Abelson Interactor Protein-1 Positively Regulates Breast Cancer Cell Proliferation, Migration, and Invasion

Chunjie Wang,1,2 Roya Navab,1,2 Vladimir Iakovlev,1 Yan Leng,4 Jinyi Zhang,4 Ming-Sound Tsao,1,2 Katherine Siminovitch,4 David R. McCready,5 and Susan J. Done1,2,3

1Division of Applied Molecular Oncology, Ontario Cancer Institute; Departments of 2Laboratory Medicine and Pathobiology and 3Medical Biophysics, University of Toronto; 4The Samuel Lunenfeld Research Institute, Mount Sinai Hospital; and 5Department of Surgical Oncology, University HealthNetwork, Toronto, Ontario, Canada

Abstract Introduction Abelson interactor protein-1 (ABI-1) is an adaptor Breast cancer is the most common cancer in North American protein involved in actin reorganization and lamellipodia women and a frequent cause of female cancer mortality. Most formation. It forms a macromolecular complex deaths from breast cancer are due to metastases that are resistant containing Hspc300/WASP family verprolin-homologous to conventional therapies. The metastatic process involves a proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 sequence of events, which includes detachment from the pri- or Abl/ABI-1/WASP family verprolin-homologous mary tumor, invasion into the vascular system, extravasation, proteins 2 in response to Rho family-dependent stimuli. and finally the formation of a metastatic deposit in the target Due to its role in cell mobility, we hypothesized that organ. The migration of cancerous cells is the key step in this ABI-1 has a role in invasion and metastasis. In the process. The intrinsic forces and mechanisms controlling cancer present study, we found that weakly invasive cell migration in response to various stimuli remain largely breast cancer cell lines (MCF-7, T47D, MDA-MB-468, unknown. SKBR3, and CAMA1) express lower levels of ABI-1 Actin polymerization and lamellipodia formation are believed compared with highly invasive breast cancer cell lines to play critical roles in cell migration during metastasis (1). (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), Abelson interactor protein-1 (ABI-1) is an adaptor protein which exhibit high ABI-1 levels. Using RNA interference, involved in actin reorganization and lamellipodia formation, and ABI-1 was stably down-regulated in MDA-MB-231, its function in cell spreading and migration via WASP family which resulted in decreased cell proliferation and verprolin-homologous proteins 2 (WAVE2) is also established anchorage-dependent colony formation and (2-4). Immunofluorescence of both endogenous and green abrogation of lamellipodia formation on fibronectin. fluorescent protein–tagged ABI-1 protein has indicated that Down-regulation of ABI-1 decreased invasiveness and ABI-1 is localized to sites of actin polymerization. These include migration ability and decreased adhesion on collagen IV platelet-derived growth factor–induced membrane ruffles and and actin polymerization in MDA-MB-231 cells. the tips of lamellipodia and filopodia at the cellular leading Additionally, compared with control parental cells, edge (5). Down-regulation of ABI-1 in cells by RNA inter- ABI-1 small interfering RNA–transfected cells showed ference indicates that ABI-1 is required for the formation of decreased levels of phospho-PDK1, phospho-Raf, platelet-derived growth factor–induced membrane ruffles, Rac- phospho-AKT, total AKT, and AKT1. These data suggest dependent actin remodeling, cell spreading, and migration that ABI-1 plays an important role in the spread of (2, 6). Further, loss of ABI-1 results in the down-regulation breast cancer and that this role may be mediated of WAVE2, nucleosome assembly protein 1 (Nap1), and PIR121 via the phosphatidylinositol 3-kinase pathway. protein levels (3). The Rho GTPase is an important coordinator in (Mol Cancer Res 2007;5(10):1031–9) cellular response necessary for cell migration (1). Recent inves- tigations have shown that an ABI-1–based macromolecular complex (Hspc300/WAVE2/ABI-1/Nap1/PIR121 and/or Abl/ ABI-1/WAVE2) mediates signaling transduction between Rho family proteins and the actin cytoskeleton by activating Arp2/3 Received 11/28/06; revised 5/16/07; accepted 6/6/07. (2, 3). The macromolecular complex colocalizes at the leading Grant support: Gattuso Chair in Breast Surgical Oncology (D.R. McCready). The costs of publication of this article were defrayed in part by the payment of edge of the lamellipodia protrusion in response to numerous page charges. This article must therefore be hereby marked advertisement in Rho family-dependent stimuli. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Although the function of ABI-1 has been clearly established Requests for reprints: Susan J. Done, Division of Applied Molecular Oncology, Ontario Cancer Institute, 610 University Avenue, Room 10-717, Toronto, Ontario, in actin reorganization, its role in cancer progression and meta- Canada M5G 2M9. Phone: 416-946-2337; Fax: 416-340-5517. E-mail: sdone@ stasis remains ill defined. ABI-1 is located at 10p11.2 in uhnres.utoronto.ca Copyright D 2007 American Association for Cancer Research. humans and is a homologue to the mouse abelson interactor 1 doi:10.1158/1541-7786.MCR-06-0391 gene (7). In human acute myelogenous leukemia, ABI-1 was

