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Carcinogenesis. 2009 December; 30(12): 2109–2116.
Published online 2009 October 20. doi:  10.1093/carcin/bgp251
PMCID: PMC2792316

Abl interactor 1 regulates Src-Id1-matrix metalloproteinase 9 axis and is required for invadopodia formation, extracellular matrix degradation and tumor growth of human breast cancer cells


Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.


Tumor cell metastasis is a multi-step process involving the loss of cell adhesion, increase in cell motility and invasion, as well as active intravasation and extravasation through the lymphatics or blood vessels. To achieve metastasis, tumor cells must remodel their cytoskeleton to form specialized structures for adhesion, invasion and directional movement. In many metastatic cancer cell lines, a specialized adhesive/invasive structure, called an invadopodium, has been observed and found to occur in good correlation with the invasive phenotype of the cancer cells (14). Inhibition of invadopodia assembly by knocking down the expression of the key regulatory proteins results in a significant reduction of invasive activities of breast cancer cells (58).

Similar to other cell-matrix contact structures, such as focal adhesions, invadopodia appear as membrane-associated F-actin-rich complexes whose assembly involves active actin polymerization/depolymerization and recruitment of adhesion molecules (4,911). However, invadopodia can be distinguished from classical adhesion structures by their ability to degrade extracellular matrix (ECM). Consistent with this functional property, matrix metalloproteinases (MMPs), the enzymes for ECM degradation, are often found in association with invadopodia. It has been reported that the membrane-bound type 1 metalloproteinase (MT1-MMP, also named MMP-14), MMP-2, MMP-9 and a disintegrin and metalloproteases are recruited and enriched in invadopodia (7,1215). The ability to recruit adhesion molecules and ECM degradation enzymes makes invadopodia special structures capable of ‘sticking to ECM and digging a hole’. Moreover, given the importance of ECM degradation enzymes in the regulation of tumor angiogenesis and the tumor microenvironment (16), it is possible that invadopodia enriched with ECM degradation enzymes may play a critical role in tumor progression.

The mechanism by which tumor cells regulate invadopodia formation remains largely unknown. It has been shown that members of the Src family of non-receptor tyrosine kinases play a key role in regulation of invadopodia assembly (1722). Src has been shown to interact with adhesion molecules as well as the regulatory molecules important for regulation of cytoskeleton remodeling. Src is also involved in the regulation of ECM degradation enzymes. More recently, it has been reported that Src plays a critical role in regulation of the inhibitor of differentiation protein 1 (Id1), a basic helix-loop-helix (HLH) protein implicated in regulation of gene transcription (23,24). Unlike most HLH transcription factors, which contain a site-specific DNA-binding domain, Id1 does not possess a DNA-binding domain. It can, however, form inactive heterodimers with intact HLH transcription factors and, by doing so, function as a dominant-negative regulator of HLH transcription factors (23). Increased expression of Id1 has been shown to associate with decreased cell differentiation and enhanced cell proliferation (23). Recently, it has been shown that the Id1 plays a key role in regulation of MMP-9 expression in Bcr-Abl-positive leukemic cells (25).

In addition to the Src signaling, actin polymerization has also been shown as a critical step for invadopodia assembly (4,11,26). Proteins involved in actin nucleation, such as the members of the Wiskott–Aldrich syndrome protein (WASP) family and Arp2/3, were found enriched in invadopodia (11,26). WASP family proteins, which consist of WASP, neural Wiskott–Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous protein (WAVE) 1, 2 and 3, are regulated by the small GTP-binding proteins Rac and Cdc 42 (27,28). WASP family proteins also interact with many other regulatory molecules including Abl interactor 1 (Abi1) (2831). For example, WAVE proteins form a complex with Abi1, Nck-associated protein 1 (Nap1), specifically Rac-associated (Sra) protein and hematopoietic stem progenitor cell 300 (Hspc 300) (30,31). In this complex, Abi1 plays a central role in holding the complex together, whereas Sra provides a binding site for active Rac (31,32). The micromolecular complex (Abi–WAVE complex) regulates initiation of actin polymerization in response to Rac activation (31). In addition to WASP family proteins, Abi1 also binds to a variety of other molecules, including those involved in the signal transductions of small GTP-binding protein Rac and PI3 kinase (3336). The ability to interact with diverse signaling pathways places Abi1 at a central position in the signaling network that regulates cell motility and proliferation. Deregulation of the Abi1 pathway in tumor cells has been reported (3741), and in vitro studies support a role of this pathway in cancer cell migration and proliferation (37,40,41). However, it remains unclear whether the Abi1 pathway contributes to tumor progression in vivo and how Abi1 functions in tumor cells. Given the importance of Abi1 in the regulation of actin cytoskeleton remodeling, we investigated the possibility that this pathway is involved in the assembly of invadopodia in metastatic tumor cells. We report here that Abi1 is found in the invadopodia and is required for the formation of invadopodia in the metastatic human breast cancer cell line, MDA-MB-231. Significantly, the knockdown of Abi1 expression in MDA-MB-231 cells inhibited the Src-Id1-MMP-9 pathway and impeded tumor growth in xenograft mouse model.

