THÈSE En vue de l'obtention du DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE Délivré par l'Université Toulouse III - Paul Sabatier Discipline ou spécialité : Gènes Cellules & Développement Présentée et soutenue par Mahta MAZAHERI Le 26 juin 2009 à 14H30 Salle de conférence de l'IEFG (IBCG) Titre : Molecular basis of anti-hormonal treatment and resistance in breast cancer JURY Dr. Sophie DOISNEAU-SIXOU, Professeur UPS,Toulouse, Président Dr. Michel RENOIR, Directeur de Recherche CNRS, Châtenay-Malabry, Rapporteur Dr. Dany CHALBOS, Directeur de Recherche INSERM, Montpellier, Rapporteur Dr. Annie VALETTE, Directeur de Recherche CNRS, Toulouse, Examinatrice Ecole doctorale : Biologie-Santé-Biotechnologies Unité de recherche : LBME, UMR 5099 Directeur(s) de Thèse : Dr. Kerstin BYSTRICKY/ Dr. Hélène Richard-Foy Rapporteurs : Dr. M. RENOIR / Dr. D. CHALBOS / Dr.S.KHOSHBIN Acknowledgment I would like to express my gratitude to all those who gave me the possibility to complete this thesis. First of all, I would like to begin with my promotore Dr. Hélène Richard- Foy (who left us in 2007), thank you for welcoming me in your Team, giving me the opportunity to develop this experience abroad and challenging me every day to make me a better Scientist. I am extremely thankful to Dr. Kerstin Bystricky for giving me the opportunity to continue my experience under your supervision; thanks for your kindness, support and encouragement during my thesis. Furthermore, I would like to thank the members of Jury: Dr. Sophie Doisneau-Sixou, Dr. Michel Renoir, Dr. Dany Chalbos, Dr. Saadi Khochbin and Dr. Annie Valette for taking the time to assess my manuscript. Your advices were greatly appreciated. I would like to thanks the members of our research group; my dear friends: Stephanie, Imen, silvia, Mathieu, Anne-Claire and Isabelle; thank you for the good times and nice company, I want to say that it was a real pleasure to work with you. Exchanging tips in the lab, discussing results in group meeting, chatting in the corridors… In the future, if you’re in the neighbourhood don’t hesitate to visit me. Especially, I would like to give my special thanks to my family; my husband, Kazem and my daughter, Sahel; your patient love enabled me to complete this work. Juin 2009 Mahta Summary Breast cancer is the most common type of malignancy among women in the world. Approximately 70% of breast tumours express the estrogen receptor alpha (ERα) and are considered hormone-responsive. Endocrine therapies have long been the treatment of choice. However, the estrogen- like agonist effect and development of resistance of the available selective estrogen receptor modulator such as tamoxifen require developing new treatments that act through different mechanisms. The objective of our study is to design tools that can help to understand the molecular mechanisms involved in ligand-dependent modulation or degradation of ERα. We selected a set of anti-estrogens with different structures and compared their effect on: 1). ERα degradation. 2). Intra-cellular localisation of ERα. 3). Regulation of transcription of ERα- endogenous target genes. 4). Regulation of transcription in the mutants of ERα. Using this mechanistic study we could classify the tested anti-estrogens into three groups based on their function: SERM, SERD and a new group for EM-652. SERM (selective estrogen receptor modulator) include compounds such as OH-tamoxifen and RU39411, that stabilise ERα, that re-localize ERα into the nucleus upon binding, that increase transcriptional activity in mutants affecting the recruitment of cofactors or the binding of their side chain and that lack inhibitory capacities of the basal expression of endogenous genes. SERD (selective estrogen receptor modulator) include compounds such as ICI182580 or RU58668 that induce nuclear proteasome-dependent degradation ERα which occur in large nuclear foci that colocalize with the proteasome and that inhibit basal gene expression of the endogenous progesterone receptor gene (PGR). Finally, EM-652 was found to affect ERα degradation and localisation similarly to SERM but inhibited basal gene expression of the endogenous PGR. This approach can be used to screen the newly designed compounds based on specific antiestrogen structural features INTRODUCTION ...................................................................................................................... 5 PART I: GENERAL HISTORY OF BREAST CANCER......................................................... 6 1. Definition and history of breast cancer .......................................................................... 9 2. Epidemiology ................................................................................................................. 9 3. The Biology of Breast Cancer ...................................................................................... 11 3.1. Critical periods of susceptibility to mammary carcinogen: ...................................... 11 3.2. Biological Characteristics of Critical Periods ........................................................... 12 4. Breast Carcinogenesis .................................................................................................. 12 4.1. Risk Factors ............................................................................................................... 15 4.2 Breast cancer classification ........................................................................................ 17 4.3. Prognostic and predictive markers ............................................................................ 18 5. Breast cancer treatment ................................................................................................ 19 5.1. Surgery ...................................................................................................................... 20 5.2. Chemotherapy ........................................................................................................... 20 5.3. Radiotherapy ............................................................................................................. 21 5.4. Hormonal therapy ...................................................................................................... 21 5.4a. Anti-estrogens...................................................................................................... 22 5.5. Other targeted therapies ........................................................................................ 23 PART II: ESTROGENS AND THEIR NUCLEAR RECEPTORS ......................................... 24 1. Transport and Metabolism of Estrogens .......................................................... 26 Physiologic actions of estrogens .............................................................................. 26 2. Actions on Breast Tissue .................................................................................. 27 3. Estrogen and carcinogenesis ............................................................................ 28 4. Estrogen Receptors ........................................................................................... 30 4.1. Estrogen Receptor isoforms and their structure ................................................... 30 4.2. Different variants of ERs...................................................................................... 32 5. Tissue distribution of ERs ................................................................................ 33 6. ERs expression in breast cancer ....................................................................... 34 PART III: MOLECULAR BASIS FOR TRANSCRIPTIONAL ACTIVITY OF THE ESTROGEN REcEPTOR alpha ............................................................................................... 36 1. ERα localization ....................................................................................................... 36 2. ERα activation and functional pathways .................................................................. 36 3. Ligand-dependent pathways ..................................................................................... 36 3.1. Genomic pathways .................................................................................................... 36 3.1a. Classical or direct pathway .................................................................................. 36 3.1b. Non- classical or indirect pathways .................................................................... 37 3.2. Non genomic pathway ............................................................................................... 38 4. Ligand independent pathways .................................................................................. 38 5. Transcriptional coregulators of ERα ........................................................................ 39 6. Coregulators and chromatin remodelling ................................................................. 40 6.1. Co-activators ............................................................................................................. 41 6.2. Co-repressors ............................................................................................................. 41 7. Activation of ERα through phophorylation.............................................................. 42 8. ER turn over and ligand dependent degradation of ERα .......................................... 43 PARTR IV: ANTIESTROGEN SIGNALING AND RESISTANCE TO ENDOCRINE THERAPY ............................................................................................................................... 45 1. Selective Estrogen Receptor Modulators (SERM)........................................................... 46 Tamoxifen ........................................................................................................................ 47 Metabolism and pharmacokinetic of tamoxifen ............................................................... 47 2. Selective Estrogen Receptor Down-regulator (SERD) .................................................... 48 1 ICI 164384........................................................................................................................ 48 ICI 182780........................................................................................................................ 48 3. Molecular mechanism of anti-estrogen activity ............................................................... 49 4. Transcriptional activity .................................................................................................... 51 4.1. Transcription activation function 1(AF-1) ................................................................ 51 4.2. Transcriptional activation function 2(AF-2) ............................................................. 51 5. Structural basis of agonism and antagonism .................................................................... 52 5.1. Flexibility of the LBD: .............................................................................................. 52 5.2. Role of H12 in antiestrogen signalling ...................................................................... 54 6. The factors affecting antiestrogenic activity .................................................................... 56 7. Breast cancer and endocrine resistance ............................................................................ 57 8. Tamoxifen treatment and resistance to endocrine treatment ............................................ 57 8.1. Loss of ERα expression and function........................................................................ 58 8.2. ERβ subtype and its alteration in expression ............................................................ 58 8.3. The role of nuclear receptor coregulators ................................................................. 59 8.4. ER pathway cross-talk with growth factor and cellular kinase pathways ................. 60 AIM OF PRESENT STUDY ................................................................................................... 61 RESULTS................................................................................................................................. 62 PUBLICATION N°1 ................................................................................................................ 63 A molecular approach to predict selective estrogen receptor modulators from down regulators .................................................................................................................................................. 64 Abstract ............................................................................................................................ 65 Introduction ...................................................................................................................... 66 Materials and Methods ..................................................................................................... 68 Results .............................................................................................................................. 73 Discusssion ....................................................................................................................... 79 Figures and Legends......................................................................................................... 83 References ........................................................................................................................ 90 PUBLICATION N°2 ............................................................................................................... 93 Ligands specify estrogen receptor alpha nuclear localization and degradation ....................... 94 Abstract ............................................................................................................................ 95 Introduction ...................................................................................................................... 95 Materials and Methods ..................................................................................................... 97 Results ............................................................................................................................ 101 Discussion ...................................................................................................................... 108 Figures and Legends....................................................................................................... 111 References ...................................................................................................................... 120 ONGOING RESULTS ........................................................................................................... 122 CONCLUSION AND PERSPECTIVES ............................................................................... 123 REFERENCES ....................................................................................................................... 126 2 List of abbreviations AF1: Transcription activation factor 1 AF2: Transcription activation factor 1 AIB1: Amplified In Breast Cancer 1 AP1: Activating Protein 1 CREB: cAMP Response Element-Binding DCIS: ductal carcinoma in situ DES: Diethyl Stilbestrol ER: Estrogen Receptor E1: Estrone E2: 17-β-Estradiol E3: Estriol EGF: Epidermal Growth Factor EGFR: Epidermal Growth Factor Receptor ERE: Estrogen Response Element ERK: Extra cellular signal Regulated Kinase FISH: Fluorescence In Situ Hybridization GF: Growth Factor HAT: Histone Acetyl Transferase HDAC: Histone deacetylases 3 Hsp: Heat shock protein HER2: Human Epidermal growth factor Receptor 2 IGF-1: Insulin like Gowth Factor1 LBD: Ligand Binding Domain MAPK: Mitogen Activated Protein Kinase MISS: Membrane Initiated Steroid Signaling N-CoR: Nuclear receptor Corepressor NISS: Nuclear Initiated Steroid Signalling 4OHT: 4-Hydroxytamoxifen PR: SP1: Progesterone Receptor Specific Protein-1 SERM: Selective Estrogen Receptor Modulator SERD: Selective Estrogen Receptor Downregulator SRC: Steroid Receptor Coactivator SMRT: Silencing Mediator for Retinoid and Thyroid receptors TGF: Transforming Growth Factor TEB: Terminal End Buds TDLU: Terminal Duct Lobular Units WT: Wild Type 4 INTRODUCTION 5 PART I: GENERAL HISTORY OF BREAST CANCER The mammary gland is a complex organ that begins development early in gestation and culminates in the postpartum lactation of the adult female. The extensive research currently being performed on the human genome and molecular biology will certainly contribute to further elucidate the role of factors involved in formation, differentiation, and development of the mammary gland. This may provide important insights into causes, treatment, and potential prevention of mammary gland abnormalities such as breast cancer. Given that breast cancer strikes 10% of women in the world, these developments may have enormous implications for the future of medicine (Brisken, 2002; Nguyen et al., 1995; Polyak, 2001). Breast development occurs in distinct stages throughout a woman's life. Human breast tissue begins to develop in the sixth week of fetal life. Breast tissue initially develops along the lines of the armpits and extends to the groin (this is called the milk ridge). By the ninth week of fetal life, it regresses to the chest area, leaving two breast buds on the upper half of the chest, In the neonate, the mammary glands, with their relatively simple architecture, remain quiescent until puberty (Naccarato et al., 2000). Female breasts do not begin growing until puberty when the breasts will begin to respond to hormonal changes in the body. Specifically, the production of two hormones, estrogen and progesterone, signal the development of the glandular breast tissue. The ductal branches that are formed during embryogenesis grow and divide to form branching ductal bundles with terminal end buds (TEB). The TEBs are major site of proliferation, and at menarche the terminal duct lobular units (TDLUs) develop from this site but remain in an arresting state until the onset of pregnancy and lactation (Vogel et al., 1981). Development initiated at the onset of puberty is generally complete by 20 years of age. If pregnancy occurs, with accelerated development of the TDLUs, the number of epithelial cells and alveoli within the lobules increases in preparation for lactation (Hovey et al., 2002) at which point the glands complete their differentiation and reach functional maturity. 6 Following lactation, there is massive apoptosis and remodelling of the tissue that will then resemble, once again, the gland in its non-pregnant state (Allan et al., 2004; Furth et al., 1997). The complex branching structure of lobules is lined by three epithelial cell types, ductal and alveolar luminal cells, and myoepithelial cells. The ductal and alveolar cells constitute the inner layer of ducts and the lobuloalveolar units, respectively, and each is surrounded by a basal layer of myoepithelial cells. All three epithelial cell types have recently been demonstrated to originate from a common multipotent stem cell (Shackleton et al., 2006; Stingl et al., 2006) The glands complete their differentiation and reach functional maturity under the influence of sustained increase in the levels of circulating progesterone, estrogens, prolactin and placental lactogen. . Figure 1: Life cycle of breast development in the nulliparous woman. The breast is primarily composed of lobules type 1, with some progression to type 2, and only minimal formation of lobules type 3 during sexual maturity, which regresses to lobules type 1 at menopause. 