Estrogen Receptor A Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis Aejaz Sayeed, Santhi D. Konduri, Wensheng Liu, Sanjay Bansal, Fengzhi Li, and Gokul M. Das Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York Abstract 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 transcrip- tional 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 activa- tion. 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] Introduction In response to various extracellular and intracellular signals, p53 mediates cellular processes, such as cell cycle arrest, apoptosis, senescence, and differentiation, depending on the signal and the cellular context (1–3). There is compelling evidence that transcrip- tional regulation by p53 is required to execute most of its functions, enabling it to be a tumor suppressor, although transcription- independent mechanisms can also contribute to p53 function (4, 5). Most studies have focused on transcriptional activation by p53 because of the strong association observed between target gene activation and tumor suppression. However, increasing evidence suggests that transcriptional repression of various target genes by p53 also plays an important role in tumor suppression (6). For example, Survivin and multidrug resistance gene 1 (MDR1 ) belong to a group of genes targeted for transcriptional repression by p53 (7–9). The Survivin gene promoter contains a p53-binding site with four 5-bp consensus sequence 5¶-PuPuPuC(A/T)-3¶ repeated in two pairs, each arranged as inverted repeats (head to head orientation such as )( )() with a spacer of 3 bp between the two 10-bp repeats (7). MDR1 promoter, on the other hand, has an atypical p53-binding site with the consensus sequence repeated in a head to tail ()) ))) orientation with a 12-bp spacer between the two pairs of repeats (8). Survivin is a member of the inhibitor of apoptosis (IAP) family. It is expressed in fetal tissues and in most types of cancer, but the expression is very low in normal adult tissues (10, 11). Unlike other members of the IAP family, Survivin can inhibit apoptosis and elicit drug/radiation resistance as well as positively regulate progression through the cell cycle. MDR1 gene encodes P-glycoprotein (PGP), an energy-dependent drug efflux pump. An increase in PGP is largely responsible for the emergence of multidrug-resistant cells (12). Estrogen receptor (ER) a and ERh are members of the superfamily of nuclear receptors (13, 14). These receptors mediate the effects of the ligand 17h-estradiol (E2) by functioning as transcriptional regulators that access various target gene pro- moters either by directly binding to specific estrogen response elements within the promoter or indirectly by interacting with other transcriptional regulators bound to the promoter. Further, several cases of ligand-independent activation of ERa mediated by its phosphorylation by various signaling pathways have been reported (15). Besides its function as a transcriptional regulator, ERa can also mediate several nongenomic effects of estrogen, including mobilization of intracellular calcium, production of cyclic AMP, activation of mitogen-activated protein kinase signaling pathway, increased phosphatidylinositol 3-kinase activity leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase, activation of membrane tyrosine kinase receptors, and phosphorylation of SRC homology–containing domain (16). Both ERa (13, 17–20) and p53 (21, 22) play a pivotal role in normal mammary development and in breast oncogenesis. Besides mutations in the p53 gene, a variety of molecular abnormalities in signaling pathways upstream and downstream of p53 can alter its function in breast cancer (21, 22). Further, the balance between opposing cellular signaling pathways impinging on p53 affects its ability to function as a tumor suppressor. A body of accumulating evidence suggests the possibility of a cross-talk between pathways mediated by ERa and p53. For example, treatment of mice with placental hormones resulted in the nuclear accumulation and activation of p53 in response to DNA damage (23). Further, in Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for A. Sayeed: California Pacific Medical Center, San Francisco, CA 94107. Current address for S.D. Konduri: M. D. Anderson Cancer Center Orlando, Cancer Research Institute, Orlando, FL 32806. Requests for reprints: Gokul M. Das, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-8542; Fax: 716-845-8857; E-mail: gokul.das@roswellpark.org. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3724 Cancer Res 2007; 67: (16). 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