Regulation of Estrogen Receptor-alpha Mediated Gene Expression and Endocrine Resistance Through Estrogen Receptor-alpha Phosphorylation and Micro-RNA in Breast Cancer PDF Download
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Author: Kyuri Kim
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Languages : en
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Estrogens are associated with the development and progression of breast cancer in addition to their role in normal reproductive physiology, and estrogen receptors (ER) mediate the actions of estrogen in target tissues by regulating the expression of numerous biologically important target genes. The progression of human breast cancer and the development of resistance to endocrine therapies are thought to be associated with ER phosphorylation. We generated multiple combinations of ER phospho-mutants, at residues serine 104, 106, 118, 167, 236, and 305, and examined their impact on receptor half-life, the agonist and antagonist balance of selective estrogen receptor modulators (SERMs) and selective estrogen receptor downregulators (SERDs), the regulation of ER transcriptional activity, and stimulation of cell proliferation in response to estradiol and SERMs/SERD. We showed that changes in ER affecting the phosphorylation status of the receptor greatly impact receptor function and differential SERM and SERD modulated cellular responses that could contribute to resistance to endocrine therapies in breast cancer. We also studied the regulation of microRNAs (miRNAs) by estradiol and growth factors through ER and extracellular signal-regulated kinase 2 (ERK2) in order to understand their physiological impact on breast cancer. We identified nine miRNA- encoding genes harboring overlapping ER and ERK2 binding sites close to their transcription start sites, which require ER and ERK2 for transcriptional induction as well as estradiol- mediated miRNA regulation. We then identified TP63, a target of miR-101, miR-190 and miR- 196a2, and showed that TP63 plays an important role in estradiol- or growth factor-mediated cellular response in breast cancer cells (MCF-7 and MDA-MB-231) by increasing tumor cell growth and in vitro invasion mainly controlled by miR-196a2 action. These results suggest a tumor-suppressive role of miR-196a2 in regulating TP63 expression and the aggressive behavior of breast cancers.
Author: Kyuri Kim
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Languages : en
Pages :
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Book Description
Estrogens are associated with the development and progression of breast cancer in addition to their role in normal reproductive physiology, and estrogen receptors (ER) mediate the actions of estrogen in target tissues by regulating the expression of numerous biologically important target genes. The progression of human breast cancer and the development of resistance to endocrine therapies are thought to be associated with ER phosphorylation. We generated multiple combinations of ER phospho-mutants, at residues serine 104, 106, 118, 167, 236, and 305, and examined their impact on receptor half-life, the agonist and antagonist balance of selective estrogen receptor modulators (SERMs) and selective estrogen receptor downregulators (SERDs), the regulation of ER transcriptional activity, and stimulation of cell proliferation in response to estradiol and SERMs/SERD. We showed that changes in ER affecting the phosphorylation status of the receptor greatly impact receptor function and differential SERM and SERD modulated cellular responses that could contribute to resistance to endocrine therapies in breast cancer. We also studied the regulation of microRNAs (miRNAs) by estradiol and growth factors through ER and extracellular signal-regulated kinase 2 (ERK2) in order to understand their physiological impact on breast cancer. We identified nine miRNA- encoding genes harboring overlapping ER and ERK2 binding sites close to their transcription start sites, which require ER and ERK2 for transcriptional induction as well as estradiol- mediated miRNA regulation. We then identified TP63, a target of miR-101, miR-190 and miR- 196a2, and showed that TP63 plays an important role in estradiol- or growth factor-mediated cellular response in breast cancer cells (MCF-7 and MDA-MB-231) by increasing tumor cell growth and in vitro invasion mainly controlled by miR-196a2 action. These results suggest a tumor-suppressive role of miR-196a2 in regulating TP63 expression and the aggressive behavior of breast cancers.
