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Published on: May 19, 2016
Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through
Mario A Cepeda1, Caitlin L Evered1, Jacob J H Pelling1
1Department of Biology, Faculty of Science, University of Western Ontario, BGS room 3053, 1151 Richmond St N, London, ON, N6A 5B7, Canada.
Abstract:
Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14) is a unique protease that cleaves extracellular proteins, activates proMMPs, and initiates intracellular signalling. MCF-7 cells are non-invasive and deficient in MT1-MMP, MMP-2, and MMP-9 expression. We created an MCF-7 cell line (C2) that stably produces active MT1-MMP and demonstrated increased ERK1/2 phosphorylation. MAPK inhibition in this cell line showed an inverse relationship in MMP-2 and MMP-9 transcripts where levels of these genes increased and decreased, respectively. Using invasive MDA-MB 231 cells that endogenously produce MT1-MMP and have naturally high pERK levels, we demonstrated the identical inverse relationship between MMP-2 and -9 transcript and protein levels, suggesting that this novel relationship is conserved amongst MT1-MMP positive breast cancer cells. To further analyze the relationship between MMP-2 and -9 levels, we chemically inhibited activation and catalytic activity of MT1-MMP using a furin and MMP inhibitor, respectively, to show that interference with the functions of MT1-MMP induced changes in MMP-2 and 9 transcript levels that were always inverse of each other, and likely mediated by differential transcriptional activity of the NF-κB transcription factor. Furthermore, we analyzed the functional consequences of these expression changes to show MMP, and in particular ERK, inhibition decreased migration and invasion using 2D culture, and inhibits the formation of an invasive phenotype in Matrigel 3D culture. This study demonstrated a novel inverse transcriptional relationship between MMP-2 and -9 levels and MT1-MMP activity that have functional consequences, and also showed that increases in the levels of MMPs does not necessarily correlate with an invasive phenotype.
Insights
Membrane type-1 matrix metalloproteinase (MT1-MMP) activity inversely regulates MMP-2 and MMP-9 expression in breast cancer cells. This novel relationship impacts cell migration and invasion, challenging the direct correlation between MMP levels and invasiveness.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key protease involved in extracellular matrix degradation and intracellular signaling.
- MCF-7 cells, a non-invasive breast cancer line, lack MT1-MMP, MMP-2, and MMP-9 expression, serving as a model to study MT1-MMP's role.
- MT1-MMP influences cell behavior, but its precise regulatory mechanisms on other matrix metalloproteinases (MMPs) and invasive phenotypes are not fully understood.
Purpose of the Study:
- To investigate the relationship between MT1-MMP activity and the expression of MMP-2 and MMP-9 in breast cancer cells.
- To elucidate the signaling pathways, including ERK1/2 and NF-κB, involved in this regulatory network.
- To determine the functional consequences of MT1-MMP-mediated MMP expression changes on cell migration and invasion.
Main Methods:
- Generation of a stable MT1-MMP-expressing MCF-7 cell line (C2).
- Pharmacological inhibition of MAPK/ERK and MT1-MMP (furin and catalytic activity inhibitors).
- Analysis of MMP-2 and MMP-9 transcript and protein levels via RT-PCR and Western blotting.
- Assessment of cell migration and invasion using 2D and 3D Matrigel culture models.
Main Results:
- Stable MT1-MMP expression in MCF-7 cells increased ERK1/2 phosphorylation.
- An inverse transcriptional relationship was observed between MMP-2 and MMP-9 levels, conserved in invasive MDA-MB 231 cells.
- Inhibition of MT1-MMP activity or ERK signaling altered MMP-2 and MMP-9 transcript levels, suggesting NF-κB involvement.
- MT1-MMP and ERK activity inhibition reduced cell migration and invasion in vitro.
Conclusions:
- MT1-MMP activity establishes a novel inverse transcriptional regulation between MMP-2 and MMP-9 in breast cancer cells.
- This regulatory axis, potentially mediated by NF-κB and impacting ERK signaling, influences cell invasion.
- Elevated MMP levels do not always correlate with an invasive phenotype, highlighting the complexity of MT1-MMP's role.
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