A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.     

PtdIns(3,4)P2 instigates focal adhesions to generate podosomes

Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4) P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.Key words: podosome, invadopodium, focal adhesion, Tks5, PtdIns(3,4)P2, N-WASP     

Sequential signals toward podosome formation in NIH-src cells

Podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with PtdIns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after PtdIns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by PtdIns(3,4)P2.     

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.     

SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediate trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) during invadopodium formation and tumor cell invasion

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.     

Metalloproteinase MT1-MMP islets act as memory devices for podosome reemergence

Podosomes are dynamic cell adhesions that are also sites of extracellular matrix degradation, through recruitment of matrix-lytic enzymes, particularly of matrix metalloproteinases. Using total internal reflection fluorescence microscopy, we show that the membrane-bound metalloproteinase MT1-MMP is enriched not only at podosomes but also at distinct “islets” embedded in the plasma membrane of primary human macrophages. MT1-MMP islets become apparent upon podosome dissolution and persist beyond podosome lifetime. Importantly, the majority of MT1-MMP islets are reused as sites of podosome reemergence. siRNA-mediated knockdown and recomplementation analyses show that islet formation is based on the cytoplasmic tail of MT1-MMP and its ability to bind the subcortical actin cytoskeleton. Collectively, our data reveal a previously unrecognized phase in the podosome life cycle and identify a structural function of MT1-MMP that is independent of its proteolytic activity. MT1-MMP islets thus act as cellular memory devices that enable efficient and localized reformation of podosomes, ensuring coordinated matrix degradation and invasion.     

Src-dependent phosphorylation of ASAP1 regulates podosomes

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.     

Cell migration and invasion in human disease: the Tks adaptor proteins

Cell invasion plays a central role in a wide variety of biological phenomena and is the cause of tumour growth and metastasis. Understanding the biochemical mechanisms that control cell invasion is one of the major goals of our laboratory. Podosomes and invadopodia are specialized cellular structures present in cells with physiological or pathological invasive behaviours. These transient structures are localized at the ventral cell surface, contain an array of different proteins and facilitate cell-substrate adhesion, as well as the local proteolytic activity necessary for extracellular matrix remodelling and subsequent cellular invasion. We have shown previously that the adaptor proteins and Src substrates Tks4 and Tks5 are required for podosome and invadopodia formation, for cancer cell invasion in vitro, and for tumour growth in vivo. We have also defined a role for the Tks-mediated generation of ROS (reactive oxygen species) in both podosome and invadopodia formation, and invasive behaviour. Tks4 and Tks5 are also required for proper embryonic development, probably because of their roles in cell migration. Finally, we recently implicated podosome formation as part of the synthetic phenotype of vascular smooth muscle cells. Inhibitors of podosome and invadopodia formation might have utility in the treatment of vascular diseases and cancer. We have therefore developed a high-content cell-based high-throughput screening assay that allows us to identify inhibitors and activators of podosome/invadopodia formation. We have used this assay to screen for small-molecule inhibitors and defined novel regulators of invadopodia formation. In the present paper, I review these recent findings.     

Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells

Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.     

c-Src-mediated phosphorylation of NoxA1 and Tks4 induces the reactive oxygen species (ROS)-dependent formation of functional invadopodia in human colon cancer cells

The NADPH oxidase family, consisting of Nox1-5 and Duox1-2, catalyzes the regulated formation of reactive oxygen species (ROS). Highly expressed in the colon, Nox1 needs the organizer subunit NoxO1 and the activator subunit NoxA1 for its activity. The tyrosine kinase c-Src is necessary for the formation of invadopodia, phosphotyrosine-rich structures which degrade the extracellular matrix (ECM). Many Src substrates are invadopodia components, including the novel Nox1 organizer Tks4 and Tks5 proteins. Nox1-dependent ROS generation is necessary for the maintenance of functional invadopodia in human colon cancer cells. However, the signals and the molecular machinery involved in the redox-dependent regulation of invadopodia formation remain unclear. Here, we show that the interaction of NoxA1 and Tks proteins is dependent on Src activity. Interestingly, the abolishment of Src-mediated phosphorylation of Tyr110 on NoxA1 and of Tyr508 on Tks4 blocks their binding and decreases Nox1-dependent ROS generation. The contemporary presence of Tks4 and NoxA1 unphosphorylable mutants blocks SrcYF-induced invadopodia formation and ECM degradation, while the overexpression of Tks4 and NoxA1 phosphomimetic mutants rescues this phenotype. Taken together, these results elucidate the role of c-Src activity on the formation of invadopodia and may provide insight into the mechanisms of tumor formation in colon cancers.     

