Estrogen-stimulated Cell Proliferation Research Articles

C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells.

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP-seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBPα favors promoter-enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer-promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer.

CDCA8 is a key mediator of estrogen-stimulated cell proliferation in breast cancer cells

Endocrine therapy is effective in the early stage of breast cancer treatment, and most tumor cells will gain the ability to proliferate under residual amounts of estrogen, which will cause the recurrence of the disease. The role of cell division cycle associated 8 (CDCA8) in Estradiol (E2)-stimulated breast cancer cells growth is investigated in this research. CDCA8 showed higher mRNA expression in E2-stimulated MCF7 and T47D cells, and such an increase could also be observed in tumor samples. CDCA8 shRNA inhibited the survival and growth detected by cell number and colony formation, while promoted cell cycle G1 phase arrest determined with flow cytometry, which coordinated with a decrease in E2-induced molecules, namely Cyclin D1 (CCND1), B-Cell CLL/Lymphoma 2 (BCL2), and an increase in apoptosis-related molecules, such as cyclin-dependent kinase inhibitor 1a (P21) and cyclin-dependent kinase inhibitor 1b (P27). Kaplan-Meier plot analysis indicated that higher CDCA8 expression was positively associated with poor prognosis with a probability lower than 0.4 at the five-year interval (p = 0.035). All of these suggest that CDCA8 is a key mediator of estrogen-stimulated breast cancer cell growth and survival, which can be utilized as a novel target in breast cancer treatment.

Histone acetyltransferase MOF is involved in suppression of endometrial cancer and maintenance of ERα stability

Histone acetyltransferase MOF is involved in active transcription regulation through histone H4K16 acetylation. MOF is downexpressed in a number of human tumors, but biological function of MOF in endometrial cancer has not been fully defined. The estrogen receptor α (ERα) is a transcription factor that regulates estrogen-stimulated cell proliferation in hormone-responsive tumors. However, ERα expression is decreased in grade III ECa samples and high expression of ERα is associated with long disease-free survival in ECa. The molecular mechanism for these observations is still unclear. Here we demonstrate knockdown of MOF promotes ECa cell growth and proliferation in vitro and in vivo. Clinical evidence indicates that expression MOF is decreased and positively correlated with that of ERα in ECa tissues. Furthermore, MOF physically interacts with ERα and modulates ERα stability in ECa cells. In addition, MOF modulates expression of a subset of endogenous genes regulated by ERα. Taken together, our results define MOF as a potential tumor suppressor in ECa participates in maintenance of ERα protein stability and regulation of ERα action.

SHARPIN stabilizes estrogen receptor α and promotes breast cancer cell proliferation.

Estrogen receptor α is expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression. In our study, we identified the novel E3 ubiquitin ligase SHARPIN function to facilitate ERα signaling. SHARPIN is highly expressed in human breast cancer and correlates with ERα protein level by immunohistochemistry. SHARPIN expression level correlates with poor prognosis in ERα positive breast cancer patients. SHARPIN depletion based RNA-sequence data shows that ERα signaling is a potential SHARPIN target. SHARPIN depletion significantly decreases ERα protein level, ERα target genes expression and estrogen response element activity in breast cancer cells, while SHARPIN overexpression could reverse these effects. SHARPIN depletion significantly decreases estrogen stimulated cell proliferation in breast cancer cells, which effect could be further rescued by ERα overexpression. Further mechanistic study reveals that SHARPIN mainly localizes in the cytosol and interacts with ERα both in the cytosol and the nuclear. SHARPIN regulates ERα signaling through protein stability, not through gene expression. SHARPIN stabilizes ERα protein via prohibiting ERα protein poly-ubiquitination. Further study shows that SHARPIN could facilitate the mono-ubiquitinaiton of ERα at K302/303 sites and facilitate ERE luciferase activity. Together, our findings propose a novel ERα modulation mechanism in supporting breast cancer cell growth, in which SHARPIN could be one suitable target for development of novel therapy for ERα positive breast cancer.

Role of GPER on proliferation, migration and invasion in ligand-independent manner in human ovarian cancer cell line SKOV3.

