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HAL Id: tel-01968068

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Deciphering the Contribution of RhoGTPases

Dependent Signaling Pathways to the Collective

Invasion of Colorectal Carcinoma

Fotine Libanje

To cite this version:

Fotine Libanje. Deciphering the Contribution of RhoGTPases Dependent Signaling Pathways to the Collective Invasion of Colorectal Carcinoma. Cellular Biology. Université Paris Saclay (COmUE), 2017. English. �NNT : 2017SACLS503�. �tel-01968068�

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NNT : 2017SACLS503

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CBMS Cancérologie : biologie - médecine - santé

Spécialité de doctorat : Aspect moléculaire et cellulaire de la biologie

Par

Mme Fotine LIBANJE

Deciphering the contribution of Rho-GTPases dependent signaling pathways to the

collective invasion of colorectal carcinoma

Etude de la contribution des voies de signalisation dépendantes des Rho-GTPases

à l’invasion collective des carcinomes colorectaux

Thèse présentée et soutenue à Villejuif, le 8.12.17 : Composition du Jury :

Dr, Boucheix, Claude Directeur de Recherche, Institut Andre Lwoff Président du Jury Dr, Etienne-Manneville, Sandrine Directrice de Recherche, Institut Pasteur Rapportrice Dr, Wang, Xiaobo Chargé de Recherche, Centre de Biologie Rapporteur Dr, Matic Vignjevic, Danijela Directrice de Recherche, Institut Curie Examinatrice Dr, Jaulin, Fanny Chargée de Recherche, Gustave Roussy Directrice de thèse Dr, Bertoglio, Jacques Chercheur Emerite, Gustave Roussy Co-directeur de thèse

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Je souhaite adresser mes sincères remerciements aux membres de mon jury de thèse : Le Dr Claude Boucheix pour avoir accepté de présider le jury. Le Dr. Sandrine Etienne‐Manneville, le Dr. Daniela Vignjevic‐Matic et le Dr. Wang Xiaobo pour avoir accepté d’être rapporteur et examinateur de ma thèse et de prendre le temps de lire et juger mon manuscrit et mon travail de thèse. Je suis honorée de votre participation à la finalisation de mon travail de thèse. I would like to sincerely thank the members of my thesis committee : The Dr. Claude Boucheix for accepting to chair the committee. The Dr. Sandrine Etienne‐Manneville, the Dr. Vignjevic‐Matic Daniela and the Dr. Wang Xiaobo for agreeing and devoting time to the task of reviewing my thesis manuscript and my PhD work. I am grateful for your contribution to the completion of my PhD work.

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Je souhaite remercier ma directrice de thèse, le Dr. Fanny Jaulin, pour m’avoir accueillie dans son laboratoire et m’avoir donnée l’opportunité de réaliser ce travail de thèse. Cette expérience a été riche autant au point de vue scientifique qu’au point de vue personnelle. Je souhaite remercier le Dr. Jacques Bertoglio pour avoir accepté de co‐diriger mon travail de thèse. Je remercie les membres actuels du Laboratoire le Dr. Joel Raingeaud et Zajac Olivier et Charlotte Canet Jourdan, et les anciens membres qui ont contribué à la conduite de mon travail de thèse. Je remercie mes collaborateurs, dont les membres de l’équipe du Dr. Geri Kreitzer.

J’adresse ma profonde gratitude à Olivier Zajac, Nadia Elkhatib, Francesco Baschieri, Enzo Bresteau, Joel Raingeaud et Charlotte Canet‐Jourdan sans qui ce manuscrit n’aurait jamais vu le jour.

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1 Colorectal Carcinoma (CRC): cancer of the intestinal epithelium ... 1 1.1 Architecture of the intestinal epithelium ... 1 1.2 Colorecatal carcinoma ... 17 1.3 CRC dissemination: a collective route to metastasis? ... 20 2 Cancer cell invasion ... 25 2.1 Cell motility in the organism ... 25 2.2 Cancer cell invasion ... 25 2.3 Molecular and cellular mechanisms of collective invasion ... 28 2.4 Cancer cell invasion determinants ... 43 3 Small RhoGTPases signaling pathways ... 46 3.1 The RhoGTPase signaling pathway ... 46 3.2 RhoGTPases effectors ... 47 3.3 Guanine nuclotide exchange factors (GEFs) ... 60 3.4 GTPases activating proteins (GAPs) ... 69 3.5 Rho‐GDP dissociation inhibitors (RhoGDI) ... 69 3.6 RhoGTPases in cancer ... 70 PhD study aim ... 71

CHAPTER 2: RESULTS

KIF17 regulates RhoA‐dependent actin remodeling at epithelial cell‐cell adhesions ………...73 ROCK inhibition triggers the collective invasion of colorectal carcinomas through the Guanine nucleotide exchange factor FARP2……….88

Collective epithelial‐based metastatic cascade in colorectal carcinoma patients………..142

CHAPTER 3: DISCUSSION

CRC cancer cells keep their epithelial architecture to invade collectively…………193

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ROCK triggers collective invasion of glandular CRC………..196 FARP2 mediates ROCK/Rac1 crosstalk in leader cell ……….199 ROCK activity promotes collective propulsive mode of invasion……….203 ROCK as a therapeutic target to prevent CRC metastatic dissemination?...203

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A. Anatomy of the gastrointestinal system.

Basement membrane Muc osa Subm uc osa Musc ul a ri s Se rosa Goblet Cells i i Intestinal gland Intestinal crypt Lamina propria

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Lumen of the colon

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actin belt

basal lamina junctionnal

complex

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Figure 2 . Cell-cell junctions

(A) Differents types on cell-cell junction in epithelial cells.

