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PNAS 109 (44): 18060-18065

Copyright © 2012 by the National Academy of Sciences.

Estrogen receptor prevents p53-dependent apoptosis in breast cancer

Shannon T. Baileya,b,c,1, Hyunjin Shina,d,1, Thomas Westerlinga,b,c, Xiaole Shirley Liua,d, and Myles Browna,b,c,2

aCenter for Functional Cancer Epigenetics and bDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; cDepartment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and dDepartment of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA 02215


Figure 01
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Fig. 1.. E2 and tamoxifen protect MCF7 cells against doxorubicin- and nutlin-mediated cell death. MCF7 cells were seeded in hormone-depleted medium containing vehicle (EtOH), E2, fulvestrant, or tamoxifen for 2 d and then treated with and without doxorubicin (A) or nutlin (B) for 3 d. Afterward, the cell viability was measured. The data shown were performed in triplicate and are representative of three independent experiments. These data demonstrate that E2 is protective against doxorubicin- and nutlin-mediated apoptosis in breast cancer cells. *P < 0.05 compared with doxorubicin alone treatment; **P < 0.005 compared with nutlin alone treatment.

 

Figure 02
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Fig. 2.. ER and p53 regulate a common subset of genes that is associated with poor outcome in patients with breast cancer. (A) We compared the 12-h doxorubicin-mediated gene expression profile in MCF7 cells, which was generated by using Affymetrix Human Genome U133A 2.0 arrays, with 12-h E2-mediated gene expression and found that 179 genes were shared by both stimuli. (B) We clustered these genes and found two main clusters: cluster 1 contained genes that were up-regulated by doxorubicin and down-regulated by E2, and cluster 2 comprised genes that were up-regulated by E2 and down-regulated by doxorubicin. (C) The 179 commonly regulated genes were interrogated by using Oncomine Concepts analysis (Compendia Biosciences) against publicly available primary breast tumor datasets, and significant associations were graphically represented in an interaction network by using Cytoscape (http://www.cytoscape.org). In this network, a node represents a dataset, and each edge represents a significant association with P < 0.01 and an odds ratio > 4. The 179 genes (shown in purple) demonstrated significant association with patient datasets exhibiting metastasis (blue circle), death (green circles), and recurrence (red circles) within 1, 3, or 5 y. The node size is proportional to the number of associated genes in each dataset. The van de Vijver dataset is shown on the right as an example of genes up-regulated in patients with recurrence at 3 y.

 

Figure 03
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Fig. 3.. ER and p53 regulate a common set of gene targets. Circos plot demonstrating the ER and p53 cistromes, their relative genomic location, genes found within 100 kB of each transcription factor, and the clusters in which they reside. The outer hashes (shown in red) demonstrate the relative genomic location of the sites comprising the ER cistrome. The purple hashes display the location of the p53 cistrome. Genes possessing both an ER and p53 binding sites are also shown in red.

 

Figure 04
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Fig. 4.. p53 activation combined with ER depletion leads to greater target gene expression. (A) Heat map demonstrating the relative expression level of each of the 36 regulated genes from gene expression microarray analysis of MCF7 cells treated with doxorubicin and E2. (B) ER loss leads to more efficient putative apoptotic gene expression. MCF7 cells were treated with doxorubicin alone or in combination with E2, fulvestrant, and tamoxifen to evaluate the expression of putative apoptosis target genes. Cotreatment with fulvestrant led to greater expression of these apoptotic genes, whereas treatment with tamoxifen inhibited in a manner similar to E2. (C) Similar effects were observed when treating with nutlin. (D and E) MCF7 cells were hormone starved and stimulated with doxorubicin (D) or nutlin (E). After a 4-h treatment, E2 was added, and a targeted p53 ChIP assay was performed. No difference was observed in the p53 recruitment level in response to doxorubicin (Left) or nutlin (Right) in the presence of E2. (F) Protein lysate from MCF7 cells treated under ChIP assay conditions were assayed by immunoblot for the level of p53 protein expression, and β-actin was blotted as a control.

 

Figure 05
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Fig. 5.. Kaplan-Meier analysis of patient datasets. Kaplan-Meier analysis reveals that the common regulated genes are predictors of relapse-free survival. To determine the role of these genes in predicting relapse-free survival in patients with ER+ breast cancer, we interrogated breast cancer datasets using the clusters 1 and 2 genes containing p53 and ER binding sites. (A) Kaplan-Meier analysis reveals that the cluster 1 genes that regulated by p53 and ER demonstrate better survival when they are highly expressed in patients with ER+ breast cancer. (B) The opposite result is observed when the p53 and ER-regulated cluster 2 genes are examined.

 


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