G protein-coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERβ. In ERα-negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand-independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co-expression with ERα and ERβ, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERβ by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP-induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c-fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP-2) and MMP-9. These findings establish that GPER ligand-independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERβ. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer.

Differential expression of estrogen receptor α and β isoforms in multiple and solitary leiomyomas

Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E2) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E2 concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes.

Blockade of estrogen-stimulated proliferation by a constitutively-active prolactin receptor having lower expression in invasive ductal carcinoma

A comprehensive understanding of prolactin's (PRL's) role in breast cancer is complicated by disparate roles for alternatively-spliced PRL receptors (PRLR) and crosstalk between PRL and estrogen signaling. Among PRLRs, the short form 1b (SF1b) inhibits PRL-stimulated cell proliferation. In addition to ligand-dependent PRLRs, constitutively-active varieties, missing the S2 region of the extracellular domain (ΔS2), naturally occur. Expression analysis of the ΔS2 version of SF1b (ΔS2SF1b) showed higher expression in histologically-normal contiguous tissue versus invasive ductal carcinoma. To determine the function of ΔS2SF1b, a T47D breast cancer line with inducible expression was produced. Induction of ΔS2SF1b blocked estrogen-stimulated cell proliferation. Unlike intact SF1b, induction of ΔS2SF1b had no effect on PRL-mediated activation of Stat5a. However induction inhibited estrogen's stimulatory effects on serine-118 phosphorylation of estrogen receptor α, serine-473 phosphorylation of Akt, serine-9 phosphorylation of GSK3β, and c-myc expression. In addition, induction of ΔS2SF1b increased expression of the cell cycle-inhibiting protein, p21. Thus, increased expression of ΔS2SF1b, such as we demonstrate occurs with the selective PRLR modulator, S179D PRL, would create a physiological state in which estrogen-stimulated proliferation was inhibited, but differentiative responses to PRL were maintained.

Estrogen stimulates cell proliferation and regulates the expression of proteins in C-type natriuretic peptide signaling pathway during chondrogenesis in ATDC5 cells

To investigate the effect of estrogen on cell proliferation and expression of proteins of C-type natriuretic peptide (CNP), natriuretic peptides B receptor (NPR-B) and natriuretic peptides C receptor (NPR-C) in ATDC5 cells during chondrogenesis. ATDC5 cells were induced for differentiation with insulin 10 µg/ml (day 0), and were started to be investigated on day 6. They were incubated with: (1) Estradiol (E2) at different concentrations (10(-11)-10(-5) mol/L) for 24 hours (for studying cell proliferation), or for 48 hours (for studying CNP, NPR-B and NPR-C protein expression); (2) E2 (10(-8) mol/L) for 24, 48, 72, 96 and 120 h (for studying cell proliferation), or for 24, 48, 72 and 96 hours (for studying CNP, NPR-B and NPR-C protein expression); (3) E2 (10(-8) mol/L) , and/or ICI 182782 (estrogen receptor antagonist ) (10(-7) mol/L) for 24 hours (for studying cell proliferation). ATDC5 cells proliferation were determined by MTT (OD value). Western-blotting was performed to identify the protein levels of CNP, NPR-B and NPR-C. (1) After incubation with E2 (10(-11)-10(-5) mol/L) for 24 h, ATD5 cell number increased with the increasing E2 concentration, peak in E2 concentrations of 10(-9) and 10(-8) mol/L (0.56 ± 0.06 and 0.52 ± 0.02, P < 0.05 and <0.01, respectively) , while significantly decreased in E2 (10(-5) mol/L) (0.30 ± 0.02) compared with DMSO-control (0.38 ± 0.02) (P < 0.05). After incubation with E2 (10(-11)-10(-5) mol/L) for 48 h, the protein level of CNP, NPR-B and NPR-C increased significantly, with the greatest effect seen at a concentration of 10(-10) mol/L E2 for CNP and NPR-B, 10(-9) mol/L E2 for NPR-C (P < 0.05). (2) After incubation with E2 (10(-8) mol/L) for 24 to 96 hours: (1) The cell number in each of the four time points was significantly increased compared with DMSO-control, with the greatest effect in 48 h (0.030 ± 0.003) (P < 0.05 or <0.01, respectively). While the cell number at 120 h was similar to that in DMSO-control. (2) The protein level of CNP increased significantly at 24 h (P < 0.05), seemed to be increased at 48 h and 72 h and decreased at 96 h. Both NPR-B and NPR-C level seemed to be increased at 24 h (P = 0.060 and 0.055, respectively) and seemed to decrease at 48 h, with decreasing significantly at both 72 h and 96 h (P < 0.05). (3) After incubation for 24 h, there was significant difference among the cell number of the four groups (P < 0.05). Cell number of group E2 (0.470 ± 0.032) was increased compared with group (E2+ICI) (0.410 ± 0.018), both being increased compared with group DMSO-control (0.370 ± 0.011, P < 0.05, respectively). There was no difference in cell number between group ICI 182782(0.360 ± 0.035) and group DMSO-control. E2 promotes the proliferation of ATDC5 cells i.e. chondrogenesis via estrogen receptor mediated mechanism, in both concentration-dependent and time-dependent manner. E2 (10(-11)-10(-8) mol/L) up-regulates protein expression of CNP, NPR-B and NPR-C of ATDC5 cells during chondrogenesis, and regulate the expression of the three proteins mentioned above positively or negatively at different time point, which implied that estrogen is one of the regulators of CNP signaling pathway.

