Affiliations of authors: Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology (CCC, JLS, MLK), School of Medicine (STC), College of Medicine, National Taiwan University, Taipei, Taiwan; Departments of Internal Medicine (JYS), Pathology (YMJ), and Surgery (BZL), National Taiwan University Hospital, Taipei; Institute of Anatomy, School of Medicine, National Yang-Ming University, Taipei (YPC); Department of Internal Medicine, National Taiwan University Hospital, National Health Research Institutes, and Institute of Biomedical Sciences, Taipei (PCY).
Correspondence to: Min-Liang Kuo, PhD, Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan (e-mail: toxkml{at}ha.mc.ntu.edu.tw)
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ABSTRACT |
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INTRODUCTION |
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Connective tissue growth factor (CTGF, also known as CCN2) is a member of the CCN family, which includes CTGF, cysteine-rich 61 (Cyr61, also known as CCN1), and nephroblastoma overexpressed (Nov, also known as CCN3), as well as Wisp-1/elm1 (CCN4), Wisp-2/rCop1 (CCN5), and Wisp-3 (CCN6) (16,17). The CTGF gene encodes a 38-kd cysteine-rich heparin-binding protein, first identified as a mitogen found in conditioned medium from human umbilical vein endothelial cells (18). CTGF is a secreted growth factor that can bind to integrins on the cell surface (16). CTGF mRNA is expressed in adult human heart, brain, placenta, liver, muscle, kidney, and lung (19) and may have physiologic functions in these human tissues. CTGF is involved in chondrocyte growth, proliferation (20,21), and differentiation (2022); endothelial cell proliferation and migration (23,24); and regulation of apoptosis in human breast cancer cells and aortic smooth muscle cells (25,26). The level of CTGF protein is increased in patients with various human diseases, including systemic scleroderma (27,28), atherosclerosis (29), renal diseases (30,31), and hepatic fibrosis in biliary atresia (32). Although elevated CTGF expression has been observed in breast cancers (33), pancreatic cancers (34), melanomas (35), and chondrosarcomas (36), a definitive role of CTGF in the development or progression of these human cancers has not yet been established.
Recent findings indicated that several members of the CCN family can affect tumor development and progression. For example, Cyr61 (CCN1) overexpression appears to inhibit the tumorigenicity of NSCLC cells in SCID mice (37). Wisp-1 (CCN4) expression appears to reduce the in vitro invasive ability of NSCLC cells (38) and the in vivo metastasis of mouse melanoma cells (39). Thus, CCN proteins appear to display different biologic properties in distinct types of cancer. Our goal in this study was to elucidate the role of CTGF and its possible downstream effector collapsin response mediator protein 1 (CRMP-1) in the metastasis and invasion of human lung adenocarcinoma.
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MATERIALS AND METHODS |
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Lung adenocarcinoma cells were grown in RPMI-1640 medium with 10% fetal bovine serum and 2 mM L-glutamine (all from Life Technologies, Rockville, MD) at 37 °C in a humidified atmosphere of 5% CO2/95% air. Lung adenocarcinoma cell lines CL1-3 and CL1-5 are sublines that were selected from parental CL1-0 cultures with a Matrigel-coated polycarbonate membrane (Collaborative Biomedical, BD Biosciences, Bedford, MA) in a Transwell invasion chamber as described previously (40). Other lung adenocarcinoma cell lines usedA549, H928, and NICH520were obtained from American Type Culture Collection (Manassas, VA). Adherent cells were detached from the culture dishes with trypsin/EDTA (Sigma, Deisenhofen, Germany).
Reagents
The CRMP-1 antisense oligonucleotide sequence was 5'-ATACCTCCGGGAATAACCAT-3'. This sequence did not have substantial homology with any other sequence in the BLAST databases from the National Center for Biotechnology Information. The CRMP-1 sense control oligonucleotide sequence was 5'-TATGGAGGCCCTTATTGGTA-3'. Anti-v
3 integrin functional blocking antibodies (product LM609) and anti-
v
5 integrin functional blocking antibodies (product B1F6) were obtained from Sigma.
