ABSTRACT
ADAMTS-1 is an extracellular protease with critical roles in organogenesis and angiogenesis. Here we demonstrate a functional convergence of ADAMTS-1 and the transmembrane heparan sulfate proteoglycan syndecan-4 in influencing adhesion, migration and angiogenesis. Knockdown of ADAMTS-1 in endothelial cells resulted in a parallel reduction in cell surface syndecan-4, attributable to increased matrix metalloproteinase-9 (MMP9) activity. Knockdown of either ADAMTS-1 or syndecan-4 increased cellular responses to vascular endothelial growth factor A isoform VEGFA164, and increased ex vivo aortic ring microvessel sprouting. On fibronectin, knockdown of either protein enhanced migration and promoted formation of long α5 integrin-containing fibrillar adhesions. However, integrin α5 null cells still showed increased migration in response to ADAMTS-1 and syndecan-4 siRNA treatment. Plating of naïve endothelial cells on cell-conditioned matrix from ADAMTS-1 and syndecan-4 knockdown cells demonstrated that the altered adhesive behaviour was matrix dependent, and this correlated with a lack of expression of fibulin-1: an extracellular matrix co-factor for ADAMTS-1 that is known to inhibit migration. These findings support the notion that ADAMTS-1 and syndecan-4 are functionally interconnected in regulating cell migration and angiogenesis, via collaboration with MMP9 and fibulin-1.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Syndecan-4 , Vascular Endothelial Growth Factor A , ADAMTS1 Protein , Animals , Cell Adhesion , Cell Movement , Endothelial Cells , Humans , Mice , Neovascularization, Pathologic , Syndecan-1 , Syndecan-2 , Syndecan-4/geneticsABSTRACT
BACKGROUND: Unsupervised learning methods, such as Hierarchical Cluster Analysis, are commonly used for the analysis of genomic platform data. Unfortunately, such approaches ignore the well-documented heterogeneous composition of prostate cancer samples. Our aim is to use more sophisticated analytical approaches to deconvolute the structure of prostate cancer transcriptome data, providing novel clinically actionable information for this disease. METHODS: We apply an unsupervised model called Latent Process Decomposition (LPD), which can handle heterogeneity within individual cancer samples, to genome-wide expression data from eight prostate cancer clinical series, including 1,785 malignant samples with the clinical endpoints of PSA failure and metastasis. RESULTS: We show that PSA failure is correlated with the level of an expression signature called DESNT (HR = 1.52, 95% CI = [1.36, 1.7], P = 9.0 × 10-14, Cox model), and that patients with a majority DESNT signature have an increased metastatic risk (X2 test, P = 0.0017, and P = 0.0019). In addition, we develop a stratification framework that incorporates DESNT and identifies three novel molecular subtypes of prostate cancer. CONCLUSIONS: These results highlight the importance of using more complex approaches for the analysis of genomic data, may assist drug targeting, and have allowed the construction of a nomogram combining DESNT with other clinical factors for use in clinical management.
Subject(s)
Biomarkers, Tumor/blood , Gene Expression Profiling/statistics & numerical data , Prostatic Neoplasms/genetics , Transcriptome/genetics , Gene Expression Regulation, Neoplastic/genetics , Genomics/statistics & numerical data , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Prognosis , Progression-Free Survival , Proportional Hazards Models , Prostate-Specific Antigen/blood , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathology , Risk Assessment , Risk FactorsABSTRACT
Integrin ß3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro- or anti-angiogenic depends on the context in which it is expressed. To understand precisely ß3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the ß3-dependent adhesome. We show that depletion of ß3-integrin in this cell type leads to changes in microtubule behaviour that control cell migration. ß3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2-driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when ß3-integrin levels are reduced.
Subject(s)
Cell Adhesion/genetics , Cell Movement/genetics , Integrin beta3/genetics , Animals , Annexin A2/genetics , Chromosomal Proteins, Non-Histone/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular , Gene Expression Regulation/genetics , Humans , Mass Spectrometry , Mice , Microtubules/genetics , Microtubules/pathology , Neoplasms/genetics , Neoplasms/pathology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , rac1 GTP-Binding Protein/geneticsABSTRACT
Improvements in the understanding of the metabolic cross-talk between cancer and its microenvironment are expected to lead to novel therapeutic approaches. Acute myeloid leukemia (AML) cells have increased mitochondria compared with nonmalignant CD34+ hematopoietic progenitor cells. Furthermore, contrary to the Warburg hypothesis, AML relies on oxidative phosphorylation to generate adenosine triphosphate. Here we report that in human AML, NOX2 generates superoxide, which stimulates bone marrow stromal cells (BMSC) to AML blast transfer of mitochondria through AML-derived tunneling nanotubes. Moreover, inhibition of NOX2 was able to prevent mitochondrial transfer, increase AML apoptosis, and improve NSG AML mouse survival. Although mitochondrial transfer from BMSC to nonmalignant CD34+ cells occurs in response to oxidative stress, NOX2 inhibition had no detectable effect on nonmalignant CD34+ cell survival. Taken together, we identify tumor-specific dependence on NOX2-driven mitochondrial transfer as a novel therapeutic strategy in AML.