ERβ- and prostaglandin E2-regulated pathways integrate cell proliferation via Ras-like and estrogen-regulated growth inhibitor in endometriosis.

In endometriosis, stromal and epithelial cells from the endometrium form extrauterine lesions and persist in response to estrogen (E2) and prostaglandin E2 (PGE2). Stromal cells produce excessive quantities of estrogen and PGE2 in a feed-forward manner. However, it is unknown how estrogen stimulates cell proliferation and survival for the establishment and persistence of disease. Previous studies suggest that estrogen receptor-β (ERβ) is strikingly overexpressed in endometriotic stromal cells. Thus, we integrated genome-wide ERβ binding data from previously published studies in breast cells and gene expression profiles in human endometriosis and endometrial tissues (total sample number = 81) and identified Ras-like, estrogen-regulated, growth inhibitor (RERG) as an ERβ target. Estradiol potently induced RERG mRNA and protein levels in primary endometriotic stromal cells. Chromatin immunoprecipitation demonstrated E2-induced enrichment of ERβ at the RERG promoter region. PGE2 via protein kinase A phosphorylated RERG and enhanced the nuclear translocation of RERG. RERG induced the proliferation of primary endometriotic cells. Overall, we demonstrated that E2/ERβ and PGE2 integrate at RERG, leading to increased endometriotic cell proliferation and represents a novel candidate for therapeutic intervention.

Identification of miR-26 as a key mediator of estrogen stimulated cell proliferation by targeting CHD1, GREB1 and KPNA2.

IntroductionEstrogen signaling is pivotal in the progression of estrogen receptor positive breast cancer primarily by the regulation of cell survival and proliferation. Micro (mi)RNAs have been demonstrated to be regulated by estrogen to mediate estrogenic effects. Herein, we determined the role of estrogen regulated miR-26 and its underlying molecular mechanisms associated with estrogen receptor (ER)+ breast cancer proliferation.MethodsThe expression of miR-26a and miR-26b was evaluated by real-time quantitative (RT)-PCR. The expression of miR-26a or miR-26b was modulated in ER+ breast cancer cells (MCF-7 and T47D) and tumor cell growth in vitro and an in vivo xenograft model was determined. Bioinformatics analyses were utilized to screen for estrogen responsive genes, which were also predicted to be targeted by miR-26. Luciferase reporter assays were performed to confirm miR-26 regulation of the 3' UTR of target genes. The levels of miR-26 target genes (CHD1, GREB1 and KPNA2) were evaluated by western blotting and immunohistochemistry.ResultsEstrogen reduced the expression of miR-26a and miR-26b in ER+ breast cancer cells. Forced expression of miR-26a or miR-26b significantly inhibited the estrogen stimulated growth of ER+ breast cancer cells and tumor growth in xenograft models, whereas miR-26a/b depletion increased the growth of ER+ breast cancer cells in the absence of estrogen treatment. Screening of estrogen responsive genes, which were also predicted to be targeted by miR-26, identified GREB1 and nine other genes (AGPAT5, AMMECR1, CHD1, ERLIN1, HSPA8, KPNA2, MREG, NARG1, and PLOD2). Further verification has identified nine genes (AGPAT5, CHD1, ERLIN1, GREB1, HSPA8, KPNA2, MREG, NARG1 and PLOD2) which were directly targeted by miR-26 via their 3′ UTR. Functional screening suggested only three estrogen regulated miR-26 target genes (CHD1, GREB1 and KPNA2) were involved in the regulation of estrogen promoted cell proliferation. Depletion of either CHD1, GREB1 or KPNA2 significantly abrogated the enhanced growth of ER+ breast cancer cells due to miR-26 depletion. We further demonstrated that estrogen stimulated c-MYC expression was both sufficient and necessary for the diminished expression of miR-26a and miR-26b.ConclusionsWe have identified a novel estrogen/MYC/miR-26 axis that mediates estrogen stimulated cell growth via CHD1, GREB1 and KPNA2.

