Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057-1469, USA.
Experimental Cell Research (Impact Factor: 3.25). 06/2006; 312(8):1240-53. DOI: 10.1016/j.yexcr.2005.12.012
Source: PubMed


Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.

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    • "Tyrosyl phosphorylation of cortactin upon PRL stimulation observed in TMX2-28 cells which was lacking in T47D cells may explain why TMX2-28 cells are more invasive than T47D cells. Bowden et al demonstrated that cortactin colocalizes with phospho-tyrosine in complexes termed " invadopodia complexes " [38]. Increasing the amount of phospho-tyrosine at these cortactin-rich invadopodia increased proteolytic activity in these areas, suggesting that increased tyrosyl phosphorylation of cortactin in invadopodia contributes to cell invasion. "
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    ABSTRACT: The hormone/cytokine prolactin (PRL) is implicated in breast cancer cell invasion and metastasis. PRL-induced pathways are mediated by two non-receptor tyrosine kinases, JAK2 and Src. We previously demonstrated that prolactin stimulates invasion of breast cancer cells TMX2-28 through JAK2 and its target serine/threonine kinase PAK1. We hypothesize herein that the actin-binding protein cortactin, a protein involved in invadopodia formation and cell invasion, is activated by PRL. We demonstrate that TMX2-28 cells are more invasive than T47D breast cancer cells in response to PRL. We determine that cortactin is tyrosyl phosphorylated in response to PRL in a time and dose-dependent manner in TMX2-28 cells, but not in T47D cells. Furthermore, we show that PRL mediates cortactin tyrosyl phosphorylation via Src, but not JAK2. Finally, we demonstrate that maximal PRL-mediated TMX2-28 cell invasion requires both Src and JAK2 kinase activity, while T47D cell invasion is JAK2- but not Src-dependent. Thus PRL may induce cell invasion via two pathways: through a JAK2/PAK1 mediated pathway that we have previously demonstrated, and Src-dependent activation and tyrosyl phosphorylation of cortactin. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 06/2015; 463(4). DOI:10.1016/j.bbrc.2015.05.116 · 2.30 Impact Factor
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    • "Invadopodia are relatively dynamic, actin rich, proteolytic cellular structures formed by invasive cancer cells (Bowden et al., 2006; Buccione et al., 2009; Linder, 2009; Linder and Aepfelbacher, 2003) (Figure 2). Invadopodia can be from a few hundred nanometers to several microns wide and can be up to 8 micrometers if the underlying matrix is thick enough (Baldassarre et al., 2003). "
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    ABSTRACT: The leading cause of death in cancer patients is metastasis. Invasion is an integral part of metastasis and is carried out by proteolytic structures called invadopodia at the cellular level. In this introductory review, we start by evaluating the definition of invadopodia. While presenting the upstream signaling events involved, we integrate current models on invadopodia. In addition, we discuss the significance of invadopodia in 2D and 3D and in vivo. We finally point out technical challenges and conclude with open questions in the field.
    Turkish Journal of Biology 11/2014; 38(6):740-747. DOI:10.3906/biy-1404-110 · 1.34 Impact Factor
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    • "Maybe that is one of the reasons why SFN inhibited cell migration and invasion in U87MG and U373MG cells. Studies showed that CD44 was related to the pseudopodia formation [45], [46], and CD44 participated in many important cellular processes, including adhesion and motility [47]. CD44v6 is an important member of the cell adhesion molecule CD44 family, and plays a crucial role in tumor invasion [48]. "
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    ABSTRACT: Glioblastoma has highly invasive potential, which might result in poor prognosis and therapeutic failure. Hence, the key we study is to find effective therapies to repress migration and invasion. Sulforaphane (SFN) was demonstrated to inhibit cell growth in a variety of tumors. Here, we will further investigate whether SFN inhibits migration and invasion and find the possible mechanisms in human glioblastoma U87MG and U373MG cells. First, the optimal time and dose of SFN for migration and invasion study were determined via cell viability and cell morphological assay. Further, scratch assay and transwell invasion assay were employed to investigate the effect of SFN on migration and invasion. Meanwhile, Western blots were used to detect the molecular linkage among invasion related proteins phosphorylated ERK1/2, matrix metalloproteinase-2 (MMP-2) and CD44v6. Furthermore, Gelatin zymography was performed to detect the inhibition of MMP-2 activation. In addition, ERK1/2 blocker PD98059 (25 µM) was integrated to find the link between activated ERK1/2 and invasion, MMP-2 and CD44v6. The results showed that SFN (20 µM) remarkably reduced the formation of cell pseudopodia, indicating that SFN might inhibit cell motility. As expected, scratch assay and transwell invasion assay showed that SFN inhibited glioblastoma cell migration and invasion. Western blot and Gelatin zymography showed that SFN phosphorylated ERK1/2 in a sustained way, which contributed to the downregulated MMP-2 expression and activity, and the upregulated CD44v6 expression. These molecular interactions resulted in the inhibition of cell invasion. SFN inhibited migration and invasion processes. Furthermore, SFN inhibited invasion via activating ERK1/2 in a sustained way. The accumulated ERK1/2 activation downregulated MMP-2 expression and decreased its activity and upregulated CD44v6. SFN might be a potential therapeutic agent by activating ERK1/2 signaling against human glioblastoma.
    PLoS ONE 02/2014; 9(2):e90520. DOI:10.1371/journal.pone.0090520 · 3.23 Impact Factor
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