Research ArticleCancer

Tks5-Dependent, Nox-Mediated Generation of Reactive Oxygen Species Is Necessary for Invadopodia Formation

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Science Signaling  15 Sep 2009:
Vol. 2, Issue 88, pp. ra53
DOI: 10.1126/scisignal.2000368

Abstract

Invadopodia are actin-rich membrane protrusions of cancer cells that facilitate pericellular proteolysis and invasive behavior. We show here that reactive oxygen species (ROS) generated by the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) system are necessary for invadopodia formation and function. Knockdown of the invadopodia protein Tks5 [tyrosine kinase substrate with five Src homology 3 (SH3) domains], which is structurally related to the Nox component p47phox, reduces total ROS abundance in cancer cells. Furthermore, Tks5 and p22phox can associate with each other, suggesting that Tks5 is part of the Nox complex. Tyrosine phosphorylation of Tks5 and Tks4, but not other Src substrates, is reduced by Nox inhibition. We propose that Tks5 facilitates the production of ROS necessary for invadopodia formation, and that in turn ROS modulate Tks5 tyrosine phosphorylation in a positive feedback loop.

Introduction

Podosomes and invadopodia are related cellular structures present in cells with physiologically or pathologically invasive behaviors, respectively (15). In two-dimensional tissue culture systems, both podosomes and invadopodia appear as fine actin-rich protrusions of the ventral plasma membrane. A defining feature of both podosomes and invadopodia is the presence of pericellular proteolytic activity: Matrix metalloproteases, ADAM (a disintegrin and metalloprotease) family proteases, cathepsins, and the urokinase plasminogen activator system are all associated with these structures (610). Both podosomes and invadopodia are believed to coordinate attachment to the extracellular matrix (ECM) with its degradation, facilitating migration and invasion. Macrophages, endothelial cells, vascular smooth muscle cells, and osteoclasts all form podosomes in response to activating stimuli. In contrast, many human cancer cells, including breast cancers, melanoma, squamous cell carcinomas of the head and neck, and glioblastomas, appear to form invadopodia constitutively (6, 9, 1114). The ability of human cancer cells to form invadopodia correlates with their invasiveness, both in vitro and in vivo (1518).

Reactive oxygen species (ROS), particularly superoxide and peroxide, can be produced in various ways (19). For instance, ROS are produced in the mitochondria as a by-product of the oxidative phosphorylation-dependent production of adenosine 5′-triphosphate (20) and in the endoplasmic reticulum as a consequence of protein misfolding (21). In cancer cells, mitochondrial dysfunction and metabolic stress can lead to increased ROS production, with subsequent DNA damage and, in some cases, apoptosis (2224). ROS production can also be of physiological benefit. For example, high amounts of ROS produced by activated phagocytic cells play an important role in host defense (25), and low amounts of ROS are important for cell motility and proliferation of nonphagocytic cells (2630). Several studies have described ROS production in cancer cells, driven by tumorigenic signals such as activated Src or Ras, overexpressed epidermal growth factor (EGF), or hepatocyte growth factor (HGF) receptors, and correlated it with survival, invasion, and metastatic growth in vivo (3134). Possible mechanisms through which ROS may promote tumorigenesis include increased synthesis of matrix metalloproteases (MMPs), amplification of signal transduction cascades by inhibition of protein tyrosine phosphatases (PTPases), and activation of protein kinase C (3538).

The NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) complex is one of the major cellular systems for the catalytic generation of ROS (3941). In phagocytes, this system comprises a membrane-localized complex of gp91phox (also known as Nox2) and p22phox, as well as cytosolic regulatory proteins, including p47phox (also known as NoxO2), p67phox (NoxA2), and the small guanosine triphosphatase (GTPase) Rac2. Activating stimuli, such as bacteria, lead to the phosphorylation of p47phox, eliciting a conformational change that allows the translocation of a complex including p47phox, p67phox, and Rac2 to the membrane to associate with Nox2. Rac, together with p67phox, catalyzes electron transfer from NADPH to FAD (flavin adenine dinucleotide) with concomitant superoxide production. Nox2 protein is largely restricted to phagocytic cells and the vasculature, and thus cannot mediate ROS production in all cell types. More recently, the homologous enzymes Nox1, Nox3, and Nox4 and the more distantly related Nox5, Duox1, and Duox2 were described, and their expression patterns were elucidated (4244). A p47phox homolog, NoxO1, and a p67phox homolog, NoxA1, have also been described (45), although NoxO1 is not expressed in all cell types capable of generating ROS by means of NADPH oxidases. Nox1 has been implicated in ROS production in colonic epithelium and is also found in colon cancer cell lines (4650). Nox4 is found more broadly, and several cancers carry this enzyme, particularly melanomas, glioblastomas, and pancreatic adenocarcinomas (42, 5153).

The Src substrate Tks5 [tyrosine kinase substrate with five Src homology 3 (SH3) domains] is a large adaptor or scaffolding protein with no obvious catalytic activity (54). Rather, it contains an N-terminal PX (phox homology) domain and five SH3 domains, as well as several polyproline motifs and two Src phosphorylation sites. Tks5 is cytoplasmic in nontransformed cells, but localizes to invadopodia in Src-transformed mouse fibroblasts (Src-3T3) and human cancer cells (55). Reduction of Tks5 abundance with short hairpin RNA (shRNA) results in the loss of invadopodia formation and invasive behavior in vitro (56) and reduced tumorigenicity in vivo (18). In addition, the combination of Tks5 and Src in epithelial cells is sufficient to generate invadopodia (56). A close ortholog of Tks5, called Tks4 because it consists of one PX and four SH3 domains, is also required for functional invadopodia formation (57). We therefore consider that the Tks adaptor proteins play essential roles as invadopodia scaffold proteins.

