When triple negative breast cancer (TNBC) are analyzed by gene expression profiling different subclasses are identified, at least one characterized by genes related to immune signaling mechanisms supporting the role of these genes in the cancers. In an earlier study we observed differences in TNBC cell lines with respect to their expression of the cytokine IL32. Our analyses showed that certain cell lines expressed higher levels of the cytokine compared to others. Because TNBC are heterogeneous and immune-related genes appear to play a pivotal role in these cancers, we chose to examine the transcriptomes of the different cell lines based on IL32 expression. We performed group analyses of TNBC cell lines demonstrating high IL32 compared to low IL32 levels and identified IL32, GATA3, MYBL1, ETS1, PTX3 and TMEM158 as differentially associated with a subpopulation of TNBC. The six candidate genes were validated experimental and in different patient datasets. The genes distinguished a subset of TNBC from other TNBC, and TNBC from normal, luminal A, luminal B, and HER2 patient samples. The current project serves as a preliminary study in which we outline the discovery and validation of our list of six candidate genes.

Purpose: The MYBL1 gene is a strong transcriptional activator, associated with cell cycle signaling and differentiation. Data show the gene is overexpressed in triple negative breast cancers. Considering the possibility that MYBL1 might be involved in events associated with the pathogenesis of these cancers, we sought to identify genes associated with MYBL1 expression in triple negative breast cancer. Methods: shRNA lentiviral knockdown was used to down-regulate the MYBL1 gene. Microarray analyses were used to identify genes either directly or indirectly affected by targeting MYBL1 knockdown. Data analyses was performed utilizing Affymetrix TAC 4.0, Chip X transcription factor analyses, Target Scan miRNA analyses, and STRING analyses was used to determine protein: protein interaction and pathway analyses. Web Gestalt and Gene Ontology were used to determine pathway and gene-set enrichments. Publicly available patient and cell line datasets were retrieved and processed using resources available in Gene Expression Omnibus and Oncomine. The polymerase chain reaction and western analyses were used to determine transcript and protein levels, respectively. Results: Knockdown of MYBL1 in a triple negative breast cell line led to down-regulation of MYBL2, TCF19, KIF18b along with an enrichment of cell cycle signaling genes. Gene expression analyses show that MYBL1, MYBL2, TCF19 and KIF18b display a similar pattern of expression in breast cell lines and many of the archival patient datasets examined. Conclusion: TNBC is a heterogeneous subtype, so these data suggest that cancers that over-express MYBL1, express MYBL2, TCF19 and KIF18b. Bioinformatic analyses suggest MYBL1 regulates MYBL2 which leads to regulation of TCF19 and KIF18b.

Purpose: The drug Zoledronic acid (ZOL) has been extensively studied as a therapeutic strategy to treat breast cancers. Previous data show the triple negative breast (TNBC) samples are particularly sensitive to killing following exposure to the drug. The goal of this current study was to examine the effect of ZOL on TNBC and identify genes that might contribute to this sensitivity.Methods: A cell line model was used to perform experiments to determine the dose effect of ZOL on TNBC and the genes differentially expressed following drug exposure. Following treatment, the mode of death was established and the transcriptomes of the cells were examined via microarray and differentially expressed genes were identified and validated via transcript and protein expression analyses.Results: Data show that TNBC cells are sensitive to killing following ZOL, with cell death occurring via the autophagy mechanism. Data also show an enrichment in dysregulation in signalling events related to cell cycle regulation. Previous studies have shown involvement of the cell cycle regulator CDKN1A/p21. We observed similar involvement of CDKN1A/p21, but in addition we found down-regulation of the mitotic serine/threonine kinase AURKA gene. Conclusion:In autophagy associated cell death in TNBC, ZOL functions via cell cycle-mediated signalling events related to regulation of CDKN1A proteinase inhibitor and down-regulation of AURKA kinase.

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The cMYB gene belongs to family of proto-oncogenes all of which function as strong transcriptional activators. The cMYB gene is being considered as a therapeutic target for receptor-positive breast cancer patients. Previous data show that estrogen receptor 1 (ESR1) gene regulates cMYB in MCF7 cells by regulating transcriptional elongation of the gene. These data are substantiated by knockdown studies that show that silencing ESR1 leads to downregulation of cMYB. In addition to cMYB, a substantial number of other genes are affected following silencing of ESR1. Theory is that analyses of the datasets will lead to identification of genes associated with cMYB expression, and further characterization of cMYB in breast cancers. For the current study, we analyzed two different knockdown datasets; one in which ESR1 was silenced and another in which cMYB was silenced. The goal of the study was to identify genes dysregulated because of their association with cMYB. We cross-referenced the differentially expressed genes from the ESR1 knockdown study with top candidates from a cMYB knockdown dataset, and identified LONRF2, DOK7, MSX2 and KCNMB4 genes. We suggest that these genes be considered for their contribution to our understanding of the cMYB signaling mechanisms in luminal breast cancer.of the MCF7 model. The genes included LONRF2, DOK7, MSX2 and KCNMB4. LONRF2 is a LON peptidase N-terminal domain and the RING finger domain gene known to be involved in protein-protein and protein-DNA binding interactions associated with a number of different signaling events [11]. DOK7 (downstream of tyrosine kinase 7; docking 7) gene is essential to neuromuscular synaptogenesis and thought to activate muscle-specific receptor kinase [12]. MSX2 is a muscle homeobox gene related to regulation of bone development and regulation of signaling events between survival and apoptosis [13]. The KCNMB4 (Potassium Calcium-Activated Channel Subfamily M Regulatory Beta Subunit 4) gene is fundamental to the control of smooth muscle tone and neuronal excitability [14]. At this point, it's unclear as to the relationship between the LONRF2, DOK7, MSX2 and KCNMB4 and cMYB. The four genes appear to be affected by conditions that silence cMYB, and further validation of their concurrent expression with cMYB should help in understanding the relationship between the signaling mechanisms. Methods Cell linesThe MCF7 (luminal, receptor positive) and MDA MB231 (triple negative; receptor negative) cell lines were utilized in this study. The Larkins T (2018) Identification of genes differentially expressed with cMYB in luminal breast cancers