Isolation of a Novel Chiral Phytoestrogen Breviflavone B from &lt;i&gt;Epimedium&lt;/i&gt; Herb by a New Approach of Liquid Chromatography

Breviflavone B is a novel chiral phytoestrogen that mimics the biological functions of 17β-estradiol (E2), an estrogen which is naturally produced in our body. It has exhibited a much higher estrogen receptor (ER) bioactivity than E2 or other phytoestrogens. Breviflavone B specifically activates ER at a low dose without activating other steroid receptors, and inhibits estrogen-stimulated breast cancer cell proliferation at high doses, making it a potential treatment option for breast cancer patients. In this report, a new liquid chromatography aprroach has been developed to isolate Breviflavone B from Epimedium Brevicornum herbal extracts. After passing through DIAION HP20 resin, the ethanolic extract of Epimedium Brevicornum was firstly fractionalized by a Diol column, then further separated by a C18 column using preparative HPLC, and finally purified by an ODS-MCR-HPS column in HPLC under gradient mode. In each stage of separation, the ER bioactivities of the each fraction were measured by estrogen receptor-driven reporter gene bioassays to guide the chromatographic fractionations. The results showed that the new combination of Diol and C18 and ODS-MCR-HPS column chromatography provided better separation selectivity than our previously-reported C18 column chromatography for isolation of Breviflavon B from Epemidium herbs. Pure Breviflavon B was successfully isolated from the ethanolic extract of Epimedium Brevicornum herb via this new approach of chromatographic fractionations guided by the bioassays.

Abstract 67: Characterization of the role of KLF9 transcription factor in breast cancer estrogen response

Abstract Kruppel-like factor 9 (KLF9) is a member of the KLF family of transcription factors. Previous studies report significant KLF member involvement in breast cancer estrogen response, notably KLF4 [1, 2] and KLF5 [3, 4]. KLF9 has been shown in uterine cancer cells to function as an estrogen-dependent regulator of the estrogen response pathway [5]. We aim to evaluate the role of KLF9 in breast cancer cell estrogen response. We have performed studies demonstrating that KLF9 exhibits an early-activated estrogen response. Five sites have been identified upstream of the KLF9 gene that interact with estrogen receptor alpha (ERα) [6-8]; we observe ERα enrichment at three of these sites that is estradiol-dependent. Though KLF9 has been shown to act as an essential element in estrogen response in the uterus, KLF9 response in breast cancer cells has yet to be characterized. To study the role of KLF9 in estrogen-mediated responses in transcriptional and proliferative activity, we have manipulated the level of KLF9 expression in MCF-7 breast cancer cells. These cells have been shown to exhibit a clear transcriptional response to estradiol in luciferase reporter assays. We have optimized RNAi conditions to achieve significant knockdown of the KLF9 gene in MCF-7 cells. Additionally, we have produced and tested a KLF9 overexpression vector construct that significantly upregulates expression of KLF9 in this cell line. These tools will be used to more extensively characterize the role of KLF9 in MCF7 cell estrogen-stimulated transcription and proliferation. We are measuring transcriptional response to estrogen signaling with luciferase reporter assays and estrogen stimulated cell proliferation with BrdU proliferation assays. Elucidating KLF9 involvement in E2-mediated breast cancer cell signaling and response, therefore, is an important component of understanding of the regulatory mechanisms behind estrogen response in breast cancer. 1. Akaogi K et al. (2009). Oncogene 28(32): 2894. 2. Quintana AM et al. (2011). BMC Cancer 11: 30. 3. Guo P et al. (2010). International Journal of Cancer 126(1): 81. 4. Zhao KW et al. (2011). Biochemical Journal 437(2): 323. 5. Velarde MC et al. (2007). Molecular Endocrinology 21(12): 2988. 6. Carroll JS et al. (2006). Nature Genetics 38(11): 1289. 7. Hua S et al. (2008). Molecular Systems Biology 4:188. 8. Fullwood MJ et al. (2009). Nature 462(7269): 58. 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 67. doi:1538-7445.AM2012-67

