Pharmacoproteomic analysis reveals that metapristone (RU486 metabolite) intervenes E-cadherin and vimentin to realize cancer metastasis chemoprevention

Metapristone is the most predominant biological active metabolite of mifepristone, and being developed as a novel cancer metastasis chemopreventive agent by us. Despite its prominent metastasis chemopreventive effect, the underlying mechanism remains elusive. Our study, for the first time, demonstrated that metapristone had the ability to prevent breast cancer cells from migration, invasion, and interfere with their adhesion to endothelial cells. To explore the underlying mechanism of metapristone, we employed the iTRAQ technique to assess the effect of metapristone on MDA-MB-231 cells. In total, 5,145 proteins were identified, of which, 311 proteins showed significant differences in metapristone-treated cells compared to the control group (P-value < 0.05). Bioinformatic analysis showed many differentially expressed proteins (DEPs) functionally associated with post-translational modification, chaperones, translation, transcription, replication, signal transduction, etc. Importantly, many of the DEPs, such as E-cadherin, vimentin, TGF-β receptor I/II, smad2/3, β-catenin, caveolin, and dystroglycan were associated with TGF-β and Wnt signaling pathways, which were also linked to epithelial-to-mesenchymal transition (EMT) process. Further validation of the epithelial marker “E-caderin” and mesenchymal marker “vimetin” were carried out using immunoblot and immunofluorescence. These results have revealed a novel mechanism that metapristone-mediated metastasis chemoprevention is through intervening the EMT-related signaling pathways.


Metapristone inhibits cell migration, adhesion, and invasion. Would healing assay was conducted
with MDA-MB-231 cells to examine the effect of metapristone on cell motility. As shown in Fig. 1B, cellular migration was controlled in a concentration-dependent manner by metapristone, being inhibited by up to 15%, 23% and 43% at 10, 50 and 75 μM, respectively (P < 0.01). Metapristone inhibited cell motility and wound closure at concentrations lower than its IC 50 , suggesting its specific inhibition on cell migration.
Tumor cells adhesion to the ECM is a fundamental step in cancer metastasis, the adherence of MDA-MB-231 cells to HUVECs was assessed to determine whether metapristone can regulate cell adhesion at a non-cytotoxic concentration. Ten fields of each well were randomly selected, and the adhered spots were counted. Compared with the control, the adhesion rate of MDA-MB-231 cells was 84, 68 and 39%, respectively, at 10, 50 and 75 μM of metapristone (Fig. 1C). Metapristone markedly and in a concentration-dependent manner inhibited the adherence of MDA-MB-231 cells to endothelial monolayers, indicating that it may fit into a new class of therapy for the reduction of risk factors of cancer metastasis.
It is well known that MDA-MB-231 cells have strong invasion properties in matrigel. In this study, we investigate the inhibitory effect of metapristone on cell invasion using a transwell system coated with matrigel. We found that treatment with metapristone for 24 h significantly inhibited MDA-MB-231 cells invasion through the transwell membrane. When metapristone was added at 10, 50 and 75 μM, the inhibitory effects were much more obvious compared to that of untreated group, with the inhibition rate of 48.52%, 60.06% and 82.88%, respectively (Fig. 1D).
Overview of quantitative proteomics. The iTRAQ analysis was performed on the purified protein extracts from MDA-MB-231 cells with or without metapristone treatment to understand the mechanism of metapristone-mediated anti-metastasis mechanism on the cellular and molecular level (Fig. 2). In total, 440,119 spectra were obtained from the iTRAQ-LC-MS/MS proteomic analysis. After data filtering to eliminate low-scoring spectra, a total of 93,114 unique spectra that met the strict confidence criteria for identification were matched to 5,145 unique proteins, of which, 311 proteins showed significant differences in metapristone-treated cells (P-value < 0.05). The detailed information including protein accession number, identified peptide number, protein score, sequence coverage, and regulation (fold change) for these identified proteins is shown in Table 1 and 2. Among these differentially expressed proteins (DEPs), 163 proteins were up-regulated (Table 1) and 148 proteins were down-regulated (Table 2). Then, GO analysis was conducted with the GSEABase package from R (http://www.r-project.org/) statistical platform 15 . Genes were classified in three major groups: the biological process, cellular component, and molecular function ( Fig. 3A-C). Approximately 50.94% of the altered proteins were binding proteins, 27.52% were catalytic and 3.91% were enzyme regulators. In addition, we performed COG function prediction and classified these 311 positive proteins into 18 functional categories (Fig. 4).
