Encyclopedia of Cancer, 2015
Podosomes and invadopodia
Invadosomes – a single name that is used for podosomes and invadopodia, of which both are specialized F-actin-based adhesive structures formed mostly as cell protrusions at sites of cell–extracellular matrix (ECM) contacts on the ventral membrane of a variety of cell types. It is assumed that invadosomes are referred to as podosomes when they are found in normal cells and as invadopodia when they are found in cancer cells. Invadosomes have the ability to degrade ECM components by regulating the local release and activation of proteases that promote the ability of cells to cross tissue barriers, and this is what mainly distinguishes them from other cell-matrix contact structures such as focal adhesions, lamellipodia, or filopodia.
Invadosomes were first discovered in the 1980s in Rous sarcoma virus (v-Src)-transformed chicken embryo fibroblasts and initially described as rosette-shape adhesions with matrix-degrading ability. Subsequently, they were found in normal human cells such as osteoclasts, dendritic cells, macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs) and then defined as “podosomes”. In 1989, podosome-like structures were described in cancer cells by Chen and were termed “invadopodia” to accent their invasive nature.
Podosomes and invadopodia share many similarities in organization, composition, and function. In cell culture, they both appear as spot-like structures (Fig. 1) with a dense filamentous actin (F-actin) core that are enriched in a set of proteins involved in actin dynamics (cortactin, Arp2/3, formins, the Wiskott–Aldrich syndrome proteins WASp and N-WASp, WIP, or a-actinin) and cell adhesion processes (integrins, non-integrin receptors, paxillin, and zyxin) as well as in the ECM remodeling (matrix metalloproteinases, sheddases, serine proteases, and cathepsins).
Despite these strong similarities, podosomes and invadopodia have some differences that distinguish them from each other. First, they differ in size and abundance. Podosomes are relatively small, approx- imately 1 mm in diameter and 0.4 mm in height, and are present in large numbers (>100 per cell). In contrast, invadopodia are larger, 8 mm diameter and 5 mm height, and are less abundant (1–10 per cell).
Second, podosomes and invadopodia differ markedly in their dynamics. Podosomes are highly dynamic and have a lifetime of several minutes, while invadopodia are considered more stable and can persist for over one hour. Podosomes and invadopodia show also a divergent architecture. Podosomes are composed of a central actin core typically surrounded by a specialized ring region that is enriched in adhesion plaque proteins such as vinculin, talin, or kindlin, whereas invadopodia lack this surrounding ring structure. Moreover, invadopodia-producing cells generally only form punctate actin contacts, and podosomes tend to be organized into higher-ordered structures, such as podosome rosettes and podosome belts. Finally, they differ in their dependence on mediators that transmit upstream signals involved in actin dynamics. They have also unique differentiation markers such as non-catalytic region of tyrosine kinase adaptor protein 1 (Nck1) or growth factor receptor-bound protein 2 (Grb2) which specifically localize to invadopodia or podosomes, respectively.
Fig. 1. Schematic diagram of invadosomal protrusions formed on the ventral surface of the cell membrane and in vitro FITC- gelatin degradation by cancer cell invadopodia. Matrix degradation is accompanied by loss of the label (dark areas). N nucleus, ECM extracellular matrix
Invadosome formation and signaling
Assembly and maturation of invadosomes is a dynamic and multistep process. Their formation is initiated by the occurrence of F-actin foci as a result of enhanced actin polymerization in the response to environmental cues. In invadosomes, the initiator of actin assembly is N-WASP–Arp2/ 3–cortactin–dynamin complex that acts in cooperation with various specific proteins including fascin, Tks5/FISH, or Grb2 (Fig. 2). The mechanism by which this complex promotes actin polymerization in invadosomes involves cortactin-dependent recruitment and activation of cofilin and signaling adaptor protein Nck1. The recruitment of both cofilin and Nck1 via cortactin tyrosine phosphorylation is necessary to achieve successful transition from a precursor form to a fully maturated invadosome that is able to degrade ECM.
A number of various signals are able to stimulate invadosomes, among them colony-stimulating factor- 1 (CSF1) or platelet-derived growth factor (PDGF), epidermal growth factor (EGF), heparin-binding (HB) EGF, hepatocyte growth factor/scatter factor (HGF), tumor necrosis factor a (TNF-a), as well as vascular endothelial growth factor (VEGF) and transforming growth factor b (TGF-b). A novel pathway for invadosome initiation that has emerged more recently involves microRNA (miRNA) control. It was found that the loss of miR-143/miR-145 results in formation of podosomes in smooth muscle cells (SMCs).
The signal transduction initiates upstream events through the activation of various signaling pathways including the phosphatidylinositol 3-phosphate kinase (PI3K), the protein kinase C (PKC), and the nonreceptor tyrosine kinase Src as well as small and large GTPases such as RhoA, Cdc42, or dynamin. All of these factors serve as well-known activators of actin cytoskeleton reorganization and membrane trafficking. Uncontrolled activation of signaling pathways perturbs focal adhesion integrity and triggers local actin nucleation and polymerization and consequently leads to the formation of branched actin network and generates the driving force for membrane protrusions. Actin polymerization is also necessary for ECM degradation activity in invadosomes. One of the crucial invadosomal proteinases, membrane type-1 matrix metalloproteinase (MT1-MMP), is recruited and localized to the cell membrane in sites of active actin turnover. MT1-MMP docking to invadosomes is not required for invadosome formation but determines their degradative capabilities. The delivery of MT1-MMP and other proteases to invadosomes involves microtubule-dependent trafficking of proteinase-containing vesicles and/or specialized secretory vesicles, known as the trans-Golgi network (TGN), and is tightly controlled through adhesion-dependent signals which are believed to play a crucial role in invadosome maturation but not initiation processes.