Materials and methods

Cell culture and transfection

The MDA-MB-231 cells were obtained from American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin in a humidified air, 5% CO2 atmosphere.

To test the role of Src tyrosine kinase in the regulation of invadopodia formation, MDA-MB-231 cells were starved in serum-free DMEM medium for 24 h. The Src tyrosine kinase inhibitor, PP2, or equivalent volume of dimethyl sulfoxide as a control was then added to a final concentration of 10 μM. After 8 h of pre-treatment, FBS was added to a final concentration of 10%, and cells were incubated at 37°C in a humidified 5% CO2 atmosphere for additional 16 h. At the end of the incubation, cells were fixed and stained for fluorescence microscopy analysis. To determine the role of Src in the regulation of Id1 and MMP-9 expression, 2 × 105 MDA-MB-231 cells were grown in six-well plate in DMEM containing 10% FBS for overnight in a 37°C, 5% CO2 incubator. The cells were then washed twice with phosphate-buffered saline (PBS) and incubated in the same incubator with 1 ml serum-free DMEM for 24 h in the presence or absence of 10 μM PP2. At the end of incubation, the media were collected, concentrated and analyzed by gelatin zymography analysis. The cells were harvested for western blot analysis and an aliquot of cells were counted by trypan blue exclusion test for cell viability. Under this condition, >90% cells treated with PP2 are viable.

Lipofectamine-mediated transfection of MDA-MB-231 cells was performed following manufacturer's instructions (Invitrogen, Carlsbad, CA). Cells were plated in six-well plates 24 h prior to transfection and 4 μg of plasmid DNA was used for each transfection. To knockdown the expression of Abi1, a MSCV-based pSM2 retroviral vector expressing the short hairpin RNA (shRNA) that specifically targets Abi1 transcripts (targeting sequences: 5′-GGTGCAATCATTTATGTTA-3′) and a control pSM2 vector expressing non-silencing shRNA were purchased from Open Biosystems (Huntsville, AL) and used for stable transfection of MDA-MB-231 cells. Forty-eight hours after transfection, the stable transfectants were selected by puromycin (1 μg/ml). The individual puromycin-resistant clones were picked in 3–4 weeks. These clones were analyzed by western blot for Abi1 expression and the clones that show dramatic reduction in Abi1 expression were chosen for further studies.

To analyze the subcellular localization of Abi1 in MDA-MB-231 cells and to test the effect of overexpression of Abi1 on MMP9 production, two MSCV retroviral vectors encoding either green fluorescence protein (GFP)-Abi1 fusion protein or GFP alone, as described previously (41), were used for both transient and stable tansfections. In transient experiment, 48 h after transfection, the cells were either lysed and subjected to western blot analysis or, for subcellular localization studies, fixed in 4% paraformaldehyde in PBS for 10 min and subjected to fluorescence microscopy analysis. The stable transfectants were selected and isolated as described for Abi1-knockdown transfectants.

Antibodies and reagents

The rabbit anti-Sra polyclonal antibodies were generated in conjunction with Affinity BioReagents (Golden, CO) using the peptide with sequences corresponding to human Sra-1 1192–1203 (DGKDEIIKNVPLKKM) as the antigen. The preparation of rabbit polyclonal antibodies against Abi1 has been described previously (38,42). The polyclonal antibodies against N-WASP, WAVE2 and c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against cortactin (Catalog #05-180) and Nap1 (Catalog #07-515) were obtained from Upstate Biotechnology (Lake Placid, NY). The antibodies against phospho-Src family (Tyr 416) and MMP-9 were purchased from Cell Signaling Technology (Danvers, MA) and Millipore (Billerica, MA), respectively. The monoclonal anti-β-actin antibody and the protease inhibitor cocktail were purchased from Sigma (Saint Louis, MO). The monoclonal antibody against mouse/human Id1 was purchased from Biocheck (Foster City, CA). The Alexa-conjugated phalloidin and secondary antibodies were purchased from Molecular Probes (Eugene, OR).

Immunocytochemistry and fluorescence microscopy

The cells cultured on fibronectin-coated coverslips were fixed in 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 8 min. The non-specific binding was blocked by incubation of the coverslips with 3% bovine serum albumin in PBS, and this was followed by incubation with primary antibodies in 1% bovine serum albumin/PBS. After extensive washing with PBS, cells were incubated with appropriate Alexa-conjugated secondary antibodies. To visualize F-actin structures and nuclei, the cells were stained with Alexa-conjugated phalloidin and 4′,6-diamidino-2-phenylindole, dihydrochloride, respectively, as described previously (34). The slides were examined under a Nikon Eclipse TE2000-U fluorescence microscope, and the images were captured and analyzed using Nikon NIS Elements imaging software.