Tks5 recruits AFAP-110, p190RhoGAP, and cortactin for podosome formation

Podosome formation in vascular smooth muscle cells is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the molecular mechanism that underlies their specific recruitment to podosomes remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. We show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5 mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. By expressing a catalytically inactive point mutant and by siRNA-mediated expression knock-down we also provide evidence that p190RhoGAP is required for podosome formation. Together our findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation.     

Inhibition of β1 integrin induces its association with MT1-MMP and decreases MT1-MMP internalization and cellular invasiveness

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.     

PtdIns(3,4)P2 instigates focal adhesions to generate podosomes

Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation, and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions, and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization, and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4)P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.     

Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.     

Proactive for invasion: Reuse of matrix metalloproteinase for structural memory

Migratory cells translocate membrane type-1 matrix metalloproteinase (MT1-MMP) to podosomes or invadosomes to break extracellular matrix barriers. In this issue, El Azzouzi et al. (2016. J. Cell. Biol. http://dx.doi.org/10.1083/jcb.201510043) describe an unexpected function for the MT1-MMP cytoplasmic domain in imprinting spatial memory for podosome reformation via assembly in membrane islets.Invasion of most normal and cancer cells across basement membranes and collagen-rich interstitial tissues involves degradation of the ECM by membrane type-1 matrix metalloproteinase (MT1-MMP/MMP14; Willis et al., 2013). To fulfill this activity, MT1-MMP is transported to podosomes, the specialized ECM-degrading membrane protrusions found in highly migratory cells such as activated macrophages, osteoclasts, endothelial cells, and smooth muscle cells (Murphy and Courtneidge, 2011). In cancer cells, MT1-MMP is transported to ECM-degrading invasive structures called invadopodia (Poincloux et al., 2009). Both these membrane protrusions, collectively called invadosomes, are composed of an actin-rich core surrounded by scaffold and adhesion proteins, and numerous mechanisms of invadosome assembly, maturation, and dynamics have been identified (Poincloux et al., 2009; Murphy and Courtneidge, 2011). MT1-MMP activity is regulated at multiple levels to achieve targeted ECM degradation, cell surface protein processing, and protease activation (Sato et al., 1994; Osenkowski et al., 2004; Sugiyama et al., 2013; Willis et al., 2013; Itoh, 2015). Potential regulatory functions of MT1-MMP toward the cytoskeleton have, however, remained unclear. In this issue, El Azzouzi et al. describe an unexpected and novel function for MT1-MMP that goes beyond its traditional proteolytic activity: they show that MT1-MMP accumulates in membrane islets that provide macrophages with spatial information, or memory, in sites of podosome dissolution so as to enable efficient podosome reassembly.El Azzouzi et al. (2016) first used total internal reflection fluorescence microscopy and a pH-sensitive version of MT1-MMP devised to fluoresce only when the MT1-MMP ectodomain is exposed to the extracellular environment’s pH. With this approach, they show that, on the ventral surface of cultured human macrophages, MT1-MMP is localized at two different membrane compartments: underneath the podosome core, as previously suggested based on matrix degradation and colocalization with podosome markers, and in distinct islets devoid of other podosome components, CD44, or integrin-mediated adhesion to the ECM (Fig. 1; Osiak et al., 2005). MT1-MMP islets were dependent on intact cortical actin, but became more apparent and persisted after podosome disruption by pharmacological perturbation of key components of podosome assembly and maturation, such as integrin adhesion, Src kinase activity, and the Arp2/3 complex essential for actin nucleation and branched actin cytoskeleton. Podosomes often reemerge at sites of previous podosome localization, and El Azzouzi et al. (2016) hypothesized that MT1-MMP islets might mark sites of podosome formation. They treated cells with an Arp2/3 inhibitor to disrupt podosomes and induce the appearance of MT1-MMP membrane clusters, and used time-lapse imaging to track what happens upon washout and podosome reformation. Interestingly, they show that these novel MT1-MMP structures serve as remarkably immobile cell membrane anchors capable of rerecruiting the podosomal actin cores/scaffolds to the same islets.Open in a separate windowFigure 1.MT1-MMP islets as memory sites for podosome reformation. Migratory cells translocate MT1-MMP (red) to podosomes or invadosomes to degrade the ECM (green fibers). These membrane structures are composed of an actin-rich core (brown) surrounded by adhesion and scaffold proteins (beige) such as integrins (blue). El Azzouzi et al. (2016) show a function for MT1-MMP accumulation in membrane ”islets” (1), where they imprint spatial memory for podosome reemergence after podosome disassembly (2). Unlike dynamic mature podosomes (3), MT1-MMP assembles in stable islets via anchorage to cortical actin. Future work in the fields of inflammation, cancer, and angiogenesis will need to address the nature of the cytoskeletal dynamics mediating islet formation, the involvement of microtubules in islet formation, the exact islet protein composition, and the relevance of these memory sites to 2D or 3D environments and to other cell types beyond macrophages, including endothelial cells and invasive cancer cells.Further, by expressing mutant MT1-MMP proteins in cells silenced for the endogenous proteinase and using a podosome reformation assay (based on pharmacological dissolution of podosomes via Src inhibition, followed by podosome reformation after washout), El Azzouzi et al. (2016) pinpointed the region of MT1-MMP critical for islet formation, the LLY-sequence in its cytoplasmic domain. Moreover, when attached to the membrane by the MT1-MMP transmembrane domain, the 20–amino acid cytoplasmic tail appeared necessary and sufficient to form the islets. Considering the LLY sequence–dependent actin-binding ability of MT1-MMP (Yu et al., 2012) coupled with the observed necessity of cortical actin for islet appearance and podosome reformation, the direct interaction with unbranched cortical actin was suggested by the authors as a likely decisive mechanism for the remarkable MT1-MMP islet stabilization in podosome-free areas, although a possible indirect interaction was not ruled out. Actin binding through the MT1-MMP cytosolic tail was likewise suggested as a potential means for podosome rerecruitment by MT1-MMP memory islets.Although cortical actin is instrumental for the emergence of the spatially and temporally stable MT1-MMP islets upon