P5-01-15: The Functional Role of the Estrogen-Regulated Gene GREB1: Characterization of a Novel GREB1 Knockout Mouse Model.

Abstract Background: Gene regulated in breast cancer 1 (GREB1) was initially discovered in breast cancers as an estrogen-regulated gene that mediates estrogen-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. However, little is known of the functional role of GREB1 protein in normal breast tissue or breast cancers. Methods: To address this unknown role, our laboratory designed and created a novel Greb1 Knockout Mouse model (C57/bl MEL Greb1 KO). This constitutive model results in the loss of Greb1 mRNA and protein expression in cells where expression of Cre recombinase promotes the cleavage of exon 1 and intron 1 of the gene encoding Greb1. ROSA26 Cre C57/b1 MEL Greb1 KO mice heterozygous for the floxed Greb1 allele were crossed to generate experimental litters. Initial experiments were designed to evaluate if the complete loss of Greb1 expression in offspring homozygous for the floxed Greb1 allele was lethal during gestation. Experimental litters were tail clipped and genotyped using gDNA and genotype-specific PCR. Results: Offspring homozygous for the floxed Greb1 allele were identified in expected Mendelian ratios with wild type and heterozygous siblings. Loss of Greb1 expression was confirmed using RT-PCR, in situ hybridization and immunoblotting. Loss of both Greb1 alleles was not observed to be lethal during gestation for either male or female pups. Preliminary gross observation of these homozygous KO mice revealed no overt anatomical differences, however, they were 25–30% smaller than their heterozygous and wild-type siblings. Breeding experiments are underway to determine the fertility of crossbred Greb1 homozygous KO mice. Imaging experiments and necropsy with histochemical analysis of tissues will reveal any alteration in architecture and function. These findings will be summarized in this presentation. Discussion: As GREB1 has been identified as an estrogen-regulated gene involved in breast cancer cell proliferation and a potential target for new therapeutic strategies, it is important to understand the contribution of GREB1 to the differentiation, development and function of normal tissues as well as in breast cancers. Characterization of this novel Greb1 KO mouse model will provide answers to these functional questions surrounding GREB1. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-15.

Nuclear estrogen receptor-mediated Notch signaling and GPR30-mediated PI3K/AKT signaling in the regulation of endometrial cancer cell proliferation

To elucidate the mechanisms of nuclear estrogen receptor (ER)-mediated and G protein-coupled receptor 30 (GPR30)-mediated signaling in the regulation of proliferation in ER-positive and ER-negative endometrial cancer cells, two human endometrial carcinoma cell lines, Ishikawa (ER-positive) and KLE (ER-negative), were used. PCR and Western blot analyses were used to determine the effects of estrogen stimulation on the activation of Notch and GPR30-PI3K/AKT signaling. Cell growth was investigated using MTT assays. Overexpression of ER in ER-negative cells was achieved by plasmid transfection and was used to investigate the effects on cellular growth and Notch signaling. GPR30-mediated signaling was evaluated using siRNA interference. Estrogen stimulated cell proliferation in both cell lines, it activated Notch signaling in ER-positive Ishikawa cells, but not in ER-negative KLE cells. Blockade of this signaling by a Notch inhibitor resulted in partial arrest of estrogen-induced cell proliferation in Ishikawa cells. Overexpression of ER in KLE cells restored estrogen-enhanced Notch signaling and further promoted cell growth. GPR30, as a new G-protein-coupled estrogen receptor, was detected in both cell lines, but was stronger in ER-negative KLE cells. Depletion of GPR30 in KLE cells abolished estrogen-induced PI3K/AKT signaling activation and resulted in inhibition of cell proliferation. Conclusively, regulation of proliferation in nuclear ER-positive endometrial cancer cells is mediated by both ER-Notch signaling and GPR30-PI3K/AKT signaling, whereas only the latter pathway is involved in the regulation of growth in nuclear ER-negative endometrial cancer cells.

