Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

Immature natural killer cells promote progression of triple-negative breast cancer.

Natural killer (NK) cells are cytotoxic lymphocytes that accumulate within the tumor microenvironment and are generally considered to be antitumorigenic. Using single-cell RNA sequencing and functional analysis of multiple triple-negative breast cancer (TNBC) and basal tumor samples, we observed a unique subcluster of Socs3highCD11b-CD27- immature NK cells that were present only in TNBC samples. These tumor-infiltrating NK cells expressed a reduced cytotoxic granzyme signature and, in mice, were responsible for activating cancer stem cells through Wnt signaling. NK cell-mediated activation of these cancer stem cells subsequently enhanced tumor progression in mice, whereas depletion of NK cells or Wnt ligand secretion from NK cells by LGK-974 decreased tumor progression. In addition, NK cell depletion or inhibition of their function improved anti-programmed cell death ligand 1 (PD-L1) antibody or chemotherapy response in mice with TNBC. Furthermore, tumor samples from patients with TNBC and non-TNBC revealed that increased numbers of CD56bright NK cells were present in TNBC tumors and were correlated to poor overall survival in patients with TNBC. Together, our findings identify a population of protumorigenic NK cells that may be exploited for both diagnostic and therapeutic strategies to improve outcomes for patients with TNBC.

Therapeutic Role of Tamoxifen for Triple-Negative Breast Cancer: Leveraging the Interaction Between ERß and Mutant p53.

The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERß) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERß in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERß protein expression and anti-proliferative interaction between mutant p53 and ERß were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERß+TNBC, especially in the setting of brain metastasis.

Estrogen Receptor-Beta2 (ERß2)-Mutant p53-FOXM1 Axis: A Novel Driver of Proliferation, Chemoresistance, and Disease Progression in High Grade Serous Ovarian Cancer (HGSOC).

High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer. Prevalence (~96%) of mutant p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERß) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unsettled. However, whether there is functional interaction between ERß and mutant p53 in HGSOC is unknown. ERß1 and ERß2 mRNA and protein analysis in HGSOC cell lines demonstrated that ERß2 is the predominant isoform in HGSOC. Specificity of ERß2 antibody was ascertained using cells depleted of ERß2 and ERß1 separately with isoform-specific siRNAs. ERß2-mutant p53 interaction in cell lines was confirmed by co-immunoprecipitation and in situ proximity ligation assay (PLA). Expression levels of ERß2, ERα, p53, and FOXM1 proteins and ERß2-mutant p53 interaction in patient tumors were determined by immunohistochemistry (IHC) and PLA, respectively. ERß2 levels correlate positively with FOXM1 levels and negatively with progression-free survival (PFS) and overall survival (OS). Quantitative chromatin immunoprecipitation (qChIP) and mRNA expression analysis revealed that ERß2 and mutant p53 co-dependently regulated FOXM1 gene transcription. The combination of ERß2-specific siRNA and PRIMA-1MET that converts mutant p53 to wild type conformation increased apoptosis. Our work provides the first evidence for a novel ERß2-mutant p53-FOXM1 axis that can be exploited for new therapeutic strategies against HGSOC.

Estrogen Receptor-Alpha and p53 Status as Regulators of AMPK and mTOR in Luminal Breast Cancer.

Luminal breast cancer (LBC) driven by dysregulated estrogen receptor-alpha (ERα) signaling accounts for 70% of the breast cancer cases diagnosed. Although endocrine therapy (ET) is effective against LBC, about one-third of these patients fail to respond to therapy owing to acquired or inherent resistance mechanisms. Aberrant signaling via ERα, oncogenes, growth factor receptors, and mutations in tumor suppressors such as p53 impinge on downstream regulators such as AMPK and mTOR. While both AMPK and mTOR have been reported to play important roles in determining sensitivity of LBC to ET, how the ERα-p53 crosstalk impinges on regulation of AMPK and mTOR, thereby influencing therapeutic efficacy remains unknown. Here, we have addressed this important issue using isogenic breast cancer cell lines, siRNA-mediated RNA knockdown, and different modes of drug treatments. Interaction of p53 with ERα and AMPK was determined by in situ proximity ligation assay (PLA), and endogenous gene transcripts were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Further, the effect of concurrent and sequential administration of Fulvestrant-Everolimus combination on colony formation was determined. The studies showed that in cells expressing wild type p53, as well as in cells devoid of p53, ERα represses AMPK, whereas in cells harboring mutant p53, repression of AMPK is sustained even in the absence of ERα. AMPK is a major negative regulator of mTOR, and to our knowledge, this is the first study on the contribution of AMPK-dependent regulation of mTOR by ERα. Furthermore, the studies revealed that independent of the p53 mutation status, combination of Fulvestrant and Everolimus may be a viable first line therapeutic strategy for potentially delaying resistance of ERα+/HER2- LBC to ET.

TP53 Status as a Determinant of Pro- vs Anti-Tumorigenic Effects of Estrogen Receptor-Beta in Breast Cancer.

