ERα-positive Human Breast Cancer Research Articles

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

Bisphenol A exposure induces metastatic aggression in low metastatic MCF-7 cells via PGC-1α mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity

AimsThe frequency of estrogen receptor alpha (ERα)-positive breast cancers and their metastatic progression is prevalent in females globally. Aberrant interaction of estrogen-like endocrine-disrupting chemicals (EDCs) is highly implicated in breast carcinogenesis. Studies have shown that single or acute exposures of weak EDCs such as bisphenol A (BPA) may not have a substantial pro-carcinogenic/metastatic effect. However, repeated exposure to EDCs is expected to strongly induce carcinogenic/metastatic progression, which remains to be studied. Main methodsLow metastatic ERα-positive human breast cancer cells (MCF-7) were exposed to nanomolar doses of BPA every 24 h (up to 200 days) to study the effect of repeated exposure on metastatic potential. Following the designated treatment of BPA, markers of epithelial-mesenchymal transition (EMT), migration and invasion, mitochondrial biogenesis, ATP levels, and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) knockdown assays were performed. Key findingsA repeated exposure of low dose BPA induced stable epithelial-mesenchymal plasticity in MCF-7 cells to augment migration and invasion in the ERα-dependent pathway. Repeated exposures of BPA increased the levels of several mesenchymal markers such as N-cadherin, vimentin, cluster of differentiation 44 (CD44), slug, and alpha-smooth muscle actin (α-SMA), whereas reduced the level of E-cadherin drastically. BPA-induced mitochondrial biogenesis favored metastatic aggression by fulfilling bioenergetics demand via PGC-1α/NRF1/ERRα signaling. Knockdown of PGC-1α resulted in suppressing both mitochondrial biogenesis and EMT in BPA exposed MCF-7 cells. SignificanceRepeated exposures of low dose BPA may induce metastatic aggression in ERα-positive breast cancer cells via PGC-1α-mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity.

Detection of Estrogen Receptor Alpha and Assessment of Fulvestrant Activity in MCF-7 Tumor Spheroids Using Microfluidics and SERS.

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity.

HDAC3-ERα Selectively Regulates TNF-α-Induced Apoptotic Cell Death in MCF-7 Human Breast Cancer Cells via the p53 Signaling Pathway.

Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3–ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3–ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3–ERα complex and substitution of the occupancy on the promoter by the p53–p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3–ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.

Cytotoxic Activity on MCF-7 Cells and in Silico Study of Sapogenin Steroids From Trigonella foenum-graecum L.

Trigonella foenum-graecum (TFG) is one of medicinal plants containing several steroidal sapogenins, such as diosgenin, yamogenin, gitogenin, tigogenin and trigoneoside, and also alkaloid trigonellin and some flavonoids such as vitexin, isovitexin, orientin, isoorientin, which has many activities, such as antidiabetic, estrogenic, and also anticancer. As phytoestrogen, TFG was predicted to have potency as Selective Estrogen Receptor Modulators (SERMs) which is used for hormonal-dependent breast cancer treatment. This experiment was carried out to investigate interaction of some sapogenin steroids and flavonoids in TFG to estrogen receptor alpha (ERα) and its activity to breast cancer cell line as confirmation. In silico prediction was carried out to investigate their estrogenic activity by analyzing their binding affinity to ERα using AutoDock Vina program. In vitro activity confirmation of TFG extract and its fractions were carried out using MTT assay on Erα-positive human breast cancer cell line, MCF-7. Results showed that free binding energies of diosgenin and yamogenin were -6.4 kcal/mol, estradiol was -6.0 kcal/mol, and tamoxifen was -5.1 kcal/mol. While cytotoxicity assay showed that ethyl acetate fraction gave the lowest IC50 of 41.81 ppm, with total steroid content of 20.03 ppm. From these results, we can conclude that diosgenin and yamogenin have greater binding affinity to ERα comparing to estradiol and tamoxifen. In vitro assay confirmation showed that ethyl acetate fraction has cytotoxic effect on MCF-7 cells.Keywords: Trigonella foenum-graecum, sapogenin steroids, MCF-7, estrogen receptor alpha, binding affinity

Neuroglobin overexpression induced by the 17β-Estradiol-Estrogen receptor-α Pathway reduces the sensitivity of MCF-7 Breast cancer cell to paclitaxel.

