDF-1{alpha} levels in culture medium also increased correspondingly with the plated human pleural mesothelial cell numbers (Figure 2D). Therefore, we concluded that human pleural mesothelial cells most likely induce chemotaxis of NSCLC cells via SDF-1{alpha}/CXCR4 axis.

SDF-1{alpha} Stimulation on NSCLC Cell Lines Activated p44/42 MAP Kinase via a PI-3 Kinase–Dependent Mechansim
As shown in Figure 3, 5 min after stimulation with SDF-1{alpha} (10 ng/ml), p44/p42 MAP kinase protein was phosphorylated, and this continued for 15 min. This phosphorylation was confirmed in all NSCLC cell lines, and there was no difference of phosphorylation time among all cell lines (data not shown). P38 and JNK MAP kinase proteins were not activated by the stimulation with SDF-1{alpha} in all cell lines. This p44/42 MAP kinase activation was inhibited by adding PTX, a G-coupled–protein receptor inhibitor. PI-3 kinase is known to be involved in CXCR4 intracellular signaling and can activate p44/42 MAP kinase protein in many cell lines (21). Therefore, we used LY294002, an inhibitor of PI-3 kinase, to investigate whether p44/42 MAP kinase activation is also dependent on PI-3 kinase in NSCLC cell lines. As shown in Figure 3B, activation of p44/42 MAP kinase protein was inhibited by adding LY294002. These results were confirmed in all cell lines. We repeated this experiment three times in each cell line and obtained the same results.



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  		Figure 3. Western blotting analysis showing activation of p44/42 MAP kinase by stimulation with SDF-1{alpha}. Phosphorylation of p44/42 MAP kinase was seen 5 min after the stimulation with SDF-1{alpha}, and continued for 15 min (A). This p44/42 activation was inhibited by adding PTX or LY294002 (B). These results were seen in all NSCLC cell lines. Data are representative of three individual experiments.

 
SDF-1{alpha}–Induced Chemotaxis of NSCLC Cell Lines was Dependent on PI-3 Kinase and p44/42 MAP Kinase
To examine whether the NSCLC cell line chemotaxis induced by SDF-1{alpha} is dependent on the G-coupled protein receptor, PI-3 kinase, or p44/42 MAP kinase, we used PTX, PD90859, and inhibitor of p44/42 MAP kinase and LY294002. As shown in Figure 4, PTX, PD90859, and LY294002 significantly reduced the chemotaxis induced by SDF-1{alpha} in all cell lines.



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  		Figure 4. Inhibition of chemotaxis by adding PTX (open bars), PD90859 (vertically striped bars), and LY294002 (diagonally striped bars). PTX, PD90859, and LY294002 significantly reduced chemotaxis induced by SDF-1{alpha} in all NSCLC cell lines. *P < 0.001 by Bonferroni-Dunn with one-way factorical ANOVA test. Filled bars, 10 ng/ml.

 
SDF-1{alpha} Level Was Elevated in Malignant PE
To examine whether the SDF-1{alpha}/CXCR4 axis contributes to malignant PE, we measured SDF-1{alpha} levels in malignant PE of patients with NSCLC. As shown in Figure 5, the SDF-1{alpha} level was significantly higher in malignant PE (mean ± SD = 2,040.1 ± 1041.5 pg/ml) than in transudate PE (mean ± SD = 237.1 ± 248.7 pg/ml). There was no significant difference between SDF-1{alpha} levels in malignant PE and nonmalignant exudate PE (mean ± SD = 1,202.5 ± 925.2 pg/ml). SDF-1{alpha} levels in malignant PE showed a significant positive causative relationship with PE volume (r = 0.5987, P < 0.01). We determined PE volume by measuring the total volume of thoracentesis. Despite treatment with drainage and antineoplastic drugs, 13 patients with NSCLC developed recurrent malignant PE, and their SDF-1{alpha} levels in the first malignant PE was higher than in patients with NSCLC without recurrent malignant PE (recurrent: 2,917.5 ± 819.9 pg/ml, nonrecurrent: 1,659.8 ± 893.2 pg/ml, P < 0.01, Student's t test). Sensitivity and specificity were 61.5% and 83.3%, respectively (cutoff = 2,500 ng/ml). Sensitivity and specificity using pH of PE was 45.5% and 75.0%, respectively (cutoff = 7.3).