found fused with mixed lineage leukemia gene in acute was observed in ABI-1 down-regulated cells compared with the myelogenous leukemia with t(10;11)(p11.2;q23) (7-9). Chro- parental control (Fig. 2B). This assured that the later observed mosomal gains on 10p have been associated with a more changes were not secondary to apoptosis. aggressive breast cancer phenotype (10). In this investigation, we used human breast cancer cells to ABI-1 Down-Regulation Induces Morphologic Changes in determine whether ABI-1 is involved in cell proliferation and MDA-MB-231 migration. Down-regulation of ABI-1 in the breast cancer cell Down-regulation of ABI-1 had a significant effect on the line MDA-MB-231 resulted in decreased cell proliferation, morphology of MDA-MB-231 cells (Fig. 2C). The control cells anchorage-dependent colony formation, migration, and inva- exhibited mainly tripolar forms with long tapered cell processes sion in response to fibronectin stimulation, suggesting an im- and abundant cytoplasm. ABI-1 down-regulated cells showed portant role for ABI-1 in breast cancer invasion and metastasis. less cohesion, reduced amounts of cytoplasm, and a reduced number of cell processes. Most cells were rounded with some Results bipolar morphology and shortened cell processes with bulbous ends. Expression of ABI-1, WAVE 2, PIR121, and Nap1 in Breast Cancer Cell Lines Nine breast cancer cell lines with varying degrees of ABI-1 Is Required for Lamellipodia Formation and invasiveness were selected (MDA-MB-231, MDA-MB-157, Adhesion in MDA-MB-231 BT549, Hs578T, MCF-7, T47D, MDA-MB-468, SKBR3, and As actin polymerization has also been shown to be involved CAMA1; ref. 11). Levels of ABI-1 and other members of the in cell adhesion, we further explored the roles of ABI-1 in the macromolecular complex, such as WAVE2, PIR121, and Nap1, attachment to collagen IV. Down-regulation of ABI-1 correlated were determined in the four highly invasive and five less with decreased adhesion of MDA-MB-231 cells to collagen IV invasive cell lines by Western blot analysis (Fig. 1A). An ABI-1 (P < 0.001; Fig. 2D). ABI-1 down-regulated cells formed only doublet was detected with molecular weight between 64 and short irregular bulbous projections compared with the control 82 kDa. This likely represents two of the three isoforms of cells that formed lamellipodia after plating on fibronectin- ABI-1: p68 and p72 (12). COS cells overexpressing ABI-1 coated coverslips (Fig. 2E). were used as a positive control (data not shown). The results showed that weakly invasive breast cancer cell lines (MCF-7, ABI-1 Down-Regulation Decreases Proliferation and T47D, MDA-MB-468, SKBR3, and CAMA1) had moderate to Colony Formation in MDA-MB-231 Cells low expression levels of ABI-1 in contrast to highly invasive To study whether ABI-1 affects breast cancer proliferation, breast cancer cell lines (MDA-MB-231, MDA-MB-157, cell growth kinetics and colony formation were used. The cell BT549, and Hs578T), which showed high expression levels growth kinetics indicated that down-regulation of ABI-1 had a of ABI-1 (P < 0.01; Fig. 1B). Other members of the significant negative effect on the proliferation of MDA-MB- macromolecular complex, WAVE2 and PIR121, were expressed 231 cells resulting in decreased cell numbers (Fig. 3A). Not at significantly lower levels in the low invasive cell line group only was there a significant decrease (P < 0.001) in colony (P < 0.05 and 0.01, respectively; Fig. 1A). Nap1 showed a formation activity observed in ABI-1 siRNA–transfected similar trend as well (P = 0.16). These results suggest that MDA-MB-231 cells (Fig. 3B) but also the size of the colonies these proteins may have an effect on the invasive potential was reduced (Fig. 3B, inset). of breast cancer cells. ABI-1 showed the largest difference, which is consistent with our hypothesis of its involvement at ABI-1 Down-Regulation Results in Cell Growth Arrest in several steps in the signal transduction leading to actin G0-G1 and Delayed Entry into G2-M polymerization. The MDA-MB-231 breast cancer cell line, To understand how ABI-1 affects cell proliferation, cell with one of the highest ABI-1 expression levels, was used in the doubling times were calculated in control cells and ABI-1 subsequent studies. down-regulated cells and found to be 29.8 and 41.4 h, respectively (P < 0.01). To further investigate how ABI-1 Establishment of ABI-1 Stably Down-Regulated MDA- regulates the cell cycle, cells were starved for 30 h and then MB-231 Clones cultured in 10% FCS for 21 h and the cell cycle was analyzed pSi-ABI-1 and its control vector SiSc were used to transfect by fluorescence-activated cell sorting. The cell cycle analysis MDA-MB-231. Six stably transfected knockdown clones and showed that cell growth was arrested in G0-G1 phase and entry 12 control clones were generated. Down-regulation of ABI-1 into G2-M phase was delayed in ABI-1 down-regulated MDA- protein in the ABI-1 small interfering RNA (siRNA) clones MB-231 cells. This was seen by a significant increase of cells in was further confirmed by Western blot analysis (Fig. 2A). The G0-G1 phase and a decreased number of cells in G2-M phase expression of ABI-1 was inhibited >90% compared with (P < 0.001; Fig. 3C and D). Similar results were also obtained control and nontransfected MDA-MB-231. by starving cells for 48 h (data not shown).

