UNIVERSITE LIBRE DE BRUXELLES FACULTE DE MEDECINE

FACULTE DE MEDECINE

Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM)

Promoteur de Thèse : Dr. C. Maenhaut Co-Promoteur de Thèse : Pr. J.E Dumont

Thèse présentée en vue de l’obtention du grade académique de Docteur en Sciences Biomédicales

Laurent Delys Novembre 2007

Gene expression profiles of Papillary and Anaplastic

Thyroid Carcinomas

Gene expression profiles of Papillary and Anaplastic Thyroid Carcinomas

Promoteur de Thèse : Dr. C. Maenhaut Co-Promoteur de Thèse : Pr. J.E Dumont

Membres du jury :

Pr. Magali Waelbroeck (Présidente) Pr. Laurence Leenhardt

Pr. Marie-Christine Many Pr. Daniel Glinoer

Dr. Pierre Heimann

Dr. Christos Sotiriou

Voilà maintenant 5 ans que je suis arrivé à l’IRIBHM et le temps est passé à une vitesse vertigineuse. Je me vois encore arrivé dans le bureau de Carine pour qu’elle me propose un sujet de thèse, ou encore le premier jour effectif de ma thèse où j’étais stressé et me demandait si j’en étais capable. Que de chemin parcouru depuis lors ! J’ai vraiment l’impression d’avoir énormément évolué intellectuellement. Cette gymnastique de l’esprit, je la dois à l’IRI avec ses personnes enrichissantes et ses différents séminaires, où il est vrai, on se force parfois pour y aller. Tout ceci m’a rendu plus fort et plus mûr.

Je pars maintenant pour d’autres horizons, mais il est certain que cette période de 5 ans passée avec vous restera comme l’une des toutes bonnes périodes de ma vie. Merci.

Et si je suis arrivé à l’IRI, c’est tout d’abord grâce à l’accueil des professeurs G.Vassart et J.E Dumont dans leur institut. Mr Vassart, vous avez déclaré un jour que votre porte était toujours ouverte aux doctorants en cas de problème. J’ai pu le constater à quelques reprises et j’ai apprécié nos franches discussions. Mr Dumont, comment vous remerciez en quelques mots alors que vous m’avez tant apporté ? Cette thèse n’aurait jamais pu être réalisée sans votre aide. D’autre part, j’espère que vos connaissances

« gigantissimes » auront un peu déteint sur moi. Enfin, je ne suis pas prêt d’oublier votre bonne humeur et votre rire légendaire.

Carine, j’ai réellement apprécié ta disponibilité tout au long de cette thèse et ta manière de diriger tes doctorants. Tu nous soutiens toujours dans nos initiatives et tu nous fais confiance. Tu es également prête à te remettre en question et j’ai apprécié nos discussions sincères. J’espère que mes futurs patrons seront tous comme toi ! Mille mercis.

La vie à l’IRI n’aurait jamais été aussi agréable sans les nombreuses personnes se réunissant au C4.124 à l’heure du midi. Entre les discussions culinaires et d’actualités, et nos fous rires, nous discutions de tout à l’exception, à mon grand regret, de sport. Il faut dire que nous, les mâles, étions en minorité. Quitte à paraître goujat, je vais donc commencer pour nous remercier, nous les « gibiers », pour avoir supporté cette horde de filles dont leurs discussions et leurs réactions m’échappaient parfois. Fabrice, tu es le premier doctorant que j’ai rencontré en arrivant à l’IRI. Le contact est tout de suite passé et tu étais parfois le seul de mon avis contre toutes ces filles. Merci pour ce soutien durant les premières années de ma thèse ! Nicolas, la force tranquille du groupe.

Tu es doté d’une intelligence rare et tu es toujours prêt à aider et à rendre des petits

services. Je te souhaite vraiment de réaliser une belle thèse et beaucoup de courage, … à

à mieux comprendre cette alchimie qui fait que nous sommes si différents, et si complémentaires à la fois. Alors parce que j’ai appris durant ces 5 années que les

« truites » sont souvent jalouses entre elles (vous ne saviez pas que je vous appelais comme cela ?), je vais donc vous remerciez par ordre alphabétique pour éviter toute embrouille. Aline, grâce à toi, j’ai la preuve qu’on peut être plus hyperkinétique que moi ! Tu ne tiens pas en place, c’est incroyable ! De plus, avec toi, pas besoin de montre : à 12h précise, tu rentrais dans le bureau avec un air mi interrogateur mi pitié et une soupe en main et tu posais la question que tout le monde attendait : on mange ? Christine, que la fin de ta thèse est déjà loin ! Mes souvenirs sont assez confus mais je garderai de toi l’image d’une personne épanouie. Delphine, ton sourire, ta bonne humeur et tes pas dansants hantent encore les murs du C4.124 ! D’autre part, je pense que tu es responsable de l’apparition de mes premiers cheveux blancs, mais bon, je te pardonne (lol)… Geneviève, la dernière venue. Tu as, à la fin de ma thèse, amener une petite touche supplémentaire de bonne humeur. Julie, tu es responsable de la plus grande traîtrise que j’ai eu à subir depuis que je suis né (loup-garou). Je te retiens et garde secrètement ma revanche… Sinon, je pense que tu es la personne qui me cerne le mieux à l’IRI. Tu vois tout de suite quand je ne suis pas au top et tu as toujours été là pour me remonter le moral. Merci et donne toi à fond pour la fin de ta thèse. Nathalie, tu es la première à m’avoir fait découvrir les voyages pas chers (UCPA) et à me montrer Reference Manager. Merci. Sandra, je pense que tu possèdes le sourire le plus large de l’institut, ce qui fait de toi une personne unique avec qui il est agréable de discuter.

Sandrine, tu seras toujours pour moi quelqu’un à deux faces. La première, celle du matin, qui est calme et réservée, et la deuxième, celle du soir, qui jouit d’une explosivité et d’une joie de vivre à couper le souffle ! Sara, je t’ai connu très timide au début et puis, petit à petit, tu as pris tes marques pour te faire une place dans le groupe. J’ai appris à te connaître et ai toujours apprécié ton extrême gentillesse. Sheela, la réservée du groupe.

Malgré cela, tu sais exactement ce que tu veux et c’est une qualité que j’apprécie énormément. Nos discussions sur les voyages me manqueront. Wilma, les journées de

« franglais » ont été très enrichissantes et je t’en remercie.

Vincent et David, vos connaissances en bioinformatique, totalement obscures pour moi, auront été cruciales pour la réalisation de ma thèse. Merci à vous. Vincent, j’admire l’enthousiasme avec lequel tu mènes ta double vie, séparée entre les sciences et le cinéma.

Je tiens également à remercier Stéphane S., qui nous rappelait les notions de physiques

élémentaires par des expériences amusantes tout en ayant une banane en bouche,

Isabelle, qui m’a suggéré l’une ou l’autre expérience intéressante et Sarah, avec qui je

pouvais toujours discuter de n’importe quel sujet. Chantal, je suis impressionné par ta

rapidité à réaliser des manips et ton envie de toujours rendre service. Mille mercis pour

ton aide technique.

discutais de sport ! Tu m’as préparé aux 20 km et m’as permis d’atteindre mes objectifs.