7 Figure 2: Life cycle of breast development in the parous woman. The breast undergoes a complete cycle of development through the formation of lobules type 4 with pregnancy and lactation. These regress to lobules type 3 after weaning, and to lobules type 1 at menopause (Russo and Russo, 1998). By 40 years of age, the mammary glands begin to atrophy. During and after menopause, the altered hormonal environment leads to a senescent state, with involution of the glandular component and replacement with connective tissue and fat. Figure 3: Breast profile. Top: A ducts B lobules C dilated section of duct to hold milk D nipple G chest wall/rib cage Botton: A normal duct cells B basement membrane C lumen (center of duct) 8 E fat F pectoralis major muscle 1. Definition and history of breast cancer Breast cancer is a major public health problem in the world; there are many texts and references that attempt to define breast cancer. The simplest definition is from the National Cancer Institute (NCI). According to the NCI, breast cancer is a “cancer that forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk)”. It occurs in both men and women, although male breast cancer is rare. 2. Epidemiology Breast cancer was recognized by the ancient egyptians as long ago as 1600 BC. However, over the past 50 years it has become a major health problem affecting as many as one in eight women during their lifetime. The burden of breast cancer is increasing in both developed and developing countries, and in many of the regions of the world. It is now the most frequently occurring malignant disease in women. Each year the disease is diagnosed in over one million women worldwide and is the cause of death in over 400,000 women. Breast cancer can occur in men, although the incidence is much lower, amounting to around 1% of all breast cancers. Overall the incidence of breast cancer rises with age, increasing rapidly during the fourth decade of life and continuing to increase thereafter, but more slowly in the fifth, sixth and seventh decades. In the USA, 75% of new breast cancers are diagnosed in women aged 50 years or older, and the lifetime risk of breast cancer diagnosis is approximately 12.5%. The incidence rates for breast cancer are similar in North America and the majority of other western industrialized countries. In France, this pathology is the first diagnosed cancer for women, and according to data from the Ministère de la Santé (2003), each year there are 42 000 estimated new cases and 11 640 deaths. In Japan and other Far Eastern countries, however, absolute incidence rates are lower for each age band and overall Japanese women are five times less likely to develop breast cancer than American women. Epidemiologic studies show a significant decrease in mortality rate of breast cancer and this could probably be the result of earlier diagnosis, but also from a better therapeutic care particularly in the adjuvant treatments. Most cases of breast cancers are sporadic, however, 9 twin studies have shown that heritable factors may cause 20-30% of all breast cancers (Lichtenstein et al., 2000). Mutations within BRCA1 and BRCA2 genes are common among women with a strong family history of breast cancer; they account for at most 3-8% of all breast cancer cases. It is important to underline that 70% of breast cancers are hormonedependent; they express estrogen receptor (ER) and their proliferation is influenced by estrogens. Frequency, severity and impact of this pathology on different aspects of human life make breast cancer a priority in research and justifies the efforts to prevent and to improve available treatments. Estimated New Cases Estimated Deaths Male Prostate Female Breast Male Lung & bronchus Female Lung & bronchus 186,320 (25%) 182,460 (26%) 90,810 (31%) 71,030 (26%) Lung & bronchus Lung & bronchus Prostate Breast 114,690 (15%) Colon & rectum 100,330 (14%) Colon & rectum 28,660 (10%) Colon & rectum 40,480 (15%) Colon & rectum 77,250 (10%) Urinary bladder 71,560 (10%) Uterine corpus 24,260 (8%) Pancreas 25,700 (9%) Pancreas 51,230 (7%) Non-Hodgkin lymphoma 40,100 (6%) Non-Hodgkin lymphoma 17,500 (6%) Liver & intrahepatic bile duct 16,790 (6%) Ovary 35,450 (5%) Melanoma of the skin 30,670 (4%) Thyroid 12,570 (4%) Leukemia 15,520 (6%) Non-Hodgkin lymphoma 34,950 (5%) Kidney & renal pelvis 28,410 (4%) Melanoma of the skin 12,460 (4%) Esophagus 9,370 (3%) Leukemia 33,130 (4%) Oral cavity & pharynx 27,530 (4%) Ovary 11,250 (4%) Urinary bladder 9,250 (3%) Uterine corpus 25,310 (3%) Leukemia 21,650 (3%) Kidney & renal pelvis 9,950 (3%) Non-Hodgkin lymphoma 7,470 (3%) Liver & intrahepatic bile duct 25,180 (3%) Pancreas 21,260 (3%) Leukemia 9,790 (3%) Kidney & renal pelvis 5,840 (2%) Brain & other nervous.system 18,770 (3%) All sites 745,180 (100%) 19,090 (3%) All sites 692,000 (100%) 8,100 (3%) All sites 294,120 (100%) 5,650 (2%) All sites 271,530 (100%) Table I: Leading Sites of New Cancer Cases and Deaths – 2008 10 *Excludes basal and squamous cell skin cancers and in situ carcinoma except urinary bladder ©2008, American Cancer Society,Inc. 3. The Biology of Breast Cancer Unlike other tissues in the body as the liver and the heart that are formed at birth, breast tissue in newborns consists only of a tiny duct. At puberty, in response to hormones (like estrogen that is secreted by the ovary), the breast duct grows rapidly into a tree-like structure composed of many ducts. Most breast development occurs between puberty and a woman’s first pregnancy. The immature breast cells, called “stem cells”, divide rapidly during puberty. The cells in the immature, developing breast are not very efficient at repairing mutations and they are more likely to bind carcinogens (cancer causing agents). The female hormone estrogen stimulates breast cell division. This division can increase the risk of damaging DNA. Furthermore, not fully matured breast cells in girls and young women are not as efficient at repairing DNA damage as mature breast cells; therefore it is important to reduce the exposure of young women and girls to carcinogens that might damage DNA during this phase of rapid breast development. Some evidence also suggests that breast feeding further reduces the breast cells’ sensitivity to mutations. Milk producing cells are fully mature and less sensitive to DNA damage than immature undifferentiated cells. Therefore, susceptibility to mutations declines in the breast cells of women who have had an early full-term pregnancy (University, 1997). 3.1. Critical periods of susceptibility to mammary carcinogen: *Birth to 4 years *Puberty *End of Puberty to 1st full-term pregnancy 11 3.2. Biological Characteristics of Critical Periods * Rapid cell division * Breast cells have higher proportion of "stem cells" * Mutations can be passed on if not repaired * Stem cells are more susceptible to carcinogens This susceptibility decreases after first full term pregnancy and the reasons could be: * Fewer stem cells * Less cell division * More cells are differentiated * Differentiated cells repair DNA more efficiently * Differentiated cells bind carcinogens more weakly than stem cells (Original hypothesis by Drs. Irma and Jose Russo )(University, 1997). 4. Breast Carcinogenesis Breast cancer, like other forms of cancer is widely perceived as a heterogeneous disorder with markedly different biological properties from normal cells. Cancerous cells develop from healthy cells, constantly changing under the influence of hormones and growth factors, in a complex process called malignant transformation. These are caused by a series of clonally selected genetic changes in key tumour-suppressor genes or oncogenes (Feinberg et al., 2006) (Olsson, 2000). It has been proposed that the process of breast cancer tumourigenesis is best described by a multi-step progression model (Beckmann et al., 1997), in which the normal epithelium develops via hyperplasia and carcinoma in situ into an invasive cancer, which can disseminate via the lymph and the vascular system. Initiation: The first step in cancer development is initiation. The development of a tumour is associated with the acquisition of genetic and epigenetic alterations that modify normal growth control and survival pathways (Albertson, 2006; Polyak, 2007 ). 12 This phase is characterized by accumulation of mutations that lead to the overexpression of pro-oncogenic factors. Genetic mutations that can lead to breast cancer have been experimentally linked to estrogen exposure (Cavalieri et al., 2006). The change in the cell’s genetic material occurs spontaneously or is brought on by carcinogens including ionizing radiation, many chemicals, viruses, radiation, and sunlight. A long list of genes has been reported for their implication in breast cancer tumourigenesis in which the most important are: Oncogenes; oncogenes refer to genes whose alteration causes gain of function. A frequent anomaly for oncogenes is amplification. Examples of amplification include erb-B2 which is a member of EGF receptor superfamily, cmyc coding for a protein important in apoptosis, and finally ccndI that codes cyclin D1, a regulator of G1/S transition in cell cycle (Osborne et al., 2004). This anomaly is found in more than 15% of breast cancers. Oncogenic activation through gene amplification of erb-B2 (HER-2) occurs in about 20% of primary breast cancers. Erb-B2 encodes a transmembrane tyrosine kinase growth factor receptor and its activation via a variety of pathways is involved in proliferation and angiogenesis, alteres cell-cell interactions, increases cell motility, metastases, and resistance to apoptosis. Tumour suppressor genes (anti-oncogene): inactivating tumour suppressor genes as p53, Rb, PTEN, p16, BRCA1 and BRCA2 induce tumour development. For example Tp53 gene (coding pour p53) which is Located at 17p13p53 loucus (Lacroix et al., 2006) is damaged or missing in most of human cancers including breast cancer. Loss of heterozygosity (LOH) of theTp53 gene was shown to be a common event in primary breast carcinomas (Davidoff et al., 1991). One function of this gene is to keep cells with damaged DNA from entering the cell cycle. The Tp53 gene can tell a normal cell with DNA damage to stop proliferating and repair the damage. In cancer cells, p53 recognizes damaged DNA and tells the cell to "commit suicide" (apoptosis). If the p53 gene is damaged and loses its function, cells with damaged 13 DNA continue to divide when normally they would have been removed through apoptosis. This is why the p53 gene has been termed "The Guardian of the Genome." Promotion: The second and final step in the development of cancer is promotion. Subsequent tumour progression is driven by the accumulation of additional genetic changes combined with clonal expansion and selection (Polyak, 2007 ). When cells enter the second stage of promotion, they gain their independence and lose their capacity for intercellular communication which leads to unregulated cell proliferation (Eccles, 2001; Hynes and Lane, 2001; Lane et al., 2001). Spread: Breast cancer usually spreads first to the nearby lymph nodes, and later to distant sites. Cancers can also spread via the blood stream. Common sites of metastases include the liver, the lung, bone, and the brain (Chambers et al., 2002). Of course estrogen receptors (ERs) play an important role in breast carcinogenesis. Overexpression of ERα is frequently observed in early stages of breast cancer. ERs are the regulatory proteins essentially localized in the nucleus, but also sometimes in the cytoplasmic membrane. The estrogen receptor regulates gene expression by both estrogen-dependent and estrogen-independent mechanisms leading to activation of gene transcription (Hayashi et al., 2003). Schematically, in breast cancer cells, the ER-hormone complex influences different pathways by: *Stimulation of synthesis and activity of certain growth factors (EGF, IGF-1, TGFα), resulting in cell proliferation. *Stimulation of activity of oncogenes (c-myc, c-myb, c-fos) interfering with cell proliferation and apoptosis. *Stimulation of protease synthesis (cathepsin D, UPA-1) contributing in metastatic processes by degradation of extra cellular matrix. 14 4.1. Risk Factors Gender: it is about 100 times more common in women than in men (Hulka and Moorman, 2001) (American Cancer Society 2009) Age: one of the best documented risk factor for breast cancer is age. The chance of getting breast cancer goes up as a woman gets older. About two-third of cases are diagnosed in women aged 55 or older (McPherson et al., 2000). From 2002-2006, the median age at diagnosis for cancer of the breast was 61 years of age. Approximately 0% was diagnosed under age 20; 1.9% between 20 and 34; 10.5% between 35 and 44; 22.5% between 45 and 54; 23.7% between 55 and 64; 19.6% between 65 and 74; 16.2% between 75 and 84; and 5.5% 85+ years of age. http//seer.cancer.gov/csr/1975_2006 Genetic background and family history: genetic predisposition is a growing knowledge suggesting that pattern of risk can be defined precisely person by person (Singletary, 2003). Genetic susceptibility to breast cancer is conferred by a large number of genes and as (Ripperger et al., 2008) which we can classify in inherited breast cancer to three groups; * Highly penetrant genes (BRCA1, BRCA2, TP53, STK11, PTEN, CDH1). Although these mutations are highly penetrant, they have been well characterized. For this group, genetic counselling and genetic testing are available and could allow appropriate screening and prophylactic measures. *The intermediate penetrance breast cancer susceptibility genes (ATM, CHEK2, BRIP1, BRAD1, and PALB2). In the literature there is no particular suggestion to performe genetic testing for these mutations as a screening strategy. *The low penetrance breast cancer susceptibility alleles (FGFR2). Family history: many studies have attempted to define the risk associated with a positive family history. It has been proposed that approximately 10-20% of breast cancers are due to 15 genetic predisposition (McPherson et al., 2000; Singletary, 2003). To date it is clear that degree of risk is in relation with the type of relative affected (first or second degree), the age at which the relative developed cancer, and the number of relatives affected. For example BRCA1 and BRCA2 genes are , germlin mutation associated with an inherited susceptibility to breast and ovarian cancer (Brody and Biesecker, 1998). Women with a strong family history of breast and/or ovarian cancer should be offered counselling to determine if genetic testing is appropriate. Families with four or more relatives affected with either breast or ovarian cancer in three generations and one living affected relative have a high risk to develop cancer (Fig.4). Studies suggest that prophylactic removal of the breasts and/or ovaries in BRCA1 and BRCA2 mutation carriers decreases considerably the risk of breast cancer(McPherson et al., 2000). Figure 4: Family tree of family with genetically inherited breast cancer (McPherson et al., 2000). An example of family tree or history that could be used for assessing familial breast cancer risk. Lifestyle and environmental factors: no factors have yet been identified that have a major effect on the risk of breast cancer (Singletary, 2003). However, there are several factors that may have a limited effect; these are diet, weight, alcohol intake. 16 Reproductive factors: women who began menstruating before the age of 12 have a relative risk for invasive breast cancer of 1.3 compared to those who began after the age of 15. On the other hand, those who have not reache menopause until age 55, have a relative risk of 1.22 compared to those who have menopause before age 45. Women who have no children, or who had their first child after age 30, have a higher risk of breast cancer (Singletary, 2003). 4.2 Breast cancer classification Currently using TNM classification for breast tumours is according to their extension of tumours. The key elements in this classification are: tumour size, presence of metastatic regional lymph node, and distance metastases (Veronesi et al., 2006). However, for making a treatment decision, the knowledge of several other biological factors as ER, PgR, HER2 overexpression or amplification is required. Advances in the biology of cancer and in technical progress such as microarray have confirmed that breast cancer is a heterogeneous disease with diversity in responsiveness in treatment which may explain differences in evolution of patients (Perou et al., 2000). Breast cancers can be subdivided into at least five sub-groups based on their gene expression patterns (Sorlie et al., 2001) (Fig.5). In this study the tumours were separated into two main branches: the groups of the left branch are characterized by low to absent ER gene expression. The groups in the right branch demonstrated the highest level of ERα gene expression. ER negative groups include: 1. Basal like or triple negative sub-type, 2. ERBB21 sub-type that presents an amplification of HER2gene, and 3. Normal breast-like group. Luminal/ER positive group includes: 1. Luminal sub-type A 2. Luminal sub-type B 3. Luminal sub-type C. thereby in this study the genome wide expression patterns of tumours are a representation of the biology of the tumours and thus, relating gene expression patterns to clinical outcomes is a key issue in understanding this diversity. 17 Figure 5: Molecular classification of breast cancer (Sorlie et al., 2001). Gene expression patterns of experimental samples including carsinomas, benign tumours and normal tissuses, analyzed by hierarchical clustering. 4.3. Prognostic and predictive markers In recent years mortality of breast cancer regressed probably as a result of widespread screening, earlier detection and advances in adjuvant treatment. However, adjuvant systemic therapy has associated risks and it would be useful to be able to optimally select patients most likely to benefit. A prognostic factor is any measurement factor available at the time of surgery that correlates with overall survival of patient without a systemic adjuvant therapy. In 18 contrast a predictive factor is a measurement associated with a response to a given therapy. Some factors are both prognostic and predictive (Cianfrocca and Goldstein, 2004) (Table. 2). Table 2: A summary of prognostic and predictive factors in breast cancer In practice: Over half of breast tumour express ERα and around 70% of these respond to anti-estrogen treatment while an absence of ER expression is associated with a poor prognosis (Ali and Coombes, 2000). Thus a analysis of steroid receptor status has become the standard of care for patients with breast cancer. The testing of breast cancer specimens for (erb-B2) HER-2/neu status has now achieved a standard role for the management of breast cancer; HER-2/neu overexpression identified by immunohistochemistry/ gene amplification detected by FISH, has been consistently associated with higher grade and extensive forms of ductal carcinoma in situ (Ross et al., 2003). Knowing if a cancer is HER2-positive directs the choice of treatment and patients could then benefit from another treatment called Herceptin (a monoclonal antibody against the HER2 protein). 5. Breast cancer treatment Different types of treatments exist for patients with breast cancer. Some of them are currently used in clinic and some others are in the evaluating phase. The standard types of treatment are 19 surgery, radiation therapy, chemotherapy and hormonal therapy. New types of treatment that are being studied in clinical trials, for exemple as monoclonal antibodies, are uses as adjuvant therapy, and other targeted therapies. The goal of different modalities is to control local extensions of disease or metastases; Thus surgery and radiotherapy will aim a regional control of disease while the objective of the systemic therapy as chemotherapy, hormonal therapy and other form of targeted therapies is to control of metastases. 5.1. Surgery: Surgery is the primary treatment of breast cancer. For a local treatment, different modes of operations are used for most women with breast cancer. The choice of different strategies of operation depends on different factors such as size and location of the breast tumour as well as the type and the stage of breast cancer. 5.2. Chemotherapy: The purpose of chemotherapy and other systemic treatments is to eliminate all cancer cells that may have spread from where the cancer started to another part of the body. The oncologist uses this treatment for three purposes: -Adjuvant therapy: chemotherapy is given after the initial surgery to prevent coming back of cancer. -Neo-adjuvant: is used for large breast cancer before surgery to shrink tumours and to ease surgery. -Tteatment metastatic cancer. There is different chemotherapy drugs used in breast cancer treatment. Cyclophosphamide, epirubicin, fluorouracil (5FU), methotrexate, paclitaxel (Taxol), doxorubicin (Adriamycin®),docetaxel (Taxotere®) are commonly used. These drugs are usually used in combination regimens as FEC that means a combination of 5FU, epirubicin and cyclophosphamide or AC meaning doxorubicin (Adriamycin®) and cyclophosphamide. 20 There is no clinically useful molecular predictor of response to any cytotoxic drug used in the treatment of breast cancer. However, certain studies propose a predictive model in chemotherapy (Ayers et al., 2004). 5.3. Radiotherapy: Radiotherapy reduces local relapse, with a relative risk reduction (Cuzick, 2005). Radiotherapy could be used in different ways: 1. after surgery to reduce remaining cancer cells / to treat the lymph node. 2. before surgery to reduce tumour size. 5.4. Hormonal therapy: For the first time in 1896 a surgeon called Beatson, reported that breast cancer regression can occur in response to oophorectomy in premenopausal women (Beatson, 1896). This report was the first recognition of hormone-dependent tumours. In 1930s, estrogens were isolated and their implications in rodent mammary tumours were described (Benson, 2008). The identification of the estrogen receptor (ER) in 1966 by Jensen (Jensen, 1966) provided a mechanism to describe specificity of estrogen action at the target site, and a target was identified to develop new drugs for the treatment and prevention of breast cancer. In addition, a test was established to predict the outcome of antihormonal therapy in breast cancer (Jensen and Jordan, 2003). Hormonal therapy only started in the 1970s with the widely prescribed anti-estrogen tamoxifen (Ward, 1973). In premenopausal women, the ovaries are the principle source of estradiol that acts on distal target tissues. In contrast in postmenopausal women estrogen is produced in extra gonadal sites (as adipose tissues) rich of aromatases from conversion of androgen produced in the adrenal gland, and functions locally at these sites (Simpson, 2003). In clinic there are three possible levels for hormonal therapy: At the hypothalamus - pituitary axis; this kind of hormone therapy is used normally for premenopause women by prescription the analogues of LH-RH. 21 By competition with the estrogen receptor level; using anti-estrogens. By inhibition of aromatases for menopausal patients. 5.4a. Anti-estrogens Anti-estrogens are the competitive inhibitors of estrogens. To date there are two groups of anti-estrogens; SERMs for Selective Estrogen Receptor Modulators and SERDs for Selective Estrogen Receptor Downregulators. The ubiquity of ER explains the diversity of effects obtained in adition to anti-hormonal action. Tamoxifen: Tamoxifen, (Nolvadex), the oldest SERM in continuous use in the clinic. Tamoxifen is the standard choice in adjuvant treatment of ER positive breast cancer (Jordan, 2003). It is a non steroidal compound with a complex mechanism of action and contradictory effects depending on tissue. In the mammary gland and in mammary tumours, it acts as an antagonist, while in some tissues, such as bone, it is an agonist. Tamoxifen could be prescribed at every age regardless of menopause status. According to NSABP (The National Surgical Adjuvant Breast and Bowel Project: a clinical trials cooperative group supported by the NIH) (MOON, 2005), tamoxifen is linked to a 43% reduction in cumulative risk of invasive breast cancer. In a similar manner it reduces the cumulative risk of non-invasive breast cancer by 37%. This study confirms that tamoxifen reduces the risk of breast cancer in all subgroups indpendently of history and predicted risk for breast cancer. The clinician must be very vigilant to side effects generated by tamoxifen. The most common are menopausal symptoms including hot flashes, vaginal dryness, low libido, mood swings and nausea. Tamoxifen increases the risk of endometrial cancer and thrombo emboli accidents. The unwanted side effects of tamoxifen led to search for an anti-estrogen more potent and more specific such as toremifen or raloxifen. Pure anti-estrogens: Other molecules with no estrogenic activity have been developped such as ICI 164,384 and ICI 182,780. ICI 182,780 (Faslodex or Fulvestrant) is a steroidal anti- 22 estrogen with an unique and different mechanism of action from tamoxifen since it is the first anti-estrogen without partial agonistic activity. To date Faslodex is admitted as second line of treatment in patients that develop a resistance to tamoxifen or an undesirable effect of tamoxifen. This agent is reclassified as SERD that will be detailed later. 5.5. Other targeted therapies In recent years the following targeted therapies have been developed: In breast cancer the best example is trastuzumab (Herceptin). The recombinant humanized anti-HER2 monoclonal antibody, that becomes FDA (US Food and Drug Administration) approved in 2006, and is recommended for patients with breast cancer demonstrating 3 fold overexpression of HER2 by immunohistochemistry or amplification of the HER2 gene by fluorescence in situ hybridization (FISH). A 52 % reduction in the risk of recurrence is reported when it is in combination (http://www.cancer.gov/search/ViewClinicalTrial). 