Viability of ABI-1 Down-Regulated Cells ABI-1 Down-Regulation Decreases Migration and Invasion To determine whether ABI-1 down-regulation affects cell of MDA-MB-231 Cells viability, we did a viability assay at several time points while We evaluated the migration of ABI-1 down-regulated cells were cultured with 10% FCS and 0% FCS. No difference MDA-MB-231 cells and their parental control cells on collagen

Mol Cancer Res 2007;5(10). October 2007 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. ABI-1 Regulates Proliferation, Migration, and Invasion 1033

FIGURE 1. ABI-1, WAVE2, PIR121, and Nap1 show a variety of expression levels in different breast cancer cell lines. A. Total protein (25 Ag) was separated by a 4% to 12% SDS-NuPage gradient gel and transferred to Hybond-P polyvinylidene difluoride membrane and blotted with the antibodies indicated. H, highly invasive breast cancer cell lines; L, weakly (low) invasive breast cancer cell lines. Images were scanned by Bio-Rad scanner and quantified by Quantity One software. B. ABI-1, WAVE2, and PIR121 have significantly higher levels of expression in highly invasive breast cancer cell lines compared with weakly (low) invasive breast cancer cell lines. Columns, mean of a group; bars, SE. **, P < 0.01; *, P < 0.05, unequal t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

