T H È S

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TH HÈ ÈS SE E
En vue de l'obtention du
D
DO OC CT TO OR RA AT T D DE E L L’ ’U UN NI IV VE ER RS SI IT TÉ É D DE E T TO OU UL LO OU US SE E
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

1
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