Although paclitaxel (Taxol) is an active chemotherapeutic agent for the treatment of breast cancer, not all breast tumors are sensitive to this drug. In particular, there is a wide agreement on the low sensitivity of estrogen receptor (ER) α-positive breast cancer to paclitaxel treatment. However, the ERα-based insensitivity to paclitaxel is still elusive. Here, the effect of the E2/ERα-dependent upregulation of neuroglobin (NGB), an antiapoptotic globin, on the reduced sensitivity of breast cancer cells to paclitaxel-induced apoptosis has been evaluated in ERα-containing MCF-7 cells. The E2 pretreatment enhances the ERα activity and significantly impairs paclitaxel-induced apoptosis as evaluated by Annexin V assay and PARP-1 cleavage. NGB displays a pivotal role in the E2/ERα-induced antiapoptotic pathway to abrogate paclitaxel-induced cell death in stable NGB-silenced MCF-7 cell clones. Moreover, in the absence of the active ERα, paclitaxel significantly reduces the NGB cell content. In conclusion, these results highlight the involvement of ERα activation and of E2/ERα-dependent NGB upregulation in the insensitivity of MCF-7 to paclitaxel. These novel findings could have important implications in the development of targeted therapeutics for overcoming paclitaxel insensitivity in ERα-positive human breast cancer. © 2016 IUBMB Life, 68(8):645-651, 2016.

Estrogen receptor alpha inhibits senescence-like phenotype and facilitates transformation induced by oncogenic ras in human mammary epithelial cells.

Exposure to estrogen has long been associated with an increased risk of developing breast cancer. However, how estrogen signaling promotes breast carcinogenesis remains elusive. Senescence is known as an important protective response to oncogenic events. We aimed to elucidate the role of estrogen receptor alpha (ERα) on senescence in transformed human mammary epithelial cells and breast cancer cells. Our results show that ectopic expression of oncoprotein H-ras-V12 in immortalized human mammary epithelial cells (HMEC) significantly inhibited the phosphorylation of the retinoblastoma protein (Rb) and increased the activity of the senescence-associated beta-galactosidase (SA-β-Gal). These senescence-like phenotypes were reversed by ectopic expression of ERα. Similar inhibition of the H-ras-V12-induced SA-β-Gal activity by ERα was also observed in the human mammary epithelial MCF-10A cells. Co-expression of ERα and H-ras-V12 resulted in HMEC anchorage-independent growth in vitro and tumor formation in vivo. Furthermore, inhibition of ERα expression induced senescence-like phenotypes in ERα positive human breast cancer cells such as increased activity of SA-β-Gal, decreased phosphorylation of RB, and loss of mitogenic activity. Thus, the suppression of cellular senescence induced by oncogenic signals may be a major mechanism by which ERα promotes breast carcinogenesis.

Triptolide inhibits human breast cancer MCF-7 cell growth via downregulation of the ERα-mediated signaling pathway.

