TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par l'Université Toulouse III - Paul Sabatier

Discipline ou spécialité : Gènes Cellules & Développement

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

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

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 anti-

estrogen structural features

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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

2

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

3

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

4

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: Progesterone Receptor

SP1: 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

5

INTRODUCTION

6

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.

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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.

8

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 E fat F pectoralis major muscle G chest wall/rib cage Botton: A normal duct cells B basement membrane C lumen (center of duct)

9

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,

10

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 hormone-

dependent; 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 Female Male Female Prostate Breast Lung & bronchus Lung & bronchus

186,320 (25%) 182,460 (26%) 90,810 (31%) 71,030 (26%)

Lung & bronchus Lung & bronchus Prostate Breast

114,690 (15%) 100,330 (14%) 28,660 (10%) 40,480 (15%) Colon & rectum Colon & rectum Colon & rectum Colon & rectum

77,250 (10%) 71,560 (10%) 24,260 (8%) 25,700 (9%) Urinary bladder Uterine corpus Pancreas Pancreas

51,230 (7%) 40,100 (6%) 17,500 (6%) 16,790 (6%) Non-Hodgkin lymphoma Non-Hodgkin lymphoma Liver & intrahepatic bile duct Ovary

35,450 (5%) 30,670 (4%) 12,570 (4%) 15,520 (6%) Melanoma of the skin Thyroid Leukemia Non-Hodgkin lymphoma

34,950 (5%) 28,410 (4%) 12,460 (4%) 9,370 (3%) Kidney & renal pelvis Melanoma of the skin Esophagus Leukemia

33,130 (4%) 27,530 (4%) 11,250 (4%) 9,250 (3%) Oral cavity & pharynx Ovary Urinary bladder Uterine corpus

25,310 (3%) 21,650 (3%) 9,950 (3%) 7,470 (3%) Leukemia Kidney & renal pelvis Non-Hodgkin lymphoma Liver & intrahepatic bile duct

25,180 (3%) 21,260 (3%) 9,790 (3%) 5,840 (2%) Pancreas Leukemia Kidney & renal pelvis Brain & other nervous.system

18,770 (3%) 19,090 (3%) 8,100 (3%) 5,650 (2%) All sites All sites All sites All sites 745,180 (100%) 692,000 (100%) 294,120 (100%) 271,530 (100%)

Table I: Leading Sites of New Cancer Cases and Deaths – 2008

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*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

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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 ).

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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, c-

myc 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

14

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. Over-

expression 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.

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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

16

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.

17

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.

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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

19

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

20

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.

21

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.

22

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-

23

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 with chemotherapy

(http://www.cancer.gov/search/ViewClinicalTrial).

24

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

25

(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).

26

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)

27

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.)

28

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

29

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).

30

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 (Robinson-

Rechavi 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,

31

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).

32

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

33

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

34

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 ERα-

negative. 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.

35

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.

36

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’ (Klein-

Hitpaß 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

37

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.

38

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 S-

palmitoylation 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

39

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

40

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.

41

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).

42

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).

43

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 ICI-

induced 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

44

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).

45

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).

46

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

47

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 anti-

cancer 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).

48

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).

49

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

50

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).

51

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).

52

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 three-

dimensional 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).

53

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 ER-

ligand 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)

54

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).

55

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)

56

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).

57

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:

58

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

59

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.

60

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.

61

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.

62

RESULTS

63

PUBLICATION N° 1

64

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 : kerstin@ibcg.biotoul.fr

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.

65

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.

66

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

67

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.

68

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 estrogen-

responsive 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% charcoal-

stripped 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.

69

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

70

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 Galacto-

Light 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

71

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 RNAse-

free 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.

72

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.

73

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 anti-

hormones 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.,

74

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.

75

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.

76

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

77

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 side-

chain. 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

78

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 anti-

estrogens: 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

79

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

80

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).

81

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 MDA-

MB231cells (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

82

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.

