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© The Author 2006. Published by Oxford University Press.
ARTICLE |
Effect of Connective Tissue Growth Factor on Hypoxia-Inducible Factor 1
Degradation and Tumor Angiogenesis
Affiliations of authors: Angiogenesis Research Center, Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (CCC, MLK); Departments of Surgery (MTL, BRL, KJC), Pathology (YMJ), Pediatrics (STC), and Dermatology (CYC), National Taiwan University Hospital, Taipei, Taiwan; Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan (RJC); Department of Internal Medicine, National Taiwan University Hospital, National Health Research Institutes, and Institute of Biomedical Sciences, Taipei, Taiwan (PCY).
Correspondence to: Min-Liang Kuo, PhD, Angiogenesis Research Center, 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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Background: Connective tissue growth factor (CTGF) inhibits the metastatic activity of human lung cancer cells in a mouse model; however, the mechanism of this modulation is unclear. We investigated the role of angiogenesis in this process. Methods: CL1-5 and A549 human lung adenocarcinoma cells were stably transfected with vectors containing CTGF or hypoxia-inducible factor (HIF) 1
or with vector controls. Transfected cells were injected into nude mice (n = 10 per group), and tumor growth, metastasis, and mouse survival were measured. Excised xenograft tumors and primary human lung adenocarcinomas (n = 24) were subjected to immunohistochemistry with antibodies to the endothelial cell marker CD31 and to CTGF. Expression of HIF-1 and vascular endothelial growth factor (VEGF) A was assessed in vitro by using reporter gene assays. Cells were transiently transfected with HIF-1 mutant and antisense arrest-defective 1 protein (ARD-1), and HIF-1 acetylation was assayed by immunoprecipitation. All statistical tests were two-sided. Results: Xenograft tumors derived from CTGF transfectants grew more slowly than those from control-transfected cells and had reduced expression of HIF-1 and VEGF-A, vascularization (as assessed by CD31 expression), and metastasis (all P<.001). Xenograft tumors derived from CTGF-overexpressing cells that were transfected with HIF-1 had higher VEGF-A expression than CTGF-overexpressing xenografts. Mice with CTGF/HIF-1 xenografts had lower survival than mice carrying CTGF-overexpressing xenografts (CL1-5/Neo, mean = 69.6 days, 95% confidence interval [CI] = 53.9 to 85.3 days versus CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days; P = .001, CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days versus CL1-5/CTGF/HIF-1, mean = 81.7 days, 95% CI = 66.5 to 96.9 days; P = .011, CL1-5/Neo, mean = 69.6 days, 95% CI = 53.9 to 85.3 days versus CL1-5/CTGF/HIF-1, mean = 81.7 days, 95% CI = 66.5 to 96.9 days; P = .122). Tumors of patients with the same disease stage but with high CTGF protein expression had reduced microvessel density compared with tumors with low expression. Transfection with antisense-ARD1 decreased the level of acetylated HIF-1 and restored HIF-1 and VEGF-A expression in CTGF-overexpressing cells. Conclusion: CTGF inhibition of metastasis involves the inhibition of VEGF-A–dependent angiogenesis, possibly by promoting HIF-1 protein degradation.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Angiogenesis, the formation of new blood vessels, is essential for tumor growth and metastasis (1–4). Angiogenesis is strictly controlled by a highly coordinated process that is regulated by many molecules. Among them is vascular endothelial growth factor (VEGF), the major regulator of tumor-associated angiogenesis in lung adenocarcinoma, which can promote tumor growth and metastasis (5). It has previously been reported that VEGF-A is the most predominant angiogenic factor in human lung cancer (6,7) and that VEGF-A protein expression is strongly associated with many types of cancer progression and with clinical outcome (8). Inhibition of VEGF reduces angiogenesis and tumor growth in vivo (9). Conversely, VEGF overexpression is associated with increased microvessel density, tumor metastasis, and poor prognosis (10–16).
Hypoxia-inducible factor (HIF) 1 is a transcription factor that regulates the supply of blood to tissues through its effects on VEGF expression (17). HIF-1 activity is induced by hypoxia in almost all cell types; however, under normoxic conditions, the protein is sensitive to ubiquitin-dependent degradation. HIF-1 protein accumulation is reduced under hypoxic conditions by posttranslational modification and binding to the von Hippel-Lindau (pVHL) tumor suppressor protein (18). HIF-1 binds to pVHL only after it is hydroxylated by HIF-prolyl hydroxylase (PHD1) (19–21) and acetylated by arrest-defective 1 protein (ARD1) acetyltransferase (22). It is well documented that posttranslational modification (i.e., hydroxylation and acetylation) of the HIF-1 protein promotes its association with pVHL and its subsequent degradation (23–25). In particular, this association is triggered by PHD's hydroxylation of prolines and is promoted by ARD1 acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation domain. HIF-1 protein levels also increase in response to growth factor stimulation by mechanisms that differ from those that occur under hypoxic conditions. These oxygen-independent mechanisms are not clearly understood, but they are thought to involve growth factors, cytokines, and other signaling molecules that stimulate synthesis of HIF-1 or decrease its degradation (26–31).
One possible mechanism involves the activity of connective tissue growth factor (CTGF). CTGF is as an extracellular matrix–associated signaling molecule that binds directly with moieties in the pericellular environment. It has recently been identified as a regulator of angiogenesis (32–34) and has many functions in normal and pathologic processes (33–41). Although CTGF was originally purified from medium conditioned by human umbilical vein endothelial cells (42), it can be produced by and acts on many cell types, including fibroblasts, smooth muscle, endothelial, neural, and cancer cells (43–49). CTGF can influence several functions of endothelial cells, including in vitro proliferation and tube formation (33,40), cell adhesion and migration (34), and induction of angiogenesis in vivo (33–34,41). However, it has also been reported that CTGF inhibits angiogenesis in vitro through an interaction with VEGF (42). Angiogenic activity of VEGF was restored after the CTGF–VEGF complex was digested by matrix metalloproteinase 3 or -7 (43). Therefore, CTGF may play an inhibitory role through VEGF-induced angiogenesis during embryonic development, tissue maintenance, and the pathological processes of various diseases. Although CTGF itself is an angiogenesis factor, its interaction with other molecules may alter its function.