Western blot validation of the proteomics analysis. Following the database search and classification of proteins, many differentially expressed proteins were reported to be involved in epithelial-to-mesenchymal transition (EMT), such as E-cadherin, vimentin, syndecan-1, β -catenin, dystroglycan, Smad2/3, glutaredoxin, TGF-β receptor, and so on. Western blots were performed on some selected proteins (E-cadherin, vimentin, β -catenin, and Smad 2) to further verify the iTRAQ results (Fig. 5B,C). While vimentin, one mesenchymal cell marker, was down-regulated by metapristone treatment, E-cadherin, one epithelial cell marker, strengthened with the increasing concentration of metapristone. Moreover, the expression of phosphorylation of Smad 2 was also found to be decreased by metapristone treatment. Notably, the western blot images correlated very well and thus confirmed the iTRAQ data obtained.
Metapristone impedes EMT in MDA-MB-231cells in vitro. Epithelial to mesenchymal transition (EMT) and extracellular matrix degradation are critical for the initiation and progression of tumor invasion. As   shown in Fig. 5A, MDA-MB-231 cells initially exhibited a typical mesenchymal-like morphology with long and narrow stretch, while cells under the treatment of metapristone showed epithelial-like morphology with relatively round extension on the plastic surface. Furthermore, we sought to determine whether metapristone could inhibit Epithelial-mesenchymal transition by regulating EMT-related markers, such as vimentin (mesenchymal-specific marker) and E-cadherin (epithelial-specific marker). As shown in Fig. 5D, up-regulated E-cadherin accumulated in the cell to cell junctions after metapristone treatment. Accordingly, the significantly reduced expression of the mesenchymal-specific marker vimentin was observed in the presence of metapristone.

Discussion
Breast cancer metastasis accounts for the lethality of the disease and therefore there is an urgent need to develop new chemopreventives to inhibit cancer cell metastasis 16,17 . Experimental, epidemiological, and clinical data from the last three decades have each supported the hypothesis that oral contraceptive, such as mifepristone, possesses anticancer properties 18,19 . Then metapristone, the most predominant biological active metabolite of mifepristone, is being developed as a novel cancer metastasis chemopreventive agent by us.
Metastasis is a hallmark of cancer and the leading cause of mortality among cancer patients. The first step in metastasis is the migration of cancer cells away from the primary tumor, a process called tumor invasion 20 . Therefore, much research effortin recent years has been directed toward disruption of this step of the metastatic process 21,22 . In this study, we chose MDA-MB-231 cells with high metastatic potential to explore the effects of metapristone on the metastatic activity of human breast cancer cells. We showed that metapristone markedly inhibited their migratory (Fig. 1B) and invasive (Fig. 1D) abilities of MDA-MB-231 cells at low concentrations. Adhesion of cancer cells to ECM or vascular endothelium is also a crucial starting point of metastasis 23 . Here, we also found that metapristone markedly and in a concentration-dependent manner inhibited the adherence of MDA-MB-231 cells to endothelial monolayers. Collectively, these results suggested that metapristone had the ability to inhibit breast cancer cells metastasis. However, the underlying mechanism remains elusive.
Pharmacoproteomic, especially quantitative pharmacoproteomics, has been emerging as a powerful tool in cancer research, providing a unique avenue to investigate direct drug targets at a functional level 24,25 . Here, we have demonstrated the ability of the isobaric tags to detect and quantify differences in expression levels of proteins between metapristone-treated and untreated MDA-MB-231 cells that reflect functions associated with cancer cells metastasis. Temporal iTRAQ analysis identified 311 proteins as differentially expressed, with 163 as up-regulated (Table 1) and 148 as down-regulated (Table 2). Followed by GO analysis and KEGG pathway analysis, we established their potentially functional classification for the first time: there are 249 pathways, including metabolic, oxidative phosphorylation, p53, MAPK, Wnt, focal adhesion, VEGF, TGF-beta signaling pathways and so on (Table 3). Importantly, some of these pathways were reported to be linked to epithelial-to-mesenchymal transition (EMT) process, which was related with cancer carcinogenesis, prognosis and especially metastasis 14,26 .
