Estrogen receptor α (ERα) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERα binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERα suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERα represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor p21 Waf1/Cip1 (CDKN1A) gene, a prototypic transcriptional target of p53. While 17β-estradiol (E2) enhanced ERα binding to p53 and inhibited p21 transcription, antiestrogens decreased ERα recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERα target gene, pS2/TFF1. These results suggest that ERα uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERα binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response.nuclear receptor corepressor | mammary epithelial cells | mammospheres | tumor suppressor protein | tamoxifen therapy

BackgroundAnti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC).MethodsESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided.ResultsESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02).ConclusionsTP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA) to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G0/G1-phase and significantly delayed the G1-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

In order to understand dementia and other ailments associated with high altitude hypoxia, adult Sprague Dawley male rats were exposed to simulated conditions of high altitude (7,500 m above sea level, 59 mmHg) for a period of 5 days and analyzed for changes in neuronal proteome by 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein extracts obtained from the brain cortex and hippocampus of the hypoxic rats were separated by 2-D gel electrophoresis. Differentially expressed proteins (analysis by 2-D gel analysis software, Bio-2D, Vilber-Lourmat, France and Delta2d, Decodon, Germany) were subjected to matrix-assisted laser desorption/ionization time-of-flight analysis. Among the proteins identified, the spot corresponding to pI 5.4 and molecular weight 21 kDa, identified as phosphatidylethanolamine binding protein (PEBP1), was consistently lowered (54%) in hypoxic cortex samples. PEBP1, also known as Raf kinase inhibitor protein, is a precursor of hippocampus cholinergic neurostimulatory peptide (HCNP). Western blot analysis revealed elevated phospho-extracellular signal-regulated kinase in hypoxic rat cortex samples, indicating activation of Raf/mitogen-activated protein kinase pathway under hypoxia. Lowered HCNP levels leading to 23% decrease in choline acetyltransferase and 63% increase in acetylcholinesterase activity were detected in hypoxic rat brain cortex, while no significant change was noted in hippocampus. Since PEBP1 is lowered in a number of neurological disorders associated with dementia, we speculate that lowered expression of PEBP1 might be responsible for dementia associated with high-altitude hypoxia. Further studies targeting PEBP1 might give clues about signaling pathways associated with hypoxia and dementia.