Matrix metalloproteinases at a glance.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMP activity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease.

Matrix Metalloproteinases Shape the Tumor Microenvironment in Cancer Progression.

Cancer progression with uncontrolled tumor growth, local invasion, and metastasis depends largely on the proteolytic activity of numerous matrix metalloproteinases (MMPs), which affect tissue integrity, immune cell recruitment, and tissue turnover by degrading extracellular matrix (ECM) components and by releasing matrikines, cell surface-bound cytokines, growth factors, or their receptors. Among the MMPs, MMP-14 is the driving force behind extracellular matrix and tissue destruction during cancer invasion and metastasis. MMP-14 also influences both intercellular as well as cell-matrix communication by regulating the activity of many plasma membrane-anchored and extracellular proteins. Cancer cells and other cells of the tumor stroma, embedded in a common extracellular matrix, interact with their matrix by means of various adhesive structures, of which particularly invadopodia are capable to remodel the matrix through spatially and temporally finely tuned proteolysis. As a deeper understanding of the underlying functional mechanisms is beneficial for the development of new prognostic and predictive markers and for targeted therapies, this review examined the current knowledge of the interplay of the various MMPs in the cancer context on the protein, subcellular, and cellular level with a focus on MMP14.

Cell Migration in Microfluidic Devices: Invadosomes Formation in Confined Environments.

The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis, allowing the experimentalists to extend their playground to single cells and single molecules, with high throughput and resolution which were inconceivable few decades ago. In particular, microfluidic devices are profoundly changing the conventional way of studying the cell motility and cell migratory dynamics. In this chapter we will furnish a comprehensive view of the advancements made in the research domain of confinement-induced cell migration, thanks to the use of microfluidic devices. The chapter is subdivided in three parts. Each section will be addressing one of the fundamental questions that the microfluidic technology is contributing to unravel: (i) where cell migration takes place, (ii) why cells migrate and, (iii) how the cells migrate. The first introductory part is devoted to a thumbnail, and partially historical, description of microfluidics and its impact in biological sciences. Stress will be put on two aspects of the devices fabrication process, which are crucial for biological applications: materials used and coating methods. The second paragraph concerns the cell migration induced by environmental cues: chemical, leading to chemotaxis, mechanical, at the basis of mechanotaxis, and electrical, which induces electrotaxis. Each of them will be addressed separately, highlighting the fundamental role of microfluidics in providing the well-controlled experimental conditions where cell migration can be induced, investigated and ultimately understood. The third part of the chapter is entirely dedicated to how the cells move in confined environments. Invadosomes (the joint name for podosomes and invadopodia) are cell protrusion that contribute actively to cell migration or invasion. The formation of invadosomes under confinement is a research topic that only recently has caught the attention of the scientific community: microfluidic design is helping shaping the future direction of this emerging field of research.

Feel the force: Podosomes in mechanosensing.

Cells interact with their environment through highly localized contact structures. Podosomes represent a subgroup of cell-matrix contacts, which is especially prominent in cells of the monocytic lineage such as monocytes, macrophages and dendritic cells, but also in a variety of other cell types. Comparable to other adhesion structures, podosomes feature a complex architecture, which forms the basis for their extensive repertoire of sensory and effector functions. These functions are mainly linked to interactions with the extracellular matrix and comprise well known properties such as cell-matrix adhesion and extracellular matrix degradation. A more recent discovery is the ability of podosomes to act as mechanosensory devices, by detecting rigidity and topography of the substratum. In this review, we focus especially on the molecular events involved in mechanosensing by podosomes, the structural elements of podosomes that enable this function, as well as the intra- and extracellular signals generated downstream of podosome mechanosensing.

Hypoxia Promotes Invadosome Formation by Lung Fibroblasts.

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

There and back again: Intracellular trafficking, release and recycling of matrix metalloproteinases.

Matrix metalloproteinases are a family of zinc-dependent endopeptidases that are involved in a large variety of proteolytic processes in physiological and pathological scenarios, including immune cell surveillance, tissue homeostasis, or tumor cell metastasis. This is based on their ability to cleave a plethora of substrates that include components of the extracellular matrix, but also cell surface-associated and intracellular proteins. Accordingly, a tight regulatory web has evolved that closely regulates spatiotemporal activity of specific MMPs. An often underappreciated mechanism of MMP regulation involves their trafficking to and from specific subcellular sites that require MMP activity only for a certain period. In this review, we focus on the current knowledge of MMP intracellular trafficking, their secretion or surface exposure, as well as their recycling back from the cell surface. We discuss molecular mechanisms that enable these steps, in particular microtubule-dependent motility of vesicles that is driven by molecular motors and directed by vesicle regulatory proteins. Finally, we also point out open questions in the field of MMP motility that may become important in the future.

