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

Abstract

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.

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-IP), as indicated. The immunoprecipitates, along with total cell lysate (TCL), were separated on 8% SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes and subjected to western blot (WB) analysis using the indicated antibodies. (B) Abi1 colocalizes with invadopodia. The MDA-MB-231 cells were incubated with anti-Abi1 specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (green). The cells were then counterstained with Alexa-conjugated phalloidin to visualize F-actin (red). Abi1 location in invadopodia is shown by an arrow in the merged image (merge); bar: 10 μm. (C) Abi1 is found in ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were incubated with Abi1 antibody and subsequently stained with Alexa-conjugated secondary antibody (red). Degradation is indicated as dark patches within the fluorescent monolayer (upper panel). Abi1 is found in degraded area, as shown by arrows. (D) Expression of GFP and GFP–Abi1 in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with plasmids expressing GFP alone or GFP-tagged Abi1, as indicated. Total cell lysate containing 50 μg protein was separated on SDS–PAGE and analyzed by western blot using indicated antibodies. (E) GFP–Abi1 is found in invadopodia-like structures. The MDA-MB-231 cells expressing GFP–Abi1 were counterstained with Alexa-conjugated phalloidin (red). The subcellular localization of GFP–Abi1 and F-actin was examined by fluorescence microscopy. GFP–Abi1 was found in F-actin-enriched puncta, as indicated by arrow. (F) F-actin-enriched puncta colocalize with ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were stained with Alexa-conjugated phalloidin (red) and examined by fluorescence microscopy. Degradation is indicated as dark patches within the fluorescent monolayer. The F-actin-enriched puncta locate in the degraded area, as indicated by arrows; bar: 10 μm. (G) GFP–Abi1 colocalizes with cortactin. The MDA-MB-231 cells expressing GFP–Abi1 were incubated with anti-cortactin-specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (red). The subcellular localization of GFP–Abi1 and cortactin was examined by fluorescence microscopy. The colocalization of Abi1 with cortactin is shown by the merged image (merge). The arrows indicate Abi1, invadopodia and their colocalization; bar: 10 μm.

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 ctrl) or Abi1 shRNA (231kd1 and 231kd2) were separated on SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and subjected to western blot analysis using anti-Abi1-specific antibodies (upper panel), as well as anti-β-actin antibody (lower panel). (B) Abi1 knockdown impaired invadopodia formation. The MDA-MB-231 cells transfected with control shRNA (231 ctrl) and Abi1 shRNA (231 Abi1kd) were stained with Alexa-conjugated phalloidin and examined by fluorescence microscopy. The cells with invadopodia structures were counted, calculated against total cells in three randomly picked areas and presented in the lower panel as mean percentage ± SD. The upper panel shows a representative field visualized by fluorescence microscopy. The invadopodia are indicated by arrows; bar: 10 μm (C) Knockdown of Abi1 expression decreased ECM degradation ability of MDA-MB-231 cells. The MDA-MB-231 cells transfected with control shRNA (231 ctrl) and Abi1 shRNA (231 Abi1KD) were tested over a period of 48 h for their abilities to degrade a thin layer of FITC-gelatin coated onto glass coverslips. Degradation is indicated as dark patches within the fluorescent monolayer (upper panel). The degraded area was calculated against fluorescent monolayer in three randomly picked fields and presented as the mean percentage ± SD of the digested area (lower panel).

