Transcription profiling by array of human breast cancer cell line BT-474 with annexin A1 over-expression or silenced with siRNA to demonstrate design of multi-condition experiments
ABSTRACT: Multiple-condition experiment was desinged to be any number of conditions in an experiment without replicate observations for microarray and used to identify genes differentially expressed between different pairs of conditions (treatments). In this study we used breast cancer stable cell lines for overexpressing and silencing annexin A1 (ANXA1), which belongs to a family of -dependent phospholipid binding proteins and are preferentially located on the cytosolic face of the plasma membrane. Cell lines overexpressing ANXA1 (MDA_MB-453/cDNA) were generated by introducing retroviral vectors containing ANXA1 cDNA (pBabe/ANXA1 cDNA) into breast cancer cell line MDA-MB-453 (a low expressor of ANXA1). Breast cancer cell line BT-474, a high expressor of ANXA1, was infected with ANXA1 siRNA-plasmid viruses to knockdown ANAXAI expressor (BT-474/siRNA) where nucleotides corresponding to siRNA were synthesized and ligated into the pLNCX retroviral vector [35,36]. We also used a pLNCX/U6 empty vector to infect BT-474 and obtained an empty vector expressor. Therefore, 5 breast cancer cell lines (MDA_MB-453, MDA_MB-453/cDNA, BT-474, BT-474/siRNA, and BT-474/U6) are attributed to two genotypes: MDA_MB-453 and BT-474. MCE was performed for microarray analysis with these 5 breast cancer cell lines, that is, only one sample was drawn from each breast cancer cell line.
Project description:The complexity of gene regulation has created obstacles to defining mechanisms that establish the patterns of gene expression characteristic of the different clinical phenotypes of breast cancer. Transcription factor TFAP2C plays a critical role in the regulation of both estrogen receptor-alpha (ERα) and c-ErbB2/HER2 (Her2). Herein, we performed chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in four breast cancer cell lines representing different clinical phenotypes. Comparing the genomic binding sites for TFAP2C in the various cell lines, we identified that glutathione peroxidase (GPX1) is regulated by TFAP2C through an AP-2 regulatory region in the promoter of the GPX1 gene. Knock-down of TFAP2C, but not the related factor TFAP2A, resulted in an abrogation of GPX1 expression. Selenium-dependent GPX activity correlated with endogenous GPX1 expression, and overexpression of exogenous GPX1 induced GPX activity and significantly increased resistance to tert-butyl hydroperoxide. Methylation of the CpG island encompassing the AP-2 regulatory region was identified in cell lines where TFAP2C failed to bind the GPX1 promoter and GPX1 expression was unresponsive to TFAP2C. Furthermore, in cell lines where GPX1 promoter methylation was associated with gene silencing, treatment with 5-aza-dC (an inhibitor of DNA methylation) resulted in activation of GPX1 RNA and protein expression. Methylation of the GPX1 promoter was identified in approximately 20% of primary breast cancers and a highly significant correlation between TFAP2C and GPX1 expression was confirmed when considering only those tumors with an unmethylated promoter, whereas the related factor, TFAP2A, failed to demonstrate a correlation. The results demonstrate that TFAP2C regulates the expression of GPX1, which influences the redox state and sensitivity to oxidative stress induced by peroxides. Given the established role of GPX1 in breast cancer, the results provide an important mechanism for TFAP2C to further influence oncogenesis and progression of breast carcinoma cells. 4 ChIP-Seq data for TFAP2C in human breast carcinoma cell lines MCF-7, BT-474, MDA-MB-453 and SKBR-3.
Project description:Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Here we report on the large-scale use of protein folding stability measurements to characterize different subtypes of breast cancer using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique. Protein folding stability differences were studied in a comparison of two luminal breast cancer subtypes, luminal-A and -B (i.e., MCF-7 and BT-474 cells, respectively), and in a comparison of a luminal-A and basal subtype of the disease (i.e., MCF-7 and MDA-MB-468 cells, respectively). The 242 and 445 protein hits identified with altered stabilities in these comparative analyses, included a large fraction with no significant expression level changes. This suggests thermodynamic stability measurements create a new avenue for protein biomarker discovery. A number of the identified protein hits are known from other biochemical studies to play a role in tumorigenesis and cancer progression. This not only substantiates the biological significance of the protein hits identified using the SILAC-SPROX approach, but it also helps elucidate the molecular basis for their dysregulation and/or dysfunction in cancer.
