Comparative analyses of MYBL1 knockdown in non-tumor and triple negative breast cancer cells PROJECT SUMMARY The MYBL1 gene belongs to the MYB family of genes, which includes c-MYB and MYBL2. The genes are proto-oncogenes that function as strong transcriptional activators involved in proliferation, differentiation and cell cycle signaling processes, all which are key events in tumor progression. Studies showed that truncated c-MYB is an oncogene and as such, the gene was studied as a therapeutic target for breast cancers. Less is known about MYBL1 and MYBL2. Both play substantial roles in cell cycle progression and are overexpressed in different cancers, including breast cancers. Published results from the Player laboratory showed MYBL1 overexpressed in a subpopulation of triple negative breast cancers (TNBC). In a separate comprehensive supervised network analyses of luminal breast cancers, MYBL1 overexpression was shown to correlate with poor disease prognosis. Based on these studies the principal investigator‘s (PI) laboratory chose to study the MYBL1 gene with an interest in characterizing the gene in non-tumor TN compared to TNBC and identifying genes affected by its expression in the cancers. The experimental approach is to knockdown the MYBL1 gene in the samples, perform microarray analyses to identify genes differentially affected by the processes, followed by further analyses to authenticate candidate genes identified during the process. As a proof of concept, MYBL1 gene was down-regulated in a TNBC cell line. The candidate genes affected by decreasing MYBL1 expression include MYBL2 and an enrichment in cell cycle signaling genes some of which are novel. This observation is consistent with those which show co-expression of MYBL1 and MYBL2 and substantiate the ability of MYBL1 to regulate expression of MYBL2. The experiments outlined in the current study should broaden our understanding of signaling events in triple negative breast samples. For Aim 1, the investigators will continue knockdown studies of MYBL1 in TNBC and include an additional TNBC and a non-tumor TN sample as comparisons. These data will allow the PI to identify differential patterns of regulation by MYBL1 in non-tumor compared to triple negative cancers in efforts to identify genes and processes that might be associated with tumor progression. Because TNBC are heterogeneous, this study is confined to subtype B, MYBL1 expressing samples. Reliable signature genes will be identified, and pathway analyses performed. The hypothesis is that expression of MYBL1 in the different tissues result in different signaling mechanisms. Select differentially expressed genes will be examined using tissue microarrays with defined receptor status and survival data and compared to the MYBL1 gene expression pattern. For Aim 2, the proliferative capacity and invasive potential of the various knockdown samples will be compared. For Aim 3 the tumorigenic properties of control untreated, shRNA sc...