Nischarin regulates focal adhesion and Invadopodia formation in breast cancer cells

Human breast cancer MDA-MB-231, MDA-MB-231 Nischarin, MCF7 shScramble, MCF7 GFP Nisch, MCF7 shNisch, WT-Nischarin mouse embryonic fibroblasts (MEFs), HET-Nischarin MEFs, Null Nischarin MEFs, HEK293, Cos7, and NIH3T3 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) at 37 °C, 5% CO₂ supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Waltham, MA). MCF10A cells were grown in DMEM/F12 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Waltham, MA). For wound healing assays the cells were seeded on a 6-well plate and allowed to attach for 12 h. Then, a wound was created with a 200 μl tip and imaging was done at various time points. Percentage of wound closure was obtained using Image J. For live cell imaging, different subsets of 10,000 MDA-MB-231 cells were plated onto gelatin-coated 35-mm glass bottom dishes (2D) (MatTek) or NIH3T3 fibroblast-derived matrices (3D) overnight. The cells were cultured at 37 °C with 5% CO2 using a Live Cell Environmental Chamber (NEUE Group, Ontario, NY). Five random acquisition points were pre-determined for each experimental well, each pre-set location was photographed every 10 min for a period of 10 h using an Olympus IX71 microscope. Cell position and average speed were determined and calculated using Slidebook software.

Twenty human breast cancer surgical specimens, thirteen malignant and seven non-cancerous tissues were obtained as frozen tissue sections from the Southern Division (Birmingham, AL), Eastern Division (Philadelphia, PA), and Mid-Western Division (Columbus, OH) of the Cooperative Human Tissue Network.

Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen, Carlsband, CA). cDNA was generated from 2μg of RNA using the Invitrogen/Applied Biosystems/ABl High Capacity cDNA Reverse Transcriptase Kit (Carlsband, CA). For gel detection, samples were run on a 3% agarose gel. Quantitative real-time PCR (qRT-PCR) was performed with 2× SYBR green mix (Roche, Basel, Switzerland) according to the manufacturer’s instructions. ΔCt was calculated as Ct (sample)-Ct (βactin or GAPDH). Relative expression was calculated as ∆∆Cq (2^-ΔCt(sample)/ 2^-ΔCt (control cell line).

The invasion assays were performed using Tanswell invasion chambers (Corning, Corning, NY) coated with 10 μg/ml Fibronectin (bottom) and matrigel (top). MCF10A cells were transferred to the top of the chamber with the indicated media. After incubation for 24 h at 37 °C in an atmosphere containing 5% CO₂, invaded cells on the lower surface were stained with crystal violet stain and counted under a light microscope.

For Nischarin-Cortactin binding experiments, HEK293 cells were transiently transfected with 4 μg of Flag-Cortactin and Myc-Nischarin using Lipofectamine® Reagent 2000 (Invitrogen, Carlsband, CA). As a control, 3 μg Myc-βgal was cotransfected with Flag-Cortactin. Forty-eight hours later, cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 0.3 M sucrose, 100 mM KCl, and 1 mM CaCl) with protease inhibitors (1 mM PMSF, 2 μg/ml aprotinin, and 5 μg/ml leupeptin). The lysates were immunoprecipitated with appropriate antibodies and pulled down by Protein G Sepharose beads (Uppsala, Sweden). Proteins were then immunoblotted for Myc and Flag antibodies.

Antibodies and dilutions were used as follows: mouse monoclonal anti-Nischarin (BD Biosciences, San Diego, CA; 1:1000), rabbit polyclonal anti-human Fibronectin (Sigma, St. Louis, MO; 1:1000), rabbit polyclonal anti-Cortactin (Santa Cruz, Dallas, TX; 1:3000), rat anti-Flag (Stratagene, San Diego, CA; 1:5000), mouse monoclonal anti-human C-myc (BioXCell, West Lebanon, NH; 1:2500), mouse monoclonal anti-Paxillin (BD Biosciences, San Diego, CA; 1:5000), mouse monoclonal anti-HIC5 (BD Biosciences, San Diego, CA; 1:5000), mouse monoclonal anti-CRP1 (BD Biosciences, San Diego, CA; 1:1000), mouse monoclonal anti-MCAM (BD Biosciences, San Diego, CA; 1:1000), mouse monoclonal anti-Zyxin (Santa Cruz, Dallas, TX; 1:100), rabbit polyclonal anti-VASP (Sigma, St. Louis, MO; 1:100), rabbit polyclonal anti-Integrin α5 (EMD Millipore, Billerica, MA; 1:100) and mouse monoclonal anti-Vinculin (Sigma, St. Louis, MO; 1:5000). The SNAKA51 antibodies (1:100) were a gift from Kenneth Yamada.