A comparison of both the sequence and the overall topology of p47phox and Tks5 reveals intriguing similarities (45, 54, 58). Each has an N-terminal PX domain followed by SH3 domains (two in the case of p47phox and five in the case of Tks5). In addition, the first two SH3 domains of Tks5 have been predicted to form a tandem SH3 domain similar to those of p47phox (59). In the latter case, it is this tandem SH3 domain that mediates the association with a proline-rich region (PRR) in p22phox (60). Both p47phox and Tks5 probably arose from the same primordial ancestor, and it is noteworthy that the simple chordate Ciona intestinalis has several nox genes but a single tks-like gene consisting of a PX domain and three SH3 domains (45). The similarities between Tks5 and p47phox, and the accumulating evidence of a role for ROS in cancer invasion, led us to investigate whether ROS were required for invadopodia formation and whether Tks5 was directly involved in ROS production.

Results

ROS are required for, and localized to, invadopodia

We first asked whether ROS are required for invadopodia formation, function, or both. For these experiments, we used NIH 3T3 cells transformed with activated Src (Src-3T3 cells), a mouse cell line characterized by numerous invadopodia (often referred to as podosomes in this cell background) arranged in characteristic rings or rosettes (56). These cells, which exhibit highly invasive behavior, provide a good model system for studying cell invasion, because podosome formation in normal cells and invadopodia formation in human cancer cells depend on Src (1). When Src-3T3 cells were incubated with the nonspecific antioxidant N-acetyl cysteine (NAC), we observed a marked and dose-dependent loss of invadopodia (Fig. 1A). We next used the flavoprotein inhibitor, diphenyleneiodonium chloride (DPI), whose predominant effect is to inhibit the Nox family of NADPH oxidases. DPI also inhibited invadopodia formation, whereas the lysyl oxidase inhibitor 3-aminopropionitrile did not (3-APN; Fig. 1A). We also found that DPI treatment markedly inhibited podosome formation in IC-21 cells (Fig. 1B), a mouse peritoneal macrophage cell line known to elaborate podosomes (61), consistent with a role for ROS in the control of both podosome and invadopodia formation.

Fig. 1

ROS are required for invadopodia and podosome formation. (A) NAC and DPI inhibit invadopodia formation in Src-3T3 cells. The diagram shows a cell (blue) attached to the ECM (green). Podosomes and invadopodia appear as F-actin–rich protrusions inside the matrix (arrows) that sometimes arrange into rings to form rosettes (inset). Src-3T3 cells were plated onto coverslips, treated with vehicle (DMSO, control), NAC (10 mM), DPI (20 μM), and 3-APN (300 μM) and processed for F-actin staining. Top, percentage of cells with invadopodia rosettes at different concentrations of NAC and DPI. Mean values and SEM of a representative experiment are shown in the graphs. Bottom, representative images of cells after the indicated treatments. (B) DPI inhibits podosome formation in macrophages. Mouse macrophages (IC-21) were incubated with DMSO (control), 20 μM DPI, or 300 μM 3-APN and processed for F-actin staining to visualize podosomes (arrows). (C) DPI reduces F-actin content of cells. Src-3T3 cells treated with DMSO (control) or 20 μM DPI were incubated with low phalloidin concentration (0.05 U/ml). Both coverslips were imaged with the same exposure times. (D) FAK localization is affected by DPI treatment. Control and 20 μM DPI–treated Src-3T3 cells were costained with FAK antibody (green) and phalloidin (red). Scale bar, 10 μm. For the indicated concentration of compound with respect to control, *P < 0.05, **P < 0.0005, ***P < 0.00005 for NAC treatment, and *** P < 0.005, ** P < 0.0005 for DPI treatment (Student’s t test).

Some punctate actin staining remained in the DPI-treated Src-3T3 cells. To determine the nature of these puncta, we performed additional staining. Phalloidin titration revealed a marked reduction in the actin concentration in these structures (Fig. 1C). We also noted that the puncta contained Tks5 and cortactin, but not MT1-MMP (fig. S1). In vehicle [dimethyl sulfoxide (DMSO)]–treated cells, focal adhesion kinase (FAK) was localized to the rims of the rosettes, but in DPI-treated cells, it was found in focal contact-like structures and was not present in the actin puncta (Fig. 1D).

To determine the role of ROS in invadopodia-mediated degradation of the ECM, we plated Src-3T3 cells onto fluorescent gelatin-coated coverslips, treated them with DPI, and measured matrix degradation 6 hours later. DPI significantly reduced degradation (Fig. 2A) in a concentration-dependent manner. Because these data suggested that ROS were required for invasive behavior, we tested the effect of NAC and DPI on the ability of Src-3T3 cells to move through Matrigel-coated Transwell chambers. Both NAC and DPI elicited concentration-dependent inhibition of invasion in this assay (Fig. 2B). Together, these data suggest that ROS are required for the invasive behavior of Src-3T3 cells. Some ROS have short half-lives and act in a spatially restricted manner. Therefore, we incubated Src-3T3 cells with the ROS sensor CM-DCF-DA and visualized them with differential interference microscopy (DIC) and fluorescence microscopy to determine the subcellular localization of the ROS. Although much of the ROS was nuclear or perinuclear, some were present in the rosettes of invadopodia (Fig. 2C), suggesting that ROS might be generated at these sites or might function there to facilitate invasive behavior.

Fig. 2

ROS are required for gelatin degradation and invasion and ROS are localized to invadopodia. (A) DPI inhibits gelatin degradation. Src-3T3 cells were plated on fluorescently labeled gelatin-coated coverslips and treated with 20 μM DPI 1.5 hours later. Cells were processed for F-actin staining 5.5 hours after treatment. Left: representative images (from areas containing comparable numbers of cells) of gelatin degradation (dark areas). Right: quantitation of gelatin degradation (percent degraded area normalized to cell number) in the absence (NT) or presence of the indicated amounts of DPI. Mean values and SEM of a representative experiment are shown in the graphs. (B) NAC and DPI inhibit Matrigel invasion. Src-3T3 cells were treated with inhibitors and assayed for Matrigel invasion as described in Materials and Methods. (C) Some ROS localize to invadopodia. Src-3T3 cells were incubated with the ROS probe CM-DCF-DA and visualized under DIC and fluorescence (DCF-DA) microscopy. Images of two representative cells are shown. Arrows indicate rosettes of invadopodia. Scale bar, 20 μm in (A), 5 μm in (C). P values were calculated with Student’s t test.