Small GTPases all over invadosomes.

Cell invasion is associated with numerous patho-physiologic states including cell development and metastatic dissemination. This process couples the activation of cell motility with the capacity to degrade the extracellular matrix, thereby permitting cells to pass through basal membranes. Invasion is sustained by the actions of invadosomes, an ensemble of subcellular structures with high functional homology. Invadosomes are 3D acto-adhesive structures that can also mediate local extracellular matrix degradation through the controlled delivery of proteases. Intracellular RHO GTPases play a central role in the regulation of invadosomes where their complex interplay regulates multiple invadosome functions. This review aims to provide an overview of the synergistic activities of the small GTPases in invadosome biology. This broad-based review also reinforces the importance of the spatiotemporal regulation of small GTPases and the impact of this process on invadosome dynamics.

Collagen and Discoidin Domain Receptor 1 Partnership: A Multifaceted Role in the Regulation of Breast Carcinoma Cell Phenotype.

Type I collagen, the major components of breast interstitial stroma, is able to regulate breast carcinoma cell behavior. Discoidin domain receptor 1 (DDR1) is a type I collagen receptor playing a key role in this process. In fact, collagen/DDR1 axis is able to trigger the downregulation of cell proliferation and the activation of BIK-mediated apoptosis pathway. The aim of this review is to discuss the role of two important factors that regulate these processes. The first factor is the level of DDR1 expression. DDR1 is highly expressed in epithelial-like breast carcinoma cells, but poorly in basal-like ones. Moreover, DDR1 undergoes cleavage by MT1-MMP, which is highly expressed in basal-like breast carcinoma cells. The second factor is type I collagen remodeling since DDR1 activation depends on its fibrillar organization. Collagen remodeling is involved in the regulation of cell proliferation and apoptosis through age- and proteolysis-related modifications.

Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-ß1, using engineered gratings with different width and depth. We first report that TGF-ß1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-ß1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

Dense fibrillar collagen is a master activator of invadopodia.

Tumor stroma is characterized by abnormal accumulation of dense fibrillar collagen, which promotes tumor progression and metastasis. However, the effect of desmoplastic collagen on cells has been unclear. Our recent findings demonstrate that dense fibrillar collagen activates a novel phosphosignaling mechanism for robust induction of invadopodia in tumor cells and normal fibroblasts.

TGF-ß1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking.

Transforming growth factor-ß1 (TGF-ß1) is an important player in chronic liver diseases inducing fibrogenesis and hepatocellular carcinoma (HCC) development. TGF-ß1 promotes pleiotropic modifications at the cellular and matrix microenvironment levels. TGF-ß1 was described to enhance production of type I collagen and its associated cross-linking enzyme, the lysyl oxidase-like2 (LOXL2). In addition, TGF-ß1 and type I collagen are potent inducers of invadosomes. Indeed, type I collagen fibers induce the formation of active linear invadosomes through the discoidin domain receptor 1 (DDR1). The goal of our study was to address the role of TGF-ß1 in collagen cross-linking and its impact on the formation of linear invadosomes in liver cancer cells. We first report a significant correlation between expressions of TGF-ß1, and type I collagen, LOXL2, DDR1 and MT1-MMP in human HCCs. We demonstrate that TGF-ß1 promotes a Smad4-dependent up-regulation of DDR1, together with LOXL2, in cultured HCC cells. Moreover, we show that LOXL2-induced collagen cross-linking enhances linear invadosome formation. Altogether, our data demonstrate that TGF-ß1 favors linear invadosome formation through the expressions of both the inducers, such as collagen and LOXL2, and the components such as DDR1 and MT1-MMP of linear invadosomes in cancer cells. Meanwhile, our data uncover a new TGF-ß1-dependent regulation of DDR1 expression.

Type I collagen fibrils and discoidin domain receptor 1 set invadosomes straight.

Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. We recently demonstrated that the collagen sensor discoidin domain receptor 1 (DDR1) interacts with type I collagen fibrils to allow proteolysis-based cancer cell invasion through the formation of a new class of invadosomes, termed linear invadosomes.

Importance of RhoGTPases in formation, characteristics, and functions of invadosomes.

Podosomes and invadopodia (collectively known as invadosomes) are specialized plasma-membrane actin-based microdomains that combine adhesive properties with matrix degrading and/or mechanosensor activities. These organelles have been extensively studied in vitro and current concerted efforts aim at establishing their physiological relevance and subsequent association with human diseases. Proper functioning of the bone, immune, and vascular systems is likely to depend on these structures while their occurrence in cancer cells appears to be linked to tumor metastasis. The elucidation of the mechanisms driving invadosome assembly is a prerequisite to understanding their role in vivo and ultimately to controlling their functions. Adhesive and soluble ligands act via transmembrane receptors that propagate signals to the cytoskeleton via small G proteins of the Rho family, assisted by tyrosine kinases and scaffold proteins to induce invadosome formation and rearrangements. Oncogene expression and cell-cell interactions may also trigger their assembly. Manipulation of the signals that regulate invadosome formation and dynamics could therefore be a strategy to interfere with their functions in a multitude of pathological settings, such as excessive bone breakdown, infections, vascular remodeling, transendothelial diapedesis, and metastasis.