In vitro ECM degradation assay

MDA-MB-231 cells stably transfected with control and Abi1 shRNA were seeded onto coverslips that were coated with fluorescein isothiocyanate (FITC)-conjugated gelatin as well as with fibronectin for an in vitro ECM degradation assay, as described previously (43). Briefly, glass coverslips were coated overnight with 100 μg/ml FITC-labeled gelatin in PBS and fixed with 0.5% glutaraldehyde. After washing six times with PBS, the coverslips were incubated with 50 μg/ml fibronectin at room temperature for 1 h. Coverslips were then washed once with 70% ethanol/PBS and once with medium. The MDA-MB-231 cells were then added to the coverslips and incubated at 37°C in an incubator with 5% CO2 for up to 24 h. Cells were fixed, permeabilized and incubated with 1% bovine serum albumin to block non-specific staining and the F-actin was stained with Alexa-conjugated phalloidin. The coverslips were examined using a Nikon Eclipse TE2000-U fluorescent microscope. The images were captured and analyzed, and the percentage of total area that was degraded was calculated using Nikon NIS Elements imaging software.

Cell migration and proliferation assays

To examine and compare cell migratory abilities, the MDA-MB-231 cells transfected with retroviral vectors expressing control shRNA and Abi1 shRNA were starved for 24 h in serum-free DMEM prior to harvest. The cells were then harvested, and 5 × 104 cells were added to the fibronectin-coated inserts in 24-well transwell plates containing 0.6 ml DMEM + 10% FBS in the bottom chambers. After incubating 24 h at 37°C in a CO2 incubator, the cells on the top of each insert were removed by wiping with a cotton swab, and the cells on the other side of the insert membrane were fixed, stained and counted under a microscope. At least six fields were counted for each insert.

Cell proliferation assay was performed as described (40). Briefly, the MDA-MB-231 cells stably transfected with control and Abi1 shRNA were plated at a density of 1 × 104 per well of six-well plates in triplicate in DMEM containing 10%FBS. Cells were harvested at days 1, 2, 3 and 4, respectively, and counted.

Biochemical assays

Immunoprecipitation and western blot analyses were performed as described previously (41). Briefly, cells were lysed in lysis buffer (20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH7.2, 150 mM NaCl, 1% Triton X-100 and 10% glycerol) and incubated with appropriate antibodies bound to Sepharose beads. The immunoprecipitates were separated on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, transferred to nitrocellulose and immunoblotted with appropriate primary and secondary antibodies.

The gelatin zymography analysis was performed, as described previously (44), to determine the activities of secreted gelatinases in cell culture supernatants. In brief, the MDA-MB-231 cells transfected with retroviral vectors expressing control and Abi1 shRNA were plated at a density of 2 × 105 cells per well in six-well plates. After overnight incubation, the cells were washed twice with PBS, and 1 ml of serum-free medium was added to each well. After a 24 h incubation, the conditioned medium was collected, concentrated to 40–50 μl with Amicon Ultra centrifugal filter devices (Millipore) and mixed with non-reducing sample buffer (250 mM Tris, pH 6.8, 25% glycerol, 10% SDS and 0.01% bromophenol blue). The proteins in the conditioned media were separated on an 8% SDS–polyacrylamide gel electrophoresis gel containing 0.25% gelatin as a substrate. After electrophoresis, the gel was washed in 2.5% Triton X-100 to remove SDS and allow the proteins to renature. The gel was then incubated overnight at 37°C in developing buffer (50 mM Tris, pH 7.6 and 10 mM CaCl2). This was followed by staining with 0.5% Coomassie Blue R250 in 50% methanol/10% acetic acid and then sequential destaining in 50% methanol/10% acetic acid and in water. Clear zones of gelatin lysis against a blue background stain represented gelatinase activities.

Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) was performed using human MMP-9 primers (forward: 5′-CGGCTTGCCCTGGTGCAGT-3′, reverse: 5′-CGTCCTGGGTGTAG AGTCTCTCG-3′) and SYBR Green Master Mix (Applied Biosystems, Foster City, CA). Briefly, total RNAs were isolated from cells using RNeasy mini kit (QIAGEN, Valencia, CA) and the complementary DNAs were subsequently generated using SuperScript III First-strand Synthesis System (Invitrogen, Carlsbad, CA). The polymerase chain reactions (PCRs) began with 10 min at 95°C for AmpliTaq Gold activation, followed by 40 cycles at 95°C for 15 s for denature and then 60°C for 1 min for annealing/extension. The RT–PCR was performed on MyiQ single color real-time PCR detection system (Bio-Rad, Hercules, CA). Relative quantification was done using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous housekeeping transcript control (forward primer: 5′-AGTCAGCCGCATCTTCTT-3′, reverse primer: 5′-CGCCCAATACGACCAAAT-3′).

The result of real-time quantitative PCR was validated by traditional RT–PCR. Gene-specific primers for human MMP-9 (forward: 5′-TGGGCTACGTGACCTATGACAT-3′, reverse: 5′-GCCCAGCCCACCTCCACTCCTC-3′) were used for detection of MMP-9 transcript. PCR products were separated on a 1% agarose gel and imaged under UV light. The expression of GAPDH (forward primer: 5′-GGGAGCCAAAAGGGTCATCATCTC-3′, reverse primer: 5′-CCATGCCAGTGAGCTTCCCGTTC-3′) was chosen as an internal control.