To prepare coverslips for matrix deposition, 2 ml of 0.2% gelatin was added to the coverslips and incubated for 1 h at 37 °C. The gelatin was aspirated and 2 ml of cold PBS was added to wash the coverslip. After aspiration of the PBS, 2 ml of 1% glutaraldehyde was added and incubated at 37 °C for 30 min. The coverslips were washed three times for 5 min each with cold PBS. 2 ml of 1 M ethanolamine was then added and incubated for 30 min at room temperature. The three PBS washes were repeated again to remove all traces of ethanolamine. 2 ml of matrix media (DMEM + 10% FBS + P/S + Anti-Anti +50μg/ml ascorbic acid) was then added. Cells were plated on the coverslips in 2 ml of matrix media. These cells were cultured for 24 h. After 24 h, the medium was aspirated and replaced with fresh matrix medium every day for six days. On day 6, the medium was carefully aspirated and the matrix was decellularized. Curvelet transform plus FIRE (CT-FIRE) algorithm was used to analyze fiber width and number.

Coverslips were cleaned with 20% nitric acid and coated with poly-L-lysine in a 24-well plate. The poly-L-lysine was fixed with 0.5% glutaraldehyde before adding the Oregon green Gelatin (Life Technologies, Calsbad, CA). Residual reactive groups were quenched by sodium borohydride. 4 X 10³ cells were plated and incubated at 37 °C. Images were visualized by confocal microscopy.

The tumor suppressor Nischarin has previously been shown to inhibit cell migration in breast cancer cells [14]. To confirm that Nischarin prevents cancer cell migration, we performed a time course wound healing assay with our previously published MDA-MB-231 and MDA-MB-231 Nischarin cells [11] for up to 24 h (Fig. 1a). Quantitation of the percentage of wound closure at 0, 3, 6, 9, 12, and 24 h revealed that MDA-MB-231 Nischarin cells have an overall reduced cell migration and the effect is more robust at 9 and 12 h (Fig. 1b). To further validate these findings, we explored whether Nischarin is still able to slow down the migration of cells attached in a 3D environment. We tracked live cell migration of cells seeded on a thin 2D gelatin layer or 3D fibroblast-derived matrices for a period of ten hours (Fig. 1c). MDA-MB-231 Nisch cells had a reduction in average speed on both 2D and 3D environments when compared to 231 cells (Fig. 1d). These experiments confirm our previously published work showing that Nischarin reduces cell migration.

Although previous findings showed that Nischarin exerts this migratory inhibition by interacting with other proteins, such as LIMK and PAK1 [11], the effects of these interactions on the entire FA machinery are unknown. FAs are the protein complexes that link the cell cytoskeleton to the ECM and their presence is necessary for cell attachment [15]. Tyrosine phosphorylation is a key event that occurs prior to the recruitment of other FA proteins [15]. To assess the effects of Nischarin on the FA machinery, we performed qRT-PCR and Western Blots of twelve key focal adhesion proteins. Out of twelve, we found five genes with consistently significant decreases in expression (Fig. 2). Paxillin is one of the first proteins to arrive at a nascent focal adhesion site [16]. Nischarin-expressing MDA-MB-231 and MCF7 cells have a significant reduction of Paxillin RNA and protein compared to Nisch-lacking cells (Fig. 2a-c). The ability of FA proteins to form protein-protein interactions is necessary for the formation of stable FAs. HIC-5 is a Paxillin-related protein that coordinates multiple protein-protein interactions in the early stages of FA development [15, 17]. MDA-MB-231 and MCF7 cells expressing Nisch have significantly less HIC-5 RNA and protein when compared to MDA-MB-231 cells (Fig. 2a-c).

In the intermediate stages of FA development, Actin filament bundling must occur in order for there to be a stable protrusion. Cysteine-rich protein 1 (CRP1) is a protein that regulates Actin filament bundling by binding to the Actin-bundling protein α-Actinin [18]. We also found reduced expression of these proteins in the presence of Nischarin (Fig. 2a-c). Finally, we assessed the expression of the Melanoma Cell Adhesion Molecule (MCAM), also known as, CD146, which is considered a marker for mesenchymal cells [19]. Nisch-expressing cells had a reduction in MCAM expression (Fig. 2a-c). To determine whether this decrease of FA protein expression is specific to cancer cells, we transiently transfected Cos7 cells (monkey kidney) with Myc or Myc-Nischarin and examined expression of the proteins. The presence of Nisch did not alter expression of any one of the FA proteins in Cos7 cells but only in the MDA-MB-231 cancer cells (Fig. 2d). These data demonstrate that the presence of Nischarin leads to a decrease in the expression of some FA proteins in cancer cells only.

