Neuromedin B and PD168368 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Tocris Bioscience (Minneapolis, MN, USA), respectively. PD168368 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM and DMSO was used as a solvent control for all in vitro experiments and in vivo assays involving treatment with PD168368. Antibodies for phospho-mTOR, mTOR, phospho-p70S6K, p70S6K, phospho-AKT, AKT, phospho-GSK3β, GSK3β, phospho-4EBP1 and 4EBP1 were obtained from Cell Signaling Technology (Danvers, MA, USA). Human NMB-R antibody and α-tubulin antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BioGenex (Fremont, CA, USA), respectively.

MDA-MB-231, MDA-MB-468 and MCF-7 cells from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin-streptomycin (Gibco-BRL) at 37°C in a humidified atmosphere containing 5% CO₂.

In order to compare the NMB-R mRNA expression in normal breast tissues and invasive cancer tissues, we analyzed publicly available gene expression datasets of human samples (accession no. GSE10797, http://www.ncbi.nlm.nih.gov/geo/). The differential expression of NMB-R mRNA expression was identified by web-based application GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) and the Kolmogorov-Smirnov test was used to determine significant differences.

Total RNA was isolated from MDA-MB-231 cells with a TRIZol reagent kit (Life Technologies, Carlsbad, CA, USA). cDNA synthesis was performed using 2 μg of total RNA with a reverse transcription kit (Promega, Madison, WI, USA). The forward and reverse oligonucleotide primers for PCR were as follows: β-actin, 5′-GACTACCTCATGAAGATC-3′ and 5′-GATCCACATCTGCTGGAA-3′; NMB-R, 5′-CAGAAGTGGCTCGCATCAGT-3′ and 5′-GCTGTTGAAATGCCTCCTGA-3′; E-cadherin, 5′-AACATGGTTCAGATCAAATC-3′ and 5′-AAGCTTGAAGATCGGAGGATTATCG-3′; vimentin, 5′-TGGCACGTCTTGACCTTGAA-3′ and 5′-GGTCATCGTGATGCTGAGAA-3′; Snail, 5′-CTGGGCGCCCTGAACATGCA-3′ and 5′-GGCTTCTCCCCCGTGTGAGTTCTA-3′.

Real-time PCR quantification was performed using SYBR-Green (LightCycler; Roche Applied Science). Cycling parameters consisted of 1 cycle of 95°C for 10 min, followed by amplification for 30 cycles of 95°C for 10 sec, 57°C for 5 sec, and 72°C for 7 sec. Subsequently, a melting curve program was applied with continuous fluorescence measurement. The entire cycling process including data analysis took less than 1 h and was monitored using the LightCycler software program (version 4.0). The forward and reverse oligonucleotide primers for real-time PCR were designed as follows: β-actin, 5′-ACTCTTCCAGCCTTCCTTCC-3′ and 5′-TGTTGGCGTACAGGTCTTTG-3′; NMB-R, 5′-GGGGTTTCCGTGTTCACTCT-3′ and 5′-CAGGAAGATTGTGTGCGCTT-3′.

Harvested cells were lysed in a buffer containing 40 mM Tris-HCl, 10 mM EDTA, 120 mM NaCl, 0.1% Nonidet P-40, and a protease inhibitor cocktail (Sigma-Aldrich). Samples containing equal amounts of protein (30 μg/lane) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membrane was blocked with 5% skim milk in PBS or TBS containing 0.1% Tween-20 for 1 h at room temperature and probed with the appropriate antibodies. The signal was developed using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences).

Transwell polycarbonate membrane inserts with 8 μm pores were coated with 10 μg gelatin. MCF-7 or MDA-MB-231 cells were suspended in DMEM at a concentration of 1×10⁵ cells/100 μl, and were added to the upper chamber. NMB (5 μg/ml) or PD168368 (5 μM) in DMEM was added into the lower chamber. Migratory MCF-7 or MDA-MB-231 cells appearing on the lower side of the chamber were fixed by careful immersion of the filter into methanol for 1 min, stained with hematoxylin-eosin (H&E) solution and counted in three random fields per well. Each experiment was performed in duplicate and three separate experiments were performed for each group.

