Transformed MDCK cells secrete elevated MMP1 that generates LAMA5 fragments promoting endothelial cell angiogenesis
journal contributionposted on 2021-08-05, 07:31 authored by SK Gopal, David GreeningDavid Greening, H-J Zhu, Richard SimpsonRichard Simpson, RA Mathias
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 â 'MMP1). MMP1 silencing diminished 21D1 cell migration, invasion and anchorage-independent growth in vitro. Additionally, 21D1 â '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 â '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.
Authors are supported, in part, by the National Health and Medical Research Council of Australia program grants APP487922 (RJS), project grant APP433619 (H-JZ), and Early Career CJ Martin Fellowship APP1037043 (RAM). H-JZ is also supported by the Melbourne Research Grant Support Scheme (The University of Melbourne). SKG is supported by a La Trobe University Postgraduate Scholarship. We acknowledge the La Trobe University-Comprehensive Proteomics Platform for providing infrastructure and expertise for Capability A: Protein Identification & Quantitation.
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EPITHELIAL-MESENCHYMAL TRANSITIONMATRIX METALLOPROTEINASESCANCERCell Line, TumorAnimalsMice, Inbred NODDogsHumansMice, SCIDCell Transformation, NeoplasticNeovascularization, PathologicPeptide FragmentsLamininProteomeNeoplasm TransplantationCell ProliferationCell MovementMaleMatrix Metalloproteinase 1Gene Knockdown TechniquesEpithelial-Mesenchymal TransitionHuman Umbilical Vein Endothelial CellsProteolysisMadin Darby Canine Kidney CellsLAMA5