83

Figures and Legends

OHHO

OHHO

S

F

O

FF

FF

OHHO

O

S

FF

FF

F

O O

OHHO

O

N

OH

O

N

HOOH

O

O

N

17 -estradiol Faslodex RU 58,668

RU 39,411 4-hydroxytamoxifen EM-652

A

Mazaheri et al Fig 1

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.

84

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.

B

0

50

100

150

E2 ICI EM RU 39 RU 58

%of

the

cont

rol

Vehicle E2 OHT ICI EM RU39 RU58

Vehicle OHT

ERα

GAPDH

C

0

50

100

150

ALLN

ALLN

ALLN

vehicleE2 ICI

%of

the

cont

rol

Vehicle - LMB ALLN - LMB ALLN - LMB ALLN

E2 ICI RU58

RU 58

ERα

GAPDH

LMB

LMB

LMB- --

85

0

20

40

60

80

100

120

7.7

8.0 5.0 12.2

n=3

- + - + - + - +E2

WT K362A D351Y L540QER -

Fold

indu

ctio

n

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.

86

D351Y

noitcudnIdloF

0

20

40

60

80

100

120

EtOH E2 Tam ICI EM RU39 RU58

EtOH E2 OHT ICI EM RU39 RU58

EtOH E2 Tam ICI EM RU39 RU58

WT

noitcudnIdloF

0

20

40

60

80

100

120

EtOH E2 ICI EM RU39 RU58OHT

noitcudnIdloF

0

20

40

60

80

100

120

EtOH E2 Tam ICI EM RU39 RU58

EtOH E2 OHT ICI EM RU39 RU58

K362A L540QnoitcudnI

dloF

0

20

40

60

80

100

120

EtOH E2 Tam ICI EM RU39 RU58

EtOH E2 OHT ICI EM RU39 RU58

GAPDH

Vehicle E2 ICI EM RU39 RU58OHT

A B

C D

E

ERERERER

ER

ERER

ER

L540Q Clone

ER

-

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

87

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).

88

0

1

2

3

4

5

6

7

8

EtOH E2 ICI RU39 EM RU58

Treatment(16 hours)

Rela

tive

leve

lofm

RNA

TGFαPRpS2

OHT

0

0,2

0,4

0,6

0,8

1

1,2

1,4

EtOH OHT RU39 EM ICI RU58

R ela

tive

leve

l of m

R NA

Treatment (16 hours)

TGFα

PR

pS2

_____________________________________________________________________

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.

89

Data shown are the average of three independent experiments; error bars represent ±

S.E. mean.Values for untreated cells (EtOH) are set at 1.

90

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PUBLICATION N° 2

94

Ligands specify estrogen receptor alpha nuclear localization and degradation

Silvia Kocanova*, Mahta Mazaheri*, Stephanie Caze-Subra and Kerstin Bystricky#

1Université de Toulouse ; UPS ; Laboratoire de Biologie Moléculaire Eucaryote; F-

31062 Toulouse, France 2CNRS; LBME; F-31000 Toulouse, France

*equally contributed

#corresponding : kerstin@ibcg.biotoul.fr

keywords: estrogen receptor, breast cancer, nuclear localization, hormone,

antiestrogens, proteasome

95

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), 4-

hydroxytamoxifen (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

96

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 transcription-

initiation 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).

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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

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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 ice-

cold 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

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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.

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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% charcoal-

stripped fetal serum and next treated for 3h with drugs as indicated above. To block

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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 X-

100 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 TBS-

T, 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

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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

103

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 MCF-

7 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

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ERα (GFP-ERα and endogenous ERα) in untreated cells. In the presence of E2 both GFP-

ERα 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

105

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,

106

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

107

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-

108

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 SERD-

dependent 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). Pre-

treatment 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

109

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 ICI-

induced 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

110

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

111

Figures and Legends

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

A

ER

GAPDH

1h 16h4h

EtOH

E2 OHTIC

IRU39

RU58EtO

HE2 OHT

ICI

RU39RU58

EtOH

E2 OHTIC

IRU39

RU58

Re

lativ

e E

Rpr

otei

n le

vels

0

0.2

0.4

0.6

0.8

1

1.2

1.4

EtOH E2 OHT ICI RU39 RU58

1h4h

16h

112

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.

