Effect of Connective Tissue Growth Factor on Hypoxia-Inducible Factor 1α Degradation and Tumor Angiogenesis

Abstract

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. 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. 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. CTGF inhibition of metastasis involves the inhibition of VEGF-A-dependent angiogenesis, possibly by promoting HIF-1alpha protein degradation.