Mais bon, je ne suis pas aussi fou que toi car jamais je ne ferai un iron man ! Hakim, un grand merci pour m’avoir initié aux expériences qRT-PCR malgré ton planning surchargé. Frédérick, tu es quelqu’un que j’apprécie énormément, d’une part parce que tu me rappelles mon chanteur préféré, et d’autre part parce que malgré les nombreux problèmes techniques que tu as rencontré, tu ne renonces jamais. Tu es un modèle pour moi ! Anne, je ne me souviens pas t’avoir vu un seul jour de mauvaise humeur. Tu agrémentais mes passages au 6

et je t’en remercie.

Merci aussi à Tanja, Séverine, Barbara, Thalie, Maria, Françoise, Agnès, Daniel, Vanessa C., Audrey, Colette, Xavier P., Milutin, Jing, Song, Vanessa V., Xavier D., Jingwei, Bruno et tous les autres qui ont fait de l’IRI un espace où il fait bon vivre et travailler.

Gerry, I would like to thank you for having given me the opportunity to spend some time in your lab in Swansea. I have experienced the Welsh culture and have improved my English. Many thanks to Sarah and Steve for your kindness and the time you spent showing me around your country.

J’ai également une pensée pour les personnes qui permettent à l’IRI de fonctionner correctement. Merci à Claude, Danielle, Joelle, Cathy, Christian, Diana, Johan, Joseph et Yves pour avoir apporter leur soutien logistique, informatique ou autre.

Je remercie mes parents qui m’ont toujours soutenu et cru en moi tout au long de mes études et mes grands-parents qui ont permis la réalisation de mes études universitaires.

Ricarda, ta présence à mes côtés depuis maintenant 10 ans m’a permis de m’améliorer et

de me remettre en question quand cela était nécessaire. Tu m’as toujours soutenu et ton

amour me comble chaque jour de joie.

Résumé

Les tumeurs thyroïdiennes constituent les tumeurs endocrines les plus fréquentes. Parmi celles-ci, on distingue les adénomes, tumeurs bénignes et encapsulées, et les carcinomes, tumeurs malignes.

Ceux-ci sont eux-mêmes subdivisés, principalement sur base histologique, en carcinomes papillaires ou folliculaires, qui conservent certaines caractéristiques de différenciation des cellules thyroïdiennes initiales dont ils dérivent, et qui peuvent évoluer en carcinomes anaplasiques, totalement dédifférenciés. Les carcinomes différenciés de la thyroïde sont généralement de bon pronostic, contrairement aux cancers anaplasiques qui sont nettement plus agressifs, avec un taux de survie à 5 ans inférieur à 5%.

La technologie des microarrays permet d’analyser simultanément l’expression de milliers de gènes dans différentes cellules et différentes conditions physiologiques, pathologiques ou toxicologiques. Au cours de cette thèse de doctorat, nous avons déterminé le profil d’expression génique des carcinomes papillaires de la thyroïde à l’aide de la technique des microarrays en utilisant une plateforme contenant plus de 8000 gènes. Douze des 26 cancers papillaires étudiés étaient issus de patients habitant la région de Tchernobyl lors de l’explosion de la centrale nucléaire de 1986 et sont considérés comme des cancers radio-induits. Les 14 tumeurs restantes proviennent de patients habitant la France. Leur étiologie n’étant pas connue, ils sont considérés comme des cancers sporadiques.

La réalisation de ces expériences nous a permis d’identifier des signatures moléculaires entre des sous-types de cancers papillaires. Premièrement, nous avons montré que malgré un profil d’expression génique global similaire entre les cancers papillaires sporadiques et radio-induits, une signature multigénique permet de les séparer, indiquant que des subtiles différences existent entre les deux types de tumeurs. Deux autres signatures indépendantes, l’une liée aux agents étiologiques présumés de ces tumeurs (radiation vs. H

O

), l’autre liée aux mécanismes de recombinaison homologue de l’ADN, permettent également de séparer les cancers post- Tchernobyl des cancers sporadiques. Nous avons interprété ces résultats comme une différence de susceptibilité à l’irradiation entre ces deux types de tumeurs. D’autre part, nous avons pu identifier une liste de gènes permettant de séparer les cancers papillaires à variante classique des autres sous-types de cancers papillaires. L’analyse de cette liste de gènes a permis de mettre en relation cette signature avec l’important remodelage de cette variante histologique par rapport aux autres.

Ces expériences ont aussi abouti à l’obtention d’une liste de gènes différentiellement exprimés entre les cancers papillaires et leur tissu normal adjacent. Une analyse minutieuse de cette liste à l’aide d’outils statistiques a permis de mieux comprendre la physiopathologie de ces tumeurs et d’aboutir à différentes conclusions : (1) un changement de population cellulaire est observé, avec une surexpression de gènes liés à la réponse immune, reflétant l’infiltration lymphocytaire de ces tumeurs par rapport au tissu normal adjacent (2) la voie de signalisation JNK est activée par surexpression de ses composants (3) la voie de signalisation de l’EGF, également par une surexpression de ses composants, complémente les altérations génétiques des cancers papillaires pour l’activation constitutive de la voie ERK1/2 (4) une sousexpression des gènes de réponse précoce est observée (5) une surexpression de nombreuses protéases, d’inhibiteurs de protéases et de protéines de la matrice extracellulaire permet d’expliquer l’important remodelage des cancers papillaires (6) le profil d’expression génique des cancers papillaires peut être corrélé avec un mode de migration collectif de ces tumeurs.

Finalement, dans la dernière partie de la thèse, nous avons déterminé le profil d’expression génique des cancers anaplasiques de la thyroïde et l’avons comparé à celui des cancers papillaires.

Nous avons montré que les deux types de tumeurs présentent des profils moléculaires globaux

distincts, reflétant leur comportement tumoral très différent.

AIT apical I^- transporter APS ammonium persulfate ATC anaplastic thyroid carcinoma ATF-1 activating transcription factor-1 BrdU bromodeoxyuridine

BSA bovin serum albumin

cAMP cyclic adenosin 3’, 5’-monophosphate CRE cAMP responsive element

CREB CRE-binding protein

CREM cAMP responsive element modulator DAG 1, 2 diacylglycerol

DAVID database for annotation, visualization and integrated discovery

DIT 3,5-diiodotyrosines

DTC differentiated thyroid carcinoma DTT 1,4-dithiotreitol

ECM extracellular matrix EGF epidermal growth factor

ERK extracellular signal-regulated kinase FAP familial adenomatous polyposis FBS fœtal bovin serum

FdU fluorodeoxyuridine FGF fibroblast growth factor FISH fluorescent in situ hybridization FNAC fine-needle aspiration cytology FOXO forkhead

FTC follicular thyroid carcinoma FVPTC follicular variant of PTC GAB GRB2-associated binding protein GEF guanine nucleotide-exchange factor GO gene ontology

GPCR receptors coupled to G-proteins GPLS generalized partial least square GRB2 growth-factor-receptor-bound-2 HGF hepatocyte growth factor

IEGs immediate early genes IGF insulin growth factor IGF-1 insulin like growth factor IP3 inositol 1, 4, 5 triphosphate

IPTG isopropyl β-D-1-thiogalactopyranoside JNK c-jun N-terminal kinase

kDa kilo Dalton

LKSVM linear kernel support vector machines LNM lymph node metastases

LZ leucine zipper

MAPK mitogen-activated protein kinases MDS Multidimensional scaling

MIT 3-monoiodotyrosines MKK MAPK kinase

NIS Na

/I

symporter PA pool of adjacent tissue

PAI plasminogen activator inhibitor PAM prediction analysis of microarrays PBS Phosphate Buffered Saline