Because the mechanisms of CTGF's antiangiogenic activity are not fully understood, in this study, we examined the function of CTGF and its possible downstream effectors on metastasis and angiogenesis of cancer cells. We analyzed the effects of CTGF on tumorigenicity, angiogenesis, and metastasis both in vitro and in vivo using human lung adenocarcinoma cell lines in which CTGF expression is increased or inhibited. In a previous study (44), we showed that the level of CTGF protein is inversely associated with tumor status and metastasis. Using these models, we also investigated the mechanistic relationships among CTGF, HIF-1, and VEGF in the growth and neovascularization of human lung adenocarcinomas.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cell Culture and Treatment
CL1-0 and CL1-5 lung adenocarcinoma cell lines were established by selection of increasingly invasive cancer cell populations from a clonal cell line of human lung adenocarcinoma, CL1. CL1-0 is the parent cell line, and CL1-5 is a subline that was selected from CL1-0 cells cultured on a polycarbonate membrane coated with Matrigel (Collaborative Biomedical, Becton Dickinson Labware, Bedford, MA) in a Transwell invasion chamber. A549, H928, and NICH330 lung adenocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured according to the supplier's recommendations. Cells were grown in RPMI 1640 medium (Life Technologies, Rockville, MD) with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (Life Technologies) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For routine culture and for harvesting, adherent cells (A549 and CL1-5) were detached from culture dishes with 0.05% trypsin–EDTA (Sigma, Deisenhofen, Germany). For experiments with the protease inhibitor, MG132, cells were cultured in serum-free media overnight before treatment with 10 µM MG132 (Sigma) for 2 hours or as indicated.
Expression Constructs
Plasmids containing the CTGF expression construct, ARD1, and antisense (AS) ARD1 were constructed by cloning the human CTGF cDNA (GenBank accession number xm-037056), human ARD1 cDNA (NM-003491), and modified ARD1 cDNA, respectively, into the pcDNA3.1 eukaryotic expression vector, which also expresses a neomycin (Neo) resistance gene. siCTGF (a small-interfering RNA oligonucleotide targeted against the 379- to 397-bp region at the 3' end of exon 2 of murine CTGF) was cloned into the pU6.1 expression vector (a kind gift from Dr. Mien-chie Hung). Plasmids containing HIF-1 and dominant-negative (DN) HIF-1 and the pCEP4 expression vector were purchased from Invitrogen (Rockville, MD). Mutant HIF-1 K532N and luciferase reporters including hypoxia response element (HRE), 1.5-kb wild-type VEGF-A promoter, and 1.2-kb HIF-1–deleted VEGF-A promoter constructs were gifts from Dr. Shuang-En Chuang.
Transfections
CL1-0 cells were stably transfected with a vector control (CL1-0/Neo) or siCTGF (CL1-0/siCTGF), and A549 and CL1-5 cells were stably transfected with vector control (A549/Neo and CL1-5/Neo), the CTGF construct alone (A549/CTGF and CL1-5/CTGF), or the HIF-1 construct (CL1-5/CTGF/HIF-1) using the TransFast transfection reagent (Promega, Madison, WI) according to the manufacturer's instructions. The transfection efficiency was 40%–60%. Cells were replated 48 hours after transfection in RPMI 1640–10% FBS and 0.8 mg/mL of gentamicin (G418; Life Technologies). G418-resistant clones were selected. Transient transfections were performed as above using the TransFast reagent (Promega), and transfected cells were cultured for 48 hours before assays were performed.
Protein Stability Assay, Immunoprecipitation, and Western Blot Analysis
For protein stability assays, CL1-5/Neo and CTGF transfectants were seeded in 6-cm dishes overnight and incubated in serum-free media for 24 hours. After adding 10 µM cycloheximide or dimethyl sulfoxide (vehicle control) for the indicated times, cells were immediately harvested. For immunoprecipitations and western blot analyses, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-base, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4). Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL).
For immunoprecipitations, lysates (equal amounts of protein) of CL1-5/Neo, CL1-5/CTGF, A549 cells transiently transfected with the CTGF expression construct, and CL1-5/CTGF and A549/CTGF cells transiently transfected with AS-ARD1 were incubated for 2 hours at 4 °C with gentle rotation with rabbit anti-human HIF-1, rabbit anti-human pVHL, or rabbit anti-human acetylated protein (4G12) antibodies immobilized onto protein A–Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Beads were washed twice with IP buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1% NP40, 10% glycerol, 1 mg/mL bovine serum albumin, 20 mM Tris, pH 8.0), boiled in sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer, and centrifuged (12 000g, 4 °C for 15 minutes). Supernatants were immediately subjected to western blot analysis.
For western blot analyses, proteins (40 µg) were separated by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked in a solution of 5% nonfat dry milk in PBS-T (0.1% Tween 20, 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, pH 7.4) and then probed with rabbit anti-human CTGF, rabbit anti-human VEGF-A, rabbit anti-human HIF-1, rabbit anti-human HIF-1
, rabbit anti-human VHL, mouse anti-human -actin, rabbit anti-human ubiquitin, mouse anti-human -tubulin (Santa Cruz Biotechnology), or rabbit anti-human acetylated protein (4G12) (Upstate, Charlottesville, VA) antibodies (1 : 100 in PBS-T) overnight at 4 °C. The membranes were washed three times with PBS-T (0.05% Tween 20) and then incubated with horseradish peroxidase–conjugated polyclonal goat anti-rabbit or rabbit anti-mouse secondary antibodies (1 : 5000) for 30 minutes. Antibody–protein complexes were detected using the Enhanced Chemiluminescence Kit (Amersham Pharmacia Biotech, Piscataway, NJ) and were visualized by exposing membranes to Kodak X-Omat Blue film (PerkinElmer, Los Angeles, CA). Where indicated, the membranes were stripped with Restore Western Blot Stripping Buffer (Pierce) and reprobed with another antibody. The signal intensities were quantified by densitometry (UVP, Upland, CA).
