Identification of new cancer-associated genes/proteins, characterization of their expression vari-ation, the interactomics-based assessment of differentially expressed genes/proteins (DEGs/DEPs), and understanding the tumorigenic pathways and biological processes involved in BC genesis and progression are necessary and possible by rapid and recent advances in bioin-formatics and molecular profiling strategies. Taking into account the opinion of other authors, as well as based on our own team’s in vitro studies, we sustain that JTB protein might be consid-ered as a tumor biomarker for BC and should be studied as a target for BC therapy. In this study we have identified the differentially expressed proteins (DEPs), carcinogenic pathways and bio-logical processes associated with JTB silencing, using 2D-PAGE coupled with nano-liquid chro-matography tandem mass spectrometry (nLC-MS/MS) proteomics applied to MCF7 breast cancer cell line, for complementing and completing our previous results based on SDS-PAGE, as well as in-solution proteomics of MCF7 cells transfected for JTB downregulation. The functions of significant DEPs have been analysed using GSEA and KEGG analysis. Almost all DEPs exert protumorigenic effects in JTBlow condition, sustaining the tumor suppressive function of JTB. Thus, the identified DEPs are involved in several signaling and metabolic pathways that exert protumorigenic roles: EMT, ERK/MAPK, PI3K/AKT, Wnt/β-catenin, mTOR, C-MYC, NF-κB, IFN-γ and IFN-α response, UPR, and glycolysis/gluconeogenesis. These pathways sustain cancer cell growth, adhesion, survival, proliferation, invasion, metastasis, resistance to apoptosis, cytoskeleton reorganization, maintenance of stemness, metabolic reprogramming, survival into a hostile environment, and a poor clinical outocome. In conclusion, JTB silencing might increase the neoplastic phenotype and behavior of MCF7 BC cell line.