In vivo tumorigenesis studies

All animals were maintained in compliance with the guidelines of the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center under approved protocols. Six- to eight-week-old female NOD–SCID mice were used for all studies. For the mammary fat pad orthotopic xenograft model, 1 × 106 cells in 50 μl PBS were injected into the mammary fat pad of 6-week-old female NOD–SCID mice and the growth of primary tumors was monitored bi-weekly by measuring tumor diameters using calipers. Twelve weeks after tumor cell inoculation, the mice were killed and the size and weight of each tumor was determined. Tumor tissues were either fixed in 10% buffered formalin solution or embedded in optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA) and examined histologically.

Statistical analyses

Descriptive statistics were generated for all quantitative data with the presentation of mean ± SD. The statistical significance of comparisons between experimental groups was tested using the Student's t-test with Microsoft Excel software and P < 0.05 was considered as statistically significant.


Abi1 is found in the invadopodia of MDA-MB-231 human breast cancer cells

Abi1 was expressed in the metastatic breast cancer cell line, MDA-MB-231, and was found to complex with Sra, Nap1, WAVE2 and N-WASP (Figure 1A). As reported previously (5,8,15), MDA-MB-231 cells develop invadopodia that are enriched with F-actin (Figure 1B and E). Abi1 was found in the F-actin-enriched invadopodia, as demonstrated by indirect immunofluorescence staining using anti-Abi1-specific antibody (Figure 1B). To confirm that Abi1 locates in invadopodia, three additional experiments were performed. First, because the invadopodia are characterized by their ability to degrade local ECM, we examined whether the punctate structures stained positively for Abi1 are also the sites where the active ECM degradation occurs. MDA-MB-231 cells grown on the slide coated with FITC-conjugated gelatin produce dark holes/areas (Figure 1C, gelatin). These dark holes/areas are generated due to active degradation of FITC-gelatin by invadopodia and therefore are often associated with invadopodia (15). As shown in Figure 1C, Abi1 was found in the ECM degradation sites. Second, we constructed a retroviral vector expressing GFP-tagged Abi1. This expression vector was introduced into MDA-MB-231 cells and the expression of GFP–Abi1 in stably transfected cells was examined by western blot (Figure 1D). The expression of GFP–Abi1 was also demonstrated by fluorescence microscopy analysis (Figure 1E). As shown in Figure 1E, GFP–Abi1 was found in the F-actin-enriched puncta similar to those described as invadopodia (5,8,15,4547). These F-actin-enriched puncta are probably invadopodia because they were found in the ECM degradation sites (Figure 1F). Our third experiment is to determine if the GFP–Abi1 colocalizes with markers for invadopodia. Cortactin has been shown to locate to the core area of the invadopodia and has been frequently used as a marker for invadopodia in many tumor cells including MDA-MB-231 (6,8,21,44,47). We therefore examined whether GFP–Abi1 colocalizes with cortactin in MDA-MB-231 cells. The MDA-MB-231 cells expressing GFP–Abi1 were analyzed by indirect immunofluorescence staining with a specific anti-cortactin antibody followed by fluorescence microscopy. As described previously (48), the cortactin displays a punctate distribution in MDA-MB-231 cells in a pattern similar to F-actin-enriched puncta (Figure 1G). GFP–Abi1 was found to colocalize with cortactin (Figure 1G).

Fig. 1.
Abi1 is found in invadopodia in MDA-MB-231 cells. (A) Expression and complex formation of Abi1 with Sra, Nap1, WAVE2 and N-WASP. The MDA-MB-231 cells were lysed and immunoprecipitated (IP) with pre-immune serum (Pre-IP) or anti-Abi1-specific antibodies ...

Abi1 silencing inhibited invadopodia formation and ECM degradation of MDA-MB-231 cells

To determine if Abi1 is required for assembly of invadopodia in MDA-MB-231 cells, we knocked down the expression of Abi1 in these cells by shRNA-mediated gene silencing. A retroviral vector expressing Abi1 shRNA was introduced into MDA-MB-231 cells, and its effect on Abi1 expression was examined. The expression of Abi1 was reduced by ~80% in two individual clones (231kd1 and 231kd2) stably transfected by the retroviral construct expressing Abi1 shRNA compared with the cells transfected by the retroviral construct expressing scrambled shRNA (231 control; Figure 2A). In agreement with the previous reports (49), knockdown of the expression of Abi1 resulted in a reduction of WAVE proteins (data not shown). The decreased expression of Abi1 was also accompanied with a ~70% reduction of the invadopodia-positive cells (Figure 2B), a result consistent with a role for Abi1 in the regulation of invadopodia formation.

Fig. 2.
Knockdown of Abi1 expression in MDA-MB-231 cells impaired invadopodia formation and ECM degradation. (A) Knockdown of Abi1 expression in MDA-MB-231 cells. Total lysates of the MDA-MB-231 cells transfected with the plasmids expressing a control shRNA (231 ...

