Mechanisms of Estrogen Receptor Antagonism Toward P53 and Its Implications in Breast Cancer Therapeutic Response and Stem Cell Regulation

Home , Estrogen receptor alpha

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

Santhi D. Konduria,1,2, Rajesh Medisettya,1, Wensheng Liua,3, Benny Abraham Kaipparettub,c,4, Pratima Srivastavaa,5, Hiltrud Brauchb,c, Peter Fritzb,c, Wendy M. Swetziga, Amanda E. Gardnerd, Sohaib A. Khand, and Gokul M. Dasa,6

aDepartment of Pharmacology and Therapeutics, Center for Genetics and Pharmacology, Roswell Park Cancer Institute, Buffalo, NY 14263; bDr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, D 70376 Stuttgart, Germany; cUniversity Tuebingen, D 72074 Tuebingen, Germany; and dDepartment of Cancer and Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 45267

Communicated by Elwood V. Jensen, Vontz Center for Molecular Studies, Cincinnati, OH, June 30, 2010 (received for review June 17, 2010) Estrogen receptor α (ERα) plays an important role in the onset and and greatly contributes to the onset and progression of breast progression of breast cancer, whereas p53 functions as a major cancer (6, 7). tumor suppressor. We previously reported that ERα binds to p53, Similar to ERα, tumor suppressor p53 also plays a central role resulting in inhibition of transcriptional regulation by p53. Here, in many cellular processes, such as cell cycle regulation, apoptosis, we report on the molecular mechanisms by which ERα suppresses senescence, and differentiation (8–10). Although these functions p53’s transactivation function. Sequential ChIP assays demon- of p53 are essential to maintain normal cellular homeostasis, if left strated that ERα represses p53-mediated transcriptional activation uncontrolled, they can lead to deleterious consequences. As such, in human breast cancer cells by recruiting nuclear receptor core- mutations in the p53 gene and aberrations in the p53 signaling pressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). pathway pave the way to tumorigenesis (11). Extensive research RNAi-mediated down-regulation of NCoR resulted in increased over the past two decades has placed p53 at the hub of a very endogenous expression of the cyclin-dependent kinase (CDK)- complex network of signaling pathways that integrate a variety of inhibitor p21Waf1/Cip1 (CDKN1A) gene, a prototypic transcriptional intracellular and extracellular inputs. It is becoming increasingly CELL BIOLOGY target of p53. While 17β-estradiol (E2) enhanced ERα binding to clear that the opposing effects of various components of the p53 p53 and inhibited p21 transcription, antiestrogens decreased ERα signaling network must be carefully balanced to correctly regulate recruitment and induced transcription. The effects of estrogen p53 function and induce the appropriate cellular response (11, 12). and antiestrogens on p21 transcription were diametrically oppo- One such modulator of p53 signaling is ERα. We have reported site to their known effects on the conventional ERE-containing that in ER-positive human breast cancer cells, as well as in a mouse ERα target gene, pS2/TFF1. These results suggest that ERα uses xenograft model, ERα binds to p53 and represses its transcriptional dual strategies to promote abnormal cellular proliferation: en- function (13–15), resulting in inhibition of p53-mediated cell cycle hancing the transcription of ERE-containing proproliferative genes arrest (14) and apoptosis (15). and repressing the transcription of p53-responsive antiprolifera- Compared with many other cancer types, p53 mutations are not tive genes. Importantly, ERα binds to p53 and inhibits transcrip- very frequent in breast cancer. Approximately 80% of breast can- tional activation by p53 in stem/progenitor cell-containing murine cers express wild-type p53. Moreover, 70–80% of breast cancers are mammospheres, suggesting a potential role for the ER–p53 inter- also ER-positive, and the majority of these express wild-type p53, action in mammary tissue homeostasis and cancer formation. albeit functionally debilitated. Interestingly, most ERα-negative Furthermore, retrospective studies analyzing response to tamoxi- breast tumors express mutant p53 (16–18). Therefore, it is likely fen therapy in a subset of patients with ER-positive breast cancer that abrogation of the p53 signaling pathway is a major event in expressing either wild-type or mutant p53 suggest that the pres- both ERα-positive and -negative tumors; in the former, wild-type ence of wild-type p53 is an important determinant of positive p53 is functionally repressed by ERα, and in the latter, p53 is therapeutic response. inactivated by mutations. ERα is the primary target of endocrine therapy in breast cancer. However, a major clinical obstacle is that nuclear receptor corepressor | mammary epithelial cells | mammospheres | a large fraction of patients with ERα-positive breast tumors do not tumor suppressor protein | tamoxifen therapy respond to tamoxifen therapy (19–21). The presence of wild-type p53 has been reported to have a positive impact on the therapeu- ells integrate multiple pathways to elicit responses to various tic response and prognosis of breast cancer patients (17, 22–24). Csignals, depending on the cellular context and tissue micro- environment. Estrogen receptor α (ERα) is a ligand-dependent transcriptional regulator that, when bound to estrogen response Author contributions: S.D.K., R.M., W.L., B.A.K., H.B., S.A.K., and G.M.D. designed re- elements (EREs) on target gene promoters, regulates transcription search; S.D.K., R.M., W.L., B.A.K., P.S., P.F., W.M.S., and A.E.G. performed research; S.A.K. contributed new reagents/analytic tools; S.D.K., R.M., W.L., B.A.K., H.B., S.A.K., via estrogen-dependent recruitment of steroid receptor coac- and G.M.D. analyzed data; and G.M.D. wrote the paper. tivators (SRCs), such as SRC1, SRC2/DRIP1/TIF2/NCoA2/, and The authors declare no conﬂict of interest. SRC3/AIB1/TRAM1/pCIP/RAC3/ACTR, followed by chromatin 1 – S.D.K., and