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  		Figure 5. Comparison of SDF-1{alpha} levels between malignant PE of patients with NSCLC, nonmalignant exudate PE, and transudate PE. The SDF-1{alpha} levels in malignant PE were significantly higher than in transudate PE (A; P < 0.01 by Bonferroni-Dunn with one-way factorial ANOVA test). SDF-1{alpha} levels in malignant PE showed a significant and positive correlation with pleural effusion volumes ([B] r = 0.5987, P < 0.01 by Pearson's correlation coefficient test).

 
Immonocytochemistry for CXCR4 of Cancer Cells in PE
CXCR4 expression on cancer cells in malignant PE of patients with NSCLC was also confirmed by immunocytochemical staining (Figure 6A). Immunocytochemical staining was performed on 12 patients with NSCLC, and all patients showed CXCR4 expression on cancer cells in malignant PE.



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  		Figure 6. Expression of CXCR4 on cancer cells in malignant PE and SDF-1{alpha} on mesothelial cells of the pleura. Cancer cells on malignant PE of patients with NSCLC stained positive for CXCR4 (A; original magnification: x300; a: stained by anti-CXCR4 antibody, b: stained by control mouse IgG antibody). Pleural mesothelial cells stained positive for SDF-1{alpha} (B; original magnification: x150; c: stained with anti-SDF-1{alpha} antibody, d: stained with control mouse IgG antibody).

 
Immunohistochemistry for SDF-1{alpha} of Pleural Tissue
Mesothelial cell are capable of producing SDF-1 (22); therefore, we performed immunohistochemical staining for SDF-1{alpha} on pleural tissue using autopsy specimens of three patients with NSCLC. As shown in Figure 6B, pleural mesothelial cells expressed SDF-1{alpha}. We obtained the same result from all three patients.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed the functional CXCR4 expression on NSCLC cell lines and PI-3 kinase–dependent activation of p44/42 MAP kinase as the CXCR4 signaling pathway in NSCLC cell lines. Human pleural mesothelial cells could induce chemotaxis of NSCLC cells. In addition, using clinical samples, we showed that the CXCR4 expression on NSCLC cells increased SDF-1{alpha} levels in PE and the expression of SDF-1{alpha} in pleural mesothelial cells, a target organ of cancer cells in pleuritis carcinomatosa. The SDF-1{alpha} level in malignant PE showed a significant and positive causative relationship with effusion volume. Taken together, we think that SDF-1{alpha}/CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space. Disseminated cancer cells can block the drainage of pleural space, and this eventually leads to PE. Therefore, we assert the possibility that the SDF-1{alpha}/CXCR4 axis may be involved in the formation of malignant PE.

Cell migration plays an essential role in a variety of physiologic and pathologic events, including wound healing, inflammation, immune response, embryogenesis, organogenesis, angiogenesis, and tumor invasion and metastasis (9, 23). Chemokines, a family of low-molecular-weight chemotactic proteins that have the potential to induce directional migration of cells, are grouped into four subfamilies—CXC ({alpha}), CC (ß), C ({gamma}), and CX3C ({delta}) chemokines—based on the difference in the spacing of conserved cysteine residues (9). SDF-1{alpha}, initially identified as a growth-stimulating factor for B-cell progenitors (24), is a member of the CXC chemokine subfamily that is expressed in a broad range of tissues including the pancreas, spleen, ovary, and small intestine (but not in peripheral blood leukocytes) (25) and has multiple biological activities toward diverse cell types (9). SDF-1{alpha} has chemotactic activity toward several cell types, including T lymphocytes, monocytes, pre-B lymphocytes, dendritic cells, and hematopoietic cells (21, 26–32). The chemotactic activity of SDF-1{alpha} is mediated by a seven-transmembrane G protein–coupled receptor, CXCR4, which is also known as a coreceptor for T cell–tropic human immunodeficiency virus strains (27). Targeted disruption of the SDF-1{alpha} gene or the CXCR4 gene in mice revealed that SDF-1{alpha} and CXCR4 have critical functions in the fetal development of the hematopoietic, cardiovascular, and cerebellar systems (33, 34). More recently, it has been shown that CXCR4 and SDF-1{alpha} are highly expressed in breast cancer cells and in organs representing the major areas of breast cancer metastasis, respectively, and that SDF-1{alpha} promotes metastasis by stimulating the migration of breast cancer cells (13). Concerning lung cancer, Kijima and coworkers (35) reported that the CXCR4 receptor is functionally expressed in small cell lung cancer cells and regulates their migration, adhesion, and morphologic change in cooperation with the stem cell factor/c-Kit pathway. Furthermore, Phillips and colleagues (17) reported that CXCR4 is also functionally expressed in NSCLC cells and regulates its metastasis. Our results support their findings and strengthen the possibility that the SDF-1{alpha}/CXCR4 axis contributes to the pathogenesis, especially in determining the metastatic destination, of NSCLC.