Proliferation Assays
For proliferation assays, CL1-5/CTGF, A549/CTGF, CL1-5/Neo, A549/Neo, CL1-0/siCTGF, and CL1-0/Neo cells were seeded at 105 cells/well in RPMI medium in six-well plates in triplicate. The cells were harvested by detachment with trypsin–EDTA at 0, 48, 72, and 96 hours and stained with 0.05% trypan blue. Unstained, i.e., viable, cells were counted using a Coulter counter (Coulter Electronics, Luton, UK).
Preparation of Conditioned Medium
CTGF or vector transfectants were plated in normal culture media and grown to confluence. Cells were then incubated in RPMI medium (without FBS) and 2 mM L-glutamine for 48 hours. Conditioned medium was collected fresh and cleared of cellular debris by using low-speed centrifugation (1500g, 4 °C for 10 minutes).
Mouse Models
For models of lung adenocarcinoma to measure tumorigenicity, 8-week-old male BALB/c nude mice (n = 90; National Defense Medical Center, Taipei, Taiwan) were acclimated for 1–2 weeks while caged in groups of five. The mice were housed in pathogen-free conditions and fed a diet of animal chow and water throughout the experiment. Mice were randomized to one of two groups and were injected subcutaneously with 1) CL1-5/CTGF cells (n = 10), A549/CTGF cells (n = 10), CL1-0/siCTGF cells (n = 10), or CL1-5, A549, and CL1-0 cells transfected with vector controls (n = 10, each); 2) CL1-5/CTGF cells (n = 10), CL1-5/CTGF/HIF-1 cells (n = 10), or CL1-5 cells transfected with pCEP4 (n = 10) (107 cells in phosphate-buffered saline [PBS] and Matrigel, vol/vol = 1 : 1). Tumors were measured with calipers every other day, starting on day 8 after injection, when they had become palpable and visible. Tumor volumes were calculated using the equation: width2 x length x 0.5. The first mouse, which was in the CL1-5/Neo group, died on day 32 after injection. Therefore, all mice were anesthetized and killed by overdose with anesthetic on day 32. Subcutaneous tumors were surgically excised, weighed, and photographed, and a portion of each tumor was placed in 10% formalin for paraffin embedding or was snap-frozen in Optimum Cutting Temperature solution (Miles, Elkhart, IN) in preparation for subsequent immunohistochemical analysis.
For metastasis models, mice were injected subcutaneously with CL1-5/Neo or CL1-5/CTGF cells (107 cells in PBS and Matrigel, vol/vol = 1 : 1). Our preliminary study in this animal model (data not shown) had indicated that mice injected subcutaneously with CL1-5 cells developed many lung metastasis nodules by 4 months. Therefore, after 4 months, mice were killed by overdose with anesthetic and all organs were examined for metastasis formation. The lungs were removed, weighed, and fixed in 10% formalin. The number of lung tumor metastases was counted under a dissecting microscope.
For angiogenesis models, Matrigel plugs (100 µL) (Becton Dickinson Labware, Bedford, MA), either alone or with 5 µg of immunoglobulin G (IgG) or human VEGF-A–specific goat polyclonal IgG–neutralizing antibody (R&D Systems, Minneapolis, MN), were presoaked in 50 µL of concentrated (50x) conditioned medium from CL1-0/Neo, CL1-0/siCTGF, CL1-5/Neo, or CL1-5/CTGF cells. Eight-week-old female BALB/c nude mice (n = 10 per condition) were then injected subcutaneously into the midabdominal regions with the plugs. After 7 days, when vessels in vivo had matured (44), the mice were killed by overdose with anesthetic and the Matrigel plugs were removed and photographed. Blood vessels were quantified by measuring plug hemoglobin (at an optical density at 540 nm) with the Drabkin Reagent Kit 525 (Sigma). All mouse studies were performed using protocols approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University.
Immunohistochemistry to Measure CTGF Expression and Angiogenesis
Tissue sections (4 µm) for immunostaining were obtained from formalin-fixed and paraffin-embedded primary tumors of 24 lung adenocarcinoma patients (who provided written permission) or from the tumors mentioned above, and protocols were approved by the ethics committee of National Taiwan University Hospital. Sections from mice were fixed in acetone and stained overnight with monoclonal anti–mouse CD31/PECAM-1 antibody (BD Biosciences, Franklin Lakes, NJ) at a dilution of 1 : 100 at 4 °C. Sections from patients were stained with anti–human CTGF antibody (1 : 100; Santa Cruz Biotechnology) or anti–human CD31 antibody (BD Biosciences). The samples were washed three times in PBS and incubated with polyclonal goat anti-mouse IgG biotin–labeled secondary antibodies (Vector Laboratories, Burlingame, CA) at a dilution of 1 : 500 for 1 hour at room temperature. Bound antibodies were detected using an ABC kit (Vector Laboratories). The tissues were stained with diaminobenzidine, washed, counterstained with Delafield's hematoxylin, dehydrated, treated with xylene, and mounted onto cover slides for microscopy. All negative controls (no primary antibody) showed low background staining (data not shown).
Microvessel density was determined in anti-CD31–stained human tumors. Entire sections were scanned under low magnification and vascularization was graded subjectively by blinded observers. Three random highly vascularized areas per tumor were then evaluated at high magnification (x400). Any red-stained endothelial cell cluster that was distinct from the adjacent microvessels, tumor cells, or other stromal cells was counted as one microvessel. The total number of microvessels was determined for each area, and the average number of vessels was recorded for each tumor. Slides were stained and analyzed by a pathologist (Dr. Yung-Ming Jeng) who was blinded to the experimental group and had not observed the immunohistochemical staining procedure. CTGF staining intensity was scored from 0 (no expression) to 3 (highest-intensity staining). Immunostaining was then 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 in less than 10% of the cells (staining intensity score = 1), and high expression was defined as positive immunostaining in 10%–50% (staining intensity score = 2) or more than 50% of the cells (staining intensity score = 3).
Reverse Transcription–Polymerase Chain Reaction
RNA was isolated using TRIzol (Invitrogen, Rockville, MD) from transfected lung adenocarcinoma cells, and reverse transcription was performed in a final reaction volume of 20 µL containing 5 µg of total RNA in Moloney murine leukemia virus reverse transcriptase buffer (10 mM dithiothreitol, all four deoxynucleoside 5'-triphosphates [dNTPs; each at 2.5 mM], 1 µg of [dT]12–18 primer, and 200 U of Moloney murine leukemia virus 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 min.