BACKGROUND: Anti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC). METHODS: ESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided. RESULTS: ESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02). CONCLUSIONS: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

Metabolic Reprogramming in Breast Cancer and Its Therapeutic Implications.

Current standard-of-care (SOC) therapy for breast cancer includes targeted therapies such as endocrine therapy for estrogen receptor-alpha (ERα) positive; anti-HER2 monoclonal antibodies for human epidermal growth factor receptor-2 (HER2)-enriched; and general chemotherapy for triple negative breast cancer (TNBC) subtypes. These therapies frequently fail due to acquired or inherent resistance. Altered metabolism has been recognized as one of the major mechanisms underlying therapeutic resistance. There are several cues that dictate metabolic reprogramming that also account for the tumors' metabolic plasticity. For metabolic therapy to be efficacious there is a need to understand the metabolic underpinnings of the different subtypes of breast cancer as well as the role the SOC treatments play in targeting the metabolic phenotype. Understanding the mechanism will allow us to identify potential therapeutic vulnerabilities. There are some very interesting questions being tackled by researchers today as they pertain to altered metabolism in breast cancer. What are the metabolic differences between the different subtypes of breast cancer? Do cancer cells have a metabolic pathway preference based on the site and stage of metastasis? How do the cell-intrinsic and -extrinsic cues dictate the metabolic phenotype? How do the nucleus and mitochondria coordinately regulate metabolism? How does sensitivity or resistance to SOC affect metabolic reprogramming and vice-versa? This review addresses these issues along with the latest updates in the field of breast cancer metabolism.

A comprehensive analysis of coregulator recruitment, androgen receptor function and gene expression in prostate cancer.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0-57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.

Estrogen receptor alpha (ERα/ESR1) mediates the p53-independent overexpression of MDM4/MDMX and MDM2 in human breast cancer.

MDM2 and MDM4 are heterodimeric, non-redundant oncoproteins that potently inhibit the p53 tumor suppressor protein. MDM2 and MDM4 also enhance the tumorigenicity of breast cancer cells in in vitro and in vivo models and are overexpressed in primary human breast cancers. Prior studies have characterized Estrogen Receptor Alpha (ERα/ESR1) as a regulator of MDM2 expression and an MDM2- and p53-interacting protein. However, similar crosstalk between ERα and MDM4 has not been investigated. Moreover, signaling pathways that mediate the overexpression of MDM4 in human breast cancer remain to be elucidated. Using the Cancer Genome Atlas (TCGA) breast invasive carcinoma patient cohort, we have analyzed correlations between ERα status and MDM4 and MDM2 expression in primary, treatment-naïve, invasive breast carcinoma samples. We report that the expression of MDM4 and MDM2 is elevated in primary human breast cancers of luminal A/B subtypes and associates with ERα-positive disease, independently of p53 mutation status. Furthermore, in cell culture models, ERα positively regulates MDM4 and MDM2 expression via p53-independent mechanisms, and these effects can be blocked by the clinically-relevant endocrine therapies fulvestrant and tamoxifen. Additionally, ERα also positively regulates p53 expression. Lastly, we report that endogenous MDM4 negatively regulates ERα expression and forms a protein complex with ERα in breast cancer cell lines and primary human breast tumor tissue. This suggests direct signaling crosstalk and negative feedback loops between ERα and MDM4 expression in breast cancer cells. Collectively, these novel findings implicate ERα as a central component of the p53-MDM2-MDM4 signaling axis in human breast cancer.

Pathway-centric integrative analysis identifies RRM2 as a prognostic marker in breast cancer associated with poor survival and tamoxifen resistance.

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2-enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa.

Tumor suppressor p53 and estrogen receptors in nuclear-mitochondrial communication.

Several gene transcription regulators considered solely localized within the nuclear compartment are being reported to be present in the mitochondria as well. There is growing interest in the role of mitochondria in regulating cellular metabolism in normal and disease states. Various findings demonstrate the importance of crosstalk between nuclear and mitochondrial genomes, transcriptomes, and proteomes in regulating cellular functions. Both tumor suppressor p53 and estrogen receptor (ER) were originally characterized as nuclear transcription factors. In addition to their individual roles as regulators of various genes, these two proteins interact resulting in major cellular consequences. In addition to its nuclear role, p53 has been localized to the mitochondria where it executes various transcription-independent functions. Likewise, ERs are reported to be present in mitochondria; however their functional roles remain to be clearly defined. In this review, we provide an integrated view of the current knowledge of nuclear and mitochondrial p53 and ERs and how it relates to normal and pathological physiology.

Transcriptional inhibition of p21WAF1/CIP1 gene (CDKN1) expression by survivin is at least partially p53-dependent: evidence for survivin acting as a transcription factor or co-factor.

Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21(WAF1/CIP1) by p53 plays an important role in p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21(WAF1/CIP1) expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21(WAF1/CIP1) protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21(WAF1/CIP1) expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21(WAF1/CIP1) promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21(WAF1/CIP1) promoter leading to the inhibition of p21(WAF1/CIP1) expression at least in part by neutralizing p53-mediated transcriptional activation of the p21 gene.

Estrogen-mediated upregulation of Noxa is associated with cell cycle progression in estrogen receptor-positive breast cancer cells.

Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17ß-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA) to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G(0)/G(1)-phase and significantly delayed the G(1)-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

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

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.

Disruption of estrogen receptor alpha-p53 interaction in breast tumors: a novel mechanism underlying the anti-tumor effect of radiation therapy.

Inactivation of tumor suppressor p53 is one of the most frequent events in cancer. Unlike many other cancers, however, p53 gene mutations are infrequent in breast cancers, as about 80% of breast tumors contain wild type p53. The mechanisms underlying functional inactivation of wild type p53 in breast cancer have remained elusive. Besides, how p53 gets activated in breast tumors subjected to radiation therapy remains unknown. We recently reported that in MCF-7 breast cancer cells, estrogen receptor alpha (ERalpha) directly binds to p53 and represses its function. Furthermore, the ERalpha-p53 interaction was disrupted by ionizing radiation. These observations have important translational implications especially as there are no reliable cellular or molecular criteria for rational radiotherapy for breast cancer. Here we report our studies towards addressing this important issue, using an MCF-7 breast cancer xenograft model in mice. Radiation effectively inhibits growth of these tumors and stabilizes p53, but has no observable effect on ERalpha protein level. Importantly, chromatin immunoprecipitation (ChIP) assays demonstrated that ERalpha interacts with p53 bound to endogenous target gene promoters in tumors in vivo, and this interaction is considerably reduced in response to radiotherapy although p53 level is increased. Concomitant with its effect on ERalpha-p53 interaction, radiation increases p53-mediated transcriptional activation of several target genes and increases p53-mediated transcriptional repression of survivin. Our studies show that disruption of ERalpha-p53 interaction in vivo resulting in restoration of functional p53 is a cellular response to radiation. Radiation could be affecting ERalpha and/or p53 directly or it could be influencing other proteins associated with the ERalpha-p53 complex. To the best of our knowledge, this is the first report on analysis of DNA-protein-protein interaction occurring on endogenous gene promoters in vivo in breast tumor tissues. These findings suggest that alleviating the inhibitory effect of ERalpha on p53 could be one of the molecular mechanisms underlying activation of p53 by radiation in breast tumors, and therefore, could be exploited to develop more effective ways of combining radiation therapy with systemic therapies such as hormonal therapy and chemotherapy.

Regulation of estrogenic effects by beclin 1 in breast cancer cells.

Beclin 1 is an essential mediator of autophagy and a regulator of cell growth and cell death. We examined the effect of Beclin 1 overexpression on the action of estradiol (E(2)) and two antiestrogens, raloxifene and 4-hydroxytamoxifen, in estrogen receptor alpha (ERalpha)-positive MCF-7 breast cancer cells. [(3)H]-thymidine incorporation studies showed that Beclin 1-overexpressing cells (MCF-7 x beclin) had a lower proliferative response to E(2) compared with cells transfected with vector control (MCF-7 x control). There was only a 35% increase in [(3)H]-thymidine incorporation, after 24 hours of E(2) treatment of MCF-7 x beclin cells compared with untreated cells, whereas this increase was 2-fold for MCF-7 x control cells. E(2)-induced changes in the expression of early-response genes were examined by real-time quantitiative PCR. There were significant differences in the pattern of expression of E(2)-induced genes c-myc, c-fos, Erg-1, and Nur77 between MCF-7 x beclin and MCF-7 x control cells two hours after treatment. Although E(2)-induced growth of MCF-7 x control cells was completely inhibited by 500 nmol/L raloxifene or 500 nmol/L 4-hydroxytamoxifen, these concentrations of antiestrogens had no significant effect on the growth of MCF-7 x beclin cells. Confocal microscopic and coimmunoprecipitation studies showed evidence for colocalization and association of Beclin 1 and ERalpha. In addition, E(2) caused a decrease in Akt phosphorylation in MCF-7 x beclin cells, compared with a 3-fold increase in MCF-7 cells, five minutes after treatment. These results indicate that Beclin 1 can down-regulate estrogenic signaling and growth response, and contribute to the development of antiestrogen resistance. This observation might be useful to define and overcome antiestrogen resistance of breast cancer.

Estrogen-mediated downregulation of CD24 in breast cancer cells.

We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ER alpha negative MDA-MB-231 cells suggesting that ER alpha was necessary. This was further confirmed by ER alpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ER alpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ER alpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ER alpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ER alpha and histone deacetylases.

Estrogen receptor alpha inhibits p53-mediated transcriptional repression: implications for the regulation of apoptosis.

Estrogen receptor alpha (ERalpha) 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 ERalpha 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 ERalpha inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERalpha interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERalpha binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERalpha is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERalpha to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERalpha to repress p53-mediated transcriptional activation. Further, RNA interference-mediated knockdown of ERalpha 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 ERalpha opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer.

Estrogen receptor-alpha binds p53 tumor suppressor protein directly and represses its function.

Estrogen receptor-alpha (ERalpha) 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 ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha 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 ERalpha and p53. Ionizing radiation together with ERalpha knock down results in 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 pro-proliferative role of ERalpha.