Estrogen receptor prevents p53-dependent apoptosis in breast cancer - PubMed (original) (raw)

Estrogen receptor prevents p53-dependent apoptosis in breast cancer

Shannon T Bailey et al. Proc Natl Acad Sci U S A. 2012.

Abstract

More than two-thirds of breast cancers express the estrogen receptor (ER) and depend on estrogen for growth and survival. Therapies targeting ER function, including aromatase inhibitors that block the production of estrogens and ER antagonists that alter ER transcriptional activity, play a central role in the treatment of ER+ breast cancers of all stages. In contrast to ER- breast cancers, which frequently harbor mutations in the p53 tumor suppressor, ER+ breast cancers are predominantly wild type for p53. Despite harboring wild-type p53, ER+ breast cancer cells are resistant to chemotherapy-induced apoptosis in the presence of estrogen. Using genome-wide approaches, we have addressed the mechanism by which ER antagonizes the proapoptotic function of p53. Interestingly, both ER agonists such as estradiol and the selective ER modulator (SERM) tamoxifen promote p53 antagonism. In contrast, the full ER antagonist fulvestrant blocks the ability of ER to inhibit p53-mediated cell death. This inhibition works through a mechanism involving the modulation of a subset of p53 and ER target genes that can predict the relapse-free survival of patients with ER+ breast cancer. These findings suggest an improved strategy for the treatment of ER+ breast cancer using antagonists that completely block ER action together with drugs that activate p53-mediated cell death.

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Conflict of interest statement

Conflict of interest statement: M.B. serves as a consultant to Novartis and receives sponsored research support from Novartis and Pfizer.

Figures

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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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