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Transcription profiling of human MCF7 time-series upon RITA treatment

ABSTRACT: Targeting “oncogene addiction” is a promising strategy for anti-cancer therapy. Here, we report a potent inhibition of crucial oncogenes by p53 upon reactivation with small molecule RITA in vitro and in vivo. RITA-activated p53 unleashes transcriptional repression of anti-apoptotic proteins Mcl-1, Bcl-2, MAP4, and survivin, blocks Akt pathway on several levels and downregulates c-Myc, cyclin E and B-catenin. p53 ablates c-Myc expression via several mechanisms at transcriptional and posttranscriptional level. We show that transrepression of oncogenes correlated with higher level of p53 bound to chromatin-bound p53 than transactivation of pro-apoptotic targets. Inhibition of oncogenes by p53 reduces the cell’s ability to buffer pro-apoptotic signals and elicits robust apoptosis. Our study highlights the role of transcriptional repression for p53-mediated tumor suppression. Experiment Overall Design: Breast carcinoma cell-line MCF7 was treated with the small-molecule p53 activator RITA for 2h, 8h, 16h and 24h.

ORGANISM(S): Homo sapiens

SUBMITTER: Grinkevich VV

PROVIDER: S-ECPF-GEOD-13291 | biostudies-other | 2009 May

REPOSITORIES: biostudies-other

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Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation. Experiment Overall Design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturer’s protocols.

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Transcription profiling of human MCF7 time-series upon RITA treatment

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