podosome dissolution in macrophages and direct actin–MT1-MMP interaction has been proven in vitro and suggested as a means for retaining MT1-MMP in invadopodia, a Src-regulated interaction between MT1-MMP’s cytoplasmic domain and the actin-binding scaffold protein palladin has also been shown to regulate MT1-MMP targeting into invadopodia (Yu et al., 2012; von Nandelstadh et al., 2014). Moreover, the LLY sequence in MT1-MMP’s cytoplasmic tail harbors a Src substrate sequence and mediates an interaction between MT1-MMP and AP-2 that is important for MT1-MMP internalization and dynamics in cell migration and invasion (Uekita et al., 2001; Nyalendo et al., 2007). Intriguingly, El Azzouzi et al. (2016) did not find evidence of involvement of dynamin-dependent membrane trafficking events in the ability of MT1-MMP islets to function as memory sites. However, their results after treatment with the microtubule inhibitor nocodazole indicated that although the islets themselves remained intact, podosome reappearance was mislocalized, suggesting that microtubules contribute by as yet undefined mechanisms to the ability of MT1-MMP islets to provide spatial memory and to facilitate podosome reassembly. Therefore, further identification of drivers and specific regulatory mechanisms of the actin–MT1-MMP interaction dynamics in podosomes, of the stable actin–MT1-MMP interaction and structures in podosome-free areas, and of microtubule-dependent podosome reassembly will be of interest.A striking observation of this study is that MT1-MMP islets do not display degradative activity in matrix degradation assays. In addition, inhibition of the proteolytic activity of MT1-MMP through pharmacological agents or via an inactivating mutation did not impact islet appearance or podosome reemergence at sites of MT1-MMP clustering. Overall, on the extracellular side of the plasma membrane, the apparent lack of contact and degradation of the ECM as well as the relatively minor impact of the N-terminal MT1-MMP ectodomain on islet formation and podosome reemergence are peculiar features of the MT1-MMP islets. However, El Azzouzi et al. (2016) show evidence for somewhat impaired islet formation in cells expressing an MT1-MMP mutant lacking the entire ectodomain, and they demonstrate that endogenous MT1-MMP must be silenced for the LLY MT1-MMP mutant to disrupt islet localization. Based on these results, the authors suggest the possible influence of MT1-MMP oligomerization and of MT1-MMP–ECM binding on islet recruitment and stabilization. Nevertheless, these observations altogether indicate that the adhesive and degradative activities of MT1-MMP memory islets toward the ECM are minor and, intriguingly, do not influence the structure or function of these islets as currently characterized in 2D cultures.Furthermore, the aforementioned results raise questions about the possible contribution of the different molecular forms of MT1-MMP (e.g., cleaved or uncleaved and inhibitor bound or not) to the stabilization and podosome reassembly function of MT1-MMP islets. In cells and conditions in which MT1-MMP activity is high, MT1-MMP turnover is typically fast via autocatalytic cleavage or shedding of the N-terminal catalytic domain (Lehti et al., 1998; Yana and Weiss, 2000; Itoh et al., 2001; Osenkowski et al., 2004). After interaction with inhibitors such as tissue inhibitors of metalloproteinases, active endocytosed MT1-MMP may dissociate from the bound inhibitor to be recycled to the plasma membrane (Jiang et al., 2001; Remacle et al., 2003). However, in the absence of interaction with a protease inhibitor or collagen/matrix substrate, MT1-MMP oligomerization facilitates MT1-MMP turnover via autocatalytic inactivating cleavage (Itoh et al., 2001; Lehti et al., 2002; Osenkowski et al., 2004). In the current study, El Azzouzi et al. (2016) used MT1-MMP proteins with a pH sensor inserted N-terminally to the transmembrane domain, so that the probe is located extracellularly on the surface-exposed protease. The fluorescence signal from these constructs is not expected to be affected by proteolytic processing or shedding of the catalytic domain, so it is unclear whether the MT1-MMP proteins clustered in islets are cleaved or not. However, FRAP results showed that the turnover of MT1-MMP molecules within the islets is relatively slow. It thus remains to be clarified if and how the proteolytically active or possibly processed or protease inhibitor–bound inactive forms of MT1-MMP are stabilized in these MT1-MMP islets.As posodomes are highly dynamic protrusions, their rapid turnover implicates a constant disassembly at the rear and formation at the front of migrating macrophages. Assembly and dissassembly are known to depend on Arp2/3-mediated actin nucleation and fission of preexisting podosomes, respectively (Linder et al., 2000). Both of these mechanisms may contribute to podosome reassembly at MT1-MMP memory sites. Considering that these islets are laterally immobile and overall stable in at least unpolarized cells, it is unclear how migrating cells coordinate their actin and microtubule cytoskeletons for podosome reassembly at the front using islets formed upon podosome dissolution at the rear of the cell (Fig. 1). Moreover, the structural and functional features of MT1-MMP islets in the scenario of 3D cell–ECM microenvironments is intriguing and will need to be investigated at high resolution, as cytoskeletal dynamics, cell polarity, and matrix stiffness greatly differ in 3D tissues and matrices from the simple 2D setting of cultured cells, and all are known to influence cell behavior. Although the transient nature of these MT1-MMP islets in bridging podosome disassembly and reassembly exemplifies and reflects the efficiency of podosome reusage, probing the protein composition of these islets as well as the dynamics of podosome reassembly will likely be challenging. Future studies comparing MT1-MMP state, dynamics, reuse, and turnover in different types of invadosomes, islets, and other subcellular compartments will be instrumental to better understand how cells integrate the different types of MT1-MMP membrane structures and cell–ECM communication with other cellular signals and with drivers of cytoskeletal dynamics.The identification of the molecular mechanisms of structural and functional podosome memory are not only relevant to the fields of macrophage biology and inflammation but also more broadly to those of tissue invasion and matrix remodeling. For instance, endothelial cells, smooth muscle cells, and cancer cells are also known to target MT1-MMP to podosomes or related invadosomes. Examining MT1-MMP memory in such specialized subcellular compartments will be interesting beyond the podosome field, as the podosome counterparts in cancer cells may display and use MT1-MMP or other metalloproteinases in a similar manner. By shedding light on the mechanisms of dynamic protrusion formation and function, this paper not only opens new avenues of investigation into the cellular structures marking protrusion sites as “memory devices” but also brings about a new concept to the fields of cell invasion, angiogenesis, and cancer.     