Subject(s)
Leukemia, Myeloid, Acute/pathology , Membrane Glycoproteins/metabolism , Mesenchymal Stem Cells/pathology , Mitochondria/pathology , NADPH Oxidases/metabolism , Superoxides/metabolism , Animals , Antigens, CD34/metabolism , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Leukemia, Myeloid, Acute/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mitochondria/metabolism , NADPH Oxidase 2 , Oxidative Stress , Reactive Oxygen Species/metabolism , Tumor Cells, CulturedABSTRACT
Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment.
Subject(s)
Adipocytes/pathology , Bone Marrow Cells/pathology , Leukemia, Myeloid, Acute/pathology , Tumor Microenvironment/physiology , Adipocytes/metabolism , Adult , Aged , Aged, 80 and over , Animals , Blotting, Western , Bone Marrow Cells/metabolism , Coculture Techniques , Fatty Acid-Binding Proteins/metabolism , Female , Flow Cytometry , Heterografts , Humans , Immunohistochemistry , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Middle Aged , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS. METHODS: Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (ß6-1089) cell lines, were used to assess MMP-8 expression and function. ß6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6ß4 integrin to hemidesmosomes (HD), TGF-ß signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models. RESULTS: Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in ß6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in ß6-1089 led to greater localisation of α6ß4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-ß signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-ß signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in ß6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001). CONCLUSIONS: These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-ß signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.
Subject(s)
Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/metabolism , Carcinoma, Intraductal, Noninfiltrating/genetics , Carcinoma, Intraductal, Noninfiltrating/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Epithelial Cells/metabolism , Matrix Metalloproteinase 8/deficiency , Biomarkers, Tumor , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Cell Adhesion , Cell Line, Transformed , Cell Line, Tumor , Cell Movement , Cell Survival , Female , Gene Expression , Gene Knockdown Techniques , Humans , Immunohistochemistry , Integrin alpha6beta4/metabolism , Matrix Metalloproteinase 8/genetics , Matrix Metalloproteinase 8/metabolism , Matrix Metalloproteinase 9/metabolism , Paracrine Communication , Protein Transport , Proteolysis , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
INTRODUCTION: Matrix metalloproteinase-8 (MMP-8; neutrophil collagenase) is an important regulator of innate immunity that has oncosuppressive actions in numerous tumor types. METHODS: We have intercrossed Mmp8-null mice with the Polyoma virus middle T oncogene-driven (MMTV-PyMT) mouse model of mammary cancer to explore the effects of loss of MMP-8 on the incidence and progression of mammary carcinomas. RESULTS: In this aggressive mouse model of breast cancer, loss of MMP-8 accelerated tumor onset even further, such that 90% of MMTV-PyMT; Mmp8-null female mice were tumor-bearing at the time of weaning. Throughout the 14 weeks of the model, tumor burden increased in homozygous Mmp8-null mice compared to Mmp8-wild-type and -heterozygote animals. Likewise, lung metastasis dramatically increased in the MMTV-PyMT; Mmp8-null mice. Immunohistochemistry revealed that tumors in wild-type, Mmp8-heterozygotes and -null animals had similar vascular density at 8 weeks, but at 10 weeks Mmp8-wild-type tumors had a lower vascularity than their heterozygote and null counterparts. No differences in macrophage infiltration were apparent throughout primary tumor development, though at 10 weeks a drop in neutrophil infiltrates was observed in Mmp8-wild-type tumors. Using quantitative real-time RT-PCR, we tracked the expression of the entire Mmp and Timp gene families, observing a significant decrease in Mmp3 expression in Mmp8-null tumors compared to wild-type and heterozygotes throughout the time course of the model, which was confirmed at the protein level. CONCLUSIONS: These findings provide novel insight into the suppressive action of MMP-8 on mammary tumorigenesis and metastasis, and indicate that the loss of MMP-8 likely has pleiotropic effects on innate immunity and angiogenesis that are reflected in changes in the protease web.