NADPH oxidases are involved in invadopodia formation

Because DPI is not totally selective for Nox enzymes, we used another method to investigate the possible involvement of Nox family enzymes in invadopodia formation and function. All Nox family enzymes (with the possible exception of Nox5, which is not found in the mouse) require p22phox for their catalytic activity (62, 63); thus, we determined the effects of p22phox knockdown with small interfering RNA (siRNA). Src-3T3 cells transfected with a pool of p22phox siRNAs had significantly reduced numbers of invadopodia (Fig. 3A). To control for off-target effects, we tested individual siRNAs and found a direct correlation between p22phox knockdown and the inhibition of invadopodia formation (fig. S2). We also found that gelatin degradation was inhibited by p22phox knockdown (Fig. 3B). We used reverse transcription–polymerase chain reaction (RT-PCR) analysis to determine which Nox enzymes are expressed in Src-3T3 cells. We identified the messenger RNAs (mRNA) that encode Nox1, Nox3, and Nox4, but not that for Nox2 (table S1). Because targeting of Nox4 to different subcellular compartments has been reported to modulate ROS-mediated signaling (64, 65), we focused on this isoform. Using confocal microscopy, we observed association of Nox4 with invadopodia, as judged by its colocalization with F-actin (Fig. 3C). Moreover, Nox4 knockdown with a lentivirally encoded shRNA sequence (Fig. 3D) reduced invadopodia formation and gelatin degradation. Similar effects on invadopodia formation were observed with siRNA directed against Nox4 (fig. S3). Knockdown was quantitated by RT-PCR or quantitative PCR (qPCR) in each case (figs. S2 and S3).

Fig. 3

The Nox system is involved in invadopodia formation and function in Src-3T3 cells. (A) p22phox knockdown decreases invadopodia number. Src-3T3 cells were treated with transfection reagent alone (mock) or with a pool of p22phox siRNAs (p22 knockdown) and assayed for invadopodia formation. Representative images are shown on the left (F-actin staining) and quantitation of invadopodia (percentage of cells with rosettes) on the right. Mean values and SEM of a representative experiment are shown in the graphs. (B) p22phox knockdown decreases gelatin degradation. Src-3T3 cells were treated with transfection reagent alone (mock) or a mixture of p22phox siRNAs (p22 knockdown) and assayed for gelatin degradation. Representative images obtained from areas of similar cell density are shown on the left, and quantitation of degradation (percent of degraded area normalized to cell number) on the right. The knockdown (KD) of p22phox is shown in fig. S2. (C) Nox4 localizes to invadopodia. Src-3T3 cells were fixed and stained with phalloidin (green) or with an antibody specific for Nox4 (red) and processed for confocal microscopy. Arrowheads point to rosettes showing colocalization of F-actin and Nox4. A merged image is shown on the right. (D) Nox4 is required for invadopodia formation (top, F-actin) and gelatin degradation [bottom, fluorescein isothiocyanate (FITC)-labeled gelatin (FITC-gelatin)]. Src-3T3 cells were infected with lentiviruses expressing either a control (scrambled) or Nox4 shRNA (Nox4 knockdown) and assayed for invadopodia formation or gelatin degradation as in (A) and (B). Nox4 KD is shown in fig. S3. Scale bars, 5 μm in (A), (C), and (D), upper row, and 20 μm in (B) and (D), lower row. *P < 0.05, **P < 0.005, ***P < 0.00001 as calculated by Student’s t test.

Invadopodia are present in many types of cancer cells but have been particularly well studied in melanoma, head and neck, and breast cancer cells. DPI inhibited invadopodia formation and gelatin degradation in human SCC61 (squamous cell carcinoma of the head and neck), and C8161.9 (melanoma) cells (Fig. 4, A and B). Similar effects of DPI on invadopodia formation were obtained with the human breast cancer line Bt549 and the melanoma RPMI-7951 (Fig. 4C).

Fig. 4

Nox-generated ROS are required for invadopodia formation and function in human cancer cells. (A) DPI inhibits invadopodia formation and gelatin degradation in the human cancer cell lines SCC61 and C8161.9. Cells were grown on gelatin-coated coverslips, treated with DMSO or 20 μM DPI, and then assayed for invadopodia formation (F-actin) and gelatin degradation (FITC-gelatin). (B) Quantitation of the gelatin degradation (percent degraded area normalized to cell number) for the indicated cell lines. (C) DPI inhibits invadopodia formation in BT549 and RPMI-7951 cells. Cells were grown on coverslips, treated with DMSO or 20 μM DPI, and then assayed for invadopodia formation by phalloidin staining (F-actin). Arrows, invadopodia. Scale bars, 4 μm in (A), upper row; 20 μm in (A), lower row; and 2 μm in C. *P < 0.01, **P < 0.0005 (Student’s t test).

siRNA-mediated knockdown of p22phox in SCC61 reduced invadopodia number and decreased gelatin degradation (Fig. 5A and fig. S4). RT-PCR analysis indicated that SCC61 expresses nox1, nox2, and nox4 (table S1). In contrast, and in keeping with the literature on melanoma cells (51), C8161.9 expressed predominantly nox4. We found that siRNA-mediated knockdown of either p22 or Nox4 reduced invadopodia formation in C8161.9 cells (Fig. 5, B and C), concomitant with its reduction of mRNA (fig. S4).

