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.

Extracellular matrix (ECM) remodeling is essential for facilitating developmental processes. ECM remodeling, accomplished by matrix metalloproteinases (MMPs), is regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). While the TIMP N-terminal domain is involved in inhibition of MMP activity, the C-terminal domain exhibits cell-signaling activity, which is TIMP and cell type dependent. We have previously examined the distinct roles of the Xenopus laevis TIMP-2 and -3 C-terminal domains during development and here examined the unique roles of TIMP-1 N- and C-terminal domains in early X. laevis embryos. mRNA microinjection was used to overexpress full-length TIMP-1 or its individual N- or C-terminal domains in embryos. Full-length and C-terminal TIMP-1 resulted in increased lethality compared to N-terminal TIMP-1. Overexpression of C-terminal TIMP-1 resulted in significant decreases in mRNA levels of proteolytic genes including TIMP-2, RECK, MMP-2, and MMP-9, corresponding to decreases in MMP-2 and -9 protein levels, as well as decreased MMP-2 and MMP-9 activities. These trends were not observed with the N-terminus. Our research suggests that the individual domains of TIMP-1 are capable of playing distinct roles in regulating the ECM proteolytic network during development and that the unique functions of these domains are moderated in the endogenous full-length TIMP-1 molecule.

The membrane bound matrix metalloproteinase MT1-MMP plays roles in modulating cell movement, independent of its abilities to remodel the extracellular matrix. Unlike many MMPs, MT1-MMP is activated in the Golgi prior to secretion by a pro-protein convertase, primarily furin. Regulation of the activation of pro-MT1-MMP has been methodically investigated, as altering the level of the active protein has broad implications in both activating other pro-MMPs, including pro-MMP-2, and many subsequent remodelling events. Our previous work in MCF-7 cells has demonstrated that modest, and not extremely high, levels of active MT1-MMP manifests into altered cell morphology and movement. At this low but optimal amount of MT1-MMP protein, changes to MT1-MMP levels are always mirrored by MMP-9 and pERK levels, and always opposite to MMP-2 levels. In this study, stable expression of the furin inhibitor α1-antitrypsin Portland (α1-PDX) in MDA-MB-231 cells increased overall MT1-MMP levels, but cells maintained a 21% proportion of pro-MT1-MMP. The increase in MT1-MMP was mirrored by increases in MMP-9 and pERK, but a decrease in MMP-2. These changes were associated with increased NF-κB transcription. In vitro analysis showed that α1-PDX decreased cell protrusions and migration, and this manifested as decreased tumourigenesis when examined in vivo using a chick CAM assay.

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Peroxisomes are organelles that are ubiquitously found in all eukaryotic cells. Enzymes within their lumen are responsible for a variety of processes including the metabolism of fatty acids and eradication (neutralization) of free radicals. Peroxisomes are dynamic organelles, able to alter their numbers in response to a variety of different metabolic and cell-specific cues. Changes in peroxisome numbers can occur through division of preexisting peroxisomes or through de novo biogenesis from the ER. Proteins such as the Pex11 family of peroxins have been implicated as regulatory factors involved in peroxisome division. Division of peroxisomes involves elongation and membrane constriction followed by fission, which requires Pex11β. The regulation of peroxisome numbers in different cell types and tissues is variable and poorly understood. Here, we examine how knockdown of Pex11β affects peroxisomal genes, proteins, and peroxisome numbers in A6 kidney epithelial cells derived from Xenopus laevis. Pex11β morpholino use subsequently decreased mRNA levels of Pex1, PMP70, and PPARγ. Moreover, the Pex11β morpholino decreased PMP70 protein levels and PMP70-positive structures. Furthermore, the marker GFP-SKL revealed fewer peroxisome-like structures. These decreases resulted in increased levels of H2O2 and cellular and mitochondrial reactive oxygen species as measured by Amplex Red, DCFDA, and MitoTracker assays, respectively.