The epithelial-to-mesenchymal transition (EMT) has been considered as the initiation process of cancer metastasis, when non-invasive and non-metastatic tumor cells lose their epithelial phenotype, acquire invasive properties, infiltrate surrounding tissues and metastasize to secondary sites 27,28 .Turning an epithelial cell into a mesenchymal cell requires loss of epithelial polarity, alteration in cellular architecture and acquisition of migrationcapacity 29 . It has also been described that during EMT, the epithelial cells acquire mesenchymal morphology, hence the expression of epithelial markers decreases and the expression of mesenchymal markers increases 30,31 . Here, we found that MDA-MB-231 cells initially exhibited a typical mesenchymal-like morphology with long and narrow stretch, while under the treatment of metapristone, cells showed epithelial-like morphology with relatively round extension on the plastic surface (Fig. 5A). We also found that metapristone-treatment resulted in decreased expression of mesenchymal marker "vimentin" and increased expression of epithelial marker "E-cadherin" in MDA-MB-231. Vimentin is a well-known metastasis marker and therapeutic target, as inhibiting vimentin function reduces the ability of cells to migrate 32 . Some anti-cancer drugs that are currently used in the clinic directly target vimentin such as "silibinin" 33 and "withaferin A" 34 . One of the hallmarks of EMT is the functional loss of E-cadherin, which is thought to be a metastatic suppressor during tumor progression 35 . E-cadherin, encoded by the gene CDH1, is a transmembrane glycoprotein responsible for calcium-dependent cell-to-cell adhesion. E-cadherin plays a pivotal role in cadherin-catenin-cytoskeleton complexes, and it grants anti-invasive and anti-migratory properties to epithelial cells 36,37 . Our results suggest that metapristone inhibits cell migration, adhesion and invasion in highly metastatic human breast cancer cells, maybe in part, through the regulation of significant EMT-related markers which then leads to reversal of EMT.
Epithelial-to-mesenchymal transition, the process closely related to tumor development, is often regulated by a variety of signaling pathways and cytokines [12][13][14]26 . In this work, we performed KEGG pathway analysis based on the differential expressed proteins in MDA-MB-231 cells under metapristone-treated and untreated. We found some DEPs, including TGF β receptor I/II, Smad 2/3, RhoA, and Glutaredoxin, were related with Transforming Growth Factor β (TGF β ) signaling pathway. TGF β signaling pathway has been characterized as an important inducer of EMT via several downstream signaling moleculars 13 . TGF β signals via formation of a heterotetrameric complex of TGF β receptor I/II (TGF β RI/RII), in which the active TGF β RII phosphorylates and activates the TGF β RI at the plasma membrane 38,39 . This conformational switch allows activated TGF β RI to interact with Smad2/3 through their MH2 domain. The activated type I receptor then propagates the signal to the nucleus by phosphorylating Smad 2 and Smad 3. Then, Smad2/3 can directly or indirectly regulate gene expression by controlling epigenetic processes, such as chromatin remodeling or by maintaining promoter DNA methylation, which is critical in silencing epithelial gene expression in cells that have undergone EMT 40 . Meanwhile, there exists a non-Smad pathway induced by TGF β 41 . In this non-Smad pathway, TGF β RII phosphorylates PAR6 (partitioning-defective protein 6), then inactivates the epithelial polarity complex, as well as activating of the small GTPase RhoA, which is contribute to cell invasion leading to breast cancer metastasis 42  Furthermore, glutaredoxin (Grx), an anti-oxidant enzyme, was reported to play an important role in intervening TGF β -induced EMT process by reducing ROS generation in intracellular and suppressing the expression of mesenchymal markers 43 . Our results demonstrated that metapristone significantly inhibited the protein expression levels of TGF β RI/RII, RhoA, Smad 2/3, and up-regulated the expression level of glutaredoxin, implying that metapristone maybe in part, reverse EMT through attenuating TGFβ signaling pathway in MDA-MB-231 cells.