(B) E-Cadherin can dimerize and form trans-homophilic interactions to form cadherin clusters. Ca2+ ions are required to stiffen the extracellular domain and are essential to form homophilic interactions. The E-cadherin intracellular domain contains binding sites for the catenins p120 and β-catenin, thereby forming the cadherin–catenin complex. p120 catenin links cadherin to microtubules and is also important to prevent cadherin endocytosis and degradation. β-Catenin binds α-catenin, which in turn binds actin and several actin-associated proteins, including α-actinin, vinculin, and formin-1. The cadherin–catenin complex also binds many other proteins, including signaling proteins, and cell surface receptors

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Figure 3. Integrin architecture and schematic representation of integrin activation

(A)The contacts between the extracellular, the transmembrane and cytoplasmic domains keep the integrin in its bent inactive conformation. Separation of the integrin extracellular, transmembrane and cytoplasmic domains occurs during integrin activation, resulting in an extended integrin conformation. There are two directions of activating integrins. In “inside-out” signaling, an intracellular activator, such as talin or kindlin, bind to the cytoplasmic integrin tail, leading to conformational changes that increase the affinity of integrin for the extracellular ligands. Integrins also serve to transmit the information from the extracellular environment into the cell via “outside-in” signaling. Binding of integrins to the extracellular ligands change the conformation of integrin and contributes to integrin clustering. Adapted from Shattil et al, Nature Reviews Mol Cell Biol 2010.

(B)Outline of adhesive signaling in migration. Integrin ligation induces the nucleation of different signaling elements. The major categories (kinases, non-catalytic adaptor proteins and actin-binding proteins) are shown. These categories can influence the recruitment and/or activation of other components of adhesions (represented by red arrows). Most migratory

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Figure 4 . The actin cytoskeleton. Actin polymerization and regulatory proteins

(A) Ribbon and space-filling models of the actin molecule. Binding site for ATP is depicted in blue in the center of the molecule.

(B) The electron micrograph shows an actin filament seed decorated with myosin II heads and elongated with ATP-actin. Schematic view of the actin filament polymerization and treadmilling. Actin monomers coupled with ATP are preferentially added to the barbed end, while monomers coupled with ADP disassociate from the filament at the pointed end.

(C) Nucleation of actin filaments by Arp2/3 complex. Nucleation-promoting factors such as WASP bind an actin monomer and the Arp2/3 complex. Binding to the side of filament completes activation, and the barbed end of the daughter filament grows from Arp2/3 complex.

(D) Nucleation and elongation by formins. Formins initiate polymerization from free actin monomers and remain associated with the growing barbed end. Profilin-actin binds to formin and transfers actin onto barbed end of the filament.

(E) Actin-binding proteins and their function. ADF/cofilin and profilin bind monomers and regulate actin polymerization; capping proteins bind to and block addition of monomers at barbed ends; cofilin and gelsolin sever filaments; cross-linking proteins assemble networks and bundles actin filaments.

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Figure 5. Structure of myosin II

A. The subunit and domain structure of non-muscle myosin II (NM II), which forms a dimer through interactions between the α-helical coiled-coil rod domains. The globular head domain contains the actin-binding regions and the enzymatic Mg2+-ATPase motor domains. The essential light chains (ELCs) and the regulatory light chains (RLCs) bind to the heavy chains at the lever arms that link the head and rod domains. In the absence of RLC phosphorylation, NM II forms a compact molecule through a head to tail interaction. This results in an assembly- incompetent form (10S; left) that is unable to associate with other NM II dimers. On RLC phosphorylation, the 10S structure unfolds and becomes an assembly-competent form (6S). S-1 is a fragment of NM II that contains the motor domain and neck but lacks the rod domain and is unable to dimerize. Heavy meromyosin (HMM) is a fragment that contains the motor domain, neck and enough of the rod to effect dimerization. B. myosin II molecules assemble into bipolar filaments through interactions between their rod domains. These filaments bind to actin through their head domains and the ATPase activity of the head enables a conformational change that moves actin filaments in an anti-parallel manner. Bipolar myosin filaments link actin filaments together in thick bundles that form cellular s tructures such as stress fibers. Adpated from Vicentre-Manzaranes et al, 2009.

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Figure 6 . Microtubules and microtubule motor proteins

A-Microtubule dynamic

B- Motor protein walking on microtubule C-members of the kinesin superfamily

en fr

HAL Id: tel-01968068

https://tel.archives-ouvertes.fr/tel-01968068

Deciphering the Contribution of RhoGTPases

Dependent Signaling Pathways to the Collective

Invasion of Colorectal Carcinoma

Fotine Libanje

To cite this version:

T

HESE DE DOCTORAT

DE

L’U

NIVERSITE

P

ARIS

-S

ACLAY

PREPAREE A

L

’U

NIVERSITE

P

ARIS

-

SUD

E

D

° 582

CBMS Cancérologie : biologie - médecine - santé

Spécialité de doctorat : Aspect moléculaire et cellulaire de la biologie

Mme Fotine LIBANJE

Deciphering the contribution of Rho-GTPases dependent signaling pathways to the

collective invasion of colorectal carcinoma

Etude de la contribution des voies de signalisation dépendantes des Rho-GTPases

à l’invasion collective des carcinomes colorectaux

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CHAPTER 2: RESULTS

CHAPTER 3: DISCUSSION

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