Integrin Function Blocking With Specific Antibodies
CL1-5 cells stably transfected with either the control vector (pcDNA3) or the full-length wild-type CTGF vector (CTGF/wt) were treated with anti-v
3 antibody (LM609), anti-
v
5 antibody (B1F6), or immunoglobulin G (IgG) control antibody (each at 10 µg/mL) for 24 hours. After treatment, cells were washed with phosphate-buffered saline (PBS) containing 5 mM EDTA and 1 mM sodium orthovanadate and then scraped into a lysis buffer (20 mM Tris-HCl [pH 8.0], 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and aprotinin at 0.15 U/mL), and the mixture was incubated for 30 minutes on ice. The lysed cells were centrifuged at 14 500g for 30 minutes at 4 °C, and the supernatant was collected. Proteins in the supernatant were quantified by spectrophotometry, and then CRMP-1 and
-tubulin proteins were assessed by western blot analysis.
Western Blot Analysis
Proteins in the total cell lysate (40 µg of protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels and electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore, Bedford, MA). After the blot was blocked in a solution of 5% skim milk, 0.1% Tween 20, and PBS, membrane-bound proteins were probed with primary antibodies against CTGF, Cyr61, -actin, or
-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) or against CRMP-1 (41). The membrane was washed and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 minutes. Antibody-bound protein bands were detected with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and photographed with Kodak X-Omat Blue autoradiography film (Perkin Elmer Life Sciences, Boston, MA).
Reverse Transcription-Polymerase Chain Reaction
Reverse transcription of RNA isolated from cells was performed in a final reaction volume of 20 µL containing 5 µg of total RNA in Moloney murine leukemia virus (MMLV) reverse transcriptase buffer (Promega, Madison, WI), which consists of 10 mM dithiothreitol, all four deoxynucleoside 5'-triphosphates (dNTPs; each at 2.5 mM), 1 µg of (dT)1218 primer, and 200 U of MMLV reverse transcriptase (Promega). The reaction mixture was incubated at 37 °C for 2 hours, and the reaction was terminated by heating at 70 °C for 10 minutes. One microliter of the reaction mixture was then amplified by polymerase chain reaction (PCR) with the following pairs of primers: CTGF primers, 5'-GCTTACCGACTGGAAGACACGTT-3' (sense) and 5'-TCATGCCATGTCTCCGTACATC-3' (antisense), to produce a 500-base-pair (bp) fragment of the CTGF gene; Cyr61 primers, 5'-CGAGGTGGAGTTGACGAGAAAC-3' (sense) and 5'-AGGACTGGATCATCATGACGTTCT-3' (antisense) to produce a 450-bp fragment of the Cyr61 gene; CRMP-1 primers, 5'-CCACGATGATCATTGACCATGT-3' (sense) and 5'-AGGGAGTAATCACAGCAGGATTTG-3' (antisense) to produce a 200-bp fragment of the CRMP-1 gene; and -actin primers, 5'-GATGATGATATCGCCGCGCT-3' (sense) and 5'-TGGGTCATCTTCTCGCGGTT-3' (antisense) to produce a 320-bp fragment of the
-actin gene, which was used as the internal control. The PCR amplification was carried out in a reaction buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, all four dNTPs (each at 167 µM), 2.5 U of Taq DNA polymerase, and 0.1 µM primers. The reactions were performed in a Biometra Thermoblock (Biometra, Hamburg, Germany) with the following program: denaturing for 1 minute at 95 °C, annealing for 1 minute at 58 °C, and elongating for 1 minute at 72 °C for a total of 23 cycles; the final extension took place at 72 °C for 10 minutes. Equal volumes of each PCR sample were subjected to electrophoresis in a 1% agarose gel, which was then stained with ethidium bromide and photographed under UV illumination.
Construction of CTGF/wt Expression Plasmid and CTGF Deletion Mutants
Total RNA was extracted from CL1-0 cells. CTGF cDNA was cloned and amplified by reverse transcription-polymerase chain reaction with primers 5'-ATGACCGCCGCCAGTATGG-3' and 5'-TCATGCCATGTCTCCGTACATCTT-3' (PubMed serial number XM-037056), and the product was subcloned into a pcDNA3/V5-His TOPO TA vector (Invitrogen, San Diego, CA). The CTGF-expression vector (CTGF/wt) was used in transient and stable transfections of human lung adenocarcinoma cells in vitro and in vivo. Three serial deletion mutants of CTGF were generated by deleting the CT domain; the CT and TSP-1 domains; or the CT, TSP-1, and VWC domains. These constructs were designated CTGF/d3, CTGF/d2, or CTGF/d1, respectively. Deletion constructs were generated with the reverse primer 5'-CGGAATTCAACCATGACCGCCGCCAGT-3' in combination with the forward primers 5'-GCTCTAGATCAGATGCACTTTTTGCCCTTC-3', 5'-GCTCTAGATCAGTCTGGGCCAAACGTGTCT-3', and 5'-GCTCTAGATCAGCAGGGAGCACCATCTTTG-3', respectively.