23 with chemotherapy PART II: ESTROGENS AND THEIR NUCLEAR RECEPTORS The existence and effect of estrogen were established from 1923 to 1938, estrogens comprise a group of steroid compounds; structurally related and hormonally active molecules that regulate critical cellular signalling pathways and by doing so, control cell proliferation, differentiation and homeostasis (Cheskis et al., 2007) . The naturally existing estrogens, 17β-estradiol (E2), estrone (E1), and estriol (E3), are C18 steroids derived from cholesterol. Estrone is a weaker estrogenic compound than estradiol, and in postmenopausal women, estrone is more produced than estradiol ( Fig 6). Estradiol (E2) Estrone (E1) Estriol (E3) Figure 6: Endogenous estrogens Estrogens are synthesized during steroidogenesis. Estrogens are produced primarily by developing follicles in the ovaries, the corpus luteum, and the placenta during pregnancy 24 (Simpson et al., 1997). Some estrogens are also produced in smaller amounts by other tissues such as the liver, adrenal glands, and the breasts. These secondary sources of estrogen are especially important in postmenopausal women. Estrogen biosynthesis is catalysed by aromatases (aromatase cytochrome P450), the product of the CYP19 gene, which is a member of the cytochrome P450 superfamily of genes (Simpson et al., 1997). Synthesis of estrogens starts in theca interna cells in the ovary. By the synthesis of androstenedione from cholesterol. Androstenedione is a substance of moderate androgenic activity. This compound crosses the basal membrane into the surrounding granulosa cells, where it is converted to estrone or estradiol, either immediately or through testosterone. The conversion of testosterone to estradiol and of androstenedione to estrone, is catalyzed by the aromatase enzyme. Estradiol levels vary through the menstrual cycle, with highest levels just before ovulation. Estrogens are present in both men and women. They are usually present at significantly higher levels in women of reproductive age. They promote the development of female secondary sex characteristics, such as breast, and are also involved in the thickening of the endometrium and other aspects of regulating the menstrual cycle. After menopause, adipose tissue becomes the main source of oestrogen (Grodin JM, 1973). Therefore, in the post-reproductive years, the degree of a woman’s estrogenisation is mainly determined by the extent of her adiposity. This is of clinical importance since corpulent women are relatively protected against osteoporosis (Melton, 1997), and the incidence of Alzheimer’s disease is lower in more corpulent postmenopausal women than in their slimmer counterparts (Simpson, 2000). Conversely, obesity is positively correlated with breast cancer risk (Huang et al., 1997). 25 1. Transport and Metabolism of Estrogens In the serum, estradiol reversibly binds to sex-hormone–binding globulin (Andersson, 1974). Estrogens are metabolized by sulfation or glucuronidation, and the conjugates are excreted into the bile or urine. Estrogens are also metabolized by hydroxylation and subsequent methylation to form catechol and methoxylated estrogens. Hydroxylation of estrogens yields 2-hydroxyestrogens, 4-hydroxyestrogens, and 16 a-hydroxyestrogens (catechol estrogens), among which 4-hydroxyestrone and 16 a-hydroxyestradiol are considered as carcinogenic. A range of synthetic and natural substances have been identified that also possess estrogenic activity (Fang et al., 2001). - Plant products with estrogenic activity are called phytoestrogens - Synthetic estrogens Phytoestrogens, which include lignans and isoflavones (e.g. genistein and daidzein), are estrogen-like compounds which occur naturally in many plants and fungi and which are biologically active in humans, Epidemiological studies showed a protective effect against breast cancer (Messina et al., 1997). DES (diethyl stilbestrol) was first synthesized estrogen in early 1938 by Leon Golberg, and a report of its synthesis was published in Nature in 1971 it was found to be a teratogen when given to pregnant women. Physiologic actions of estrogens: In the Central Nervous System, experimental studies on different animal models have shown that estrogen is neuroprotective. The mechanisms involved in the neuroprotective effects of estrogen are still unclear. Anti-oxidant effects, activation of different membrane-associated intracellular signalling pathways, and activation of classical nuclear estrogen receptors (ERs) 26 could contribute to neuroprotection. Interactions with neurotrophins and other growth factors may also be important for the neuroprotective effects of estradiol (Cardona-Gómez et al., 2001). Some epidemiologic data suggest that in postmenopausal women, estrogen deficiency is associated with a decline in cognitive function and an increased risk of Alzheimer’s disease (Henderson, 1997). Estrogens are arterial vasodilators and have cardioprotective actions. In the liver, estrogens stimulate the uptake of serum lipoproteins as well as the production of coagulation factors. Estrogens also prevent and reverse osteoporosis and increase cell viability in various tissues. In addition, estrogens stimulate the growth and development of the reproductive system. When applied topically, estrogens increase skin turgor and collagen production and reduce the depth of wrinkles (Fig 7). 2. Actions on Breast Tissue The ovarian steroids, estrogen and progesterone, are known to play a vital role in the staged development of the mammary gland, acting through specific nuclear receptors on target cells. These cells which represent less than 20% of the epithelium, express estrogen receptor alpha (ERα) and progesterone receptor (PR), both are known to be located in the luminal epithelia of the ductal and lobular structures (Petersen et al., 1987). The lobular units of the terminal ducts of the breast tissue of young women are highly responsive to estrogen and estrogens stimulate the growth and differentiation of the ductal epithelium, induce mitotic activity of ductal cylindric cells, and stimulate the growth of connective tissue (Porter, 1974.) 27 Figure 7: Physiologic actions of estrogens (Gruber et al., 2002). 3. Estrogen and carcinogenesis Since Sir George Beatson observed in 1896 that breast tumours in premenopausal women sometimes regressed after oophorectomy, numerous investigations have established that estrogen stimulates the growth of breast cancer cells. Mechanisms of carcinogenesis in the breast cancer caused by estrogen include the metabolism of estrogen to genotoxic, mutagenic metabolites and stimulation of tissue growth. Together these processes cause initiation, promotion, and progression of carcinogenesis (Yager and Davidson, 2006). In 28 postmenopausal women, extraovarian sex hormone production plays an important role in hormone-related diseases, such as breast and endometrial cancers (see Table 3). Figure 8: Pathways for Estrogen carcinogenesis (Yager and Davidson, 2006) Table 3: The carcinogenic events associated with estrogens (Yager and Davidson, 2006). 29 4. Estrogen Receptors The biological actions of estrogens are manifested in cells expressing a specific high-affinity estrogen receptor (ER). ER has two subtypes; ERα and ERβ each encoded by a separate gene (ESR1 and ESR2 respectively). They belong to the superfamily of the nuclear receptors (Pearce and Jordan, 2004). Other members of this family include receptors for other hydrophobic molecules such as steroid hormones (e.g.glucocoticoids progesteron, mineralocorticoid, androgens, vitamin D3,..), retinoic acids, and thyroid hormones (RobinsonRechavi et al., 2003). ER acts as a ligand-dependent transcription factor in most target tissues (Means et al., 1972). 4.1. Estrogen Receptor isoforms and their structure In the late 1950s, Jenson and Jacobsen (Jensen and Jacobson, 1962) demonstrated the existence of a receptor molecule that could bind to 17β-estradiol (Jensen and Jordan, 2003). ERα is the first estrogen receptor cloned from MCF-7 human breast cancer cells in 1980s (Green et al., 1986). ERα gene (ESR1) is located on chromosome 6 at 6q25.1locus, composed of 8 exons coding for a protein with 595 amino acids (66Kda). In addition, several ERα splicing variants have been characterized (Murphy et al., 1997). In 1996, a second receptor was reported from the rat prostate, ERβ (Kuiper et al., 1996b). ERβ is encoded by a distinct gene (ESR2) with 8 exons, located on chromosome 14q22-24. ERβ is a protein with 530 acid amine (60KDa) Different studies have demonstrated conservation of the regions denoted from A through F within the structure of ERs (Gronemeyer, 1991) (Fig.9) A/B domain: The N terminal A-B domain contains an activation function 1 (AF1), a constitutive activation function contributing to the ligand independent transcriptional activity of the ER. This domain is one of the least conserved domains between ERα and ERβ, 30 exhibiting only 30% identity (Ogawa et al., 1998). Functional studies have shown a lack of AF1 activity in ERβ (Hall and McDonnell, 1999) and this could explain at least, in part, the weak transcriptional activity of ERβ on certain promoters (McInerney et al., 1998). C domain: The DNA binding domain is the most highly conserved region between ERα and ERβ, with 96% identity. The C region contains two zinc finger structures each resulting from the coordination of one Zn++ to four cysteine residues. This allowes both receptors to bind to similar target sites (Schwabe et al., 1990). D domain: this hinge region which contains, the nuclear localization signal, is not well conserved between the receptors. E domain: It is the more complex domain of ER, it contains the ligand binding domain (LBD), a coregulator binding surface, the dimerization domain, HSP 90 binding domain, a second nuclear localization signal, and activation function 2, AF2 in contrast to AF1 is a ligand-dependent activation function. This domain exhibits 53% sequence identity between two ERs (Gronemeyer, 1991; Tora et al., 1989). This difference in homology could explain a subtle difference in ligand binding specifity (Kuiper et al., 1997). Despite the slight difference in the affinity of ERα and ERβ for E2, both receptors are considered to have a similar affinity for E2. However, differences were observed for other ligands, such as antiestrogens (Pearce and Jordan, 2004) (Table 4). Table 4: The relative binding affinity of various ligands for ERα ERβ (Kuiper et al., 1997). 31 Difference in LBD sequence of the two ERs led to synthesis of molecules that function as selective agonists or antagonists for ERα or ERβ such as TAS-180 (SR16234) which could be a pure antagonist on ERα and a partial antagonist on ERβ (Sun et al., 1999; Yamamoto et al., 2005). F domain: The C terminal of ERs shares 18% sequence homology between ERα and ERβ. The role of this domain of ER is uncertain, but there is evidence suggesting that the F domain has different role in the activity of ER α and β subtypes and it is in part responsible for the difference in the biological activity of the two ER sub-types. For example on the AP-1 site, ERβ deleted for the F domain is activated by tamoxifen while wt ERβ is activated only by raloxifene. In ERα the F domain is activated by E2 and tamoxifen but not by raloxifene and in ERα deleted for the F domain the receptor is activated by raloxifene (Skafar and Zhao, 2008). The differences between the F domains of the ER alpha and beta subtypes and among the other members of the nuclear hormone receptor superfamily may offer opportunities for selective control of the activity of these proteins. . Figure 9 : ER isoforms and their function and homology(adopted from (Zhenlin Bai, 2009). 4.2. Different variants of ERs Several splice variants have been described for both receptor subtypes, but whether all the variants are expressed as functional proteins with biological functions is not clear. Flourio et 32 al. characterized a 46 KDa isoform (hERα46) that lacks the first 173 amino acids of the 66KDa of ERα (Flourio et al., 2000 ) (Fig 10). In addition, several other ERα splicing variants have been isolated and identified in different cell lines (Poola et al., 2000). Figure 10: Schematic representation of estrogen receptor (ERα) isoforms (adopted from Heldring et al., 2007). Unlike ERα, several splice variants of ERβ are expressed in tissues. Characterization of the functional isoform pattern in human samples are not complete, but several experiments demonstrated that estrogen signalling could be modulated by ERβ isoforms differentially leading to an important impact on target gene regulation. For example, the hERβ2 isoform (ERβ cx) (Fig. 11) with a 26aa insertion in the LBD has no transcriptional activity because it is not capable to bind ligands or coactivators (Ogawa et al., 1998). The existence of different isoforms of ERβ complicates the interpretation of the studies performed to understand the prognostic and predictive role of ERβ in breast cancer. Figure 11: Estrogen receptor (ERβ) isoforms (Heldring et al., 2007). 5. Tissue distribution of ERs ERs are widely expressed in different tissues. Using the techniques as RT-PCR, Northern blot, immunohistochemistry and in situ hybridization, ERα and ERβ were shown to localize in the brain, the cardiovascular system, the breast, the urogenital tract also in bones (Kuiper et 33 al., 1997). However, there is specific main subtype expression in certain tissues: the main ER subtype in colon is ERβ, whereas ERα is the predominant isoform in the liver. Different distribution of ER, will determine in part, particular effect of ligands. ERβ is the predominant form in the normal mammary gland and benign breast disease. During the highest proliferative phase of the breast, i.e. pregnancy, there is very expression of ERα and high expression of ERβ. 6. ERs expression in breast cancer In cell-based studied, ERα appears to be predominant in cell proliferation, and ERβ was suggested to act as a protective factor against breast cancer development (Fox et al., 2008). In breast tumors, ERα expression increases several-fold compared to normal tissue. In low-grade ductal carcinoma in situ (DCIS) 75% of cells express ERα. And in high-grade DCIS 30% of the cells express low levels of ERα. In 1987, Peterson et. al found that the human mammary gland containes a small but distinct population of ERα-positive cells, comprising approximately 7% of the total epithelial cell population. Stromal cells were found to be ERαnegative. Moreover, on the average, 87% of the ERα-positive cells were luminal epithelial cells in ductal and lobular structures. The factors during carcinogenesis that generate the ERα+ and ERα- breast cancer phenotype remain unknown. Dontu et.al proposed a model in which ER+ breast tumors arise from either ERα- or ERα+ progenitors while ERα- tumors arise from stem cells or early ERα- progenitor cells (Dontu et al., 2004) (Fig. 12). Thereby they confirmed cellular heterogeneity within the tumor and phenotypic diversity in different patients. 34 Figure 12: Mammary development, carcinogenesis and ER expression (adopted from Dontu et al., 2004); carcinogenesis could be the result of mutations in various progenitor cells. This figure demonstrates that breast tumours could be classified into three sub-types based on the cell of origin. Type 1 or ERα- tumours, type 2 or ERα+ and finally type 3 or heterogeneous tumours. These groups display different molecular signatures and clinical behaviours. For example in the first group these tumours display a ‘basal’ phenotype and histologically, they are poorly differentiated. 35 PART III: MOLECULAR BASIS FOR TRANSCRIPTIONAL ACTIVITY OF THE ESTROGEN RECEPTOR ALPHA 1. ERα localization Using various techniques including immunocytochemical studies it was shown that ERα exists almost exclusively in the nucleus both in presence or absence of hormone (Monje et al., 2001). In studies based on GFP-ER fusions, Marduva et al (Maruvada et al., 2003) have shown that a small proportion of unliganded ERα exists in the cytoplasm, with dynamic shuttling between the cytoplasm and nucleus in living cells. And this shuttling of ERα is markedly affected by estrogen treatment. 2. ERα activation and functional pathways Unliganded ERα exists in the cytoplasm and the nucleus, and is in a complex with chaperone proteins such as heat shock proteins (HSP) especially HSP70 et HSP90 (Reid et al., 2002). Estrogen binding mediates conformational ERα modifications causing dissociation of HSPs, receptor phosphorylation, dimerization and recruitment of coactivator proteins to E2-ERα complexes which lead to activation of distinct pathways by which ERα regulates transcriptional activity of target genes. 3. Ligand-dependent pathways 3.1. Genomic pathways: 3.1a. Classical or direct pathway: According to the classical genomic pathway, after ligand binding, the dimerised receptor complex binds directly to a specific sequence in the regulatory region of target genes called EREs (estrogen response element), thus controlling their level of transcription (Fig. 13A). EREs consist of a palindromic sequence of 13 base pairs: 5’-GGTCAnnnTGACC-3’ (KleinHitpaß et al., 1986). It was reported that not all estrogen regulated genes have a perfect ERE sequence and that EREs could be imperfect or more than one ERE could be present in the 36 regulatory sequence. As shown in (Fig. 13) the nature of promoter sequence has important consequences on gene regulation and tissue specific responses to ER ligands. 3.1b. Non- classical or indirect pathways: In addition to direct interaction of ERα with an ERE, ERα can activate transcription of genes whose promoters do not possess an ERE, by indirect protein/protein interactions. ERα functions as a transcriptional cofactor and binds to other transcription factors such as fos/jun activator protein 1 (AP-1) (Kushner et al., 2000), specifity protein 1 SP1(Saville et al., 2000), and NFκb (Nilsson et al., 2001) (Fig.13). Genomic pathways are also referred to as nuclear initiated steroid signalling (NISS). A) B) C) D) Figure 13: Different transcriptional mechanisms triggered by E2-ER complexes (genomic pathways) (Nilsson et al., 2001). In panel A the classical interaction of the activated receptor with ERE on DNA is shown. The other three panels present indirect effects of estrogen receptor on transcription, which occur through protein-protein interactions with the SP1 (B), AP-1 (C), and NFκb (D): it is one of the few cases in which E2-ER acts as a repressor of transcription. 37 3.2. Non genomic pathway It is clear that ERs are mainly located in nucleus however data exist suggest that a small pool of ERα exists near the membrane (Razandi et al., 1999). Membrane associated ERα can trigger a variety of signal transduction events in seconds to a few minutes. These events include the stimulation of cAMP, calcium flux, phospholipase C activation, and inositol phosphate generation (Razandi et al., 2004). Recently it has been proposed that Spalmitoylation allows ERα localization with the plasma membrane, where it associates with caveolin-1. Upon E2 stimulation, ERα dissociates from caveolin-1 allowing the activation of rapid signals relevant for cell proliferation. In contrast to ERα, E2 increases ERβ association with caveolin-1 and activates the pro-apoptotic cascade (Marino and Ascenzi, 2008). This “nongenomic” ERα action is also called membrane initiated steroid signalling (MISS) (Ilka Nemere, 2003) (Fig.14). Figure 14: Non-genomic ERα signaling pathway (Heldring et al., 2007). The ligand activates a receptor, possibly associated with the membrane Signaling cascades are subsequently initiated via second messengers (SM) that affect ion channels or increase nitric oxide levels in the cytoplasm, and this ultimately leads to a rapid physiological response without gene regulation. 4. Ligand independent pathways : (Growth factor signalling pathway) In addition to conventional ligand-dependent regulation of ERα, many studies have shown a cross-talk between ERα and signal transduction pathways. A considerable number of studies have documented the fact that growth factors (eg epidermal growth factor [EGF], insulin-like 38 growth factor), cAMP and other agents (eg. dopamine) can stimulate the activity of ERα (S Katzenellenbogen and A Katzenellenbogen, 2000). In this case other signalling molecules such as growth factors could activate ERα by phosphorylation of ERα in the AF1 domain (Dutertre and Smith, 2003). AF-1 is phosphorylated by extracellular signal-regulated kinases as mitogen-activated protein kinase (MAPK). It has been shown that the MAPK stimulation of ERα activity involves the phosphorylation not only of S118 but also of S104 and S106 (Thomas et al., 2008) (Fig. 15). MAPK-mediated hyperphosphorylation of ERα at these sites may contribute to resistance to tamoxifen in breast cancer. Figure 15 : Growth factor signalling pathway (Heldring et al., 2007). Growth factor activated kinases phosphorylate ERs and thereby activate them to dimerize and to bind to DNA thereby regulating gene expression. 5. Transcriptional coregulators of ERα Nuclear receptor coregulators are molecules required for efficient function of nuclear receptors (O'Malley and McKenna, 2008). About 300 coregulators have been reported (http://www.NURSA.org). Transcriptional activity of ERα is not only regulated by hormones but also by regulatory proteins called coactivators and corepressors. Coregulators are recruited in complexes to ERα in a ligand dependent manner. Coregulators such as histone acetyl transferases (HAT) or deacetylases (HDAC), lead to chromatin remodeling and create a transcriptionally permissive or nonpermissive environment (McKenna et al., 1999). It has been demonstrated that 39 coregulators are involved in endocrine related cancers such as breast and uterine cancer (Lonard et al., 2007; Rajesh R. Singh, 2005). 6. Coregulators and chromatin remodelling Eukaryotic chromatin is a dynamic structure that is modified and remodelled in response to cellular signalling. The nucleosome is the basic unit of chromatin composed of an octamer of core histones (an H4/H3 tetramer and two H2A/H2B dimers), around which 147 bp DNA are wrapped. The free N-terminal tails of the core histones protrude from the core octamer. The positively charged, arginine and lysine rich N-terminal amino acid extensions are subjected to various posttranslational modifications (Luger et al., 1997). It has been proposed that transfer of acetyl groups to the terminal amino group of lysine residues of histones H2A, H2B, H3, and H4, by HAT activity of certain coregulators results in disruption of the interaction between neighboring nucleosomes. This loss of density of chromatin facilitates the access of transcriptional machinery to the promoter. In contrast, recruitment of corepressors with HDAC activity results in loss of the acetyl groups, stabilising the nucleosome contact and reducing accessibility of the promoter to transcription factors (Fig. 16). Figure 16: HAT (acetyltransferase) and HDAC (deacetylase) activity of coregolators result in change of nucleosomes stability. 