One microliter of the reaction mixture was amplified by polymerase chain reaction (PCR) with the following pairs of primers: VEGF-A, 5'-AGCTACTGCCATCCAATCGC-3' (sense) and 5'-GGGCGAATCCAATTCCAAGAG-3' (antisense); VEGF-C, 5-CAGTTACGGTCTGTGTCCAGTGTAG-3 (sense) and 5-GGACACACATGGAGGTTTAAAGAAG-3 (antisense) to produce a 300-bp fragment of the VEGF-C gene; ARD1, 5'-AGGTTGTTCGATATGGTGAG-3' (sense) and 5'-TCTGCTACAGGGAAAACAGT-3' (antisense); prolyl hydroxylase (PHD)1, 5'-ATGGACAGCCCGTGCCAGCCGCA-3' (sense) and 5'-CGCAGCTCACCACCATCCTGCCC-3' (antisense); PHD2, 5'-ATGGCCAGTGACAGCGGC-3' (sense) and 5'-CAACGGCTTGGTCTGCCC-3' (antisense); PHD3, 5'-ATGCCTCTGGGACACATC-3' (sense) and 5'-TCAGTCTTTAGCAAGAGCA-3' (antisense); and -actin, 5'-GATGATGATATCGCCGCGCT-3' (sense) and 5'-TGGGTCATCTTCTCGCGGTT-3' (antisense) to produce a 320-bp fragment of the -actin gene, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCACCCATGGCAAATTCCATGGCA-3' (sense), 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (antisense), 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, Tampa, FL) using the following protocol: denaturing, 1 minute at 95 °C; annealing, 1 minute at 58 °C; and elongation, 1 minute at 72 °C for a total of 23 cycles, with a final extension of 72 °C for 10 minutes. Equal volumes of PCR samples were subjected to electrophoresis in a 1% agarose gel, which was then stained with 0.1% ethidium bromide and photographed under ultraviolet illumination.
VEGF-A Enzyme-Linked Immunosorbent Assay
To determine VEGF-A concentrations, CTGF transfectants were plated at similar densities (30%–40% confluence) and incubated for 48 hours in RPMI 1640–3% FBS. VEGF-A protein concentrations were measured in 3 mL of conditioned medium collected from the cells using an enzyme-linked immunosorbent assay (ELISA) kit specific for the 165-amino-acid form of human VEGF-A (Biosource International, Camarillo, CA), per the manufacturer's protocol. A VEGF-A standard concentration curve was derived using reagents provided in the kit. VEGF-A levels were expressed in picograms.
Immunofluorescence
CL1-5/Neo and CL1-5/CTGF cells were plated at 105 cells/well and grown on degreased glass coverslips to 60%–80% confluence in RPMI 1640–10% FBS, fixed in methanol/acetic acid (3 : 1, vol/vol) for 30 minutes at 4 °C, and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. The cells were then rinsed with PBS and incubated for 1 hour in PBS–5% FBS at room temperature. The cells were then incubated at 4 °C overnight with anti–HIF-1 polyclonal antibody (1 : 100 in PBS; Santa Cruz Biotechnology). The cells were next washed twice in PBS and incubated with a secondary fluorescein isothiocyanate–conjugated antibody (1 : 200; Sigma) for 1 hour at room temperature. After washing six times in cold PBS, cells were counterstained with 0.1% Hoechst 33258 (Molecular Probes, Eugene, Oregon), Invitrogen, Rockville, MD), coverslips were inverted onto glass slides using Mowiol (Calbiochem) mounting medium. The slides were then observed using a fluorescence microscope (Leica DMR, Stuttgart, Germany).
Luciferase Assays
CL1-5 cells were seeded at 105/well in six-well plates in triplicate and transfected with pGL2-basic vector, which contains a luciferase reporter construct, pGL2–1.5-kb VEGF-A, containing the 1.5-kb region of the VEGF-A promoter, pGL2–1.2-kb/HIF-1 VEGF-A, containing the 1.2-kb region of the VEGF-A promoter mutant for HIF1 binding, and HRE, which was generously donated by Dr. Min-Chie Hung. After transfection, the medium was replaced and cells were cultured for 48 hours. The cells were harvested with passive buffer provided and luciferase activity was determined using a Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's instructions.
Statistical Analysis
The two-tailed Student's t test was used for simple comparison of two values where appropriate. Pearson chi-square tests and Student's t tests were used to compare the clinicopathologic characteristics of tumors (and patients) with high and low CTGF expression. Survival of mice carrying tumors with high and low expression of CTGF and HIF-1 was analyzed using the Kaplan–Meier method. Variables were retained in the model if the associated two-sided P was .10 or less. All statistical tests were two-sided. P<.05 was considered statistically significant for all tests. The SPSS software package (version 10.0; SPSS, Chicago, IL) was used for all statistical analyses.
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RESULTS |
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CTGF Expression and Neovascularization in Primary and Metastatic Tumors
To determine the role of CTGF in tumor angiogenesis, we transfected A549 and CL1-5 human lung adenocarcinoma cell lines with a CTGF-overexpressing vector and transfected CL1-0 lung adenocarcinoma cell line with a small-interfering CTGF (siCTGF) RNA-expressing vector. A549 and CL1-5 cells express very low levels of endogenous CTGF protein, and CL1-0 cells express high levels (45). CTGF protein expression in cells transfected with the CTGF construct was three- to fourfold higher than in cells transfected with empty vector (45), and cells transfected with the siCTGF construct had lower levels of endogenous CTGF protein than cells transfected with empty vector (Fig. 1, A, top). The in vitro growth properties of these cells, as determined by trypan blue exclusion assay, suggested that neither overexpression nor knockdown of CTGF affected tumor cell proliferation (Fig. 1, A, bottom).