Abstract 3101: RET, an estrogen-dependent proto-oncogene, is required for the proliferation of ER-positive breast cancer cells

Abstract Background: RET is a member of the cadherin superfamily, which encodes a receptor tyrosine kinase. RET is a transforming proto-oncogene associated with many endocrine carcinomas. Regulation of RET expression is very complex. The RET promoter can be regulated by many transcription factors, including retinoic acid receptor, Sp1, Nkx2-1, and others. Using a transcriptional profiling assay, our laboratory previously identified RET as an estrogen-induced gene in breast cancer cells. In this study, we investigated the molecular mechanism by which estrogen regulates RET expression in breast cancer, and assessed the function of RET proto-oncogene in breast cancer. Methods: RET expression in breast cancer cell lines was analyzed by Q-RT-PCR, and RET expression in tissue specimens was retrieved from the oncomine database. Transcriptional regulation of RET promoter and distal enhancer elements was analyzed by luciferase assay and chromatin immunoprecipitation (ChIP) after treatment with vehicle or estrogen. Cell proliferation was measured by MTS assay and cell counting. Results: RET was highly expressed in estrogen receptor (ER)-positive breast cancer cell lines and breast cancer tissue specimens compared to ER-negative ones. Expression of RET was induced robustly by estrogen, and this induction was dependent on ER via a primary transcriptional regulatory mechanism, as determined by pretreatment of cycloheximide, ER siRNA, or the pure ER-antagonist, fulvestrant. The proximal promoter of RET gene was not responsive to estrogen treatment and ER did not bind to the proximal promoter. ChIP analysis showed that of nine potential ER binding sites in the RET gene locus, only three bind ER with different efficiency. A 1kb DNA fragment, which is located about 50kb upstream of the transcriptional start site, had an extremely high affinity to ER. When inserted in front of the promoter-luciferase cassette, only this 1kb fragment, but not other eight fragments, enhanced estrogen inducibility and therefore functions as an enhancer acting in cis. This enhancer region has two half estrogen responsive elements (EREs) and a full ERE, which are critical for estrogen-induced RET expression. In a functional assay, siRNA knockdown of RET inhibited estrogen-stimulated cell proliferation in ER-positive breast cancer cells, but not in ER-negative ones. Conclusion: The RET proto-oncogene is induced by estrogen treatment and is required for the proliferation of ER-positive breast cancers. Estrogen-induced RET expression is facilitated by ER binding to a distal enhancer containing novel EREs. This pathway of estrogen regulation of RET gene expression and cell proliferation may be targeted in the future for better treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2011-3101

Estrogen receptors, antiestrogens, and non-small cell lung cancer

This review considers data on expression of different types of estrogen receptors (ERα and ERβ) in in vitro cultured cells of non-small cell lung cancer and also in human and animal lung tumors. Estrogens are shown to play an important role in genesis and development of non-small cell lung cancer because the estrogen-stimulated cell proliferation as well as antiestrogen-caused inhibition of proliferation occurred only in the cells expressing different types of estrogen receptors. In general, the situation is similar to that observed in breast cancer, but in the cells of non-small cell lung cancer not ERα are expressed in more than half of cases but ERβ. Just estrogen receptors β play the crucial role in inducing cell proliferation in response to estrogens, and ERβ is a prognostic marker of a favorable course of non-small cell lung cancer. Data on the interactions between ER and EGFR signaling pathways, as well as on the additive antitumor effect of antiestrogens (tamoxifen and fulvestrant) combined with tyrosine kinase inhibitors (gefitinib, erlotinib, and vandetanib) are considered. The review also includes data on the influence of estrogens on genesis and development of lung cancer in humans and animals and the frequency of ERα and ERβ expression in non-small cell lung cancer in tissues from patients of the two sexes. Problems of quantitative determination of α and β estrogen receptors in the tumor cells are also discussed.