Previous studies have shown that invadopodia function as specialized invasive structures that degrade ECM (4). Since the knockdown of Abi1 expression in MDA-MB-231 cells resulted in a reduction in invadopodia formation, we set out to determine if it also affects the ability of MDA-MB-231 cells to degrade fluorescence-labeled gelatin layers on slides. As shown in Figure 2C, the gelatin degradation activity of MDA-MB-231 Abi1-knockdown (Abi1KD) cells was reduced markedly compared with that of MDA-MB-231 control cells.

Abi1 is involved in the regulation of MMP-9 expression in MDA-MB-231 cells

Degradation of ECM requires MMP activity. The finding that the knockdown of Abi1 expression impaired the ability of MDA-MB-231 to degrade ECM prompted us to examine and compare the activities of MMPs produced by MDA-MB-231 Abi1KD cells and MDA-MB-231 control cells. The conditioned medium collected from control and Abi1KD MDA-MB-231 cells was analyzed by gelatin zymography. Consistent with previous reports (50), the gelatin zymography detected a major enzyme band with a molecular mass ~92 kDa and a minor enzyme band with molecular masse ~72 kDa, respectively, in the conditioned medium from control MDA-MB-231 cells (Figure 3A). Based on previous reports (50), it is probably that the 92 kDa band represents pro-MMP-9, whereas the 72 kDa band represents MMP-2. Remarkably, the 92 kDa enzyme, presumably MMP-9, is reduced in conditioned medium from Abi1KD cells compared with that from control cells (Figure 3A). Thus, the data suggest a role of Abi1 in regulation of MMP-9 production in MDA-MB-231 cells. In line with this notion, we found that, by both RT–PCR and quantitative RT–PCR analyses, the MMP-9 messenger RNA level is markedly reduced in Abi1-knockdown cells compared with control cells (Figure 3B and C). Furthermore, expression of a transgene encoding for GFP–Abi1 in MDA-MB-231 cells resulted in an increase of MMP-9 protein level and enzyme activity in conditioned media (Figure 3D). Collectively, these data show that Abi1 is involved in the regulation of MMP-9 expression in MDA-MB-231 breast cancer cells and this regulation occurs, at least in part, at messenger RNA level.

Fig. 3.
Abi1 is involved in the regulation of MMP-9 expression. (A) Abi1 gene silencing decreased MMP-9 production. The conditioned media collected from equal numbers of control (231 Ctrl) and Abi1-knockdown cells (231kd1 and 231kd2) were concentrated, separated ...

Activation of Src tyrosine kinase is essential for the regulation of Id1/MMP-9 expression and formation of invadopodia in MDA-MB-231 cells

It is widely believed that invadopodia formation is regulated by Src tyrosine kinases (15,20,21). Gautschi et al. (24) reported recently that the Src signaling is also crucial for regulation of the expression of Id1, a basic HLH transcription factor implicated in the control of MMP-9 gene expression. To determine the mechanism by which Abi1 regulates invadopodia formation and MMP-9 expression, we first investigated the role of the Src signaling in regulation of these cellular processes in MDA-MB-231 cells. Src is expressed and activated in MDA-MB-231 cells, as evidenced by western blot analysis using the antibodies that recognize either c-Src or active Src, which is phosphorylated on tyrosine 416 (Figure 4A). Treatment of MDA-MB-231 cells with PP2, a pharmacological inhibitor of Src protein tyrosine kinase, inhibited Src activation and this was accompanied by a reduction of Id1 expression (Figure 4A). In addition, the treatment of MDA-MB-231 cells with PP2 decreased MMP-9 production (Figure 4B). Furthermore, the PP2 treatment resulted in a 65% reduction of invadopodia formation in MDA-MB-231 cells (Figure 4C). Taken together, these data strongly suggest that the Src signaling plays a key role in regulation of Id1 and MMP-9 expression as well as invadopodia formation in MDA-MB-231 cells.

Fig. 4.
Src tyrosine kinase is required for regulation of Id1 and MMP-9 expression. (A) Pharmacological inhibition of Src activity downregulates Id1 expression. MDA-MB-231 cells treated with 10 μM Src-inhibitor PP2 (PP2), dimethyl sulfoxide as a control ...

Abi1 silencing inhibited the Src-Id1 signaling and impaired MDA-MB-231 cell migration and proliferation

Next, we examined whether Abi1 silencing in MDA-MB-231 cells affects Src-Id1 signaling. As shown in Figure 5A, Src activation is inhibited in Abi1KD cells compared with control cells (Figure 5A, left panel), suggesting that Abi1 is essential for Src activation in MDA-MB-231 cells. Furthermore, knockdown of Abi1 expression resulted in a significant reduction of Id1 expression in MDA-MB-231 cells (Figure 5A, right panel). Thus, these data provide the first evidence supporting a role of Abi1 in regulation of Src-Id1 axis in human breast cancer cells.