Fig. 2. Schematic diagram of the invadosome. The organization and key components involved in actin polymerization and the ECM degradation
Several classic markers and specific invadosomal modulators are known to localize at lipid rafts, highly ordered, cholesterol-rich, and detergent-resistant membrane microdomains, that may act as platforms for sorting and focalizing these essential components at the sites of invadosome formation and their activity. Lipid rafts are highly present in invadosomes and depletion of plasma membrane cholesterol with methyl-b-cyclodextrin (MbCD) which disrupts lipid rafts has been shown to impair formation of invadosomes in both normal and cancer cells.
Biological functions of invadosomes
The main function of invadosomes is to degrade extracellular matrix. This is achieved by accumulation and release of cell surface and secreted proteases at invadosomes. These proteases include metalloproteases such as MMPs and ADAMs (a disintegrin and metalloproteinase/sheddases), serine proteases such as DPP4 (dipeptidyl peptidase 4/CD26) and FAP-a (fibroblast-activating protease a/ seprase), and cathepsin proteases such as cathepsin B. Invadosome-associated proteolytic activity is important for cleavage of both ECM and non-ECM targets, including latent growth factors and various membrane receptors, for example, integrins. Membrane-associated, type-I transmembrane MMP (MT1-MMP) and secreted gelatinases, MMP-2 and MMP-9, have emerged as key proteases that are involved in invadosome-related matrix degradation in a variety of normal and cancer cell types. MMPs are able to cleave most of the structural components of the ECM, such as collagens, laminin, and fibronectin as well as many cell surface molecules. ADAMs which are able to cleave various growth factors, cytokines, receptors, and adhesion molecules have been localized to invadosomes of Src-transformed fibroblasts, where they interact with b1 integrins and participate in the shedding of the EGFR ligands. DPP4 and FAP-a form complexes involved in the ECM degradation by DPP4-gelatin- binding domain at invadopodia in cells with a highly infiltrative behavior. Cathepsins are activated in the extracellular space and participate in degradation of matrix components such as plasminogen, plasmin, or tenascin and were found to be tightly associated with both podosomes and invadopodia.
Invadosomes are able to regulate cell migration by adhering to and reorganizing the local matrix substratum and therefore they may infiltrate surrounding tissue environment in both physiological and pathological conditions including different stages of embryonic and tissue development, inflammation, wound-healing, and cancer development and metastasis.
The podosomes serve to support a physiological role of osteoclasts as organelles that mediate bone resorption. Osteoclast podosomes are critically involved in the formation and maintenance of the sealing zone, an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers, during the growth and repair of bones. Podosomes which are formed by macrophages are believed to be important for the initiation and mediation of immune responses due to podosome-associated proteolytic activity which is required for macrophage migration and tissue infiltra- tion. In patients with Wiskott–Aldrich (WAS) syndrome, an X-chromosome-linked hereditary immune deficiency, podosomes do not develop. The clinical manifestation of this disorder is assigned to abnormal trafficking of immune cells, including macrophages, in the organism that confirms a role of podosomes in macrophage tissue transmigration. It is also known that macrophages which form defective podosomes manifest a marked impairment in mobilizing of immune cells in response to inflammation, despite normal expression and activity of metalloproteases. Podosome formation in vascular smooth muscle cells was found to be necessary for tissue reorganization and repair processes during vascular development in response to injury. It is known that podosome deficiency leads to both reduced bone resorption and impaired innate immunity. The presence of podosomes in myoblasts indicates that also precursor cells of striated muscles utilize podosome-related ECM degradation and cell migration during muscle develop- ment and repair. Moreover, a new study now implicates podosomes in facilitating cell membrane juxtaposition during myoblast fusion. Podosomes formed by tumor-associated macrophages (TAMs) were found to play supportive roles for cancer cell invasion and metastasis.
Invadopodia are believed to be the key cellular structures that regulate cancer cell invasion across various settings and are involved in cell dissemination to the distant sites in many cancers. Invadopodium- mediated pericellular proteolysis not only results in the ECM remodeling but also in production of proangiogenic factors such as vascular endothelial growth factor (VEGF) which is necessary for tumor growth.
The growing interest in invadosomes is related to their significant role in the initiation and regulation of cell-mediated extracellular matrix degradation, a rate-limiting step of multiple pathological processes mostly related to cancer progression but also to other human diseases such as genetic diseases Wiskott–Aldrich syndrome and Frank–ter Haar syndrome, as well as atherosclerosis or osteopetrosis. Although invadosomes are very dynamic, multifaceted structures, it is expected that their key regulators would be good targets for pharmacological attack. The plasma membrane components such as lipid rafts which mediate signals important for actin reorganization as well as membrane protease focalization could promote further research on factors that modulate or disrupt membrane rafts as potential drugs for the treatment of diseases that are a consequence of invadosome-dependent abnormal cell migration.
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