40 6.1. Co-activators During the last decade many coactivators associated with the ER were identified. These include the p160 family and CREB binding proteins (Table 5). Co-activators are proteins that enhance transcriptional activity by remodelling local chromatin structure locally allowing regulatory regions to be accessible to the transcriptional machinery. These proteins have a common LXXLL motif (Kong et al., 2003) and, after binding to the AF-2 domain of ERα, could function as HATs (Histone acetyl transferase) (McDonnell and Norris, 2002) or facilitate recruitment of other factors with enzymatic activity. Table 5: ERα interacting Coactivators (Hall and McDonnell, 2005). 6.2. Co-repressors The role of corepressors is a reduction of ERα-mediated transcriptional activity. Some of these possess HDAC activity and after deacetylation of histones, they lead to condensation of chromatin and to transcriptional repression. Corepressors can inhibit transcription in different ways: by competition in binding with coactivators, by inhibition of ERα dimerisation or by inhibition of ERα binding to DNA (Hall and McDonnell, 2005) (Table.6). 41 Table 6: Corepressors interacting with ERα (Hall and McDonnell, 2005). 7. Activation of ERα through phophorylation Estrogen binding regulates ERα dimerization, intracellular localization and stability. Additionally, estrogen binding stimulates ERα phosphorylation at several sites. The best studied of these is serine 118 (S118) (Le Goff et al., 1994). S118 phosphorylation can be affected by extracellular signal regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK). ERα is also phosphorylated on other amino acid residues. For example, in response to estradiol binding, human ERα is phosphorylated on Ser-104 and Ser-106 but to a lesser extent than Ser-118 (Lannigan, 2003). In response to activation of the mitogen-activated protein kinase pathway (induced by growth factors such as (EGF) and factor (IGF)), phosphorylation occurs on Ser-118 and Ser-167 (Kato et al., 1995) (Lannigan, 2003). These serine residues are all located within the activation function 1 region of the N-terminal domain of ERα. In contrast, activation of protein kinase A increases the phosphorylation of Ser-236, which is located in the DNA-binding domain (Fig.17). 42 Figure 17: phosphorilation sites of ERα (Lannigan, 2003). 8. ER turn over and ligand dependent degradation of ERα ERα is a short lived protein and its concentration is strictly regulated. Eckert et al showed that in MCF-7 ERα turns over rapidly, with a half-life of 3-5 h, in the presence or absence of estradiol or antiestrogen (Eckert et al., 1984). Alarid et al. described that the half-life of ERα was only 1 h to 3 h in the presence of estrogen and that ERα was degraded by a proteasome dependent pathway (Alarid et al., 1999). It has been demonstrated that different ligands exert differential effects on steady-state levels of ERα (Wijayaratne and McDonnell, 2001). Aalthough E2 and a pure antagonist, ICI 182, 780 degrade ERα, the mechanism involved in this degradation is not exactly similar. E2-mediated ERα degradation was shown to be dependent on other factors such as coactivator recruitment or transcription, whereas ICIinduced degradation of ER is independent of these processes (Reid et al., 2003). The role of different domains ligand- induced degradation of ERα has been investigated. For example, ligand binding to the C-terminus of ER results in variable effects on proteolysis. Agonist and antagonists induce degradation while certain ligands such as tamoxifen lead to 43 only minor degradation of ERα. Thus it has been concluded that ERα degradation could be induced by a conformation the ligand binding domain (Alarid, 2006). There is the evidence that N-terminus of ERα has an important role in ligand dependent degradation of ERα. AF-1 domain function is modulated by phosphorylation induced by ligand binding. Inhibiting this phosphorylation of AF-1 prevents estrogen induced degradation of ERα (Callige et al., 2005). 44 PARTR IV: ANTIESTROGEN SIGNALING AND RESISTANCE TO ENDOCRINE THERAPY Estrogen plays a very important role in initiation and progression of breast tumours via their receptors. The majority of tumours express ERα s and thereby inhibit the ER-signalling pathway by anti-estrogens is a valuable option in treatment of women with ER-positive (ER+) breast cancer. The benefits of estrogen deprivation (by oophorectomy), for the first time was described in 1896 by Beatson as an option for breast cancer therapy. Today, the main strategies for disrupting estrogen-dependent growth of breast tumours are non-surgical and are based on the reduction of circulating estrogen levels in the body or blocking ERα signalling by targeting ERα with different antiestrogens (Nicholson and Johnston, 2005). The first molecule described in the literature with antiestrogenic property was MER-25 ( Lerner et al. 1958). This first non-steroidal anti-estrogen did not become a clinically useful agent because of toxicity and low potency (Lerner, 1981). The idea of competitive hormone therapy truly emerged when, in 1960s, a failed post-coital contraceptive, ICI 46,474 (Tamoxifen) was reinvented as a potential targeted therapy for adjuvant treatment and prevention of oestrogen receptor positive breast cancer (Harper and Walpole, 1967). Although tamoxifen has improved survival of many patients certain side effects, such as uterine carcinogenesis are common. Frequently patients develop resistance to tamoxifen which motivate researcher to determine the exact mechanism of antiestrogen action and to try to find new agents with better tolerance and efficacy in resistant cases. Anti-hormonal options for treatment of breast cancer in clinical data exist for two groups of agents: the selective estrogen receptor modulators (SERM) and the selective estrogen receptor down-regulator (SERD). 45 1. Selective Estrogen Receptor Modulators (SERM) The decision to use anti-hormonal therapy is based on precise prognostics. The most important indication for anti-estrogen treatment of patients is ERα status (Buzdar and Hortobagyi, 1998). The term SERM, prototype as Tamoxifene, indicates that these compounds are not only estrogen antagonists, but that they can have a tissue selective activity: they are agonists in certain tissues (liver, cardiovascular system, and bone), mixed agonist/antagonist (as tamoxifen in uterus)and antagonist in other tissues (brain, breast) (Katzenellenbogen and Katzenellenbogen, 2002). There are sub-divisions in this group: ‘tamoxifen-like’ (eg toremefine, droloxifene and idoxifen) and ’fixed-ring’ (eg raloxifene, arzoxifene, acolbifen) (Johnston, 2005) (Fig.18) . Figure 18: Chemical structure of SERMs. Tamoxifen like and fixed-ring 46 Among SERMs tamoxifen is used as the gold standard in the treatment of women with ERα positive breast cancer. Tamoxifen ICI 46,474 renovated as Tamoxifen is a trans isomer of triphenylethylene that was discovered in the Imperial Chemical Industries (ICI) Ltd (now Astrazeneca) (Jordan, 2008). Tamoxifen was approved by the U.S. Food and Drug Administration (FDA) in 1977 for treatment of patients with hormone-sensitive breast cancers. Metabolism and pharmacokinetic of tamoxifen The trans-tamoxifen is the form administered to the patients because this isomer has more affinity to ER than cis isomers. Lateral chain: dimethylaminoethoxide, and trans configuration are reported crucial for anti-estrogenic activity of tamoxifen (Jordan, 1984). It was accepted that tamoxifen is extensively metabolized by cytochrome P450 and, in the liver, into two principal metabolites: the N-desmethyltamoxifen (NDT), a compound with low affinity for ERα but a long biological half-life, or a minor metabolite 4-hydroxy-tamoxifen (4OHT) with high binding affinity for ERα but with a short half life (Coezy, 1982). Although it has been assumed that 4OHT is the primary means by which tamoxifen exerts its anticancer effect, another metabolite of tamoxifen called endoxifen was discovered which forms in liver by cytochrome P450 by an enzymatic system from NDT. Endoxifen is equivalent to 4OHT in its ability to bind to ERα and suppression of breast cancer progression (Jordan, 2007). 4 OH-tam binds to ERα with a similar affinity to E2; wille NDT bind with a significantly affinity. These molecules inhibit estrogen actions on mammary tissues in a competitive manner by binding to ER. As adjuvant therapy or for prevention of recurrence, in most cases, tamoxifen is used routinely at 20 mg/daily for 5 years (Gradishar, 2004). 47 2. Selective Estrogen Receptor Down-regulator (SERD) Patients with hormone-responsive breast cancer following progression or relapse after tamoxifen therapy may receive a second anti-estrogenic agent called ”Fulvestrant”or Faslodex that was approved by the FDA in 2002 (Bross et al., 2002). Fulvestrant is a 7α- alkylsulphinyl analogue of 17β-estradiol that not only structurally but also in terms of molecular activity is distinct from tamoxifen (Dowsett et al., 2005). In fact, Fulvestrant (ICI 182, 780) is a pure (ERα) antagonist that binds to ERα and blocks function of both AF1 and AF2 via a rapid downregulation of cellular levels of the ERα and thus described as a SERD for selective estrogen receptor downregulator (Howell et al., 2004a; Wijayaratne and McDonnell, 2001). Fulvestrant and a group of anti-estrogens were described first as pure anti-estrogens (see schematic representation below); they are the compounds that have no estrogen-like properties in laboratory assays, to date less than ten distinct compounds have been discovered (Hermenegildo and Cano, 2000). The main compounds that have been demonstrated to have pure antiestrogenic activity in the laboratory are: ICI 164384: the first pure anti-estrogen, is a 7α-alkylamine derivative of 17β-estradiol discovered in 1987 by Wakeling and Bowler ICI 182780: also called fulvestrant or Faslodex®. This compound was developed from ICI 164 384 to improve the bioavailability and the biological profile of its activity but this agent actually used in clinic is poorly soluble with a low activity after oral administration and thus requires intramuscular injection (250 mg monthly). 48 There are other agents discovered and described as pure anti-estrogens such as RU58 688, EM-800 (active metabolite EM-652). To date certain of these compounds are reclassified as SERDs, meaning that they could decrease cellular ERα levels to a similar extent ICI 164 384 and ICI 182 780. 3. Molecular mechanism of anti-estrogen activity It has been demonstrated that in the presence of an agonist such as estradiol, estrogen diffuses into the cell and binds to ERα. Estrogen binding causes a conformational change that results in coactivator recruitment, and subsequent DNA binding the E2-ERα complex to promoter 49 region in upstream of genes regulated by estrogens influencing cellular phenotype and then degradation of ERα by the proteasome (see Part III). In the presence of a SERM such as tamoxifen, the situation changes.Tamoxifen functions as a competitive antiestrogen to block the action of estrogen. Tamoxifen binds to ligand binding domain (LBD) of the ERα and causes a ligand induced perturbation of receptor structure that leads to binding of corepressors (or coactivators) on the external surface of this complex. The recruited factors differ from the ones that bind to E2-ERα complexes. SERM-ERα can repress gene expression of ER target genes.In the presence of ICI 182,780 (a SERD) ligand induces a rapid relocalisation of ER to an insoluble compartment in nucleus and degradation of ERα (Callige et al., 2005; Stenoien et al., 2001). Table 7: Mode of ER-ligand action (Howell et al., 2004b). 50 4. Transcriptional activity The transcriptional activity of ERα is mediated by two acidic domains; AF1 and AF2. The AFs function in a synergic manner but may also function independently in certain cell and promoter contexts and they could be regulated differentially by ligand (Tora et al., 1989; Tzukerman et al., 1994a). 4.1. Transcription activation function 1(AF-1): The AF-1 is located in the N-terminal A/B domain of ER (Moras and Gronemeyer, 1998). ERα and ERβ have different AF-1 domains. In ERβ AF-1 is incomplete and its activity is weak (Hall and McDonnell, 1999). It has been proposed that the partial agonist activity of Tamoxifen (in certain cells and promoter contexts) relies on AF-1 (Tzukerman et al., 1994b). ICI blocks AF-1 by affecting dimerization, DNA-binding and targeting ERα for degradation (Dauvois et al., 1992; Fawell et al., 1990 ). In addition to the known AF-2 coactivators that enhance receptor activity by interacting with the LBD, AF-1 coactivators have also been described (Hall and McDonnell, 2005). Endoh et al have characterized a specific AF-1 coactivator, p68, that potentiates the AF-1 ERα activity in response to estrogen and tamoxifen, thereby providing another alternative mechanism that may lead to partial agonist activity of tamoxifen (Endoh et al., 1999). 4.2. Transcriptional activation function 2(AF-2): Ligand dependent activation of transcription by ERs is mediated by interaction of coactivators with the AF-2 domain. Through mutational analysis combined to crystallographic studies it was demonstrated that receptor-coactivator interactions are mediated through ERα helix12 and the LXXLL motif of coactivators (Hall and McDonnell, 2005). Tamoxifen acts by blocking AF-2 activity so it is an antagonist in cells where AF-2 is dominant and a partial agonist where AF-1 is dominant (Pearce and Jordan, 2004). ICI blocks AF-2 as well as AF-1 activities. The transcriptional activities of both AF-1 and AF-2 are dependent on the recruitment of coregulators. In most promoter contexts a functional synergism between AF-1 and AF-2 has been described (Kraus et al., 1995) (Fig.19). 51 Figure 19: Transcriptional activity of AF-1 and AF-2 (Dowsett et al., 2005). 5. Structural basis of agonism and antagonism 5.1. Flexibility of the LBD: The LBD is a multifunctional domain. A series of structural studies have described the threedimensional structure of LBD as a polypeptide chain folded into a canonical α-helical sandwich (Wurtz et al., 1996). It is composed of 12 helices (H1-H12) that are arranged into three anti-parallel layers (Pike et al., 2000) and it has been demonstrated that the ligand binding cavity has a remarkable plasticity. Brzozowski et al (Brzozowski et al., 1997) were the first to reveal the crystal structure of the ERα LBD in complex with E2 and raloxifene (RAL). They indicated that E2 and RAL bind at the same site within ERα. Pike et al. also demonstrated that despite similar ligand orientation for E2, ICI, and RAL in the LBD different spatial conformations were formed due to different chemical structures of ligands (Pike et al., 2001) (Fig 20). 52 Figure 20 Superposition of orientation of E2 (cyan), ICI (green), and RAL (purple) in LBD (Pike et al., 2001). Several researchers have shown that different ligands interact with the same residues in the LBD with a preferential binding mode for distal hydroxyl of ER ligands (Pike et al., 2001). Similar orientation for distal side chains in α or β positions of different ligands in the ERligand complex were shown. Figure 21: The interaction of different ligands with critical amino acids in the ERα LBD: Above E2 and raloxifene with ERα (Brzozowski et al., 1997), below ICI within the ERβ (Pike et al., 2001) 53 5.2. Role of H12 in antiestrogen signalling Ligand- dependent transcriptional activation function (AF-2) is located in a conformationally dynamic region of the LBD. The key element for AF-2 conformational changes after binding to ligands is a short helical region (Helix 12; H12) located at the C-terminus of the LBD. The orientation of H12 is remarkably sensitive to the nature of the ligand bound: in the presence of estradiol H12 lies over the ligand binding cavity, thereby generating a hydrophobic groove formed by H3, H4, H5 and H12. Competent AF-2 is then able to recruit the coactivators via a leucine-rich LXXLL motif (NR-box), that is stabilized within the groove by a charge clamp involving residues as Lys 362 thereby facilitating transcriptional activation (Kong et al., 2003) (Fig. 22). Figure 22: The three-dimensional protein structure of the E2-ERα LBD complex including H12 (blue cylinder) and hydrophobic residues (yellow) (Brzozowski et al., 1997). In contrast, in the presence of antagonists, as described by Brzozowski et al for ERα-Ral or by Shiau for ERα-OHT, the bulky side chain is too long to fit in the binding pocket. It is displaced by H12 that lies along a groove between H3 and H5. Thus AF2 is incorrectly formed and coactivator binding is blocked. In this position H12 mimics the hydrophobic interaction of a NR-box peptide with the static region of the groove with a stretch of residues (residue 540 and 544) that resembles an NR-box (Shiau et al., 1998) (Fig.23). 54 Figure 23: Left the three-dimensional protein structure of the RAL-ERα LBD complex including H12 (green cylinder) and hydrophobic residues (yellow). Right: structural representation of ERα LBD-Ral and NR-box binding site (Brzozowski et al., 1997; Kong et al., 2003). The only crystal structure of a SERD bound to an ER is a structure in complex with ICI164,384 and the rat ERβ LBD (Pike et al., 2001). In this structure, ICI is long alkylamido side chain binds along the AF-2 recruitment site, preventing H12 to adopt the position described above. H12 is not associated with the rest of LBD. This disordered conformation may lead to full antagonism, possibly through disruption of the receptor structure leading to degradation in the cell (Kong et al., 2003) (Fig 24). Figure 24:ERβ LBD and ICI (Pike et al., 2001) 55 Figure 25: A schematic presentation of modulation of ER by ligands and H12 (Heldring et al., 2007) 6. The factors affecting antiestrogenic activity It has been demonstrated that several compounds can act in a tissue-specific manner. The best example is tamoxifen, for which an estrogen like agonist activity in the endometrium and bone occurs simultaneously with an estrogen antagonist activity in breast (Diel, 2002). The mechanisms by which the same compound can exert tissue-specific agonist and antagonist actions are still being investigated. One possible explanation for the selectivity of compounds may be differential expression of ERα coregulators and transcription factors (Ball et al., 2009; Smith and O'Malley, 2004). Each of these factors could contribute to cell specific effects. One major determinant of cell type selectivity is the expression level of coregulators in different tissues (Smith and O'Malley, 2004). This was demonstrated by the observation that the agonist effect of tamoxifen in endometrial cells was due to the high expression of SRC-1 (Shang and Brown, 2002). AIB1 or SRC3, which function as a coactivator to enhance transcription of genes, play an important role in the function of ERα. These coactivators were reported to be overexpressed in 65% of breast cancers. In patients, this high expression level of AIB1 is correlated with resistance to endocrine therapy which could thus be explained by increased agonistic activity of drug-ERα complex (Schiff and Osborne, 2005). 56 7. Breast cancer and endocrine resistance The molecules that are used for treatment of tumours expressing ERα have shown their therapeutic efficacy for many years. At present anti-estrogen therapy is the most effective treatment for women with ERα-positive breast cancers. Unfortunately many patients with a considerable level of ERα develop resistance to endocrine therapy described as primary or de novo resistance, and all of them, after a certain period of treatment, acquire resistance to therapy. The mechanisms of these resistances are not exactly understood. To date considerable effort is made to identify the pathways and other factors that are implicated acquired resistance in order to develop new agents to overcome endocrine resistance. 8. Tamoxifen treatment and resistance to endocrine treatment Tamoxifen is the most administrated treatment of estrogen positive breast cancer that dramatically improved survival of patients. However, a very important obstacle is intrinsic or acquired resistance to this therapy, almost 50% of patients with ERα-positive tumour fail to respond to tamoxifen and furthermore the patients who initially have a good response to tamoxifen, acquire resistance (Howell et al., 1995; Osborne, 1998). Several studies in breast xenograft models have been performed which demonstrate that initial growth suppression of the tumour by tamoxifen is followed by reactivation of tumour proliferation stimulated by the drug. Since this cell growth could be fully inhibited by Fulvestrant, there are arguments that the resistance/stimulated growth of these tumours may be mediated by ERα. The antagonistic activity of tamoxifen was proposed to be changed to an agonist effect (Berstein et al., 2004; Osborne et al., 1995; Yao et al., 2000). Different mechanisms are involved in resistance to tamoxifen: 57 8.1. Loss of ERα expression and function Lack of ERα and PgR expression is the most dominant form of de-novo resistance. More than 90% of ER/PR negative tumours do not respond to antiestrogens (Clarke et al., 2003). There is no strong evidence for loss of ERα expression in ERα positive tumours that acquire antiestrogen resistance. In cell cultures, there are a few examples where cells switch to an ERα negative phenotype through two major mechanisms: population remodelling and transcriptional repression of ERα expression (Graham et al., 1992; van den Berg et al., 1989 ). Multiple mechanisms involving silencing of ERα have been identified. They include mutations within the open reading frame and transcriptional silencing by DNA methylation of the promoter (Yamashito, 2008). Function of the ERα could be disturbed by mutating of the ESR1 gene that encodes ERα or the generation of splice variants. The presence of ERα transcripts with deletions in several exons has been described in breast cancer cell lines and normal- and malignant breast tissue samples. While the exact function of these splice variants is not established, it has been hypothesized that the splice variant mRNAs may result in proteins that differ in activity (Poola et al., 2000). For example Fuqua et al. have reported that co-expression of an exon 5 estrogen receptor (ER) variant, originally identified in ERα-negative tumours, with wild typeERα resulted in resistance to the growth-inhibitory effects of tamoxifen in MCF-7 cells (Suzanne, 1994).. A naturally occurring ERα mutant has been described that recognised tamoxifen as an agonist (Levenson et al., 1997). These two last points (splice variant and ER mutant) are awaiting approval for clinical relevance. 