Project description:Tumor formation hinges on the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand the minimum genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed, or contain a single or double knock-in (DKI) of mutant PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC- target gene signature. This gene signature was mediated in part by the bromodomain-containing protein, 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced the MYC transcript and increased the MYC-negatively regulated target, NDRG1. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR/Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and overexpression of BRD9 partially rescued the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncogene cooperation, and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS- driven transformation.
Project description:We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.
Project description:We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer. 2 cell lines (H1047R and WT), 4 time points (0, 6, 12, 24 hours), 3 replicates
Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating gene expression profiles of overexpressing cells and normal MCF-10A cells.
Project description:We profiled gene expression and splicing changes in MCF-10A human mammary epithelial cells expressing MYC fused to a portion of estrogen receptor (MYC-ER) (Eilers et al., 1989; Littlewood et al., 1995). We performed RNA-seq, in triplicate, on 3D-grown MCF-10A MYC-ER cells at 0, 8, and 24 hours (h) after 4-OHT-induced MYC activation. As a control for 4-OHT-induced effects, 3D-grown parental MCF-10A cells lacking the MYC-ER fusion protein were treated with 4-OHT at the same timepoints.
Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating gene expression profiles of overexpressing cells and normal MCF-10A cells. MCF-10A cells were infected with MBD2 lentivirus in order to increase MBD2 expression. Total RNA was extracted from both infected and non-infected cells and hybridized to Affymetrix gene expression microarrays. Three technical replicates were hybridized for infected and non-infected cells.
Project description:A comparison of different energetics based techniques for the characterization of two mammalian breast cell lines, MCF-7 a luminal A breast cancer cell line and MCF-10A a normal human breast cell line. The techniques of stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and conventional expression level analyses were compared and the relative advantages and disadvantages are discussed.
Project description:To detect gene expression changes in skeletal muscle caused by EVs from mMCF-10A/miR-122 cells, we analyzed RNA isolated from the GA muscle of EV-treated female NOD scid gamma (NSG) mice. Mice had received tail-vein injections of EVs twice a week for 5 weeks (~10 ug EV per injection). Gene expression in muscle from mice treated with MCF-10A/miR-122-derived EVs.
Project description:To compare gene expression changes in skeletal muscle caused by EVs from normal (MCF-10A) and cancer (MDA-MB-231) cells, we analyzed RNA isolated from the GA muscle of EV-treated female NOD scid gamma (NSG) mice. Mice had received tail-vein injections of EVs twice a week for 5 weeks (~10 ug EV per injection). Gene expression in muscle from mice treated with MDA-MB-231-derived EVs was compared to mice treated with MCF-10A-derived EVs.
Project description:To study the function of 14-3-3ζ, we established MCF-10A human mammary epithelial cells transduced with 14-3-3ζ (10A.ζ) and vector (10A.Vec) We performed gene expression profiling on 10A.ζ cells and 10A.Vec cells, and normalized to profiling of MCF-10A parental cells