Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Studies of the interactions between melatonin and 2 Hz, 0.3 mT PEMF on the proliferation and invasion of human breast cancer cells

Interactions between the hormone melatonin at pharmacological concentrations (10(-3) M) and 2 Hz, 0.3 mT pulsed electromagnetic fields (PEMF) on the proliferation and invasion of human breast cancer cells were studied in vitro. Three types of human breast cancer cells were used in this study: MDA-MB-435, MDA-MB-231, and MCF-7. Results showed that cellular growth of MDA-MB-231 cells, which were reported to be lowly metastatic, and MCF-7 cells, which were reported to be nonmetastatic, were both significantly reduced by melatonin regardless of the presence of the field. Results also showed that MDA-MB-435 and MDA-MB-231 cells were invasive, with MDA-MB-231 cells being more invasive than the MDA-MB-435 cells for both unexposed and experimental-PEMF groups. In addition, invasion studies showed that MCF-7 cells were not invasive and that melatonin did not have any effects on the invasion of these cells, with or without the PEMF. It is also suggested that since metastasis requires growth and invasion into tissue, anti-invasion agents can be used in conjunction with melatonin to prevent formation of secondary metastases. The overall studies suggest that PEMF at 2 Hz, 0.3 mT does not influence cancer metastasis; while having clinical merit in the healing of soft tissue injury, this field has shown no influence on cancer cells as 60 Hz power line fields have.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.     

Interaction of retinoids and tamoxifen on the inhibition of human mammary carcinoma cell proliferation

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 microM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

Pattern of serum immunoreactivity against breast cancer cell lysates may predict severity of disease in breast cancer patients

Humoral tumor-specific immunity has been investigated as a potential tool to identify tumor-associated antigens and evaluate cancer diagnosis and prognosis. Using SDS-PAGE and western blotting techniques we investigated the humoral immune response against tumor cell antigens in 36 breast cancer patients, 17 node-positive (NP) and 19 node-negative (NN). As a source of antigens, we prepared protein lysates from four breast cancer cell lines (AU565, BT474, MCF-7 and MDA-MB-231) which in vitro exhibit different features of invasion, estrogen receptor/progesterone receptor status and HER2/neu expression thereby potentially representing mild to aggressive forms of clinical disease. A higher number of immunocomplexes Ag–Ab were formed when serum from NN patients was immunoreacted against lysates from AU565 and MCF-7 in comparison to serum from NP patients (P < 0.01). BT474 cells were not a good antigenic source. MDA-MB-231 cells could not significantly discriminate between NN and NP patients since both groups showed higher amounts of reactivity against the lysate. However, comparative analysis of protein preparations purified from MCF-7 and MDA-MB-231 cells and immunodetected concomitantly with the same serum samples showed that serum from patients with cancers with worse prognosis (stage, nodality, HER2/neu and hormonal status) reacted more intensely to proteins purified from the relatively more invasive cell line MDA-MB-231 compared to MCF-7. These findings suggest that the study of serum antibody reactivity to antigens purified from breast cancer cell lines with different invasive properties should be further investigated for its potential in providing beneficial prognostic information in breast cancer. Supported by the United States Military Cancer Institute and the Department of Clinical Investigation at Walter Reed Army Medical Center. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

Stimulation of growth of human breast cancer cell lines in culture by linoleic acid

Linoleic acid, an omega-6 unsaturated fatty acid, stimulated growth of the MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Responses of the estrogen-independent MDA-MB-231 cells both in serum-free medium and with 1% fetal bovine serum added were positively correlated with linoleic acid concentration over the entire range examined (5-750 ng/ml). Growth stimulation of the estrogen-responsive MCF-7 cell line was maximal at a LA concentration of 500 ng/ml when cultured in 1% fetal bovine serum-containing medium with added estradiol. Linoleic acid had no mitogenic effect on three human cancer cell lines derived from sites other than breast, or on untransformed 3T3 cells.     

Toll-Like Receptor 4 Prompts Human Breast Cancer Cells Invasiveness via Lipopolysaccharide Stimulation and Is Overexpressed in Patients with Lymph Node Metastasis

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.     

Clotrimazole preferentially inhibits human breast cancer cell proliferation, viability and glycolysis

Background

Clotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles.

Methodology/Principal Findings

Three cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with K_i values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231.

Conclusions/Significance

Clotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 μM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

Production of sulfated proteoglycans by human breast cancer cell lines: Binding to fibroblast growth factor-2

The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [³⁵S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (K_av 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (K_av 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind ¹²⁵I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties. J. Cell. Biochem. 64:605–617. © 1997 Wiley-Liss, Inc.     

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca²⁺ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca²⁺ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca²⁺ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.     

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

Background  

Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone.     

Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion

Abelson interactor protein-1 (ABI-1) is an adaptor protein involved in actin reorganization and lamellipodia formation. It forms a macromolecular complex containing Hspc300/WASP family verprolin-homologous proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 or Abl/ABI-1/WASP family verprolin-homologous proteins 2 in response to Rho family-dependent stimuli. Due to its role in cell mobility, we hypothesized that ABI-1 has a role in invasion and metastasis. In the present study, we found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, SKBR3, and CAMA1) express lower levels of ABI-1 compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), which exhibit high ABI-1 levels. Using RNA interference, ABI-1 was stably down-regulated in MDA-MB-231, which resulted in decreased cell proliferation and anchorage-dependent colony formation and abrogation of lamellipodia formation on fibronectin. Down-regulation of ABI-1 decreased invasiveness and migration ability and decreased adhesion on collagen IV and actin polymerization in MDA-MB-231 cells. Additionally, compared with control parental cells, ABI-1 small interfering RNA-transfected cells showed decreased levels of phospho-PDK1, phospho-Raf, phospho-AKT, total AKT, and AKT1. These data suggest that ABI-1 plays an important role in the spread of breast cancer and that this role may be mediated via the phosphatidylinositol 3-kinase pathway.