To investigate the anticancer mechanisms of triptolide, a diterpenoid isolated from the plant Tripterygium wilfordii Hook F, against human breast cancer cells and the involvement of the estrogen receptor-α (ERα)-mediated signaling pathway in particular. Human breast cancer ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells were tested. PrestoBlue assay was used to evaluate the cell viability. The levels of ERα mRNA and protein were detected with real-time PCR and immunoblotting, respectively. Mouse models of MCF-7 or MDA-MB-231 xenograft tumors were treated with triptolide (0.4 mg·kg(-1)·d(-1), po) or a selective estrogen receptor modulator tamoxifen (mg·kg(-1)·d(-1), po) for 3 weeks, and the tumor weight and volume were measured. Triptolide (5-200 nmol/L) dose-dependently inhibited the viability of both MCF-7 and MDA-MB-231 cells, with a more potent inhibition on MCF-7 cells. Knockdown of ERα in MCF-7 cells by siRNA significantly attenuated the cytotoxicity of triptolide, whereas overexpression of ERα in MDA-MB-231 cells markedly enhanced the cytotoxicity. Triptolide dose-dependently decreased the expression of ERα in MCF-7 cells and MCF-7 xenograft tumors. Furthermore, treatment of MCF-7 cells with triptolide inhibited the phosphorylation of ERK1/2 in dose- and time-dependent manners. In the mice xenografted with MCF-7 cells, treatment with triptolide or tamoxifen resulted in significant reduction in the tumor weight and volume. Similar effects were not obtained in the mice xenografted with MDA-MB-231 cells. The anticancer activity of triptolide against ERα-positive human breast cancer is partially mediated by downregulation of the ERα-mediated signaling pathway.

A kinetic model identifies phosphorylated estrogen receptor‐α (ERα) as a critical regulator of ERα dynamics in breast cancer

Receptor levels are a key mechanism by which cells regulate their response to stimuli. The levels of estrogen receptor-α (ERα) impact breast cancer cell proliferation and are used to predict prognosis and sensitivity to endocrine therapy. Despite the clinical application of this information, it remains unclear how different cellular processes interact as a system to control ERα levels. To address this question, experimental results from the ERα-positive human breast cancer cell line (MCF-7) treated with 17-β-estradiol or vehicle control were used to develop a mass-action kinetic model of ERα regulation. Model analysis determined that RNA dynamics could be captured through phosphorylated ERα (pERα)-dependent feedback on transcription. Experimental analysis confirmed that pERα-S118 binds to the estrogen receptor-1 (ESR1) promoter, suggesting that pERα can feedback on ESR1 transcription. Protein dynamics required a separate mechanism in which the degradation rate for pERα was 8.3-fold higher than nonphosphorylated ERα. Using a model with both mechanisms, the root mean square error was 0.078. Sensitivity analysis of this combined model determined that while multiple mechanisms regulate ERα levels, pERα-dependent feedback elicited the strongest effect. Combined, our computational and experimental results identify phosphorylation of ERα as a critical decision point that coordinates the cellular circuitry to regulate ERα levels.

Abstract LB-183: Y-box binding protein-1 YB-1 negatively regulates ERα expression accompanying by enhanced HER2/ErbB2 expression in breast cancer