Although our understanding of the biological role of SDF-1{alpha} has greatly increased in recent years, relatively little is known about the signaling pathways that may mediate these effects. SDF-1{alpha} has been shown to elicit an increase in calcium flux in a number of settings (28, 36) and has also been reported to stimulate phosphorylation of p44/42 MAP kinase in leukemic T cell lines, T cell clones, and a pre–B cell lymphoma cell line (37–40). SDF-1{alpha} stimulation also can induce PI-3-kinase activity (38, 40). A previous study demonstrated that PI-3 kinase and its metabolic products play an important role in signaling pathways related to chemotaxis (41). Sotsios and associates indicated that PI-3 kinase inhibitors prevent SDF-1{alpha}–stimulated activation of p44/42 MAP kinase, implying an upstream requirement for PI-3 kinase activation. To our knowledge, our study is the first report to show involvement of PI-3 kinase–dependent p44/42 MAP kinase activation as the downstream of the SDF-1{alpha}/CXCR4 signaling pathway in the NSCLC cell line.

We suggest that the SDF-1{alpha}/CXCR4 axis is involved in the development of malignant PE in NSCLC. Lung cancer is the leading cause of malignant PE and at least 25% of all patients with lung cancer will develop malignant PE at some point in the course of the disease (1, 42). Treatment of malignant PE in NSCLC consists of drainage by chest tube and induction of pleural sclerosis by injection of antibiotics, antiseptics, or antineoplastics (42–44). The results, unfortunately, are variable, because the procedure does not prolong survival and prediction of recurrence is also difficult (42). The sensitivity and specificity of SDF-1{alpha} levels of 250 pg/ml for prediction of recurrent PE were 61.5% and 83.3%, respectively, and were better than those of pH of PE, a classical marker of PE prognosis. Reported sensitivity and specificity of pleural effusion pH for the prediction of recurrent PE are not so favorable either, and are almost the same as our results (45–47). Therefore, we encourage considering measurement of SDF-1{alpha} in PE as one of the predictive markers for the recurrence of malignant PE in NSCLC.

Also, in our study, blocking the SDF-1{alpha}/CXCR4 signaling cascade resulted in migration inhibition of cancer cells. Furthermore, SDF-1{alpha} stimulation also enhanced tyrosine phosphorylation of focal adhesion complex components (including Pyk-2, paxillin, and Crk), increased nuclear factor-{kappa}B activity, and induced PI-3 kinase activity associated with antiphosphotyrosine immunoprecipitates (38, 40). Thus, SDF-1{alpha} signaling is associated with the signaling pathways that may mediate cell growth, migration, and transcriptional activation. Control of NSCLC cell dissemination may be possible by controlling the SDF-1{alpha}/CXCR4 axis. Yet on the other hand, this axis also exists in normal cells such as leukocytes (5, 8, 9, 15). It is not clear from this study whether this axis is also activated in normal tissue during the development of malignant PE. Therefore, we consider further studies addressing this point are necessary to clarify the molecular mechanism of the dissemination of NSCLC cells into pleural space.


   Acknowledgments
 
The authors acknowledge Dr. Carole Galligan and Dr. Teizo Yoshimura (Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD) for their technical help. They thank Dr. Joeji Wakimoto for his pathologic diagnosis (Department of Pathology, National Minami-Kyushu Hospital, Kagoshima, Japan). They also wish special thanks to Mrs. Rumi Matsuyama and Dr. Arlene Ng (Third Department of Internal Medicine, Kagoshima University Faculty of Medicine, Kagoshima, Japan) for their invaluable help in this study.


   Footnotes
 
* These authors contributed equally to this study. Back

Received in original form September 15, 2003

Received in final form November 24, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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