8.2. ERβ subtype and its alteration in expression Both the original ERα and the more recently identified ERβ and its splicing variants are expressed in many breast cancers. ER protein stability is regulated differently: in breast cancer ERα and ERβ protein levels are decreased by E2 but increased by tamoxifène (Pearce 58 et al., 2003). ICI treatment results in ERα degradation and ERβ stabilisation.Using specific treatments may thus result in an altered ERα/ERβ ratio. A number of groups have reported results regarding possible functions of ERβ that show signalling by a third isoform in different ways depending on ligand or promoter structures. For example binding of several SERMs, including tamoxifen resulted in agonistic activity of ERβ. Also ICI can activate ERβ mediated transcription through AP-1, a pattern of regulation different from some other antiestrogrns. To date, it is clear that ERα/ ERβ ratios change during carcinogenesis. ERα expression is increased and ERβ is decreased in early breast cancer, whereas expression of both declines in more invasive cancer (Fox et al., 2008). 8.3. The role of nuclear receptor coregulators Several investigations have described the role of coregulators that can significantly influence ERα -mediated transcription. In breast cancer, the AIB1 gene has been reported to be amplified in 4 out of 5 ERα -positive breast cancer cell lines and to over-expressed in the majority of primary breast cancers (Anzick et al., 1997). Shang et al demonstrated that estrogenic activity of tamoxifen in the uterus requires a high level of steroid receptor coactivator 1 (SRC-1) expression. He concluded that cell type and promoter-specific differences in coregulator recruitment may determine the cellular response to tamoxifène (Shang and Brown, 2002). The corepressors N-CoR and SMRT interact more strongly with ERα in the absence of E2 or in the presence of tamoxifen. In a nude mouse model growing MCF-7 cells, acquired resistance to tamoxifen was found correlated with reduction of N-CoR level (Lavinsky et al., 1998). Thus, gain of coactivator function or loss of corepressor function or a shift in balance of coregulators may be playing an important role in resistance to antiestrogens such as tamoxifen. 59 8.4. ER pathway cross-talk with growth factor and cellular kinase pathways Expression of several growth factors and their receptors is regulated by estrogen. Growth factors (TGFα, EGF, IGF-1) have been shown to activate ERα signalling via the MAP kinase cascade (Nicholson et al., 1999; Smith, 1998). Experimental and clinical evidence suggest that overexpression of growth factor receptors in breast cancer especially those of the EGFR/HER2 family is associated with resistance to endocrine therapy in particular to tamoxifen (Massarweh and Schiff, 2007). King et al. reported that a member of the tyrosine kinase proto-oncogene family known as c-erbB2 or HER2, was amplified in human mammary carcinoma (King et al., 1985). Currently, it is estimated that the HER-2 gene is amplified in 20% of breast cancers, and that hyper activity of HER2 signalling may lead to a complete loss of ER expression as a mechanism of resistance to endocrine therapy (Massarweh and Schiff, 2006). Recently Hurtado and co -workers proposed that PAX-2 a general transcriptional repressor of ERBB2 play an important role in endocrine responsiveness. They showed that PAX2 and AIB compete for binding and regulation of the ERBB2 gene. An increase in AIB1 levels blocks PAX2 binding and ERBB2 gene repression thereby reversing antiproliferative effects of tamoxifen (Hurtado et al., 2008). Activation of different kinases such as MAPK, Protein Kinase A (PKA) have been shown to enhance tamoxifen resistance in breast tumours. PKA-mediated phosphorilation of S305 in the hinge region of ERα reorient tamoxifen bound ERα towards its coactivator SRC-1, this orientation results in recruitment of RNA polymerase II and ERα -transcription in presence of tamoxifen (Zwart et al., 2007). Endocrine resistance is a multifactorial problem and various mechanisms may account for insensitivity to tamoxifen. To date few of these mechanisms are validated in clinical features of tamoxifen resistant breast cancer as the combined elevation of AIB1 and ERBB2 pathways, and the molecular details of these events remain elusive. 60 AIM OF PRESENT STUDY The proposed project was part of a larger project in the context of resistance to hormonal therapy of breast cancer (Resisth network; GSO Cancéropôle, financially supported by INCA). The aim of this study was to gain further insight into the mechanism of antiestrogen activity and thereby to design tools that can help to understand the molecular mechanisms involved in ligand-dependent modulation or ERα down regulation The specific aims of this thesis were to: 1. Establish a molecular and cellular system to discriminate rapidly SERM from SERD. 2. Investigate the impact of nature of ligand on intracellular localisation of ERα. 3. Design the antiestrogens with modified structure and study their effect on localization of ERα degradation of ERα and their effect on activation of estrogen regulated genes. 61 RESULTS 62 PUBLICATION N° 1 63 A molecular approach to predict selective estrogen receptor modulators from down regulators Mahta Mazaheri, Clarisse Loulergue-Majorel, Anne-Claire Lavigne, Kerstin Bystricky # and Helene Richard-Foy 1, Université de Toulouse ; UPS ; Laboratoire de Biologie Moléculaire Eucaryote ; F-31062 Toulouse, France 2 CNRS; LBME; F-31000 Toulouse, France #corresponding : [email protected] Key words: Endocrine therapy, Breast cancer, Estrogen receptor, anti-estrogen, ICI 182,780 Abbreviations: ER, estrogen receptor; OHT, 4-hydroxytamoxifen; SERM, Selective Estrogen Receptor Modulator; SERD, Selective Estrogen Receptor Down regulator; ICI, ICI 182780. 64 Abstract ___________________________________________________________________________ Breast cancer is most common type of malignancy among women in the world. Approximately 60% of breast tumours express the oestrogen receptor alpha (ERα) and are considered hormone-responsive. Thus endocrine therapies have long been the treatment of choice. However, the estrogen-like agonist effect of the available selective estrogen receptor modulators such as tamoxifen and the development of resistances require the development of new treatments that act through different mechanisms. The objective of our study is to design tools that can help to understand the molecular mechanisms involved in ligand-dependent modulation or degradation of ERα. It has been proposed that ligand-dependent ERα degradation of antagonist may result from the presence of a long aliphatic side chain on a steroidal core, thus we selected the following compounds: RU 39411, RU 58668, which dervatives of 17β-estradiol, but different side-chains: RU 39411 has a dimethyl-amino-ethoxy-phenyl side chain similar to that of Tamoxifen, while RU 58668 has a neutral hydropfobic side chain similar to that of Faslodex. The third compound, EM 652, has a phenylcoumarine core that mimics the steroid backbone of oestradiol while the side chain is similar to that of Raloxifen. Fulvestrant and RU 58668 induced degradation of ERα, while RU 39411 and EM 652 have the same effect as Tamoxifene and stabilized ERα. Three mutants of ERα, L540Q, K362A and D351Y were generated and used in transient transfection assays in MDA-MB231cells. The effect of the ligands on gene expression of a luciferase reporter gene under the control of an estrogen inducible promoter and ligand-induced degradation of ERα were measured. Our results demonstrate that antiestrogens can be classified into two groups based on structure and function: Tamoxifen and RU39411 fall into a first group, and Faslodex and RU58668 into a second group. Interestingly EM 652 has an intermediate behavior. 65 We have used molecular studies to screen and classify antiestrogen compounds into SERM or SERD with the aim to exploit the structural characteristics of anti-estrogens to create new compounds with different lateral side chains for the study of the relationship between structure and activity of antiestrogens. Introduction ___________________________________________________________________________ Approximately 60% of breast tumours express the estrogen receptor ERα and are thus considered hormone-responsive. Endocrine therapy having usually fewer side effects than chemotherapy, it is recommended as treatment of choice for women with ERα-positive breast cancer. Endocrine therapies are based on the use of synthetic agents which can inhibit the proliferative action of estrogens (Clarke et al., 2001) (Katzenellenbogen et al., 2000) (O'Regan et al., 1998). Among anti-hormonal options anti-estrogens are compounds which compete with estrogens for binding to estrogen receptors ERα and ERβ (Green and Chambon, 1988; Kuiper et al., 1996a) particularly efficient. The biological action of ERα in the presence different ligands has been studied for a long time, In the absence of ligand, the majority of ER resides in the nucleus bound to an inhibitory heat-shock protein, HSP90 (DeFranco, 2002; McDonnell and Norris, 2002). Ligand binding induces a conformational change of the ER leading to displacement of the HSP90 heat-shock protein, ER dimerization and recruitment of coactivators or corepressors upon interaction with DNA response elements within the regulatory sequence of target genes (Heldring et al., 2007; O'Malley and McKenna, 2008) Tamoxifen has been the first-line endocrine therapy in estrogen receptor positive tumors for nearly three decades in premenopausal patients as well as for postmenopausal women (Gradishar, 2004). However, frequent relapses while on tamoxifen and the long term toxicities of the drug stimulated research to find safer and more effective endocrine strategies. Two 66 major classes of molecules have been developed: selective estrogen receptor modulators (SERM) such as Tamoxifene and selective estrogen receptor downregulator (SERD) such as a pure antiestrogen Faslodex. Crystallographic studies demonstrated that agonist or antagonists bind to the same site within the core of the LBD of ERα but with different binding modes. Comparison of the estrogen receptor-LBD complexed to the antiestrogens like tamoxifen or raloxifene has demonstrated that the bulky side chain of antiestrogens can not be accommodated within the LBD. The resulting structures differ from that observed in the presence of estrogen by the position of the C-terminal helix 12 (H12), which in the presence of agonist sits above the ligand binding cavity and contributes to coactivator recruitment (Brzozowski et al., 1997; Shiau et al., 1998). In the antagonist-bound structures, the side chains of antiestrogens force H12 to reposition over the coactivator binding groove, preventing interaction with coactivators and promoting the interaction with corepressors. This results in a transcriptionally inactive complex that blocks ER induction of genes important for cell proliferation (Johnston, 1997). It has been postulated that in the presence of Faslodex (ICI) this disordered conformation may lead to full antagonism, either through disruption of the AF1 and AF2 site or through destabilizing the receptor structure leading to degradation in the cell (Kong et al., 2003). The ICI- ERα complex is rapidly sequestered and degraded in a salt-insoluble compartment in the nuclear matrix (Balan et al., 2006; Callige et al., 2005). Although faslodex is an efficient treatment for patients who failed after endocrine therapy, and in metastatic breast cancer its poor pharmacodynamic properties and lack of oral bioavailability have limited its clinical utility (Howell, 2000; Robertson et al., 2005) We have used these mechanistic studies to clarify the underlying mechanism of action of several antiestrogens belonging to different classes with the aim to develop an effective molecular screen for newly designed pharmaceutical properties of different compounds. 67 Materials and Methods ___________________________________________________________________________ Cell lines and cell culture The MDA-MB-231 cell line was from the American Type Culture Collection (Manassas, VA) and grown in DMEM containing phenol red, 10% heat-inactivated fetal calf serum (Invitrogen), and 50 µg/ml gentamycin (Invitrogen). MELN cells (Balaguer et al., 1999) estrogen-sensitive human breast cancer cells (MCF-7) stably transfected with the estrogenresponsive gene (ERE-βGlo-Luc-SVNeo). They were cultured in DMEM/F12 medium with GlutaMax™ I containing phenol red, supplemented with 50 µg/ml gentamicin (Invitrogen), 1 mg/ml G418 sulphate (Promega, Leiden, The Netherlands) and 10% heat-inactivated fetal calf serum (Invitrogen). The cell line was maintained in an incubator at 37°C, at a relative humidity of 95% and with a CO2 concentration of 10%. To study the effects of estrogens and anti-estrogens the cells were progressively deprived in serum as follows: they were first grown for 3 days in medium without phenol red and antibiotic, containing 5% charcoalstripped serum (SSH) Cells were treated or not with 10 nM estradiol or 1µM of anti-estrogen for the indicated times. Chemicals 17β-estradiol (E2) and 4-hydroxy tamoxifen (4OHT) were purchased from Sigma-Aldrich (St. Louis, MO). ICI 182,780 (ICI) was purchased from Zeneca Pharmaceuticals. RU39411 (RU 39) and RU 58668 (RU 58) were gifts from Dr. Michel Renoir (CNRS/ Châtenay-Malabry) , and EM-652 (EM) was provided by Dr. Labrie (U Canada). Stock solutions of E2, Tamoxifen (4OHT), ICI, EM, RU 39 and RU58 were prepared in absolute ethanol. Leptomycin B (LMB) was purchased from Sigma and Calpain Inhibitort I (ALLN) was purchased from Calbiochem. 68 Antibodies Anti-human ERα (HC-20: sc-533) and ERα (H-184: sc-7207) were purchased from Santa Cruz Biotechnology. Anti-GAPDH was purchased from Chemicon International. Mutagenesis Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The ER PSG5 plasmid (HEGO, kindly provided by P Chambon) was used as a template for PCR. Primers used were: A-K362A-R (5'-ATC AAC TGG GCG GCG CGC GTG CCA GGC TTT TG -3' and 5'-ACA AAG CCT GGC ACG CGC GCC GCC CAG TTG ATC -3'), L540Q (5'-CTA TGA CCT GCA GCT GGA GAT GCT GGA -3' and 5'-TCC AGC ATC TCC AGC TGC AGG TCA TAG -3'), A350V-D351Y (5'-CTG ACC AAC CTG ATG TAC AGG GAG CTG GTT C -3' and 5'-GAA CCA GCT CCC TGT ATA CCA GGT TGG TCA G -3'). Purified DNA was sequenced to check for the presence of the mutation. A larger scale DNA preparation was used to sequence the entire cDNAs of the ERα and mutants. Each mutant was then cloned into the pSG5puro plasmid using the NotI and SacI site flanking the ER cDNA. Transient Transfection Experiments and Luciferase Assays MDA-MB-231 cells were grown for 3 days in phenol red-free medium, containing 5% dextran-coated charcoal-treated fetal calf serum (FCS). Cells were then transiently cotransfected with vectors expressing either wild-type ERα or the mutants (0.5 µg), along with an ERE-luciferase plasmid (0.35 µg) and pCH110 (0.05 µg) that expresses the Galactosidase under the control of a SV40 promoter was used to normalise for tansfection efficiency. For the Western Blot experiments performed after transient transfection, pSV40 luciferase internal vector (0.1 µg) was cotransfected either wild-type ERα or the mutants (0.5 69 µg). It was used to determine transfection efficiency for each sample. MDA-MB-231 cells were plated at 2 ×104 per 12-well plates and grown for 1 day in phenol red-free medium, containing 5% FCS, before the transfection using FuGENE® HD Transfection Reagent (Roche Applied BioSystems, MA), according to the manufacturer's instructions. Two days after transfection, cells were treated with the appropriate compound for 3 or 24 hours (as indicated in figures) and harvested. At the end of the incubation period, the remaining medium was removed. The cells were washed once with cold PBS and lysed in 60 µl reporter lysis buffer (Promega, E397A Leiden, and The Netherlands). After shaking plates for 20 min, plates were frozen (−80°C) for minimum 1 h and maximum 1 week. After thawing the plates, luminescence was measured using a luminometer (Luminoskan) after injection of 50 µl luciferase assays reagent (Promega, E397A Leiden, The Netherlands) in each well, according to the manufacturer's instruction, on a Centro LB 960 (Berthold). Results are expressed as relative light units (RLU). -Galactosidase activity was measured using 10 µl of each sample and the GalactoLight Plus detection system (Roche Applied Biosystems, Bedford, MA). Final data are reported as relative light units, which is the luciferase reading divided by the -galactosidase reading. For western blotting, protein concentrations were measured using the Amido Schwartz technique and normalize with luciferase activity. For each condition, average luciferase activity was calculated from the data obtained from 2-3 independent wells and the experiments were repeated at least 3 times. Statistical analysis was performed using Student’s t test analysis. Cell extracts and Western blots MDA-MB-231 and MELN cells grown 12-well plates and were treated as indicated, washed with PBS and collected by centrifugation. Total cell lysates were prepared by resuspending the cells of each well in 100 µl lysis buffer (50 mM Tris pH 6.8, 2% SDS, 5% glycerol, 2 mM 70 EDTA, 1.25% β-mercaptoethanol, 0.004% Bromophenol blue). The samples were boiled for 20 min at 95°C and cleared by centrifugation at 12 000×g for 10 min. The protein concentration was determined by the Amido Schwartz assay when the samples contained SDS. Samples were subjected to PAGE on a 10% SDS-polyacrylamide gel in 25 mM Tris– HCl, 200 mM glycine, pH 8.3, 0.1% SDS and the proteins transferred onto nitrocellulose membrane. Western blot analysis was performed as previously described (Giamarchi2002) using rabbit polyclonal ERα antibodies diluted to 1 µg/ml (HC20 or H-184 Santa Cruz Biotechnology, Inc.). Western blots were quantified using the TINA PC-Base Software from FUJI. qRT-PCR experiments RNA extraction MCF-7 cells were grown in phenol red-free Dulbecco’s (DMEM)/F12;(invitrogen) supplemented with 5% charcoal-dextran-stripped fetal bovine serum(FBS) for 3 days and then cells were treated with10nM of E2, 1µM of anti-estrogens, or vehicle for 16h. Total RNAs were extracted using TRIzol Reagent (invitrogen), the quality of RNA samples were ensured by electrophoresis in an agarose gel followed by ethidium bromide staining, where the 18S and 28S RNA bands could be visualised under UV light.Quantification of RNA was performed by spectrophotometry at 260 nm. RNA samples were stored in RNAsefree distilled water and at 80°C. cDNA synthesis 1 - 5 µg of total RNA was reverse transcribed in a final volume of 20 µl using SuperScriptrTM III Reverse Transcriptase manufacture. cDNA was stored at 80°C. 71 Real-time PCR amplification All target transcripts were detected using quantitative real time RT-PCR (SyberGreen) assays. The experiments were performed on a Mastercycler ep realplex device; and TBP was used as endogenous control for normalization of the data. The following primer pairs were used to amplify cDNA after reverse transcription experiment: TBP: 5’-CGGCTGTTTAACTTCGCTTTC-3 5’-CCAGCACACTCTTCTCAGCA-3’; pS2 /TFF 1: 5’-CCCCTGGTGCTTCTATCCTAAT-3’ 5’-CAGATCCCTGCAGAAGTGTCTA-3’; PGR: 5’-CTTAATCAACTAGGCGAGAG-3’ 5’-AAGCTCATCCAAGAATACTG-3’; TGFα; 5’-ATCTCTGGCAGTGCTGTCCCT-3’ 5’-CTTGCTGCCACTCAGAAACA-3’; The thermal cycling condition comprised 2 min at 55°C and 2 min at 95°C followed by 40 cycles at an appropriate annealing temperature depending on the primer set for1 min. To quantify the result obtained we employed the comparative Ct method using qBASE software. 72 Results ___________________________________________________________________________ Relationship between molecular structure of ERα ligands and ERα stability ER binds a variety of ligands including agonists such E2, SERMs such tamoxifen and raloxifen (Shiau et al., 1998) and SERDs such as faslodex. In all cases the receptor-ligand interaction induces a distinctive conformation of the ligand-binding domain (LBD) of the ER. The conformation of this part of the protein determines recruitment of transcriptional coregulators, thereby regulating target gene transcription (Pike et al., 2001). The first objective of this study was to define the characteristics of ligands that are important for cellular fate of ERα. We selected several compounds with different structures (Figure 1A) that have previously been described as pure anti-estrogens or SERMs – describe more for review see (Anstead et al., 1997). We first examined the stability of ERα in MELN cells, a cell line derived from MCF-7 that stably express a luciferase reporter gene driven by an estrogen reponse element containing promoter(Balaguer et al., 1999) following treatment with these compounds. ERα content from MELN whole cell extracts after 3h treatment with hormones or antihormones was analyzed by Western blotting. We found that the pure antagonists ICI 182780 (ICI) and RU 58668(RU58) induced rapid degradation of ERα. In contrast, in the presence of RU 39411 (RU39) and EM652 ERα was not degraded as well as ICI and RU58 after 3h. This behavior was similar to that of 4OHT which stabilized ER after three hours of treatment. These results show that functional interaction of ERα with ICI and RU58 promotes ER degradation, while binding to 4OHT, EM652 and RU39 stabilizes the ERα (Figure 1.b). It was previously proposed that ERα degradation occurs in the cytoplasm in the presence of a natural ligand (E2), but, in the presence a full antiestrogen such as ICI ER is rapidly degraded, at least partially, in a nuclear compartment in a proteasome dependent manner (Callige et al., 73 2005; Marsaud et al., 2003; Nawaz et al., 1999). We investigated the effect of LeptomycineB (LMB), an inhibitor of CRM1-dependent nuclear export, and ALLN, an inhibitor of the proteasome, on ligand-dependent degradation of ERα. MELN cells were pretreated with LMB or ALLN for 30 min. As shown in Figure1.c E2 induced partial ER turnover was blocked by ALLN but not by LMB suggesting that nuclear export is not required for ER degrdadation. Furthermore, LMB treatment did not affect ICI induced loss of ERα, while ALLN clearly stabilized ICI induced ERα degradation. Similarly, LMB treatment had no effect on RU 58 induced ERα degradation while ALLN blocked it, suggesting that in the presence of RU58 ERα is degraded in the nucleus by a nuclear proteasome as has been described for proteolysis of ICI-bound ERα. Thus, analyzing the extent and cellular location of ERα degradation offers a simple test that allows discrimination of two classes of antiestrogens: SERMs which stabilize ERα and SERDs which induce rapid proteasome dependent degradadation of ERα in a nuclear compartment. The crystal structures of the LBD of the estrogen receptors (ERα and ERβ) bound to different ligands (E2, tamoxifen, raloxifen, diethylstilbestrol, ICI 164,384) (Brzozowski et al., 1997; Pike et al., 2001; Shiau et al., 1998) have reveal that ligands of different sizes, shapes and side chains induce a spectrum of conformational states (Katzenellenbogen and Katzenellenbogen, 2002) and repositioning of helix H12 of the LBD. The resulting AF-2 surfaces that modulate recruitment of cofactors are directly dependent on the structure of the bound ligand. Thus, we next investigated whether mutating specific residues that either have a role in interaction between ERα and various ligands, in stabilizing of H12, in antagonist position or in recruitment of coactivators would influence the fate of ERα and its ability to stimulate transcription. 