Abstract [Purpose] YB-1 containing a cold-shock domain is a potent oncogene for breast cancer (Bergmann et al., Cancer Res, 2005). Our previous studies have demonstrated that YB-1 plays pivotal roles not only in acquisition of global drug resistance and cell proliferation (Kuwano et al., Mol. Cancer Ther., 2004), but also in expression of HER2/ErbB2 in breast cancer cells (Fujii et al., Cancer Res., 2008) and stomach cancer cells (Shibata et al., Mol. Cancer Ther., 2013) in culture and clinical samples. In our present study, we first asked whether ERα affect YB-1-induced HER2 expression in breast cancer cells, and then asked whether YB-1 activation could be biostatistically linked to HER2 and/or ER expression in breast cancer patients. [Results] Is this study, we first examined the role of YB-1 in expression of HER2 and ERα by using ERα-positive and -negative breast cancer cell lines in culture, and we next examined whether YB-1 expression in nucleus could be biostatistically associated with HER2 or ERα expression by using clinical specimens of breast cancer. We observed novel findings as follows. [1] Knockdown of YB-1 markedly reduced HER2/ErbB2 expression in ERα positive human breast cancer cells, but only slightly reduced HER2/ErbB2 expression in ERα-negative breast cancer cells. [2] Nuclear YB-1 was forced expression by the tetracycline-induced YB-1/Tet-On system. Nuclear YB-1 expression enhanced HER2 expression and decreased ERα expression in ERα-positive cancer cells. By contrast, in ERα-negative breast cancer cells, there was no apparent increase in HER2 expression by YB-1. [3] Exogenous introduction of ERα cDNA resulted in suppression of HER2 expression dependent on YB-1 in ERα negative cells. Furthermore, treatment with tamoxifen stimulated YB-1-dependent HER2 expression in ERα-positive cells. [4] We further examined expression of YB-1, ERα, PgR and HER2 by immunohistochemical (IHC) analysis in clinical samples of breast cancer patients who had received no treatments by hormonal and chemical therapeutics. Based on expression levels of various factors, nuclear YB-1 expression was significantly correlated with HER2 expression, but negatively correlated with ERα expression in tumors of postmenopausal patients (n=114). By contrast, such significant association of YB-1 expression with ERα or HER2 expression was not seen in tumors of premenopausal patients (n=57). [Conclusions] YB-1 positively regulated HER2 expression and negatively ERα expression in ERα positive breast cancer cells, and the presence of ERα seems to be required for YB-1-dependent HER2 expression. Furthermore, in breast tumors of post-menopausal patients, nuclear localization of YB-1 was positively correlated with HER2 expression and negatively correlated with ERα expression. This study may contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Ryuji Takahashi, Kosuke Watari, Yuichi Murakami, Kimitoshi Kohno, Ken-ichi Ito, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Y-box binding protein-1 YB-1 negatively regulates ERα expression accompanying by enhanced HER2/ErbB2 expression in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-183. doi:10.1158/1538-7445.AM2014-LB-183

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

α-Mangostin-induced apoptosis is mediated by estrogen receptor α in human breast cancer cells

In this study, we evaluated the effects of α-mangostin on cell growth inhibition and induction of apoptosis in MCF-7 ERα-positive human breast cancer cells. Our results showed that α-mangostin inhibited MCF-7 cell proliferation whereas ERα-negative MDA-MB-231 cells were less sensitive to the agent. Additionally, α-mangostin effectively induced apoptosis as evidenced by the appearance of apoptotic nuclei observed with Hoechst 33258 staining and evaluation of sub-G1 DNA contents by flow cytometry. α-Mangostin also activated caspases-8, -9, and -7; increased the protein levels of Bax, p53, and cytosolic cytochrome c; and induced PARP cleavage while reducing Bid and Bcl-2 protein expression. In addition, apoptosis-inducing factor (AIF) was transported from mitochondria to the cytosol after α-mangostin treatment. α-mangostin also induced apoptosis in 17-β-estradiol (E2)-stimulated MCF-7 cells in parallel with the non-stimulated cells. Moreover, treatment with 10μM α-mangostin for 48h specifically decreased the expression of ERα and pS2, an estrogen-responsive gene, in MCF-7 cells. Furthermore, knockdown of ERα expression in MCF-7 cells with siRNA attenuated α-mangostin-induced cell growth inhibition and caspase-7 activation. These results suggest that ERα is required for α-mangostin-induced growth inhibition and apoptosis in human breast cancer cells. Therefore, α-mangostin may be used to prevent and treat of ER-positive breast cancer.

NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells

BackgroundThe placenta-specific 1 (PLAC1) gene encodes a membrane-associated protein which is selectively expressed in the placental syncytiotrophoblast and in murine fetal tissues during embryonic development. In contrast to its transcriptional repression in all other adult normal tissues, PLAC1 is frequently activated and highly expressed in a variety of human cancers, in particular breast cancer, where it associates with estrogen receptor α (ERα) positivity. In a previous study, we showed that ERα-signaling in breast cancer cells transactivates PLAC1 expression in a non-classical pathway. As the members of the p160/nuclear receptor co-activator (NCOA) family, NCOA1, NCOA2 and NCOA3 are known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation we asked if these proteins are involved in the ERα-mediated transactivation of PLAC1 in breast cancer cells.MethodsApplying quantitative real-time RT-PCR (qRT-PCR), Western Blot analysis and chromatin immunoprecipitation, we analyzed the involvement of NCOA1, NCOA2, NCOA3 in the ERα-mediated transactivation of PLAC1 in the breast cancer cell lines MCF-7 and SK-BR-3. RNAi-mediated silencing of NCOA3, qRT-PCR, Western blot analysis and ERα activation assays were used to examine the role of NCOA3 in the ERα-mediated regulation of PLAC1 in further detail. Transcript expression of NCOA3 and PLAC1 in 48 human breast cancer samples was examined by qRT-PCR and statistical analysis was performed using Student’s t-test.ResultsWe detected selective recruitment of NCOA3 but not NCOA1 or NCOA2 to the PLAC1 promoter only in ERα-positive MCF-7 cells but not in ERα-negative SK-BR-3 breast cancer cells. In addition, we demonstrate that silencing of NCOA3 results in a remarkable decrease of PLAC1 expression levels in MCF-7 cells which cannot be restored by treatment with estradiol (E2). Moreover, significant higher transcript levels of PLAC1 were found only in ERα-positive human breast cancer samples which also show a NCOA3 overexpression.ConclusionsIn this study, we identified NCOA3 as a selective co-activator of ERα-mediated transactivation of PLAC1 in MCF-7 breast cancer cells. Our data introduce PLAC1 as novel target gene of NCOA3 in breast cancer, supporting the important role of both factors in breast cancer biology.

Synthesis of Novel Estrogen Receptor Antagonists Using Metal-Catalyzed Coupling Reactions and Characterization of Their Biological Activity

Estrogen receptor (ER) antagonists are valuable in the treatment of ER-positive human breast cancer. In this study, we designed and synthesized nine new derivatives of 17β-estradiol (E2) with a bulky side chain attached to its C-7α position, and determined their ER antagonistic activity using in vitro bioassays. Four of the derivatives showed a strong inhibition of ERα transactivation activity in a luciferase reporter assay and blocked ERα interactions with coactivators. Similarly, these derivatives also strongly inhibited the growth of the ERα-positive human breast cancer cells. Computational docking analysis was conducted to model the interaction of these antagonists with the human ERα and showed that they could tightly bind to the ERα in a manner similar to that of ICI-182,780, a pure ER antagonist. These results provide an example that attachment of a bulky side chain to the C-7α position of E2 can produce ER antagonists with ER affinity comparable to that of ICI-182,780.

RNA sequencing of MCF-7 breast cancer cells identifies novel estrogen-responsive genes with functional estrogen receptor-binding sites in the vicinity of their transcription start sites.

Estrogen receptor α (ERα) is a key transcription factor in breast cancer, which plays an essential role in the pathophysiology of the disease by regulating the expression of various target genes. In the present study, we performed deep RNA sequencing (RNA-seq) as an unbiased high-throughput technique for comprehensive transcriptome analysis in ERα-positive human breast cancer MCF-7 cells, to facilitate the elucidation of ERα regulatory gene networks. From the 17,336 mapped RefSeq genes from the sequenced fragments of the cell samples treated with estrogen time dependently, substantial numbers of sequence reads were observed in 3,386 genes (>100 tags per million reads per sample at any of the six time points studied). ERα occupancy within and in the proximal regions of the genes (<10-kb upstream and downstream regions) was significantly enriched in the subgroup of the 3,386 genes compared to the whole 17,336 RefSeq genes. Of the 3,386 genes, we focused on 29 genes, which included ERα occupancy adjacent to their transcription start sites and whose expression was estrogen dependently altered by >3-fold. Knockdown studies using siRNAs specific to the 29 genes validated that prototypic ERα targets V-myc myelocytomatosis viral oncogene homolog and cyclin D1 promote both proliferation and migration of MCF-7 cells and further identified novel candidate ERα targets EIF3A and tumor protein D52-like 1, which will also facilitate the proliferation or migration of MCF-7 cells. Taken together, the present findings provide a valuable dataset that will elucidate ERα regulatory mechanisms in breast cancer biology, based on the integrative analysis of RNA-seq combined with the genome-wide information for ERα occupancy.