PBST PBS tween

PDGF platelet-derived growth factor PDK1 phosphatidylinositol-dependent kinase

PI3K Phosphatidylinositol-3-kinase PIP2 phosphatidylinositol-4,5-biphosphate PKB proteine kinase B

PLC phospholipase C

PTC papillary thyroid carcinoma PtdIns(3,4,5)P

phosphatidylinositol- PTEN phosphatase with tensin homology RF random forest

RT room temperature

RTK tyrosine kinase receptor

SAM Statistical Significance of Microarray SDS sodium dodecyl sulfate

SFK Src family kinase SH2 Src Homology 2

SNPs single nucleotide polymorphisms SOS son-of-sevenless

T3 triiodothyronin T4 tetraiodothyronin TBS tris buffered saline TBST TBS tween

TEMED tetra methyl ethylene diamine Tg thyroglobulin

ThOX thyroid oxydase

TIMP tissue inhibitor for metalloproteinases TPA 12-O-tetradecanoylphorbol-13-acetate tPA tissue-type plasminogen activator TPO thyroperoxidase

TRH Thyrotropin Releasing Hormone TSH thyroid stimulating hormone

TSHR thyroid stimulating hormone receptor uPA urokinase-type plasminogen activator WHO World health organization

XGAL 5-bromo-4-chloro-3-indolyl-b-D-

galactopyranoside

1

Table of contents

TABLE OF CONTENTS ... 1

CHAPTER I. INTRODUCTION ... 4

I. S

:

... 4

I.1 Receptors ... 5

I.1.1 Receptors with a tyrosine kinase activity ... 5

I.1.2 Receptors coupled to G-proteins (GPCR) ... 6

I.2 Second messengers ... 7

I.2.1 Second messengers derived from phosphatidylinositol-4,5-biphosphate... 7

I.2.2 Cyclic AMP ... 7

I.3 Downstream signal transduction pathways ... 8

I.3.1 The PI3K/Akt signaling pathway ... 8

I.3.2 Signaling cascade induced by cAMP ... 9

I.3.3 The mitogen-activated protein kinase signaling pathways... 10

I.3.3.1 The ERK1/2 signaling pathway ... 11

I.3.3.2 The JNK signaling pathway... 12

I.3.3.3 The p38 MAPKs signaling pathway ... 12

I.4 Nuclear responses: example of the transcription factors AP1 ... 13

II. T

... 14

II.1 Introduction... 14

II.2 The multistep process of tumorigenesis ... 14

II.3 Behavior of metastatic cells ... 15

II.3.1 General view ... 15

II.3.2 Processes involved in tumoral invasion ... 16

II.3.2.1 ECM remodelling ... 16

II.3.2.2 Diversity of tumor invasion mechanisms ... 17

II.3.2.3 Integrin signaling ... 18

III. T

... 20

III.1 Introduction ... 20

III.2 Thyroid hormone synthesis ... 21

III.3 Regulation of the thyroid cell... 22

III.4 Control of thyroid-specific gene expression ... 23

III.5 Control of growth and differentiation ... 23

IV. T

... 25

IV.1 Introduction ... 25

IV.2 The autonomous thyroid adenomas ... 26

IV.3 The thyroid carcinomas ... 26

IV.3.1 Differentiated Thyroid Carcinomas (DTC) ... 27

IV.3.1.1 Papillary thyroid carcinomas ... 28

IV.3.1.2 Follicular thyroid carcinoma (FTC)... 29

IV.3.2 Anaplastic thyroid carcinoma (ATC) ... 30

IV.3.3 Medullary thyroid carcinoma (MTC) ... 31

IV.4 The multi-step process of thyroid carcinogenesis... 31

IV.5 Etiology of thyroid cancers... 32

IV.6 Genetic alterations commonly found in thyroid cancers ... 33

IV.6.1 Chromosomal rearrangements ... 33

IV.6.1.1 The RET/PTC rearrangement ... 33

IV.6.1.2 Rearrangements involving TRK ... 35

IV.6.1.3 The AKAP9-BRAF fusion ... 35

IV.6.1.4 PAX8-PPARγ rearrangement... 36

IV.6.2 Point mutations ... 36

IV.6.2.1 The BRAF mutations ... 36

IV.6.2.2 RAS mutations ... 37

IV.6.2.3 p53 mutations ... 38

IV.6.2.4 β-catenin mutations ... 38

IV.6.3 Constitutive activation of the MAPK in PTCs ... 39

IV.6.4 Mutations along the PI3K/Akt signaling pathway in thyroid tumors... 40

V. T

... 41

V.1 Principle ... 41

V.2 Analysis of microarray data ... 42

V.2.1 Unsupervised methods ... 43

V.2.2 Supervised methods ... 43

CHAPTER II. AIM OF THE WORK... 45

CHAPTER III. RESULTS ... 47

I. D

RNA

.. 47

I.1 Optimization of an RNA amplification protocol ... 47

I.2 Combination of an amplification protocol with a cDNA labelling protocol .... 48

I.3 Validation of our protocol ... 49

I.4 Conclusion ... 50

II. T

DNA

... 51

III. I

PTC ... 54

III.1 Characterization of the molecular signature discriminating the classical papillary variant from the other forms of PTC ... 54

III.2 Sporadic and post-Chernobyl PTC are distinguishable on the basis of a subset of genes... 56

IV. G

... 65

V. S

PTC ... 76

V.1 Gene expression profiles revealed a high proportion of genes involved in integrin signalling cascade ... 76

V.2 Expression of focal adhesion kinase in PTC ... 77

V.3 Conclusion ... 79

3 VI. I

TPC1

PCCL3

CELLS

.. ... 80

VI.1 Principle of the experiments... 80

VI.2 Preparation of mediums ... 80

VI.2.1 Preparation of control 2H medium ... 81

VI.2.2 Preparation of 2H medium containing the potential paracrine factors ... 81

VI.2.3 Experiment allowing to obtain quiescent PCCL3 cells ready to be stimulated... 82

V.3 Cell proliferation measurements... 83

The BrdU incorporation experiments were performed 4 times. Results are shown below... 83

VI.4 Conclusion ... 84

VII. S

ATC

... 86

CHAPITRE IV. GENERAL DISCUSSION AND PERSPECTIVES ... 89

CHAPTER V. MATERIAL AND METHODS ... 94

I. M

... 94

I.1 Cell lines... 94

I.2 Culture mediums ... 94

I.3 Solutions ... 95

I.3.1 Protein extraction and quantification... 95

I.3.2 Western blotting ... 96

I.3.3 Silver nitrate staining... 98

II. M

... 99

II.1 Proteins manipulations ... 99

II.1.1 Extraction and quantification of proteins ... 99

II.1.2 Western blotting ... 100

II.1.3 Silver Nitrate staining... 100

II.2 Cell lines manipulations ... 101

II.2.1 Trypsinisation... 101

II.2.2 Bromodeoxyuridine staining and indirect immunofluorescence ... 101

II.3 Microarray analyses ... 102

II.3.1 Experiments on Agilent cDNA Microarray slides ... 102

II.3.2 Experiments on Affymetrix slides... 103

II.4 Real-time RT-PCR experiments ... 103

CHAPTER VI. BIBLIOGRAPHY ... 105

Chapter I : Introduction

4

Chapter I. Introduction

I. Signaling cascades involved in physiological and pathological conditions:

generalities

Survival of a multicellular organism depends of a wide network of intercellular communications that coordinates growth, differentiation and metabolism of the numerous cells forming tissues and organs. Incapacities to regulate these functions can lead to an altered phenotype, and eventually to cancer.