Fig. 5

NADPH oxidases are required for invadopodia formation and function. (A) p22phox knockdown reduces invadopodia number and FITC-gelatin degradation in SCC61 cells. Left: SCC61 cells were transfected with scrambled control or a p22phox siRNA (p22 knock down) and assayed for invadopodia formation by phalloidin staining (F-actin) and gelatin degradation (FITC-gelatin). Right: quantitation of number of invadopodia per cell (average and SEM values) and quantitation of gelatin degradation (percent degraded area normalized to cell number) of representative experiments. Knockdown of p22phox is shown in fig. S4. (B) p22phox knockdown reduces invadopodia number in C8161.9 cells. Left: C8161.9 cells were infected with scrambled control or a p22phox shRNA (p22 knockdown) and assayed for invadopodia formation by phalloidin staining (F-actin). Right: quantitation of percentage of cells with invadopodia (average and SEM values of a representative experiment). Knockdown of p22phox is shown in fig. S4. (C) Nox4 is required for invadopodia formation in C8161.9 cells. Cells were transfected with scrambled or Nox4-specific siRNAs (Nox4 knockdown) and assayed for invadopodia formation by phalloidin staining (F-actin). No invadopodia were detected in any Nox4 knockdown cells. Nox4 knockdown is shown in fig. S4. Scale bars, 4 μm in (A), upper row; 20 μm in (A), lower row; and 10 μm in (B) and (C). *P < 0.05, **P < 0.01, ***P < 0.0001 (Student’s t test).

Tks5 is required for ROS formation

Both Tks5 and p47phox have N-terminal PX domains followed by SH3 domains (54). Furthermore, the PX and the tandem SH3 domains of these two proteins have similar sequences. We therefore hypothesized that Tks5, like p47phox, might be directly involved in ROS production. We tested this hypothesis in two different assay systems. In the first, we infected cells with scrambled or tks5-specific shRNA-encoding lentiviruses, incubated them with CM-DCF-DA to measure intracellular oxidants, and quantitated the data with fluorescence-activated cell sorting (FACS) (Fig. 6A). In the second, we measured oxidant production in control and tks5-siRNA–transfected Src-3T3 cells, using a luminol-based chemiluminescence assay that measures both intracellular and extracellular reactive oxygen (Fig. 6B). In both cases, we observed a significant decrease in total cellular ROS with Tks5 knockdown. We obtained similar results using the luminol assay with SCC61 cells (Fig. 6C). In all cases, the decrease in ROS concentration achieved by decreasing Tks5 abundance was the same as that achieved by inhibiting Nox, either with DPI or with p22phox siRNA (see fig. S4E for p22phox knockdown and Fig. 5, A to C, for Tks5 protein abundance). In the accompanying manuscript, Gianni et al. show that Tks5 can act as an organizer protein for Nox1 and Nox3, but not Nox4 (66). This is in keeping with studies in the literature, which suggest that Nox4 does not require organizer proteins (67). However, we found that the increase in ROS production in B16-F10 melanoma cells transfected with Nox4 and p22phox requires Tks5 (Fig. 6D and fig. S5D). Together, these data suggest that the majority of Nox-dependent ROS production in Src-3T3 cells and also in cancer cells depends on Tks5.

Fig. 6

Tks5 is required for ROS production. (A) Tks5 knockdown reduces ROS in Src-3T3 cells as measured by CM-DCF-DA. Left: 3T3 and Src-3T3 cells were infected with control (scr) or Tks5-specific (KD) shRNAs, incubated with CM-DCF-DA to detect ROS, and analyzed by FACS (left). Right: mean fluorescence values from graph on the left. Knockdown of Tks5 is shown in fig. S5. (B) Tks5 knockdown reduces ROS in Src-3T3 cells as measured by luminol chemiluminescence. Src-3T3 cells were transfected with control (scr), Tks5 (Tks5 KD), or p22phox (p22 KD)-specific siRNAs, and ROS concentrations were quantitated by a luminol-based chemiluminescence assay. Knockdown of p22phox and Tks5 is shown in figs. S4 and S5, respectively. (C) Tks5 knockdown reduces ROS in SCC61 cells as measured by luminol chemiluminescence. SCC61 cells were transfected with control (scr) or a pool of Tks5-specific siRNAs (Tks5 KD). One hour before the assay, the control cells were incubated with either DMSO or 20 μM DPI, and ROS were quantitated by a luminol-based chemiluminescence assay. Tks5 knockdown is shown in fig. S5. (D) Nox4-mediated ROS production requires Tks5. B16-F10 melanoma cells were infected with lentiviruses expressing scrambled or Tks5-specific shRNAs and transfected with complementary DNAs for Nox4 and p22phox 24 hours later. Forty-eight hours after transfection, cells were incubated with CM-DCF-DA and ROS were determined by FACS. Mean relative fluorescence values are shown on the right. Analysis of Tks5, Nox4, and p22phox abundance is shown in fig. S5. Mean values and SEM of a representative experiment are shown in the graphs of middle panels. *P < 0.005, **P < 0.0005 (Student’s t test).

