Research ArticleExperimental Studies

Role of ARHGAP24 in ADP Ribosylation Factor 6 (ARF6)-dependent Pseudopod Formation in Human Breast Carcinoma Cells

SHOTA UEHARA, KOJI SAITO, HISAYO ASAMI and YASUTAKA OHTA
Anticancer Research September 2017, 37 (9) 4837-4844;

Abstract

Background/Aim: The small GTPase ADP ribosylation factor 6 (ARF6) promotes carcinoma cell invasion and metastasis through remodeling of actin cytoskeleton and formation of pseudopod that is regulated by RAC. RHO GTPase activating protein 24 (ARHGAP24), a RAC-specific GTPase activating protein, binds to activated ARF6 and is recruited to the plasma membrane. The aim of the present study was to demonstrate if ARHGAP24 is involved in the ARF6-mediated formation of pseudopods in breast carcinoma cells. Materials and Methods: The formation of pseudopods induced by activated ARF6 was monitored using MDA-MB-231 human breast carcinoma cells. The effect of knockdown of endogenous ARHGAP24 by siRNA was examined. Results: Knockdown of ARHGAP24 in MDA-MB-231 carcinoma cells increased the lifespan of pseudopods to retract, which resulted in increased length of pseudopods induced by activated ARF6. ARHGAP24 required a binding site of ARF6 to achieve ARF6-dependent actin remodeling. Conclusion: ARHGAP24 may regulate pseudopod formation downstream of activated ARF6 in MDA-MB-231 human breast carcinoma cells.

Metastasis is a major clinical problem in cancer and abnormal cell migration and invasion is intimately involved in cancer metastasis (1, 2). Cancer cells extend actin-rich surface protrusions (pseudopods) to migrate and actin remodeling is essential for cell shape change and migration (3). RHO family small GTPases control actin cytoskeletal dynamics to induce pseudopod formation in cells. For example, RHOA promotes the formation of actin stress fibers and focal adhesions, RAC induces lamellae, and CDC42 stimulates the formation of filopodia (4-6). Previous studies have demonstrated altered expression of RHO GTPases and their regulators in various cancers (7).

The ADP ribosylation factor (ARF6) protein is a small GTPase of the RAS superfamily and is a component of signaling cascades promoting cancer invasion and metastasis (8-11). ARF6 down-regulates E-cadherin expression at cell–cell junctions and up-regulates recycling of β1 integrin (12, 13). Importantly, ARF6 contributes to cancer cell malignancy through regulation of formation of invadopodia and cancer cell migration (14, 15). ARF6 regulates endocytosis and recycling of plasma membrane proteins, and also regulates cortical actin cytoskeleton. Previous studies have demonstrated that ARF6-dependent actin remodeling is mediated by RAC (16-20).

ARHGAP24 (also called FilGAP) is a RHO GTPase-activating protein (GAP) and binds to actin filament crosslinking protein filamin A (21-31). The GAP activity of ARHGAP24 is specific for RAC. ARHGAP24 is phosphorylated by RHO-associated protein kinase (ROCK), an effector of RHOA, and recruited to plasma membranes by binding to activated ARF6 (25, 27). ARHGAP24 binds to ARF6 through its pleckstrin-homology (PH) domain and inactivates RAC as a downstream effector of ARF6 (25). We previously showed that ARHGAP24 may regulate carcinoma cell invasion and metastasis by controlling cell shape change during migration (23). Moreover, pathological studies have shown that expression level of ARHGAP24 may affect behavior of human B-cell lymphoma and astrocytoma (30, 31).

In this study, we examined the role of ARHGAP24 downstream of activated ARF6 in actin remodeling and cell shape in MDA-MB-231 human breast carcinoma cells.