Fig. 5.
Abi1 knockdown in MDA-MB-231 cells inhibited Src activation, Id1 expression, cell migration and proliferation. (A) Knockdown of Abi1 expression inhibited Src activation and Id1 expression. Left panel shows MDA-MB-231 control and Abi1KD cells were lysed ...

Src interacts with a network of intracellular pathways and is a key regulator of many cellular functions including cell migration and proliferation. The ability of Abi1 to regulate Src activation in MDA-MB-231 cells suggests that this pathway may play an important role in breast cancer cell migration and proliferation. To test this, we performed the Boyden chamber migration assay as well as cell proliferation assay. As shown in Figure 5B, Abi1 silencing inhibited MDA-MB-231 cell migration. Cell proliferation assay reveals that knockdown of Abi1 expression also impaired proliferation of MDA-MB-231 cells (Figure 5C).

Knockdown of Abi1 expression slowed tumor growth of MDA-MB-231 breast cancer cells

To determine if Abi1 is required for tumor growth in vivo, the MDA-MB-231 control and Abi1KD cells were injected into the mammary fat pad in NOD–SCID mice. Tumor growth was monitored bi-weekly. Tumor became evident in 4 weeks in mice implanted with control MDA-MB-231 cells and grew to a mean size of 1.35 ± 0.1 cm in diameter in 12 weeks (Figure 6A). In contrast, no tumor was detected in mice implanted with Abi1KD cells in 4 weeks and by 12 weeks, tumor grew to a mean size of 0.68 ± 0.2 cm in diameter. We also examined tumor weights 10–12 weeks post-implantation. As shown in Figure 6B, the MDA-MB-231 control cells developed tumors with a mean weight of 0.44 ± 0.08 g. In contrast, the MDA-MB-231 Abi1KD cells developed much smaller tumors with a mean weight of 0.06 ± 0.05 g (P < 0.01).

Fig. 6.
Knockdown of Abi1 expression markedly slowed tumor growth of MDA-MB-231 breast cancer cells. (A) Tumor development in murine mammary fat pad xenograft model. MDA-MB-231 control and Abi1KD cells were implanted in the mammary fat pads of NOD–SCID ...


In this study, we identified a novel function of Abi1 in the regulation of invadopodia formation and Src-Id1-MMP-9 pathway in MDA-MB-231 human breast cancer cell line. We show that Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of Abi1 gene expression in these cells impaired the formation of invadopodia and inhibited Src activation and Id1/MMP9 expression. More importantly, we found that the suppression of Abi1 expression in MDA-MB-231 breast cancer cells resulted in a drastic inhibition of tumor development in xenograft mouse model. Knockdown of Abi1 expression not only reduced ECM degradation, cell migration and proliferation in vitro but also slowed tumor growth in vivo. These findings indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as an important therapeutic target for treatment of human breast cancer.

Invadopodia are actin-based membrane protrusions in which the multiple cellular processes such as branched actin assembly, cell signaling and adhesion and secretion of proteases spatially converge to promote remodeling of the ECM (4). Accordingly, blockade of the formation of invadopodia in cancer cells is often associated with defects in ECM degradation, cell migration and invasion. This was the case in MDA-MB-231 Abi1KD cells, where the downregulated Abi1 expression was accompanied by decreased invadopodia formation and significant reductions in ECM degradation and cell migration. There are at least two possible pathways that may be utilized by Abi1 to regulate invadopodia formation. First, Abi1 may regulate invadopodia formation by promoting actin polymerization, a cellular process absolutely required for assembly of invadopodia in tumor cells (4). Recent studies have shown that Abi1 exerts its function by forming a complex with WASP family proteins and thereby regulating the actin nucleation promoting activity of these proteins (2931). Interestingly, although both WAVE and N-WASP are expressed in MDA-MB-231 cells and interact with Abi1 (Figure 1A), we found that only N-WASP colocalizes with invadopodia (X.S. and Z.D., data not shown). This is in agreement with a previous report by Yamaguchi et al. (11) in which N-WASP, but not WAVE, was found to colocalize with invadopodia and was required for invadopodia formation in metastatic breast cancer cells. Taken together, these studies suggest that Abi1 may affect invadopodia formation in part by regulating the actin nucleation activity of N-WASP.

Second, our studies also suggest that Abi1 may regulate invadopodia formation through Src tyrosine kinase pathway. In addition to actin polymerization, the Src family of tyrosine kinases appears to play a central role in the regulation of invadopodia formation in MDA-MB-231 cells (4,15,21). Consistently, inhibition of Src kinase activity in these cells resulted in an inhibition of invadopodia formation. We found that the knockdown of Abi1 expression in MDA-MB-231 cells also inhibited Src activation. Thus, the decreased invadopodia formation correlated well with an inhibition of Src kinase activity in MDA-MB-231 Abi1KD cells, suggesting that the inhibitory effect of Abi1 knockdown on invadopodia formation may be mediated in part by the Src pathway.