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Languages : en
Pages : 230
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Estrogen receptor-alpha (ER[alpha]) is a member of nuclear receptor superfamily of transcription factors. It is known to regulate carcinogenic gene expression programs that are involved in the development and progression of breast cancer. The transcriptional function of ER[alpha] is mediated by a C-terminal AF2 and an N-terminal AF1 activation domains. Ligand-dependent AF2 activity is well-characterized and serves as a basis for hormonal therapy for breast cancer. In contrast, structural and functional mechanisms governing AF1 functions remain poorly understood. AF1 activity of ER[alpha] is regulated by phosphorylation stemming from hormone, peptide growth factors, and second messenger pathways. Paradoxically, phosphorylation results in contrasting responses (differentiation and growth, protein stability and degradation, agonist and antagonist activities). How phosphorylation translates into diverse outcome is not clearly understood. The work presented in this thesis has uncovered a post-translation modification beyond phosphorylation that regulates the function and fate of ER[alpha]. I found that phosphorylation-dependent prolyl cis/trans isomerase, Pin1, causes structural changes at the AF1 region of ER[alpha]. These local changes allosterically regulate DNA binding and dimerization activities, enhancing overall ER[alpha] transcriptional function. Pin1 also stabilizes ER[alpha] protein by blocking its ubiquitination and degradation by the proteasome. Further studies in understanding the role of Pin1 in breast cancer led us to uncover the importance of Pin1 in proliferation of ER[alpha]-positive breast cancer cells and mammary tumors in rodent models. Pin1 overexpression was sufficient to overcome the antagonistic effects of tamoxifen and also contributed to tamoxifen resistance in breast cancer cells. Finally, the clinical relevance of Pin1 activity was confirmed by our findings in human breast tumors, where Pin1 levels were correlated with ER[alpha] protein levels, and ER[alpha]-positive tumor patients with high Pin1 levels had poor overall survival. Overall, the findings in this thesis have identified a new regulatory mechanism governing ER[alpha] AF1 function in breast cancer and discovered Pin1 as an important component modulating ER[alpha] protein levels and transactivation functions.
Author: David Kaiwen Lung
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Category :
Languages : en
Pages : 0
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Estrogen receptor [alpha] (ER) plays a critical role in the growth and survival of breast cancer, which has made it an important target for endocrine therapies that ultimately inhibit its transcriptional function. However, in advanced stages of breast cancer, endocrine therapies decline in effectiveness, despite the majority of breast cancers maintaining their ER-positive status during disease progression. Two potentially key contributors to endocrine therapy resistance are the tumor microenvironment (TME) and the emergence of [ESR1] mutations that confer constitutive ER activity. To mediate endocrine therapy resistance, the TME and [ESR1] mutations affect the expression and function of the receptor, but it is unclear how these extrinsic and intrinsic factors co-exist to ultimately affect breast cancer cell behavior. In the work presented in this thesis, my goal focused on determining how the bone marrow microenvironment, the most common site of breast cancer metastasis, regulates ER expression and activity, and how these paracrine interactions affect cells with [ESR1] mutations. I determined that conditioned media (CM) from cancer-associated bone marrow stromal cells (BMSCs) and the BMSC cell line HS5 primarily transcriptionally repress [ESR1] expression to decrease overall ER expression in ER-positive breast cancer cell models MCF7 and T47D. Transcriptional repression of [ESR1] by HS5-CM involved rapid eviction of RNA polymerase II (Pol II) and potential inhibition of p300 activation on two major regulatory elements of [ESR1], the proximal promoter and a distal enhancer (ENH1). Additionally, HS5-CM treatment decreased the active enhancer mark H3K27Ac on ENH1, implicating ENH1 as a central regulatory element for driving [ESR1] transcriptional repression. BMSC-CM also caused co-repression of several neighboring genes within a 300 kb locus in addition to [ESR1]. Further studies assessed the impact of ER downregulation on the ER transactivation pathway by BMSCs. Despite detection of ER phosphorylation at serine 118 (pS118-ER) by HS5-CM, no increase in ER occupancy above basal levels was observed on strong ER binding sites nor changes in ERE activity. HS5-CM also repressed activated ER target genes, suggesting BMSCs have an overall repressive effect on ER transcriptional activity. In MCF7 cells expressing the [ESR1] mutations D538G or Y537S, HS5-CM was also able to significantly downregulate ER expression. However, activation of ER target genes remained significantly higher in cells expressing these mutations relative to cells expressing wild-type ER, despite treatment with HS5-CM. Furthermore, knockdown of a central co-activator p300 produced similar results with maintenance of significantly elevated ER target gene expression relative to cells expressing wild-type receptor. Together, these findings suggest that the TME affects breast cancer cell behavior by decreasing ER expression, potentially allowing other stimulated signaling pathways to control cell growth and survival. However, [ESR1] mutations appear to overcome the repressive effects of the TME on ER expression and transcriptional activity as well as the need for the co-activator p300 to mediate its transcriptional activity, demonstrating these mutations allow ER to maintain control over cancer cell behavior. These results ultimately contribute to our limited knowledge of the relationship between the TME and ER and provide the basis for our understanding on how [ESR1] mutations are affected by the metastatic TME.
