Inhibition Of MT1-MMP Proteolytic Function And ERK1/2 Signalling Influences Breast Cancer Cell Migration And Invasion Through Changes In MMP-2 And MMP-9 Expression

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a multifunctional protease that invokes changes extracellularly via cleavages of ECM substrates, and intracellularly through induction of cell signalling cascades, both to influence cell behaviour and motility. The effects of MT1-MMP activation, MMP catalytic activity, and ERK1/2 signalling were examined by chemically or genetically altering each parameter. Regardless of treatment, expression of the two gelatinases, MMP-2 and -9, were always altered in an inverse relationship. This work proposes a pathway through active MT1-MMP initiation of ERK1/2 phosphorylation and subsequent targeting the NF-κB transcription factor, to ultimately influence MMP-9 expression. Modulation of MT1-MMP activation, activity, or downstream signalling, all resulted in decreased invasive potential in MDA-MB-231 breast cancer cells. In a chicken embryo tumour model, only untreated MDA-MB-231 cells resulted in tumour vascularization and complete wound closure. Furthermore, these results suggest that cells lacking active MT1-MMP, phospho-ERK1/2, or adequate MMP-9, have considerably reduced invasive potential

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

Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

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 proMMPs, 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

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

Background: 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. 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. Methods: MCF-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. Results: In 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. Conclusions: This 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

Negotiation in strategy making teams : group support systems and the process of cognitive change

This paper reports on the use of a Group Support System (GSS) to explore at a micro level some of the processes manifested when a group is negotiating strategy-processes of social and psychological negotiation. It is based on data from a series of interventions with senior management teams of three operating companies comprising a multi-national organization, and with a joint meeting subsequently involving all of the previous participants. The meetings were concerned with negotiating a new strategy for the global organization. The research involved the analysis of detailed time series data logs that exist as a result of using a GSS that is a reflection of cognitive theory

Alzheimer’s disease cerebrospinal fluid biomarkers are not influenced by gravity drip or aspiration extraction methodology

Introduction: Cerebrospinal fluid (CSF) biomarkers, although of established utility in the diagnostic evaluation of Alzheimer's disease (AD), are known to be sensitive to variation based on pre-analytical sample processing. We assessed whether gravity droplet collection versus syringe aspiration was another factor influencing CSF biomarker analyte concentrations and reproducibility. Methods: Standardized lumbar puncture using small calibre atraumatic spinal needles and CSF collection using gravity fed collection followed by syringe aspirated extraction was performed in a sample of elderly individuals participating in a large long-term observational research trial. Analyte assay concentrations were compared. Results: For the 44 total paired samples of gravity collection and aspiration, reproducibility was high for biomarker CSF analyte assay concentrations (concordance correlation [95%CI]: beta-amyloid1-42 (Aβ42) 0.83 [0.71 - 0.90]), t-tau 0.99 [0.98 - 0.99], and phosphorylated tau (p-tau) 0.82 [95 % CI 0.71 - 0.89]) and Bonferroni corrected paired sample t-tests showed no significant differences (group means (SD): Aβ42 366.5 (86.8) vs 354.3 (82.6), p = 0.10; t-tau 83.9 (46.6) vs 84.7 (47.4) p = 0.49; p-tau 43.5 (22.8) vs 40.0 (17.7), p = 0.05). The mean duration of collection was 10.9 minutes for gravity collection and <1 minute for aspiration. Conclusions: Our results demonstrate that aspiration of CSF is comparable to gravity droplet collection for AD biomarker analyses but could considerably accelerate throughput and improve the procedural tolerability for assessment of CSF biomarkers

Alzheimer\u27s Disease cerebrospinal fluid biomarkers are not influenced by gravity drip or aspiration extraction methodology

Introduction Cerebrospinal fluid (CSF) biomarkers, although of established utility in the diagnostic evaluation of Alzheimer’s disease (AD), are known to be sensitive to variation based on pre-analytical sample processing. We assessed whether gravity droplet collection versus syringe aspiration was another factor influencing CSF biomarker analyte concentrations and reproducibility. Methods Standardized lumbar puncture using small calibre atraumatic spinal needles and CSF collection using gravity fed collection followed by syringe aspirated extraction was performed in a sample of elderly individuals participating in a large long-term observational research trial. Analyte assay concentrations were compared. Results For the 44 total paired samples of gravity collection and aspiration, reproducibility was high for biomarker CSF analyte assay concentrations (concordance correlation [95%CI]: beta-amyloid1-42 (Aβ42) 0.83 [0.71 - 0.90]), t-tau 0.99 [0.98 - 0.99], and phosphorylated tau (p-tau) 0.82 [95 % CI 0.71 - 0.89]) and Bonferroni corrected paired sample t-tests showed no significant differences (group means (SD): Aβ42 366.5 (86.8) vs 354.3 (82.6), p = 0.10; t-tau 83.9 (46.6) vs 84.7 (47.4) p = 0.49; p-tau 43.5 (22.8) vs 40.0 (17.7), p = 0.05). The mean duration of collection was 10.9 minutes for gravity collection andaspiration. Conclusions Our results demonstrate that aspiration of CSF is comparable to gravity droplet collection for AD biomarker analyses but could considerably accelerate throughput and improve the procedural tolerability for assessment of CSF biomarkers. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

Advancing specificity in delirium: The delirium subtyping initiative

BACKGROUND: Delirium, a common syndrome with heterogeneous etiologies and clinical presentations, is associated with poor long-term outcomes. Recording and analyzing all delirium equally could be hindering the field's understanding of pathophysiology and identification of targeted treatments. Current delirium subtyping methods reflect clinically evident features but likely do not account for underlying biology. METHODS: The Delirium Subtyping Initiative (DSI) held three sessions with an international panel of 25 experts. RESULTS: Meeting participants suggest further characterization of delirium features to complement the existing Diagnostic and Statistical Manual of Mental Disorders Fifth Edition Text Revision diagnostic criteria. These should span the range of delirium-spectrum syndromes and be measured consistently across studies. Clinical features should be recorded in conjunction with biospecimen collection, where feasible, in a standardized way, to determine temporal associations of biology coincident with clinical fluctuations. DISCUSSION: The DSI made recommendations spanning the breadth of delirium research including clinical features, study planning, data collection, and data analysis for characterization of candidate delirium subtypes. HIGHLIGHTS: Delirium features must be clearly defined, standardized, and operationalized. Large datasets incorporating both clinical and biomarker variables should be analyzed together. Delirium screening should incorporate communication and reasoning