74 Transcription efficiency but not E2 mediated agonist activity of an estrogen responsive reporter gene is reduced by K362A, D351Y and L540Q point mutations of ERα We created a series of receptor mutations by site directed mutagenesis (see materials and methods) of pSG5 (HEGO)-ERα. The transcriptional activity of a luciferase reporter and the stability of ERα were assayed in the presence of the selected ligands. These specific residues were chosen because they appear to participate in the interaction of ERα with ligands and coregulators or in conformational changes of ERα in the presence of various antiestrogens (Montano et al., 1996). Crystallographic studies have demontrstaed that Asp351 plays an important role in anchoring the ligand to protein. For example upon ER-Raloxifene complex formation, Asp351 makes a hydrogen bond with piperazine ring nitrogen (Brzozowski et al., 1997) or in the presence hydroxy-tamoxifen, Asp351 makes a salt bridge with methylamino group of side chain (Shiau et al., 1998). Lys362 is a highly conserved residue which is required for efficient E2-dependent recruitment of certain coactivators. It is buried by induced repositioning of H12 in the presence of raloxifen or tamoxifen (Kong et al., 2003). Leu-540 which is part of the helix H12 is thought to play a role in regulation of ER ligand recognition or transcriptional activation. Although, these ER mutants have previously been tested for their role in transcription activation, a majority of studies was not performed in breast cancer cell lines and were based on receptor constructs under the control of promoters which contained multiple ER binding sites unlike the structure of natural ER responsive promoters (Pearce et al., 2003). We first tested transcriptional activity of the ERα mutants by transient co-transfection of ER negative MDA-MB 231 cells with the mutant ER cDNA and a luciferase reporter gene under the control of a single ERE containing promoter. Compared to the wt ERα, all mutants displayed a much lower basal transcription in the presence of vehicle control ethanol. 75 However, 10nM E2 treatment resulted in a significant induction of luciferase activity relative to the EtOH control levels (Fig 2). The 12-fold induction of luciferase activity observed with L540Q mutant was greater than the 7.7 fold observed for wt ER. The K362A mutant displayed an intermediate level of induction, and D351Y reached a small, but significant 5-fold induction. These results demonstrate that ERα mutants are perfectly capable of activating transcription when bound to E2. However, weak basal transcription may indicate that the residues mutated reduce ER binding to its target sequence or may hamper its interaction with co-activators necessary for efficient transcription initiation. The effect of the different antiestrogens on the activation of a luciferase reporter gene and on the stability of mutated ERα transiently transfected in MDA-MB231. 4OHT and RU39 binding to wt ERα stimulated luciferase transcription 1.5 - 2 fold, while ICI and RU58 had a weak inhibitory effect. In Figure 3A we show that compared to wt ER, the D351Y mutant ER increased agonistic activity of 4OHT, EM, and RU39 to 2.5 - 4.5 folds. Even with ICI and RU 58 a 1.5 -2 fold induction of transcription was measured (Fig 3.B). The K362R mutant also significantly induced luciferase transcription in the presence of 4OHT and RU39. Binding of ICI and RU58 to this ERα mutant had no notable effect on transcription. EM bound ER wt inhibited transcription to the same extent as ICI which corroborates earlier reports that EM652 was a pure antagonist (Labrie et al., 1999). Curiously, both ER mutants, D351Y and K362R, become agonist upon treatment with EM652 (Figure 3B and C). Luciferase transcription is stimulated 3 fold by the D351Y ER bound to EM652 which is equivalent to the stimulation measured in the presence of 4OHT. Activation of transcription is less pronounced by the K362R mutant, but still about 2 fold. Changes in the conformation of 76 the mutated AF2 domain upon EM652 binding to these mutants may alter recruitment of cofactors. Interestingly, when bound to the L540Q mutant of ER all ligands appear to partially induce transcription, albeit to a lesser extent than E2, with only 3 - 5 fold compared to 12 fold by E2. Notably, ICI and RU58 exhibit significant agonist activity (Fig 3D). Thus, the effect of antiestrogens appears to depend on the conformation of ERα. In order to further characterize the effect of selected compounds on the transiently transfected wt and mutant ERα, protein levels after an acute (3h) treatment were compared between each of the transiently transfected cell lines. Loading was controlled based on SV40-LUC expression. We found that wt ERα-ICI and ERα-RU58 complexes were rapidly degraded, while 4OHT and EM652 stabilized wt ER protein levels (Fig 3A). This behavior was also true for ER mutated at residues Asp351 and Lys362 (Figure 3B and C). However, treating cells with EM652 had a different effect on these ER mutants than on ER wt. Indeed, in the presence of EM652 wt ER was stabilized but ER mutated at either D351 or K362 was degraded almost to the same extent as in the presence of ICI or RU58. These mutated residues may alter folding of ER when bound to EM since the aliphatic side chain of EM forms a bond with D351 similarly to Raloxifen (Bentrem et al., 2001). Replacing Asp with Tyr may release the sidechain. Such a loose, long side-chain is reminiscent of the side chain of ICI and targets ER to the proteasome. Strikingly, the L540Q point mutation of ERα confered resistance to degradation in the presence of ICI and RU58 (Figure 3D). To further evaluate of this mutant in a reproducible manner, we have generated a stable transfectant of L540Q in MDA-MB 231 cells and ERα protein levels were compared in the presence of different ligands (figure 3E). We did not observe significant changes in ER protein levels for any ligand tested confirming the result of 77 transient transfection assays. This observation provides further evidence that Leu540 is a crucial amino acid for antagonist activity and for ERα stability. In conclusion, using a mutational study we could discriminate different classes of antiestrogens: OHT and RU39 belong to the same group (SERM) and ICI and RU58 to another group (SERD). For EM652 the situation is less clear, based on studies with a wt ER, we would conclude that EM652 is a SERM because we did not observe ERα degradation as seen with our standard reference of SERD, ICI, but at the same time, inhibition of transcription was more in line with SERD-like activities. The opposite was true for the mutated ER which was degraded in the presence of EM652 which in turn sustained transcription activation similar to OHT and RU39. EM652 may be a SERM with a pure anti-estrogenic activity (Labrie et al., 2001) or a in other words, a compound that could modulate ER target gene transcription due to an alteration of the conformational change of ER by its somewhat intermediate side-chain properties. Our study and others indicate the important role of Leu 540 in stabilizing of H12 in antagonist position in presence of 4OHT and that its mutation reverses the pharmacology of ICI. (Pearce et al., 2003; Shiau et al., 1998). Variations in transcription of endogenous TGFα, PGR and TFF1/PS2 differentiate selected antiestrogen compounds Using quantitative RT-PCR We analysed mRNA expression levels of three well characterized estrogen responsive genes whose regulation is relevant for endocrine therapy: transforming growth factor alpha (TGFα), progestrone receptor (PGR), and pS2/TFF1, after 16 hour of treatment with different compounds in MCF7 cells. E2 stimulates transcription of TGFα, PGR and TFF1 genes with respectively a 2 and 6-7 and 2-3 fold increase in expression compared to untreated cells. In presence the anti-estrogens these effects were reduced compared to those produced by E2. However, but interestingly, the 78 three genes responded differently to selected compounds: the effects were comparable for OHT and RU39 on the one hand and for EM, ICI and RU58 on the other hand. TGFα expression was reduced 50-60% by SERMS, but only ~20% by SERDs. Similarly, Ps2/TFF1 mRNA levels were reduced in the presence of SERMs and SERDs, 20-35% and ~40% repectively. Thus transcription regulation of TGFα and PS2/TFF1 in MCF7 cells did not unambiguously discriminate the antiestrogens used in this study. Interestingly, expression levels of PGR were hardly affected after 16h treatment with SERMs, but decreased significantly (60-70%) in the presence of EM652, ICI and RU58. Reduction of basal transcription by SERDs suggest that basal transcription of PGR requires the presence of ERα, possibly through its function as a docking partner in growth factor mediated transcription activation (Baron et al., 2007).We note that analysis of endogenous gene expression in MCF7 cells show similar effects for EM652, ICI and RU58. Discusssion ___________________________________________________________________________ We describe a molecular approach to classify modulators of ERα function and to predict the effect of antiestrogens based on structural features. There is great interest in developing novel anti-estrogens that could be used in patients that exhibit unwanted side-effects or resistances to the commonly used tamoxifen. This endeavour is a dual challenge: chemical synthesis and feasibility at acceptable cost of new compounds on the one hand, effective simple screens on the other hand. Recently, Dai and co-workers (Dai et al., 2008) presented a biochemical method based on hydrogen/deuterium exchange mass spectrometry (HDX-MS) to predict the functional impact of SERMs in a tissue specific manner. Such high throughput tests offer unprecedented opportunities to screen now generation compounds. Yet, once identified, these compounds still need to be tested on biological samples. We describe a set of readily available 79 molecular techniques useful in predicting both effectiveness and functional outcome of steroid compounds interacting with ER. Existing antiestrogens are described as partial or mixed antagonists, and full or pure antagonists, and more recently SERM or SERD anti-estrogens. Two compounds are readily used to treat patients: tamoxifen (Jordan, 2003) is categorized as a SERM to describe its mixed agonist/ antagonist activity that is tissue and promoter dependent (Shang and Brown, 2002), and faslodex (ICI 182,780) a promising agent which is a steroidal pure antiestrogen commonly presented as SERD prototype (Bross et al., 2002; Dauvois et al., 1993). Great efforts have been made to clarify the exact mechanism of antiestrogen action. It has been established that the pharmacological effects of ERα ligands are determined by spatial conformation of ERα following interaction (or binding) with ligand (Brzozowski et al., 1997; Pike et al., 1999; Shiau et al., 1998). It was proposed that ligand binding cavity of ERα has a remarkable plasticity with a preferential binding mode for distal hydroxyl of ERα ligands (Pike et al., 2001) showing similar orientation for distal side chains in α or β positions of different ligands in the ERα-ligand complex (Figure 2c). The LBD of ERα undergoes dynamic conformational changes upon ligand binding. These changes involve the position of helix 12 and are characteristic of specific members within an antiestrogen class (Métivier et al., 2002; Tamrazi et al., 2003). We have used a set of anti-estrogens with different sizes and positions of side chains to evaluate their effect on ERα. Using mutational analysis we observed that Asp351 is an important residue for ERα stability and may play a role in the interaction of the proteasome with the AF1 domain of ERα. The D351Y mutation increased DNA binding of ERα-ligands suggesting that increased agonist activity could partly be caused by a decrease in ERα degradation. Thus the amino acid at position 351 is critical for anti-estrogenic activity of compounds that we could group as SERMs (Figure 1B). 80 Lys 362 was demonstrated to have an important role not only in agonist- ERα complex but also in antagonist- ERα complex; in agonist conformation (DES-ERα) Lys362 interact with side chain of the residue of coactivator and in antagonist conformation (OHT-ERα) the side chain of Lys362 has capping Interaction with helix 12 residues (Shiau et al., 1998). Thus considering that Mutation Lys 362 in Ala, should prevent the capping interaction; in our study we observed that in K362A mutation increased agonist activity although its more important for compound with mixed agonist/antagonist activity (OHT, RU39) (Fig 3.c) It seems that this residue is discriminator between anti-estrogenswith pure properties and mixed properties. L540Q is a very important residue in ERα stability. Pearce and Martin (Martin et al., 2003; Pearce et al., 2003) have reported that L540Q ERα was no longer degraded in the presence of ICI and that Helix 12 is important for maintaining ER protein regulation in complex with ligands. Increased in transcription activation of reporter genes in this mutant may result from increased in ERα level and stability. We show here that the L540Q point mutation of ERα confers resistance to degradation also in the presence of RU58. Thus, following antiestrogen treatment, partial agonist activity appears to correlate with stabilization of cellular ER protein levels. This observation is not true for EM652. EM652, a nonsteroidal anti-estrogen that has been classified as pur antiestrogen because there are no estrogenic properties in rodent uterus (Martel et al., 1998). Our results appear to be in agreement with this classification since EM652 inhibited luciferase expression in the presence of ERwt in co-transfected MDAMB231cells (Figure 3) and EM652 reduced endogenous gene expression inlcuding PGR expression in MCF7 cells (Figure 4). Yet, paradoxically, EM-ERwt complexes were not degraded in these cells (Figure 1B and 4). EM652 has an alkylamino side chain that interacts 81 with D351. Binding of EM652 to ER mutated at positions D351 as well as K362 induced degradation of ER in our transient transfection assays (Figure 3). These mutated residues may alter folding of ER when bound to EM652 since the aliphatic side chain of EM forms a bond with D351 similarly to Raloxifen (Bentrem et al., 2001). Replacing Asp with Tyr may release the side-chain. Such a loose, long side-chain is reminiscent of the side chain of ICI and targets ER to the proteasome. Gene Expression Profiles of different ligands could be used for predicting a compound’s in vivo pharmacological effect. The most important indicator of response to endocrine therapy is the presence of estrogen receptors and progesterone receptors in tumours the identification the growth factor (as TGFα) that affects proliferation of target cells revolutionized the concept of hormonal regulation. In summary we have compared the activity of a series of anti-estrogens to improving existing molecular classification and conception demonstrating that direct molecular approaches on mammary tumor cells in culture represent a good tool for further screening and developing novel compounds for use in hormone-sensitive breast cancers. Acknowledgements We acknowledge financial support from the Institut National du Cancer (INCa), Resisth network, the Association pour la Recherche sur le Cancer and La Ligue pour le Cancer, comité du Gers. 82 Figures and Legends F F F Mazaheri et al Fig 1 F A F F F F F F S O O S O O HO OH 17 -estradiol HO OH OH HO Faslodex RU 58,668 N N N O O O OH HO OH O HO OH RU 39,411 4-hydroxytamoxifen EM-652 A) The chemical structure of various oestrogen receptor ligands The compound RU 58668 is a steroid substituted in the 11β position with a long hydrophobic side chain. This produces the same spatial arrangement for a side chain as the 7α substitution (Faslodex) relative to the plane of steroid nucleus. RU39411 has a steroid core, with a side chain similar to that of Tamoxifen, and EM652 a benzopyran derivative with a phenylcoumarine core that mimics the steroid backbone of oestradiol with a side chain similar to that of Raloxifen. 83 B C E2 OHT ICI EM RU39 RU58 RU58 Vehicle ERα ERα GAPDH GAPDH - LMB ALLN - LMB ALLN - LMB - ALLN 150 % of the control 150 100 50 0 100 50 EM RU 39 RU 58 - - ALLN ICI ALLN OHT LMB E2 ALLN 0 Vehicle LMB % of the control ICI LMB Vehicle E2 vehicle E2 ICI B) Stability of ERα in the presence of different ligands. Western blot analysis of ERα protein levels. MELN cells were treated with vehicle (EtOH), estradiol (10nM; E2), 4OH-Tamoxifen (1µM; 4OHT), ICI 182,780 (1µM; ICI), EM 652 (1µM; EM), RU 39,411 (1µM; RU39), and RU 58,668 (1µM; RU58) for 3h, 2 µg of whole cell extract was loaded onto SDS-PAGE gel. GAPDH served as a loading control .Quantification as percentages of the value of the sample treated with ethanol of three independent experiments. C) Proteasome dependent ligand degradation of ERα. MELN cells were pretreated or not with 10nM LMB or 100µM ALLN for 30 min and then treated with vehicle (EtOH), E2 (10nM), ICI 182,780(1µM), and RU 58668(1µM) for 3h. 2µg of total protein was loaded; ERα and GAPDH (internal control) detection by Western blotting. Quantification as percentage of the value of the sample treated with ALLN+E2 (100%) of two independent experiments. 84 RU 58 120 n=3 100 Fold induction 80 60 7.7 40 20 8.0 0 - E2 ER - + WT - 5.0 + - K362A + D351Y 12.2 - + L540Q Figure 2: Transcriptional activation of a luciferase reporter by ERα mutants ER-negative MDA-MB231 cells were co- transfected (as described in materials and methods) with 350ng of pSG5-HEG0 expressing wt ERα or K362A, D351Y, L540Q mutants of ERα., 500ng of an ERE- luciferase reporter plasmid and with 50 ng of pCH110 (β-galactosidase). 24 h post-transfection, cells were treated with E2 (10nM) for 24h. Luciferase activities were measured and results were normalized for transfection efficiency using β-galactosidase activity. 85 B A 120 120 WT 80 60 40 80 60 40 20 20 0 0 EtOH E2 Tam ICI EtOH OHT ICI E2 EM EtOH RU39 RU58 EM RU39 RU58 EtOH ER E2 E2 Tam OHT ICI ICI EM RU39 RU58 EM RU39 RU58 ER C D 120 120 K362A 80 60 40 ER 80 60 40 20 20 0 L540Q 100 Fold Induct ion 100 Fold Induct ion D351Y 100 Fold Induct ion Fold Induct ion 100 0 EtOH E2 Tam ICI EM EtOH OHT ICI EM RU39 RU58 E2 RU39 RU58 EtOH E2 Tam ICI EM RU39 RU58 EtOH E2 OHT ICI EM RU39 RU58 ER ER E L540Q Clone Vehicle E2 OHT ICI EM RU39 RU58 ER GAPDH Figure 3: Key Point mutations in the ligand binding domain of ER change SERM or SERD properties ER-negative MDA-MB231 cells were transiently transfected as described in in materials and methods. 24 h post- transfection, cells were treated with the EtOH, E2 (10nM), 4OHT (1µM), ICI 182,780(1µM), EM652 (1µM), RU 39411(1µM), RU58668 (1µM). Transcriptional activities were measured after 24h, and results were normalized for transfection efficiency 86 using β-galactosidase activity (A-D). ERα protein expression was analysezd after 3h treatment by western blotting and amount of protein loaded was normalized for transfection efficiency with luciferase activities from a co-transfection of SV4-lucvector (200ng) instead of pCH110. A) Wt ERα, B) D351Y mutant, C) K362A mutant, D) L540Q mutant E) The L540Q mutant were derived from MDA-MB231 cells that stably express ERα (see materials and methods). ER proteinα expression was analyzed by western blotting in presence or absence of ligands as in (A-E). 87 8 7 Relative level of mRNA 6 5 TGFα 4 PR pS2 3 2 1 0 EtOH E2 OHT RU39 ICI EM RU58 Treatment(16 hours) 1,4 Relative level of mRNA 1,2 1 0,8 TGF α PR pS2 0,6 0,4 0,2 0 EtOH OHT RU39 EM ICI RU58 Treatment (16 hours) _____________________________________________________________________ Figure 4: Regulation of endogenous gene expression by different ligands PGR, TFF1/pS2 and TGFα mRNA expression was quantified by RT-PCR in MCF-7 cells Following 16h treatment with E2 (10nM), OHT (1µM), ICI, RU39 (1µM), or RU58 (1µM) mRNA expression levels relative to the control TBP gene are shown. 88 Data shown are the average of three independent experiments; error bars represent ± S.E. mean.Values for untreated cells (EtOH) are set at 1. 89 References ___________________________________________________________________________ Anstead, G. M., K. E. Carlson, et al. (1997). 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Cell 95(7): 927-37. 91 Tamrazi, A., K. E. Carlson, et al. (2003). "Molecular Sensors of Estrogen Receptor Conformations and Dynamics." Molecular Endocrinology 17(12): 2593-2602. 