In addition to the TGF β signaling pathway, the Wnt signaling pathway also plays an important role in EMT 44,45 . Wnt pathway contributes to EMT by activating β -catenin, and then activating Snail, which in turn suppresses epithelial markers expression like E-cadherin 45,46 . Meanwhile, Caveolin-1 (CAV1), the principal structural protein of the cholesterol-rich plasma membrane invaginations, could induce EMT process through Wnt/β -catenin pathway to promote cancer metastasis 47,48 . Caveolin-1 is also an important regulator of cell polarity and directional movement 49 . The decreases in caveolin-1 expression follows classically described cellular changes associated with MET (including changes in cell morphology and expression of the E-cadherins and fibronectin) 50 . Our studies show that metapristone inhibits cell growth, and reverses EMT in conjunction with the activation of E-cadherin, and the inactivation of β -catenin and Caveolin-1 in MDA-MB-231 cells, implying that the MET potential of metapristone maybe related with Wnt signaling pathway.
In conclusion, our data show that metapristone inhibits migration, adhesion, and invasion abilities of the breast cancer cells. The pharmacoproteomic study reveals that metapristone intervenes EMT-related signaling pathways, such as TGF-β and Wnt signaling pathways, in conjunction with the activation of E-cadherin and glutaredoxin and inactivation of vimentin, TGFβ RI/RII, Smad2/3, RhoA, β -catenin and Caveolin-1 (Fig. 6).

No.
Score % Cov   These findings imply that the application of metapristone is a possible new method to control EMT, which contributes to metastatic processes in breast cancer. Our results suggest that knowledge of the putative pharmacoproteomic mechanisms will promote better use of existing drugs and facilitate the conception of new therapies and new drug development.

Materials and Methods
Cell culture, antibodies and reagents. MDA-MB-231 human breast cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in ATCC-formulated Leibovitz's L-15 Medium (Catalog No. . Cells were supplemented with heat inactivated fetal bovine serum to a final concentration of 10%, and incubated at 37 °C in a free gas exchange with atmospheric air. Mouse monoclonal anti-vimentin (ab8978), -E-cadherin (ab1416), -β -actin antibodies (ab6276), goat anti-rabbit (ab150077) and goat anti-mouse (ab150115) antibodies were all obtained from Abcam Corporation.
In vitro cytotoxicity studies. The cytotoxicity of metapristone was investigated by the MTT assayas described previously by this lab 51,52 . Briefly, MDA-MB-231 cells were seeded into 96-well plates at a density of 1 × 10 4 cells/well, and then incubated at 37 °C in a humidified atmosphere with 100% air. After overnight incubation, the cells were treated with different concentrations of metapristone for 24 h. Culture medium was used as a blank control. Then, cells were incubation with the MTT solution (5 mg/ml) in the medium without phenol red and serum for another 4 h. The MTT-formazan formed by metabolically viable cells was dissolved in 150 μl of dimethyl sulfoxide (DMSO). Cell viability was determined by detecting the absorbance at 565 nm using an infinite M200 Pro microplate reader (Tecan, Switzerland). The absorbance of untreated cells was considered as 100%. Each sample was assayed in triplicate in three independent experiments.
Wound healing assay. Migration of MDA-MB-231 cells was investigated in the in vitro wound-healing assay as described previously by this lab 3,51 . The MDA-MB-231 cells were seeded in 6-well plate; once confluent, 10 μg/ml mitomycin C was added. The scratch wound was generated in the surface of the plate using a pipette tip, followed by extensive washing with serum-free medium to remove cell debris. DMSO (final concentration: 0.1%) as vehicle control was added after wounding. Cells were then cultured and allowed to migrate into the wound area for up to 24 h at 37 °C. At indicated time points, motility was quantified by measuring the average extent of wound closure. Each well was counted under a light microscope (Zeiss, Germany) at a magnification of 10 × and then photographed.
