Mario Cepeda

Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

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.

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels.

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.

Knockdown of Pex11β reveals its pivotal role in regulating peroxisomal genes, numbers, and ROS levels in Xenopus laevis A6 cells.

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.

The cytoplasmic domain of MT1-MMP is dispensable for migration augmentation but necessary to mediate viability of MCF-7 breast cancer cells

Abstract Membrane‐type‐1 Matrix Metalloproteinase (MT1‐MMP) is a multifunctional protease that regulates ECM degradation, proMMP‐2 activation, and varied cellular processes including migration and viability. MT1‐MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1‐MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1‐MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF‐7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1‐MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1‐MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1‐MMP function in a manner required for cell survival, but is dispensable for cell migration. HighlightsAltering the cytoplasmic domain (CD) of MT1‐MMP affects its maturation and activity.MT1‐MMP CD alterations affect cell viability but not cell motility.The CD of MT1‐MMP enhances cell viability during serum‐free 3D culture.The CD of MT1‐MMP mediates tumour vascularization in vivo.

Analysis of Xenopus laevis RECK and Its Relationship to Other Vertebrate RECK Sequences

Aims: Reversion - inducing cysteine - rich protein with Kazal motifs (RECK) is a membrane - anchored protein that regulates cell migration by both inhibiting matrix metalloproteinases and modulating cellular pathways. Our study is the first to clone and examine the expression pattern of reck during early Xenopus laevis development. Study Design : Expression and sequence analysis.

Functional Characterization of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) N- and C-Terminal Domains during Xenopus laevis Development

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.