Plasmids and Transient Transfection
The CTGF expression vectors were transiently transfected into CL1-5 and A549 cells with TransFast transfection reagent (Promega). Briefly, 3 µg of plasmid DNA (CTGF/wt, CTGF/d3, CTGF/d2, CTGF/d1, or pcDNA3) and 8 µg of transfection reagent were mixed, and the transfection protocol was carried out according to the manufacturer's instructions (Promega). One hour after transfection, the cells were cultured in normal complete medium for another 8 hours. The transfected cells were harvested and subjected to an invasion assay and western blot analysis.
Stable Transfected Clone Selection
Purified plasmid DNA (3 µg) was transfected into CL1-5 and A549 cells with TransFast transfection reagent. Twenty-four hours after transfection, stable transfectants were selected in Gentamicin (G418; Life Technologies) at a concentration of 600 µg/mL. Thereafter, the selection medium was replaced every 3 days. After 2 weeks of selection in G418, clones of resistant cells were isolated and allowed to proliferate in medium containing G418 at 100 µg/mL. Integration of transfected plasmid DNA was confirmed by reverse transcription-polymerase chain reaction and western blot analysis.
In Vitro Cell Growth Assay
Control vector (pcDNA3), the respective CTGF/wt expressing vector, and several truncated deletion plasmids (3 µg/µL) were added to 6-cm dishes initially containing 105 cells per well. At regular intervals, cells were trypsinized and resuspended, and the cell numbers were counted with a hemacytometer.
Boyden Chamber Assays
For invasion assays, we used modified Boyden chambers with filter inserts (pore size, 8 µm) coated with Matrigel (40 µg; Collaborative Biomedical, Becton Dickinson Labware) in 24-well dishes (Nucleopore, Pleasanton, CA). Approximately 2.5 x 104 cells in 100 µL of complete RPMI-1640 medium were placed in the upper chamber, and 1 mL of the same medium was placed in the lower chamber. After 48 hours in culture, cells were fixed in methanol for 15 minutes and then stained with 0.05% crystal violet in PBS for 15 minutes. 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 viewed and counted under a microscope (type 090-135.001; Leica Microsystems, Wetzlar, Germany). Each clone was plated in triplicate in each experiment, and each experiment was repeated at least three times.
Antisense Oligonucleotide Experiments
CRMP-1 antisense or sense oligonucleotides were introduced into stable transfected clones CL1-5/neo and CL1-5/CTGF by incubation with Lipofectamine (TransFast transfection reagent; Promega) (oligonucleotide/Lipofectamine ratio = 10 : 1 [wt/wt]) in starvation medium for 8 hours. After the indicated time, cells were transferred to complete RPMI-1640 medium for another 48 hours. Total cell extracts were then collected and subjected to western blot analysis for CRMP-1 and -tubulin proteins. In the invasion experiments, CL1-5/neo and CL1-5/CTGF cells were transfected with CRMP-1 antisense (or sense) oligonucleotides by use of Lipofectamine in starvation medium. After 8 hours of incubation, cells were trypsinized; 2.5 x 104 cells were seeded in the upper chamber, and 1 mL of the RPMI-1640 medium was placed in the lower chamber. After another 48 hours, cells were fixed and counted as described above.
Experimental Metastasis
Cells were washed and resuspended in PBS. Subsequently, a single-cell suspension containing 106 cells in 0.1 mL of PBS was injected into the lateral tail vein of 6-week-old SCID mice (supplied by the animal center in the College of Medicine, National Taiwan University, Taipei, Taiwan). Mice were killed after 8 weeks. (Our preliminary study in this animal model indicated that CL1-5 cells developed numerous lung metastasis nodules by 8 weeks.) All organs were examined for metastasis formation. The lungs were removed, weighed, and fixed in 10% formalin. The number of lung tumor colonies was counted under a dissecting microscope. Representative lung tumors were removed, fixed, and embedded in paraffin. Embedded tissue was sectioned into 4-µm sections, and the sections were stained with hematoxylin-eosin for histologic analysis. All animal work was performed under protocols approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University.