IV–coated Transwell filters. There was a decrease of f5-fold and in the normal mammary cell line MCF 10A (13, 14). in the number of migrating cells in ABI-1 down-regulated Considering this reported AKT involvement in cell migration MDA-MB-231 compared with the parental control (P < 0.05; and our results in ABI-1 down-regulated MDA-MB-231 cells, Fig. 4A and B). The invasive ability of ABI-1 down-regulated we decided to assess phosphatidylinositol 3-kinase (PI3K)/AKT cells was only f20% of parental control cells using a Matrigel pathway proteins in ABI-1 down-regulated cells at basal culture invasion assay (P < 0.05; Fig. 4C). We did cell counts after conditions. Immunoblotting with antibodies to phospho-PDK1, 48 h of starvation to estimate the proliferation rate in conditions phospho-Raf, phospho-AKT, and total AKT showed that ABI-1 similar to the invasion assay. The cellularity of ABI-1 down- down-regulation resulted in a constant striking decrease in these regulated cells was reduced by 16% compared with control. proteins. Further AKT isoform analysis showed that it was This difference is too small to account for the 80% reduction in AKT1 rather than AKT2 that was abolished in ABI-1 down- measured invasive ability. Thus, it indicates that levels of ABI-1 regulated cells (Fig. 5A). affect motility and invasiveness of the cancer cells. ABI-1 Down-Regulation Results in Reduced Levels of Phosphatidylinositol 3-Kinase/AKT Pathway Is Affected WAVE2, PIR121, and Nap1 by ABI-1 Down-Regulation ABI-1 is a member of the macromolecular complex Recent evidence from different groups suggests that AKT1 Hspc300/WAVE2/ABI-1/Nap1/PIR121, which is unstable with- may negatively regulate migration both in breast cancer cells out ABI-1 (3). Because its role in breast cancer is unclear,

FIGURE 2. A. Selection of the ABI-1 down-regulated MDA-MB-231 clones by Western blot. B. Viability assay done using Vi-CELL XR cell viability analyzer at several time points while cells were cultured with 10% FCS and 0% FCS. ABI-1 siRNA cells showed no difference in viability compared with control; therefore, observed changes were not due to apoptosis. C. ABI-1 down-regulation induced morphologic changes in MDA-MB-231. Cells were cultured in 10% FCS with antibiotics and images were acquired by an Axiovert 200 M microscope (siRNA clone 9 shown; Zeiss). Magnification, Â100. D. ABI- 1 down-regulated cells showed a significant decrease in adhesion to collagen IV. Cells (5 Â 104) were plated onto 24-well plates coated with collagen IV and incubated for 1 h at 37jC. Images before and after PBS rinsing were acquired and analyzed using the Image-Pro Plus software. Results are represented as relative numbers in comparison with parental control. Columns, mean of three independent experiments in siRNA clones 9, 11, and 15; bars, SE. **, P < 0.001, two-sided t test. E. ABI-1 down-regulation interrupted lamellipodia formation of MDA-MB-231. Cells were plated on a 10 Ag/mL fibronectin precoated glass coverslip in medium free of FCS for 18 h and then fixed with 3.7% formaldehyde/PBS solution and stained for filamentous actin with rhodamine-coupled phalloidin (1:40 dilution) for 30 min and mounted to standard slides with antifading mounting medium containing 4¶,6-diamidino- 2-phenylindole. Images were acquired with a Zeiss LSM 510 confocal microscope (siRNA clones 9, 11, and 15 studied, clone 11 shown).

we assessed the levels of the complex members in ABI-1 down- their roles in actin dynamics (2, 16, 17). There is relatively little regulated cells in comparison with their parental controls. There information on the role of ABI-1 in cancer cell motility, an was a significant reduction of WAVE2, PIR121, and Nap1 levels important step in metastasis. As actin polymerization is in ABI-1 down-regulated cells (clones 9, 11, and 15; Fig. 5B). involved in cell migration, it is important to explore whether ABI-1 plays a role in cell migration. It is also not clear if ABI-1 Discussion affects cell proliferation. Thus, we investigated whether ABI-1 ABI-1, being an adaptor protein, is involved in actin has a role in proliferation, formation of lamellipodia, and cell reorganization and lamellipodia formation by forming a migration in breast cancer cell lines. To the best of our know- macromolecular complex containing Hspc300/WAVE2/ABI-1/ ledge, this is the first investigation of the expression and func- Nap1/PIR121 or Abl/ABI-1/WAVE2 to mediate signal trans- tion of ABI-1 in breast cancer cells. duction from Rac to the Arp2/3 complex (15). Currently, most As a first step to understanding the involvement of ABI-1 in studies have focused on the interacting partners of ABI-1 and cancer cells, we measured expression levels of ABI-1 in nine