Materials and Methods

Cell culture and transfection. MDA-MB-231 Human breast cancer cell line and HEK293 Human embryonic kidney cell line were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), and 50 units/ml penicillin/streptomycin at 37°C with 5% CO2. Cells were transfected with plasmid DNA for 24 h or ARHGAP24 siRNA for 48 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For co-transfection of plasmid DNA and ARHGAP24 siRNA, cells were first transfected with siRNA for 24 h and then co-transfected with plasmid DNA, followed by additional culture for 24 h. MDA-MB-231 cells were cultured on poly-L-lysine-coated coverslips. In some experiments, the cells were cultured on coverslips coated with collagen type I or fibronectin.

Antibodies and regents. Mouse anti-hepatitis A (HA) monoclonal (12CA5), anti-α-tubulin monoclonal and rabbit anti-FLAG polyclonal antibodies were purchased from Sigma-Aldrich. Mouse anti-ARF6 monoclonal antibody (3A-1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-green fluorescent protein (GFP) polyclonal antibody was purchased from MBL (Nagoya, Japan). Rabbit anti-ARHGAP24 polyclonal antibody was prepared as described previously (21). Secondary antibodies conjugated to Alexa Fluor 488 or 568 or Alexa Fluor 568-phalloidin (Invitrogen) and Hoechst 33258 (Dojido Laboratries, Kumamoto, Japan) were also purchased from commercial sources.

The pIRES2-AcGFP (Clonetech, Palo Alto, CA, USA) and pFLAG-CMV-6c (Sigma-Aldrich) plasmids encoding ARF6 Q67L were generated as follows; the ARF6 Q67L coding sequence was polymerase chain reaction (PCR)-amplified using HA-tagged ARF6 Q67L construct (pcDNA3-HA-ARF6 Q67L) (25) as a template and the PCR products were inserted into the pIRES2-AcGFP or pFLAG-CMV-6c vector using the EcoRI site and sequenced. MDA-MB-231 cells transfected with ARF6 Q67L-IRES-AcGFP plasmid produce both ARF6 Q67L and GFP proteins and the transfected cells are easily identified using GFP expression as a marker.

The FLAG-tagged ARHGAP24 constructs [wild type (WT), ΔPH, R39C, see below] and HA-tagged ARHGAP24 siRNA-resistant construct (rKD) were as described previously (23,25). The ARHGAP24 ΔPH (rKD) coding sequence was amplified by PCR and cloned into the pCMV5-HA vector using the EcoRI site. The HA-tagged ARHGAP24 R39C (rKD) construct was generated by introducing point mutations using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) as described previously (23). siRNA oligonucleotide duplexes targeting human ARHGAP24 was purchased from Invitrogen. The targeting sequence was: 5’-CAGUGGUAAAUUACAACCUCCUCAA-3’ (nt 770-794). This siRNA was found to significantly reduce the expression of endogenous ARHGAP24 in MDA-MB-231 cells (23).

Immunofluorescence and microscopic observation. MDA-MB-231 cells plated on coverslips were washed with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS at room temperature for 10 min. The fixed cells were then permeabilized with 0.5% Triton X-100 in PBS for 10 min, then incubated with blocking buffer [10% Blocking one (Nacarai tesque, Kyoto, Japan) in PBS] for 30 min, and immunostained with primary antibodies in blocking buffer for 1 h at room temperature. Cells were then washed with PBS and incubated with Alexa Fluor dye-labeled secondary antibodies (Invitrogen) in blocking buffer for 1 h at room temperature. For visualization of F-actin and nuclei, Alexa Fluor 568-palloidin and Hoechst 33258 were added with secondary antibodies. Cells were washed with PBS and mounted on glass slides then covered with AquaPolyMount (Polysciences, Warrington, PA, USA) and coverslipped. Cells were observed under an Olympus IX73 fluorescence microscope with 40× or 60× objective (Olympus, Tokyo, Japan). Images were acquired by HCImage Live (Hamamatsu photonics, Hamamatsu, Japan) and analyzed by ImageJ (National Institutes of Health, NIH, Bethesda, MD, USA) to measure the length of pseudopods and the elongation factor of cells (the ratio of maximum length to width).

