Research paperSignificance of kinase activity in the dynamic invadosome
Introduction
Cell migration is an essential physiological process in development, wound healing and immune system-mediated protection from pathogens. Many different cell types must migrate through tissues and basement membranes as part of their normal function or in response to environmental cues. Methods of cell migration vary depending on the cell type and extracellular environment; cells can move collectively or as single entities, using amoeboid or mesenchymal modes of migration (Friedl and Wolf, 2010). Cancer cells make use of various migration methods to pathologically invade tissues and disseminate from primary tumours, forming new tumours around the body in a process called metastasis (Bravo-Cordero et al., 2012).
In order to facilitate migration, tissue cells can adhere to the extracellular matrix (ECM), produce force via the modulation of the actomyosin cytoskeleton, and degrade the surrounding matrix and basement membranes blocking the path of migration (Parsons et al., 2010). A key feature of migrating cells is cell-matrix adhesions, of which there are different classes with varying functions. This review will focus on two closely related cell-matrix adhesions: podosomes and invadopodia, collectively termed invadosomes (Murphy and Courtneidge, 2011). These are small multi-molecular complexes formed at the plasma membrane, linking the actin cytoskeleton to the extracellular membrane via integrin receptors. While some types of adhesion such as focal adhesions predominantly function to anchor the cell to the ECM, invadosomes are utilised for cell invasion.
Invadosomes consist of a filamentous actin (f-actin) core which contains proteins regulating actin nucleation, elongation and branching, and a ring structure of integrins and integrin-associated proteins (Murphy and Courtneidge, 2011, Schachtner et al., 2013b). Integrins provide a transmembrane link between the actin cytoskeleton and the ECM; in invadosomes, proteins within the ring structure such as paxillin, talin and vinculin link integrins to the f-actin core. As well as cell-matrix adhesion, invadosomes are sites of matrix degradation. At invadosomes, cells secrete various matrix metalloproteases (MMPs) to degrade ECM proteins and basement membranes and consequently facilitate cell migration through tissues (Saltel et al., 2011). Invadosomes also exhibit rigidity sensing capabilities (Alexander et al., 2008, Collin et al., 2008, Labernadie et al., 2014). Atomic force microscopy revealed that podosomes exert traction on the substrate as well as being protrusive (Labernadie et al., 2014) and thus are sensitive to matrix rigidity, suggesting that they play a role in both outside-in and inside-out mechanosensing (Collin et al., 2008).
Invadosomes are formed by many different cell types in response to different signalling events. Monocyte derived cells including macrophages, immature dendritic cells, osteoclasts and megakaryocytes constitutively form podosomes in order to carry out their inherent immune or cell specific functions (Dovas and Cox, 2011, Schachtner et al., 2013b). Macrophages and dendritic cells produce podosomes in order to adhere to the ECM and migrate through tissues towards sites of infection or tissue damage; osteoclast podosomes enable bone remodeling via the formation of podosome superstructures called sealing zones, which act to confine the area of bone resorption; megakaryocyte podosomes penetrate basement membranes to allow proplatelet arm extension into blood vessels for platelet release (Schachtner et al., 2013a). In all these cell types, podosome assembly occurs upon adhesion to ECM proteins and engagement of integrin receptors. Matrix stiffness therefore plays an important role in podosome assembly, and can also affect podosome lifetime and invasiveness (Collin et al., 2008). Clinically, the importance of podosomes is highlighted in Wiskott-Aldrich syndrome, a well-described immunodeficiency disorder (Kirchhausen, 1998, Thrasher and Burns, 2010). Additionally, osteopetrosis is characterised by increased bone mass due to impaired osteoclast-mediated bone resorption (Tolar et al., 2004).