different breast cancer cell lines. The results showed that highly but also inhibited MDA-MB-231 cell adhesion on collagen IV. invasive breast cancer cells had much higher expression levels These results support the previously reported data on the of ABI-1 than weakly invasive breast cancer cells. To further involvement of ABI-1 in the formation of lamellipodia. elucidate the functions of ABI-1 in breast cancer cells, we Actin reorganization plays a critical role in leading cell mi- down-regulated ABI-1 expression in MDA-MB-231 cells by gration. A previous report showed that ABI-1 down-regulation RNA interference. Our results showed that knockdown of ABI- in B16F1 melanoma cells impairs migration (2). More recently, 1 expression not only abrogated the formation of lamellipodia data from different groups have suggested that the ABI-1/

FIGURE 3. ABI-1 down-regulation inhibits cell proliferation and colony formation. A. Proliferation kinetics. Cells (1 Â 104) were plated onto six-well plates and total cells were harvested and counted at indicated times. Points, mean of three independent experiments in siRNA clones 2, 9, 11, and 15; bars, SE. **, P < 0.001, two-sided t test. B. ABI-1 down-regulation resulted in decreased colony formation. Anchorage-dependent colony formation was accomplished as described in Materials and Methods. Plating efficiencies are expressed as colony number to total plated cell numbers (colony number/plated cell number). Columns, mean; bars, SE. **, P < 0.001, two-sided t test. Inset, representative photos of anchorage-dependent colonies of ABI-1 siRNA – transfected and control vector – transfected MDA-MB-231 cells (results of three independent experiments in siRNA clones 2, 9, 11, and 15). C. Representative cell cycle profiles of ABI-1 down-regulated cells and their parental control cells. The cells were subjected to starvation with medium free of FCS for 30 h followed by culturing in medium containing 10% FCS for 21 h. The cells were collected, counted, and fixed with ice-cold 80% ethanol for 60 min and then stained with 1 mL propidium iodide/RNase staining buffer for nuclear DNA. Cells were acquired by FACScan CellQuest software and analyzed with ModFit II software as described in Materials and Methods. Data are percentage of gated total cells. D. ABI-1 down-regulation results in cell growth arrest in G0-G1 and delayed entry into G2-M. Results are expressed as a percentage of total gated cells. Columns, mean of three independent experiments in siRNA clones 2, 9, 11, and 15; bars, SE. **, P < 0.001, two-sided t test.

that loss of ABI-1 may play a role in human prostatic adenocarcinoma and the oncogenesis of Bcr-Abl–positive leukemia (23, 24). However, experiments with Drosophila suggest that both fly and human ABI-1 can enhance the ability of Abl to

FIGURE 4. The migration and invasion ability of breast cancer cells is positively related to the ABI-1 levels. A. Representative photos of migration assay. Magnification, Â100. B. ABI-1 down-regulation in MDA-MB-231 results in decreased migration in comparison with their parental control cells. C. ABI-1 down-regulation in MDA-MB-231 results in decreased invasion compared with their parental control cells. The total number of migrating or invasive cells was counted in the entire field. Results are expressed as percentage of parental control cells. B and C. Columns, mean of three independent experiments in siRNA clones 2, 9, and 11; bars, SE. *, P < 0.05. Viable cell count after 48-h starvation was done to estimate the effect of proliferation rate in similar conditions on invasion and migration assays (ABI-siRNA was 84% of control; mean, 0.38 Â 106 and 0.32 Â 106 cells/mL for control and siRNA clones, respectively). It showed that the observed change in migration ability was not due to decreased proliferation.