R.M. contributed equally to this work. histone acetylation (1 3). On the other hand, when antiestrogens 2 α Present address: Cancer Research Institute, MD Anderson Cancer Center Orlando, Orlando, such as tamoxifen are bound to ER ,variouscorepressors,in- FL 32827. cluding nuclear receptor corepressor (NCoR) and silencing medi- 3Present address: Department of Pediatrics, State University of New York, Buffalo, NY 14214. ator for retinoid and thyroid hormone receptors (SMRT), are 4Present address: Department of Molecular and Human Genetics, Baylor College of Med- recruited. This is followed by recruitment of histone deacetylases icine, Houston, TX 77030. α (HDACs), leading to transcriptional repression (4, 5). ER sig- 5Present address: Department of Drug Metabolism and Pharmacokinetics, Lupin Research naling plays an important role in many tissues, including the Park, Pune 411042, India. mammary gland, where its expression is required for normal gland 6To whom correspondence should be addressed. E-mail: [email protected]. development and the preservation of adult mammary gland func- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tion.Whenderegulated,ERα becomes abnormally proproliferative 1073/pnas.1009575107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1009575107 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 However, the cellular and molecular mechanisms by which p53 Importantly, HDAC1 was present in the p53/ERα/NCoR/SMRT influences endocrine therapy efficacy remain elusive. complex on the p21 promoter (Fig. 1A). NCoR, SMRT, and Recent progress in the identification and characterization of HDAC1 were not recruited to the p21 promoter when ERα was normal and tumor stem and progenitor cells in murine and human knocked down, suggesting that ERα is required for their re- mammary gland tissue has triggered an intense effort to explore the cruitment (Fig. 1B). ERα knockdown did not affect NCoR, mechanisms that regulate the balance between symmetric and SMRT, or HDAC1 protein expression levels (Fig. 1C) but, as asymmetric division of these cells (25, 26). Disabling p53 increases expected, resulted in the loss of ERα on the promoter (Fig. 1D), stem cell production by favoring symmetric division over asymmetric further supporting the role of ERα in recruiting the corepressors division, suggesting an important role for p53 in mammary tissue and HDAC1 to the p21 promoter. Treatment with E2 enhanced homeostasis and cancer formation (25). Importantly, a fraction of the recruitment of HDAC1 and NCoR to the p21 promoter mammospheres derived from normal murine and human mammary but did not affect SMRT recruitment (Fig. 1E). Furthermore, tissue and breast cancer tissue display stem/progenitor cell char- knocking down NCoR resulted in transcriptional activation of an acteristics and express ERα (27–29). However, whether ERα dis- exogenously transfected p21 promoter plasmid (Fig. 1F) and ables wild-type p53 in these cells remains to be elucidated. increased endogenous expression of p21 mRNA and protein In the present study, we examined the mechanisms by which (Fig. 1 G and H). Collectively, these data suggest that ERα- ERα binds to p53 on p53 target gene promoters and represses mediated inhibition of p53 transcriptional activity is NCoR-de- p53-mediated transcriptional activation. We provide evidence pendent. Furthermore, the importance of NCoR in the ERα–p53 that ERα recruits corepressors when bound to p53 and show that repressor complex is strengthened by the finding that, although the ERα–p53 interaction is relevant to normal mammary stem the ERα-L539A mutant is capable of accessing the p53 target cell regulation and to breast cancer patient response to anti- promoter via p53 (Fig. S1B), it is defective in repressing p53 (14), estrogen therapy. as this mutant is less efficient in recruiting NCoR compared with wild-type ERα (Fig. S1C). Results p53-Bound ERα Recruits Transcriptional Corepressors and HDAC1 to Effects of Estrogen and Antiestrogens Cofactor Recruitment and the p21Waf1/Cip1 (CDKN1A) Gene Promoter. We have previously Transcription of the p53 Target Gene p21 Are Diametrically Opposite demonstrated that ERα binds to p53, leading to functional re- to Their Effects on the ERE-Containing ERα Target Gene pS2. We pression of p53 transcriptional activity (13–15). To explore the previously observed that ionizing radiation (known to affect the mechanisms by which ERα inhibits p53-mediated transcriptional posttranslational modification and activation status of p53) dis- activation of the p21 gene, a prototypic p53-target gene, we rupts the ERα–p53 interaction (13, 14). Therefore, we wanted to performed conventional and sequential site-specific ChIP assays explore whether estrogens or antiestrogens, which bind to ERα to analyze whether ERα recruits corepressors to the p53-binding and modulate ERα function, affect ERα’s ability to bind to p53. site of the p21 promoter. For sequential ChIP assays, chromatin Indeed, quantitative ChIP (qChIP) analysis showed that E2 from human MCF-7 breast cancer cell extract was immunopre- treatment increased ERα binding to the p21 promoter, whereas cipitated using anti-p53 antibody, reimmunoprecipitated a sec- tamoxifen and fulvestrant (ICI 182780) disrupted the interaction ond time with anti-ERα antibody, and reimmunoprecipitated (Fig. 2A). These effects were not caused by changes in p53 a third time with antibodies against NCoR, SMRT, RIP 140, or or ERα protein levels following E2 and antiestrogen treatment HDAC1. NCoR and SMRT were found in a complex with ERα (Fig. 2B). A time-course experiment in MCF-7 cells showed that and p53 on the p21 promoter; however, the amount of RIP 140 E2 repressed endogenous p21 transcription, whereas tamoxifen in the complex was very low, reflecting low levels of this co- and fulvestrant increased p21 transcription (Fig. 2C). Impor- repressor in the complex or a relatively weak antibody (Fig. 1A). tantly, when measured in parallel by quantitative real-time PCR