During ROS production in phagocytes, serine phosphorylation of C-terminal sequences of p47phox releases an intramolecular inhibitory interaction involving the tandem SH3 domains (68). This enables p47phox recruitment to membranes through association of its PX domain with phosphatidylinositol phospholipids, and association of the tandem SH3 domains with p22phox. Because of the similarities between p47phox and the N-terminal half of Tks5, we tested whether Tks5 could associate with p22phox. To do this, we cotransfected 293T cells with p22phox using myc-tagged full-length Tks5 or with a construct containing the first 390 amino acids of Tks5 (390) encompassing the PX domain and the first two SH3 domains (Fig. 7A). We found coimmunoprecipitation of p22phox with both forms of Tks5 (Fig. 7B). These data suggest that the first two SH3 domains of Tks5 might mediate the association. To explore this, we repeated the cotransfections, this time with wild-type Tks5 or constructs with point mutations in each SH3 domain independently, designated M1, M2, M3, M4, and M5 (Fig. 7C). We detected a reduction in p22phox binding to the M1 mutant and, to a lesser extent, to the M2 mutant. To map the association site in p22phox, we tested the proline-156 to glutamine (P156Q) mutant of p22phox, which is in the PRR that mediates the association with p47phox and NoxO1 SH3 domains. This is a naturally occurring mutant, described in a person with chronic granulomatous disease, that acts as a dominant-negative inhibitor of Nox-dependent ROS production (69). We found decreased association of this mutant with Tks5 compared to that of wild-type p22phox (Fig. 7D). Finally, we measured the effects on association of combining the P156Q p22phox mutant with mutation of both of the first two SH3 domains (M1M2 mutant) of Tks5 (Fig. 7E). The combined effect on complex formation of both p22phox and Tks5 mutants was greater than that of either mutant alone. Together, these data suggest that Tks5 participates in Nox-mediated ROS production at least in part through association with p22phox.

Fig. 7

Tks5 and p22phox can associate. (A) Schematic of Tks5, p47phox, and the Tks5 truncation mutant 390. (B) Cotransfection of Tks5 and p22phox. 293T cells transfected with the indicated plasmids were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies shown. Top, immunoprecipitation and immunoblotting of Tks5; bottom, p22phox in whole-cell lysates; and middle, immunoblot of Tks5 immunoprecipitates with p22phox antibody. (C) Tks5 mutants containing point mutations in the ligand-binding surface of each SH3 domain (M1 to M5) were tested for p22phox association as in (B). (D) Wild-type and P156Q versions of p22phox were tested for their ability to bind Tks5. (E) Tks5 and p22phox mutants were tested in combination for the ability to coimmunoprecipitate. The relative intensity of the p22phox bands is shown in each case at the bottom of the corresponding lane (pixel intensity, arbitrary units) as calculated with Odyssey software. Numbers on the left of each panel show the position of the corresponding molecular weight markers.

The effect of invadopodia-localized ROS

One effect of ROS, particularly hydrogen peroxide, is to transiently inhibit the catalytic activity of some tyrosine, dual-specificity, and lipid phosphatases through conversion of the sulfhydryl group of the catalytic cysteine residue to sulfenic acid (70). Because many of the tyrosine-phosphorylated proteins in invadopodia are Src substrates, we hypothesized that their phosphorylation might be ROS sensitive. To test this possibility, we incubated Src-3T3 cells with DPI overnight, then lysed cells, immunoprecipitated several Src substrates, and immunoblotted with antibodies against phosphotyrosine (Fig. 8A and fig. S6). Tyrosine phosphorylation of most of the proteins we tested, including cortactin, Stat3, p190RhoGAP, and Nck, was largely unaffected by DPI. In contrast, there was a marked reduction in the phosphotyrosine content of Tks5 and Tks4. Tyrosine phosphorylation of both Tks4 and Tks5 is necessary for invadopodia formation (57, 71); suggesting that ROS might act locally to promote invadopodia formation by regulating PTP activity. Consistent with this, we detected the tyrosine phosphatase PTP-PEST in invadopodia (Fig. 8B). We transfected Src-3T3 cells with a pool of siRNAs targeting PTP-PEST or with a scrambled control siRNA. PTP-PEST knockdown increased the number of rosettes per cell, compared to that in control cells (Fig. 8C). Rosette number varies from cell to cell, but in our experience usually does not exceed five per cell. To evaluate rosette number in the knockdown cells, we cotransfected cells with a fluorescent reporter oligo and evaluated the phenotype of reporter-positive cells. Of the 10 PTP-PEST siRNA–transfected cells we evaluated, eight had more than 20 invadopodia rosettes, compared to only one of nine cells transfected with nontargeting siRNA. These data suggest that PTP-PEST may play a role in the formation of invadopodia or the control of rosette formation or both.

Fig. 8

Effects of ROS on tyrosine phosphorylation and role of PTP-PEST in invadopodia formation. (A) Tyrosine phosphorylation of various Src substrates. Src-3T3 cells were incubated with DMSO or DPI, then lysed and immunoblotted whole-cell lysate (WCL) or immunoprecipitated (IP) with antibodies directed against phosphotyrosine (left and middle) and antibodies against the indicated proteins (right). Quantification of the relative phosphotyrosine concentrations by densitometry is shown in fig. S6. (B) Localization of PTP-PEST to invadopodia. Src-3T3 cells were co-stained with phalloidin (F-actin) and with a PTP-PEST antibody. (C) Knockdown of PTP-PEST increases invadopodia number. Src-3T3 cells were transfected with scrambled or PTP-PEST pooled siRNAs, along with a fluorescent reporter, and stained with phalloidin 72 hours later. In each case, the arrow indicates one cell positive for the reporter in a field of otherwise reporter-negative cells. Scale bars, 4 μm.

Discussion

We used chemical inhibition and siRNA-mediated p22phox knockdown to show that Nox-generated ROS are required for the formation and function of invadopodia, both in mouse fibrosarcoma cells and in human cancer cells. This prompted us to determine the expression of Nox family members in cancer cells. We focused these analyses on Nox 1, 2, 3, and 4. We did not analyze the expression of Nox5, Duox1, and Duox2 because these enzymes do not require p22phox for oxidant production (62, 63). Src-3T3 cells express Nox1, Nox3, and Nox4, but not Nox2. Of the human cancer cells, C8161.9 melanoma cells predominantly expressed Nox4, consistent with previous studies on melanomas (51), whereas SCC61 head-and-neck cancer cells expressed Nox1, Nox2, and Nox4. We found that Nox4 contributed to the invasive phenotype in Src-3T3 and C8161.9 cells. It was present in invadopodia, and its knockdown reduced invadopodia formation. Nox1 is the most abundant Nox in colon cancer cells and is localized to invadopodia in a Tks4-dependent manner (66). It is possible that different cell types require different Nox proteins, or that optimal invadopodia formation requires multiple Nox family members.