92 PUBLICATION N° 2 93 Ligands specify estrogen receptor alpha nuclear localization and degradation Silvia Kocanova*, Mahta Mazaheri*, Stephanie Caze-Subra and Kerstin Bystricky# 1 Université de Toulouse ; UPS ; Laboratoire de Biologie Moléculaire Eucaryote; F- 31062 Toulouse, France 2 CNRS; LBME; F-31000 Toulouse, France *equally contributed #corresponding : [email protected] keywords: estrogen receptor, breast cancer, nuclear localization, hormone, antiestrogens, proteasome 94 Abstract Intracellular distribution of the oestrogen receptor alpha (ERα), a member of the nuclear-receptor superfamily, changes upon agonists or antagonist binding. We demonstrate a tight correlation between the nature of ligands, ERα protein turnover and cellular localization of ERα in a stable clone of MCF-7 cells in the presence of a variety of ligands. Predominantly nuclear in MCF-7 cells both in the absence and presence of ligands, GFP-ERα formed numerous nuclear foci upon addition of agonist, 17β-estradiol (E2), and pure antagonists (selective estrogen regulator disruptor; SERD), ICI 182,780 or RU58668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM), 4hydroxytamoxifen (OHT) or RU39411, diffuse staining persisted. Cell fractionation analyses revealed endogenous ERα and GFP-ERα predominantly in the nuclear fraction. Overall ERα protein levels were reduced after E2 treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and ERα accumulated in a nuclear insoluble fraction. mRNA levels of ESR1 were largely reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERD induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Introduction The estrogen receptor alpha (ERα) is a member of the steroid nuclear receptor family. In the presence of estradiol, ERα undergoes a conformational change leading to association with ERα target genes via direct binding to regulatory elements and modulation of their expression. Estrogens have a proliferative effect on various tissues, including the breast. Thus ERα plays a key role in mammary tumour development. In mammary cells, the effects of 95 17β-estradiol can be antagonized by compounds such as 4-hydroxytamoxifen (OHT), a tamoxifen metabolite that is a selective estrogen receptor modulator (SERM), and Fulvestrant/ICI 182,780 (ICI), a selective estrogen receptor disruptor (SERD). OHT has a partial agonist activity, depending on the tissue and response examined while ICI compounds are totally devoid of agonist activity in the models studied to date (Jordan, 1984; Katzenellenbogen and Katzenellenbogen, 2002). ERα-OHT complexes accumulate in nuclei and ICI treatment provokes rapid degradation of the ERα-ICI complex by the nuclear proteasome (Wijayaratne and McDonnell, 2001). ERα is downregulated in the presence of E2, its cognate ligand, through the ubiquitin/proteasome (Ub/26S) pathway (Alarid et al., 1999). Although ER-mediated transcription and proteasome-mediated degradation are linked (Lonard et al., 2000), transcription per se is not required for ER degradation and assembly of the transcriptioninitiation complex is sufficient to target ER for degradation by the nuclear fraction of the proteasome (Callige et al., 2005). In immunocytochemical studies it was shown that ERα exists almost exclusively in the nucleus both in presence or absence of hormone (Monje et al., 2001). Marduva et al (Maruvada et al., 2003) have shown that a small proportion of unliganded ERα exists in the cytoplasm, with a dynamic shuttling between the cytoplasm and nucleus in living cells. And this shuttling of ERα is markedly affected by estrogen treatment. Estradiol (E2). It has been shown that upon binding, E2 induces degradation of ERα, which is related to its ability to rapidly activate transcription (Jensen et al., 1969). Interestingly, two chemically different SERDs (ICI and RU58668) competitively inhibit estradiol mediated activation by ERα and induce a rapid down-regulation of the receptor (Bross et al., 2003; Buzdar and Robertson, 2006). In contrast, in the presence of tamoxifen ERα protein levels increased, although its initial mechanism of function is similar to the one observed for SERD's (Wittmann et al., 2007). 96 The purpose of the present study was to determine the impact of different ligands on nucleocytoplasmic shuttling of ERα and to examine the relation between localization and proteolysis, two mechanisms involved in ERα regulation in MCF-7 cells. To achieve this goal, we examined the relationship between ERα protein concentration, subnuclear localization with relationship to the proteasome and the level of ERα gene transcription simultaneously in presence two classes of antiestrogens. Materials and Methods Reagents 17β-estradiol (E2), tamoxifen (OHT) and Leptomycine B (LMB) were purchased from Sigma-Aldrich (St. Louis, MO). ICI 182,780 (ICI) was purchased from Zeneca Pharmaceuticals. RU39,411 (RU39) and RU58,668 (RU58) were kindly provided by Dr. J.M. Renoir (Paris, France). Stock solutions of E2, OHT, ICI, RU39 and RU58 were prepared in ethanol. Stock solution of LMB was prepared in methanol. The solution of proteasome inhibitor ALLN was purchased from Calbiochem. Rabbit polyclonal anti-ERα (HC-20), rabbit polyclonal anti-lamin A (H-102), rabbit polyclonal anti-cytokeratine 18 (H-80) were purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal anti-GAPDH (MAB374) was purchased from Chemicon International, mouse monoclonal anti-GFP from Roche, mouse monoclonal anti-α-tubulin (clone DM1A) from Sigma-Aldrich. Mouse monoclonal anti-20S proteasome subunit α2 (clone MCP21) was gift from Dr. M.P. Bousquet (IPBS, Toulouse, France). All cell culture products were obtained from Invitrogen. Cell culture and Generation of stable GFP-ERα cell line 97 Human breast cancer cell lines were maintained in Dulbecco's modified Eagle's medium F-12 (DMEM F-12) with Glutamax containing 50 µg/ml gentamicin, 1 mM sodium pyruvate and 10% heat-inactivated fetal calf serum. All cells were grown at 37°C in a humidified atmosphere containing 5% CO2. The stably transfected GFP-ERα reporter SK19 cell line was generated from ERα-positive breast cancer MCF-7 cells (ATCC). 2nd passage cells were transfected with a GFP-ERα expression vector (pEGFP-C2-hERα) using FuGENE® HB Transfection Reagent (Roche Applied Science) and G418 resistant clones were selected at the concentration 1 mg/ml. GFP-ERα expressing clones were isolated, ERα protein expression in response to estradiol and to anti-estrogens was quantified using fluorescence microscopy and western blot. Expression of ERα-regulated genes was tested by qRT-PCR and compared to gene-expression regulation in MCF-7 cells (data not shown). The clone SK19 in which GFP-ERα behavior was comparable to the one in MCF-7 cells was selected for further study. To study the effects of estrogens and anti-estrogens, cells were grown for 3 days in medium containing phenol red-free DMEM F-12 supplemented with 5% charcoal-stripped fetal calf serum, without gentamicin and sodium pyruvate. Cells were subsequently treated or not with 10 nM E2, 1 µM ICI, 1 µM OHT, 1 µM RU39, 1 µM RU58 for the indicated times. To study ERα degradation by the proteasome, cells were pre-treated 30 min with 100 µM ALLN, a proteasome inhibitor, or 10 nM LMB, a nuclear export inhibitor. Cell extracts and Western blots MCF-7 cells grown in 6-well plates and were treated as indicated, washed with icecold PBS and collected by centrifugation. Total cell lysates were prepared by resuspending the cells in 100 µl lysis buffer (50 mM Tris pH=6.8, 2% SDS, 5% glycerol, 2 mM EDTA, 1.25% β-mercaptoethanol, 0.004% Bromophenol blue). The samples were boiled for 20 min at 95°C and cleared by centrifugation at 12 000×g for 10 min. The protein concentration was 98 determined by the Amido schwartz assay when the samples contained SDS. Samples were subjected to SDS-PAGE and the proteins transferred onto nitrocellulose membrane. Western blot analysis was performed as previously described (Giamarchi 2002) using ERα and GAPDH antibodies. qRT-PCR expriments Total RNAs were extracted using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. 1-5 µg of total RNA was reverse transcribed in a final volume of 20 µl using SuperScriptTM III Reverse Transcriptase (Invitrogen). cDNA was stored at -80°C. All target transcripts were detected using quantitative RT-PCR (SYBRGreen SuperMix, Invitrogen) assays on a Mastercycler Realplex device (Eppendorf) using TBP gene as endogenous control for normalization of the data. The following primer pairs were used for amplification: TBP: 5’-CGGCTGTTTAACTTCGCTTTC-3’ 5’-CCAGCACACTCTTCTCAGCA-3’ ESR1: 5’-TGGAGATCTTCGACATGCTG-3’ 5’-TCCAGAGACTTCAGGGTGCT-3’ GREB1: 5’-GTGGTAGCCGAGTGGACAAT-3’ 5’-AAACCCGTCTGTGGTACAGC-3’ The thermal cycling condition comprised 2 min at 55°C and 2 min at 95°C followed by 40 cycles at an appropriate annealing temperature depending on the primer set for 1 min. The results were analyzed using Mastercycler Realplex and qBASE software. 99 Cell fractionation Three hours after incubation with ERα ligands, SK19 cells were washed with ice-cold PBS, scraped and centrifuged at 1,500 rpm for 5 min at 4°C. The pellets were resuspended in 150 µl digitonin lysis buffer containing 1% digitonin and 1 mM EDTA in PBS, immediately centrifuged at 13,000 rpm for 20 min at 4°C, to obtain the cytosolic fraction. The pellets were resuspended in 150 µl HEPES lysis buffer containing 1% Triton X-100, 10% glycerol, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 1 mM Na3VO4 and 50 mM NaF in HEPES buffer (25 mM HEPES, 0.3 M NaCl, 1.5 mM MgCl2, 20 mM β-glycerol-phosphate, 2 mM EDTA, 2 mM EGTA and 1 mM DTT), kept 15 min on ice and centrifuged at 13,000 rpm for 15 min at 4°C, to obtain the soluble nuclear fraction. The last pellets were resuspended in 100 µl of a third buffer containing 95% Laemmli buffer and 5% β-mercaptoethanol and incubated 5 min on ice before boiling them for 20 min at 95°C to obtain the insoluble nuclear fraction. The different fractions were stored at -80°C until use. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad). Immunofluorescence and Fluorescence microscopy Before imaging SK19 cells were grown for 3 days on coverslips in DMEM without phenol red, containing 5% charcoal-stripped fetal serum. After 3 days, cells were treated for 1h with the following ligands: 10 nM E2, 1 µM ICI, 1 µM OHT, 1 µM RU39, 1µM RU58. The cells were then washed twice with PBS, fixed in 4% paraformaldehyde/PBS for 10 min at room temperature, subsequently permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature counterstained with DAPI and mounted on microscope slides. To study co-localization of ERα and proteasome by immunofluorescence, SK19 cells were grown for 3 days on coverslips in DMEM without phenol red, containing 5% charcoalstripped fetal serum and next treated for 3h with drugs as indicated above. To block 100 proteasome-mediated ERα degradation, the cells were incubated 30 min with 100 µM ALLN prior to treatment with ICI and RU58. Before immunostaining, the cells were fixed in 4% paraformaldehyde/PBS for 30 minutes at room temperature, washed three times in PBS, quenched in 75 mM NH4Cl containing 20mM glycine and permeabilized with 0.5% Triton X100 in PBS for 30 minutes. Next, the cells were washed with PBS, blocked for 1h at room temperature in 5% dry milk in TBS-T (20mM TRIS-HCl, 150mM NaCl, 0.1% Tween 20, pH=7.4) and incubated overnight at 4°C with anti-20S proteasome antibody at final concentration 2 µg/ml in 5% dry milk in TBS-T followed, after washing, by incubation with the Alexa Fluor® 647 goat anti-mouse secondary antibody (1:1000, Invitrogen, Molecular Probes) for 90 min in the dark at room temperature. Finaly, the cells were washed with TBST, counterstained with DAPI and mounted on microscope slides. The cells were examined by fluorescence microscopy using an Olympus IX-81 microscope, equipped with a CoolSNAPHQ camera (Roper Scientific) and imaged through an Olympus oil-immersion objective 100x PLANAPO NA1.4. Images were recorded and deconvolved using Metamorph software (Universal Imaging). All images were processed for presentation using Adobe Photoshop 9.0.2. Results Ligands regulate ERα protein levels and transcription rates independently We first examined the kinetics of ERα protein turnover in MCF-7 cells following treatment with estradiol (E2), two SERMs (OHT and RU39) and two SERDs (ICI and RU58). Treatment of MCF-7 cells with 10 nM E2, 1 µM ICI and 1µM RU58 rapidly induced massive decrease in ERα protein levels as detected by western blot (Figure 1A). Time course experiments showed that 1h after E2 induction, the detected amount of ERα protein accounted for only 40% of ERα levels detected before treatment; after 4h ERα levels were as 101 low as 20% of the quantity of ERα present in untreated cells and after 16h ERα protein remained at a level equivalent to the one observed 1h after addition of E2, ICI or RU58 (Fig. 1A). Treatment of MCF-7 cells with OHT or RU39 (Figure 1A), two compounds classified as SERM, only slightly reduced ERα protein levels at the initial 1h time-point. ERα protein levels were almost equivalent to the ones detected in untreated cells after 4h treatment with OHT and 16h treatment with RU39. We thus demonstrate that both SERMs stabilize ERα protein levels compared to 60-80% loss observed in the presence of E2 or SERDs. To assess whether changes in protein levels reflected variations in ERα protein stability or are modulated by transcription of the ESR1 gene which codes for ERα, we quantified ERα mRNA accumulated after 16h treatment with the different compounds (Figure 1B). ESR1 mRNA expression was greatly reduced in all samples subjected to ERα ligands. In the presence of estradiol (E2), only 40% of ESR1 mRNA could be recovered. Similarly, treatment with SERMs and SERDs lead to relative ESR1 expression levels equivalent to 45%60% of ESR1 mRNA in untreated MCF-7 cells. Despite the down-regulated ERα protein level readily detectable after 1h and significant after 16h (Figure 1A), we observed that the presence of E2 results in 7 to 10 fold induction of an ERα-target gene GREB1 compared to mock treated cells (Figure 1C). GREB1 transcription was inhibited by SERMs and SERDs (>40% reduction; Figure 1C). These results were expected since E2 is an agonist of this ERα target gene expression, while SERMs and SERDs are antiestrogens and thus repress GREB1 transcription in ERα positive mammary tumour cells. Thus, in MCF-7 cells, variations in ERα protein levels do not correlate with ESR1 transcription in the presence of ligands. We note that the decrease in ERα protein levels is more pronounced after treatment with SERDs than after addition of E2, while the effect of hormone and SERDs on mRNA accumulation is comparable. These results indicate that reduction of ERα protein levels following treatment with SERDs cannot be solely attributed 102 to decreased ESR1 mRNA levels. SERDs apparently act both on transcription of the ESR1 gene and on ERα protein turnover. In contrast, despite reduced ESR1 expression levels, ERα protein accumulated.. after 16h treatment with SERMs suggesting that binding to SERMs stabilizes the ERα. However, from these data we cannot exclude that reduced transcription of ESR1 directly influences cellular ERα protein concentrations in the presence of E2. Ligands directly affect intracellular distribution and stability of ERα SERMs and SERDs can be distinguished based on molecular mechanisms (Mazaheri et al., in preparation). To unambiguously determine localization of the estrogen receptor and its intracellular trafficking in response to treatment with various ligands we established a MCF-7 cell line stably expressing GFP-ERα from a CMV promoter. It was previously shown that transiently expressed GFP-ERα is functional using an estrogen response element driven luciferase reporter gene (Htun et al., 1999). Inducing expression of GFP-ERα in MCF-7 cells did reportedly not alter cell cycle progression and GFP-ERα participated in estrogen target gene regulation similarly to endogenous ERα (Zhao et al., 2002). We tagged the N-terminus of the human ERα with the S65T variant of GFP for transfection and stable integration in MCF-7 cells. Several clones were recovered and screened for total GFP-ERα protein content after cells were treated with E2, OHT or ICI using fluorescence microscopy and western blots. Here, we selected a MCF-7 derived clone (SK19) expressing GFP-ERα in which changes in endogenous ERα protein levels in response to a 4h treatment with E2, OHT and ICI were identical to the ones observed in MCF-7 cells (compare lanes labeled ERα in MCF7 and SK19 cells in Figure 2A). In addition, mRNA expression levels of some ERα target genes was verified in the selected clone SK19 and compared to gene expression levels in MCF-7 cells (data not shown). In SK19 cells, GFP-ERα protein accounted for 50% of total 103 ERα (GFP-ERα and endogenous ERα) in untreated cells. In the presence of E2 both GFPERα and endogenous ERα protein levels are reduced similarly to MCF-7 cells. The CMV promoter being insensitive to E2 and antiestrogens, GFP-ERα protein levels are unlikely to be transcriptionally regulated. This observation together with the results shown in Figure 1 provides evidence that ERα protein turnover is regulated directly by binding of the receptor to ligands and subsequent degradation. Several studies have demonstrated a mainly nuclear localization of GFP-ERα either in transiently transfected mammary tumour cell lines (Htun et al., 1999) or Hela cells (Stenoien et al., 2001; Stenoien et al., 2000b) and in MCF-7 cells expressing GFP-ERα from an inducible promoter (Zhao et al., 2002). These microscopy based observations are in contrast with cellular fractionation studies which have regularly indicated that large amounts of ERα, in the absence or the presence of ligands, associate with the cytoplasmic fraction (Wittmann et al., 2007). It has been proposed that the relative amount of cytoplasmic ERα is indicative of the mechanism of action of certain antiestrogens (Wittmann et al., 2007). Commonly used cell fractionation protocols include a detergent based extraction step. Or ERα and other nuclear receptors such as the glucocorticoid receptor (GR) are easily extracted from the nucleus in the presence of low concentrations of detergents such as NP40 (data not shown; V.Marsaud and H. Richard-Foy, unpublished observations). As a consequence, enrichment of ERα or GR in the cytoplasm likely results from the extraction protocol rather than a specific behavior of nuclear receptors. We used a digitonin based cell fractionation protocol to determine the distribution of unbound and ligand-bound ERα and GFP-ERα in different cellular compartments (Figure 2B). Effectiveness of the fractionation protocol (for details see Materials and Methods) was confirmed using loading controls, lamin A for the nuclear fractions, cytokeratin 18 for the nuclear and cytoplasmic fractions, and alpha-tubulin for the cytoplasmic fraction (Figure 2B). Treatment of cells with E2 and various antiestrogens did not 104 affect cellular distribution of these proteins. We found that the endogenous ERα associates predominantly with the nuclear fraction in our SK19 cells. In untreated cells, the part of ERα retained in the cytoplasm corresponds to 22% of total endogenous ERα detected using the HC-20 antibody (Figure 2B, lane 1). Similarly, the bulk of GFP-ERα, detected using either the HC-20 antibody or an antibody directed against GFP, was found in the nucleus. Following addition of E2, we note an overall decrease in ERα protein levels that could mainly be attributed to a reduction in nuclear ERα (Fig. 2B, lanes 4-6). Treatment of SK19 cells with SERDs, ICI or RU58 lead to a decrease in overall ERα protein levels as shown for MCF-7 cells in Figure 1A. Notably, the remaining ERα was concentrated in the nuclear insoluble fraction in the presence of either ICI or RU58 (Fig. 2B, lanes 12 and 18) suggesting that the nuclear soluble fraction is rapidly degraded. In contrast, we found that treatment with OHT and RU39 (Fig. 2B, lanes 7- 9 and 13- 15) reflected a cellular distribution similar to one observed in untreated cells (Fig. 2B, lanes 1- 3) with high ERα protein concentration in nuclear soluble compartment. Our cellular fractionation protocol is robust since the effects of various ligands are reproducible inside each category: OHT and RU39 induce the same effect on ERα protein distribution and this effect is distinct from the one of ICI and RU58. In addition, we show that occupation of different cellular compartments by GFP-ERα reflects the localization of endogenous ERα. Relative amounts of GFP-ERα in each fraction correlated with its presence as detected by fluorescence imaging (see below). Ligands induce specific intracellular relocalization of GFP-ERα GFP-ERα can be visualized in SK19 cells using conventional wide-field microscopy. SK19 cells were cultured on conventional glass microscopy coverslips in phenol-red free media for 3 days (culture conditions were identical to conditions used for cell fractionation, 105 immunoblotting or RNA extraction priori to RT-qPCR). Figure 3 shows representative images of SK19 cells treated or not with E2, SERMs and SERDs. We note that in our SK19 cell line GFP-ERα is excluded from the nucleoli, as previously observed for the cellular distribution of endogenous ERα in MCF-7 cells (King and Greene, 1984; Welshons et al., 1988) and of transiently transfected GFP-ERα (Htun et al., 1999; Stenoien et al., 2000a), under all conditions tested. Furthermore, exposure times were identical for all conditions examined by fluorescence microscopy. In untreated cells, ERα is uniformly distributed in the nucleus (compare GFP-ERα fluorescence (Fig. 3b), to the DAPI nuclear stain in Figure 3a). A linear scan across the entire field including cytoplasm and nucleus (Fig. 3c) shows that the cytoplasmic GFP-ERα fluorescence is barely above background (~12% of the maximum fluorescence intensity detected in the nucleus) which is in correlation with our cellular fractionation observations (Fig 2B, lane 1). In the presence of E2, GFP-ERα rapidly relocalized to accumulate in numerous large foci scattered through the nucleoplasm (Figure 3e). In E2 treated cells, no GFP-ERα fluorescence could be detected in the cytoplasm (see linescan Fig. 3f). In contrast, after 1h treatment with SERMs, OHT or RU39, we did not observe any intranuclear relocalization of GFP-ERα. GFP-ERα staining remained diffuse with fluorescence intensity comparable to mock cells (Fig 3b, n, r and corresponding linescans Fig 3c, o, s). However, again, no cytoplasmic GFP-ERα could be detected. Upon exposure to SERDs, both ICI and RU58, GFP-ERα formed large intranuclear foci, reminiscent to the ones observed in the presence of E2 (Fig. 3h, k and e). The maximum fluorescence intensity measured after E2 and ICI treatments decreased by 20-40% as compared to untreated or SERM treated cells (Fig. 3f and 3i compare to 3c and/or 3o, 3s). The effects of ICI and RU58 were indistinguishable suggesting that both molecules operate via similar molecular mechanisms despite significant 106 structural differences (Wittmann B.M. et al, 2007, Cancer Res,). Again, we find that SERMs and SERDs can readily be classified using molecular and imaging approaches. Proteasome-dependent degradation of ERα bound to E2 or SERDs ERα is a short-lived protein (a half-life ∼ 4-5 hours for ligand-free ERα and ∼ 3h for ligand-bound ERα) whose cellular expression is