Short Communication
A new role for cortactin in invadopodia: Regulation of protease secretion

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Abstract

Invadopodia are actin-dependent organelles that function in the invasion and remodeling of the extracellular matrix (ECM) by tumor cells. Cortactin, a regulator of the Arp2/3 complex, is of particular importance in invadopodia function. While most of the focus has been on the possible role of cortactin in actin assembly for direct formation of actin-rich invadopodia puncta, our recent data suggest that the primary role of cortactin in invadopodia is to promote protease secretion. In this manuscript, we review our previous work and present new data showing that cortactin is essential for both the localization of key invadopodia matrix metalloproteinases (MMPs) to actin-positive puncta at the cell–ECM interface and for ECM degradation induced by overexpression of MT1-MMP-GFP. Based on these data and results from the literature, we propose potential mechanisms by which cortactin may link vesicular trafficking and dynamic branched actin assembly to regulate protease secretion for invadopodia-associated ECM degradation.

Introduction

Invasion and metastasis is dependent on the ability of tumor cells to remodel and degrade the extracellular matrix (ECM) (Hoon et al., 2006; Pantel and Brakenhoff, 2004). In vitro, many invasive cancer cell lines have been shown to form specialized structures termed invadopodia for this process. Invadopodia are actin-based protrusions on the basal surface of invading cells that serve as centers in which cellular processes such as branched actin assembly, cell signaling and adhesion, and secretion of proteases spatially converge to promote remodeling of the ECM (Linder, 2007; Weaver, 2006).

Invadopodia were first identified in src-transformed cells and subsequent studies have shown that the formation of invadopodia is dependent on src tyrosine kinase signaling (Chen et al., 1985; Linder, 2007; Weaver, 2006). A number of src substrates, such as cortactin, Tks5/FISH, p130Cas, dynamin2, and N-WASp (Baldassarre et al., 2003; Bowden et al., 1999; Mizutani et al., 2002; Seals et al., 2005; Weaver, 2006; Yamaguchi et al., 2005), also localize to invadopodia and/or the related structures, podosomes (Linder, 2007). These downstream targets presumably function to coordinate the activities of the actin cytoskeleton, focal adhesions, protease activity, and membrane dynamics to the site of invadopodia formation.

Cortactin, an actin assembly protein that functions in both the activation and stabilization phases of branched actin assembly by the Arp2/3 complex (Tehrani et al., 2007; Uruno et al., 2001; Weaver et al., 2001), has been a focus of particular attention in the invadopodia field. Cortactin is present at sites of dynamic actin assembly in cellular protrusions such as lamellipodia and invadopodia (Yamaguchi and Condeelis, 2007); however, the precise role of cortactin in these processes is under much debate. Cortactin overexpression enhances cell migration (Bryce et al., 2005; Hill et al., 2006; Huang et al., 1998; Patel et al., 1998), and cells with cortactin knockdown by siRNA show defects in two-dimensional migration as well as invasion through Matrigel-coated transwell filters (Bryce et al., 2005; Hill et al., 2006; Rothschild et al., 2006; van Rossum et al., 2006). Cortactin has also been shown to have a major role in the function of invadopodia (Artym et al., 2006; Bowden et al., 1999; Clark et al., 2007) and the related structures, podosomes (Tehrani et al., 2006; Webb et al., 2006, Webb et al., 2007). Bowden et al. (1999) first demonstrated that cortactin is important for invadopodia function, since microinjection with neutralizing antibodies against cortactin blocked degradation of the underlying ECM (Bowden et al., 1999). That study also showed that cortactin is enriched in invadopodia in a complex with paxillin and protein kinase Cμ/protein kinase D. A recent study by Artym et al. (2006) proposed a step-wise model of invadopodia formation. Their live-cell imaging studies, combined with fixed cell analyses, indicate that cortactin and actin are early markers of invadopodia, followed quickly by accumulation of MT1-MMP in the developing invadopodia. Ongoing accumulation of actin, cortactin, and MT1-MMP coincides with matrix degradation (Artym et al., 2006). Using siRNA, Artym et al. (2006) found that downregulation of cortactin decreases the number of actin/cortactin-rich invadopodia puncta along with associated ECM degradation. Based on this work, on studies in podosomes (Tehrani et al., 2006; Webb et al., 2006, Webb et al., 2007), and the important role of cortactin in branched actin assembly (Uruno et al., 2001; Weaver et al., 2001), much of the focus in the field has been on the putative role for cortactin in actin assembly that takes place on site at podosomes and invadopodia.