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We further investigated the effects of CTGF on tumor growth in a xenograft tumor model in which mice were injected subcutaneously with these stably transfected cell clones. Palpable tumors were first detected in all mice by day 8 after injection. At day 32, tumors in mice injected with CL1-5/Neo cells were larger than tumors in mice injected with CTGF-transfected clones (CL1-5/Neo, mean = 567 mm3, 95% confidence interval [CI] = 538 mm3 to 595 mm3; CL1-5/CTGF, mean = 18.7 mm3, 95% CI = 6.2 mm3 to 24.6 mm3; P = .001; A549/Neo, mean = 733 mm3, 95% CI = 601 mm3 to 909 mm3; A549/CTGF, mean = 40.4 mm3, 95% CI = 0 mm3 to 100 mm3; P = .001, Fig. 1, B). By contrast, CL1-0/siCTGF clones generated larger tumors than the CL1-0/Neo variant (CL1-0/Neo, mean = 33.5 mm3, 95% CI = 27.6 mm3 to 40.1 mm3; CL1-0/siCTGF, mean = 327 mm3, 95% CI = 366 mm3 to 437 mm3; P = .001, Fig. 1, B). Staining with anti-CD31 (PECAM-1) revealed that microvessel density was lower in the xenograft tumors derived from CL1-5/CTGF cells than those derived from CL1-5/Neo cells (CL1-5/Neo, mean = 119 vessels per field, 95% CI = 115 to 123; CL1-5/CTGF, mean = 45 vessels per field, 95% CI = 38.7 to 51.3; P = .027, Fig. 1, C).
To measure metastasis, we injected severe combined immunodeficiency mice subcutaneously with CTGF-transfected clones and counted the number of lung metastases 4 months later. CTGF expression effectively reduced the metastatic tumor burden of the mice (number of metastatic nodules >.25 cm in diameter, CL1-5/Neo, mean = 33.2, 95% CI = 25.5 to 41.0 versus CL1-5/CTGF, mean = 7.0, 95% CI = 3.3 to 10.7, P = .001; number of metastatic nodules <.25 cm in diameter, CL1-5/Neo, mean = 35.2, 95% CI = 23.7 to 46.8 versus CL1-5/CTGF, mean = 11.5, 95% CI = 8. 7 to 14.3; P = .001, Fig. 1, D). Also, immunohistochemical analysis demonstrated that the CD31-positive vessels were detected only in macrometastatic nodules (i.e., those >.25 cm in diameter), whereas few or none were observed in the micrometastatic (<.25 cm) lesions (data not shown). These data suggest that CTGF overexpression inhibits the progression of pulmonary micrometastases to macrometastases. To our knowledge, CTGF inhibits invasion in human lung cancer (45), and we observed lung metastasis after 4 months in mice that were subcutaneously injected with cancer cells, not in mice that were subjected to in situ implantation. These results suggest that the number of macro- and micronodules in the CTGF transfectant group decreased because of the invasion inhibition of CTGF. Nevertheless, to our knowledge, this is the first report that CTGF could influence the microvessel density and the progression of pulmonary micrometastases to macrometastases.
To determine whether the suggestion that CTGF overexpression may inhibit tumor growth in both primary and metastatic sites, possibly due to inhibition of angiogenesis, may hold true in human lung adenocarcinoma, we examined primary microvessel density in sections from tumors of 24 lung adenocarcinoma patients. Tumors with high CTGF expression (n = 14) had lower microvessel density than tumors with low CTGF expression (n = 10) (tumors with high CTGF expression, mean density = 32.8 vessels per field, 95% CI = 15.4 to 50.2, versus tumors with low CTGF expression, mean density = 70.4 vessels per field, 95% CI = 59.5 to 121; P = .009, Fig. 1, E).
CTGF, Angiogenesis, and VEGF-A Expression
VEGF-A protein and mRNA expression were lower in CTGF-overexpressing CL1-5 and A549 cells than in cells transfected with empty vector (Fig. 2, A). VEGF-A protein and mRNA expression were also increased in CL1-0 cells that were transfected with siCTGF constructs. To measure levels of secreted VEGF-A protein, we analyzed the conditioned media of CTGF transfectants. Consistent with the above findings, ELISA analysis showed that less VEGF-A protein was secreted by CTGF-transfected CL1-5 or A549 cells than vector controls; in contrast, CL1-0/siCTGF cells secreted the most VEGF-A (CL1-5/Neo clones, mean = 2210 pg/mL, 95% CI = 2051 pg/mL to 2368 pg/mL versus CL1-5/CTGF, mean = 1321 pg/mL, 95% CI = 1229 pg/mL to 1413 pg/mL; P = .049; A549/Neo clones, mean = 2015 pg/mL, 95% CI = 1829 pg/mL to 2201 pg/mL versus A549/CTGF, mean = 1202 pg/mL, 95% CI = 1070 pg/mL to 1334 pg/mL, P = .049; CL1-0/Neo, mean = 1110 pg/mL, 95% CI = 882 pg/mL to 1337 pg/mL versus CL1-0/siCTGF, mean = 1680 pg/mL, 95% CI = 1454 pg/mL to 1906 pg/mL, P = .049; Fig. 2, B).
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To test whether VEGF-A is involved directly in the CTGF-mediated inhibition of angiogenesis, mice were injected subcutaneously with Matrigel plugs that had been presoaked in conditioned media of CL1-0/siCTGF or CL1-0/Neo cells, i.e., media that contained different amounts of the VEGF-A protein. As expected, the angiogenic activity in the conditioned media from CL1-0 clones expressing siCTGF was higher (as demonstrated by high levels of hemoglobin in the Matrigel plug, Fig. 2, C, top) than that of CL1-0 vector controls (Fig. 2, C, bottom). Of interest, adding VEGF-A–blocking antibody, but not nonspecific IgG, decreased the angiogenic activity of the conditioned media from CL1-0/siCTGF cells (Fig. 2, C). Conditioned media from CL1-5/Neo cells had much stronger angiogenic activity than that from CL1-5/CTGF cells (Fig. 2, C). These data thus indicate that the CTGF may inhibit angiogenesis by decreasing the amount of functional VEGF-A in tumor cells.