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,andp53inMCF-7 cells transfected with NS or NCoR siRNAs.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009575107 Konduri et al. Downloaded by guest on September 28, 2021 neither coactivator SRC1 nor SRC2 was recruited to the p21 promoter in the presence of E2; however, as expected, both were recruited to the pS2 promoter (Fig. 3C). These observations suggest that besides regulating ERE-containing target genes, ERα can also effectively regulate p53 target genes by inﬂuencing the transcriptional function of p53 (Fig. 3D). Interestingly, repression of p53-mediated transcriptional activation by ERα appears to be gene-selective, as it fails to repress some p53 target genes tested, suggesting the importance of promoter context (Fig. S2).

ERα–p53 Interaction Exists in Mammary Stem Cells. Mammary epithelial stem cells (SCs) are considered to be primary targets in the onset and progression of breast cancer, and several recent reports demonstrate that p53 is a key regulator of stem cell division (26). Although activation of wild-type p53 in breast cancer stem cells (CSCs) has been reported to reverse this process (25), the causes of p53 inactivation remain unclear. Mammary epithelial cells α– Fig. 2. Estrogen enhances and antiestrogens disrupt the ER p53 in- form spherical colonies termed “mammospheres” when cultured teraction on the p21 promoter, leading to diametrically opposite effects on on nonadherent surfaces, and these mammospheres are enriched transcription of p21 compared with that of pS2. (A) qChIP was performed to α– in cells with functional characteristics of stem/progenitor cells analyze the ER p53 interaction on the p21 promoter in MCF-7 cells. Cells α were grown in media with dextran-coated charcoal-treated FBS for 4 d and (30). To test whether ER suppresses wild-type p53 in mammo- treated with E2 (10 nM) with or without 1 μM 4-hydroxy tamoxifen or ICI spheres, we derived mouse mammospheres (25, 31) from mam- fl/fl 182780 for 3 h. (B) Western analysis of ERα and p53 protein levels in lysates mary epithelial cells isolated from C57BL/6 ERα mice (32) of MCF-7 cells treated with E2, E2 plus tamoxifen, or E2 plus ICI 182780. (Fig. S3A). Compared with mouse mammary epithelial cells, the (C and D) The effects of estrogen and antiestrogens on p21 and pS2 tran- mammospheres expressed very high levels of Sca-1, but relatively scription, respectively, in MCF-7 cells, as assayed by qPCR (value = mean ± SD; low levels of cytokeratin 14, cytokeratin 18, E-cadherin, and P < P < * 0.05; *** 0.001). integrin α 6 (Itga6) (Fig. 4A). The expression pattern of these CELL BIOLOGY markers is a characteristic of cultured murine mammospheres – (qPCR) in the same cells, both E2 and antiestrogens elicited capable of repopulating the entire mammary gland (33 35). As the mammospheres also expressed both p53 and ERα (Fig. 4B), diametrically opposite effects on transcription of pS2/TFF2, we used micro-ChIP assays to analyze whether ERα and p53 a prototypic ERE-containing ERα target gene (Fig. 2D), com- cooccupy the p53 binding site of the p21 promoter and whether pared with their effects on p21 transcription. Therefore, we ERα occupancy suppresses p21 transcription in these cells. In- tested whether these opposite effects on p21 and pS2 gene α α deed, as is the case with MCF-7 cells, ER binds to the p53 site of transcription were caused by differential recruitment of ER , the p21 promoter but not to a nonspecific (NS) site without any corepressors, or coactivators to the p21 and pS2 gene promoters. p53-binding sequence (Fig. 4C). Subsequent deletion of the Although p53 recruitment to the p53 binding site of the p21 floxed ERα gene using Cre-recombinase–expressing lentivirus promoter was not considerably affected by E2 or antiestrogens, resulted in a decrease in ERα transcript levels and a consider- α ER recruitment was enhanced by E2 and reduced by anti- able increase in p21 levels, suggesting that loss of ERα induces A estrogens (Fig. 3 ). However, in the case of the pS2 promoter, p53 activity in stem cell-containing mammospheres (Fig. 4D). E2 enhanced ERα binding to the ERE in both the presence and absence of antiestrogens, as expected (Fig. 3A). Similarly, re- Importance of p53 Status in the Response of ER-Positive Breast cruitment of NCoR to the p21 promoter has a pattern different Cancer Patients to Tamoxifen Therapy. Based on the data obtained from that of its recruitment to the pS2 promoter. E2 enhanced from our cell culture models and mouse xenograft studies (13– NCoR recruitment to the p53 binding site of the 21 promoter, 15), we postulated that one of the determinants for responsive- whereas tamoxifen and fulvestrant increased NCoR recruitment ness to antiestrogen therapy could be the ability of antiestrogens to the ERE site of the pS2 promoter (Fig. 3B). Interestingly, to reactivate p53 by disrupting the ERα–p53 interaction. To test

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).

