Understanding the role of Bcl-3 in triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents an aggressive subtype associated with progression to metastasis and poor prognosis. The NFkB co-factor B-cell lymphoma 3 (Bcl-3) is a proto-oncogene commonly dysregulated in a range of cancers and has been shown to be up-regulated in metastatic breast cancer lesions. In this study we investigated the therapeutic potential of targeting Bcl-3 in TNBCs. Knockdown of Bcl-3 with siRNA in a panel of TNBC cell lines resulted in reduced cellular proliferation, collective migration, single-cell motility, NFkB-targeted transcriptional activity and increased apoptosis-mediated cell death. Ectopic overexpression of wild type (WT) Bcl-3 protein in TNBC cells resulted in increased proliferation, single cell motility, and NF-kB activity; however, substantially increased apoptosis-mediated cell death was also observed, resulting in an overall reduced cell turnover. Affymetrix gene expression profiling of MDA-MB-231-Luc cells treated with Bcl-3 siRNA revealed enrichment of up-regulated genes associated with BCL2-mediated apoptosis regulators (MCL1, TP53, BAX, BAK1, BCL2); as well as enrichment of down- regulated genes associated with migration including Rho GTPase signalling (CDC42, RHOB, CFL2, ROCK1, PAK1, PFN2). In a murine in vivo xenograft metastasis model, MDA-MB-231 cells treated with Bcl-3 siRNA were injected via the tail vein, resulting in significantly fewer pulmonary metastatic lesions at experimental endpoint when compared with controls. Tail vein injection of MDA-MB-231 cells overexpressing WT Bcl-3 resulted in significantly increased pulmonary and hepatic metastatic lesions when compared with controls, as well as colonisation at novel distal organs including the heart, mammary gland and bone marrow (femur). Pharmacological inhibition of Bcl-3 with novel small molecule inhibitors, JS6 and CB1, were able to recapitulate Bcl3 siRNA responses in functional assays. Affymetrix gene expression profiling of MDA-MB-231-Luc cells treated with CB1 revealed a significant proportion of the genes that responded to siRNA treatment also responded to this pharmacological inhibitor (including TFGB1, INPP4B, IL1B, IL24), highlighting mechanistic similarities. In a murine in vivo xenograft tail vein metastasis model, daily treatment of CB1 significantly inhibited early metastatic seeding events and reduced average metastatic burden, as well increased the proportion of disease free animals at experimental endpoint when compared to controls. The findings from this study highlighted Bcl-3 as a promising therapeutic target in TNBCs and we were able to demonstrate that the novel Bcl-3 inhibitor, CB1 functioned as an effective anti-metastatic agent in murine models of advanced TNBC