Patients and Specimens
Lung adenocarcinoma specimens were obtained from a total of 78 consecutive patients who underwent surgical resection at the National Taiwan University Hospital from September 1, 1993, to August 31, 1997. Patients who had previous history of cancers or had been treated with neoadjuvant chemotherapy and/or radiation therapy were not included in this study. Only patients who provided lung adenocarcinoma specimens were included in this study. Paraffin-embedded, formalin-fixed surgical specimens were collected for immunohistochemical staining for CTGF. The group consisted of 39 men and 39 women with an age of 62 ± 11 years (mean ± standard deviation). Written informed consent was obtained from all patients. The histologic identification of lung cancer was determined as recommended by the World Health Organization (42). Tumor size, local invasion, and lymph node metastasis were determined at pathologic examination. The final disease stage was determined by a combination of surgical and pathologic findings, according to the current tumor-node-metastasis staging system for lung cancer (43). Follow-up data were obtained from the patients medical charts and from our tumor registry service. The survival time of patients was calculated from the date of surgery to the date of death. The relapse time was calculated from the date of surgery to the date of local recurrence or distant metastasis. Median follow-up was 37.4 months (range = 1117 months).
Immunohistochemistry
After rehydration, 4-µm sections of paraffin-embedded tissue on glass slides were incubated in 3% hydrogen peroxide to block the endogenous peroxidase activity. After trypsinization, sections were blocked by incubation in 3% bovine serum albumin in PBS. The primary antibody, a polyclonal goat anti-human CTGF antibody (Santa Cruz Biotechnology) was applied to the slides at a dilution of 1 : 100 (diluted in 3% bovine serum albumin) and incubated at 4 °C overnight. After three washes in PBS, the samples were treated with donkey anti-goat IgG biotin-labeled secondary antibodies (Vector Laboratories, Burlingame, CA) at a dilution of 1 : 500 (diluted in 0.05% PBS-Tween 20) for 1 hour at room temperature. Bound antibodies were detected with an ABC kit (Vector Laboratories). The slides were stained with diaminobenzidine, washed, counterstained with Delafield's hematoxylin, dehydrated, treated with xylene, and mounted. A scoring system was devised to assign a staining intensity score for CTGF expression from 0 (no expression) to 3 (highest intensity staining). Immunostaining was classified into one of two groups according to both intensity and extent: low expression was defined as no staining present (staining intensity score = 0) or positive staining detected in less than or equal to 10% of the cells (staining intensity score = 1) and high expression was defined as positive immunostaining present in 10%50% of the cells (staining intensity score = 2) or more than 50% of the cells (staining intensity score = 3).
Statistical Analysis
Statistical evaluation of the data was performed with a two-tailed Student's t test for simple comparison between two values when appropriate. All statistical analyses were performed with SPSS, version 10.0 (SPSS, Chicago, IL). Pearson chi-square tests and Student's t tests were used to compare the clinicopathologic characteristics of tumors (and patients) with high expression of CTGF and those with low expression of CTGF. Survival was analyzed by the Kaplan-Meier method, and the log-rank test was used to test the difference in relapse time and survival between patients with tumors that had high expression of CTGF and those with low expression of CTGF. The median survival times with 95% confidence intervals (CIs) were calculated as described (44). Multivariable analyses with the Cox proportional hazards model were used to estimate the simultaneous effects of prognostic factors on survival (45). After confirmation that the data met the assumptions for a proportional hazards analysis, stepwise selection was used. Variables were retained in the model if the associated two-sided P values were .10 or less. All statistical tests were two-sided. P values of less than .05 were considered statistically significant.