Our studies provide the first evidence that the Abi1 signaling is critical for the regulation of Id1/MMP-9 expression in MDA-MB-231 cells. It is most probably that Abi1 regulates the expression of Id1 and MMP-9 through the Src signaling because: (i) Src has been shown to positively regulate Id1 expression in a panel of lung, breast, prostate and colon cancer cell lines and a Src-responsive regulatory element has been identified in the Id1 promoter region (24); (ii) pharmacological inhibition of Src decreased the expression of Id1 and MMP-9 (Figure 4) and (iii) Abi1 silencing inhibited not only Id1 and MMP-9 expression but also Src activation (Figure 5). On the other hand, Id1 itself is a regulator of gene transcription. Earlier studies by Nieborowska-Skorska et al. (25) demonstrated that Id1 regulates MMP-9 gene expression by promoting transactivation of the MMP-9 promoter. Based on these data, we propose that a signaling flow from Abi1 to Src-ID1-MMP-9 axis may exist and function in MDA-MB-231 cells.

Collectively, our studies support a hypothetical model in which Abi1 positively regulates Src activity through a mechanism yet to be discovered (Figure 7). The activated Src may then up-regulate the gene expression of Id1, which in turn enhances the expression of MMP-9. In addition, the activated Src may promote the formation of invadopodia. Furthermore, Abi1 may also contribute to invadopodia formation by forming a complex with WASP family proteins to enhance actin polymerization, which is absolutely required for invadopodia assembly. Id1 and MMP-9 have been shown as key players in tumor growth and progression (16,23,51). The role of invadopodia in regulation of ECM degradation, tumor cell migration and invasion has also been demonstrated (20). Therefore, by regulating invadopodia formation and Src-Id1-MMP-9 axis, Abi1 may play an important role in breast cancer progression. Thus, the work described here warrants further investigation on the role of Abi1 in breast cancer progression.

Fig. 7.
A hypothetical model of the Abi1 signaling that regulates invadopodia formation and the Src pathway in MDA-MB-231 breast cancer cells.


National Institutes of Health/National Cancer Institute (R01 CA094921 and R21 CA133597 to Z.D.); National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (K01 DK067191 to Y.T.); China Scholarship Council to C.Z.


We thank Dr Candace Myers for critical review of the manuscript.

Conflict of Interest Statement: None declared.



Abl interactor 1
Dulbecco's modified Eagle's medium
extracellular matrix
fetal bovine serum
fluorescein isothiocyanate
green fluorescence protein
inhibitor of differentiation protein 1
matrix metalloproteinase
neural Wiskott–Aldrich syndrome protein
phosphate-buffered saline
polymerase chain reaction
reverse transcription–polymerase chain reaction
sodium dodecyl sulfate
short hairpin RNA
Wiskott–Aldrich syndrome protein
WASP family verprolin-homologous protein