Abstract 573: Deguelin inhibits the expression of estrogen receptor alpha in human breast cancer cells

Abstract The estrogen receptor alpha (ERα), a nuclear receptor and transcription factor governs the action of estrogen. This hormone is the major contributor to normal breast development and breast carcinogenesis. Thus, antiestrogens are widely utilized to treat and circumvent the progression of hormone receptor positive breast cancer. However, resistance to these agents develops after sustained use and major risk factors such as enhanced susceptibility to endometrial cancer have been associated with this type of treatment. In an effort to develop natural therapeutic compounds against ERα positive breast cancer, the effect of deguelin, a rotenoid isolated from an African plant; Mundulea sericea on ERα positive human breast cancer cells was investigated. Here we report that deguelin treatment downregulated the expression of ERα protein in a dose dependent manner in MCF-7 and T47D human breast cancer cell lines as determined by immunoblotting. Quantitative RT-PCR analysis revealed that the mRNA expression of ERα target genes Cathepsin D (CTSD), Trefoil factor 1 (TFF1) and the Progesterone receptor (PGR) was also downregulated by 50% in the presence of deguelin in MCF-7 cells. Furthermore, deguelin treatment significantly inhibited the proliferation of these two cell lines. Importantly, estrogen mediated stimulation of cell growth was reduced by 30% in MCF-7 cells after deguelin treatment. Together these findings suggest that deguelin suppresses ERα signaling, paving the way for the development of a novel natural therapeutic agent against endocrine receptor positive breast cancer. Future studies are aimed at deciphering the molecular mechanisms by which deguelin adversely affects ERα function. (This work was supported by NCI CA140321). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 573. doi:1538-7445.AM2012-573

Distinct expressions of microRNAs that directly target estrogen receptor α in human breast cancer

Estrogen receptor (ER) α is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ERα-positive breast cancer. Breast cancer patients show a wide range of ERα expression levels and the levels of expression in individual patients change during disease progression and in response to systemic therapies. However, little is known about how the expression of ERα in human breast cancer is regulated. Recently, several microRNAs (miRNAs) that directly target ERα have been identified, and we previously demonstrated that miR-206 expression was downregulated in ERα-positive human breast cancer. In this study, expression levels of miRNAs that directly target ERα, including miR-18a, miR-18b, miR-22, miR-193b, miR-221/222 and miR-302c, were analyzed in human breast cancer samples by quantitative reverse transcription-PCR analysis. Correlations between the expression levels of these miRNAs and clinicopathological factors, including prognosis, were analyzed. miR-18a expression was much higher in ERα-negative than in ERα-positive tumors (P < 0.0001), with the expression levels of miR-18a not differing in ERα-positive breast cancer as a function of ERα protein level. Surprisingly, the expression levels of miR-193b and miR-221 were significantly lower in ERα-negative than in ERα-positive tumors (P = 0.0015 and P = 0.0045, respectively), and the levels of these miRNAs gradually increased as ERα protein expression increased. There was no statistically significant association between miR-22 and ERα expression, and miR-302c expression was minimal in human breast cancer samples. Prognostic analysis showed that low miR-18b expression was significantly associated with improved survival in HER2-negative breast cancer, although miR-18b expression was not correlated with ERα protein expression. Our results suggest that miRNAs that directly target ERα have distinct roles in not only regulating ERα but also regulating other target genes in human breast cancer.

Response of Estrogen Receptor-Positive Breast Cancer Tumorspheres to Antiestrogen Treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.