Tks5 knockdown was sufficient to reduce total ROS concentration in Src-3T3 and SCC61 cells, suggesting that the Nox system is the predominant source of ROS in these cells. These results further suggest that even though the organizer NoxO1 was present in both cell types, it did not make a major contribution to ROS abundance. It is possible that complexes involving NoxO1 in these cells are not active, or that Tks5 is present at a much higher relative abundance and therefore plays the dominant role in oxidant production. It is also possible that localization to invadopodia is critical for oxidant production, perhaps through recruitment of coactivators. In this regard, it may be important that the PX domain of Tks5, which is required both for functional invadopodia formation (56) and for oxidant production (66), is recruited to nascent sites of invadopodia formation through its association with PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) (55, 72). In contrast, the PX domain of NoxO1 associates preferentially with PI(4)P (phosphatidylinositol 4-phosphate), PI(5)P (phosphatidylinositol 5-phosphate), and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) and is found on several membranes (73). Regardless, our data implicate Tks5 directly in the Nox-mediated production of oxidants, which is in keeping with the observation that both Tks4 and Tks5 act as Nox organizers in reconstitution assays (66). Together, these findings are consistent with a role for the Tks adaptor proteins targeting ROS production to invadopodia.

Nox4 is required for the invasive behavior of Src-3T3 and C8161.9 cells, and the simplest model to account for its activity would be that a Nox4-p22phox complex bound to Tks5 generates ROS in invadopodia. However, Nox4 is not thought to require organizer proteins for oxidant production (67). Indeed, 293 cell cotransfection of Nox4 with Tks4 or Tks5 does not increase Nox4-mediated ROS production (66). However, we show here that transfection of Nox4 into melanoma cells increases total ROS concentration in a Tks5-dependent manner. Nox4 may be more sensitive to a requirement for organizer proteins in certain cell backgrounds, or it may have a higher affinity for the endogenous concentrations of Tks4 or Tks5 present in the 293 cells than Nox1. Alternatively, Tks5-dependent localization to invadopodia might be required to bring Nox4 into proximity with as yet unidentified activator proteins.

Nox-generated ROS may function directly in invadopodia. In particular, we found that ROS inhibition resulted in the selective inhibition of tyrosine phosphorylation of Tks4 and Tks5, but not other Src substrates in invadopodia. Tyrosine phosphorylation of these two proteins is required for functional invadopodia formation (57, 71), suggesting that ROS acts in a positive feedback loop to promote invadopodia formation. In this model, Tks5 and Tks4 are required for the focal generation of ROS, which promotes their tyrosine phosphorylation to stimulate invadopodia formation. We detected the PTPase PTP-PEST localized to the rosettes of invadopodia in Src-3T3 cells, and PTP-PEST has also been found in podosomes of osteoclasts (74). Furthermore, PTP-PEST knockdown increased the number of invadopodia rosettes in Src-3T3 cells. Control of PTP-PEST by NADPH oxidases in invadopodia would be consistent with previous studies showing its role in adhesion and migration and its regulation by Nox in endothelial cells (7577). In the future, it will be important to determine the oxidation state and substrates of PTP-PEST in invadopodia. Podosomes and invadopodia are dynamic structures, with a half-life of actin turnover in some cases of minutes. We speculate that invadopodia and podosome localization of PTPs may allow a cycle of transient activation and inactivation of their catalytic activity by ROS, causing their substrate proteins to cycle between phosphorylated and nonphosphorylated forms. This in turn could contribute to the turnover of the entire structure. It is possible that lipid phosphatases can also be reversibly regulated by ROS, and at least one phosphoinositide 5-phosphatase, synaptojanin, has been localized to invadopodia (13). Other mechanisms through which ROS might function in invadopodia formation is by direct or indirect activation of protein kinase C, Src family kinases, the MAPK signaling pathway, or MMPs, all of which promote podosome-invadopodia formation and function (5, 3638, 78).

There are several studies on the effects of ROS on cancer cell growth, survival, and invasion (3638); but few have focused directly on the possible roles of members of the Nox family. However, one study demonstrates the up-regulation of Nox4 in glioblastoma and shows that reduction in abundance enhances chemotherapy-induced apoptosis (79). Other studies describe similar findings in melanoma and pancreatic adenocarcinoma cells (51, 53). Likewise, Nox1 overexpression has been correlated with Ras mutation in colorectal cancers and is also an early event in the development of prostate cancer (47, 49, 80). Here, we link two key features of cancer cells that contribute to invasive behavior: the generation of ROS by NADPH oxidases and the formation and function of invadopodia. The invadopodia scaffold protein Tks5, which is required for tumor cell invasion in vitro and in vivo (18, 56), is a key player in this link. It participates directly in the focal generation of ROS, acts as a scaffold for the polymerization of the actin cytoskeleton, and coordinates the activity of pericellular proteases. We show here that the formation of podosomes in macrophages also depends on ROS. In the future, it will be important to determine whether the concerted action of Tks adaptor proteins and NADPH oxidases is also implicated in podosome formation and function.

Materials and Methods

Cell lines

Mouse Src-3T3 cells, RPMI-7951, C8161.9 human melanoma cells and Bt549 human breast cancer cells were grown as previously described (56). SCC61 human head and neck carcinoma cells (a gift from A. Weaver) were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (MediaTech) containing 20% fetal bovine serum (FBS; Hyclone) and hydrocortisone (0.4 μg/ml; Sigma) in 10% CO2. IC-21 mouse macrophages were grown in RPMI (MediaTech) containing 10% FBS in 5% CO2. B16-F10 mouse melanoma and human embryonic kidney (HEK) 293T cells were grown in DMEM high glucose containing 10% FBS and penicillin-streptomycin in 10% CO2. NuSerum I culture supplement is from BD.