Subject(s)
Antigens, Viral, Tumor/genetics , Mammary Neoplasms, Experimental/etiology , Mammary Neoplasms, Experimental/pathology , Mammary Tumor Virus, Mouse/genetics , Matrix Metalloproteinase 8/genetics , Retroviridae Infections/complications , Tumor Virus Infections/complications , Animals , Cell Transformation, Neoplastic , Disease Progression , Female , Inflammation Mediators/metabolism , Lung Neoplasms/secondary , Matrix Metalloproteinase 8/metabolism , Mice , Mice, Knockout , Multigene Family , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Neutrophil InfiltrationABSTRACT
The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.
Subject(s)
ADAM Proteins/physiology , Breast Neoplasms/enzymology , Liver Neoplasms/enzymology , ADAMTS Proteins , ADAMTS1 Protein , Animals , Breast Neoplasms/pathology , Cell Movement , Extracellular Matrix/enzymology , Female , Human Umbilical Vein Endothelial Cells/physiology , Humans , Liver Neoplasms/secondary , MCF-7 Cells , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/enzymology , Organ Specificity , Syndecan-4/metabolism , Tumor MicroenvironmentABSTRACT
Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.
Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Matrix Metalloproteinase 8/biosynthesis , Neoplasm Proteins/biosynthesis , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Inflammation Mediators/metabolism , Interleukin-6/genetics , Interleukin-8/genetics , Matrix Metalloproteinase 8/genetics , Neoplasm Proteins/geneticsABSTRACT
INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.
Subject(s)
Breast Neoplasms , Research , Translational Research, Biomedical , Animals , Breast Neoplasms/diagnosis , Breast Neoplasms/epidemiology , Breast Neoplasms/etiology , Breast Neoplasms/therapy , Female , HumansABSTRACT
Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.
Subject(s)
Cell Transdifferentiation , Endothelium, Vascular/metabolism , Lymphatic Vessels/metabolism , Lymphoid Progenitor Cells/metabolism , Microtubules/metabolism , Adult , Cell Differentiation , Cell Lineage , Cells, Cultured , Coculture Techniques , Collagen/metabolism , Drug Combinations , Endothelium, Vascular/pathology , Extracellular Matrix , Fibrin/metabolism , Humans , Laminin/metabolism , Lymphatic Vessels/pathology , Lymphoid Progenitor Cells/pathology , Neovascularization, Physiologic , Phenotype , Proteoglycans/metabolism , Tissue EngineeringABSTRACT
Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.
Subject(s)
Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/metabolism , Microglia/metabolism , NF-kappa B/metabolism , Transcription Factor AP-1/metabolism , Tuberculosis, Central Nervous System/metabolism , Anti-Inflammatory Agents/pharmacology , Dexamethasone/pharmacology , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Expression , Gene Expression Profiling , Humans , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/immunology , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/immunology , Microglia/immunology , Microscopy, Confocal , Monocytes/immunology , Monocytes/metabolism , Mycobacterium tuberculosis/immunology , NF-kappa B/genetics , NF-kappa B/immunology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription Factor AP-1/genetics , Transcription Factor AP-1/immunology , Tuberculosis, Central Nervous System/genetics , Tuberculosis, Central Nervous System/immunology , Up-RegulationABSTRACT
The original notion that matrix metalloproteinases (MMPs) act as tumour and metastasis-promoting enzymes by clearing a path for tumour cells to invade and metastasize has been challenged in the last decade. It has become clear that MMPs are involved in numerous steps of tumour progression and metastasis, and hence are now considered to be multifaceted proteases. Moreover, more recent experimental evidence indicates that some members of the MMP family behave as tumour-suppressor enzymes and should therefore be regarded as anti-targets in cancer therapy. The complexity of the pro- and anti-tumorigenic and -metastatic functions might partly explain why broad-spectrum MMP inhibitors failed in phase III clinical trials. This review will provide a focussed overview of the published data on the tumour-suppressive behaviour of MMPs.
Subject(s)
Enzyme Inhibitors/adverse effects , Matrix Metalloproteinases , Neoplasms/enzymology , Neovascularization, Pathologic/enzymology , Animals , Clinical Trials, Phase III as Topic , Enzyme Inhibitors/therapeutic use , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Knockout , Neoplasms/blood supply , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Neovascularization, Pathologic/geneticsABSTRACT
Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on ß1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.