Author: Tissa Thomas Manavalan
Publisher:
ISBN:
Category : Breast
Languages : en
Pages : 314
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MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level by repressing translation or stimulating mRNA degradation. In this study, I tested the hypothesis that miRNAs are differentially expressed in antiestrogen-sensitive MCF-7 versus -resistant L Y2 human breast cancer cells. Microarray analyses identified 97 miRNAs that are differentially expressed between two estrogen receptor alpha (ERa) -positive human breast cancer cell lines: endocrine-sensitive MCF-7 versus -resistant L Y2 cells under basal conditions. Opposite expression of miRs-lOa,-21, -22, -12Sb, -181, -200a, -200b, -200c, -221, and -222 was confirmed between MCF-7 and L Y2 cells. The ER antagonist ICI 182,780 (fulvestrant or Faslodex) generally blocked the effect of estradiol E2 and 4-hydroxytamoxifen (4-OHT) regulated miRs, i.e.. , miR-lOa, miR-21, miR-22, miR-200a, miR-221, and miR-222, indicating that these responses in MCF-7 cells are ER-mediated. Time dependent variation in basal (ethanol, the vehicle), E2, and 4-0HT regulation of the top 8 miRNAs was detected in MCF-7 cells. Bioinformatic analyses to impute the biological significance of the identified miRNAs by identifying their computationally predicted target genes in the human genome using TargetScan, Pic Tar, and the Sanger miRBase Targets databases was performed. Thirty six putative mRNA targets were identified. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs showing opposite expression between these two breast cancer cell lines may be involved in endocrine resistance. MiR-200 family includes two clusters i.e. miR-200 a/200 b/ 429 and miR-200c/141 encoded on chromosome 1 and chromosome 12, respectively. Lower miR-200a, miR-200 b and miR-200c expression was observed in estrogen-independent LCC1 and endocrine-resistant LCC2, LCC9, and LY2 compared to the parental, endocrine-sensitive MCF-7 human breast cancer cell line. ZEB 1 protein was found to be expressed in endocrine-resistant LY2 cells but not in endocrine-sensitive MCF-7 cells. L Y2 cells did not express E-cadherin, a ZEB 1 target which is a marker for epithelial phenotype. This is the first demonstration that L Y2 cells have undergone EMT as part of their endocrine-resistant phenotype. Concomitant with miR-200 decrease, there was an increase in ZEB 1 mRNA expression m L Y2 cells. Overexpression of miR-200b or miR-200c in LY2 cells changed the cellular morphology from a mesenchymal to an epithelial appearance and sensitized cells to inhibition by 4-0HT and fulvestrant. These studies indicate that reduced expression of miR-200 and a corresponding increase in ZEB 1 protein is an indicator of endocrine-resistance in breast cancer cells.