DNA constructs

Overlapping PCR was used to generate constructs in which a single Flag epitope tag sequence was inserted downstream of the Tks5 or Tks5 deletion constructs. The vector used for the Flag Tks5 was pCMV-Tag 4B. For Myc-tagged Tks5 and Tks5 point mutations, we used the pRK5 plasmid. Point mutations in the different SH3 domains of Tks5 (M1 = W118A, M2 = W260A, M3 = W441A, M4 = W827A, M5 = W1056A) were generated with QuikChange II site-directed mutagenesis (Stratagene). The p22phox wild-type and P156Q constructs were expressed from the pcDNA3.1 vector and were a gift from U. Knaus.

Materials

We used the following antibodies: antibodies against Nox4 (Abcam), Tks5 (1737 serum), p22phox (Santa Cruz), Myc tag (Affinity BioReagents), Stat3 (Santa Cruz), PTP-PEST (a gift from T. Mustelin), β-tubulin (Sigma), Tks4, cortactin, phosphotyrosine, RhoGAP p190, Nck, FAK, and MT1-MMP (all from Millipore). Antibody against Flag (M2 affinity gel) was from Sigma. Alexa Fluor 488– and Alexa Fluor 568–conjugated phalloidin, Alexa Fluor 568–streptavidin, and Oregon green–labeled gelatin were from Invitrogen. Biotin antibody against rabbit immunoglobulin G (IgG) was from Jackson Immuno-Research. Alexa Fluor 488 and Alexa Fluor 680 antibodies against mouse and rabbit IgG were from Invitrogen. Growth factor reduced Matrigel invasion chambers and control inserts were from BD. DPI was from Calbiochem, and luminol, horseradish peroxidase (HRP), 3-APN, and NAC were from Sigma. Crystal violet was from EMD.

RNA interference experiments

Lentiviral shRNA constructs targeting mNox4 and mTks5 were from Sigma (TRCN0000076587 and TRCN0000105734 clones, respectively). Lentiviral shRNA constructs targeting hp22phox were from Open Biosystems (clone TRCN0000064578). Lentiviruses were generated by the Burnham Institute for Medical Research Lentiviral Core Facility. The following siRNA constructs were purchased from Thermo Scientific: mp22phox (M-042497 pool and individual sequences), hp22phox (M-011020 pool and individual sequences), mNox4 (individual sequence J-058509-05), hNox4 (M-010194 pool), mTks5 (L-045361 pool), hTks5 (M-006657 pool), mPTP-PEST (M-042199 pool), and nontargeting siRNA#3 (D-001210-03-05) as a control. Reporter red fluorescent oligo (Block-iT) was purchased from Invitrogen. siRNA constructs were transfected with Lipofectamine 2000 (Invitrogen). The efficiency of RNA interference was monitored by RT-PCR or qPCR. Knockdown of Tks5 and PTP-PEST was evaluated by immunoblotting.

Fluorescence staining

Cells were fixed in 4% paraformaldehyde, blocked with 15% goat serum, and incubated with antibody against Nox4 (1:5000), PTP-PEST (1:5000), FAK (1:1000), MT1-MMP (1:500), Tks5 (1:1000), or cortactin (1:1000). Cells were washed and incubated with Alexa Fluor 488 antibody against mouse or rabbit (1:500) or, in the case of Nox4 and PTP-PEST, with biotin antibody against rabbit IgG (1:500) and streptavidin-Alexa Fluor 568 (1:500). F-Actin was stained with Alexa Fluor 488– or Alexa Fluor 568–conjugated phalloidin (1:500). For the experiment in Fig. 1C, phalloidin was titrated so that the staining of the untreated cells was in the linear range during automatic image capture, and then the inhibitor treated cells were imaged under the same exposure conditions. Nuclei were stained with Hoechst. Fluorescence microscopy images were obtained with a Zeiss Axioplan2 microscope equipped with a Zeiss Axiocam HRm CCD camera with Axiovision software. Confocal microscopy images were acquired with a Radiance 2100/AGR-3Q BioRad Multiphoton Laser Point Scanning Confocal Microscope equipped with argon and krypton lasers. Images were processed with Adobe Photoshop software.

Invadopodia formation and function assays

Src-3T3 or IC-21 cells growing on glass coverslips were treated for 6 to 8 hours with the indicated amounts of NAC or 3-APN or for 1 hour with the indicated amounts of DPI. Src-3T3 cells were transfected on glass coverslips and processed for immunofluorescence 48 to 60 hours after transfection. Human cancer cells were transfected at 50% confluence, replated 24 or 48 hours after transfection onto unlabeled gelatin-coated coverslips in growth medium containing 10% NuSerum supplement and processed for immunofluorescence after 15 to 20 hours. Lentivirus-infected cells were assayed 4 to 5 days after infection. Quantification of invadopodia was performed on phalloidin-stained samples on at least 15 randomly chosen fields representing around 150 total cells per experimental point. Src-3T3 cells containing at least one complete rosette of invadopodia were scored as positive. C8161.9 cells containing F-actin–rich invadopodia were scored as positive. Total cell numbers were calculated by scoring number or nuclei on the same field.