Two types of cell communication exist. Specialized junctions in the plasma membrane enable exchanges of small molecules and coordination of the metabolism between adjacent cells. However, this type of communication is only possible between closed cells.

In order to able communication between distant cells, extracellular messenger molecules are synthesized and secreted by some cells, and reach target cells where they trigger a specific response through a signaling cascade. Only cells possessing specific receptors for these molecular mediators will be able to respond to the signal.

Communication by extracellular molecules can be globally represented in two steps.

Firstly, an extracellular signal binds and activates a transmembrane receptor. Then, this receptor activates multiple signaling proteins in cascade, which finally reach the nucleus in order to induce modifications in gene expression leading to cellular responses.

Different classes of proteins are responsible for this signal transduction: receptors that receive the signal, second messengers (such as cAMP, DAG, IP3, Ca

) which amplify the signal, adaptors which distribute it, and effectors which induce the cellular responses.

This mechanism is simplified when the external signal can directly activate a

transcription factor (nuclear receptor) or when the receptor directly activates effectors

(for instance by phosphorylation). Because the cellular responses of some signaling

pathways are crucial for proliferation and differentiation, genetic alterations occurring

along these cascades in these different classes of proteins can lead to different

pathologies.

Chapter I : Introduction

5 Multiple signaling cascades have been described and their use and functions vary according to the cell types. Therefore, in this thesis, we will only describe receptors, second messengers and signaling cascades related to cancer cells, and focus on thyroid carcinogenesis.

I.1 Receptors

Most of the extracellular signals (growth factors, cytokines, hormones, …) are usually recognized by cells through transmembrane receptors. These proteins display at the extracellular side the binding site for the growth factor or hormone and in the cytoplasm the domains responsible for intracellular signaling. Other membrane receptors are ionic channels, such as the nicotinic receptor of acetylcholine. Binding of the ligand to these receptors enables an opening of the channel, triggering an ions flow throughout the cell.

Finally, some ligands are small hydrophobic molecules that diffuse throughout the plasma membrane. These include steroid hormones such as androgens and estrogens.

Once entered in the cell, these molecules bind intercellular receptors of the steroid family which act as transcription factors to modulate gene expression

.

I.1.1 Receptors with a tyrosine kinase activity

Tyrosine kinase receptors (RTKs) are receptors displaying an intrinsic catalytic activity

in the intracellular part of their sequence and act as enzyme when they are activated by

their ligand. Epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte

growth factor (HGF), platelet-derived growth factor (PDGF), insulin like growth factor

(IGF-1) and insulin belong to the family of growth factors that bind a RTK. These

receptors share a common structure: an extracellular N-terminal domain that binds the

ligand, a transmembrane α-helix and a cytosolic C-terminal domain with the tyrosine

kinase activity

. The insulin receptor and IGF-1 receptor are dimers constituted by 2

extracellular α-chains, each linked by a disulfur binding to a β-chain that crosses the

membrane and carries the tyrosine kinase activity

.

Chapter I : Introduction

6 The first step of the binding process for most of these RTKs is the dimerization of the receptor induced by the ligand

(Figure 1). For the insulin receptor, rather than a dimerization, ligand binding triggers an interaction between both cytoplasmic β-chains.

The dimerization leads to the autophosphorylation of the receptor on different tyrosine residues in the intracellular region

. These phosphorylations increase the activity of the receptor and create specific binding sites for proteins such as phospholipase Cγ (PLCγ), phosphatidylinositol-3-kinase (PIP3K) and adaptor proteins (i.e GRB2, Shc, …).

Interactions of these proteins with receptors are mediated by specific domains which specifically bind peptides containing these phosphotyrosines. The best characterized of these domains is the SH2 domain (Src Homology 2 domain) constituted of about 100 amino acids. Association of proteins containing SH2 domains and receptor leads to recruitment of other proteins and activation of downstream signaling pathways.

I.1.2 Receptors coupled to G-proteins (GPCR)

These receptors are characterized by 7 transmembrane α-helixes with an extracellular N- terminal domain and a cytosolic C-terminal domain. Binding of the ligand to the extracellular domain triggers a conformational modification which enables the cytosolic domain to bind to a G-protein associated with the internal face of the plasma membrane (Figure 2). Three subunits constitute a G-protein: α, β and γ. The α-subunit binds to the guanylic nucleotides that regulate the activity of the G-protein. When the receptor is not stimulated, the α-subunit of the G-protein binds GDP. After ligand stimulation, interaction between the cytosolic domain of the receptor and the G-protein leads to the exchange of GDP by GTP and the dissociation of the α- and β/γ subunits. The respective subunits then interact with their target proteins. These proteins are effectors such as adenylyl cyclase, Ras, PI3K, phospholipase Cβ and A2. Activity of the α-subunit ends by GTP hydrolysis and reassociation between the GDP-α subunit and the β/γ subunits.

Different isoforms of each subunit have been described, leading to a wide variety of G-

protein with different effects

.

Proteins

phosphorylation Ca

release from

endoplasmic reticulum Figure 3. The phosphatidylinositol-4,5-biphosphate cascade. Adapted from « Molecular Biology of the cell » by Alberts et al. 1994.

Figure 4. From Cully et al, 2006. PI3K can be activated by at least three different ways, all of which

start with activation of the RTKs by ligand binding. In one PI3K activation pathway, thanks to its SH2

domains, the 85 kDa regulatory subunit (p85) binds directly the cytosolic domain of the RTK, triggering

activation of the p110 catalytic subunit (left side of the diagram). Other PI3K signaling pathways

depend on the adaptor protein GRB2, which also binds RTKs. In the right pathway of the diagram,

GRB2 binds to the scaffolding protein GAB, which in turn can bind p85. Finally, GRB2 can also exist in

Chapter I : Introduction

7 I.2 Second messengers

I.2.1 Second messengers derived from phosphatidylinositol-4,5-biphosphate

Many signaling pathways use second messengers derived from the phospholipid phosphatidylinositol-4,5-biphosphate (PIP2). PIP2 is a minor component of the plasma membrane localized in the internal layer. A wide variety of hormones and growth factors stimulate hydrolysis of PIP2 by phospholipase C (PLC). This reaction produces two second messengers, diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3)

(Figure 3).

Hydrolysis of PIP2 is activated by receptors coupled to G-proteins and by RTKs. Indeed, PLCβ is stimulated by G-proteins

whereas PLCγ displays SH2 domains that enable association with activated RTKs

. This interaction with RTK leads to its phosphorylation and increases its catalytic activity (Figure 3).

DAG produced by hydrolysis of PIP2 activates the PKC family of serine/threonine kinases, that plays a major role in the control of growth and differentiation by phosphorylation of different proteins such as MEKK, Raf-1, EGFR

. While DAG stays associated to the plasma membrane, IP3, the second messenger induced by hydrolysis of PIP2, is released in the cytosol where it induces Ca

release from intracellular stocks.