CTGF-Mediated Inhibition of Angiogenesis and HIF-1 Expression
Because transcription factor HIF-1 regulates VEGF-A gene expression (14), we hypothesized that CTGF might act by influencing HIF-1 activity. Western blotting revealed that HIF-1 protein levels were lower in CTGF-overexpressing cells, and levels in cells transfected with siCTGF were higher than in cells transfected with empty vector (Fig. 3, A, top). Also, immunofluorescence analysis showed nuclear staining of HIF-1 (Fig. 3, A, bottom) in the cells transfected with empty vector but not in CL1-5/CTGF cells.
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To confirm the inhibitory effect of CTGF on HIF-1
activity, we transfected cells with a luciferase gene reporter driven by a promoter containing an HRE. We then assayed luciferase activity in the transfected cells. CL1-5 or A549 cells stably overexpressing CTGF showed attenuation of HRE-luc reporter activity compared with vector control transfected cells; however, CL1-0/siCTGF cells had increased HRE-driven luciferase activity compared with vector control transfected cells (Fig. 3, B).
We next determined the effect of CTGF on the transcriptional activity of the 1.5-kb VEGF-A promoter, which contains a consensus HRE site, using luciferase reporter gene assays. CL1-5/Neo cells that were cotransfected with the VEGF-A luciferase reporter gene and various concentrations of DN-HIF-1–expressing plasmid, which lacks the DNA-binding domain and competes for endogenous HIF-1, exhibited a dose-dependent decrease in VEGF-A promoter activity (Fig. 3, C). By contrast, transfection of CL1-5/CTGF cells, which show low VEGF-A promoter activity, with wild-type HIF-1–expressing vector restored the VEGF-A promoter activity (Fig. 3, C). Reverse transcription–PCR studies demonstrated that overexpressing HIF-1 in CTGF-overexpressing CL1-5 cells could rescue VEGF-A mRNA expression (Fig. 3, D, right). By contrast, transfection of DN-HIF-1 into CL1-5/Neo cells attenuated VEGF-A mRNA expression (Fig. 3, D, left). Collectively, these results suggest that CTGF inhibits VEGF-A gene expression through inhibition of HIF-1 activity and a reduction in HIF-1 protein level.
Effect of CTGF on HIF-1 Protein Degradation
Because the level of HIF-1 protein was dramatically decreased in the CTGF transfectants, we examined the effects of CTGF on HIF-1 protein stability. To test the hypothesis that increased degradation was responsible for the lower HIF-1 protein levels, we treated CL1-5/Neo and CL1-5/CTGF cells with MG132, a specific inhibitor of the 26S proteasome, and assayed HIF-1 protein levels over time. In the presence of MG132, the decreased HIF-1 protein level in CTGF-transfected cells was restored to that of CL1-5 vector controls (Fig. 4, A). Furthermore, in a cycloheximide pulse-chase experiment, the half-life of HIF-1 protein was shorter in CL1-5/CTGF cells than in CL1-5/Neo cells (half-life of CL1-5/Neo clones, mean = 55.5 minutes, 95% CI = 51.4 to 59.6 versus CL1-5/CTGF, mean = 23.2 minutes, 95% CI = 17.5 to 29.0; P = .021, Fig. 4, B). We also compared the ubiquitination pattern of HIF-1 protein in CL1-5/CTGF and CL1-5/Neo cells in the presence of MG132. As expected, the more extensively ubiquitinated HIF-1 protein was observed in stable CL1-5/CTGF transfectants and in transiently transfected CTGF-expressing A549 cells than in cells transfected with vector controls (Fig. 4, C). Thus, these results support the hypothesis that the reduction in HIF-1 protein levels seen with CTGF overexpression probably reflects enhanced HIF-1 protein degradation.
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To determine whether the CTGF-mediated HIF-1
protein degradation is due to direct interaction with the von Hippel-Lindau protein (pVHL), coimmunoprecipitation assays were conducted. After MG132 treatment, more HIF-1/pVHL complexes were detected in CL1-5/CTGF cells than in vector controls (Fig. 4, D). Furthermore, the enhanced association between HIF-1 and pVHL was not due to an increase in the expression level of pVHL protein in the CTGF-overexpressing cells (Fig. 4, D, right lower panel). These findings suggest that CTGF overexpression promotes HIF-1 degradation by facilitating the recruitment of pVHL to the HIF-1 protein.
CTGF and ARD1-Dependent Acetylation and Degradation of HIF-1
To determine the role of hydroxylation and acetylation of HIF-1 in CTGF-accelerated HIF-1 degradation, we examined PHD1, 2, 3, and ARD1 mRNA levels in CL1-5/CTGF, CL1-0/siCTGF, and cells transfected with vector controls. No substantial changes in PHD1, 2, or 3 mRNA levels in CTGF-expressing or knockdown cells were observed compared with levels in their respective controls (Fig. 5, A). However, the level of ARD1 mRNA was higher in CL1-5/CTGF cells and strongly reduced in CL1-0/siCTGF cells (Fig. 5, A) than in cells transfected with vector controls, suggesting that CTGF expression increases ARD1 gene expression. Also, CTGF overexpression stimulated HIF-1 protein acetylation, as demonstrated by immunoprecipitation using a specific antiacetylated Lys antibody (4G12) followed by western blotting with anti–HIF-1 antibody (Fig. 5, B, upper left). Interestingly, under similar immunoprecipitation and western blot conditions, transfection of CTGF-expressing A549 or CL1-5 cells with antisense (AS) ARD1 resulted in more abundant HIF-1 protein that had a lower acetylation level (Fig. 5, B, lower left). Because ARD1 acetylates HIF-1 at Lys532 (42,45), we constructed the HIF-1 mutant HIF-1/K532N, which would not be acetylated. Mutant HIF-1/K532N was transiently transfected into CL1-5/CTGF cells and CL1-5/Neo cells, and western blotting was performed with anti–HIF-1. The transfected HIF-1/K532N protein was resistant to proteolysis in CTGF-overexpressing CL1-5 clones (Fig. 5, B, upper right). To ensure that these results were not due to competition of the endogenous and K532N-HIF-1 for ubiquitin-mediated degradation, we also transfected CL1-5/CTGF cells with wild-type and K532N HIF-1 expressing plasmids at titrated doses (Fig. 5, B, lower right). HIF-1 protein level was increased in a dose-dependent manner after transfection with increasing amounts of wild-type or K532N HIF-1–expressing plasmids. However, HIF-1 protein accumulation was higher in cells transfected with K532N than in cells transfected with wild-type HIF-1 (Fig. 5, B, lower right). This observation is consistent with our previous findings (Fig. 4, C) showing that CTGF promotes HIF-1 protein degradation through acetylation.