WAVE2 complex is crucial for actin dynamics and migration in macrophages and T cells in response to various stimuli (6, 18). As expected, our results showed that the migration and invasive ability was significantly decreased in ABI-1 down-regulated MDA-MB-231 cells compared with control cells (Fig. 4A-C). In our model system, multiple levels of regulation may contribute to this phenotype. First, the loss of the direct effect of Hspc300/WAVE2/ABI-1/Nap1/PIR121 complex on its down- stream target Arp2/3 complex and actin may affect cell migration. Second, ABI-1 can affect PI3K and AKT1 through Rac and Cdc42, which results in decreased migration ability (Fig. 6). Lastly, decreased adhesion to extracellular matrix, which we found to be the result of ABI-1 suppression, may also have a role in inhibiting the migration and invasion. FIGURE 5. A. ABI-1 down-regulation in MDA-MB-231 cells results in Although the role of ABI-1 has been clearly established in decreased PI3K/AKT pathway proteins compared with their parental actin reorganization, its roles in cell growth and tumorigenesis control cells. Cells cultured at normal conditions were lysed in CytoBuster, and 20 Ag total protein was separated by a 4% to 12% SDS-NuPage remain controversial. Some reports have indicated that over- gradient gel and transferred to Hybond-P polyvinylidene difluoride expression of ABI-1 inhibits growth and transforming activity membrane and blotted with the antibodies as indicated (representative of v-Abl in NIH3T3 cells as well as epidermal growth factor– photos, numbers shown are means of three independent experiments in siRNA clones 9 and 15). P-AKT, phosphorylated AKT; P-PDK1, induced and v-Abl–induced activation of extracellular signal- phosphorylated PDK1; P-Raf, phosphorylated Raf. B. Reduced expres- regulated kinase in 293T cells (12, 19-22), which suggest a sion of the Hspc300/WAVE2/ABI-1/Nap1/PIR121 complex proteins in ABI-1 down-regulated cells. Total protein (25 Ag) loaded and blotted as potential negative regulatory role of ABI-1 on epidermal above (representative photos, three independent experiments in siRNA growth factor–mediated signaling. Other studies have found clones 9, 11, and 15).