Konduri et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 by ERα have remained unclear. Our results demonstrate that ERα accesses p53 target gene promoters by directly binding to p53, and ERα represses p53 function by recruiting NCOR, SMRT, and HDAC1. In addition to our earlier observation that p53 is necessary for recruitment of ERα to the p21 gene promoter (14), the data presented here show that ERα is necessary for the recruitment of corepressors and HDAC1, as none of them could be detected on the p53-binding site of the p21 gene promoter when ERα was knocked down. These observations, along with the fact that ERα, NCoR, SMRT, and HDAC1 were detected on the p21 promoter in a sequential ChIP assay starting with p53 antibody, strongly suggest that p53, ERα, NCoR, SMRT, and HDAC1 coexist in a single complex on the α A p53-binding site(s) of the human p21 promoter. The functional Fig. 4. ER represses p53 in murine mammary stem cells. ( ) qPCR analysis fi of the expression of Sca1, cytokeratin 14 (Ck14), cytokeratin 18 (Ck18), E- signi cance of NCoR recruitment was ascertained by the ob- cadherin (E-Cad), and integrin α 6 (Itga6) in murine mammary epithelial cells servation that transcription from an exogenously transfected (normalized to 1) and murine stem cell-containing mammospheres derived p21 promoter reporter and the endogenous p21 gene were both from the mammary glands of C57 BL/6 ERαfl/fl mice. Data are averages from increased when NCoR was knocked down. Furthermore, the three samples with SD. (B) Western analysis of ERα and p53 proteins in ERα-L539A mutant, although capable of interacting with pro- murine mammospheres and MCF-7 cells. (C) Micro-ChIP analysis of the ERα– moter-bound p53, had reduced ability to recruit NCoR to the p53 interaction on the p21 gene promoter in murine mammospheres ER–p53 complex and, therefore, was less efficient in repressing Upper ( ). Micro-ChIP analysis of a region of the p21 gene (NS) that does not p53’s transcriptional activity. Notably, ERα-L539A, which is contain any p53-binding sites (negative control) (Lower). (D)PCRanalysis fi of ERα and p21 RNA levels in C57BL/6 ERαfl/fl murine mammospheres with also de cient in recruiting coactivators and incapable of acti- or without infection with Cre-recombinase lentivirus. vating transcription of ERE-containing target genes in the presence of E2, was capable of activating the endogenous ERα- target gene pS2 in the presence of antiestrogens (42). this hypothesis, we analyzed the p53 status of 35 ER-positive Similar to NCoR, HDAC1 appears to be playing an important tamoxifen-treated breast cancer cases and 36 ER-negative cases role in the p53/ERα/NCoR/SMRT/HDAC1 complex. HDAC1 (Table S1) with a clinical follow-up of up to 176 mo. Because binds to p53 and down-regulates its transactivation function, and only mutant p53 protein is usually detectable in clinical samples, this inactivation is largely dependent on the 30 C-terminal resi- we used positive protein detection by immunohistochemical ana- dues of p53 (43). Interestingly, this same region of p53 has been lysis to determine mutant p53 status. Importantly, none of the 17 mapped as the domain that interacts with ERα (14). As HDAC1 patients whose overall survival (OVS) was positively impacted by can directly bind to ERα (44), ERα may be facilitating HDAC1 tamoxifen treatment had detectable mutant p53 protein in their binding to p53, which in turn could lead to deacetylation and tumors (in other words, their tumors expressed wild-type p53), inactivation of p53. In addition to repressing p53 function by whereas among the 18 ER-positive patients who did not respond recruiting corepressors and HDAC1, the possibility that ERα to tamoxifen therapy, 7 (39%) had tumors expressing mutant p53. recruits other HDACs or prevents recruitment of p53 coacti- Thus, the OVS of patients with ER-positive breast cancer under vators remains to be tested. Of note, HDAC1 was reported to tamoxifen treatment was significantly influenced by p53 status antagonize p53 via Sp1 in regulating p21 transcription (45). (P = 0.007) (Fig. 5A). Multivariate analysis by Cox regression Furthermore, HDAC1 and HDAC2 act in concert to repress the model revealed that p53 is a stable predictor of OVS in ER- expression of p21 and p57 and promote G1 to S phase progression positive patients [P = 0.012; relative risk 5.4; 95% confidence in mouse embryonic fibroblasts in vitro and B cells in vivo (46). interval (CI) 1.45–20.76]. In contrast, mutant p53 was not a pre- The effects of E2 and antiestrogens were diametrically opposite dictor for OVS in patients with ER-negative tumors, for whom on p21 (prototypic p53-target gene) transcription versus pS2 tamoxifen is not a standard treatment (Fig. 5B). (prototypic ER-target gene) transcription. E2 repressed p21 transcription, whereas E2 increased pS2 transcription, as expected. Discussion Consistent with this, E2 enhanced ERα and NCoR recruitment to A strong relationship of estrogen signaling and ER with p53 has the p21 promoter, whereas both tamoxifen and fulvestrant de- been documented (16, 18, 36–41), and we have reported that creased ERα and NCoR recruitment. Similar to the p21 case, E2 ERα and p53 physically interact, leading to inhibition of p53 enhanced ERα recruitment to the pS2 promoter, although the function in cell culture and xenograft models (13–15). How- transcriptional response was opposite; both tamoxifen and fulves- ever, the mechanisms underlying functional repression of p53 trant enhanced p21 transcription but reduced pS2 transcription.