Time-lapse microscopy. MDA-MB-231 cells were seeded on glass bottom dishes and transfected with pIRES2-AcGFP plasmid encoding constitutively activated ARF6 Q67L in the presence or absence of ARHGAP24 siRNA. The cells were examined under Olympus IX81 fluorescence microscope with 10× objective, and images were acquired at 37°C every 10 min for 12 h and analyzed by MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) to measure the length and lifespan of pseudopods.

Association of ARHGAP24 and ARF6. Association of ARHGAP24 and ARF6 was determined as described previously (25). Briefly, HEK293 cells were transfected with the HA-ARF6 Q67L and FLAG-ARHGAP24 constructs (WT, ΔPH, R39C). 24 h later, the cells were washed twice with PBS, suspended in 500 μl of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1.0% Nonidet P-40, protease inhibitor cocktails (Sigma-Aldrich), and 1 mM Phenylmethylsulfonyl fluoride, and homogenized. The cell lysates were precleared, and supernatant fluid was subjected to immunoprecipitation with an M2 anti-FLAG antibody coupled to Sepharose beads (Sigma-Aldrich) to precipitate transfected ARHGAP24. Immunoprecipitates were washed five times with the lysis buffer, and bound protein was detected by western blot analysis using anti-FLAG antibody for ARHGAP24 or anti-HA antibody for ARF6.

Statistical analysis. The statistical significance was accessed by two-tailed unpaired Student's t-test or Welch's t-test (see figure legends). Differences were considered to be statistically significant at a p<0.05. Error bars (standard error of the mean, S.E.M) and p-values were determined from the results of at least three experiments.

Results

Depletion of ARHGAP24 increases the length of pseudopods induced by activated ARF6 in MDA-MB-231 cells. ARF6 controls shape change and actin remodeling of carcinoma cells through regulation of RAC activity. Several RAC guanine nucleotide exchange factors were shown to be activated downstream of ARF6 (17, 20), and we showed that RACGAP activity of ARHGAP24 is regulated downstream of activated ARF6 (25, 27). Constitutively activated ARF6 Q67L stimulates lamellae formation, which is characterized by protrusions with accumulation of actin filaments (17). We, therefore, studied whether ARHGAP24 is involved in the ARF6-mediated actin remodeling and cell shape change of human breast carcinoma MDA-MB-231 cells.

To study the role of ARHGAP24, MDA-MB-231 cells were transfected with ARHGAP24-specific siRNA. Endogenous ARHGAP24 was effectively but not completely depleted in MDA-MB-231 cells after treatment with ARHGAP24 siRNA (expression less than 30%) (Figure 1A). Non-transfected control (mock) cells exhibited flat morphology and about half the cells had polarized morphology, with one or two pseudopods (Figure 1B and D). Depletion of endogenous ARHGAP24 alone increased the number of polarized cells and the length of pseudopods (Figure 1B-D). When MDA-MB-231 cells were transfected with ARF6 Q67L alone, many cells produced lamellae and long pseudopods with actin filaments at the periphery. Depletion of ARHGAP24 in cells transfected with AFR6 Q67L significantly increased in the length of pseudopods, compared to cells transfected with ARF6 Q67L alone (Figure 1B and C). Interestingly ARHGAP24 knockdown did not increase the total number of pseudopods induced by activated ARF6 (Figure 1D). These results suggest that endogenous ARHGAP24 may have a role in blocking the extension of pseudopods induced by ARF6.

Figure 1.
Figure 1.

