Estrogen receptor-␣ (ER␣) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ER␣ binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ER␣ and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ER␣ by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ER␣ and p53. Ionizing radiation together with ER␣ knock down results in an additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the proproliferative role of ER␣.As a tumor suppressor, p53 plays a central role in cellular processes such as cell cycle arrest, apoptosis, senescence, and differentiation (1, 2). Although these functions of p53 are essential to prevent cells from becoming cancerous, left uncontrolled, they can lead to consequences deleterious to normal cells. Mutations in the p53 gene or aberrations in the mechanisms to balance p53 function pave the way to tumorigenesis (3). p53 elicits its biological functions mainly by functioning as a transcriptional regulator of various cellular genes with p53-response elements. On the other hand, estrogen receptor-␣ (ER␣) 5 regulates growth and development of various tissues and promotes proliferation of breast cancer cells (4 -8). ER␣ is a transcriptional regulator that is recruited to the promoter regions of target genes directly through binding to estrogen response elements (EREs) or indirectly through other DNA-binding factors, such as AP1 and Sp1 (7, 9). The opposing functions of p53 and ER␣, while stringently controlled in normal cells, are likely disrupted in cancer cells. Various observations have alluded to the potential for a cross-talk between p53 and ER␣ signaling pathways. For example, in murine models, early exposure to 17␤-estradiol (E 2 ) and progesterone to mimic pregnancy induced nuclear p53 enabling resistance to carcinogenesis by blocking proliferation of apparently ER␣-positive cells (10). In breast cancer cells, increased expression of ER␣ led to elevated levels of p53 and MDM2, an inhibitor of p53 function (11), whereas overexpression of MDM2 enhanced the function of ER␣ (12). However, whether there is a direct link between the p53 and ER␣ pathways has remained unclear. To address this important issue, we investigated whether ER␣ directly interacts with p53 and affects its function. EXPERIMENTAL PROCEDURESCell Culture and Irradiation-MCF7 cells and Saos2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen) or 10% dextran-charcoal-t...

Estrogen receptor A (ERA) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERA binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERA inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERA interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERA binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERA is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERA to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERA to repress p53-mediated transcriptional activation. Further, RNA interference-mediated knockdown of ERA resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERA opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer. [Cancer Res 2007;67(16):7746-55]

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

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an unrecognized impact in modulating O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions. In vitro, when used at concentrations within the human therapeutic range for seizure prophylaxis, LEV decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation analysis reveals that LEV enhances p53 binding on the MGMT promoter by recruiting the mSin3A/histone deacetylase 1 (HDAC1) corepressor complex. However, LEV does not exert any MGMT inhibitory activity when the expression of either p53, mSin3A, or HDAC1 is abrogated. LEV inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide. In 4 newly diagnosed patients who had 2 craniotomies 7-14 days apart, prior to the initiation of any tumor-specific treatment, samples obtained before and after LEV treatment showed the inhibition of MGMT expression. Our results suggest that the choice of AED in patients with malignant gliomas may have an unrecognized impact in clinical practice and research trial design.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary ®ndings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sensetransfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the ®rst study to demonstrate that down-or upregulation of TFPI-2 plays a signi®cant role in the invasive behavior of human gliomas. Oncogene (2001) 20, 6938 ± 6945.