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.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009575107 Konduri et al. Downloaded by guest on September 28, 2021 These observations led us to propose a model for ERα function possibility is underway. Based on our results, future studies on (Fig. 3D). We propose that when bound to p53 on p53 target gene the ER–p53 interaction should provide insight into its role not promoters, ERα recruits corepressors in the presence of estrogen only in normal mammary gland development and breast cancer to repress transcription. Antiestrogens, on the other hand, disrupt but also in other tissues and cancers where ER and p53 are ERα-p53 interaction thereby alleviating transcriptional repression. expressed and may have preventive and therapeutic implications. This is in addition to what has been previously reported: when boundtotheEREofERtargetgenes,ERα recruits coactivators in Materials and Methods the presence of E2 to activate transcription, whereas it recruits Cell Culture. MCF-7 and Saos2 cells were maintained in DMEM supplemented corepressors in the presence of antiestrogens to repress transcrip- with 10% FBS (Invitrogen) or in DMEM media containing 10% dextran-coated tion (47, 48). Collectively, these findings suggest dual roles for ERα charcoal-treated FBS at 37 °C under 5% CO2. in promoting breast tumorigenesis: ERα increases the transcription of ERE-containing proproliferation genes and also counteracts Antibodies, Drugs, and Western Analysis. Antibodies were obtained from the p53’s ability to activate antiproliferative p53 target genes (such as following companies: anti-p53 (DO-1), -ERα (HC-20), -p21, -SMRT, -RIP140, p21, BTG2, and PUMA) and to repress proproliferative p53 target -SRC1, -SRC3, -cytokeratin-14, and -LamininA/C antibodies, and normal rab- fi α bit and mouse serum from Santa Cruz; anti-NCoR and -HDAC1 from Upstate genes (such as survivin). Our previous nding that ER represses β β both p53-mediated activation of p21 (14) and p53-mediated re- Biotechnology; and anti- -tubulin and -Actin (A2066) from Sigma-Aldrich. 17β-estradiol and 4-hydroxytamoxifen were purchased from Sigma-Aldrich, pression of survivin (15) is consistent with this model. HDAC1 and α– and ICI 182780 was purchased from Tocris Bioscience. Cell lysates were an- HDAC4, but not SRC1 or SRC3, are recruited to the ER p53 alyzed on SDS/PAGE gels, followed by Western blotting with antibodies complex on the survivin promoter, leading to inhibition of p53- against various proteins, as noted in the figure legends. Specific proteins mediated transcriptional repression (15). were detected by the enhanced chemiluminescence method (Amersham Bio- As repression of p53-mediated transcriptional activation by ERα sciences). appears to be gene-selective, promoter context likely determines the formation and/or functional consequences of the ERα–p53 com- Plasmids and siRNAs. The −1265 PCNA-luc has been previously described (14). plex. This is consistent with the importance of promoter context in PRc/CMV hp53 and −2326 p21-luc were generous gifts from A. J. Levine transcriptional outcome in general (49) and in protein–protein (Institute of Advanced Study, Princeton, NJ), and W. El-Deiry (University of interactions involving p53 (50). We observed that ERα antagonizes Pennsylvania School of Medicine, Philadelphia, PA), respectively. The pCR3.1- p53-dependent transcriptional regulation of p53 target genes by based hERα expression plasmids (ER wild-type; ER L539A) were from C. Smith