Invadopodia function assays were performed as described (81) with some modifications. For DPI-treated Src-3T3, cells were plated onto 0.2% labeled gelatin in 10% FBS-containing DMEM and allowed to attach for 1.5 hours before treatment. Cells were processed 5.5 hours after treatment. For DPI treatment of human cancer cell lines, cells were plated onto 0.2% labeled gelatin in 2.5% FBS-containing DMEM and allowed to attach for 2 hours before treatment with DPI. Cells were processed after 14 to 16 hours. For RNA interference experiments, labeled gelatin was used at 1 mg/ml and cells were incubated in 1% FBS-containing DMEM. Src-3T3 cells were processed after 16 to 18 hours and human cells after 48 hours. Quantification of gelatin degradation activity was performed on at least 15 randomly chosen fields, representing a minimum of 200 total cells scored per experimental point. Quantification of the degradation area per field was performed with ImageJ software, and the percent of degraded area per field was normalized to the number of cells on this field.

Motility and invasion assay was conducted in Matrigel invasion chambers as described (56) except that 50 to 100,000 cells were used in each assay. NAC or DPI was added to the cells at the moment of plating on the upper chamber inset.

Measurement of ROS

The luminol-based chemiluminescence assay was performed on attached cells. Cells (104 per well) were plated onto a 96-well tissue culture–treated white plastic plate (BD) and incubated overnight at 37°C. Cells were washed twice with Hanks’ balanced salt solution (HBSS) (Invitrogen) and incubated in HBSS containing 250 μM luminol and 1 U HRP. Chemiluminescence was immediately measured on a Veritas microplate luminometer (Turner Biosystems). For data normalization, a replica plate prepared in parallel in a standard 96-well tissue culture plate was processed for crystal violet staining. Cells were washed with PBS and incubated with a solution containing 0.4% crystal violet in 30% methanol for 20 min at room temperature (RT), washed three times with distilled water, and allowed to dry. Dye was extracted with 1% SDS for 30 min (RT) and optical density was measured on a plate reader. ROS measurements were expressed as relative light units (RLU) normalized to optical density units (OD).

The 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) ROS fluorescent probe was used to detect endogenous ROS concentrations. For microscopy, 105 cells were plated onto glass-bottomed six-well plates (MatTek) and incubated overnight at 37°C. Cells were washed with HBSS and incubated with 25 μM carboxy-H2DCFDA (Invitrogen) in HBSS for 45 min at 37°C in the dark. Cells were washed three times with HBSS, incubated in HBSS containing 1% FBS, and imaged immediately. Imaging was performed with an Inverted TE300 Nikon Wide Field Fluorescence Microscope equipped with Nomarski (DIC) optics, ASI 2000, and Cooled Color CCD SPOT RT Camera (Diagnostic Instruments). Images were taken with a neutral density filter and processed with Adobe Photoshop. For analysis of ROS concentrations by FACS, cells were washed three times with HBSS and incubated in HBSS containing carboxy-H2DCFDA (25 μM) for 30 min at 37°C. Cultures were then harvested with trypsin-EDTA (1 min) and resuspended in HBSS containing 10% FBS. Subsequently, cells were centrifuged for 5 min at 350g, resuspended in 250 μl of HBSS, and analyzed. The flow cytometry data set was acquired with an unmodified FACSort (BD Biosciences, San Jose). DCFDA fluorescence was detected from 515 to 545 nm.

Tks5-p22phox association assay

HEK293T cells growing in 10-cm dishes were transfected with 7 to 12 μg of DNA constructs using Lipofectamine 2000 according to the manufacturer’s instructions and harvested after 18 hours. Before lysis, cells were treated with 1 μM cytochalasin D for 1 hour, washed with PBS, trypsinized, and then lysed in ice-cold buffer B [30 mM Hepes (pH 7.6), 200 mM NaCl, 0.5 mM EDTA, 0.4% NP-40] supplemented with standard protease inhibitors, as well as the phosphatase inhibitors β-glycerophosphate (50 mM) and sodium orthovanadate (100 μM). Protein extracts were precleared with protein A-PLUS agarose beads (Sigma) for 2 hours at 4°C. The precleared extracts were incubated with beads that were blocked with 5% BSA. Lysates were subjected to immunoprecipitation with 20 μl of antibody against FLAG M2 affinity gel or 20 μl A-PLUS agarose incubated with 2 μl of antibody against Myc tag (9E10) for 2 hours at 4°C. Immunoprecipitates were subsequently washed four times with buffer B containing 250 mM NaCl and once with PBS. Proteins were eluted by heating the beads at 95°C in sample buffer, separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted with the specified antibodies.

Immunoprecipitation

Subconfluent Src-3T3 cells were incubated overnight with 0.5 μM DPI or the same volume of DMSO. Fourteen to 16 hours after treatment, cells were lysed in CHAPS-based standard lysis buffer containing protease and phosphatase inhibitors for 45 min at 4°C. Total proteins (500 to 1000 μg) were incubated for 1 to 2 hours 4°C with the corresponding antibody and 1 hour with protein A or protein G agarose beads. The product was analyzed by SDS-PAGE and standard immunoblotting conditions. Images were acquired with an infrared Odyssey Imager (LI-COR Biosciences). Processing and densitometry were performed with Adobe Photoshop.

Acknowledgments

We thank G. Bokoch and D. Gianni for helpful discussions and suggestions, U. Knaus for advice and p22 reagents, T. Mustelin for antibodies to PTP-PEST, and A. Weaver for SCC61 cells. We thank Y. Altman, J. Russo, and E. Monosov for help with ROS measurements. We are especially grateful to E. Azucena, P. Bromann, and M. Peterman for their help in the early stages of this project. Research in the Courtneidge laboratory was supported by the National Cancer Institute and the Mathers Foundation.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/2/88/ra53/DC1

Table S1. The expression of Nox family members in cancer cells.

Fig. S1. Characterization of DPI-treated Src-3T3 cells.

Fig. S2. p22 knockdown in Src-3T3 cells.

Fig. S3. Nox4 knockdown in Src-3T3 cells.

Fig. S4. Knockdown of Nox components in human cancer cells.

Fig. S5. Knockdown of Tks5 and ROS production.

Fig. S6. Relative phosphotyrosine levels of Src substrates.

References and Notes

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