Jose Javier Bravo-Cordero

Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.

Directed cell invasion and migration during metastasis.

Metastasis requires tumor cell dissemination to different organs from the primary tumor. Dissemination is a complex cell motility phenomenon that requires the molecular coordination of the protrusion, chemotaxis, invasion and contractility activities of tumor cells to achieve directed cell migration. Recent studies of the spatial and temporal activities of the small GTPases have begun to elucidate how this coordination is achieved. The direct visualization of the pathways involved in actin polymerization, invasion and directed migration in dissemination competent tumor cells will help identify the molecular basis of dissemination and allow the design and testing of more specific and selective drugs to block metastasis.

Functions of cofilin in cell locomotion and invasion

Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.

MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway

MT1‐matrix metalloproteinase (MT1‐MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1‐MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1‐MMP occurs during MDA‐MB‐231 adenocarcinoma cell migration into collagen type I three‐dimensional matrices. Polarized trafficking of MT1‐MMP is triggered by β1 integrin‐mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1‐MMP within VSV‐G/Rab8‐positive vesicles, but not in Rab11/Tf/TfRc‐positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1‐MMP exocytic traffic, collagen degradation and invasion, whereas Rab8‐ but not Rab11‐knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1‐MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1‐MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Invasive carcinoma cells use specialized actin polymerization-driven protrusions called invadopodia to degrade and possibly invade through the extracellular matrix (ECM) during metastasis. Phosphorylation of the invadopodium protein cortactin is a master switch that activates invadopodium maturation and function. Cortactin was originally identified as a hyperphosphorylated protein in v-Src-transformed cells, but the kinase or kinases that are directly responsible for cortactin phosphorylation in invadopodia remain unknown. In this study, we provide evidence that the Abl-related nonreceptor tyrosine kinase Arg mediates epidermal growth factor (EGF)-induced cortactin phosphorylation, triggering actin polymerization in invadopodia, ECM degradation, and matrix proteolysis-dependent tumor cell invasion. Both Src and Arg localize to invadopodia and are required for EGF-induced actin polymerization. Notably, Arg overexpression in Src knockdown cells can partially rescue actin polymerization in invadopodia while Src overexpression cannot compensate for loss of Arg, arguing that Src indirectly regulates invadopodium maturation through Arg activation. Our findings suggest a novel mechanism by which an EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Furthermore, they identify Arg as a novel mediator of invadopodia function and a candidate therapeutic target to inhibit tumor invasion in vivo.

Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway

Cortactin phosphorylation induces recruitment of the sodium-hydrogen exchanger NHE1 to invadopodia, resulting in pH changes that regulate cortactin-cofilin binding and invadopodium dynamics.

Specific tyrosine phosphorylation sites on cortactin regulate Nck1-dependent actin polymerization in invadopodia

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells enriched in proteins that regulate actin polymerization. The on–off regulatory switch that initiates actin polymerization in invadopodia requires phosphorylation of tyrosine residues 421, 466, and 482 on cortactin. However, it is unknown which of these cortactin tyrosine phosphorylation sites control actin polymerization. We investigated the contribution of individual tyrosine phosphorylation sites (421, 466, and 482) on cortactin to the regulation of actin polymerization in invadopodia. We provide evidence that the phosphorylation of tyrosines 421 and 466, but not 482, is required for the generation of free actin barbed ends in invadopodia. In addition, these same phosphotyrosines are important for Nck1 recruitment to invadopodia via its SH2 domain, for the direct binding of Nck1 to cortactin in vitro, and for the FRET interaction between Nck1 and cortactin in invadopodia. Furthermore, matrix proteolysis-dependent tumor cell invasion is dramatically inhibited in cells expressing a mutation in phosphotyrosine 421 or 466. Together, these results identify phosphorylation of tyrosines 421 and 466 on cortactin as the crucial residues that regulate Nck1-dependent actin polymerization in invadopodia and tumor cell invasion, and suggest that specifically blocking either tyrosine 421 or 466 phosphorylation might be effective at inhibiting tumor cell invasion in vivo.

β1 integrin regulates Arg to promote invadopodial maturation and matrix degradation.

β1 integrin is a major regulator of invadopodium maturation. Studies reveal that β1 integrin–mediated adhesion is a key upstream switch that induces Arg-dependent cortactin phosphorylation, actin polymerization, and MMP recruitment to invadopodia for extracellular matrix degradation.

Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation

Most cancer patients die as a result of metastasis, thus it is important to understand the molecular mechanisms of dissemination, including intra- and extravasation. Although the mechanisms of extravasation have been vastly studied in vitro and in vivo, the process of intravasation is still unclear. Furthermore, how cells in the tumor microenvironment facilitate tumor cell intravasation is still unknown. Using high-resolution imaging, we found that macrophages enhance tumor cell intravasation upon physical contact. Macrophage and tumor cell contact induce RhoA activity in tumor cells, triggering the formation of actin-rich degradative protrusions called invadopodia, enabling tumor cells to degrade and break through matrix barriers during tumor cell transendothelial migration. Interestingly, we show that macrophage-induced invadopodium formation and tumor cell intravasation also occur in patient-derived tumor cells and in vivo models, revealing a conserved mechanism of tumor cell intravasation. Our results illustrate a novel heterotypic cell contact-mediated signaling role for RhoA, as well as yield mechanistic insight into the ability of cells within the tumor microenvironment to facilitate steps of the metastatic cascade.

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: (1) single cells and (2) multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor.