Abstract
BackgroundMembrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete.MethodsMCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, we analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. We also utilized Matrigel 3D cell culture and avian embryos to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenecity and extravasation efficiency in vivo. ResultsIn 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenecity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein.ConclusionsThis study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0547-x) contains supplementary material, which is available to authorized users.
Highlights
Membrane Type-1 Matrix Metalloproteinase (MT1-Matrix Metalloproteinases (MMPs)) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane
Production of functional Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) in MCF-7 cells did not result in increased migratory potential We transiently transfected MCF-7 breast cancer cells with wild-type untagged MT1-MMP cDNA and assessed proMMP-2 activation, Extracellular regulated kinase (ERK) activation, migration and invasion (Fig. 1). qPCR and immunoblot analysis of MT1MMP mRNA and protein levels, respectively, of MCF-7 cells transiently transfected with MT1-MMP cDNA showed that 24-h post-transfection these cells have very high levels of MT1-MMP mRNA (~17,000 fold relative to mock transfected cells) and produced predominately the pro- and active isoform, along with degradation forms, of MT1-MMP protein (Fig. 1a)
Addition of proMMP-2 conditioned media to transfected cells demonstrated that MT1-MMP overexpression activated proMMP-2 after 12 h of incubation, shown by the presence of an intermediate band, whose activation was enhanced by the addition of low levels of Tissue inhibitor of metalloproteinases 2 (TIMP-2) (100 ng/ml) that resulted in the presence of more active MMP-2 isoform (Fig. 1b, bottom gel)
Summary
Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Matrix Metalloproteinases (MMPs) have been implicated as the central mediators of ECM remodeling throughout the metastatic cascade, Membrane Type-1 MMP (MT1-MMP), MMP-2, and −9 [5,6,7] These MMPs play a key role in the degradation of the major structural components of the basement membrane (BM), which separates the primary tumour from the endothelium and is the first main structural barrier to metastasis [8, 9]. It has been shown that MT1-MMP overexpression increases the migratory ability of breast cancer cells independent of its proteolytic activity [24, 25] The mechanism underlying this increase in migratory potential remains poorly understood, with evidence suggesting that it involves ERK activation and cleavage of the cell adhesion molecule CD44 [26, 27]. Other experimental inconsistencies regarding the source of MMPs, whether they are predominately stromal or cancer cell derived [29], and their role in mediating cancer cell growth and invasion in vivo are under intense debate [7, 9, 30, 31]
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