physically binding to promoter-bound p53 on p53 response ele- (Baylor College of Medicine, Houston, TX). The pCR3.1/p53 construct was gen- CELL BIOLOGY ments; however, it has also been reported that ERα and p53 (wild- erated by cloning full-length p53 cDNA (HindIII and XbaI fragment) from the type or mutant) bind to EREs and p53 response elements, re- pRc/CMV hp53 plasmid into the pCR3.1 vector. NCoR (pKD-v2) and control (pKD- spectively, to cooperatively enhance FLT1 transcription (40). The NegCon-v1) shRNA plasmids were purchased from Upstate Biotechnology. The p53 and ERα siRNAs (SMARTpools) were obtained from Dharmacon. combined results of these studies highlight the complexities of ER-mediated regulation of p53 transcriptional activity and suggest Luciferase Reporter Assays and Transfection of siRNAs and shRNAs. Transient that such regulation is highly context-dependent. It is likely that × 5 α α in cis transfections of MCF-7 (1.5 10 ) cells with ER or p53 siRNA were per- cooperation of ER with p53 (40) and physical interaction of formed using Lipofectamine 2000 (Invitrogen) according to the manufac- ERα with p53 (resulting in repression of p53 function) (13–15) are turer’s instructions. NS siRNA was used as a control. For reporter assays, 0.5 × both dependent upon the target genes and signaling context. 105 cells were seeded in 12-well plates and transfected with the following Moreover, wehavenot ruled out the possibility that on certain ERE- plasmids using Fugene 6 Transfection Reagent (Roche Diagnostics), accord- containing genes and some p53 target genes, p53 may repress ER ing to the manufacturer’s protocol: for MCF-7 cells, −2326 p21-luc reporter function. Alternatively, in some cases, physical interaction between plasmid and NCoR shRNA plasmid or a NS shRNA plasmid; for Saos2 cells, − ERα and p53 may result in activation of either ER or p53. Another 1265 PCNA-luc reporter plasmid and p53 expression plasmid alone or in intriguing possibility is a potential role for the ERα–p53 interaction combination with ERα wild-type or ER L539A mutant expression plasmid or in the “gain of function” by certain p53 mutants. Future experiments a molar equivalent of vector (PCR 3.1). The total amount of transfected DNA should address these plausible and interesting scenarios. was kept constant by the addition of empty plasmid. After 24 h, cells were Our micro-ChIP data show that ERα and p53 are expressed in harvested and lysed, and luciferase activity was measured using the Lucif- stem cell-containing murine mammospheres and that they in- erase Reporter Assay System (Promega). teract with one another, resulting in inhibition of p53’s ability qPCR. Total cellular RNA was isolated using the Absolutely RNA Miniprep kit to activate p21 transcription. It is likely that normal signaling μ α– (Stratagene). Up to 1 g of total RNA from individual samples was reverse mechanisms operating to regulate the ER p53 interaction in transcribed in 20 μL of reaction using the iScript cDNA synthesis kit (Bio-Rad). mammary SCs could be disrupted in breast CSCs, favoring pre- One microliter of the resulting cDNA was used in a total volume of 25 μLof dominance of ERα over p53 and symmetric over asymmetric cell PCR; qPCR was performed on an ABI Prism 7300 sequence detection system division, thereby leading to abnormal proliferation. with SYBR Green PCR Master Mix (Bio-Rad) using speciﬁc primers. Relative The conventional understanding of the genomic ERα signaling mRNA levels or DNA levels (in the case of qChIP) were calculated using the pathway is that antiestrogens block estrogen from binding to ΔΔCt method. For mRNA analysis, β-actin mRNA was used as an endogenous ERα, cause ERα to recruit transcriptional corepressors, and lead control. p53 binding to the p21 promoter was used to normalize q-ChIP. to transcriptional repression of ER target genes. Here we show Primer sequences for qPCR are described in SI Materials and Methods. that the antiestrogen tamoxifen can also disrupt the ERα–p53 inhibitory complex, resulting in reactivation of p53. This raised ChIP, qChIP, Sequential ChIP, and Micro-ChIP. ChIP assays were performed as ﬁ the possibility that the latter function of tamoxifen could be described previously (14, 15). For qChIP, DNA in the nal step was analyzed one of the determinants of response of ER-positive breast can- by qPCR. For sequential ChIP assays, cell lysates were initially incubated cer patients to tamoxifen therapy. Indeed, results from our pilot with anti-p53 antibody, and the immunocomplexes were eluted at 37 °C for 30 min in rechip buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM retrospective analysis of clinical OVS data of tamoxifen-treated Tris-HCl, pH 8.1), followed by incubation with anti-ERα antibody. Secondary patients are consistent with studies on other patient cohorts (17, – immunocomplexes were eluted in rechip buffer and incubated with anti- 22 24) and support the idea that ER-positive breast cancer bodies against NCoR, SMRT, RIP140, and HDAC1. Final immunocomplexes

patients whose tumors express wild-type p53 (as opposed to were eluted in elution buffer (500 μL 1% SDS, 0.1 M NaHCO3) and processed mutant p53) will be more responsive to tamoxifen therapy, as for DNA analysis. Micro-ChIP was performed essentially as described (51) tamoxifen will disrupt the ERα–p53 interaction, thereby reac- with some modiﬁcations. Additional details and primer sequences are de- tivating p53. A prospective clinical trial to directly verify this scribed in SI Materials and Methods.

Konduri et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Generation of Primary Murine Mammary Epithelial Cells and Mammospheres. SPSS software version 12.1. Univariate survival analysis was by the Kaplan- Primary mammary epithelial cells and mammospheres were prepared from 9- Meier method, and multivariate survival analysis was calculated by the Cox to 12-wk-old virgin female mice. Murine mammospheres were generated by proportional hazard model. Associations between ER status and clinical adapting previously described methods (25, 31). Details are in SI Materials parameters were assessed by χ2 test. All P values resulted from two-sided and Methods. tests and were considered signiﬁcant when P < 0.05.