Involvement of RHO GTPase activating protein 24 (ARHGAP24) in ADP ribosylation factor 6 (ARF6)-induced cell shape change. A: MDA-MB-231 cells were transfected with pIRES2-AcGFP-ARF6 Q67L in the absence or presence of ARHGAP24 siRNA. ARHGAP24 and ARF6 proteins were analyzed by immunoblotting using anti-ARHGAP24 and anti-ARF6. Tubulin was used as a loading control. B: MDA-MB-231 cells transfected as discribed in (A) on poly-L-lysine-coated coverslips were fixed and ARF6-expressing cells were identified by staining the cells with anti-green fluorescent protein (GFP) (green). F-actin was localized by Alexa Fluor 568-phalloidin (red). Representatively merged images are shown. Cell outlines are shown by white dotted lines. Scale bars, 30 μm. C: The maximal length of pseudopods in the cells were calculated and the data are shown as box and whisker plots (n=100 cells counted for each experiment). Statistical significance was determined by Welch's t-test. **p<0.01. D: The total number of pseudopods in the cells (n=100) were calculated and the cells were categorized as having no pseudopod (0, blue), one pseudopod (1, red), or more than two pseudopods (≥2, green).

Figure 2.
Figure 2.

Depletion of RHO GTPase activating protein 24 (ARHGAP24) in MDA-MB-231 cells increases the lifespan of pseudopods induced by activated ADP ribosylation factor 6 (ARF6). A: Time-lapse images of MDA-MB-231 cells expressing activated ARF6 Q67L. MDA-MB-231 cells were transfected with pIRES2-AcGFP-ARF6 Q67L in the absence or presence of ARHGAP24 siRNA on glass-bottom dishes. Images were acquired at 10-min intervals and are shown from a selected 60-min interval. Arrowheads indicate the cells transfected with ARF6 Q67L. Cell outlines are shown by black dotted lines. Scale bars, 30 μm. B: Lifespan of individual pseudopods. The data were calculated and expressed as the mean±S.E.M. (n=49-69 pseudopods). Statistical significance was determined by Student's t-test: *p<0.05, **p<0.01. C: Length of pseudopod over time (n=3-5 cells).

Figure 3.
Figure 3.

The pleckstrin homology (PH) domain of RHO GTPase activating protein 24 (ARHGAP24) is required for retraction of pseudopods. A: Schematic diagram of mutated ARHGAP24 constructs [wild type (WT), ΔPH, R39C]. B: HEK293 cells were transfected with FLAG-ARHGAP24 constructs and hepatitis A (HA)-ARF6 Q67L. Cell lysates were prepared, and FLAG-ARHGAP24 was immunoprecipitated (IP) using an anti-FLAG antibody coupled to Sepharose beads. The washed immunoprecipitates were immunoblotted for the presence of ARHGAP24 (anti-FLAG) and ARF6 Q67L (anti-HA). Asterisks indicate non-specific bands. C: The relative amount of bound ARF6 protein. The signal intensity of the bands was quantified by ImageJ. The amount of bound ARF6 (IP) was normalized to that of total ARF6 (cell lysates) and expressed as the mean±S.E.M. (N=3). A.U., Arbitrary units. D: MDA-MB-231 cells were transfected with FLAG-ARF6 Q67L or HA-ARHGAP24 constructs (WT, ΔPH, R39C) resistant to ARHGAP24 siRNA (rKD) in the absence or presence of ARHGAP24 siRNA. FLAG-ARF6 and ARHGAP24 proteins were analyzed by immunoblotting using anti-FLAG and anti-ARHGAP24 antibodies. Tubulin was used as a loading control. E: MDA-MB-231 cells transfected as in (D) on poly-L-lysine-coated coverslips were fixed and FLAG-ARF6 and HA-ARHGAP24 proteins were localized by staining the cells with anti-FLAG (green) and anti-HA (red) antibodies. Scale bars, 30 μm. F: Maximal length of pseudopods in the cells were calculated and the data are shown as box and whisker plots (n=100 cells). Statistical significance was determined by Welch's t-test (vs. FLAG-ARF6 Q67L-expressing cells): **p<0.01.