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RESULTS |
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To explore the possible role of CTGF in invasiveness of lung adenocarcinoma cells, we first examined the expression of CTGF in a panel of cell lines, CL1-0, CL1-3, and CL1-5, with either low or high invasive ability, as previously described (41). CTGF mRNA was highly expressed in CL1-0 cells that have low invasive and low metastatic ability but was almost undetectable in highly invasive CL1-3 and CL1-5 cells (Fig. 1, A, upper right panel). The invasive ability of CL1-3 and CL1-5 was fourfold to sixfold higher than that of CL1-0 cells (Fig. 1, A, lower panel). The level of Cyr61 mRNA, another member of the CCN family, was the same in cell lines with low or high invasive ability (Fig. 1, A, upper right panel). CL1-0 cells expressed a high level of CTGF protein, and CL1-3 and CL1-5 cells expressed extremely low levels of CTGF protein (Fig. 1, A, upper left panel); the level of Cyr61 protein appeared constant in CL1-0, CL1-3, and CL1-5 cells (Fig. 1, A, upper left panel). We tested other lung adenocarcinoma cell lines (A549, NICH520, and H928) to determine whether this relationship between the level of CTGF and invasive ability was also present. A549 cells were more invasive than NICH520 and H928 cells (Fig. 1, B, lower panel). The highly invasive A549 cells expressed a very low or undetectable level of CTGF protein, whereas the less invasive NICH520 and H928 cells expressed high levels of CTGF protein (Fig. 1, B, upper panel). Thus, expression of CTGF was inversely associated with an invasive and/or metastatic phenotype of lung adenocarcinoma cell lines. Although Cyr61 has been shown to act as a tumor suppressor in NSCLC cells (37), the same level of Cyr61 expression was detected in A549, NICH520, and H928 lung adenocarcinoma cells. Thus, Cyr61 does not appear to be associated with invasion and metastasis in lung adenocarcinoma cells.
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To clarify the direct role of CTGF expression in the invasiveness of lung adenocarcinoma cells, human CTGF cDNA expression vectors or control vectors were transfected into the highly invasive CL1-5 cells. After G418 selection, we isolated two single clones (CL1-5/CTGF-3 and CL1-5/CTGF-10), a clonal mixture (CL1-5/CTGF-M), and vector control clone (CL1-5/neo), and then we assessed the levels of CTGF expression in each. We detected 3.6-fold to 5.5-fold more CTGF protein in CTGF-overexpressing cell lines than in vector control cells (Fig. 1, C). Invasive capacity was much lower in CTGF-transfected CL1-5 cells than in CL1-5/neo control cells (Fig. 1, D) (percentage of invasive CL1-5/neo control cells: CL1-5/CTGF-M cells = 52.7% [95% CI = 23.9% to 70.8%], CL1-5/CTGF-3 cells = 44.0% [95% CI = 47.0% to 65.0%], and CL1-5/CTGF-10 cells = 54.0% [95% CI = 36.9% to 55.2%]; all P<.001). We also transfected the CTGF expression vector or the control vector into highly invasive A549 cells and examined the invasive ability of the transfected cells. After transfection and selection in G418, a pool of cells (A549/CTGF-M), two single clones (A549/CTGF-4 and A549/CTGF-5), and a vector control clone (A549/neo) were isolated, and their expression of CTGF protein was assessed by western blotting. CTGF-transfected A549 cells (A549/CTGF-M, A549/CTGF-4, and A549/CTGF-5) clearly expressed CTGF protein (Fig. 1, E, upper panel), and the invasive ability of CTGF-overexpressing A549 cells was only about 40% that of the control A549/neo cells (percentage of invasive A549/neo cells: A549/CTGF-M cells = 37.7% [95% CI = 37.0% to 87.7%], A549/CTGF-4 cells = 32.9% [95% CI = 30.9% to 88.4%], A549/CTGF-5 cells = 41.0% [95% CI = 32.7% to 85.3%]; all P<.001) (Fig. 1, E, lower panel).
To rule out the possibility that the effect of CTGF on in vitro cell invasiveness was caused by different proliferation rates among the cell lines, we compared the growth rates of CTGF-overexpressing cells with those of the corresponding vector control cells. The growth curves for the vector control cells CL1-5/neo and the CTGF-overexpressing cells CL1-5/CTGF-M, CL1-5/CTGF-3, and CL1-5/CTGF-10 were similar (Fig. 1, F), as were those for the vector control cells A549/neo and CTGF-overexpressing cells A549/CTGF-M, A549/CTGF-4, and A549/CTGF-5 (data not shown). Thus, the decreased invasiveness of these CTGF-transfected cells is apparently not caused by decreased proliferation rates.