Subject(s)
Breast Neoplasms/pathology , Cell Adhesion/physiology , Cell Movement/physiology , Colonic Neoplasms/pathology , Prostatic Neoplasms/pathology , Serine Proteinase Inhibitors/physiology , Serpins/physiology , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Culture Media, Conditioned/pharmacology , Female , Fluorescent Antibody Technique , Humans , Integrin beta1/metabolism , Male , Mutagenesis, Site-Directed , Point Mutation/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Serine Proteinase Inhibitors/chemistry , Serpins/chemistry , Tumor Cells, CulturedABSTRACT
Phase III clinical trials with cancer patients with the first generation of synthetic MMP inhibitors (MMPIs) failed due to inefficacy and adverse side effects. These results were unexpected, given the wealth of pre-clinical data implicating MMPs as cancer targets, but are attributable to the broad-spectrum activity of these early MMPIs and the limited knowledge of the variety of biological functions of MMPs at the time they were deployed. These experiences stimulated the development of a variety of highly specific synthetic MMPIs. However, the bottle-neck is the identification of true target-MMPs. Functional genetic approaches are being complicated by the existence of the 'protease web,' i.e., the dynamic interconnectivity of MMPs and other proteases, their inhibitors, and substrates that collectively establish homeostasis in signaling in healthy and disease-afflicted tissue. Therefore, even specific MMP inhibition can result in seemingly unpredictable induction of systemic protease web-associated modulations (spam), which can comprise metastasis-promoting molecules such as other proteases and cytokines. Such undesired information in local proteolytic networks or relayed systemically in the organism via the proteolytic internet needs to be understood and defined in order to design specific metastasis therapies employing highly specific MMPIs in combination with spam-filtering agents.
Subject(s)
Matrix Metalloproteinase Inhibitors , Neoplasm Metastasis/drug therapy , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Protein Processing, Post-Translational , Animals , HumansABSTRACT
The mammalian vascular system consists of two distinct, but closely related, networks: the blood vasculature (itself divided into arterial and venous networks) and the lymphatic vasculature. EC (endothelial cell) lineage specification has been proposed to be determined during embryonic development, after which the ECs are committed to their fate. However, increasing evidence suggests that ECs retain various degrees of plasticity, and have the ability to express characteristics of alternative cell lineages. Therapeutic control of endothelial plasticity will allow greater understanding of the genesis and treatment of several vascular diseases.
Subject(s)
Endothelial Cells/cytology , Animals , Blood Vessels/growth & development , Cell Lineage , Endothelial Cells/metabolism , Humans , Lymphatic Vessels/physiology , Models, Biological , PhenotypeABSTRACT
A 4-component antibody-phthalocyanine-polyethylene glycol-gold nanoparticle conjugate is described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles (4 nm) were stabilised with a self-assembled layer of a zinc-phthalocyanine derivative (photosensitiser) and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently bound to the nanoparticles via a terminal carboxy moiety on the polyethylene glycol. The nanoparticle conjugates were stable towards aggregation, and under irradiation with visible red light efficiently produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.
Subject(s)
Antibodies, Monoclonal/chemistry , Breast Neoplasms/drug therapy , Gold/chemistry , Indoles/chemistry , Metal Nanoparticles/chemistry , Photochemotherapy , Photosensitizing Agents/chemistry , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Female , Humans , Isoindoles , Photosensitizing Agents/therapeutic use , Photosensitizing Agents/toxicity , Polyethylene Glycols/chemistry , Receptor, ErbB-2/immunology , Singlet Oxygen/metabolismABSTRACT
MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.
Subject(s)
ADAM Proteins/physiology , Histocompatibility Antigens Class I/metabolism , Membrane Microdomains/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM17 Protein , Cell Line , Cell Line, Tumor , Dipeptides/pharmacology , Humans , Hydrolysis/drug effects , Membrane Microdomains/enzymology , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Solubility , Thiophenes/pharmacologyABSTRACT
The ADAM disintegrin metalloproteinases (where ADAM is 'a disintegrin and metalloproteinase') are a family of transmembrane cell-surface proteins with essential roles in adhesion and proteolytic processing in all animals. The archetypal family member is ADAM17 {also known as TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme]}, which is involved in processing pro-TNFalpha and in the activation of ligands for the EGFR [EGF (epidermal growth factor) receptor], as well as cleavage of diverse cell-surface receptors and adhesion molecules. ADAM-mediated shedding is itself influenced via cell signalling pathways. In this issue of the Biochemical Journal, Willems et al. make the observation that phorbol ester activates shedding by ADAM17 by affecting the activity of PDI (protein disulfide isomerase). They propose that PDI maintains ADAM17 in an inactive 'closed' state and PMA stimulation generates ROS (reactive oxygen species) and thus an altered redox environment, which in turn inactivates PDI and allows ADAM17 to adopt an 'open' active conformation. This activation is accompanied by changes in disulfide bonds in the ADAM17 ectodomain. This is a novel and exciting finding that could help to unlock the actions of ADAM sheddases, as well as a host of other mechanisms that rely upon rapid alterations in protein conformation on the cell surface.