Lentiviral Transduction of Mammospheres. Murine mammospheres were ACKNOWLEDGMENTS. We thank A. J. Levine (Institute of Advanced Study, transduced with Cre-recombinase lentivirus at 1 × 107 TU/mL or multiplicity Princeton, NJ), C. L. Smith (Baylor College of Medicine, Houston, TX), and of infection (MOI) 1 on day 4 of their growth and harvested on day 6 for W. S. El-Deiry (University of Pennsylvania School of Medicine, Philadelphia, subsequent molecular analysis. Cre-recombinase lentivirus was prepared as PA) for generous gifts of plasmids; R. Chakrabarti and S. Sinha (State Uni- described in SI Materials and Methods. versity of New York at Buffalo, Buffalo, NY) for help in optimization of mouse mammary epithelial cell culture and generation of mammospheres; Analysis of Clinical Breast Cancer Samples. Files of patients with breast cancer M. Ip (Roswell Park Cancer Institute, Buffalo, NY) for discussions and critical reading of the manuscript; and S. Bansal, A. Sayeed (Roswell Park Cancer (surgery performed 1986 through 1988) were retrieved from the surgical Institute, Buffalo, NY) and D. Singleton (University of Cincinnati College of pathology archives at the Dr. Margarete Fischer-Bosch-Institute. Seventy-one Medicine, Cincinnati, OH) for discussions and general help. This work was breast cancer cases were selected for the study. Breast cancer diagnosis was supported by National Cancer Institute Grant CA079911 (to G.M.D.), Susan G. ﬁ ﬁ con rmed with hematoxylin and eosin staining of paraf n-embedded tissue Komen for the Cure Foundation Grant BCTR0600180 (to G.M.D.), The Jayne α sections. Immunohistochemical analyses for ER and p53 statuses were & Phil Hubbell Family and Roswell Park Alliance Foundation Grant (to performed on 3-μm sections using Dako clone 1D5 and clone DO-7 anti- G.M.D.), National Institutes of Health Grants T32 CA059268 (to A.E.G.) bodies, respectively, in a DAKO autostainer with ChemMate En Vision K5007 and 2T32CA009072 (to W.M.S.), Robert Bosch Foundation Stuttgart (to (Dako Cytomation). Slides were evaluated by an expert pathologist who was B.A.K., H.B., and F.P.), and National Cancer Institute Cancer Center Sup- blinded to the clinical information. Statistical analysis was performed using port Grant CA016056 (to Roswell Park Cancer Institute).

1. Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functions 27. Alvi AJ, et al. (2003) Functional and molecular characterisation of mammary side of nuclear receptors. Genes Dev 14:121–141. population cells. Breast Cancer Res 5:R1–R8. 2. McKenna NJ, O’Malley BW (2002) Combinatorial control of gene expression by 28. Clarke RB, et al. (2005) A putative human breast stem cell population is enriched for nuclear receptors and coregulators. Cell 108:465–474. steroid receptor-positive cells. Dev Biol 277:443–456. 3. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M (2000) Cofactor dynamics and 29. Dontu G, El-Ashry D, Wicha MS (2004) Breast cancer, stem/progenitor cells and the sufficiency in estrogen receptor-regulated transcription. Cell 103:843–852. estrogen receptor. Trends Endocrinol Metab 15:193–197. 4. Perissi V, Jepsen K, Glass CK, Rosenfeld MG (2010) Deconstructing repression: Evolving 30. Dontu G, et al. (2003) In vitro propagation and transcriptional profiling of human models of co-repressor action. Nat Rev Genet 11:109–123. mammary stem/progenitor cells. Genes Dev 17:1253–1270. 5. Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: From bacteria 31. Pei XH, et al. (2009) CDK inhibitor p18(INK4c) is a downstream target of GATA3 and and yeast to mice and men. Nat Rev Mol Cell Biol 9:206–218. restrains mammary luminal progenitor cell proliferation and tumorigenesis. Cancer 6. Clarke RB, Anderson E, Howell A (2004) Steroid receptors in human breast cancer. Cell 15:389–401. Trends Endocrinol Metab 15:316–323. 32. Feng Y, Manka D, Wagner KU, Khan SA (2007) Estrogen receptor-alpha expression in 7. Deroo BJ, Korach KS (2006) Estrogen receptors and human disease. J Clin Invest 116: the mammary epithelium is required for ductal and alveolar morphogenesis in mice. 561–570. Proc Natl Acad Sci USA 104:14718–14723. 8. Riley T, Sontag E, Chen P, Levine A (2008) Transcriptional control of human p53- 33. Liao MJ, et al. (2007) Enrichment of a population of mammary gland cells that form regulated genes. Nat Rev Mol Cell Biol 9:402–412. mammospheres and have in vivo repopulating activity. Cancer Res 67:8131–8138. 9. Vousden KH, Prives C (2009) Blinded by the Light: The Growing Complexity of p53. 34. Welm BE, et al. (2002) Sca-1(pos) cells in the mouse mammary gland represent an Cell 137:413–431. enriched progenitor cell population. Dev Biol 245:42–56. 10. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310. 35. Pontier SM, Muller WJ (2009) Integrins in mammary-stem-cell biology and breast- 11. Oren M, et al. (2002) Regulation of p53: Intricate loops and delicate balances. cancer progression—a role in cancer stem cells? J Cell Sci 122:207–214. Biochem Pharmacol 64:865–871. 36. Díaz-Cruz ES, Furth PA (2010) Deregulated estrogen receptor alpha and p53 heterozygosity 12. Agarwal ML, Taylor WR, Chernov MV, Chernova OB, Stark GR (1998) The p53 collaborate in the development of mammary hyperplasia. Cancer Res 70:3965–3974. network. J Biol Chem 273:1–4. 37. Liu G, Schwartz JA, Brooks SC (2000) Estrogen receptor protects p53 from deactivation 13. Liu W, Ip MM, Podgorsak MB, Das GM (2009) Disruption of estrogen receptor alpha- by human double minute-2. Cancer Res 60:1810–1814. p53 interaction in breast tumors: A novel mechanism underlying the anti-tumor 38. Shirley SH, et al. (2009) Transcriptional regulation of estrogen receptor-alpha by p53 effect of radiation therapy. Breast Cancer Res Treat 115:43–50. in human breast cancer cells. Cancer Res 69:3405–3414. 14. Liu W, et al. (2006) Estrogen receptor-alpha binds p53 tumor suppressor protein 39. Sivaraman L, Conneely OM, Medina D, O’Malley BW (2001) p53 is a potential directly and represses its function. J Biol Chem 281:9837–9840. mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis. 15. Sayeed A, et al. (2007) Estrogen receptor alpha inhibits p53-mediated transcriptional Proc Natl Acad Sci USA 98:12379–12384. repression: Implications for the regulation of apoptosis. Cancer Res 67:7746–7755. 40. Menendez D, Inga A, Resnick MA (2010) Estrogen receptor acting in cis enhances WT 16. Cattoretti G, Rilke F, Andreola S, D’Amato L, Delia D (1988) P53 expression in breast and mutant p53 transactivation at canonical and noncanonical p53 target sequences. cancer. Int J Cancer 41:178–183. Proc Natl Acad Sci USA 107:1500–1505. 17. Miller LD, et al. (2005) An expression signature for p53 status in human breast cancer 41. Medina D, Kittrell FS (2003) p53 function is required for hormone-mediated predicts mutation status, transcriptional effects, and patient survival. Proc Natl Acad protection of mouse mammary tumorigenesis. Cancer Res 63:6140–6143. Sci USA 102:13550–13555. 42. Lupien M, et al. (2007) Raloxifene and ICI182,780 increase estrogen receptor-alpha 18. Olivier M, et al. (2006) The clinical value of somatic TP53 gene mutations in 1,794 association with a nuclear compartment via overlapping sets of hydrophobic amino patients with breast cancer. Clin Cancer Res 12:1157–1167. acids in activation function 2 helix 12. Mol Endocrinol 21:797–816. 19. Musgrove EA, Sutherland RL (2009) Biological determinants of endocrine resistance in 43. Juan LJ, et al. (2000) Histone deacetylases specifically down-regulate p53-dependent breast cancer. Nat Rev Cancer 9:631–643. gene activation. J Biol Chem 275:20436–20443. 20. Jordan VC, O’Malley BW (2007) Selective estrogen-receptor modulators and antihor- 44. Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and deacetylation of monal resistance in breast cancer. JClinOncol25:5815–5824. non-histone proteins. Gene 363:15–23. 21. Schiff R, Osborne CK (2005) Endocrinology and hormone therapy in breast cancer: 45. Lagger G, et al. (2003) The tumor suppressor p53 and histone deacetylase 1 are New insight into estrogen receptor-alpha function and its implication for endocrine antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene. therapy resistance in breast cancer. Breast Cancer Res 7:205–211. Mol Cell Biol 23:2669–2679. 22. Bergh J, Norberg T, Sjögren S, Lindgren A, Holmberg L (1995) Complete sequencing of 46. Yamaguchi T, et al. (2010) Histone deacetylases 1 and 2 act in concert to promote the the p53 gene provides prognostic information in breast cancer patients, particularly G1-to-S progression. Genes Dev 24:455–469. in relation to adjuvant systemic therapy and radiotherapy. Nat Med 1:1029–1034. 47. McDonnell DP, Norris JD (2002) Connections and regulation of the human estrogen 23. Yamashita H, et al. (2006) p53 protein accumulation predicts resistance to endocrine receptor. Science 296:1642–1644. therapy and decreased post-relapse survival in metastatic breast cancer. Breast Cancer 48. Shang Y, Brown M (2002) Molecular determinants for the tissue specificity of SERMs. Res 8:R48. Science 295:2465–2468. 24. Berns EM, et al. (2000) Complete sequencing of TP53 predicts poor response to 49. Das G, Hinkley CS, Herr W (1995) Basal promoter elements as a selective determinant systemic therapy of advanced breast cancer. Cancer Res 60:2155–2162. of transcriptional activator function. Nature 374:657–660. 25. Cicalese A, et al. (2009) The tumor suppressor p53 regulates polarity of self-renewing 50. Schumm K, Rocha S, Caamano J, Perkins ND (2006) Regulation of p53 tumour suppressor divisions in mammary stem cells. Cell 138:1083–1095. target gene expression by the p52 NF-kappaB subunit. EMBO J 25:4820–4832. 26. Krizhanovsky V, Lowe SW (2009) Stem cells: The promises and perils of p53. Nature 51. Dahl JA, Collas P (2008) MicroChIP—a rapid micro chromatin immunoprecipitation 460:1085–1086. assay for small cell samples and biopsies. Nucleic Acids Res 36:e15.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009575107 Konduri et al. Downloaded by guest on September 28, 2021