Invadopodia can be described as a cancer cell’s mimic of a podosome. They are formed as adhesive and invasive structures to enable migrating cancer cells to penetrate tissues during metastasis and contain many of the same molecular components as podosomes (Revach and Geiger, 2014). Despite their similarities however, invadopodia are generally observed to be longer lived, sometimes lasting several hours (Sharma et al., 2013) compared to the highly dynamic podosome which typically lasts less than ten minutes (Calle et al., 2004, Evans et al., 2003, Yamaguchi et al., 2005). Also, invadopodia protrude further from the cell body (Enderling et al., 2008) and are more degradative than podosomes (Artym et al., 2011); this is in keeping with the notion that cancer cells adapt normal cellular processes for their own pathological function (Murphy and Courtneidge, 2011). The clinical relevance of invadopodia in cancer progression has been studied in animal models (Eckert et al., 2011, Lohmer et al., 2014). The recent development of multiphoton microscopy in intravital imaging has transformed our ability to visualise cancer cell movement in vivo and confirmed the importance of invadopodia in tumour cell intravasation during metastasis (Condeelis and Segall, 2003, Gligorijevic et al., 2012).
The exact mechanisms that regulate invadosome dynamics are not fully understood, however many proteins are known to be involved and regulation is thought to be cell-type specific (Murphy and Courtneidge, 2011). Invadosome formation is initiated in response to signalling events promoting a migratory and invasive phenotype. This includes a variety of stimuli, including matrix adhesion, growth factors and cytokines (Destaing et al., 2011, Hoshino et al., 2013). At the plasma membrane, integrins and receptor kinases relay extracellular signals to downstream signalling molecules such as kinases, GTPases and adaptor proteins. These signalling pathways initiate invadosome formation via the nucleation of branched actin filaments by Wiskott-Aldrich Syndrome Protein (WASP) family members and the actin-related protein 2/3 (Arp2/3) complex; this will form the invadosome actin core (Beaty and Condeelis, 2014, Calle et al., 2008, Hurst et al., 2004, Kaverina et al., 2003, Machesky and Insall, 1998, Thrasher and Burns, 2010).
The invadosome is subsequently stabilised by elongation and cross-linking of actin filaments, and recruitment of various molecules to the region surrounding the core to make up the invadosome ring. The ring structure has been proposed to form a link between integrin receptors contacting the ECM and the intracellular actin core (Murphy and Courtneidge, 2011, Schachtner et al., 2013b). Mature, invadosomes can then recruit and secrete MMPs (Marchesin et al., 2015). Less is known about invadosome disassembly; in dendritic cells, myosin IIA has been suggested to induce contractions of the actin cytoskeleton, which disrupt the structural integrity of podosomes and lead to their dissolution (van Helden et al., 2008). Calpain, a cysteine protease, has also been linked to podosome disassembly, through its cleaving of podosome-related proteins such as talin and WASP (Calle et al., 2006, Macpherson et al., 2012).
While the invadosome life cycle can be described in various stages including formation, proteolytic activity and dissolution (Artym et al., 2006, Branch et al., 2012, Hoshino et al., 2013), the identification of regulatory factors acting at a precise stage has proved experimentally challenging due to the dynamic nature of these structures. This is further complicated by the observations that podosome f-actin cores can undergo oscillations in stiffness in response to localised actomyosin contractility (Labernadie et al., 2010) and can also undergo fission and fusion events (Evans et al., 2003). Following perturbation of a regulatory factor, the presence or absence of invadosomes can be used to determine whether this factor is necessary for the initiation and formation of the invadosome. The number of adhesions per cell allows further conclusions to be drawn: an increased number of invadosomes per cell would suggest either a promotion of invadosome formation or a defect in invadosome dissolution (Hoshino et al., 2013). Live cell imaging is crucial in determining between these stages, allowing invadosome lifetime measurements, as well as comparisons between the timing of regulatory factor localisation and recruitment of ring proteins or MMPs (Artym et al., 2006, Branch et al., 2012). Additionally, studies have assessed the size of invadosomes or tertiary structures, as well as their ability to degrade matrices such as gelatin (Beaty and Condeelis, 2014, Schachtner et al., 2013b).
