Effect of connective tissue growth factor on hypoxia-inducible factor 1alpha degradation and tumor angiogenesis.
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) 1alpha 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-1alpha and vascular endothelial growth factor (VEGF) A was assessed in vitro by using reporter gene assays. Cells were transiently transfected with HIF-1alpha mutant and antisense arrest-defective 1 protein (ARD-1), and HIF-1alpha 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-1alpha 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-1alpha had higher VEGF-A expression than CTGF-overexpressing xenografts. Mice with CTGF/HIF-1alpha 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-1alpha, 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-1alpha, 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-1alpha and restored HIF-1alpha and VEGF-A expression in CTGF-overexpressing cells.


CONCLUSIONS
CTGF inhibition of metastasis involves the inhibition of VEGF-A-dependent angiogenesis, possibly by promoting HIF-1alpha protein degradation.
Chang
Cheng-Chi
CC
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.
Lin
Ming-Tsai
MT
Lin
Been-Ren
BR
Jeng
Yung-Ming
YM
Chen
Szu-Ta
ST
Chu
Chia-Yu
CY
Chen
Robert J
RJ
Chang
King-Jen
KJ
Yang
Pan-Chyr
PC
Kuo
Min-Liang
ML
D000107:Acetylation; D000230:Adenocarcinoma / Q000098:blood supply / Q000737:chemistry* / Q000473:pathology; D020533:Angiogenesis Inhibitors / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D055513:Connective Tissue Growth Factor; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis* / Q000235:genetics; D017874:Immediate-Early Proteins / Q000032:analysis* / Q000276:immunology; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis* / Q000276:immunology; D008175:Lung Neoplasms / Q000098:blood supply / Q000737:chemistry* / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016513:Mice, SCID; D008833:Microcirculation; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis* / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D014408:Biomarkers, Tumor; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D055513:Connective Tissue Growth Factor