This increased rate of Ca

modulates the activity of many proteins such as kinases and phosphatases, often through another protein, calmodulin.

Finally, PIP2 is also the starting point of a signaling cascade that plays a key role in the survival of cells. In this cascade, PIP2 is phosphorylated by phosphatidylinositol-3-kinase (PI3K) leading to the formation of phosphatidylinositol-3,4,5-triphosphate ((PtdIns(3,4,5)P

) which act as a second messenger

(see §I.3.1 of introduction).

I.2.2 Cyclic AMP

According to the cell type, cyclic adenosine 3’,5’-monophosphate (cAMP) controls

proliferation, differentiation, secretion or cell adhesion

. Although in the 1970s cAMP

was considered as a negative regulator of proliferation, it is now well known that it

PTEN

GRB2

PIP2

SOS

Ras

MEK1/2

ERK

ERK Raf

PI3K

PIP3 PDK1

PKB

PKB FOXO1

FOXO4

FOXO3A

Transcription

Transcription

Cell-cycle arrest Proliferation

Nucleus

RTK

Cytosol

BAD

Apoptosis

GSK3

Chapter I : Introduction

8 produces a mitogenic effect from hormones and neurotransmettors in some epithelial cells such as dog and human thyrocytes, epithelial mammal cells and melanocytes

. On the other hand, cAMP inhibits proliferation of fibroblasts, macrophages and astrocytes

.

cAMP is synthesized from ATP through the action of adenylyl cyclase and can be degraded by phosphodiesterases

(Figure 2). At least ten different adenylyl cyclases have been identified. All are activated by the αs subunit of the Gs-protein and most of them are inhibited by the αi subunit of the Gi-protein. Some are activated whereas others are inhibited by the βγ complex of Gs or Gi. Their activity can also be modulated by calcium and by phosphorylation through PKC and PKA

.

I.3 Downstream signal transduction pathways

I.3.1 The PI3K/Akt signaling pathway

The phosphatidyl inositol 3-kinase (PI3K)/Akt pathway is activated downstream of a variety of extracellular signals and activation of this signaling pathway impacts a number of cellular processes including cell growth, proliferation and survival. The alteration of components of this pathway, through either activation of oncogenes or inactivation of tumor suppressors, disrupts a signaling equilibrium and can thus lead to tumorigenesis

. Three classes of phosphatidylinositol-3-kinase (PI3K) have been described, based on the homology of their sequence, their substrate preference and their regulation. Class I is the best characterized and includes enzymes phosphorylating PIP2. It is also the only class involved in carcinogenesis. Members of this class are heterodimers composed by a catalytic subunit of 110 kDa (p110) and a regulatory subunit of 85 kDa (p85).

The phosphatidylinositol-3-kinase (PI3K) signaling pathway starts with PI3K activation

by RTKs (Figure 4). PI3K activity phosphorylates and converts the second messenger

PIP2 into PIP3, which recruits and activates phosphatidylinositol-dependent kinase 1

(PDK1) (Figure 5). PDK1 in turn phosphorylates and activates protein kinase B (PKB,

also known as AKT), which phosphorylates different substrates playing crucial roles in

Figure 6. The cAMP signaling cascade. Adapted from « Molecular Cell Biology » from Baltimore et al. 1996.

nucleus

GPCR

Adenyly l

cyclase cAMP

Chapter I : Introduction

9 cell-cycle regulation and survival. This includes inhibition of the forkhead (FOXO) transcription factors which are mediators of apoptosis and cell-cycle arrest, resulting in cell proliferation and survival

. Another PKB target is BAD (pro-apoptotic protein BCL2-antagonist of cell death) which binds 14-3-3 proteins after phosphorylation, sequestering it in the cytoplasm and preventing its pro-apoptotic effects

. PKB might also indirectly stabilize the cell-cycle proteins c-Myc and cyclin D1 through the inhibition of GSK3, leading to proliferation

. The tumor-suppressor phosphatase with tensin homology (PTEN) negatively regulates PI3K signaling by dephosphorylating PIP3, converting it back to PIP2 (Figure 5).

I.3.2 Signaling cascade induced by cAMP

Regulation of gene expression by cAMP plays a crucial role in the control of proliferation, survival and differentiation in a large variety of cells.

cAMP can directly regulate CNG ionic channels (Cyclic Nucleotide-Gated) found in olfactory neurons in brain and in some non neuronal tissues

. It also activates EPAC which itself activates Rap1. Nevertheless, the majority of cAMP effects are transmitted by PKA (cAMP-dependent protein kinase). The inactive form of PKA is a tetramer constituted by 2 catalytic and 2 regulatory subunits, respectively called C and R subunits.

cAMP binds to R subunits, triggering a conformational modification leading to the

dissociation of the C subunits (Figure 6). The active free C subunits subsequently

phosphorylate serine and threonine residues of their target proteins. Increasing cAMP

levels activate the transcription of target genes that contain a specific regulatory sequence

called CRE (cAMP Responsive Element). In this case, the signal is transmitted from the

cytoplasm to the nucleus by the C subunit of PKA which can enter into the nucleus. C

subunits can then phosphorylate the transcription factors of the CREB family, such as

CREB (CRE-binding protein), CREM (cAMP responsive element Modulator) and ATF-1

(Activating transcription factor-1), leading to the activation or the repression of cAMP

inducible-genes

(Figure 6). Phosphorylation of CREB on serine 133 by PKA is

required for its interaction with CBP/p300 (CREB-binding protein), a coactivator that

interacts with many transcription factors and carrying a histone acetyl transferase

JNK1 B Raf

MEK1

ERK1 p38α

MAP3K

MAP2K

MAPK

A Raf C Raf

MEK2

ERK2 JNK2

JNK3 MEK4 MEK7

MEKK1 MEKK2 MEKK3

MEKK4 ASK1

ASK2

MEK3 MEK6

TAO2

TAO1 TAO3

MLK1

MLK2 MLK3 DLK LZK

p38β p38γ p38γ TAK1 Tpl2 Mos

RTK

Ras

Cytoskeleton

Integrin GPCR

Growth factors Drugs

ECM

Antigens

Cold O2-

Tox ins

Cellular response

Cytoplasm

Nucleus

Chapter I : Introduction

10 activity

. Different isoforms of members of the CREB family have been described and are mainly produced by alternative splicing. Some of them act as activators (CREMτ, CREB, ATF-1) and others as repressors (CREMα, β γ and CREB-2).

I.3.3 The mitogen-activated protein kinase signaling pathways

The mitogen-activated protein kinases (MAPKs) are generally expressed in all cell types, yet they function to regulate specific responses that differ from cell type to cell type.

These cascades are intensely studied, especially the extracellular signal-regulated kinases (ERK) 1/2, the c-jun N-terminal kinase (JNK) 1, 2, 3 and the p38 kinase (p38α, β, γ and δ). Reasons for such intensive studies are explained by involvement of MAPKs in the cellular responses to almost all stimuli. In general, the ERK subfamily is mainly activated by growth factors, p38 by stress factors and JNK are activated by stress- and growth- factors

. MAPK family is conserved in evolution and is involved in diverse cellular processes including proliferation, apoptosis, survival, migration and development.