Abstract PD05-05: Hormone-Independent Breast Cancer Cells Maintain an ERα — Dependent, E2F-Directed Transcriptional Program

Abstract In order to discover mechanisms of escape from hormone dependence, we established four ERα-positive human breast cancer cell lines after long-term estrogen-deprivation (LTED). Growth of MCF-7/LTED and HCC-1428/LTED cells was inhibited by the ER downregulator fulvestrant and by ER siRNA, suggesting continued dependence upon estrogen-independent ER signaling. Fulvestrant also prevented the emergence of hormone-independent MCF-7, HCC-1428, and BT-474 cells, but not ZR75- 1, MDA-361, and T47D cells. Gene expression analysis revealed that ER modulates a transcriptional program directed by the E2F proteins, a family of transcription factors derepressed by CDK4/6-mediated phosphorylation of Rb. A set of 1,003 genes with expression altered ≥1.5-fold (P&amp;lt;0.05) by fulvestrant in MCF-7/LTED and HCC-1428/LTED cells was used to query the TRANSFAC database. Five of the top ten sets of genes which share a transcription factor binding motif ± 2 kb of transcription start sites contained E2F motifs (all p&amp;lt;10-12). From these, we derived an expression signature of 24 genes with no cell cycle association. This signature was predictive of response of 48 ER+ primary tumors to 2 weeks of neoadjuvant letrozole as assessed by Ki67 IHC. In a separate set of experiments, we screened a siRNA library targeting 779 kinases and found that CDK4 is required for MCF-7/LTED cell growth. Inhibition of CDK4/6 using the small molecule PD-0332991 or CDK4 siRNA phenocopied the inhibitory effect of fulvestrant on MCF-7/LTED and HCC-1428/LTED cells, implying that the primary growth-promoting effect of ligand-independent ER is activation of the Rb/E2F pathway. CDK4 inhibition also suppressed the hormone-independent growth of 4 other ER+ breast cancer lines. We previously demonstrated that phosphatidylinositol-3 kinase (PI3K) signaling is required for the escape of ER+ breast cancer cells from hormone dependence. Thus, we postulated that treatment with the PI3K inhibitor BKM120 (Novartis) would synergize with fulvestrant to suppress hormone-independent cell growth. Indeed, these drugs exerted a synergistic inhibitory effect in 6/8 ER+ breast cancer cell lines. Finally, we injected MCF-7 cells and 14-day-release 17P-estradiol pellets into female athymic mice. After 19-29 days, mice bearing tumors ≥100 mm3 were randomized to treatment with vehicle, fulvestrant, BKM120, or the combination. While BKM120 and fulvestrant individually slowed tumor growth, the combination induced near-complete tumor regression. Fulvestrant downregulated ER and PR levels, while BKM120 decreased P-AKT-S473, P-AKT-T308, P-S6-S240/244 (by immunoblotting), and [18F]fluorodeoxyglucose (FDG) tumor uptake as measured by PET, indicative of suppressed ER and PI3K signaling, respectively. These findings collectively suggest that 1) a fraction of ER+ breast cancer cells engage ERα in a ligand-independent manner to drive cell growth, 2) pharmacological downregulation of ER may be effective in a fraction of ER+ tumor cells that escape estrogen deprivation, 3) CDK4/6 inhibition suppresses the hormone-independent growth of fulvestrant-sensitive and-insensitive ER+ breast cancer cells, and 4) resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both the ER and PI3K pathways. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-05.

Dual effects of TGF-β on ERα-mediated estrogenic transcriptional activity in breast cancer

BackgroundTGF-β resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-β regulates ERα-induced gene transcription and potential mechanisms of frequent TGF-β resistance in breast cancer.MethodsEffect of TGF-β on ERα-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERα-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays.ResultsA complex of Smad3/4 mediates TGF-β inhibition of ERα-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-β from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERα-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma.ConclusionThese results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-β suppression of ERα transactivity in breast cancer cells.