Abstract
Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in cortactin by RNA interference knockdown methods. In these cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3-positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with cortactin expression levels. MMP inhibitor treatment of control cells mimicked the cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients.
Highlights
The ability to degrade extracellular matrix (ECM) is a hallmark of invasive tumors and is thought to be essential for the movement of cancer cells through tissue barriers [1, 2]
Antibodies against matrix metalloproteinases (MMP)-2, MMP-9, and MT1-MMP were from Chemicon International; anti-Arp3 has been previously described [43]; monoclonal anticortactin antibody for immunofluorescence was 4F11 (Upstate); polyclonal anticortactin antibody for Western blotting was previously described [36]; anti–h-actin was from Sigma (AC-74); anti– cyclophilin B antibody was from Alexis Biochemicals; and anti-apolipoprotein A1 (ApoA1) antibody was a gift from Larry Swift (Vanderbilt University)
Cells were considered to be positive for invadopodia formation if they had actin-rich puncta in the cell that stained for cortactin and were overlying areas of cell-associated matrix degradation
Summary
The ability to degrade extracellular matrix (ECM) is a hallmark of invasive tumors and is thought to be essential for the movement of cancer cells through tissue barriers [1, 2]. Similar structures, termed podosomes, are formed by a number of normal cell types that need to cross tissue barriers or remodel matrix, including. Invadopodia and podosomes share many structural and functional characteristics, including a dependence on branched actin assembly and src kinase signaling, as well as common molecular components [4, 5]. Invadopodia have a number of unique aspects, including the absolute dependence on src kinase signaling, the presence of focal adhesion proteins in the same location as branched actin, and the localization of active matrix degradation to sites of invadopodia formation [4, 5]. How the diverse processes of actin assembly, signaling, and membrane trafficking for protease secretion and cell-matrix adhesion are integrated to produce a functional ECMdegrading machine is poorly understood
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