Depletion of ARHGAP24 may stabilize and increase the lifespan of pseudopods induced by activated ARF6. In order to understand in greater detail how ARHGAP24 may affect ARF6-mediated change in cell shape, we next studied the changes in cell morphology over time using time-lapse video microscopy (Figure 2). MDA-MB-231 cells were transfected with ARF6 Q67L-IRES-GFP in the absence or presence of ARHGAP24 siRNA. Transfected cells were identified using GFP signals under microscopic observation and their live cell behavior was monitored by time-lapse phase-contrast microscopy for 12 h. Compared to non-transfected control (mock) cells (Figure 2A), the cells transfected with constitutively activated ARF6 Q67L alone induced pseudopod formation with continuous extension and retraction (Figure 2A) and increased the average lifespan of pseudopods (Figure 2B). Depletion of ARHGAP24 in ARF6 Q67L-expressing cells produced longer pseudopods (Figure 2A) and significantly increased the average lifespan of pseudopods (Figure 2B). In order to further analyze the time course of elongation and retraction of pseudopods induced by ARF6, we monitored the behavior of cells with average pseudopod lifespan in control (mock) and ARHGAP24-depleted cells. We found that the time for which cells manage to maintain a stable pseudopod length was significantly increased in ARHGAP24-depleted cells, whereas the extension of pseudopods occurred at similar speed in control and ARHGAP24-depleted cells (Figure 2C). These results may suggest that ARHGAP24 could be involved in the retraction of pseudopods induced by ARF6.

Figure 4.
Figure 4.

Depletion of RHO GTPase activating protein 24 (ARHGAP24) does not require extracellular matrix to affect ADP ribosylation factor 6 (ARF6)-mediated change in cell shape. MDA-MB-231 cells were transfected with FLAG-ARF6 Q67L in the absence or presence of ARHGAP24 siRNA on coverslips coated with poly-L-lysine, collagen type I, or fibronectin. After transfection, the cells were fixed and FLAG-ARF6 protein and F-actin were localized by staining the cells with anti-FLAG for ARF6 and phalloidin for F-actin. The elongation factor was calculated as the ratio of maximum length to the width of cells and the data are expressed as the mean±S.E.M. (n=100 cells). Statistical significance was determined by the Student's t-test: **p<0.01.

Binding of ARHGAP24 to activated ARF6 may affect cell shape change induced by ARF6. We next examined if binding of ARHGAP24 to ARF6 is required for retraction of pseudopods induced by ARF6. We confirmed that full-length ARHGAP24 binds to activated ARF6 Q67L after immunoprecipitation from HEK293 cells transfected with cDNAs of both proteins (Figure 3A-C). ARHGAP24 mutant lacking PH domain (ΔPH) did not bind to ARF6 Q67L (Figure 3A-C). Therefore the PH domain of ARHGAP24 mediates a stable complex with activated ARF6 in intact cells. ARHGAP24 R39C, a mutation resulting in deficiency in binding to phosphatidylinositol 3,4,5-trisphosphate (PIP3), was co-precipitated with constitutively activated ARF Q67L when expressed in HEK293 cells (Figure 3A-C) as reported previously (25). Thus, ARHGAP24 binds to ARF6 in a PIP3-independent manner.

We introduced five silent mutations into the siRNA-targeting sequence of ARHGAP24 (WT, ΔPH, R39C) (23) and examined whether or not formation of long pseudopods that were induced by ARHGAP24 siRNA was prevented (Figure 3D). ARHGAP24 knockdown stimulated the production of long pseudopods in ARF6 Q67L-expressing cells (Figure 3E and F). Depletion of ARHGAP24 was rescued by overexpression of siRNA-resistant ARHGAP24 WT and R39C, but not ΔPH (Figure 3E and F). This demonstrates that retraction of pseudopods induced by ARF6 may require the PH domain of ARHGAP24, which is the binding site of activated ARF6.

Knockdown of ARHGAP24 does not require a particular extracellular matrix (ECM) to affect ARF6-mediated change in cell shape. Finally, we examined whether the change of cell shape is dependent on particular ECM components. MDA-MB-231 cells were co-transfected with ARHGAP24 siRNA and ARF6 Q67L on collagen type I- or fibronectin-coated coverslips. We quantified cell morphology by measuring the elongation factor of cells (the ratio of maximum length to width). The values for cells cultured on collagen type I and fibronectin were similar for cells cultured on poly-L-lysine-coated coverslis (Figure 4). These results suggest that ARHGAP24 may regulate change in cell shape independently from the ECM.