decrease levels of AKT through reduced activation of these proteins. It is also believed that there is a positive feedback loop between adhesion and Rac activation, which stimulates the recruitment and/or activation of PI3K at the plasma membrane (Fig. 6; ref. 30). Therefore, the decreased levels of the PI3K/AKT pathway proteins we observed may be the con- sequence of alterations in multiple signal transduction pathways, and conversely, it may also regulate the functions of other pathways. In summary, we have shown differential ABI-1 expression levels in several breast cancer cell lines associated with varying phenotypes. Down-regulation of ABI-1 in MDA-MB-231 resulted in decreased adhesion, cell proliferation, migration, and invasion as well as abrogated lamellipodia formation. Com- pared with control parental cells, ABI-1 siRNA–transfected MDA-MB 231 cells showed lower levels of PI3K/AKT path- FIGURE 6. ABI-1 interactions with the Sos1 and WAVE2 complexes and their relationship to Rac, actin polymerization, and PI3K. Actin way members, which may contribute to the decreased prolife- polymerization has a positive feedback effect on PI3K and Rac. RTK, ration, migration, and invasion we observed. Whether this receptor tyrosine kinase; EGF, epidermal growth factor; PDGF, platelet- surprising phenotype is a cell type–specific effect or is uni- derived growth factor; PI3K, phosphatidylinositol 3-kinase. versal in cancer cells remains to be determined. We have detected high expression of ABI-1 in some pancreatic and phosphorylate one of its substrates, Ena, consistent with a posi- prostate cancer cell lines and further investigation is under way. tive role for ABI-1 in this pathway (25). Recently, expression Workon other breast cancer cell lines is also ongoing. array studies showed that high ABI-1 expression is associated Nevertheless, these results suggest that ABI-1 could have a with liver metastasis of small cell lung cancer and paclitaxel pivotal role in breast cancer progression. Further in vivo and resistance in ovarian cancer cells, respectively (26, 27). We clinical studies are required to determine the prognostic and/or found that ABI-1 down-regulation in MDA-MB-231 cells predictive value of ABI-1 in human breast cancer. resulted in a dramatic decrease in proliferation and colony formation activity accompanied by growth arrest in G0-G1 phase and delayed entry into G2-M phase, without affecting cell Materials and Methods apoptosis and viability. This may be mediated by down- ABI-1 siRNA Vectors and Antibodies regulation of the PI3K/AKT pathway. These data are consistent pAV-ABI-1 siRNAs, spanning nucleotides 169 to 187 with a role for ABI-1 as a positive regulator in breast cancer (pAV169) and 197 to 215 (pAV197), were kind gifts from Dr. proliferation. G. Scita (Italian Foundation for Cancer Research, Institute of As an adaptor protein, ABI-1 is involved in several signaling Molecular Oncology Foundation, Milan, Italy). Rabbit poly- pathways by forming Hspc300-WAVE2-ABI-1-Nap1-PIR121 clonal anti-Nap1 and anti-PIR121 were from Dr. T. Stradal and EPS8/SOS1/ABI-1 complexes (3, 28). We observed (National Research Center for Biotechnology, Braunschweig, decreased levels of WAVE2, PIR121, and Nap1 in the cell Germany). ABI-1 siRNA vectors (pSi-ABI-1), scrambled con- lines with low ABI-1 expression (Fig. 1A). These proteins trol vectors (pSiSc), and rabbit anti-human ABI-1 were as were also down-regulated in ABI-1 knockdown cells (Fig. 5B). described previously (2). Antibodies against phospho-PDK1, These data show that the Hspc300/WAVE2/ABI-1/Nap1/ phospho-Raf, phospho-AKT, AKT, AKT1, and AKT2 were PIR121 complex is dependent on ABI-1 in breast cancer purchased from Cell Signaling Technology. Goat polyclonal cell lines, which is consistent with previous reports in other sys- anti-WAVE2 was from Santa Cruz Biotechnology. Rhodamine- tems (3). coupled phalloidin was obtained from Invitrogen Life Tech- A direct interaction between p85 and ABI-1 has been nologies. Glyceraldehyde-3-phosphate dehydrogenase was suggested to be necessary for ABI-1–dependent Rac activation obtained from Abcam. (28). Therefore, we were not surprised that levels of the PI3K/ AKT pathway proteins were significantly decreased in ABI-1 Cell Culture and Transfections knockdown MDA-MB-231 cells at normal culture conditions. MDA-MB-231 cell line (American Type Culture Collection) This may be a direct effect of ABI-1 on the PI3K/AKT was maintained in DMEM supplemented with 10% FCS pathway; however, other indirect effects are also possible. (Hyclone), 100 Ag/mL streptomycin, and 100 units/mL These indirect effects may be associated with abrogated penicillin. Cells were transfected with pSi-ABI-1 (or pAV- lamellipodia formation, which is a specific cellular region ABI-1 siRNAs) using LipofectAMINE 2000 (Invitrogen Life required for activation of various signaling proteins (Raf1, Technologies). Cells were cultured in 10-cm dishes until 70% to mitogen-activated protein/extracellular signal-regulated kinase 80% confluent and transfected with 5 Ag of pSi-ABI-1. Forty- kinase 1, extracellular signal-regulated kinase 2, AKT1, eight hours after transfection, cells were placed into selection glycogen synthase kinase-3a, glycogen synthase kinase-3h, medium containing 2 mg/mL G418 (Invitrogen Life Technol- Rb, and signal transducers and activators of transcription 3; ogies) and individual G418-resistant colonies were screened by ref. 29). Therefore, ABI-1 down-regulation may indirectly Western blotting. Control cells were transfected with pSiSc in

the same manner. ABI-1 down-regulation was confirmed by number of migrating cells or invasive cells was counted with showing at least a 90% reduction in signal intensity by Western entire fields and the results were calculated as migration/ blotting compared with control pSiSc-transfected clones. pAV- invasion rate in relation to parental control cells. Each expe- ABI-1 siRNAs were cotransfected with pSiSc for G418 selec- rimental condition was done in duplicate and repeated at least tion, which resulted in a similar phenotype to pSi-ABI-1. thrice.