Cell invasion assay. Cell invasion assay was performed using 24-well transwells (Costar, Coring Incorporated, USA), which allows cells to migrate through a polycarbonate membrane with 8-μm pore size as we described previously 52,53   TEAB (Applied Biosystems, Milan, Italy) and sonicated in ice. After centrifuging at 30000 g at 4°C, an aliquot of the supernatant was taken and protein concentration was determined using the Bradford method. Then, total protein (100 μg) of each sample was digested with Trypsin Gold (Promega, Madison, WI, USA) with the ratio of protein: trypsin = 30:1 at 37°C for 16 hours. After trypsin digestion, peptides were dried by vacuum centrifugation, reconstituted in 0.5 M TEAB, and processed according to the manufacture's protocol for 8-plex iTRAQ reagent (Applied Biosystems, Foster City,CA). The labeled peptide mixtures were pooled and dried by vacuum centrifugation, and then fractionated using Poly SULFOETHYL ATM SCX column (200 × 4.6 mm, 5 μm particle size, 200 A° pore size) by HPLC system (Shimadzu, Japan) at flow rate 1.0 ml min-1. The eluted peptides were pooled into 20 fractions, desalted with a Strata × C 18 column, concentrated to dryness using vacuum centrifuge and then reconstituted in 0.1% formic acid for LC-MS/MS analysis.

LC-ESI-MS/MS analysis based on Q EXACTIVE.
The mass spectroscopy analysis was performed using a tandem mass spectrometry (MS/MS) in an Q EXACTIVE (Thermo Fisher Scientific, San Jose, CA) coupled online to the HPLC as described before 54,55 . Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0; ion fragments were detected in the Orbitrap at a resolution of 17500. A data-dependent procedure that alternated between one MS scan followed by 15 MS/MS scan with a following Dynamic Exclusion duration of 15s. Proteins identification was performed by using Mascot search engine (Matrix Science, London, UK; version 2.3.02). For protein quantitation, it was required that a protein contains at least two unique peptides. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We only used ratios with p-values < 0.05, and only fold changes of > 1.5 were considered as significant.  Table 3. Pathway analysis of the DEPs obtained from the iTRAQ analysis. There were 249 pathways revealed. Among them, the following 33 signaling pathways were significant (P < 0.01).
Proteomic data analysis. Functional annotations of the proteins were conducted using Blast2GO program against the non-redundant protein database (NR; NCBI). The keg database (http:www.genome.jp/keg/), the COG database (http://www.ncbi.nlm.nih.gov/COG/), and GO (Gene Ontology) analyses (http://www.geneontology.org) were used to classify and group these identified proteins according to the methods reported in early literature 15,56 .
Western blot analysis. Cell lysates were collected using radio immunoprecipitation (RIPA) lysis buffer, supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific), and immunodetection of electrophoresis-resolved proteins was performed using standard protocols. The E-Cadherin, vimentin, Smad2, pSmad2, β -catenin, and β -actin antibodies were from Abcam. Immunodetection was accomplished using enhanced chemiluminescence, and data were acquired with a quantitative digital imaging system (Quantity One, Bio-Rad) allowing it to check for saturation. Overall emitted photons were quantified for each band, particularly for homogeneously the loading controls.
Immunofluorescence staining and high-content confocal imaging. MDA-MB-231 cells were cultured on a 35 mm cell culture dish (NEST, GBD-  to 50% confluence at least 2 days before carrying out the immunofluorescence assay. Cells (with or without metapristone-treatment) were first washed by phosphate buffer 3 times and then fixed by 4% paraformaldehyde for 30 minutes. One milliliter of 0.1% Triton-X-100 was subsequently added to culture cells for ten minutes to increase cell permeability. Cells were blocked for 30 minutes at room temperature with 10% goat serum followed by culturing with primary antibodies, mouse monoclonal anti-vimentin antibody and E-cadherin for 1 h at room temperature. Then cells were added with secondary antibodies, Goat Anti-Mouse IgG-FITC antibody (Boster, BA1101) and Goat Anti-Rabbit IgG-CY3 antibody (Boster, BA1032) respectively, and cultured in the dark for 1 h at room temperature. Phosphate buffer was used to wash cells for at least three times between every two contiguous steps. Confocal analysis was performed on a Leica-TCS-SP8 confocal microscope and the images were taken under the same parameter configuration.
Statistical analysis. All data were analyzed using SASS software and expressed as the mean ± SD or SE.
Statistical comparisons between different groups were performed using Student t-test. A P value of < 0.05 was considered to be statistically significant.