dynamically regulated(Eckert et al., 1984). We and others, have previously shown that ERα degradation occurs in the cytoplasm in presence of a natural ligand (E2), but, in the presence a full antiestrogen such as ICI ERα is rapidly degraded, at least partially, in a nuclear compartment in a proteasome dependent manner (Callige et al., 2005; Marsaud et al., 2003; Nawaz et al., 1999). The 26S proteasome is a large protein complex (1500-2000 kDa) present in the cytoplasm and nucleus of eukaryotic cells. The catalytic core of this multi-subunit complex, described as the 20S proteasome, contains α and β subunits (Brooks et al., 2000). To determine the proteasome-mediated ERα degradation induced by E2 and SERDs, we visualized GFP-ERα and 20S proteasome subunit α2 in SK19 cells. SK19 cells were fixed, permeabilized and subjected to indirect immunofluorescence using a monoclonal anti-20S proteasome subunit α2 primary antibody. Images acquired on an Olympus inverted wide-field microscope revealed punctate nuclear staining of proteasome subunits throughout the nucleus (Figure 4A, center panels). We did not observe any cytoplasmic staining of this proteasome subunit under our culture conditions. In the presence of E2 GFP-ERα accumulated at numerous nuclear sites that colocalized at least partially with proteasome foci (Figure 4A panel f). When cells were treated with ICI or RU58 GFP-ERα foci also significantly overlapped with accumulation sites of the 20S proteasome subunit α2 throughout the nucleus (Fig 4A, insets of panels i and o). On average, we observed larger and more frequent GFP- 107 ERα-proteasome complexes in the presence of SERDs than in the presence of E2 consistent with the fact that ERα is more readily degraded when ERα is bound to SERDs. We investigated the effect of LMB, an inhibitor of the nuclear export receptor CRM1, and of ALLN (acetyl-leucyl-leucyl-norleucinal), an inhibitor of the proteasome, on the SERDdependent degradation of ERα in SK19 cells. SK19 cells were pretreated with 10 nM LMB or 100 µM ALLN for 30 min. Figure 4B shows that LMB did not block ICI or RU58 induced ERα degradation suggesting that SERD- bound ERα is degraded in the nucleus. In contrast, ALLN inhibited ICI and RU58 induced degradation of ERα confirming that SERD-ERα complexes were degraded by the nuclear proteasome (Figure 4B, lanes 7 and 10). Pretreatment with ALLN reduced the number of contacts between GFP-ERα and proteasome foci (Figure 4A panels l and s). ICI-ERα and RU58-ERα complexes remained stably associated with the nuclear matrix (Fig. 4A, insets of j and p panels). Interestingly, in a few cells treated with either E2 or SERDs we observed a single very large site of accumulation of the 20S proteasome α2 subunit (data not shown). These sites, also called clastosomes, were reported to colocalize with the c-jun and c-fos proteins (Lafarga et al., 2002), very unstable proteins with a half lives of less than 90min. In our cells, clastosomes did not colocalize with GFP-ERα foci (data not shown) which could be explained by the higher stability of ERα compared to c-jun and/or c-fos proteins. Discussion Most members of the nuclear receptor superfamily form focal accumulations within the nucleus in response to hormone (Hager et al., 2000). Receptors undergo constant exchange between target sequences, multi-protein complexes including a variety of transcription factor, as well as subnuclear structures that are as yet poorly defined. The 108 estrogen receptor alpha is found almost exclusively in the nucleus, both in hormone stimulated and untreated cells which makes it an exception among nuclear receptors which generally translocate from the cytoplasm into the nucleus upon hormone stimulation. Hager and colleagues (Hager et al., 2000) proposed that distribution of the ERα is dependent not only on localization signals directly encoded in the receptors, but also on the nature and composition of the associated macromolecular complexes. Formation of these complexes depends on the nature of the ligand bound to ERα. Thus, as demonstrated here, ligands directly affect the nuclear fate of the receptor. We used a MCF-7 cell line stably expressing GFP-tagged human ERα to determine the localization of ligand-bound GFP-ERα in mammary tumor cells to demonstrate that few hours after treatment cellular localization of the ERα correlates with the nature of the ligand independently of its impact on transcription. In the presence of E2 and SERMs which induce binding of ERα to target sequences and subsequent formation of macromolecular complexes, the cytoplasmic fraction of E2 bound ERα rapidly translocated into the nucleus suggesting that DNA binding somehow recruits cytoplasmic ERα. In contrast, SERD bound cytoplasmic ERα was retained in the cytoplasm consistent with the fact that SERDs induce a conformational change that blocks ERα binding to DNA and leads to rapid degradation. Our data also corroborate recent observations by Long and co-workers (Long and Nephew, 2006) that ICI induced specific nuclear matrix interaction of protein-ERα complexes with cytokeratins 8 and 18 which mediate immobilization and turnover of ERα. Interaction with these proteins provoked ICIinduced cytoplasmic localization of newly synthesized ERα. A non genomic role of ERα in the cytoplasm has been proposed to play a role in acquired resistance to antiestrogens, in particular OHT (Fan et al., 2007b). Indeed, in OHT resistant cells, the ERα accumulated in the cytoplasm, suggesting that the observed SERM 109 stimulated ERα relocalization into the nucleus is necessary for anti-hormone effectiveness (through the modulation of macromolecular complexes bound to the ERα). An attractive possibility would thus reside in not only blocking non-genomic ERα function in SERM resistant tumors, but to increase ERα translocation into the nucleus. Furthermore, E2 induced focal accumulations of ERα scattered throughout the nucleus. Similarly, SERD-bound ERα also concentrated into nuclear foci. These foci frequently colocalize with the proteasome which may explain that ligand bound ERα is less dynamic, and appears more strongly associated with nuclear matrix like structures(Stenoien et al., 2001). Thus we propose a simple explanation reconciling all previous observations of ERα dynamics: ligands that allow ERα to bind its target sequence and recruit macromolecular complexes induce ERα nuclear accumulation (estradiol and SERMs); ligands that bind to ERα but do not lead to DNA binding due to conformational changes of the receptor (Mazaheri et al., in preparation) do not induce relocalization of the receptor, but accelerate its degradation (SERDs); finally, ligands that induce association of ERα with the proteasome (estradiol and SERDs) lead to focal accumulations and immobilize the ERα. It is the association with the proteasome (Sato et al., 2008) and not active degradation by the proteasome that leads to ERα sequestration. Acknowledgements Imaging was performed at the RIO microscopy facility, Toulouse. SK is supported by a postdoctoral fellowship from the Association pour la Recherche contre le Cancer (ARC). We thank Anne-Claire Lavigne for critical reading of the manuscript. We acknowledge financial support from the Institut National du Cancer (INCa), Resisth network, ARC and La Ligue pour le Cancer, comité du Gers. ANR 110 Figures and Legends A Relative ER protein levels 1.4 1h 4h 16h 1.2 1 0.8 0.6 0.4 0.2 0 EtOH O Et H E2 O HT E2 I IC 39 58 RU RU OHT O Et H ICI E2 O HT I IC RU39 39 58 RU RU RU58 O Et H E2 O HT I IC 39 58 RU RU ER GAPDH 1h 4h 16h Figure 1: Protein and mRNA levels of ligand bound ERα in MCF-7 cells A) Western blot and quantification of ERα protein levels after 1h, 4h and 16h treatment with 10 nM E2, 1 µM ICI, 1 µM RU58, 1 µM OHT and 1 µM RU39 relative to ERα protein levels in untreated (EtOH) cells. 2µg of total protein extraction were loaded. Quantification averaged from three independent experiments 111 C B Relative mRNA expression Relative mRNA expression 1.2 ESR1 1 0.8 0.6 0.4 0.2 0 EtOH E2 OHT ICI RU39 10 GREB1 8 6 4 2 0 EtOH RU58 E2 OHT ICI RU39 RU58 Figure 1: Kocanova, Mazaheri et al. B-C) Total RNA was extracted from untreated and estrogen or antiestrogens treated MCF-7 cells after 16h. Relative expression levels of the ESR1 gene and the ERα target gene GREB1 was analyzed by qRT-PCR using primer sets for ESR1, GREB1 or TBP as an internal control (see Materials and Methods). Data shown are the average of three independent experiments, error bars represent ± S.E. mean. 112 A MCF-7 EtOH E2 SK-19 OHT ICI EtOH E2 OHT ICI GFP-ER * ER GAPDH * - unspecific band Figure 2: Nuclear accumulation and degradation of the estrogen receptor alpha in SK19 cells A) Western blot quantifying protein levels of endogenous ER SK19. The stably transfected GFP-ER and GFP-ER in reporter SK19 cell line was generated from MCF-7 cells by transfection with a GFP-ERα expression vector (pEGFP-C2-hERα) using FuGENE® HB Transfection Reagent (Roche Applied Science). G418 resistant clone SK19 in which GFP-ER behavior was comparable to the one in MCF-7 cells was selected. ERα protein expression in response to estradiol and to antiestrogens was quantified using western blot. 113 B E2 vehicle GFP-ER Cyt NS NI Cyt OHT NS NI Cyt NS ICI NI Cyt NS NI * ER % of ER in fraction 24% 53% 23% 24% 47% 29% 19% 53% 28% 23% 29% 48% -Tubulin Lamin A Cytokeratin 18 vehicle GFP-ER Cyt NS 22% 55% RU39 NI Cyt RU58 NS NI Cyt 50% 31% 28% NS NI 33% 39% * ER % of ER in fraction 23% 19% -Tubulin Lamin A Cytokeratin 18 * - unspecific band B) Digitonin based cellular fractionation showing nuclear relocalization and degradation of ER and GFP-ERα SK19 cells were treated with vehicle (EtOH), E2 (10 nM), OHT (1 µM), ICI (1µM), RU39 (1µM) or RU58 (1µM) for 3h. Nuclear fractions of untreated and estrogen or antiestrogens treated cells were isolated as described under “Materials and Methods”. Nuclear content of ERα and GFP-ERα was analyzed by Western Blot using antiERα antibodies (Santa Cruz Biotechnology). The specific subcellular proteins, α-tubulin for cytoplasmic fraction (Cyt), Lamin A for nuclear soluble fraction (NS) and cytokeratin 18 for nuclear insoluble fraction (NI) indicate equal loading. Results shown are representative of at least 3 independent experiments. 114 GFP-ERα linescan Fluorescence intensity (a.u.) DAPI vehicle b E2 ICI 64 0 RU58 Fluorescence intensity (a.u.) OHT RU39 p s r 115 25 31 128 64 0 6 12 19 25 31 Distance along line (µm) 256 192 128 64 0 6 12 19 25 31 Distance along line (µm) 256 192 128 64 6 12 19 25 31 Distance along line (µm) 256 192 128 64 0 o n Fluorescence intensity (a.u.) m 19 192 l k 12 256 0 j 6 Distance along line (µm) i h Fluorescence intensity (a.u.) g 128 f e Fluorescence intensity (a.u.) d 192 c Fluorescence intensity (a.u.) a 256 6 12 19 25 31 Distance along line (µm) 256 192 128 64 0 6 12 19 25 31 Distance along line (µm) Figure 3: Estrogen, SERDs and SERMs induce distinct intracellular relocalization of ERα SK19 cells were incubated for 1h with 10 nM E2 and different antiestrogens (SERDs or SERMs). After 4h cells were fixed and stained with DAPI. The cells were examined by fluorescence microscopy using an Olympus IX-81 inverted microscope, equipped with a CoolSNAPHQ camera (Roper Scientific) and imaged through an Olympus oil-immersion objective 100x PLANAPO NA1.4. Pictures are representative of at least three independent experiments. GFP-ER forms intranuclear foci in the presence of E2, RU58 and ICI, but remains uniformly distributed in the nucleus after addition of OHT and RU39. Linescans indicate relative fluorescence intensities in the cytoplasm and the nucleus in the cells imaged using identical parameters. 116 A AL LN LM B -- RU58 AL LN B LM -- B LM -- AL LN B LM -- GFP-ER ICI AL LN E2 vehicle * ER GAPDH * - unspecific band Figure 4: Nuclear Proteasome-GFP-ERα contacts are frequent in the presence of E2 and SERDs A) SERDs degrade ERα through proteasome and in the nucleus, SK19 cells were pretreated or not with 10 nM LMB or 100 µM ALLN for 30 min and then treated with vehicle (EtOH), ICI (1µM), or RU58 (1µM) for 3h. ERα and GAPDH (internal control) detection by Western 117 blotting. 20S proteasome α2 GFP-ERα B merge a b c d e f g h i j k l m n o p r s vehicle E2 ICI ICI + ALLN RU58 RU58 + ALLN 118 B) SK19 cells were grown for 3 days on coverslips in DMEM without phenol red, containing 5% charcoal-stripped fetal serum and next treated for 3h with drugs as described in Materials and Methods. Cells were fixed and subjected to immunofluorescence using a monoclonal anti-20S proteasome subunit α2 primary antibody, followed by incubation with the Alexa Fluor® 647 goat anti-mouse secondary antibody. Right panels represent colocalization of GFP-ER and 20S proteasome (insets): untreated cells (4A,c), E2 treated cells (4A,f), SERDs treated cells without (4A,i and o) or with (4A,l and s) 100 µM ALLN. Images are representative of three independent experiments. 119 References Alarid, E. T., N. Bakopoulos, et al. (1999). 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"Characterization of stably transfected fusion protein GFPestrogen receptor-alpha in MCF-7 human breast cancer cells." J Cell Biochem 86(2): 365-75. 121 ONGOING RESULTS In this part of our study we wanted tested the molecules designed by chimists with our molecular approach. The molecules sythesized by M Poirot’s group may be patented and thus I am not able to provide their molecular structures here in order to comply by patenting restrictions. The molecules are analogs of ICI164,384 whose side chains were modified to assess its importance for pure antagonist. The steroid motif was replced by a stilbene to simplify the synthesis procedure. Here our colabators were interested in the position of the polar group on this side chain of ICI 164,384. The chemical synthesis was carried out by M Poirot at the ICR. They observed that positioning of an amide group along the side chain affects the molecules activity. 122 CONCLUSION AND PERSPECTIVES Estrogens have been recognized to play an important role in proliferation of a large proportion of breast tumours many years ago. Today it is well established that estrogens act via their receptors, and that ERα expression in a tumour represents a good prognosis. In addition, ER positive tumours are responsive to hormonal therapy. In more than half of breast tumours ERα is overexpressed and around 70% of these tumours respond to anti-estrogen therapies via the use of tamoxifen. Unfortunately, some of these tumours do not respond to endocrine therapy initially (de novo resistance), and the majority of responsive tumours eventually become resistant (acquired resistance). In most of these resistant tumours ERα is still expressed, and other options of endocrine therapy can be proposed. A second line treatment for resistant tumours is the use of antiestrogens that do not show a cross resistance. Few antiestrogens that can be used in tamoxifène acquired resistance. One of these is Faslodex, which has unfortunately poor bioavailability (intravenous administration is required) and is a costly treatment for patients. There is a great interest to developing the novel molecules which could be used as first or second line in endocrine therapy. Great efforts are being made in all areas of research, including random screening of a bank of compounds (Roussel Uclaf.) synthesis of specifically designed molecules or extraction of natural compounds. Although our understanding of estrogen receptor biology and the consequence of ligand interaction is expanding, certain aspects of liganded ER function areis not fully understood, for example; how structurally different ligands induce interaction between AF-1 and AF-2. Which may require extensive modelling based on crystallographic data to determine exact structure –function relationship of antiestrogens with ER. 123 In this study we propose a molecular approach that could be interesting for rapid discrimination between SERM and SERD. We subsequently determined that the compounds classified as SERD by our approach function in a similar manner and a grand part of their activity is dependent on proteasome dependent degradation. We confirmed our initial assumption that the position and nature of side chains of antiestroges is crucial for stability of ERα. Thereby the side chain defines a SERD. I think a molecule described as SERD is a pure antiestogen but a pure antiestrogen is not necessarily a SERD. For example: EM652 in our molecular approach is a SERM but with pure antiestrogenic activity, and it could be interesting to develop molecules similar to EM 652. Such compounds offer the exciting possibility to reduce undesirable side effects especially interesting in premenopause patients. In the second part we investigated the impact of the nature of ligands in ERα localisation and relationship of intracellular ligand-ER localisation and expression or transcription of ERα. Localization of ERα in either the nuclear or cytoplasmic compartments has functional implications (Fox et al., 2009). In one model of tamoxifen resistance, developed by long term treatment of MCF7 cells with tamoxifen, increased ER_localization to the cytoplasm/membrane was observed that correlated with increased cytoplasmic signalling (Fan et al., 2007a). It would thus be of interest to investigate ERα localisation in patients who have developed resistance to tamoxifène. A novel perspective may be to influence ER localization, restoring or re-inforcing its concentration in different cellular compartment. A lot of wrok is still require to assess feasibility of such an approach. In ER signalling three actors seem to be important: the ligand, the hormone receptors and its co-regulators. In in vitro models the role of co-activator/ co-repressor ratio in cell specific 124 response is well established and also in certain tamoxifen resistance model a high expression of co-activator as AIB1 is demonstrated; now we will continue our research not only in fundamental domain but also in clinical domain with the aim of better understanding the mechanisms of resistance to anti-estrogens. At the fundamental level we want to continue our ongoing work to determine the expression level of different class of HDAC, their intracellular localisation, in different cell types as ER/ER+ and resistance to antiestrogens and if their inhibition result in overcoming to resistance to antiestrogens (either in de novo resistance or acquired resistance). 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Environ 70% des tumeurs mammaires expriment le récepteur des œstrogènes alpha (REα) et sont considérées comme hormono-sensibles. La thérapie hormonale a longtemps été le traitement de choix. Toutefois, les effets agonistes sur le REα et le développement de la résistance des thérapies actuels, tels que le tamoxifène nécessitent le développement de nouveaux traitements qui agissent par des mécanismes distincts. L'objectif de notre étude était de concevoir des outils qui peuvent aider à comprendre les mécanismes moléculaires impliqués dans la modulation liganddépendante ou dans la dégradation du REα. Nous avons choisi quelques anti-œstrogènes avec différentes structures et nous avons comparé leurs effets sur: 1) la dégradation du REα. 2). La localisation intra-cellulaire du REα. 3). La régulation de la transcription des gènes cibles du REα 4). La régulation de la transcription dans les mutants de REα. Grace à notre appproche mécanistique, nous avons pu classer ces anti-estrogènes en trois groupes sur la base de leur fonction : SERM, SERD et un nouveau groupe (EM-652). Les SERM (selective estrogen receptor modulator) stabilisent le REα, induisent une re-localisation du REα dans le noyau, augmentent l'activité transcriptionnelle de mutants qui affectent le recrutement de cofacteurs et qui altèrent la liaison avec la chaîne latérale de l'anti-œstrogène et, enfin, manquent de pouvoir d'inhibition de l'expression basale de gènes endogènes. Les SERD (selective estrogen receptor disruptor) induisent une dégradation protéasome dépendante du REα dans le noyau en formation des foyers nucléaires qui colocalisent avec le proteasome, et inhibent l'expression basale du gène endogène du récepteur de la progestérone (PGR). Enfin, EM652 est un composé qui affecte le devenir du REα comme les SERM mais qui a un effet inhibiteur sur l’expression basale du gene endogene PGR. Cette approche peut être utilisée pour cribler de nouveaux anti-œstrogènes. Abstract Breast cancer is the most common type of malignancy among women in the world. Approximately 70% of breast tumours express the estrogen receptor alpha (ERα) and are considered hormone-responsive. Endocrine therapies have long been the treatment of choice. However, the estrogen- like agonist effect and development of resistance of the available selective estrogen receptor modulator such as tamoxifen require developing new treatments that act through different mechanisms. The objective of our study is to design tools that can help to understand the molecular mechanisms involved in ligand-dependent modulation or degradation of ERα. We selected a set of anti-estrogens with different structures and compared their effect on: 1). ERα degradation. 2). Intra-cellular localisation of ERα. 3). Regulation of transcription of ERα- endogenous target genes. 4). Regulation of transcription in the mutants of ERα. Using this mechanistic study we could classify the tested anti-estrogens into three groups based on their function: SERM, SERD and a new group for EM-652. SERM (selective estrogen receptor modulator) include compounds such as OH-tamoxifen and RU39411, that stabilise ERα, that relocalize ERα into the nucleus upon binding, that increase transcriptional activity in mutants affecting the recruitment of cofactors or the binding of their side chain and that lack inhibitory capacities of the basal expression of endogenous genes. SERD (selective estrogen receptor modulator) include compounds such as ICI182580 or RU58668, that induce nuclear proteasome-dependent degradation ERα which occur in large nuclear foci that colocalize with the proteasome and that inhibit basal gene expression of the endogenous progesterone receptor gene (PGR). Finally, EM-652 was found to affect ERα degradation and localisation similarly to SERM but inhibited basal gene expression of the endogenous PGR. This approach can be used to screen the newly designed compounds based on specific anti-estrogen structural features MOTS-CLEFS : Cancer du sein, anti-estrogènes thérapie, Résistance DICIPLINE : Gène Cellule Développement ADRESSE DU LABORATOIRE: Institut d'Exploration Fonctionnelle des Génomes (IFEG). Laboratoire de Biologie Moléculaire Eucaryote (LBME) CNRS UMR 5099, IBCG, 118 Route de narbonne 31062, Toulouse)