Mechanisms of estrogen receptor antagonism toward p53 and its implications in breast cancer therapeutic response and stem cell regulation - PubMed (original) (raw)

Mechanisms of estrogen receptor antagonism toward p53 and its implications in breast cancer therapeutic response and stem cell regulation

Santhi D Konduri et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Estrogen receptor alpha (ERalpha) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERalpha binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERalpha suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERalpha 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 17beta-estradiol (E2) enhanced ERalpha binding to p53 and inhibited p21 transcription, antiestrogens decreased ERalpha 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 ERalpha target gene, pS2/TFF1. These results suggest that ERalpha 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, ERalpha 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

ERα recruits transcriptional corepressors to repress p53-mediated transcriptional activation. (A) ChIP and sequential ChIP assays were performed on MCF-7 cells with primers specific to the p53-binding site of the p21 promoter. The primary ChIP was performed with anti-p53 antibody, and the immunoprecipitate was subjected to a second ChIP with anti-ERα antibody. The immunoprecipitate from the ERα ChIP was then subjected to the third ChIP with antibodies against HDAC1, NCoR, SMRT, and RIP140. ChIP with IgG was the negative control. (B) ChIP assay as in A with antibodies against NCoR, SMRT, HDAC1, and IgG in MCF-7 cells transfected with NS or ERα siRNAs. (C) Western analysis of protein expression of NCoR, SMRT, RIP140, HDAC1, and ERα in MCF-7 cells transfected with NS siRNA or ERα siRNA. (D) ChIP assay performed on ERα knockdown MCF-7 cells using antibodies against p53, ERα, or IgG. (E) Recruitment of NCoR, SMRT, and HDAC1 to the p53-binding site of the p21 promoter in MCF-7 cells treated with E2 (10 nM) or vehicle for 3 h, as analyzed by ChIP assay. (F) MCF-7 cells were transfected with −2326 p21-luc reporter and NCoR shRNA plasmid or a nonspecific (NS) shRNA plasmid. Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Data are averages from three samples with SD. (G) Transcription of the endogenous p21 gene in MCF-7 cells treated as in F was assayed by qPCR. Data are averages from three samples with SD. (H) Western analysis of protein expression of NCoR, ERα, and p53 in MCF-7 cells transfected with NS or NCoR siRNAs.

Fig. 2.

Fig. 2.

Estrogen enhances and antiestrogens disrupt the ERα–p53 interaction on the p21 promoter, leading to diametrically opposite effects on transcription of p21 compared with that of pS2. (A) qChIP was performed to analyze the ERα–p53 interaction on the p21 promoter in MCF-7 cells. Cells were grown in media with dextran-coated charcoal-treated FBS for 4 d and treated with E2 (10 nM) with or without 1 μM 4-hydroxy tamoxifen or ICI 182780 for 3 h. (B) Western analysis of ERα and p53 protein levels in lysates of MCF-7 cells treated with E2, E2 plus tamoxifen, or E2 plus ICI 182780. (C and D) The effects of estrogen and antiestrogens on p21 and pS2 transcription, respectively, in MCF-7 cells, as assayed by qPCR (value = mean ± SD; *P < 0.05; ***P < 0.001).

Fig. 3.

Fig. 3.

Differential effects of ERα bound to ERE-containing promoters versus p53 binding site-containing promoters. (A) ChIP using anti-p53 or anti-ERα antibodies to analyze p53 and ERα binding to endogenous p21 and pS2 gene promoters in MCF-7 cells treated with E2, E2 plus tamoxifen, or E2 plus ICI 182780. (B) ChIP assay using anti-NCoR antibody, demonstrating differential recruitment of NCoR to pS2 vs. p21 gene promoters in MCF-7 cells treated as in A. (C) ChIP assay using SRC1 and SRC3 antibodies, demonstrating differential recruitment of SRC1 and SRC3 to pS2 versus p21 gene promoters in MCF-7 cells treated as in A. (D) A model for the dual roles of ERα in promoting proliferation via direct binding to ERE elements and indirect binding to p53 binding sites (by tethering to p53).

Fig. 4.

Fig. 4.

ERα represses p53 in murine mammary stem cells. (A) qPCR analysis of the expression of Sca1, cytokeratin 14 (Ck14), cytokeratin 18 (Ck18), E-cadherin (E-Cad), and integrin α 6 (Itga6) in murine mammary epithelial cells (normalized to 1) and murine stem cell-containing mammospheres derived from the mammary glands of C57 BL/6 ERαfl/fl mice. Data are averages from three samples with SD. (B) Western analysis of ERα and p53 proteins in murine mammospheres and MCF-7 cells. (C) Micro-ChIP analysis of the ERα–p53 interaction on the p21 gene promoter in murine mammospheres (Upper). Micro-ChIP analysis of a region of the p21 gene (NS) that does not contain any p53-binding sites (negative control) (Lower). (D) PCR analysis of ERα and p21 RNA levels in C57BL/6 ERαfl/fl murine mammospheres with or without infection with Cre-recombinase lentivirus.

Fig. 5.

Fig. 5.

ERα-positive breast cancer patients with tumors expressing wild-type p53 respond better to tamoxifen therapy. (A) Kaplan-Meier analysis of the overall survival of 35 ERα-positive (28 patients had tumors with wild-type p53 and 7 patients had tumors with mutant p53), tamoxifen-treated breast cancer patients as a function of wild-type or mutant p53 expression. (B) Similar analysis as in A of 36 ERα-negative breast cancer patients (17 patients had tumors with wild-type p53 and 19 patients had tumors with mutant p53) who were not treated with tamoxifen. (C) A model for the role of the ERα–p53 interaction in tumor response to antiestrogen therapy.

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