Author: Chun Gong
Publisher:
ISBN: 9781361320174
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Languages : en
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This dissertation, "Regulation of Estrogen Receptor Alpha Expression by Translation or Degradation and the Relevance to Tamoxifen Resistance in Breast Cancer" by Chun, Gong, 龚纯, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Breast cancer is one of the most prevalent cancers affecting women worldwide. In the breast, estrogen receptor alpha (ERα), upon binding with ligands, activates gene transcription and promotes cell growth and proliferation. Tamoxifen, a selective antagonist of ERα in breast, has been proved to be effective therapeutically. In spite of this, resistance remains a prominent issue and underlying mechanisms are not yet fully understood. Aberrant regulation of ER expression at genetic and transcriptional levels has been implicated as the mechanisms accounting for tamoxifen resistance. However, regulation of ERα expression at translational level including protein synthesis and degradation has not yet been characterized and its relevance to tamoxifen resistance has not been described. At level of protein synthesis, eukaryotic translation initiation factor 4E (eIF4E) selectively enhances the translation of 4E-sensitive mRNAs which contain long and complex 5''-untraslated regions (5''-UTR). eIF4E is often over-expressed in cancers. In silico analysis revealed that ERα contained a highly structured 5''-UTR similar to reported eIF4E-sensitive mRNAs, suggesting that ERα mRNA might be eIF4Esensitive. We showed by polysome fractionation and subsequent Q-PCR quantification that the ERα mRNAs were more actively translated in the cell line expressing higher levels of eIF4E. Consistently, transient transfection of eIF4E into an ERα-positive cell line resulted in enhanced protein expression of ERα. Moreover, subcelluar fractionation showed that eIF4E was bound with ERα mRNAs in the nucleus thus participating in transportation of mRNAs from the nucleus into the cytoplasm. Therefore, eIF4E could positively modulate protein synthesis of ERα by enhancing mRNA export in the nucleus as well as translation in the cytoplasm. Their positive correlation was validated in vivo using 106 Chinese breast cancer samples (Chi-square test, p=0.004). It was also found that elevated expression of eIF4E could mediate resistance to tamoxifen treatment and enhance cell survival. This could be due to enhanced expression of ERα or activation of PI3K/Akt pathway upon eIF4E over-expression. At the level of degradation, ERα is conjugated to poly-ubiquitin chains catalyzed by multiple enzymes and degraded by 26S polysomes. Carboxyl-terminus of Hsc70- interacting protein (CHIP) is an E3 enzyme specific for ERα degradation through interaction with ERα''s ligand-binding domain (LBD). Various splicing variants of ERα have been reported and implicated in tamoxifen resistance by interfering with functions of ERα wild type. Variants ERαΔ4, ERαΔ5, ERαΔ6/7 and ERαΔ7 with different degrees of truncation in their LBDs and differential expression were detected or reported in human breast cancers. Their interactions with CHIP may be different, resulting in variations in degradation. We found that the degradation of ERαΔ6/7 through ubiquitin-proteasome pathway was impaired whilst the degradation of other variants were less affected. This finding suggests that the binding site of CHIP to ERαmight be located within the peptide sequences encoded by exon6. Furthermore, as ERαΔ6/7 plays a dominant negative role in regulating functions of ERα wild type, aborted degradation of this variant may result in accumulation of this variant in the cell, inhibiting and in
Author: Amy M. Fowler
Publisher:
ISBN:
Category :
Languages : en
Pages : 226
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Author: William R. Miller
Publisher: CRC Press
ISBN: 0203909836
Category : Medical
Languages : en
Pages : 395
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This reference evaluates and describes the latest strategies for hormone suppression and blockade in the management of early and advanced stage breast cancer and explores the effects of tamoxifen, selective estrogen receptor modulators (SERMs), aromatase inhibitors, and their combination on both breast cancers and normal tissues. Endocrine T
Author: Xiaoting Zhang
Publisher: Springer
ISBN: 331999350X
Category : Medical
Languages : en
Pages : 422
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The discovery of ER by Dr. Elwood Jensen exactly 60 years ago has not only led to the birth of a whole new vital nuclear receptor research field but also made a rapid, direct and lasting impact on the treatment and prevention of breast cancer. Since that landmark discovery, tremendous progress has been made in our understanding of the molecular functions of ER and development of targeted therapies against ER pathways for breast cancer treatment. However, there is currently no book available addressing these discoveries and recent advancement in a historical and systematic fashion. This book is intended to provide comprehensive, most up-to-date information on the history and recent advancement of ER and breast cancer by world renowned leaders in the field. These chapters include the history of the discovery of ER; physiological and pathological roles of ER; recent discovery of ER cistrome, transcriptome and its regulation of noncoding RNAs such as microRNAs and enhancer RNAs in breast cancer; development and clinical practices of the first targeted therapy Tamoxifen and other antiestrogens for breast cancer treatment; structural basis of ER and antiestrogen actions; molecular insights into endocrine resistance; the role of ER mutants, ER-beta and environmental estrogens in breast cancer; and emerging state-of-the-art therapeutic approaches currently in development to overcome treatment resistance and future perspectives. The book will provide undergraduate and graduate students, basic scientists and clinical cancer researchers, residents, fellows, as well as clinicians, oncology educators and the general public a thorough and authoritative review of these exciting topics.