The effect of CTGF overexpression on metastatic colonization was further assessed by intravenous injection of 1 x 106 cells into the lateral tail vein of male SCID mice. Each clonal cell line was injected into 10 mice. Eight weeks later, the mice were killed, and the number of metastatic tumors formed in the lungs was counted. Mice injected with CL1-5/neo or A549/neo control clones had numerous large lung metastases, whereas mice injected with CL1-5/CTGF-M or A549/CTGF-M cells had fewer and smaller metastatic nodules in the lung (Fig. 2, A). Metastatic tumors formed in the lungs by CL1-5/neo cells had the morphology of a typical adenocarcinoma (Fig. 2, B). Mice injected with CL1-5/neo cells had 77.5 metastatic lung nodules, and mice injected with CL1-5/CTGF had 12.3 metastatic lung nodules (difference = 65.2 nodules, 95% CI = 48.9 to 81.6 nodules; P<.001). Mice injected with A549/neo had 61.3 metastatic lung nodules, and mice injected with A549/CTGF had 21.7 metastatic lung nodules (difference = 39.6 nodules, 95% CI = 23.4 to 56.0 nodules; P = .003). Thus, overexpression of CTGF in CL1-5 and A549 cells suppressed the ability of these cells to form metastatic nodules in the lungs.
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CTGF is a secreted growth factor that binds to integrins on the cell surface (16). The binding of CTGF to integrins could activate intracellular pathways that regulate invasion and metastasis. It has been reported that CRMP-1, an intracellular molecule, inhibits the invasion of lung cancer cells (40). To characterize the relationship between CTGF and CRMP-1, we first used western blot analysis to determine the level of CRMP-1 protein in CL1-5/CTGF stable clones and found that CRMP-1 protein expression was threefold to sixfold higher in CTGF-overexpressing CL1-5 cells than in vector control cells (Fig. 3, A, upper panel). A similar increase of CRMP-1 protein was also seen in A549/CTGF-M, A549/CTGF-4, and A549/CTGF-5 cells (Fig. 3, A, upper panel). CL1-0 cells, which express a high level of CRMP-1 (40), also expressed a high level of CTGF (Fig. 1, A). When CL1-0 cells were transfected with an antisense CTGF expression vector or a control vector, the level of CRMP-1 protein was substantially lower in cells transfected with the antisense vector than with the control vector (Fig. 3, A, upper panel). In addition, the expression of CRMP-1 was greatly reduced in CL1-5/CTGF-M cells incubated with antibodies that specifically block the functions of integrin v
3 or
v
5 compared with a control IgG antibody (Fig. 3, A, lower panel). Thus, it appears that CTGF induces CRMP-1 expression through integrins
v
3 and
v
5.
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To further assess the role of CRMP-1 in the CTGF-mediated inhibition of cell invasion, CL1-5/CTGF-M cells were treated with 10 or 20 µM antisense or sense oligonucleotides specific for CRMP-1. The level of CRMP-1 protein was effectively reduced in CL1-5/CTGF-M cells by treatment with 20 µM CRMP-1-specific antisense oligonucleotides but not by treatment with its sense oligonucleotides (Fig. 3, C, upper panel). Treatment with antisense oligonucleotides specific for CRMP-1 did not alter the invasiveness of CL1-5/neo cells (mean cell number = 578 cells; Fig. 3, C, lower panel). In addition, treatment of CL1-5/CTGF cells with 20 µM CRMP-1-specific sense oligonucleotide also did not alter their invasion activity (mean cell number = 327 cells). However, the invasive ability of CL1-5/CTGF-M cells was statistically significantly increased by treatment with 20 µM CRMP-1 antisense oligonucleotides (mean numbers of CL1-5/CTGF cells treated with 10 µM or 20 µM CRMP-1-specific antisense oligonucleotides = 354 cells [difference from control cells = 27 cells, 95% CI of the difference = 1 to 52 cells; P<.001] and 516 cells [difference from control cells = 189 cells, 95% CI of the difference = 156 to 221 cells; P<.001]). Therefore, CTGF-mediated inhibition of invasiveness in lung adenocarcinoma cells appears to be mediated, at least in part, by enhanced CRMP-1 expression.