Studies from our laboratory on the role of cortactin in invadopodia also found that knockdown of cortactin leads to a decreased number of actin-based invadopodia puncta per cell when compared with control cells (Fig. 1; Clark et al., 2007). However, while invadopodia puncta (defined as being positive for β-actin and Arp3, two branched actin invadopodia markers) are still present in cortactin knockdown (cortactin-KD) cells at a level of 40% compared to wild-type cells, there is a striking absence of invadopodia puncta-associated ECM degradation. In addition, in cortactin-overexpressing cells there is a much larger increase in invadopodia-associated ECM degradation than for β-actin/Arp3-positive invadopodia puncta formation (see data for LZRS-CortFL cells in Clark et al. (2007), and for pRS-KD1/LZRS-CortFL cells in Fig. 1). These findings are surprising since cortactin as an actin assembly protein should affect the earlier actin puncta phase at least equally if not better than the ECM degradation phase. Inhibition of MMPs with either the broad-spectrum inhibitor GM6001 or the more specific TIMP-2 has an effect on invadopodia that is similar to cortactin-KD: fewer invadopodia puncta and abolition of ECM degradation (Clark et al., 2007). These two pieces of evidence led us to suspect that cortactin may have a role in the protease phase of invadopodia function.

Western blot analyses of whole-cell lysates from cells with cortactin-KD or cortactin overexpression revealed no change in the overall cellular levels of the key invadopodia metalloproteinases MMP2, MMP9 or MT1-MMP (Clark et al., 2007). However, since these MMPs must reach the outside of the cell to access and degrade ECM, we also quantified the level of these proteins in conditioned medium (MMP2, MMP9) or on the cell surface (MT1-MMP) as a function of cortactin expression. Indeed, secretion of all three MMPs is profoundly affected by cortactin expression (see analyses and zymograms for MMP2 and MMP9 in Fig. 2, analysis of MT1-MMP surface expression in Clark et al. (2007)), correlating well with the effect on invadopodia-associated ECM degradation (Fig. 1). Interestingly, cortactin is not only essential for secretion of MMP2 and MMP9, but overexpression boosts the levels of secreted proteases (Fig. 2; Clark et al., 2007), suggesting that cortactin can actually tune the system up or down. This finding is particularly important in light of the fact that cortactin is frequently overexpressed in cancer as a function of 11q13 amplification (Myllykangas et al., 2007; Ormandy et al., 2003; Rodrigo et al., 2000; Schuuring et al., 1992, Schuuring et al., 1993).

To further investigate the role of cortactin in the protease phase of invadopodia function, we performed experiments in which we immunolocalized MMP2, MMP9, or MT1-MMP with β-actin in control and cortactin-KD cells, using confocal microscopy to image only the cell–ECM interface where invadopodia puncta occur.

Section snippets

Cell culture and antibodies

The HNSCC cell line SCC61 was originally isolated from a tongue squamous cell carcinoma tumor and considered aggressive, as defined by lack of response to radiation therapy and the presence of tumor-positive lymph nodes (Weichselbaum et al., 1986). These cells were maintained in DMEM supplemented with 20% fetal bovine serum and 0.4 μg/ml hydrocortisone. Antibodies against MMP2, MMP9, and MT1-MMP were from Chemicon International (AB809, AB16996, and AB8345, respectively). The anti-Arp3 antibody

Results

Our published analysis of MMP secretion consisted of bulk assays, in which we determined whether MMP2 and MMP9 secretion into conditioned media (Fig. 2) or MT1-MMP cell surface levels were affected by cortactin expression (Clark et al., 2007). However, the ECM-degrading activity of cells is focused at invadopodia, as is evident by the typical fluorescent ECM degradation assay (Fig. 1). To determine whether cortactin affects the focal localization of MMPs to invadopodia puncta, we performed

Discussion

The discovery that cortactin affects secretion of invadopodia-associated proteases raises the question of where cortactin acts within the cell to regulate secretion (Fig. 5). Our recent finding that cortactin is essential not only for the secretion of key invadopodia proteases but also for a non-invadopodia protein, apolipoprotein A (Clark et al., 2007), is consistent with a general role for cortactin in secretion. Indeed, a study from the McNiven laboratory previously implicated cortactin in

Acknowledgments

The MT1-MMP-EGFP cDNA was a kind gift of Dr. Sarah Netzel-Arnett. Thanks to Dr. Susette Mueller for many fruitful discussions. This work was supported by NIH grants R01GM075126 and R21DE018244 to A.M. Weaver.

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