The MAPK signaling cascades are now well-known. The aim of the current intensive

researches is now to understand how MAPKs, which can be activated by a plethora of

stimuli, can have highly specific biological functions. The answer is in part related to the

spatio-temporal regulation of MAPKs within cells. MAPKs transmit signals by sequential

phosphorylation events. The phospho-relay system is composed of three kinase modules

(Figure 7): MAPKs are phosphorylated and activated by MAPK kinases (MKKs or

MAP2Ks); MAPK kinase kinases (MKKKs or MAP3Ks) phosphorylate and activate

MKKs

. Note that additional kinases may also be required upstream of this three-kinase

module. Whereas there are at least 11 MAPKs, there are only 7 MKKs, but at least 20

MKKKs. The different regulatory domains and motifs encoded in the different MKKKs

selectively control localization, activation and inactivation of associated MKKs and

MAPKs. In addition, scaffold proteins such as kinase suppressor of Ras, β-arrestin and

the JNK-interacting proteins organize MAPK modules in complexes with other proteins,

control trafficking and subcellular location and duration of MAPK signaling

. Thus, the

role of MKKKs in regulation of specific MAPKs and the organization of signaling

complexes by scaffolding proteins are two key elements providing a combinatorial diversity for the integration of cellular networks in the cellular response to stimuli.

Given the role of MAPKs as important mediators of cellular responses to so many extracellular signals, it is not surprising that loss of fine control of MAPK regulation resulting from mutations (such as activating Ras or Raf mutations), or changes in expression of proteins regulating MAPK signaling (such as EGF receptor overexpression), contribute to cancer.

I.3.3.1 The ERK1/2 signaling pathway

ERK1 and ERK2, sharing 83% identity, are ubiquitously expressed and are involved in many cellular responses such as cell motility, proliferation, differentiation and survival

. They are activated to varying extents by growth factors, serum, phorbol esters, cytokines, osmotic and other cell stresses

. Nevertheless, the most well defined signaling pathway from the cell membrane to ERK1/2 is that used by RTKs. Phosphorylation of these receptors results in the formation of multiprotein complexes whose organization dictates further downstream signaling events. A major function is the activation of the monomeric G protein Ras, achieved by the recruitment of adaptator proteins such as Shc and Grb2 (Figure 1). SOS then becomes engaged with the complex and induces Ras to exchange GDP for GTP. GTP-liganded Ras is able to interact with a number of effectors, including Raf isoforms. Ras binding to Raf results in conformational changes in Raf that increase its kinase activity. The increase in Raf activity leads to the phosphorylation of MEK1 and 2, the two MKKs that specifically phosphorylate and activate ERK1/2 (Figure 7).

Activated ERK1/2 may phosphorylate proteins involved in cell attachment and migration

such as paxillin and focal adhesion kinase (FAK). ERK1/2 can also enter in the nucleus

and phosphorylate transcription factors such as Elk1, c-fos and c-myc

.

Chapter I : Introduction

12 I.3.3.2 The JNK signaling pathway

The JNK are encoded by three genes: JNK1/SAPKβ, JNK2/SAPKα, JNK3/SAPKγ. The proteins share more than 85% of identity and more than 10 spliced forms have been described. JNK1 and JNK2 are expressed ubiquitously. In contrast, JNK3 has a more limited pattern of expression and is restricted to brain

.

The c-jun N-terminal kinase (JNK) pathway is activated primarily by cytokines and exposure to environmental stress. Phosphorylation of transcription factors, such as c-jun, JunB, JunD and ATF2 by JNK causes increased transcriptional activity. In each case, the sites of phosphorylation correspond to motifs located in the activation domain of the transcription factor. Activation of these transcription factors regulates the expression of specific sets of genes that mediate cell proliferation, differentiation or apoptosis. JNK proteins are involved in cytokine production, inflammatory response, stress-induced apoptosis, actin reorganization and metabolism

.

The JNK are activated by phosphorylation by 2 MKKs, MEK4 (MKK4) and MEK7 (MKK7). In certain conditions, these two proteins may also activate the p38 pathway.

These two MKKs are activated by a large group of MKKKs, such as the MEKK group (1 to 4), the ASK group (ASK1 and ASK2, also known as MAP3K5 and MAP3K6, respectively) and the mixed-lineage protein kinase group (MLK1-3, DLK and LZK) (Figure 7). Different ways can lead to activation of MKKKs: Rho proteins may mediate the activation of JNK caused by RTKs while activation of JNK by cytokine receptors appears to be mediated by the TRAF group of adaptator proteins. It is also shown that the adaptator protein Nck and the Ste20-like protein kinase NIK may mediate JNK activation by Eph receptors

.

I.3.3.3 The p38 MAPKs signaling pathway

The p38 family includes four members (α, β, γ, δ) and responds to a wide range of

extracellular stimuli, particularly cellular stresses, such as UV radiation, osmotic shock,

hypoxia, pro-inflammatory cytokines and less often growth factors.

The MKKs MEK3 and MEK6 may both be required for maximal activation of p38.

MEK3 and MEK6 are activated by numerous MKKKs, including MEKK1-4, TAO group (1 to 3) and TAK1. Activation of the transcription factors by the p38 family mediates cell proliferation, differentiation, development and response to stress (Figure 7).

I.4 Nuclear responses: example of the transcription factors AP1

Signaling cascades usually lead to activation of transcription factors inside the nucleus.

For instance, quiescent cells exposed to serum or growth factors lead to a fast and usually temporary transcription of genes called “immediate early genes” (IEGS)

. Among IEGs, a very well known family is the family of the nuclear proto oncogenes fos and jun.

The fos family includes c-fos, fosB, fra-1 and fra-2 and the jun family includes c-jun, junB and junD. Jun proteins have the possibility to homodimerize or heterodimerize with one member of the fos family to form the transcription factors AP-1

. Members of fos family cannot homodimerize. Binding between these proteins are mediated by hydrophobic interactions between their leucine zippers domain (LZ). A basic region in the complex enables the binding to the consensus DNA sequence TGACTCA called TRE (TPA responsive element)

.

AP-1 complexes are activated by many stimuli, such as mitogenic growth factors,

inflammatory cytokines, UV and radiations or other cellular stresses

. For instance,

ERK1/2 can induce phosphorylation of the Elk1/TCF transcription factor, that stimulates

the transcription of c-fos by binding to a SRE (Serum Response Element) localized in the

promoter of c-fos. This leads to an increase of the AP-1 complexes activity

. Activity

of AP-1 complexes can also be stimulated by the phosphorylation of jun by the JNK

.

AP-1 complexes bind the consensus DNA sequence TRE in the regulatory region of a

large variety of genes usually important for cellular growth, such as interleukin 2, TGFβ,

c-jun or cyclin D1

. It can also bind DNA sequences called CRE (Cyclic AMP

responsive element), normally recognized by proteins of the CREB family.

Chapter I : Introduction

14 II. The tumorigenesis process

II.1 Introduction

Cell proliferation is a very well controlled process which meets the organism needs. In a young animal, cell division and multiplication overtake apoptosis, enabling the growth of the organism. In adults, birth of new cells is compensated by apoptosis, leading to a dynamic but stationary state. A tumor, by definition, is an abnormal growth of tissue resulting from uncontrolled, progressive multiplication of cells and serving no physiological function. Tumors may be benign (not cancerous) or malignant (cancerous).