Zyxin is a protein that concentrates along the Actin cytoskeleton and localizes at FA sites [20]. We previously determined the number of FAs in Nisch positive and negative cells. Mouse embryonic fibroblasts (MEFs) are widely used for quality visualization of FAs due to their complete mesenchymal nature. We stained WT and Null Nisch MEFs [21] with Zyxin and quantitated the number of FAs using CellProfiler. Null Nischarin MEFs had an increase in the number of FAs and percentage of area covered by FAs (Fig. 3a). These quantitated images support the data in Fig. 1 that showed the decrease of migration and FA protein expression in Nischarin-expressing cells.

The Ena/VASP family of proteins play an important role in cell migration, invasion and adhesion [22]. In fact, they are capable of binding the Integrin α5 (ITGA5) cytoplasmic tail similar to Nischarin [22, 23]. We examined VASP protein expression and found that its expression is reduced in Nischarin-expressing MDA-MB-231 cells (Fig. 3b). We determined that this is cancer cell specific since addition of Nisch to Cos7 cells does not affect VASP expression (Fig. 3c). To further assess the impact of Nischarin on the number of VASP-positive FAs, we stained MDA-MB-231 and MDA-MB-231 Nisch cells with VASP and used the CellProfiler software to count the number of VASP FAs per cell (Fig. 3d). MDA-MB-231 Nisch cells had a reduction in the number of VASP FAs and percentage of area covered by VASP positive FAs. To determine whether we see the same pattern in WT and Null Nischarin MEFs, we stained them with VASP and quantitated FA numbers using Cell Profiler. Null Nischarin MEFs had an increase in the number of VASP FAs and percentage of area covered by VASP positive FAs (Additional file 1: Fig. S1). Since Nisch and VASP both bind to Integrins, we next determined whether Nischarin binds to VASP to reduce its presence in FAs. Co-Immunoprecipitation experiments between Nischarin and VASP showed that there is no interaction between the two proteins (Additional file 1: Fig. S1B). These results demonstrate that while Nischarin and VASP both bind ITGA5, they do not interact but the presence of Nisch leads to a decrease in VASP-positive FAs.

We have established that Nisch regulates FA protein expression. Integrins are membrane proteins that connect the FA protein complexes to the extracellular matrix (ECM) [15]. We have previously shown that Nischarin binds to and inhibits the expression of ITGA5 [23]. To support our previous findings, we visualized whether this Nisch-mediated ITGA5 reduction is still present in FA sites. Vinculin is often used as a marker to detect Integrin-mediated cell-matrix adhesions by immunofluorescence [24, 25]. Next, to determine whether Nischarin inhibits targeting of ITGA5 to FA sites, we stained WT and Null Nisch MEFs with Vinculin (green) and ITGA5 (red) (Fig. 3e). Merged images of Vinculin and ITGA5 showed less colocalization of ITGA5 with FAs in WT MEFs when compared to Null MEFs (Fig. 3e). We further validated the expression of ITGA5 and activation of its regulator, FAK. Western Blotting demonstrated an increase in protein expression of ITGA5 and pFAK in Null Nisch MEFs (Fig. 3f). Cancer cells exhibit increased autophosphorylation of FAK at Y397 [26]. This activated FAK is responsible for initiating the FA signaling pathway, thus allowing Integrin activation. We have established that Nisch inhibits ITGA5 expression, and thus the whole Integrin α5β1 complex is reduced [11]. For the Nisch blot, the lower band present in Null MEFs reflects the 172 amino acid deletion that produces a Null Nisch phenotype [21]. The doublet band for HET MEFs reflects the normal allele (top) and the truncated allele (bottom) [21].

Furthermore, we identified the levels of active Integrin α5β1 by immunofluorescence using the conformation-dependent ITGA5 antibody SNAKA51 [27]. There was significantly less SNAKA51 fluorescence in 231 Nis cells, indicating less active ITGA5 (Fig. 3g). This is the first time we have been able to quantify the reduction in the amount active Integrin α5β1.

Finally, we assessed whether Nischarin alters the expression of other Integrins. We performed RT-PCR experiments that showed alterations in the expression of four other Integrins in the presence of Nischarin. Integrins α1, 4, and 7 levels were increased, while Integrin α2 was decreased in the presence of Nischarin (Fig. 3h). Taken together, we have established that Nischarin regulates the expression of multiple α-Integrins.