Cdc42 and Tks5: a minimal and universal molecular signature for functional invadosomes.

Invadosomes are actin-based structures involved in extracellular-matrix degradation. Invadosomes, either known as podosomes or invadopodia, are found in an increasing number of cell types. Moreover, their overall organization and molecular composition may vary from one cell type to the other. Some are constitutive such as podosomes in hematopoietic cells whereas others are inducible. However, they share the same feature, their ability to interact and to degrade the extracellular matrix. Based on the literature and our own experiments, the aim of this study was to establish a minimal molecular definition of active invadosomes. We first highlighted that Cdc42 is the key RhoGTPase involved in invadosome formation in all described models. Using different cellular models, such as NIH-3T3, HeLa, and endothelial cells, we demonstrated that overexpression of an active form of Cdc42 is sufficient to form invadosome actin cores. Therefore, active Cdc42 must be considered not only as an inducer of filopodia, but also as an inducer of invadosomes. Depending on the expression level of Tks5, these Cdc42-dependent actin cores were endowed or not with a proteolytic activity. In fact, Tks5 overexpression rescued this activity in Tks5 low expressing cells. We thus described the adaptor protein Tks5 as a major actor of the invadosome degradation function. Surprisingly, we found that Src kinases are not always required for invadosome formation and function. These data suggest that even if Src family members are the principal kinases involved in the majority of invadosomes, it cannot be considered as a common element for all invadosome structures. We thus define a minimal and universal molecular signature of invadosome that includes Cdc42 activity and Tks5 presence in order to drive the actin machinery and the proteolytic activity of these invasive structures.

0.1 kilopascal difference for mechanophenotyping: soft matrix precisely regulates cellular architecture for invasion.

Current knowledge understands the mesenchymal cell invasion in a 3D matrix as a combined process of cell-to-matrix adhesion based cell migration and matrix remodeling. Excluding cell invasion stimulated by cytokines and chemokines, the basal cell invasion itself is a complicated process that can be regulated by matrix ligand type, density, geometry, and stiffness, etc. Understanding such a complicated biological process requires delicate dissections into simplified model studies by altering only one or two elements at a time. Past cell motility studies focusing on matrix stiffness have revealed that a stiffer matrix promotes 2D X-Y axis lateral cell motility. Here, we comment on two recent studies that report, instead of stiffer matrix, a softer matrix promotes matrix proteolysis and the formation of invadosome-like protrusions (ILPs) along the 3D Z axis. These studies also reveal that soft matrix precisely regulates such ILPs formation in the stiffness scale range of 0.1 kilopascal in normal cells. In contrast, malignant cells such as cancer cells can form ILPs in response to a much wider range of matrix stiffness. Further, different cancer cells respond to their own favorable range of matrix stiffness to spontaneously form ILPs. Thus, we hereby propose the idea of utilizing the matrix stiffness to precisely regulate ILP formation as a mechanophenotyping tool for cancer metastasis prediction and pathological diagnosis.

Invadosomes in their natural habitat.

Podosomes and invadopodia (collectively known as invadosomes) are small, F-actin-rich protrusions that are located at points of cell-ECM contacts and endow cells with invasive capabilities. So far, they have been identified in human or murine immune (myelomonocytic), vascular and cancer cells. The overarching reason for studying invadosomes is their connection to human disease. For example, macrophages and osteoclasts lacking Wiskott-Aldrich syndrome protein (WASp) are not able to form podosomes, and this leads to altered macrophage chemotaxis and defective bone resorption by osteoclasts. In contrast, the ability of cancer cells to form invadopodia is associated with high invasive and metastatic potentials. While invadosome composition, dynamics and signaling cascades leading to their assembly can be followed easily in in vitro assays, studying their contribution to pathophysiological processes in situ remains challenging. A number of recent papers have started to address this issue and describe invadosomes in situ in mouse models of cancer, cardiovascular disease and angiogenesis. In addition, in vivo invadosome homologs have been reported in developmental model systems such as C. elegans, zebrafish and sea squirt. Comparative analyses among different invasion mechanisms as they happen in their natural habitats, i.e., in situ, may provide an outline of the invadosome evolutionary history, and guide our understanding of the roles of the invasion process in pathophysiology versus development.