CTGF and other CCN proteins have a structure with several domains or modules. To determine which module participates in the induction of CRMP-1 and inhibition of invasion, we produced and characterized a series of CTGF deletion constructs. Because CTGF is a secretory protein, the signal sequence at the amino-terminal end was retained and sequences at the carboxyl-terminal end were sequentially deleted. The products were subcloned into a pcDNA3 expression vector, and isolated clones were designated CTGF/wt, CTGF/d3, CTGF/d2, and CTGF/d1 (Fig. 4, A). The invasive capacities of transiently transfected CL1-5 cells expressing the full-length CTGF (CTGF/wt) or its deletion mutants CTGF/d3, CTGF/d2, or CTGF/d1 were determined. CL1-5 cells expressing CTGF/d3, which lacks the CT module, had a high invasive capacity that was equivalent to that of CL1-5 cells expressing the control vector. The mean numbers of invasive CL1-5 cells transfected with CTGF/wt and CTGF/d3, a deletion of the CT module, were 148 cells and 385 cells (difference = 237 cells, 95% CI = 208 to 266 cells; P<.001), respectively (Fig. 4, B). CL1-5 cells transfected with CTGF/d2 or CTGF/d1 also had a relatively high invasive capacity compared with cells transfected with CTGF/wt. Western blot analysis showed statistically significantly higher expression of CRMP-1 protein in CTGF/wt-transfected CL1-5 cells than in CTGF/d3-, CTGF/d2-, or CTGF/d1-transfected CL1-5 cells, which was either very low or undetectable (Fig. 4, C). Thus, the CT module appears to be essential for CTGF-induced inhibition of tumor invasion and metastasis.
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To investigate the involvement of CTGF in the progression of human lung adenocarcinoma, normal and tumor specimens from 78 patients with lung adenocarcinoma were analyzed immunohistochemically for the expression of CTGF. Expression of CTGF protein was high (intensity level 3) in the normal lung epithelium (Fig. 5, A) and moderate to high (intensity levels 2 and 3) in stage I lung adenocarcinoma cells (Fig. 5, B). In these tumors, the protein was predominately localized to the cytoplasm. Expression of CTGF was reduced (intensity level 1) in low-grade metastatic epithelial tumor cells (Fig. 5, C). CTGF was not detected in adenocarcinoma cells but was detected in the normal fibroblast and epithelial components of the same field, which were used as the corresponding positive staining control (Fig. 5, D).
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DISCUSSION |
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We have demonstrated that the level of CTGF protein was statistically significantly higher in normal lung type I and II epithelial cells than in the majority of metastatic adenocarcinoma specimens, suggesting that the level of CTGF protein decreases during clinical disease when cells acquire the ability to grow at a metastatic site. This type of an effect has also been observed with other members of the CCN family in different types of cancer. For instance, expression of Cyr61 mRNA is higher in breast cancers but is lower in hepatoma (46), prostate cancer (47), and NSCLC (48) than their corresponding normal tissues. Although overexpression of Cyr61 in breast cancer cells increased tumor growth (49), overexpression of Cyr61 in lung cancer cells decreased tumor growth in mice (37). Expression of human Wisp-1 (CCN4) increased tumorigenesis in normal rat kidney fibroblasts (50) but decreased tumorigenesis and metastasis in melanoma cells (39) and lung cancer cells (38). Thus, the relationship between these proteins and cancer progression cannot be generalized across different types of cancers.