Project description:We report the chromatin binding sites of HOXB7 transcription factor in BT-474 breast cancer cell line using ChIP-sequencing. We validated the chromatin binding sites in BT-474, MDA-MB-361, MCF7 and T-47D breast cancer cell lines using ChIP-qPCR. The ChIP experiments have been performed using HOXB7 antibody and IgG non-specific antibody as a negative control. The direct downstream target genes of HOXB7 were identified by analyzing the expression of genes located nearby HOXB7 binding sites in HOXB7 knockdown versus control cells using qRT-PCR. Examination of chromatin binding sites of HOXB7 in BT-474 breast cancer cell line using ChIP-seq. Four parallel IgG samples were sequenced, merged together and used as a control data set. Two parallel HOXB7 ChIP samples were sequenced and merged for each replicate, AF1 and AF2. Both HOXB7 ChIP replicates (AF1 and AF2) contained approximately the same amount of reads as the merged IgG control data set.
Project description:Ablation of ERRalpha significantly delays ERBB2-induced mammary tumorigenesis and ERRalpha regulates genes of the ERBB2 amplicon. To further investigate the relationship between ERRalpha activity and RTK signaling, we mapped ERRalpha binding sites in SKBr3 cells upon EGF treatment or heregulin treatment. Inhibition of ERBB2 signaling using the RTK inhibitor lapatinib impacts on ERRalpha stability, while cells resistant to lapatinib treatment exhibit restored ERRalpha expression. We therefore mapped ERRalpha binding sites in parental (sensitive) cells (pSKBr3) as well as in lapatinib-resistant cells (LRSKBr3). ChIP-Seq analysis of ERRalpha binding profile in SKBr3 or BT-474 breast cancer cells.
Project description:two luminal cell lines were added, MCF7 and BT-474 two TNBC cell line were added, MB231 and BT-549 microRNA expression profiles were conpared between the luminal group and the TNBC group
Project description:We performed a comparision of AR binding sites as well as the histone modifications H3K27 acetylation and H3K4 monomethylation in the presence and absence of FoxA1 in the molecular apocrine breast cancer cell line, MDA-MB-453. We also probed AP2alpha binding in asynchronous MDA-MB-453 cells.
Project description:We performed androgen receptor (AR) ChIP-seq after GFP control or FOXA1 over-expression in two AR driven cancer models; LNCaP prostate cancer cell line and MDA-MB-453 molecular apocrine breast cancer cell line.
Project description:Androgen receptor (AR) is expressed in 60-70% of breast cancers independent of estrogen receptor (ER) expression, however its function in breast cancer is largely unknown. Our study identified the high level of AR in ER–/HER2+ breast tumors and andorgen and AR greatly stimulated growth of MDA-MB-453 breast cancer cells. To define the genome-wide AR binding sites, we performed AR ChIP-seq using MDA-MB-453 breast cancer cells followig stimulation of DHT. We also identified FOXA1 is a crucial AR cofactor in MDA-MB-453 cells and the FOXA cistrome showed signaficant overlap with AR at both early and late time points of DHT stimulation. AR ChIP was performed in MDA-MB-453 breast cancer cells following 5a-dihydrotestosterone (DHT) stimulation for 4h and 16h respectively. FOXA1 ChIP-seq was performed after 4h DHT stimulation in MDA-MB-453 cells.
Project description:Characterization of genomic copy number changes in breast cancer cell lines BT474, SKBR3, KPL4 and MCF7 compared to a normal human female genomic DNA reference. The experiment utilized four breast cancer cell lines; BT474, SKBR3, MCF7 and KPL4. All cell lines were grown in accordance with the distributor’s instructions. All samples were hybridized once on 1M Agilent Human Genome CGH microarrays according to manufacturers instructions. Genomic DNA pooled from healthy female donors was used as a reference in all hybridizations. DNA from cell line samples were labeled with Cy5 and DNA from reference were labeled with Cy3.