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Next, we sought to determine whether ARD1 was involved in CTGF-mediated inhibition of VEGF-A gene expression. To address this issue, we transfected the AS-ARD1-expression plasmid into CL1-5/CTGF cells, and ARD1 expression was measured 48 hours later using RT–PCR. AS-ARD1 transfection reduced the level of ARD1 mRNA compared with that in mock-transfected cells and led to a dose-dependent elevation in VEGF-A mRNA expression (Fig. 5, C, top). Also, VEGF-A gene promoter activity was decreased in CL1-5/CTGF cells; however, cotransfection with K532N HIF-1
abolished the CTGF-mediated inhibition of VEGF-A gene promoter activity (Fig. 5, C, bottom). Taken together, these data suggest that CTGF-mediated acetylation of HIF-1 protein and its subsequent degradation involves ARD-1.
Exogenous Expression of HIF-1 and CTGF-Mediated Inhibition of Tumor Growth, Angiogenesis, and Metastasis In Vivo
To determine the role of HIF-1 in CTGF-mediated inhibition of angiogenesis in primary and metastatic tumors, nude mice were injected subcutaneously with CL1-5 transfectant cells (CL1-5/Neo, CL1-5/CTGF, and CL1-5/CTGF/HIF-1). CL1-5/CTGF/HIF-1–derived tumors were larger than those derived from CL1-5/CTGF cells 32 days after injection (volume of CL1-5/Neo clones, mean = 4688 mm3, 95% CI = 3670 to 5676 mm3; CL1-5/CTGF, mean = 317 mm3, 95% CI = 120 to 513 mm3; CL1-5/CTGF/HIF-1, mean = 3898 mm3, 95% CI = 2657 to 5139 mm3; Neo versus CTGF, P = .021; CTGF versus HIF-1, P = .021; Neo versus HIF-1, P = .387. Fig. 6, A, bottom), suggesting that enhanced HIF-1 expression may antagonize the effect of CTGF on tumor growth.
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The amount of HIF-1
and VEGF-A and the number of blood vessels in tumors from the mice injected with the CL1-5/CTGF/HIF-1 cells were higher than those derived from CL1-5/CTGF cells (Fig. 6, A, top). Also, the number and weight of lung tumors in the mice carrying CL1-5/CTGF/HIF-1 xenografts were larger than those of mice carrying CL1-5/CTGF xenografts (Table 1). The above data suggest that HIF-1 reexpression in the CTGF-overexpressing clones may have effectively abolished the metastasis and angiogenesis inhibition effect of CTGF. Moreover, mice injected with CL1-5/CTGF cells survived longer than mice injected with CL1-5/Neo cells (CL1-5/Neo, mean = 69.6 days, 95% CI = 53.9 to 85.3 days versus CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days; P = .001, Fig. 6, B.) By contrast, HIF-1 reoverexpression in CL1-5/CTGF cells shortened the survival time (CL1-5/CTGF, mean = 102.1 days, 95% CI = 92.1 to 112.1 days, versus CL1-5/CTGF/HIF-1, mean = 81.7 days, 95% CI = 66.5 to 96.9 days, P = .011; CL1-5/Neo, mean = 69.6 days, 95% CI = 53.9 to 85.3 days versus CL1-5/CTGF/HIF-1, mean = 81.7 days, 95% CI = 66.5 to 96.9 days, P = .122, Fig. 6, B).
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We have shown that CTGF expression can inhibit tumor growth in primary or metastatic sites by reducing VEGF-A gene expression and its subsequent angiogenic effects in tumor cells. We also observed that the effect of CTGF on VEGF-A gene expression was mediated by accelerating HIF-1
protein degradation through ARD1-dependent acetylation. Most importantly, we have provided functional evidence that CTGF acts as an angiogenesis suppressor, inhibiting tumor growth and metastasis in mouse models of human lung adenocarcinoma.
CTGF is a multifunctional factor that engages a wide variety of biologic processes (52–58). CTGF stimulates endothelial cell proliferation and in vivo angiogenesis, but to a much lesser extent than VEGF-A at equivalent concentrations (42–43). Recombinant human CTGF protein, at physiologic concentrations of 10–100 ng/mL, has relatively weak angiogenic activity as determined by Matrigel plug assay (Chang CC, Kuo ML, unpublished data). Recently, CTGF has been identified as interacting with VEGF165 (42). By forming a complex with VEGF165, CTGF inhibits VEGF-induced angiogenesis both in vitro and in vivo (43). In this study, we confirmed that CTGF expression effectively inhibits tumor angiogenesis by reducing VEGF-A gene expression through promoting the degradation of transcription factor HIF-1.
CTGF can be expressed and secreted by several types of stromal cells, including endothelial cells, fibroblasts, and smooth muscle cells. A recent study (54) has demonstrated that engineered expression of CTGF in mouse prostate stromal fibroblasts enhanced LNCaP xenograft tumor growth, which was accompanied by an increase in microvessel density. CTGF may, under certain circumstances, regulate angiogenesis in the tumor-reactive stromal microenvironment. CTGF expression is elevated, compared with the corresponding normal tissues, in many types of cancers, including breast (55), glioma (56), and hepatoma (57), whereas it is decreased in lung adenocarcinoma (44) and colorectal cancer (58). Overexpression of CTGF in human oral squamous cell carcinoma inhibits cell proliferation as well as tumor growth in vivo (59). Our results suggest that CTGF does not alter the proliferation rate of human lung adenocarcinoma cells in vitro but inhibits neovascularization effects at both primary neoplasm sites and distal metastatic sites. Altogether, the data indicate that the complex interactions between tumor stromal cells and tumor cells may affect the role of CTGF in modulating angiogenesis.
