Mechanisms and roles of podosomes and invadopodia.

Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Cellular and Molecular Mechanisms of MT1-MMP-Dependent Cancer Cell Invasion.

Metastasis is responsible for most cancer-associated deaths. Accumulating evidence based on 3D migration models has revealed a diversity of invasive migratory schemes reflecting the plasticity of tumor cells to switch between proteolytic and nonproteolytic modes of invasion. Yet, initial stages of localized regional tumor dissemination require proteolytic remodeling of the extracellular matrix to overcome tissue barriers. Recent data indicate that surface-exposed membrane type 1-matrix metalloproteinase (MT1-MMP), belonging to a group of membrane-anchored MMPs, plays a central role in pericellular matrix degradation during basement membrane and interstitial tissue transmigration programs. In addition, a large body of work indicates that MT1-MMP is targeted to specialized actin-rich cell protrusions termed invadopodia, which are responsible for matrix degradation. This review describes the multistep assembly of actin-based invadopodia in molecular details. Mechanisms underlying MT1-MMP traffic to invadopodia through endocytosis/recycling cycles, which are key to the invasive program of carcinoma cells, are discussed.

MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.

Podosome Nucleation Is Facilitated by Multivalent Interactions between Syk and ITAM-containing Membrane Complexes.

Immune cells survey their microenvironment by forming dynamic cellular protrusions that enable chemotaxis, contacts with other cells, and phagocytosis. Podosomes are a unique type of protrusion structured by an adhesive ring of active integrins that surround an F-actin-rich core harboring degradative proteases. Although the features of podosomes, once-established, have been well defined, the steps that lead to podosome formation remain poorly understood by comparison. In this study, we report that spleen tyrosine kinase (Syk) is a critical regulator of podosome formation. Deletion of Syk or targeting its kinase activity eliminated the ability for murine macrophages to form podosomes. We found that the kinase activity of Syk was important for the phosphorylation of its substrates, HS1 and Pyk2, both of which regulate podosome formation. Additionally, before podosomes form, we report that the tandem Src homology 2 domains of Syk afforded multivalent clustering of ITAM-containing adaptors that associated with integrins to structure platforms that initiate podosomes. We therefore propose that Syk has a dual role in regulating podosomes: first, by facilitating the assembly of multivalent signaling hubs that nucleate their formation and second, by sustaining tyrosine kinase activity of the podosomes once they form against their substrates. In cells expressing recently identified gain-of-function variants of SYK, podosomes were dysregulated. These results implicate SYK in the (patho)physiological functions of podosomes in macrophages.

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

Stiffness promotes cell migration, invasion, and invadopodia in nasopharyngeal carcinoma by regulating the WT-CTTN level.

Matrix stiffness potently promotes the malignant phenotype in various biological contexts. Therefore, identification of gene expression to participate in mechanical force signals transduced into downstream biochemical signaling will contribute substantially to the advances in nasopharyngeal carcinoma (NPC) treatment. In the present study, we detected that cortactin (CTTN) played an indispensable role in matrix stiffness-induced cell migration, invasion, and invadopodia formation. Advances in cancer research have highlighted that dysregulated alternative splicing contributes to cancer progression as an oncogenic driver. However, whether WT-CTTN or splice variants (SV1-CTTN or SV2-CTTN) regulate matrix stiffness-induced malignant phenotype is largely unknown. We proved that alteration of WT-CTTN expression modulated matrix stiffness-induced cell migration, invasion, and invadopodia formation. Considering that splicing factors might drive cancer progression through positive feedback loops, we analyzed and showed how the splicing factor PTBP2 and TIA1 modulated the production of WT-CTTN. Moreover, we determined that high stiffness activated PTBP2 expression. Taken together, our findings showed that the PTBP2-WT-CTTN level increases upon stiffening and then promotes cell migration, invasion, and invadopodia formation in NPC.

Cross-talk between gastric cancer and hepatic stellate cells promotes invadopodia formation during liver metastasis.

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRß) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM.

Cancer cell extravasation requires iplectin-mediated delivery of MT1-MMP at invadopodia.

BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.

Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.

Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

Latent membrane protein 1 and macrophage-derived TNFα synergistically activate and mobilize invadopodia to drive invasion of nasopharyngeal carcinoma.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Invadopodia associated Thrombospondin-1 contributes to a post-therapy pro-invasive response in glioblastoma cells.

A critical challenge in the treatment of glioblastoma (GBM) is its highly invasive nature which promotes cell migration throughout the brain and hinders surgical resection and effective drug delivery. GBM cells demonstrate augmented invasive capabilities following exposure to the current gold standard treatment of radiotherapy (RT) and concomitant and adjuvant temozolomide (TMZ), resulting in rapid disease recurrence. Elucidating the mechanisms employed by post-treatment invasive GBM cells is critical to the development of more effective therapies. In this study, we utilized a Nanostring® Cancer Progression gene expression panel to identify candidate genes that may be involved in enhanced GBM cell invasion after treatment with clinically relevant doses of RT/TMZ. Our findings identified thrombospondin-1 (THBS1) as a pro-invasive gene that is upregulated in these cells. Immunofluorescence staining revealed that THBS1 localised within functional matrix-degrading invadopodia that formed on the surface of GBM cells. Furthermore, overexpression of THBS1 resulted in enhanced GBM cell migration and secretion of MMP-2, which was reduced with silencing of THBS1. The preliminary data demonstrates that THBS1 is associated with invadopodia in GBM cells and is likely involved in the invadopodia-mediated invasive process in GBM cells exposed to RT/TMZ treatment. Therapeutic inhibition of THBS1-mediated invadopodia activity, which facilitates GBM cell invasion, should be further investigated as a treatment for GBM.

A Model for Membrane Degradation Using a Gelatin Invadopodia Assay.

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.

PI(3,4)P2-mediated membrane tubulation promotes integrin trafficking and invasive cell migration.

Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P2 However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal.

Estrogen (17ß-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells.

Invadopodia are protrusive structures that mediate the extracellular matrix (ECM) degradation required for tumor invasion and metastasis. Rho small GTPases regulate invadopodia formation, but the molecular mechanisms of how Rho small GTPase activities are regulated at the invadopodia remain unclear. Here we have identified FilGAP, a GTPase-activating protein (GAP) for Rac1, as a negative regulator of invadopodia formation in tumor cells. Depletion of FilGAP in breast cancer cells increased ECM degradation and conversely, overexpression of FilGAP decreased it. FilGAP depletion promoted the formation of invadopodia with ECM degradation. In addition, FilGAP depletion and Rac1 overexpression increased the emergence of invadopodia induced by epidermal growth factor, whereas FilGAP overexpression suppressed it. Overexpression of GAP-deficient FilGAP mutant enhanced invadopodia emergence as well as FilGAP depletion. The pleckstrin-homology (PH) domain of FilGAP binds phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], which is distributed on membranes of the invadopodia. FilGAP localized to invadopodia in breast cancer cells on the ECM, but FilGAP mutant lacking PI(3,4)P2-binding showed low localization. Similarly, the decrease of PI(3,4)P2 production reduced the FilGAP localization. Our results suggest that FilGAP localizes to invadopodia through its PH domain binding to PI(3,4)P2 and down-regulates invadopodia formation by inactivating Rac1, inhibiting ECM degradation in invasive tumor cells.Key words: invadopodia, breast carcinoma, Rac1, FilGAP, PI(3,4)P2.