- The Wilms’ tumor 1 (WT1) gene encodes a zinc finger acting as a transcriptional activator or repressor for many genes involved in cell differentiation, growth, and apoptosis. These functions depend on the cell types, WT1 isoforms, and the status of targeted molecules. To determine the relationship between WT1 and related proteins, WT1 was silenced with siRNA in MCF-7 and MDA-MB-468 breast cancer cell lines. The protein expression patterns were analyzed by proteomics techniques: two-dimensional gel electrophoresis (2-DE) and one-dimensional gel electrophoresis (1-DE) combined with LC-MS/MS mass spectrometry. For 2-DE LC-MS/MS analysis, 27 protein spots (15 spots in siRNAneg (present WT1) and 12 spots in siRNAWT1 (without WT1)) were found to have a significant change in expression level. However, in MDA-MB-468, 70 protein spots (61 spots in siRNAneg (present WT1) and 9 spots in siRNAWT1 (without WT1)) had a significant change in expression level. While, 1-DE LC-MS/MS showed 164 proteins differentially expressed between siRNAneg and siRNAWT1 in MCF-7 and MDA-MB-468. These proteins could be classified by their functions in apoptosis, cell signaling, protein folding, metabolism, structural, cell adhesion, gene expression, transport, redoxregulation, protein degradation and unknown functions. In MCF-7, the proteins involving apoptosis were cathepsin D, apoptosis inducing factor, and apoptosis stimulating of p53 protein and were found only in silenced WT1 condition. In the presence of WT1, the following proteins involving signal transduction pathway were found: 14-3-3 epsilon, signal transducing adaptor protein 1, phospholipase C, and metabotropic glutamate 5 receptor. In MDA-MB-468, proteins involving apoptosis 3 including ALG-2 interacting protein x and apoptosis- inducing factor 1 were upregulated in the presence of WT1, while mitogaligin, an apoptosis related molecule, was identified when WT1 was silenced. On the other hand, proteins related in the signaling pathway were detected in both siRNAneg and siRNAWT1 but the type of proteins were different. For example, IBTK protein, and SH2 domain containing protein were present in siRNAWT1 condition, while the platelet derived growth factor receptor (PDGFRA), rho guanine nucleotide exchange factor 1, guanine nucleotide binding protein, and neuropolypeptide h3 were expressed in siRNAneg. From these results it may be assumed that WT1 could be related with proteins involved in apoptosis: cathepsin D, apoptosis-inducing factor 1, and apoptosis stimulating factor of p53 protein 2 and may play a role as an anti-apoptosis in MCF-7. While in the signal transduction pathway, WT1 may crosstalk with 14-3-3 epsilon, signal transducing adaptor protein 1, phospholipase C, and metabotropic glutamate receptor resulting in cell growth or cell proliferation. Thus, WT1 acts as an oncogene in MCF-7. In MDA-MB- 468, WT1 relates to mitogaligin and behaves as an anti-apoptotic molecule. Moreover, WT1 may be associated with the platelet derived growth factor receptor alpha, rho guanine nucleotide exchange factor 1 that activates proliferation via the mTOR pathway. Furthermore, WT1 may act as a negative regulator or block Raf kinase inhibitor resulting in activation of the MAPK pathway and promote metastasis. However, validation of the selected protein is necessary to confirm these hypotheses.