ER-α36, a variant of ER-α, is the estrogen receptor that mediates mitogenic estrogen signaling in breast cancer cells.

Abstract Abstract #18 The role of endogenous estrogens in breast cancer etiology has been well established. Dysregulated estrogen signaling increases the rate of cell proliferation and thus the risk of breast cancer. However, despite widespread agreement that estrogens are involved in breast cancer etiology, the molecular mechanisms of estrogen action, especially those by which estrogen signaling stimulates mammary tumorigenesis and breast cancer progression have not been well established.&amp;#x2028; Recently, we have identified and cloned a 36-kDa novel isoform of estrogen receptor α (ER-α36) that is generated from a promoter located in the first intron of the original 66-kDa ER-α (ER-α66) gene. The ER-α36 differs from the ER-α66 by lacking both transcriptional activation domains (AF-1 and AF-2) but retains the DNA-binding, dimerization and most of the ligand-binding domains. ER-α36 is mainly expressed on the plasma membrane and mediates membrane-initiated estrogen signaling that activates the MAPK/ERK pathway and stimulates cell proliferation. ER-α36 inhibits the genomic estrogen signaling mediated by the AF1 and AF2 domains of ER-α66 and ligand-dependent and -independent transactivation activities of ER-β. We designed and constructed shRNA expression vectors to express shRNAs specific for ER-α36 or ER-α66. We found that ER-positive breast cancer cells MCF7 and T47D expressing ER-α36 shRNA that successfully knockdown about 90% ER-α36 expression failed to respond to estrogen stimulation. However, estrogen strongly stimulated cell growth in MCF7 and T47D cells with ER-α66 knockdown. We found that some estrogen responsive genes such as c-Myc, CyclinD1 and progesterone receptor (PR) were induced through the extra-nuclear estrogen signaling pathway while pS2 was induced by the nuclear pathway. Our results for the first time demonstrated that ER-α36, not the original ER-α36 is the estrogen receptor that actually mediates estrogen-stimulated cell proliferation in breast cancer cells.&amp;#x2028; Among 800 cases of human breast cancer specimens examined, ER-α36 is expressed in most of the breast cancer specimens, and even in 40% of ER-negative breast cancer cases that lack expression of ER-α66. In addition, ER-α36 expressing patients responded poorly to anti-estrogen Tamoxifen treatment. In vitro study revealed that breast cancer cells that highly express ER-α36 exhibited estrogen hypersensitivity and Tamoxifen resistance, consistent with the clinical evidence that breast cancers expressing ER-α36 exhibit higher degree of malignancy and poorer survival rate.&amp;#x2028; Taken together, our results demonstrated that ER-α36 is a novel player in mitogenic estrogen signaling that plays an important role in estrogen stimulated mammary carcinogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 18.

The G Protein-Coupled Receptor GPR30 Inhibits Human Urothelial Cell Proliferation

We have previously shown that estrogen stimulates cell proliferation in both normal and transformed urothelial cells mainly through activation of the two primary estrogen receptors (ERs), ERalpha and ERbeta. A growing body of evidence suggests that estrogen also initiates nongenomic effects that cannot be explained by activation of primary ERs. In the present study, we observed that urothelial cells express high amounts of GPR30, a G protein-coupled receptor recently identified as a candidate for membrane-associated estrogen binding. Membrane- impermeable bovine serum albumin-conjugated 17beta-estradiol and the specific GPR30 agonist G-1 both inhibited urothelial cell proliferation in a concentration-dependent manner. Transient overexpression of GPR30 inhibited 17beta-estradiol (E2)-induced cell proliferation. Decreased GPR30 expression caused by specific small interfering RNA increased E2-induced cell proliferation. These results indicate that membrane-associated inhibitory effects of E2 on cell proliferation correlate with abundance of GPR30. Although E2 induced a significant increase in caspase-3/7 activity, G-1 did not, suggesting that the GPR30-mediated inhibitory effect on cell proliferation was not caused by apoptosis. Furthermore, we found that G-1 failed to induce c-fos, c-jun, and cyclin D1 expression, and GPR30 overexpression abolished E2-induced c-fos, c-jun, and cyclin D1 expression. However, inactivation of GPR30 by small interfering RNA increased c-fos, c-jun, and cyclin D1 expression. These results suggest that GPR30-mediated inhibition of urothelial cell proliferation is the result of decreased cyclin D1 by down-regulation of activation protein-1 signaling.