Cells were seeded in 6-well plates at a concentration of 1×10⁶ cells/well in 1 ml of serum-free DMEM for 6 h, until an adherent monolayer was obtained. A 10 μl pipette tip was used to create a scratch in the monolayer and the cells were washed 3 times with serum-free medium. The cells were then placed in fresh serum-free medium and treated with either NMB (5 μg/ml) or PD168368 (5 μM). Samples were taken at the beginning and after 24 h of culture in 5% CO₂ at 37°C. Images of the scratch wounds were taken and measured by ImageJ software to calculate the mean and standard deviation. Each experimental group was compared with its respective control. The experiments were repeated 3 times.

Invasion was examined in a Corning Costar Transwell system. The lower and upper sides of polycarbonate filters with 8 μm pores were coated with 0.5 mg/ml type I collagen and 0.5 mg/ml Matrigel, respectively. The lower compartment contained medium with NMB or PD168368, and MCF-7 or MDA-MB-231 cells were placed in the upper part of the Transwell apparatus. Cell invasion was determined by counting cells on each filter with an optical microscope at x40 magnification.

MDA-MB-231 cell culture dishes (24-well plates) were precoated with undiluted phenol red-free Matrigel (10 mg/ml). For each well, 1×10⁴ cells were suspended in 200 μl PBS and mixed with 100 μl of cold Matrigel (10 mg/ml). The cell suspension was added dropwise over the bottom layer to cover it. After the cell layer was completely set, culture media was added over the top. Media was changed every 2 days without disturbing the cell/matrix layer. Images were taken at the indicated times using x10 magnification for an overview and x40 magnification to document spheroid structure.

Female BALB/c-nude mice (age 8–10 weeks) were anesthetized by intraperitoneal injection of a mixture containing 30 mg/kg zoletil and 10 mg/kg xylazine (Rompun). MDA-MB-231 cells (2×10⁶ cells/0.1 ml in PBS) were injected into the left cardiac ventricle of nude mice with a 26-1G needle according to previously described methods with modifications (19). Correct injection position in the left ventricle was confirmed by the appearance of bright red blood at the hub of the needle in a pulsatile fashion. The first group of animals received 70 μl of vehicle [polyethylene glycol 400 (PEG; Sigma-Aldrich)] by intraperitoneal injection, while the second group of animals received 1.2 mg/kg injections of PD168368 in PEG by intraperitoneal injection. All mice were sacrificed 4 weeks post-tumor inoculation. Any mice showing signs of distress prior to 4 weeks were sacrificed immediately. Animals were euthanized by CO₂ asphyxiation and histological analysis was performed. Metastatic lung tissues were prepared and thin sections (4 μm) from selected areas were analyzed. After deparaffinization, H&E staining was used to evaluate morphology. This study conformed to the ethical guidelines of the Institutional Animal Care and Use Committee at Pusan National University, Korea.

Data are represented as the mean ± standard deviation obtained for at least 3 independent experiments. Statistical comparisons between groups were performed by the one-way ANOVA followed by the Student’s t-test.

We analyzed a public genomics data set deposited in the NCBI Gene Expression Omnibus (GEO) database (accession no. GSE10797) in which microarray data were compared between normal breast tissue (n=5) and invasive breast cancer (n=28) (20). This dataset showed a significant increase in NMB-R mRNA expression in invasive breast cancer tissues (Fig. 1A). This result is supported by our previous findings showing NMB-R to be highly expressed in malignant breast tissues (17,18). Based on these results, we explored the expression levels of the NMB-R in 3 human breast cancer cell lines (MDA-MB-231, MCF-7 and MDB-MB-468), by employing RT-PCR, real-time PCR and western blot analysis. As shown in Fig. 1B and 1C, examination of NMB-R gene expression in 3 breast cancer cell lines revealed that NMB-R was expressed in all of these cancer cell lines using RT-PCR and real-time PCR analysis. Moreover, the expression level of the NMB-R mRNA was higher in invasive breast cancer cells (MDA-MB-231) than in less-invasive breast cancer cells (MCF-7 and MDB-MB-468). The pattern in NMB-R expression was confirmed at the protein level in MDA-MB-231 and MCF-7 cells (Fig. 1B, lower). The following studies were designed to investigate the inhibitory role of an NMB-R antagonist in the invasive breast cancer cell line MDA-MB-231 that highly expresses NMB-R.