A malignant tumor may destroy adjacent tissues and may spread to distant anatomic sites through a process called metastasis. These malignant properties of cancers differentiate them from benign tumors, which are eventually self-limited in their growth and do not invade or metastasize. Nevertheless, additional genetic alterations in some benign tumors can transform them in malignant tumors.

II.2 The multistep process of tumorigenesis

Several lines of evidence indicate that tumorigenesis is a multistep process and that these steps reflect genetic alterations in proto-oncogenes or tumor-suppressor genes that drive the progressive transformation of normal cells into highly malignant derivatives

. A proto-oncogene is defined as a non-mutated cellular gene which may be the origin of an oncogene. An oncogene is a mutated gene which contributes to the initiation or progression of cancers by overexpression or constitutive activation of its corresponding protein. Examples of oncogenes are Ras, BRAF, β-catenin, erbB, fos and myc. A tumor- suppressor gene is a growth controlling gene that normally limits the normal growth of cells. When a tumor suppressor gene is mutated and inactivated, it fails to keep cells from proliferating. Examples of tumor-suppressor genes are p53, Rb, PTEN and p16INK4a.

Gain of function mutations in oncogenes, and loss of function mutations in tumor-

Chapter I : Introduction

15 suppressor genes only when both alleles are mutated, disrupt the regulatory circuits that control cell fate, conferring on neoplastic cells the ability to survive and proliferate, even if appropriate extracellular signals are not available

.

It is proposed that the large majority of cancer genotypes, if not all, are a manifestation of six essential alterations in cell physiology that collectively drive to malignant growth:

self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Each alteration enables cancer cells to acquire novel capabilities and favors the development and progression of the tumor

.

II.3 Behavior of metastatic cells

II.3.1 General view

Cancer cells spread throughout the body by metastasis. To have emergence of cells with

metastatic capability, both genetic and epigenetic changes have to appear in the primary

tumor. Recent findings indicate that metastatic subclones probably arise from primary

tumors that have already progressed to the invasive stage

. Several sequential and

obligatory steps have to occur in order to have metastasis formation (Figure 8). First,

cancer cells need to detach from their neightbouring cells, degrade the basement

membrane and penetrate into the interstitial stroma. Secondly, tumor cells penetrate into

blood and lymphatic vessels in a process known as intravasation. To enter into vessels,

cancer cells must traverse the endothelial basement membrane and disrupt their cell-cell

adhesion. After reaching the bloodstream, either directly or through the lymphatic system,

tumor cells often adhere to platelets and leukocytes, facilitating their circulation until the

target organs compared to isolated tumor cells

. Finally, metastatic cells exit the

bloodstream by a process known as extravasation, and start to grow in the parenchyma of

the target organ. Expansion of cancer cells in the new organ requires similar needs than in

the primary organ, including a supportive stroma and an adequate blood supply

.

II.3.2 Processes involved in tumoral invasion

Multiple acquired capabilities contribute to the invasive properties of metastatic cells.

First is the ability to move through tissues. To break away from their primary tissue, metastatic cells have to loose their proteins enabling adhesion with the adjacent nonmetastatic cells and the basement membrane, acquire a migratory phenotype, and degrade or remodel the ECMs that impose barriers to their dissemination. Secondly, metastatic cells have to induce angiogenesis in order to provide oxygen and nutrients required for their tumor growth. Indeed, cancer cells cannot grow beyond a relatively limited size unless they elicit an angiogenesis response

. Thirdly, metastatic cells have to survive in foreign microenvironnements before they colonize their target organ, and they have to survive and proliferate within the stroma of the target organ. In the next paragraphs, we discuss some aspects of the carcinogenesis process involved in progression and invasion of cancer cells.

II.3.2.1 ECM remodelling

The extracellular matrix (ECM) is a complex architecture composed of collagens, fibrillar glycoproteins and proteoglycans that play a major role in the tissue architecture and the cellular adhesion. Components of the ECM provide a large variety of specific signals that directly influence cell proliferation, migration and cell survival, mainly by their interactions with integrins (see below). Alterations of the ECM might therefore lead to cancer. It is suggested that perturbation of the tissue microenvironment may be sufficient to induce tumor formation. Moreover, tumor cell invasion and metastasis also require destruction of the ECM during local invasion, angiogenesis, intravasation and extravasation

.

These processes are mediated by multiple degradative actions of proteolytic enzymes.

These complex events need cooperation of different proteases, including aspartyl and

cysteine enzymes (mainly cathepsins) involving in intracellular proteolysis within

lysosomes, serine enzymes (the urokinase-type plasminogen activator, uPA and the

tissue-type plasminogen activator, tPA) and metal-dependent enzymes

(metalloproteinases, MMPs). Both last ones are responsible for extracellular proteolysis.

Figure 9. Diversity of tumor invasion mechanisms. From Friedl and Wolf, 2003. Individual or

collective tumor-cell migration strategies are determined by different molecular programmes

(triangles). From individual (top) to collective (bottom) movements, increased control of cell-ECM

interactions is provided by integrins and matrix-degrading proteases. Cell-cell adhesion through

cadherins and other adhesion receptors as well as cell-cell communication via gap junctions, are

specific characteristics of collective cell behaviour. Detached and disseminating cell collectives

(cluster or cohorts) are observed in epithelial cancers that retain high or intermediate levels of

differentiation, such as breast and colon cancer. Multicellular strands and sheets that do not detach are

invasive, yet rarely metastatic. These occur in some epithelial cancers, including basal-cell

carcinomas and benign vascular tumors.

These enzymes can act directly by degrading ECM or indirectly by activating other proteases, which in turn degrade the ECM

. ECM remodeling is also mediated by inhibitors of these proteases, such as cystatins for cathepsins, plasminogen activator inhibitor 1 and 2 (PAI1 and PAI2) for serine proteases and the TIMP family members (tissue inhibitor for metalloproteinases) for MMPs

.

II.3.2.2 Diversity of tumor invasion mechanisms

To spread within tissues, tumor cells use migration mechanisms that are similar, if not identical, to those occurring in normal, non-neoplastic cells during physiological processes such as embryonic morphogenesis

. To migrate, the cell body must modify its shape and stiffness to interact with the surrounding tissue structures. Hereby, the ECM provides the substrate, as well as a barrier towards the advancing cell body. In vitro and in vivo observations have shown that tumor cells infiltrate neighbouring tissue matrices by different ways. They can disseminate as individual cells (amoeboid and mesenchymal migration), referred to as “individual cell migration”, or expand in solid cell strands, sheets or clusters, called “collective migration” (Figure 9). Whereas leukaemias, lymphomas and most solid stromal tumors, such as sarcomas, disseminate via single cells, epithelial tumors commonly use collective migration mechanisms. In principle, the lower de differentiation stage, the more likely the tumor is to disperse via individual cells

. The central molecules that govern and specify such diverse migration processes are: the matrix-binding adhesion receptors, most notably those belonging to the integrin family;

matrix-degrading proteases of the MMP family and serine protease family (uPA/uPAR);

molecules that enable cell-cell adhesion and communication (Figure 9).