1. Chen WT. Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J. Exp. Zool. 1989;251:167–185. [PubMed]
2. Kelly T, et al. Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins. J. Cell. Physiol. 1994;158:299–308. [PubMed]
3. Buccione R, et al. Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell. Biol. 2004;5:647–657. [PubMed]
4. Gimona M, et al. Assembly and biological role of podosomes and invadopodia. Curr. Opin. Cell Biol. 2008;20:235–241. [PubMed]
5. Hashimoto S, et al. Requirement for Arf6 in breast cancer invasive activities. Proc. Natl Acad. Sci. USA. 2004;101:6647–6652. [PubMed]
6. Onodera Y, et al. Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities. EMBO J. 2005;24:963–973. [PubMed]
7. Seals DF, et al. The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell. 2005;7:155–165. [PubMed]
8. Nam JM, et al. CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery. EMBO J. 2007;26:647–656. [PubMed]
9. Badowski C, et al. Paxillin phosphorylation controls invadopodia/podosomes spatiotemporal organization. Mol. Biol. Cell. 2008;19:633–645. [PMC free article] [PubMed]
10. Marchisio PC, et al. Vinculin, talin, and integrins are localized at specific adhesion sites of malignant B lymphocytes. Blood. 1988;72:830–833. [PubMed]
11. Yamaguchi H, et al. Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J. Cell Biol. 2005;168:441–452. [PMC free article] [PubMed]
12. Nakahara H, et al. Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion. Proc. Natl Acad. Sci. USA. 1997;94:7959–7964. [PubMed]
13. Bourguignon LY, et al. CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. J. Cell. Physiol. 1998;176:206–215. [PubMed]
14. Chen WT, et al. Specialized surface protrusions of invasive cells, invadopodia and lamellipodia, have differential MT1-MMP, MMP-2, and TIMP-2 localization. Ann. N. Y. Acad. Sci. 1999;878:361–371. [PubMed]
15. Artym VV, et al. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function. Cancer Res. 2006;66:3034–3043. [PubMed]
16. Deryugina EI, et al. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 2006;25:9–34. [PubMed]
17. Hauck CR, et al. v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion. J. Biol. Chem. 2002;277:12487–12490. [PubMed]
18. Mizutani K, et al. Essential role of neural Wiskott-Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res. 2002;62:669–674. [PubMed]
19. Berdeaux RL, et al. Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J. Cell Biol. 2004;166:317–323. [PMC free article] [PubMed]
20. Courtneidge SA, et al. The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb. Symp. Quant. Biol. 2005;70:167–171. [PubMed]
21. Bharti S, et al. Src-dependent phosphorylation of ASAP1 regulates podosomes. Mol. Cell. Biol. 2007;27:8271–8283. [PMC free article] [PubMed]
22. Destaing O, et al. The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol. Biol. Cell. 2008;19:394–404. [PMC free article] [PubMed]
23. Sikder HA, et al. Id proteins in cell growth and tumorigenesis. Cancer Cell. 2003;3:525–530. [PubMed]
24. Gautschi O, et al. Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer. Cancer Res. 2008;68:2250–2258. [PubMed]
25. Nieborowska-Skorska M, et al. Id1 transcription inhibitor-matrix metalloproteinase 9 axis enhances invasiveness of the breakpoint cluster region/abelson tyrosine kinase-transformed leukemia cells. Cancer Res. 2006;66:4108–4116. [PubMed]
26. Linder S, et al. Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages. Proc. Natl Acad. Sci. USA. 1999;96:9648–9653. [PubMed]
27. Bompard G, et al. Regulation of WASP/WAVE proteins: making a long story short. J. Cell Biol. 2004;166:957–962. [PMC free article] [PubMed]
28. Takenawa T, et al. The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat. Rev. Mol. Cell Biol. 2007;8:37–48. [PubMed]
29. Innocenti M, et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nat. Cell Biol. 2005;7:969–976. [PubMed]
30. Eden S, et al. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature. 2002;418:790–793. [PubMed]
31. Innocenti M, et al. Abi1 is essential for the formation and activation of a WAVE2 signalling complex. Nat. Cell Biol. 2004;6:319–327. [PubMed]
32. Gautreau A, et al. Purification and architecture of the ubiquitous Wave complex. Proc. Natl Acad. Sci. USA. 2004;101:4379–4383. [PubMed]
33. Innocenti M, et al. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol. 2003;160:17–23. [PMC free article] [PubMed]
34. Scita G, et al. EPS8 and E3B1 transduce signals from Ras to Rac. Nature. 1999;401:290–293. [PubMed]
35. Innocenti M, et al. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J. Cell Biol. 2002;156:125–136. [PMC free article] [PubMed]
36. Fan PD, et al. Abl interactor 1 binds to sos and inhibits epidermal growth factor- and v-Abl-induced activation of extracellular signal-regulated kinases. Mol. Cell. Biol. 2000;20:7591–7601. [PMC free article] [PubMed]
37. Leng Y, et al. Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation. Proc. Natl Acad. Sci. USA. 2005;102:1098–1103. [PubMed]
38. Dai Z, et al. Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway. Genes Dev. 1998;12:1415–1424. [PubMed]
39. Taki T, et al. ABI-1, a human homolog to mouse Abl-interactor 1, fuses the MLL gene in acute myeloid leukemia with t(10;11)(p11.2;q23) Blood. 1998;92:1125–1130. [PubMed]
40. Wang C, et al. Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion. Mol. Cancer Res. 2007;5:1031–1039. [PubMed]
41. Li Y, et al. Bcr-Abl induces abnormal cytoskeleton remodeling, beta1 integrin clustering and increased cell adhesion to fibronectin through the Abl interactor 1 pathway. J. Cell Sci. 2007;120:1436–1446. [PMC free article] [PubMed]
42. Courtney KD, et al. Localization and phosphorylation of Abl-interactor proteins, Abi-1 and Abi-2, in the developing nervous system. Mol. Cell. Neurosci. 2000;16:244–257. [PubMed]
43. Bowden ET, et al. Invadopodia: unique methods for measurement of extracellular matrix degradation in vitro. Methods Cell Biol. 2001;63:613–627. [PubMed]
44. Clark ES, et al. Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res. 2007;67:4227–4235. [PubMed]
45. Chen WT, et al. Membrane proteases as potential diagnostic and therapeutic targets for breast malignancy. Breast Cancer Res. Treat. 1994;31:217–226. [PubMed]
46. Kelly T, et al. Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases. Clin. Exp. Metastasis. 1998;16:501–512. [PubMed]
47. Bowden ET, et al. An invasion-related complex of cortactin, paxillin and PKCmu associates with invadopodia at sites of extracellular matrix degradation. Oncogene. 1999;18:4440–4449. [PubMed]
48. Bowden ET, et al. Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells. Exp. Cell Res. 2006;312:1240–1253. [PubMed]
49. Kunda P, et al. Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr. Biol. 2003;13:1867–1875. [PubMed]
50. Olmeda D, et al. SNAI1 is required for tumor growth and lymph node metastasis of human breast carcinoma MDA-MB-231 cells. Cancer Res. 2007;67:11721–11731. [PubMed]
51. Bergers G, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2000;2:737–744. [PMC free article] [PubMed]

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