Cell Viability Assay Spreading and Adhesion Assays Cells were cultured in 10% FCS and free of FCS for Cells were plated on a 10 Ag/mL fibronectin precoated glass different time points and harvested. Cell viability assay was coverslip in medium free of FCS for 6 to 18 h, fixed with 3.7% done with Vi-CELL XR cell viability analyzer with Vi-CELL formaldehyde/PBS solution, and permeabilized with 0.1% XR 2.03 software (Beckman Coulter). Triton X-100 in PBS for 5 min. Cells were stained for filamentous actin with rhodamine-coupled phalloidin (1:40 Cell Proliferation and Clonogenic Assay dilution) for 30 min, washed briefly with PBS, and mounted to ABI-1 down-regulated MDA-MB-231 (pSi-ABI-1, clones 2, standard slides with antifading mounting medium containing ¶ 9, 11, and 15) and control MDA-MB-231 cells (pSiSc) were 4 ,6-diamidino-2-phenylindole. Images were acquired with a plated at a density of 1 Â 104 per well of six-well plates in Zeiss LSM 510 confocal microscope. Â 4 triplicate. Cells were harvested at days 1, 3, 5, and 7, For the cell adhesion assay, 5 10 cells were plated in each respectively, and counted with a Beckman Coulter cell counter well of 24-well plates coated with collagen IV (Becton j (Beckman Coulter Canada). For anchorage-dependent colony Dickinson), incubated for 1 h at 37 C, and then rinsed with formation, ABI-1 down-regulated MDA-MB-231 and control PBS four times. The remaining cells in each well were fixed in MDA-MB-231 cells were plated at a density of 100 per well of 10% buffered formalin for 20 min at room temperature and six-well plates in triplicate and cultured for 14 days in the washed with PBS, and the entire field was counted with a presence of G418. Cells were stained with 0.1% Coomassie stereomicroscope. All images were recorded and analyzed blue (Bio-Rad) in 30% methanol and 10% acetic acid. Colony- using Image-Pro Plus software (version 6.0; Media Cybernetics, forming efficiencies after plating (z50 cells per colony) were Inc.). evaluated (colony number/total plated cell numbers). Western Blotting Flow Cytometry Analysis Cells cultured at normal conditions were lysed in CytoBuster ABI-1 siRNA (clones 2, 9, 11, and 15) and control MDA- protein extraction reagent (Novagen) containing kinase and MB-231 cells were plated at a density of 3 Â 105 in 6-cm dishes phosphatase inhibitors. The supernatants were collected by Â j in triplicate and cultured overnight in medium containing 10% microcentrifugation at 10,000 g for 5 min at 4 C. For ABI-1, A FCS. The cells were subjected to starvation with medium free WAVE2, Nap1, and PIR121 detection, 25 g total protein was A of FCS for 30 h followed by culturing in medium containing used, and for all other target markers 20 g total protein was 10% FCS for 21 h. Cells were collected, counted, washed with separated by a 4% to 12% SDS-NuPage gradient gel PBS, and fixed with ice-cold 80% ethanol for 60 min. They (Invitrogen Life Technologies). Immunoblot analysis was done were washed with PBS thrice and permeabilized with 0.2% using standard methods. Images were scanned by Bio-Rad Triton X-100 for 5 min and then stained with 1 mL propidium scanner and quantified by Quantity One software (Bio-Rad). iodide/RNase staining buffer (BD Biosciences) for 60 min. Cells were acquired with a FACScan flow cytometer and Acknowledgments CellQuest software (Becton Dickinson). The cell cycle was We thankDrs. Richard Hill, Lichun Wu, Wei Liu, and Nona Arneson for their kind help and suggestions. analyzed with ModFit II LT software (Verity Software House, Inc.). References 1. Ridley AJ. Rho GTPases and cell migration. J Cell Sci 2001;114:2713 – 22. Transwell Migration and Invasion Assay 2. Leng Y, Zhang J, Badour K, et al. 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Mol Cancer Res 2007;5(10). October 2007 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Abelson Interactor Protein-1 Positively Regulates Breast Cancer Cell Proliferation, Migration, and Invasion

Chunjie Wang, Roya Navab, Vladimir Iakovlev, et al.

Mol Cancer Res 2007;5:1031-1039.

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