During progressive dedifferentiation in epithelial cancer, the conversion from

multicellular growth and invasion to mesenchymal single cell migration is termed the

epithelial-mesenchymal transition (EMT)

. The primary step is the loss of cell-junctions

via several mechanisms. These include reduced cadherin expression, loss-of-function

mutations in cadherin and deregulated functions of proteases leading to degradation of

cadherins and other cell-cell adhesion molecules. These changes in cell morphology and

Figure 10. From Guo and Giancotti, 2004. Clustering of integrins leads to activation of FAK that recruits SH2-containing proteins such as Src-family kinases (SFKs). When recruited, SFK phosphorylates P130CAS, which recruit the complex DOCK180/Crk leading to activation of Rac.

This in turn results in the activation of p21-activated kinase (PAK), Jun amino-terminal kinase (JNK) and nuclear factor κB (NF-κB). Activation of FAK also enables the recruitment of the p85 subunit of PI3K, leading to the activation of AKT/proteine kinase B (PKB) through the synthesis of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3. Finally, there are multiple pathways that result in ERK activation through integrins and FAK. This includes activation by recruiting the RAP1 guanine nucleotide-exchange factor (GEF) C3G leading to B-RAF activation through RAP1.

Another pathway involves the growth-factor-receptor-bound-2 (GRB2) and son-of-sevenless (SOS)

complex, and transactivation of the epidermal growth factor (EGF) receptor.

functions are accompanied by changes in protein expression profiles, including the loss of cytokeratins and appearance of vimentin.

The EMT is considered to be a significant step in the invasive cascade. Once the tumor has achieved the dedifferentiated stage of single-cell dissemination, metastatic spread is increased, resulting in poor prognosis

.

II.3.2.3 Integrin signaling

Integrins are a large family of receptors that mediate the adhesive interactions of the cells.

They are heterodimerics proteins composed of α and β transmembrane subunits. Sixteen α and height β different subunits have been described, leading to at least 25 different integrins, each being specific for a unique set of ligands. Most of the integrins bind to components of the ECM (such as fibronectin and collagen). Upon binding of the integrins to the ECM components, the integrins cluster and their cytoplasmic tails provide binding sites for cytoskeletal and signaling molecules

.

FAK (Focal adhesion kinase) is a nonreceptor tyrosine kinase that plays a major role in the integrin signaling. FAK was initially found to be localized to focal adhesions, providing a structural link between the ECM and the actin cytoskeleton

. After more than 15 years of investigation, many studies have shown that integrins and FAK can regulate many aspects of cell behavior other than the cytoskeleton. Signaling enzymes and adaptor proteins regulated by integrins control cell survival, proliferation, motility and differentiation.

Most integrins recruit FAK through their β-subunits (Figure 10). Integrin clustering facilitates the autophosporylation of tyrosine 397 which increases the catalytic activity of FAK. This phosphorylation is required for the recruitment of SH2-containing proteins such as Src or p85 subunit of PI3K. When recruited to the 397Y, Src mediates phosphorylation on other sites on FAK, creating additional SH2-domain binding sites.

Protein bindings to these sites result in a cascade of protein interactions that transduce

signals to many downstream pathways, including PI3K/Akt, Crk/Dock180/Rac and

Chapter I : Introduction

19 Ras/Erk. These signaling pathways exert a stringent control on cell survival, cell proliferation and cell migration (Figure 10)

.

It has been shown that integrins and RTKs exert a joint control on survival and mitogenic pathways

. This property can be explained by the fact that even if RTKs are activated, normal cells are unable to proliferate when cultured in suspension and are referred to as

“anchorage-dependent”. Normal cells need ECM adhesion through integrins for their survival and their proliferation. In contrast, tumor cells are shown to replicate without attachment to a substratum. But despite their relative anchorage independence, cancer cells still benefit from integrin signals and because integrins connected to RTKs lead to activation of important signaling pathways for cell development and proliferation, deregulations in integrins and their downstream proteins contribute to tumor initiation and progression. Activating mutations of Src-family kinases (SFKs), Ras, various guanine nucleotide-exchange factors (GEFs), AKT/PKB, B-RAF, NF-κB and c-jun, and loss-of-functions mutations of PTEN have been identified in primary tumors

. Moreover, a large number of reports show an enhanced expression of FAK mRNA and/or protein in a variety of human cancers, including invasive colon and breast cancers, metastatic prostate carcinoma and malignant melanoma

. Neoplastic cells also tend to lose integrins that secure their adhesion to the basement membrane and help them to remain in a quiescent and differentiated state. However, they maintain or overexpress integrins that foster their survival, migration and proliferation during tumor invasion and metastasis.

Unfortunately, cell-type-dependent changes in integrin signaling make it impossible to rigidly assign each of the integrins to the “anti-neoplastic” or the “pro-neoplastic”

category

. Nevertheless, dysregulated joint integrin-RTK signaling seems to play a

major role in numerous steps of tumor progression, including disruption of cell-cell

adhesion, migration of tumor cells, matrix remodeling and tumor angiogenesis

.

Chapter I : Introduction

20 III. The thyroid gland

III.1 Introduction

Thyroid is an endocrine gland with a butterfly shape as seen from the front. It is fixed by fibrous tissue to the anterior and lateral parts of the larynx and trachea (Figure 11). The weight of a thyroid from a normal nongoitrous adult is 10-20 g depending on body size and iodine supply. The major role of thyroid gland is to trap iodide in blood and synthetize the thyroid hormones, triiodothyronine (T

) and tetraiodothyronine or thyroxine (T

). T

is the active form and constitutes 10% of the production, whom 1% is an inactive isomer (rT

). Secreted in the circulatory system, thyroid hormones act on peripheral tissues in order to control different physiological functions, such as growth and the development of the central nervous system in fetus and during the first weeks of life.

They also participate to the general metabolism of the body (stimulation of proteins synthesis in all tissues, participation in lipids and carbohydrate metabolism).

The adult thyroid is composed of follicles, or acini, a spheric structure constituted by a single layer of epithelial cells, the thyrocytes (Figure 12). Follicles may be considered, from both structural and functional points of view, as the primary, or secretory, units of the organ. Thyrocytes enable to delimit a closed compartment called the follicular lumen, which contains the colloid, a sticky substance secreted by thyrocytes and mainly constituted by thyroglobulin (Tg). Tg is a glycoprotein with a high molecular weight (2 sub-units of 330 kDa each) which is synthesized by the thyrocytes and then excreted in the follicular lumen. Tg serves as a precursor for thyroid hormones synthesis

.

In addition to follicles, there are individual cells or small groups of cells that do not

extend to the follicular lumen and which may appear between follicles. These cells, or C-

cells, secrete calcitonin in response to an increase in serum calcium

. This hormone is

important in the regulation of bone resorption and lowers plasma free Ca

levels. In

adult human thyroid, they represent 1% of the cell population.

TSH

Gs

cAMPÊ

TPO TG NIS

2 Na

I

TPO

TG

TG MIT DIT

T3 T4

TG MIT DIT

T3 T4 MIT DIT

T4 T3 AA AC

NIS NIS

+

I

Basolateral

membrane Apical

membrane

Secondary lysosome T3

T4

I

O

H

O

NADPH

oxydase ThOXs

Figure 12. Synthesis and secretion of thyroid hormones. From N.Fortemaison, PhD thesis.

TSH: thyroid stimulating hormone, TSHR: TSH receptor, AC: adenylyl cyclase, NIS:

iodide Na+/I– symporter, AIT: Apical I- Transporter, TPO: thyroperoxydase, TG:

+

TSHR

Endosome Primary

lysosome