Using a combination of such analytical techniques, the key proteins that make up the structure of the invadosome have been well described (Beaty and Condeelis, 2014, Schachtner et al., 2013b), however less is known about the regulatory proteins involved. Kinases are a large group of protein enzymes which function to add phosphate groups to their substrates, resulting in extremely diverse cellular actions. Kinases often act as signal transducers within intracellular signalling cascades in order to affect cell physiology or phenotype; in relation to invadosomes, many different kinases have been implicated in the dynamics of these structures (Hoshino et al., 2013, Saykali and El-Sibai, 2014, Winograd-Katz et al., 2011). Each stage of the invadosome is tightly regulated by signalling factors influencing where and when invadosomes are formed. Kinases are integral components of these signalling pathways, and while certain phosphorylation events have been identified as important, there is much still to be explored. In this review, we aim to describe known kinase involvement in invadosome stimulation, formation, stabilisation, activity and dissolution (Fig. 1), and highlight where current knowledge is lacking.
Section snippets
Kinases in invadosome stimulation
The stimuli inducing invadosome formation are varied; they include growth factors, cytokines, ECM proteins, micro RNAs, calcium and reactive oxygen species (ROS) (Murphy and Courtneidge, 2011). The extracellular signals inducing invadosome formation are well studied (Hoshino et al., 2013), as well as the downstream involvement of Rho GTPases (Spuul et al., 2014) and the molecules responsible for the formation of branched f-actin, such as WASP/N-WASP and the Arp2/3 complex (Saykali and El-Sibai,
Src: a major regulator of invadosome formation
While the extracellular factors promoting invadosome formation are varied and complex, these signalling pathways converge on a set of crucial kinases involved in initiating the invadosome actin core. Src is considered the most important kinase in invadosome regulation, being both necessary and sufficient for podosome formation (Berdeaux et al., 2004, Sanjay et al., 2001), and functioning in multiple stages of the invadosome cycle (Fig. 1), including formation, stabilisation, MMP secretion and
Kinases in invadosome stabilisation
Invadosome stabilisation refers here to the elongation and stability of the protrusion via actin polymerisation and the cross-linking of actin filaments (Beaty and Condeelis, 2014, Murphy and Courtneidge, 2011). Since much research has focused on the simple parameters of presence or absence of invadosomes, current knowledge of factors influencing their growth and turnover is limited. Despite this, many kinases have been shown to be involved in invadosome dynamics and function, acting to
Kinases in invadosome activity
The final stage of invadosome maturity is marked by the delivery of MT1-MMP to the protrusion and the secretion of MMPs (Murphy and Courtneidge, 2011). In addition, MT1-MMP islets have recently been shown to co-localise with podosomes, persist longer than the podosome lifetime and enable podosome re-emergence in primary macrophages (El Azzouzi et al., 2016). This suggests that MT1-MMP accumulation not only regulates the activity of mature invadosomes but also influences their reformation.
In
Kinases in invadosome dissolution
Invadosome dissolution is arguably the least well studied stage of invadosome dynamics. Nonetheless, effective invadopodia and podosome turnover contribute to tumour and immune cell migration and invasion respectively. Therefore, the regulation of invadosome dissolution should not be overlooked (Murphy and Courtneidge, 2011, Schachtner et al., 2013b). Our knowledge regarding the regulation of invadosome disassembly is mainly derived from work on podosomes. In dendritic cells calpain appears to
Conclusion
This review has highlighted the involvement and importance of kinase signalling in every stage of the invadosome life cycle. Given that podosome deregulation and invadopodia function are both associated with pathophysiological states, studies investigating the potential clinical benefit of inhibiting the invadosome kinases are warranted.
It is evident that Src is not only implicated in every stage of the invadopodia life cycle but also in many other processes of tumourigenesis, such as
Acknowledgements
Work in the Jones laboratory is supported by the Medical Research Council (G1100041). E.F. is in receipt of a King’s College London Ph.D. stipend. A.P. is funded by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
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Authors contributed equally to this work.