Tissue inhibitor of metalloproteinase 3 suppresses tumor angiogenesis in matrix metalloproteinase 2-down-regulated lung cancer.
Matrix metalloproteinase-2 (MMP-2) expression is often up-regulated in advanced cancers and known to play an important role in tumor angiogenesis. We previously showed that adenoviral-mediated delivery of siRNA for MMP-2 (Ad-MMP-2-Si) inhibited lung cancer growth, angiogenesis, and metastasis. In this study, we investigated the signaling mechanisms involved in Ad-MMP-2-Si-mediated inhibition of angiogenesis. Ad-MMP-2-Si treatment inhibited neovascularization in vivo as determined by mouse dorsal air sac model, and conditioned medium from Ad-MMP-2-Si-infected A549 lung cancer cells (Ad-MMP-2-Si-CM) inhibited endothelial tube formation in vitro. Ad-MMP-2-Si-CM decreased proliferation as determined by Ki-67 immunofluorescence and induced apoptosis in endothelial cells as determined by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay. Furthermore, Ad-MMP-2-Si-CM inhibited AKT phosphorylation and induced phosphorylation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase in endothelial cells. Overexpression of constitutively active AKT reversed the Ad-MMP-2-Si-CM-mediated inhibition of tube formation and induction of ERK phosphorylation. Conversely, Ad-MMP-2-Si-CM induced tissue inhibitor of metalloproteinase (TIMP) 3 expression, and the interaction of vascular endothelial growth factor 2 and TIMP-3 was determined by coimmunoprecipitation experiments. TIMP-3 induction was mediated by ERK activation. In addition, electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1 transcription factor mediated Ad-MMP-2-Si-CM-stimulated increase of TIMP-3. Vasculature destruction was confirmed with colocalization studies with TUNEL and an endothelial marker, CD31, in tumor sections of Ad-MMP-2-Si-treated mice. Our data collectively suggest that MMP-2 inhibition induces endothelial apoptosis in vivo and inhibits endothelial tube formation. These experiments provide the first evidence that inhibition of p-AKT and induction of p-ERK1/2 are crucial events in the induction of TIMP-3-mediated endothelial apoptosis in MMP-2 inhibited lung tumors.
Program of Cancer Biology, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, USA.
Chandramu
C
Chetty
Sajani S
SS
Lakka
Praveen
P
Bhoopathi
Sateesh
S
Kunigal
Roger
R
Geiss
Jasti S
JS
Rao
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008175:Lung Neoplasms / Q000098:blood supply* / Q000201:enzymology*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D017077:Culture Media, Conditioned; D034741:RNA, Small Interfering; D019717:Tissue Inhibitor of Metalloproteinase-3; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2