Network-based strategies provided by systems biology are attractive tools for cancer therapy. Modulation of cancer networks by anticancer drugs may alter the response of malignant cells and/or drive network re-organization into the inhibition of cancer progression. Previously, using systems biology approach and cancer signaling networks, we identified top-5 highly expressed and connected proteins (HSP90AB1, CSNK2B, TK1, YWHAB and VIM) in the invasive MDA-MB-231 breast cancer cell line. Here, we have knocked down the expression of these proteins, individually or together using siRNAs. The transfected cell lines were assessed for in vitro cell growth, colony formation, migration and invasion relative to control transfected MDA-MB-231, the non-invasive MCF-7 breast carcinoma cell line and the non-tumoral mammary epithelial cell line MCF-10A. The knockdown of the top-5 upregulated connectivity hubs successfully inhibited the in vitro proliferation, colony formation, anchorage independence, migration and invasion in MDA-MB-231 cells; with minimal effects in the control transfected MDA-MB-231 cells or MCF-7 and MCF-10A cells. The in vitro validation of bioinformatics predictions regarding optimized multi-target selection for therapy suggests that protein expression levels together with protein-protein interaction network analysis may provide an optimized combinatorial target selection for a highly effective anti-metastatic precision therapy in triple-negative breast cancer. This approach increases the ability to identify not only druggable hubs as essential targets for cancer survival, but also interactions most susceptible to synergistic drug action. The data provided in this report constitute a preliminary step toward the personalized clinical application of our strategy to optimize the therapeutic use of anti-cancer drugs.