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 on 8% SDS–polyacrylamide gel electrophoresis (PAGE) gel containing 0.25% gelatin and subjected to zymography analysis. The molecule markers were indicated. Two enzyme bands, presumably pro-MMP-9 and pro-MMP-2 based on their molecular masses, are indicated. (B) Decreased MMP-9 messenger RNA (mRNA) level in Abi1KD cells. Total RNAs isolated from control and Abi1KD cells were subjected to RT–PCR analysis using primers specific for Abi1 (upper panel) or GAPDH (lower panel) as a control. (C) Analysis of MMP-9 mRNA levels in control and Abi1KD cells by real-time quantitative RT–PCR. Relative quantification was done using GAPDH mRNA level as a control. (D) Ectopic expression of GFP–Abi1 in MDA-MB-231 cells increases MMP-9 production. Upper panel shows the conditioned media collected from equal numbers of MDA-MB-231 cells expressing GFP as a control (GFP Ctrl) or GFP–Abi1 were concentrated, separated on 8% SDS–PAGE gel containing 0.25% gelatin and subjected to zymography analysis. Molecular mass marker and presumable pro-MMP-9, based on its molecular mass, are indicated. Middle panel shows the cells used for collection of conditioned media, as described above, were lysed and analyzed by western blot to show the expression of GFP–Abi1 and, as a loading control, β-actin as indicated. Lower panel shows the conditioned media from equal numbers of MDA-MB-231 cells expressing either GFP alone or GFP–Abi1, as indicated, were collected, concentrated and subjected to western blot analysis using specific antibody to MMP-9. To ensure that the conditioned media were collected from equal number of cells, the cell lysates were analyzed by western blot using β-actin antibody. Molecular mass marker and presumable pro-MMP-9 are indicated.

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 (DMSO) or no addition (MB231) were lysed and analyzed by western blot using anti-Src p416 antibody (anti-pSrc), which recognizes the active form of Src phosphorylated at tyrosine residue 416. The membrane was then stripped and reprobed with the antibody that recognizes total Src protein (anti-cSrc). The cell lysates were also analyzed by western blot using specific antibodies to Id1 (anti-Id1) or β-actin (anti-actin) as a loading control. (B) Inhibition of Src activity decreased MMP-9 production. The conditioned media from cells treated as in (A) were concentrated and subjected to zymography analysis. The 92 kDa enzyme band, presumably MMP-9 based on its molecular mass, was indicated. (C) Invadopodia formation is inhibited by Src-inhibitor PP2. Upper panel shows MDA-MB-231 cells were treated with 10 μm PP2 or dimethyl sulfoxide, as indicated. The cells were then stained with Alexa-conjugated phalloidin and examined by fluorescence microscopy. The arrows indicate invadopodia structures. The percentage of the cells with invadopodia structures in three randomly picked areas were calculated and presented as mean ± SD in lower panel; bar: 10 μm.

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 and immunoprecipitated with anti-Src antibody. The immunoprecipitates were separated on SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to western blot analysis using anti-Src p416 antibody that recognize active Src, as indicated by an arrowhead. The membrane was then stripped and reprobed with the antibody that recognizes total Src protein, as indicated by an arrow. Right panel shows control and Abi1KD cells were lysed, separated on SDS–PAGE and subjected to western blot analysis using indicated antibodies. (B) Abi1 knockdown inhibited MDA-MB-231 cell migration. A total of 5 × 10⁴ MDA-MB-231 control and Abi1KD cells were tested for their migratory abilities using the Boyden chamber migration assay. The y-axis represents the percentage of the cells that migrated to the other side of insert membranes and is the mean ± SD of triplicate inserts. The upper panel shows a representative field of the insert membranes with migrated cells; *P < 0.01. (C) Abi1 gene silencing reduced cell proliferation in MDA-MB-231 cells. A total of 1 × 10⁴ control and Abk1KD cells were grown in six-well plate for indicated time periods. Cells were harvested at the end of the time period and cell numbers in each well were counted and expressed as mean ± SD of triplicate wells.

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 mice (five mice per group), and tumor growth was monitored bi-weekly by measuring tumor diameters. (B) Tumor development was also examined by measuring tumor weights 10–12 weeks post-implantation. The y-axis represents the mean ± SD of tumor weight; *P < 0.01. A representative picture of tumors from the mice injected with control (ctrl) and Abi1KD cells (kd) is shown. The data represent one of three independent experiments.

Fig. 7.

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