The human breast cancer cell line MDA-MB-468 was purchased from American Type Culture Collection. MDA-MB-468 was cultured in DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin and 10% glutamine. Then, the cells were incubated in a 37°C incubator with 5% CO₂ (7).

MDA-MB-468 at 1×10⁵ cells were seeded in each well of 24-well culture plates and incubated in a CO₂ incubator at 37°C for 24 h. The cells were transfected using Lipofectamine^® 2000 reagent (Invitrogen) in 24-well plates with 200 nM siRNA duplexes (optimal siRNA conditions performed in preliminary study and data not shown). The siRNA against WT1 (siRNA_WT1) (Invitrogen) consisted of a mixture of two 25-nt duplexes, i.e., siRNA_WT1R88 (5′-AAATATCTCTTATTGCAGCCT GGGT-3′) and siRNA_WT1R90 (5′-TTTCACACCTGTATGTCT CCTTTGG-3′). To minimize the cytotoxicity of the reagent itself, the cells were washed once with PBS and the media was changed 6 h after transfection (7). After 72 h, the cells were harvested and the protein level was investigated by western blot analysis.

Cell pellets were harvested by trypsinization and extracted with radioimmunoprecipitation assay (RIPA) buffer (Pierce, USA). Then, the concentration of protein was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The 50 μg of protein samples were loaded to 12% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked by blocking solution [5% low fat dry milk in 1X TTBS (0.1% Tween-20, 154 mM NaCl, 48 mM Tris-base)] for 1 h and washed 5 min for three times with washing solution (1% low fat dry milk in 1X TTBS buffer). After blocking, the blot was incubated with primary antibody anti-WT1 (1:200), anti-rho-GEF (1:1,000) and anti-actin (1:1,000) antibodies (diluted with 1% low fat dry milk in 1X TTBS) for 2 h and washed 5 min for three times with washing solution. The membrane was then incubated with secondary antibody polyclonal anti-IgG rabbit (1:10,000) antibody in 1% low fat dry milk in 1X TTBS for 1 h and washed three times (10 min/wash). The proteins were visualized using a chemiluminescent detection kit (Pierce) and exposed to X-ray film (7).

The MDA-MB-468 breast cancer cell line was transfected with 200 nM of siRNA against WT1 (siRNA_WT1) compared to control (siRNA_neg) for 72 h. After transfection of the cells we detected WT1 level by western blot analysis. The results showed that knockdown of WT1 led to decrease in WT1 protein expression in MDA-MB-468 (Fig. 1A).

The quantitative proteomic, one-dimensional gel electrophoresis (1D-PAGE) was carried out to determine the protein expression patterns between siRNA_neg compared to siRNA_WT1 in MDA-MB-468. Fig. 1B1 represents the protein patterns obtained from 1D-PAGE. Lane 1 and 2 show the protein bands of siRNA_neg and siRNA_WT1, respectively. After 1D-PAGE, the gels were cut into 15 slices as shown in Fig. 1B2.

The quantification of protein from 1D-PAGE was analyzed by the DeCyder™ MS 2.0 Differential Analysis Software (GE Healthcare). The protein expressions of siRNA_WT1 and siRNA_neg were compared. The different intensity of protein expression in both conditions is shown in Venn’s diagram (Fig. 2A). These demonstrated all possible relations of protein expressions in two conditions. The protein names and their biological functions of expressed proteins found only in siRNA_WT1 are listed in Table I and the expressed proteins found only in siRNA_neg are listed in Table II. Table III shows the protein names and the biological functions of expressed proteins found in siRNA_WT1 and siRNA_neg. Rho guanine nucleotide exchange factor 1 (Rho-GEF) was selected to validate by western blot analysis. The result showed the presence of Rho-GEF only in WT1 presence in the cell (Fig. 2B).

Due to p53 mutation in MDA-MB-468 (15), the apoptosis pathway may occur via p53 independently. Notably, a novel target protein of WT1, mitogaligin, was found when WT1 was silenced. Mitogaligin is a 96 amino acid protein highly cationic and rich in tryptophan (16). This protein contains two localization signals, mitochondria and nucleus. Mitogaligin is mainly localized in mitochondria and promotes the release of cytochrome c resulting in the induction of cell death (17). Moreover, it can also be directed to the nucleus and can play a role in apoptotic properties leading to cell death (18). The STRING shows the correlation between WT1 and mitogaligin via EGFR (Fig. 3A). WT1 may act as negative regulator of mitogaligin through the EGFR leading cell death.

WT1 interacts with many genes involved in the cell signaling pathway. In the present study, the proteins involved in the cell signaling pathway, platelet-derived growth factor receptor α (PDGFRA) and Rho-GEF were found when WT1 was present in MDA-MB-468, while SH2 domain-containing protein and IBtK were found when the cell was without WT1. However, the STRING 9.05 showed that these molecules were not associated with WT1 (Fig. 3B).

The signal transduction pathway in the MDA-MB-468 breast cancer cell line was related to the mTOR signaling pathway that regulates cell growth, proliferation, differentiation and survival (19). The mTOR protein exists in two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 contains the protein raptor while mTORC2 contains the protein rictor. In the presence of growth factors, activated Akt phosphorylates and inhibits tuberous sclerosis protein 2 (Tsc2), thereby promoting the activation of Rheb. Activated Rheb (Rheb-GTP) helps activate mTORC1, which in turn stimulates cell growth. Furthermore, mTORC2 phosphorylates Akt at Ser473 and regulates the actin cytoskeleton and cell motility (20). Recently, Razmara et al demonstrated that PDGFRs are essential for multiple growth factor signaling pathways that lead to PI3K/Akt activation. The pathway from PDGFR leads to phosphorylation of Akt which is involved in both the mTORC2 and PLCγ/PKC pathways (21).

The WT1 protein has two nuclear localization domains: within zinc fingers I and within zinc fingers II and III. It is responsible for transcription and RNA processing (22). However, WT1 can be detected in the cytoplasm of various cell lines including breast cancer and shuttles the nucleus and the cytoplasm (23,24). PDGFRA is a tyrosine-protein kinase that acts as a cell surface receptor for PDGFA and plays a role in the regulation of cell proliferation and survival (25). Rho-GEF is an intracellular signaling molecule that regulates cytoskeleton organization, gene expression, cell cycle progression, cell motility and other cellular processes. It represents the activating enzymes of Rho GTPases by serving to relay a variety of signals to catalyze GDP/GTP exchange of specific Rho GTPases (26).

WT1 may be related to PDGFRA leading to activation of Akt/TSC1, TSC2/mTOR2 pathway resulting in cell growth. Moreover, WT1 may also be associated with mTOR2/Rho-GEF resulting in cell motility (Fig. 4).

Thus, WT1 plays an oncogenic role in MDA-MB-468. Moreover, when WT1 was silenced with siRNA_WT1, IBtK, SH2 domain-containing protein were upregulated. The relationship between WT1 and these proteins in the signaling pathway in MDA-MB-468 has not previously been elucidated. WT1 may behave as a negative-regulator of IBtK that binds to SH2 domain of BtK tyrosine kinase receptor resulting in IBtK inactivate leading to B-cell differentiation (27).