Potential Mechanisms of Estrogen Quinone Carcinogenesis

There is a clear association between the excessive exposure to estrogens and the development of cancer in hormone-sensitive tissues (breast, endometrium). It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. One major pathway is the extensively studied hormonal pathway, by which estrogen stimulates cell proliferation through nuclear estrogen receptor (ER)-mediated signaling, thus resulting in an increased risk of genomic mutations during DNA replication. A similar "nongenomic pathway", potentially involving newly discovered membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways. This perspective is focused on a third pathway involving the metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze o-quinone formation, so that these electrophilic/redox-active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. It appears that the endogenous estrogen quinones primarily form unstable N3-adenine or N7-guanine DNA adducts, ultimately resulting in mutagenic apurinic sites. In contrast, equine estrogen quinones, formed from estrogens present in popular hormone replacement therapy prescriptions, generate a variety of DNA lesions, including bulky stable adducts, apurinic sites, DNA strand cleavage, and oxidation of DNA bases. DNA damage induced by these equine quinones is significantly increased in cells containing ERs, leading us to hypothesize a mechanism involving ER binding/alkylation by the catchol/quinone, resulting in a "Trojan horse". The "Trojan horse" carries the highly redox-active catechol to estrogen -sensitive genes, where high amounts of reactive oxygen species are generated, causing selective DNA damage. Our data further suggest that other key protein targets for estrogen o-quinones could be redox-sensitive enzymes (i.e, GST P1-1, QR). These proteins are involved in stress response cascades that are known to contribute to the regulation of cell proliferation and apoptosis. Finally, it has been shown that catechol estrogens can transform breast epithelial cells into a tumorigenic phenotype and that these transformed cells had differential gene expression of several genes involved in oxidative stress. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

Aromatase Expression in Uterine Leiomyomata Is Regulated Primarily by Proximal Promoters I.3/II

Uterine leiomyomata are common tumors that cause irregular uterine bleeding and pregnancy loss and depend on estrogen for growth. Aromatase catalyzes the conversion of androgens to estrogens. Aromatase expression is regulated via alternatively used promoters in the placenta (I.1 and I.2a), fat (I.4, I.3, and II), bone (I.6), and gonads (II). A prostaglandin E(2)/cAMP-dependent pathway regulates coordinately the proximal promoters I.3/II, whereas glucocorticoids and cytokines regulate the distal promoter I.4. Use of each promoter gives rise to a population of aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). Uterine leiomyoma tissue, but not normal myometrium, overexpresses aromatase leading to estrogen-stimulated cell proliferation. Aromatase inhibitor treatment shrank uterine leiomyomata in a few women. Promoter I.4 was reported to regulate aromatase expression in uterine leiomyomata from a group of Japanese women. Here, we used two independent techniques to identify the promoters that regulate aromatase expression in uterine leiomyomata (n = 30) from 23 African-American, Hispanic, and white women. Rapid amplification of 5'-cDNA ends of aromatase mRNA species revealed the following distribution of promoter usage in leiomyomata: promoters I.3/II, 61.5%; I.2a, 15.4%; I.6, 15.4%; and I.4, 7.7%. Real-time PCR, which quantifies mRNA species with promoter-specific 5'-UTRs, revealed the following distribution for each 5'-UTR as a fraction of total aromatase mRNA: I.3/II, 69.6%; I.4, 7.3%; and other promoters, 23.1%. The primary in vivo aromatase promoter in leiomyoma tissues in non-Asian U.S. women is the prostaglandin E(2)/cAMP-responsive I.3/II region. Alternative signals may stimulate aromatase expression that is a common biological phenotype in uterine leiomyomata.