Integrins also stabilize invadopodia, which are protrusive cell-matrix adhesions that contain proteases for ECM degradation. In addition to proteases, these protrusions are enriched with proteins including, Cortactin, Tks4 and Tks5 [28]. To determine whether Nischarin affects invadopodia formation, we performed a gelatin degradation assay [29]. To compare degradative patterns between MDA-MB-231 and MDA-MB-231 Nischarin cells, we plated the cells on fluorescent gelatin-coated coverslips and allowed them to degrade the gelatin matrix. A greater number of MDA-MB-231 cells degraded the fluorescent substrate at both four and five hours of time (Fig. 4a-b). To determine whether this degradation also occurs on Fibronectin fibers, we stimulated NIH3T3 fibroblasts to produce Fibronectin-derived matrix (FDM). After decellularization, we plated WT- Nisch and Null-Nisch MEFs on the NIH3T3 FDMs to visualize matrix alterations. Seeding Null MEFs on the FDMs reduced the Fibronectin signal (Fig. 4c), suggesting that the Null MEFs have degraded the matrix. Taken together, Nischarin negative (Null-Nisch) cells degrade the ECM at a greater rate than WT cells.

To further asses this in vitro while still maintaining a physiologically realistic culture substrate, we generated fibroblast-derived Fibronectin-rich matrices from WT-Nisch and HET-Nisch MEFs. Fibroblasts were cultured for eight days in the presence of ascorbic acid to stimulate matrix production. At day 8, cells were decellularized and Fibronectin was detected on the fibers by confocal microscopy (Fig. 4d). WT-Nisch and HET-Nisch fibronectin fibers were quantitated by CT-FIRE and the analysis revealed that WT-Nisch fibers have a greater fiber number and fiber width than the Het-Nisch fibers (Fig. 4d). These data suggest that the HET cells that were seeded on the substrate were degrading the matrix.

Since the invadopodia are enriched with several secreted proteins, we explored the effects of secreted media from MDA-MB-231 and MDA-MB-231 Nisch cells on the invasion of moderately invasive MCF7 cells. Media from MDA-MB-231 cells increased the invasiveness of MCF7 cells, while addition of media from MDA-MB-231 Nisch cells reduced the percentage of MCF7 cells degrading the fluorescent gelatin matrix (Fig. 4e). To further explore this on non-invasive cells, we isolated media from MDA-MB-231 and MDA-MB-231 Nisch cells and cultured them with normal MCF10A cells to monitor invasion. MCF10A cells are normal, noncancerous breast epithelial cells that typically do not have the capacity to invade. Adding media from MDA-MB-231 cells significantly induced the invasiveness of MCF10A cells in transwell inserts (Fig. 4f). Culturing the cells with media from MDA-MB-231 Nisch cells significantly reduced the invasive capability of the cells. Next, we wanted to determine whether media from MDA-MB-231 Nisch cells reduced invadopodia formation. Similar to the transwell invasion assays, MCF7 cells cultured in 231 Nisch media had a reduction in the percentage of cells degrading the gelatin matrix (Fig. 4g). Taken together, these results suggest that Nisch prevents invadopodia formation, and media isolated from Nisch expressing cancer cells might have an altered protease profile.

Cortactin has been shown to be good marker for invadopodia [30] and its mRNA expression is increased in malignant human breast tissues (Fig. 5a). We investigated whether Nisch regulates formation of invadopodium through Cortactin. QRT-PCR experiments showed no significant difference in the expression of Cortactin mRNA in MDA-MB-231 versus MDA-MB-231 Nisch cells (Fig. 5b). Agarose gel visualization of Cortactin cDNA from MDA-MB-231 and MDA-MB-231 Nisch cells confirmed our findings of no significant differences (Fig. 5c). Western blot detection of Cortactin also confirmed that the protein expression was not altered by the presence of Nisch (Fig. 5d). Since we did not find any changes in Cortactin RNA or protein expression, we thought Nischarin might alter invadopodia formation by interacting with Cortactin. Coimmunoprecipitation experiments demonstrated that Nischarin does not interact with Cortactin (Fig. 5e). These data suggest that Nischarin uses another mechanism to prevent invadopodia formation.

Tks4 and Tks5 are adaptor proteins that are required for invadopodia formation [31]. RT-PCR of Tks4 and Tks5 showed a decrease in the expression of Tks4, but not Tks5 (Fig. 5f). During invadopodia formation, Tks5 recruits AFAP-110 to the future protrusion site [32]. We found decreased amounts of AFAP-110 in Nisch expression cells (Fig. 5g). Although the expression of, Cortactin, is not regulated by Nischarin, other invadopodium proteins seem to be affected.

Fourteen Matrix-Metallo Proteases (MMPs) have been noted to have increased expression in breast cancer tissue when compared to normal breast tissue [33]. To assess whether the presence of Nischarin in breast cancer cells affects MMPs, we performed reverse transcriptase PCR of several MMPs on MDA-MB-231 and MDA-MB-231 Nisch cells. We found reduced expression of MMPs 1, 2 and 9 in the Nischarin expressing cancer cells (Fig. 5h). Taken together, our findings suggest that the presence of Nischarin in breast cancer cells leads to a down regulation of MMPs 1, 2, and 9, which leads to a reduction in ECM degradation.