HIF-1, a regulator of VEGF-A, is a heterodimer of HIF-1 and HIF-1 subunits, both of which are basic helix–loop–helix transcription factors (14). HIF-1 (also known as ARNT) is a nuclear protein that is constitutively expressed, independent of O2 tension (60). In contrast to HIF-1, HIF-1 is a cytoplasmic protein and is responsive to O2 levels. In well-oxygenated cells, HIF-1 is continuously degraded by the ubiquitin–proteasome system. Degradation occurs when certain conserved prolyl or lysyl residues of HIF-1 are hydroxylated or acetylated by PHD and/or ARD1 acetyltransferase. Unexpectedly, our results reveal that CTGF expression leads to an increase in the expression of ARD1, but not of PHD 1, 2, or 3, in lung cancer cells. Under hypoxic conditions, PHD 1, 2, and 3 mRNA expression is normally regulated by HIF-1 (61–63). However, this regulatory relationship does not exist in malignant cells under normoxic conditions (64) because the expression of a broad array of genes is altered by HIF activation.
There are many ways in which HIF activation could lead to changes in tumor cell behavior. The first recognized mechanisms were enhanced angiogenesis and increased glycolytic flux (65). There are many other examples of HIF targets that are predicted to promote adaptation and survival of cells and tissues in hypoxic or nonhypoxic conditions. Several new targets of the HIF system that may be relevant to cancer biology have been recently identified, including IGF-2 (66), telomerase (67), CXCR4 (68), and SDF-1 (69). Thus, the contribution of HIF activation to aspects of tumor biology such as growth and evolution to a more aggressive phenotype is much less clear than its role in promoting angiogenesis and glycolysis. In our studies, the endogenous level of HIF-1 protein was higher in more invasive CL1-5 cells than that in low invasive parental CL1-0 cells in normoxic conditions. Under similar experimental conditions, mRNA expression of all three PHDs was unaltered in both cell lines. Therefore, the overexpression of HIF-1 protein in highly malignant cells could regulate a broad range of genes and biologic activities.
Recently, many studies (70,71) have investigated the effect of ARD1 on HIF-1 protein stability, with contradictory results. Most of these studies have concluded that ARD1 binds specifically to HIF-1 but that it has a limited role in regulating acetylation and protein stability of HIF-1. The first study, by Fisher et al. (70), showed using cDNA microarray analysis that CTGF mRNA was reduced by fourfold in ARD1 knockdown cells. This finding strongly supports a relationship between the regulation of CTGF and ARD1. Second, Asaumi et al. (72) recently used a yeast two-hybrid assay with ARD1 as bait and demonstrated that ARD1 interacts with the cytoplasmic domain of -amyloid precursor protein and affects its secretion. Thus, ARD1 may interact with certain cellular proteins and alter their functions. Our unpublished data showed that ARD1 overexpression only mildly decreased HIF-1 protein levels (Chang CC, Kuo ML). By contrast, our results clearly show that ARD1 expression is readily increased in CTGF-transfected cells, in which HIF-1 protein expression was dramatically reduced (Fig. 5, A). Transfection of CTGF-transfected cells with antisense ARD1 not only reduced ARD1 protein expression but also restored the level of HIF-1 protein (Fig. 5, C). Therefore, we hypothesize that whether ARD1 interacts with and whether the effect on HIF-1 protein stability is CTGF dependent. It is possible that certain cellular proteins are induced by CTGF and work coordinately with ARD1 to affect HIF-1 protein stability.
To our knowledge, this is the first report to show that CTGF, a multifunctional cytokine, can manipulate nuclear oncoprotein HIF-1 degradation by decreasing acetyltransferase enzyme expression. In an earlier study, we identified a potential role for CTGF in the regulation of lung adenocarcinoma tumor invasion and metastasis (44). Our experiments suggest that CTGF, an endogenous metastatic suppressor protein, inhibits primary-site and distal-organ angiogenesis and metastasis via enhanced HIF-1 degradation.
Taken together, our results reveal that CTGF accelerates HIF-1 degradation, probably at the level of protein modification, which in turn leads to repression of VEGF-A mRNA levels, and therefore inhibits lung adenocarcinoma angiogenesis and metastasis. The validated anticancer drug target protein HIF-1 may be an important mediator for initiating angiogenesis during progression of human lung adenocarcinoma. Indeed, blocking HIF-1 by using gene therapy to inhibit the interaction between HIF-1 and its transcriptional coactivator or with small molecules to enhance HIF-1 protein degradation has potential efficacy in cancer treatment (73,74). Therefore, these biologic processes offer exciting potential for therapeutic intervention.
CTGF is a multitarget functional protein in human neoplasms. It can act alone to inhibit angiogenesis or with other molecules, such as ARD1, with a different net effect. This may complicate the approaches used to study CTGF function. Furthermore, it is urgent to identify the potentially different functions of the CTGF that is released by stromal cells in the microenvironment and those of CTGF released by the cancer cell itself. In this study, we investigated the activities of various forms of CTGF, including full-length protein as well as the fragment products of digestion by matrix metalloproteinase, plasmin, or other enzymes, in human cancer progression. Because CTGF has such diverse roles in cancer metastasis, further investigation is needed.
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Supported by grants from the National Science Council of Taiwan (NSC94-2323-B-002-010, NSC94-2320-B-002-107, NSC-94-2320-B-002-012 to M.-L. Kuo), and Department of Industrial Technology, Ministry of Economic Affairs, Taipei, Taiwan (92-EC-17-A-19-S1–0016 to M.-L. Kuo).
Sponsors had no role in the study design, data collection, analysis, interpretation of the results, or the preparation of this report.
We sincerely thank Dr. Shuang-En Chuang of the Division of Cancer Research, National Health Research Institute, Taipei, Taiwan, for valuable technical suggestions, and Dr. Patricia S. Steeg, Director of Molecular Therapeutics Program, Co-Chairperson of Molecular Targets Faculty, Chief of Women's Cancers Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, USA, for careful suggestions.
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Manuscript received October 5, 2005; revised May 25, 2006; accepted June 1, 2006.
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: A Novel Mechanism for Connective Tissue Growth Factor Inhibition of Angiogenesis
J Natl Cancer Inst 2006 98: 946-948.
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