The effect of the NMB-R antagonist PD168368 on cellular behavior of breast cancer cells was determined by migration and invasion assays. Concentrations of PD168369 used in the present study showed no cytotoxic effect on human breast cancer cell lines MDA-MB-231 and MCF-7. We demonstrated that the NMB-R antagonist PD168368 clearly decreased the migratory ability of MDA-MB-231 cells in a Boyden chamber migration assay (Fig. 2A). To complement this Boyden chamber assay, we performed a wound healing migration assay to qualitatively observe the inhibitory effect of PD168368 on the motility of MDA-MB-231 cells. As shown in Fig. 2B, PD168368 treatment decreased the number of cells that migrated into the scratch wound compared to control MDA-MB-231 cells. Next, we examined the effect of PD168368 on invasion capacity of the breast cancer cells in a Matrigel-based Transwell invasion assay. Treatment with PD168368 suppressed the invasion ability of MDA-MB-231 cells (Fig. 2C). In addition, we investigated the inhibitory effects of PD168368 on NMB-induced migration and invasion of MCF-7 cells by utilizing a Boyden chamber assay. As shown in Fig. 2D, PD168368 inhibited the migration and invasion ability of MCF-7 cells induced by NMB, suggesting that PD168368 specifically inhibited NMB-induced migration and invasion in breast cancer cells.

MDA-MB-231 cells grown in 3D Matrigel culture form thorn or leg shapes, which shows the migratory and invasive properties of these cells (21). However, treatment with PD168368 attenuated these aggressive phenotypes in 3D Matrigel culture (Fig. 2E).

The epithelial-mesenchymal transition (EMT) is a crucial early event in the migration, invasion, and metastasis of cancer cells from the primary tumor site (22,23). To investigate whether PD168368 regulates metastatic features of breast cancer cells via inhibiting EMT, we assessed the effect of PD168368 on EMT marker expression in MDA-MB-231 cells by RT-PCR and real-time PCR analysis. As shown in Fig. 3A and B, PD168368 treatment resulted in upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal markers vimentin and Snail in MDA-MB-231 cells. We next investigated the inhibitory effects of PD168368 on NMB-regulated EMT marker expression in MCF-7 cells by adopting a RT-PCR and real-time PCR assay. As shown in Fig. 3C, PD168368 increased the levels of E-cadherin expression reduced by NMB, whereas PD168368 diminished the levels of vimentin and Snail expression induced by NMB. These results suggest that PD168368 inhibits EMT in breast cancer cells.

Recent studies showed that the mTOR pathway is frequently activated in breast cancer metastasis (24,25). Activation of mTOR and the subsequent phosphorylation and activation of its downstream targets p70S6K and eIF4E binding protein 1 (4EBP1) play an important role in promoting cell growth and metastasis in breast cancer (26,27). Therefore, we determined whether NMB stimulates the phosphorylation of mTOR, p70S6K and 4EBP1 in MCF-7 cells. As shown in Fig. 4A, exogenous NMB treatment increased the phosphorylation levels of mTOR, p70S6K and 4EBP1 in MCF-7 cells. Additionally, because activation of the AKT/GSK-3β pathway is emerging as a central feature of EMT (21,28), we speculated NMB regulates AKT/GSK-3β activity in breast cancer cells. Phosphorylation of AKT and GSK-3β were also significantly induced in MCF-7 cells treated with NMB, without obviously influencing total AKT and GSK-3β levels. In contrast, as shown in Fig. 4B, treatment of the MDA-MB-231 cells with PD168368 decreased phosphorylation levels of mTOR, p70S6K, 4EBP1, AKT and GSK-3β in a time-dependent manner. Next, we investigated the effect of PD168368 on NMB-induced activation of mTOR/p70S6K/4EBP1 and AKT/GSK-3β in MCF-7 cells. As shown in Fig. 4C, PD168368 effectively reduced the NMB-induced phosphorylation of mTOR/p70S6K/4EBP-1 and AKT/GSK-3β signaling.

The effect of PD168368 on breast cancer cell metastasis was further investigated in vivo by employing lung metastasis assays. Cardiac injection of MDA-MB-231 cells (2×10⁶ cells/mouse) can lead to the formation of lung metastases in mice. The mice received intraperitoneal injections with PD168368 (1.2 mg/kg) or PEG (vehicle) for 30 days. As shown in Fig. 5A, no metastatic tumor nodules were observed in lungs of PD168368-treated mice compared to PEG-injected mice. After sacrifice, lungs were collected and sectioned, and H&E staining showed that the PD168368-injected mice formed much fewer metastatic lung tumors than PEG-injected mice (Fig. 5B and C). These data showed that PD168368 significantly reduces lung metastatic potential of human breast cancer cells.