Figure 1: Kocanova, Mazaheri et al.

B

EtOH E2 OHT ICI RU39 RU58

0

0.2

0.4

0.6

0.8

1

1.2

Re

lativ

e m

RN

A e

xpre

ssio

n

ESR1

Re

lativ

e m

RN

A e

xpre

ssio

n

0

2

4

6

8

10

GREB1

EtOH E2 OHT ICI RU39 RU58

C

113

Figure 2: Nuclear accumulation and degradation of the estrogen receptor alpha

in SK19 cells

A) Western blot quantifying protein levels of endogenous ER� and GFP-ER� in

SK19. The stably transfected GFP-ER� 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.

A MCF-7 SK-19

GAPDH

GFP-ER

ER*

EtOH E2 OHT ICI EtOH E2 OHT ICI

* - unspecific band

114

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 anti-

ERα 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.

Cyt NS NI

vehicle RU39

Cyt NS NI

RU58

Cyt NS NI

-Tubulin

ER

GFP-ER

Cytokeratin 18

Lamin A

*

E2

Cyt NS NI

OHT

Cyt NS NI

ICI

Cyt NS NICyt NS NI

vehicle

-Tubulin

ER

GFP-ER

Cytokeratin 18

Lamin A

*

* - unspecific band

22% 55% 23% 19% 50% 31% 28% 33% 39% % of ER in fraction

24% 47% 29% 19% 53% 28% 23% 29% 48% % of ER in fraction 24% 53% 23%

B

115

Distance along line (µm)0 6 12 19 25 31

Fluo

resc

ence

inte

nsity

(a.u

.)64

128

192

256

Distance along line (µm)

Fluo

resc

ence

inte

nsity

(a.u

.)

0 6 12 19 25 31

64

128

192

256

vehicle

E2

ICI

RU58

OHT

RU39

DAPI GFP-ERα linescan

Fluo

resc

ence

inte

nsity

(a.u

.)

64

128

192

256

Distance along line (µm)0 6 12 19 25 31

Fluo

resc

ence

inte

nsity

(a.u

.)

64

128

192

256

Distance along line (µm)0 6 12 19 25 31

Fluo

resc

ence

inte

nsity

(a.u

.)

64

128

192

256

Distance along line (µm)0 6 12 19 25 31

Fluo

resc

ence

inte

nsity

(a.u

.)

64

128

192

256

Distance along line (µm)0 6 12 19 25 31a b c

d e

g h

j k

m n

p r

f

i

l

o

s

116

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.

117

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

A

GAPDH

ER

GFP-ER

*

* - unspecific band

E2 ICI RU58

-- LMB

ALLN -- LM

BALL

N -- LMB

ALLN

vehicle

-- LMB

ALLN

118

blotting.

vehicle

E2

ICI

ICI + ALLN

RU58

GFP-ERα 20S proteasome α2 merge

RU58 + ALLN

B a b c

d e f

g h i

j k l

m n o

p r s

119

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.

120

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122

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.

123

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.

124

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

125

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).

It would be exciting to compare results from this study to clinical trials and with patient

biopsies, and for cases developing resistance phenotype or patients with ER-negative tumours

– opening new avenues for treatment of these more aggressive and poor prognosis cases.

126

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AUTEUR : Mahta MAZAHERI TITRE : Etude des mécanismes moléculaires des traitements anti-oestrogènes et de résistance dans le cancer du sein DIRECTUR DE THESE. : Kerstin BYSTRICKY / Hélène Richard-Foy LIEU ET DATE DE SOUTENANCE : Salle de conférence de l'IEFG Résumé Le cancer du sein est le plus fréquent cancer chez les femmes dans le monde. 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 ligand-dépendante 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 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 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)