Invadopodia and podosomes in tumor invasion

Cell migration through the extracellular matrix (ECM) is necessary for cancer cells to invade adjacent tissues and metastasize to an organ distant from primary tumors. Highly invasive carcinoma cells form ECM-degrading membrane protrusions called invadopodia. Tumor-associated macrophages have been shown to promote the migratory phenotypes of carcinoma cells, and macrophages are known to form podosomes, similar structures to invadopodia. However, the role of invadopodia and podosomes in vivo remains to be determined. In this paper, we propose a model for possible functions and interactions of invadopodia and podosomes in tumor invasion, based on observations that macrophage podosomes degrade ECM and that podosome formation is regulated by colony-stimulating factor-1 signaling.     

Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.     

Establishment and validation of computational model for MT1-MMP dependent ECM degradation and intervention strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.     

Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

GEFH1 binds ASAP1 and regulates podosome formation

Invadopodia are cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing a BAR domain, is a substrate of Src. ASAP1 is required for the assembly of invadopodia and podosomes, which are Src-induced structures related to invadopodia in NIH 3T3 fibroblasts. The BAR domain of ASAP1 is required for the assembly of podosomes. Using two-hybrid screening, we have identified GEFH1, a guanine nucleotide exchange factor for RhoA, as a binding partner of the BAR domain of ASAP1. We validated the interaction of endogenous GEFH1 with ASAP1 by immunoprecipitation, and found GEFH1 colocalized with ASAP1 in podosomes. The overexpression of GEFH1 inhibited podosome assembly and ASAP1 catalytic activity as a GAP. A mutant of GEFH1 lacking the domain that binds to the BAR domain of ASAP1 was less effective. Reduced expression of GEFH1, achieved with siRNA treatment, did not affect matrix degradation by podosomes but increased the rate of podosome assembly. Based on these results, we conclude that GEFH1 is a negative regulator of podosomes.