N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Journal of Cell Science (Impact Factor: 5.43). 02/2012; 125(Pt 3):724-34. DOI: 10.1242/jcs.092726
Source: PubMed


Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.

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Available from: Bojana Gligorijevic
    • "Invadopodia presence was shown in vitro in 2D conditions, in 3D environment [18] and in vivo. Tumor cryosections from mice mammary carcinoma were used to show the presence of the invadopodial markers actin and cortactin accompanied by collagen degradation in the protrusions of cells that are adjacent to the tumor edge and to tumor blood vessels [2] [12] [19] [20]. Other recent work demonstrated that invadopodia Contents lists available at ScienceDirect journal homepage: "
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    ABSTRACT: Invadopodiaareactin-richprotrusionsformedbymesenchymallymigratingcancercells.Theyaremainly composed ofactin,actin-associatedproteins,integrinsandproteinsofsignalingmachineries.These protrusionsdisplayfocalizedproteolyticactivitytowardstheextracellularmatrix.Itiswellknownthat polymerized (F-)actinispresentinthesestructures,butthenatureoftheactinisoformhasnotbeen studied before.Wehereshowthatbothcytoplasmicactinisoforms, β- and γ-actin, arepresentinthe invadopodiaofMDA-MB-231breastcancercellsculturedona2D-surface,wheretheycolocalizewiththe invadopodialmarkercortactin.Invadopodialstructuresformedbythecellsina3D-collagenmatrixalso contain β- and γ-actin. Wedemonstratethisusingisoform-specific antibodiesandexpressionof fluor- escently-taggedactinisoforms.Additionally,usingsimultaneousexpressionofdifferentiallytagged β- and γ-actin incells,weshowthattheactinisoformsarepresenttogetherinasingleinvadopodium.Cells with anincreasedlevelof β- or γ-actin, displayasimilarincreaseinthenumberandsizeofinvadopodia in comparisontocontrolcells.Moreover,increasingthelevelofeitheractinisoformsalsoincreases invasionvelocity.
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    • "Invadopodia comprise a central actin-rich core surrounded by a group of adhesion, membrane remodeling, scaffolding, and matrix degradation proteins [12, 13]. Although most mechanistic studies regarding invadopodia formation are conducted in vitro in cell culture experiments, there is growing evidence for the importance of invadopodia in vivo in cancer metastasis formation14151617. To better dissect the broad effect of the ZEB1/miR- 200 feedback loop in cancer cell invasion, we used an in silico screening approach together with expression data from our breast cancer EMT/MET-cell line model [9], to find relevant genes besides ZEB1, that are specifically inhibited by miR-200 to prevent cell invasion. "
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    ABSTRACT: Epithelial to mesenchymal transition (EMT) is a developmental process which is aberrantly activated during cancer invasion and metastasis. Elevated expression of EMT-inducers like ZEB1 enables tumor cells to detach from the primary tumor and invade into the surrounding tissue. The main antagonist of ZEB1 in controlling EMT is the microRNA-200 family that is reciprocally linked to ZEB1 in a double negative feedback loop. Here, we further elucidate how the ZEB1/miR-200 feedback loop controls invasion of tumor cells. The process of EMT is attended by major changes in the actin cytoskeleton. Via in silico screening of genes encoding for actin interacting proteins, we identified two novel targets of miR-200c - TKS5 and MYLK (MLCK). Co-expression of both genes with ZEB1 was observed in several cancer cell lines as well as in breast cancer patients and correlated with low miR-200c levels. Depletion of TKS5 or MYLK in breast cancer cells reduced their invasive potential and their ability to form invadopodia. Whereas TKS5 is known to be a major component, we could identify MYLK as a novel player in invadopodia formation. In summary, TKS5 and MYLK represent two mediators of invasive behavior of cancer cells that are regulated by the ZEB1/miR-200 feedback loop.
    Full-text · Article · Aug 2015 · Oncotarget
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    • "Some data also support the existence of podosome-like structures within tissue collected from mice and fixed without culturing (Quintavalle et al., 2010). To dissect the function of podosomes/invadopodia in vivo, several groups disrupted their formation by genetically manipulating the genes that are crucial for their assembly, such as Tks5 (Blouw et al., 2008; Murphy et al., 2011), N-WASP (Gligorijevic et al., 2012), and Arg kinase (Gil-Henn et al., 2013). Nevertheless, evidence of podosome function in vivo is still scarce. "

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