Discussion

In this study, we showed that ARHGAP24 might play a role in ARF6-mediated actin remodeling and change in cell shape. Overexpression of constitutively activated ARF6 Q67L in human breast carcinoma MDA-MB-231 cells produced lamellae around the cell periphery. On the other hand, depletion of endogenous ARHGAP24 in ARF6 Q67L-expressing cells induced long pseudopods. This suggests that ARHGAP24 may control the length of pseudopods induced by ARF6. Previous studies have shown that ARF6 controls actin cytoskeleton through regulation of RAC (16-20). ARHGAP24 may suppress extension or stimulate retraction of pseudopods thorough down-regulation of RAC.

The time-lapse video microscopic observation revealed more details in the change of cell shape of ARHGAP24-depleted cells expressing ARF6 Q67L. The extension of protrusion occurred at similar speed between control and ARHGAP24-depleted cells, whereas the time for which cells manage to maintain a stable protrusion length was significantly increased in ARHGAP24-depleted cells. These results suggest that ARHGAP24 may be involved in the retraction of pseudopods induced by ARF6.

Our study suggests that ARHGAP24 may function downstream of ARF6 to regulate the formation of pseudopods induced by ARF6. Firstly, ARHGAP24 binds to activated GTP-bound ARF6 Q67L but not GDP-bound inactive ARF6 T27N (25). Secondly, knockdown of ARHGAP24 was rescued by WT but not mutant ARHGAP24 lacking ARF6-binding site (i.e. PH domain). Moreover, the mutant ARHGAP24 R39C, which binds to ARF6 but is deficient in PIP3 binding (25), still enabled rescue from knockdown. Although the PH domain of ARHGAP24 may bind to factors other than PIP3 and ARF6, and could affect ARF6-dependent actin remodeling, our present study strongly suggests that binding of ARHGAP24 to ARF6 is necessary to regulate the length of pseudopods induced by ARF6.

We showed that depletion of ARHGAP24 induced elongated cell shapes in cells cultured on poly-L-lysine-coated coverslips. Similar effect of ARHGAP24 knockdown was also observed from cells cultured on collagen and fibronectin. These results suggest that signals from specific extracellular matrixes do not affect production of long pseudopods induced by knockdown of ARHGAP24 (32).

It is well established that ARF6 is involved in the regulation of cancer cell migration and invasion (8-11, 14, 15). We showed that ARHGAP24 mediates breast cancer cell migration and invasion (23). Although ARHGAP24 appears to regulate ARF6-mediated change in cell shape, we were unable to determine the role of ARHGAP24 in ARF6-mediated cancer cell migration. MDA-MB-231 cells transfected with ARF6 Q67L induced actin-rich protrusions and showed dynamic cell shape changes. However, they did not migrate effectively on the substrates. The cells may need to maintain proper ARF6 activity in order to migrate, and expression of constitutively activated ARF6 may be inhibitory for cell migration (14). Further study is necessary to determine the role of ARHGAP24 in ARF6-mediated cancer cell migration.

In summary, we showed that ARHGAP24 may function to stabilize pseudopod formation downstream of ARF6 during ARF6-dependent change in cell shape. We present evidence that ARHGAP24 may control retraction of pseudopods through binding to activated ARF6.

Acknowledgements

The Authors thank K. Nakayama (Kyoto University, Japan) for HA-ARF6 constructs. This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Grant for All Kitasato Project Study.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received June 19, 2017.
  • Revision received July 11, 2017.
  • Accepted July 12, 2017.

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Role of ARHGAP24 in ADP Ribosylation Factor 6 (ARF6)-dependent Pseudopod Formation in Human Breast Carcinoma Cells
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