Author: Athéna Sklias
Publisher:
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Languages : en
Pages : 0
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Previous epidemiological and experimental studies have strongly implicated estrogens in breast cancer risk and Estrogen Receptor (ER), the transcription factor to which estrogen binds, is considered as the major molecular driver of around 70% breast cancers. The importance of the deregulated estrogen signalling is further highlighted by increasing evidence that current chemopreventive and therapeutic strategies that target hormonally responsive breast cancers frequently result in the development of resistance to anti-estrogens and metastatic progression, highlighting the need for understanding the molecular underlying mechanisms. While until recently, ER was believed to act as a stand-alone transcription factor, which can directly bind its motifs in DNA, it is now accepted that ER activity is a complex and dynamic process that requires highly concerted actions of a dozen transcriptional cofactors and various chromatin regulators at DNA. Recent studies focused on characterising ER-associated cofactors and their role in opening the chromatin provided a remarkable insight into transcriptional regulation mediated by ER. However DNA methylation and histone acetylation are poorly understood in the context of ER's dynamic binding. In this thesis, I combined a cell culture protocol adapted for studying estradiol (E2) deprivation and re-stimulation in stricto sensu in ER-positive breast cancer cells with the latest methylation array, that allowed a genome-wide interrogation of DNA methylation (including a comprehensive panel of enhancers). I further investigated histone acetylation (ChIP-seq) and transcriptome (RNA-seq) after E2 deprivation and re-stimulation to better characterise the ability of ER to coordinate gene regulation. I found that E2 deprivation and re-stimulation result in time-dependent DNA methylation changes and in histone acetylation across diverse genomic regions, many of which overlap with enhancers. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER tethering mainly through two partner TF families, AP-1 and FOX, in the proximity of enhancers that are differentially methylated and acetylated. This is the first study that comprehensively characterized DNA methylation at enhancers in response to inhibition and activation of ER signalling. The transcriptome and genome occupancy data further reinforced the notion that ER activity may orchestrate a broad transcriptional programme through regulating a limited panel of critical enhancers. Finally, the E2 re-stimulation experiments revealed that although the majority of the observed epigenetic changes induced by E2 deprivation could be largely reversed when the cells were re-stimulated we show that DNA hypermethylation and H3K27 acetylation at enhancers as well as several gene expression changes are selectively retained. The partial reversibility can be interpreted as a sign of treatment efficiency but also as a mechanism by which ER activity may contribute to endocrine resistance. This study provides entirely new information that constitutes a major advance in our understanding of the events by which ER and its cofactors mediate changes in DNA methylation and chromatin states at enhancers. These findings should open new avenues for studying role of the deregulated estrogen signalling in the mechanism underlying the “roots” of endocrine resistance that commonly develops in response to anti-estrogen therapy.
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Languages : en
Pages : 11
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Proliferation and metastasis of many breast cancers depend on the steroid hormone estrogen. The actions of estrogens are mediated by the estrogen receptors ERalpha and ERbeta. These hormone-regulated transcription factors translate the presence of estrogen into changes in gene expression. According to new findings, these receptors also act outside of the nucleus and are often found associated with the plasma membrane. In contrast to their roles in regulating cell proliferation, very little is known about the mechanisms by which estrogens promote metastasis. It has been suggested that estrogens aid this process by changing the expression of cell adhesion proteins, such as E-cadherin. However, results in our laboratory have opened the possibility that disruption of cell adhesions by estrogens involves the direct interaction of ER with cell adhesion proteins. The goal of this grant is to explore this possibility. If true, this mechanism would represent a novel example of a non-nuclear activity of the estrogen receptor, steer ongoing studies on the role of estrogens in the regulation of cellular adhesions into a new direction, and open new venues for the prevention, diagnosis and therapy of breast cancer.