CTGF-mediated increase in CRMP-1 expression was abolished by treatment with antibodies that specifically block the function of integrins v
3 and
v
5. In addition, antisense CRMP-1 oligonucleotides essentially abolished the CTGF-mediated inhibition of cell invasion. These data indicate that CRMP-1 acts downstream of CTGF and is regulated by an integrin-related signaling pathway. CRMP-1 is a member of the CRMP family, whose members are involved in axonal guidance and neuronal differentiation (51). CRMPs can function as inhibitors of axon extension during development and can decrease cell motility by the depolymerization of filamentous actin in filopodia. The level of CRMP-1 protein was much lower in more invasive CL1-5 cells than in less invasive CL1-0 cells (41). Overexpression of CRMP-1 in CL1-5 cells decreased the invasiveness of CL1-5 cells. These CRMP-1-overexpressing cells displayed a rounded and less invasive epithelial morphology with fewer filopodia containing filamentous actin (41). CTGF-transfected CL1-5 cells had the typically rounded epithelial cell morphology with less filamentous actin, but CL1-5 cells had a flat, spindle- and fibroblast-like morphology (data not shown). Hall et al. (52) and Leung et al. (53) showed that CRMP-2 can be regulated by Rho kinase and Rho GTPase and can, in turn, promote neurite outgrowth and dynamic cell shape changes. In addition, small G proteins are involved in the integrin-mediated modulation of cell migration and invasion (54). These findings lead us to suspect that CTGF affects CRMP-1 expression by modulating the activity of integrin-coupled small G proteins. Indeed, our preliminary data indicate that RhoA activity is lower in CTGF-transfected CL1-5 cells than in vector control CL1-5/neo cells (data not shown). Our study has identified a critical role of CRMP-1 in CTGF-mediated tumor invasion and metastasis and also identified the regulatory mechanism of the metastasis suppressor gene CRMP-1. Details of the CTGF-induced signaling pathway leading to the increased expression of the CRMP-1 gene are currently under investigation.
Integrins are important receptors for CCN proteins, and receptor activation may produce a variety of effects. CTGF protein can bind directly to integrins v
3 and
IIb
3 (24,55). Interaction of CTGF with integrin
v
3 promotes endothelial cell adhesion, migration, and survival and also induces angiogenesis in vivo (24). CTGF also stimulates human skin fibroblast migration and proliferation through integrin
6
1 (56). In contrast, we show that both
v
3 and
v
5 integrins were required for the inhibition of CTGF-mediated invasion. Our findings are supported by those of Soon et al. (38) that Wisp-1 (CCN4) inhibits the invasion of lung cancer cells through a pathway dependent on integrins
v
5- and
1-dependent pathway. It is puzzling that interaction of CCN proteins with the same cell surface integrins exerts contrasting biologic outcomes in different cell types. CCN proteins interact with a wide variety of cell surface glycoproteins, such as heparan sulfate proteoglycan (57), decorin (58,59), and biglycan (59), which might serve as co-receptors to modulate the signaling and function of integrins and in turn engage diverse biologic effects. In addition, CTGF protein forms complexes with various growth factors, such as vascular endothelial growth factor (60), bone morphogenetic proteins, and transforming growth factor
(61). CTGF could alter biologic functions by the selective interactions with possible co-receptors of integrins
v
3 or
v
5 or other soluble factors. Investigations are underway to identify additional factors that could interact with CTGF in human lung adenocarcinoma cells.
Members of the CCN family have high structural homology (62), sharing four conserved cysteine-rich modular domains with sequence similarity to insulin-like growth factor-binding protein, von Willebrand factor, thrombospondin, and growth factor cysteine knots (CT module). To determine which domain of CTGF is responsible for invasion inhibition, we established a series of constructs by sequential deletion of sequences at the CT module of CTGF. Cells transfected with and expressing CTGF constructs lacking the CT module could not induce increased CRMP-1 expression or inhibit cell invasion (data not shown). The CT module may contribute a dimerization motif and, in turn, bind with cell surface integrins, heparin sulfate proteoglycan, or Notch receptor (63,64), suggesting that the CT module may play a role in CTGF-mediated outside-in signaling. Interestingly, the CT module is also essential in mitosis and increases adhesive properties of fibroblasts and endothelial cells (65,66).
In summary, CTGF appears to be a suppressor of lung tumor invasion and metastasis. Our studies directly demonstrated that overexpression of CTGF not only suppressed the ability of lung adenocarcinoma cells to invade Matrigel in vitro but also strongly inhibited tumor metastasis in an animal model. At the mechanistic level, we found that CRMP-1 acts downstream of CTGF and that its regulation is mediated by integrins v
3 and
v
5. Decreased CTGF expression in tumor tissues was associated with advanced tumor stage, lymph node metastasis, early postoperative relapse, and shorter patient survival. We document a functional linkage between two metastasis suppressors, CTGF and CRMP-1, in the same tumor.
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Manuscript received July 25, 2003; revised December 26, 2003; accepted January 12, 2004.
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