Transformed MDCK cells secrete elevated MMP1 that generates LAMA5 fragments promoting endothelial cell angiogenesis.
Epithelial-mesenchymal transition (EMT) enhances the migration and invasion of cancer cells, and is regulated by various molecular mechanisms including extracellular matrix metalloproteinase (MMP) activity. Previously, we reported transformation of epithelial Madin-Darby canine kidney (MDCK) cells with oncogenic H-Ras (21D1 cells) induces EMT, and significantly elevates MMP1 expression. To explore the biological significance, in this study we characterized 21D1 cells with knocked-down MMP1 expression (21D1(-MMP1)). MMP1 silencing diminished 21D1 cell migration, invasion and anchorage-independent growth in vitro. Additionally, 21D1(-MMP1) cells displayed reduced tumour volume when grown as in vivo subcutaneous xenografts in mice. Depletion of MMP1 lowered the ability of the cellular secretome (extracellular culture medium) to influence recipient cell behaviour. For example, supplementation with 21D1 secretome elevated cell migration of recipient fibroblasts, and enhanced endothelial cell angiogenesis (vessel length and branching). By contrast, 21D1(-MMP1) secretome was less potent in both functional assays. We reveal laminin subunit alpha-5 (LAMA5) as a novel biological substrate of MMP1, that generates internal and C-terminal proteolytic fragments in 21D1 secretome. Furthermore, antibody-based inhibition of integrin αvβ3 on endothelial cells nullified the angiogenic capability of 21D1 secretome. Therefore, we report this as a new VEGF-independent mechanism that oncogenic cells may employ to promote tumour angiogenesis.
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Shashi K
SK
Gopal
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
David W
DW
Greening
Department of Surgery, The University of Melbourne, Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia.
Hong-Jian
HJ
Zhu
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
Richard J
RJ
Simpson
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.|Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.
Rommel A
RA
Mathias
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D004285:Dogs; D058750:Epithelial-Mesenchymal Transition; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells / Q000502:physiology*; D006801:Humans; D007797:Laminin / Q000502:physiology*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism*; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000201:enzymology; D010446:Peptide Fragments / Q000502:physiology; D059748:Proteolysis; D020543:Proteome / Q000378:metabolism
D007797:Laminin; D010446:Peptide Fragments; D020543:Proteome; D020781:Matrix Metalloproteinase 1